Neuropilin and angiotensin-converting enzyme 2 fusion peptide for treating viral infections

Abstract

The present disclosure relates to fusion protein compositions and methods for reducing and treating viral infections. The fusion protein comprises a polypeptide containing the b1 domain of neuropilin or a derivative or fragment thereof; the ACE2 domain of angiotensin-converting enzyme 2 or a derivative or fragment thereof; and / or an immunoglobulin domain. Both the b1 and ACE2 domains are capable of binding to the coat protein of a virus selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Retroviridae. In some embodiments, the b1 domain or a derivative or fragment thereof and / or the (ACE2) domain can be used to specifically bind to the S protein of COVID-19 particles.

Description

Technical Field

[0001] (Cross-Reference to Related Applications) This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 059,915 filed on July 31, 2020, the disclosure of which is hereby fully incorporated herein by reference.

[0002] Sequence Listing This application fully incorporates by reference the Sequence Listing titled "268824-494885_ST25.txt" (980 KB), created at 12:21 p.m. on July 30, 2021 and electronically filed herewith.

[0003] The present disclosure relates to fusion protein compositions and methods for reducing and treating viral infections, and more specifically to polypeptides comprising a combination of a neuropilin-1 (NRP1) domain, a neuropilin-2 (NRP2) domain, an angiotensin-converting enzyme 2 (ACE2) domain, and / or an immunoglobulin domain that can be used to specifically bind to a coat protein of a viral particle such as the S protein of COVID-19 virus. Background Art

[0004] Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of July 27, 2020, more than 16,100,000 cases have been reported across 188 countries and regions, resulting in more than 647,000 deaths.

[0005] Common symptoms include fever, cough, fatigue, shortness of breath, and loss of smell and taste. Most cases cause mild symptoms, but some progress to acute respiratory distress syndrome (ARDS), likely caused by cytokine storm, multiple organ dysfunction, septic shock and blood coagulation. The time from exposure to onset of symptoms is generally around 5 days, but can vary from 2 days to 14 days.

[0006] The virus is primarily transmitted between people during close contact, often through small droplets produced by coughing, sneezing, and talking. These droplets usually fall to the ground or a surface rather than travel long distances through the air. Transmission can occur through smaller droplets that can remain suspended in the air for longer periods. Less commonly, a person can become infected by touching a contaminated surface and then touching their face. It is most contagious during the first three days after the onset of symptoms, but transmission is possible before symptoms appear and from asymptomatic individuals. The standard diagnostic method is real-time reverse transcription polymerase chain reaction (rRT-PCR) from nasopharyngeal swabs.

[0007] Currently, there is no vaccine or specific antiviral treatment for COVID-19. Management includes symptomatic treatment, supportive care, isolation, and experimental measures. The World Health Organization (WHO) declared the COVID-19 outbreak a Public Health Emergency of Global Concern (PHEIC) on January 30, 2020, and a pandemic on March 11, 2020. [Overview of the Initiative] [Means for solving the problem]

[0008] In some embodiments, the disclosure provides a polypeptide comprising a neuropyrin bl domain or derivatives or fragments thereof; and an immunoglobulin domain, wherein the bl domain is capable of binding to the coat protein of a virus selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae and Retroviridae.

[0009] In other embodiments, the disclosure provides a polypeptide comprising the ACE2 domain or derivatives or fragments thereof of angiotensin-converting enzyme 2; and an immunoglobulin domain, wherein the ACE2 domain is capable of binding to the coat protein of a virus selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae and Retroviridae.

[0010] In further embodiments, the Disclosure provides polypeptides comprising the bl domain or derivatives or fragments thereof of neuropyrin; and the ACE2 domain or derivatives or fragments thereof of angiotensin-converting enzyme 2, wherein each of the bl domain and the ACE2 domain is capable of binding to the coat protein of a virus selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae and Retroviridae.

[0011] In further other embodiments, the Disclosure provides a polypeptide comprising the bl domain or derivative or fragment thereof of neuropyrin; the ACE2 domain or derivative or fragment thereof of angiotensin-converting enzyme 2; and an immunoglobulin domain, each of which the bl domain and the ACE2 domain is capable of binding to the coat protein of a virus selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae and Retroviridae.

[0012] Embodiments of these aspects of the present invention relating to a polypeptide comprising a combination of two or more domains, including the bl domain or derivatives or fragments of neuropyrin; the ACE2 domain or derivatives or fragments of angiotensin-converting enzyme 2; and an immunoglobulin domain, may include one or more of the following optional features: In some embodiments, the bl domain or derivative or fragment comprises the amino acid sequence of SEQ ID NO: SEQ ID NO: 3 (NRP1 bl) or SEQ ID NO: 11 (NRP2 ​​bl). In some embodiments, the bl domain or derivative or fragment comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the polypeptide is capable of binding to the coat protein of a coronavirus. In some embodiments, the polypeptide is capable of binding to the coat protein of COVID-19. In some embodiments, the coat protein is the S protein of COVID-19. In some embodiments, the b1 domain or derivative or fragment comprises a mutation that enhances affinity to the S protein of COVID-19 compared to a non-mutant b1 domain. In some embodiments, the b1 domain or derivative or fragment comprises a mutation at a position selected from the group consisting of E319 and K351. In some embodiments, the b1 domain contains the amino acid sequence of either SEQ ID NO: SEQ ID NO: 4 (NRP1 b1 E319A) or SEQ ID NO: 5 (NRP1 b1 K351A). In some embodiments, the polypeptide contains multiple b1 domains or derivatives or fragments thereof. In some embodiments, the b1 domain or derivative or fragment thereof further includes a linker, a neuropyrin b2 domain, or a combination thereof. In some embodiments, the b1 domain or derivative or fragment thereof is selected from the group consisting of SEQ ID NOs: 7 to 14. In some embodiments, the ACE2 domain or derivative or fragment thereof contains a sequence selected from the group consisting of SEQ ID NOs: 38 to 39. In some embodiments, the polypeptide contains multiple ACE2 domains or derivatives or fragment thereof. In some embodiments, the ACE2 domain contains a mutation at a position selected from the group consisting of F28, D30, and L79.In some embodiments, the ACE2 domain, its derivatives, or fragments include the amino acid sequence of SEQ ID NOs: SEQ ID NOs: 40 to 43. In some embodiments, the polypeptide further includes a linker between the b1 domain and the ACE2 domain. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 44 to 50. In some embodiments, the immunoglobulin domain includes an Fc domain. In some embodiments, the immunoglobulin domain is essentially composed of an Fc domain. In some embodiments, the Fc domain contains a mutation that reduces ADCC when compared to a wild-type Fc domain. In some embodiments, the mutation is located at position N297 as determined by Kabat-numbering. In some embodiments, the Fc domain contains one or more mutations that enhance affinity to FcRn when compared to a wild-type Fc domain. In some embodiments, the mutation is located at a position or combination thereof selected from the group consisting of T307, E380, and N434 as determined by Kabat-numbering. In some embodiments, the Fc domain contains a mutation that reduces affinity to Fcγ receptor subtypes when compared to a wild-type Fc domain. In some embodiments, the mutation is located at a position selected from the group consisting of L324 and L325, or a combination thereof, as determined by Kabat numbering. In some embodiments, the Fc domain is selected from the group consisting of human IgG1, human IgG2, human IgG3, human IgG4, and human IgA. In some embodiments, the Fc domain contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-31. In some embodiments, the Fc domain sequence contains the amino acid sequence of SEQ ID NOs: 23, 30, or 31.In some embodiments, the polypeptide has a configuration selected from the group consisting of: (b1), IgG1 WT, ACE2-1 polypeptide; (b1b2), IgG1(T307A / E380A / N434A), ACE2-2 polypeptide; (b1b1)-(G4S)*2-(b1b1), IgG1(N297A), ACE2-3 polypeptide; (b1b2)-(G4S)*2-(b1b2), IgG1(L324A / L325A), ACE2-4 polypeptide. ;(b1b2)-(G4S)*2-(b1b2) and b1(E319A), IgG1(N297A / T307A / E380A / N434A), ACE2-5 polypeptide; and (b1b2)-(G4S)*2-(b1b2) and b1(K351A), IgG1(L324A / L325A / T307A / E380A / N434A), ACE2-6 polypeptide. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 88-110. In some embodiments, the b1 domain is bound to the C-terminus of the Fc domain. In some embodiments, the b1 domain is bound to the N-terminus of the Fc domain. In some embodiments, the ACE2 domain is bound to the C-terminus of the Fc domain. In some embodiments, the ACE2 domain is bound to the N-terminus of the Fc domain. In some embodiments, the polypeptide further comprises a signal peptide. In some embodiments, the signal peptide includes SEQ ID NO: 51.

[0013] In some embodiments, the disclosure provides a method for generating a polypeptide disclosed herein, comprising recombinantly expressing a nucleic acid vector encoding the polypeptide in a host cell.

[0014] In some embodiments, the Disclosure provides a pharmaceutical composition comprising a polypeptide disclosed herein and a pharmaceutically acceptable excipient.

[0015] In some embodiments, the Disclosure provides a method for reducing COVID infection, which includes administering the polypeptides disclosed herein to subjects in need thereof.

[0016] In some embodiments, the Disclosure provides a method for treating a subject suffering from COVID infection, which includes administering a polypeptide disclosed herein to a subject in need.

[0017] In some embodiments, the Disclosure provides a method for preventing COVID infection, which includes administering the polypeptides disclosed herein to subjects in need.

[0018] In some embodiments, the Disclosure provides a method for reducing symptoms of COVID-19, which includes administering the polypeptides disclosed herein to subjects in need.

[0019] In some embodiments, the Disclosure provides a method for reducing the transmission of COVID-19, which includes administering the polypeptides disclosed herein to subjects in need.

[0020] Furthermore, this disclosure describes a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) domains or fragments thereof, and (b) an immunoglobulin domain, wherein the one or more mutant NRP domains result in a reduced binding of the recombinant polypeptide to heparin or heparan sulfate compared to a wild-type NRP domain.

[0021] Furthermore, this disclosure describes a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) b1 domains, NRP b2 domains or fragments thereof, and (b) an Fc domain, wherein one or more mutant NRP b1 domains, NRP b2 domains or fragments thereof are derived from NRP1 or NRP2 proteins, and one or more mutant NRP b1 domains, NRP b2 domains or fragments thereof have one or more amino substitutions selected from the group consisting of K373E, K351A, E319A, K358E, R513E, K514E, K516E, R513A, K514A, K516A, Y297A, S345A and Y353A compared to the wild-type amino acid sequence shown in SEQ ID NO: 1; and one or more amino substitutions result in a reduction in the binding of the recombinant polypeptide to heparin or heparan sulfate.

[0022] Furthermore, this disclosure includes (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP A recombinant polypeptide comprising a b2 domain (b2) or a fragment thereof, and (b) an Fc domain, wherein (a) and (b) comprise constructs having the following orientations: b1-Fc; b1b1-Fc; b1b1b1-Fc; b1-Fc; b1b1b1-Fc; b1b2-Fc; b1b2-Fc; b1b2-Fc; b1b2-Fc; Fc-b1b2; Fc-b1b2; b1-Fc-b1; b1b1-Fc-b1; b1b1b1-Fc; b1-Fc; b1b1b1-Fc; b1-Fc; b1b1b1-Fc; b1b2-Fc; b1b2-Fc; Fc-b1b2; Fc-b1b2; b1-Fc -b1;b1b1-Fc-b1;one or more b1, b2 or fragments thereof are derived from the NRP1 or NRP2 protein; one or more b1, b2 or fragments thereof have one or more amino substitutions selected from the group consisting of K373E, K351A, E319A, K358E, R513E, K514E, K516E, R513A, K514A, K516A, Y297A, S345A and Y353A compared to the wild-type amino acid sequence shown in SEQ ID NO: 1; one or more amino substitutions result in a reduction of the binding of the recombinant polypeptide to heparin or heparan sulfate; describes a recombinant polypeptide.

[0023] Furthermore, the disclosure describes a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) domains or fragments thereof, and (b) an immunoglobulin domain, wherein the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine and X1 and X2 are any amino acids.

[0024] Furthermore, the present disclosure relates to a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2) or fragments thereof, and (b) an Fc domain, wherein one or more b1, b2 or fragments thereof are derived from NRP1 or NRP2 proteins; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids; the viruses include: dengue fever; respiratory syncytial virus (RSV); hantavirus; Epstein-Barr virus (EBV); EBV (uncleaved); SARS-CoV-2 Wuhan; SARS-CoV-2 Wuhan (uncleaved); SARS-CoV-2 UK; SARS-CoV-2 India; SARS-CoV-2 Recombinant polypeptides selected from the group consisting of India (uncleaved); HCoV-OC43; MERS-CoV; MERS-CoV (uncleaved); herpes simplex virus (HSV) 1; HSV 1 (uncleaved); influenza A H5N1 virus (IAV H5N1); human papillomavirus (HPV); human metapneumovirus; and human immunodeficiency virus (HIV) are described.

[0025] Further, the present disclosure provides a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) b1 domain (b1), NRP b2 domain (b2) or fragments thereof, and (b) an Fc domain, wherein the one or more b1, b2 or fragments thereof are derived from NRP1 or NRP2 protein, the recombinant polypeptide is operable to bind to a virus having a -Z1-X1-X2-Z2-(CendR) motif, wherein Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acid; the virus has a CendR motif selected from the group consisting of: GTCTQSGERRREKR; KNTNVTLSKKRKRR; LTHKMIEESHRLRR; VSFKPPPPPSRRRR; VSFKPPPPPSRRRRGACVVY; CASYQTQTNSPRRAR; CASYQTQTNSPRRARSVASQSIIAYTMSLG; ASYQTQTNSHRRAR; ASYQTQTNSRRRAR; ASYQTQTNSRRRARSVASQSIIAY; GSGYCVDYSKNRRSR; LLEPVSISTGSRSAR; LLEPVSISTGSRSARSAIEDLLFDK; ERPRAPARSASRPRR; ERPRAPARSASRPRRPV; VLATGLRNVPQRKKR; PTTSSTSTTAKRKKR; IDMLKARVKNRVAR; AKRRVVQREKR; and AKRRVVQREKRAVGIGALFLG. The present disclosure describes said recombinant polypeptide.

[0026] Further, the present disclosure describes a recombinant polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 116, 121 to 122, 133 to 137, 148 to 149, 154, 162 and 193 to 201, or a pharmaceutically acceptable salt thereof.

[0027] Further, the present disclosure describes a recombinant polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 116, 121 to 122, 133 to 137, 148 to 149, 154, 162 and 193 to 201, or a pharmaceutically acceptable salt thereof.

[0028] The present disclosure further provides a recombinant polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 116, 121 to 122, 133 to 137, 148 to 149, 154, 162, and 193 to 201, or a pharmaceutically acceptable salt thereof.

[0029] The present disclosure further provides a method for restricting the occurrence, reducing the risk, reducing the severity, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of a recombinant polypeptide comprising (a) one or more mutated neuropilin (NRP) domain or a fragment thereof, and (b) an immunoglobulin domain, wherein the recombinant polypeptide is operable to bind to a virus having a -Z1-X1-X2-Z2-(CendR) motif, wherein Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids.

[0030] Furthermore, the present disclosure relates to a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2) or fragments thereof, and (b) a therapeutically effective amount of a recombinant polypeptide comprising an Fc domain, wherein one or more b1, b2 or fragments thereof are derived from the NRP1 or NRP2 protein; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids; the viruses include: dengue fever; respiratory syncytial virus (RSV); hantavirus; Epstein-Barr virus (EBV); EBV (uncleaved); SARS-CoV-2 Wuhan; SARS-CoV-2 Wuhan (uncleaved); SARS-CoV-2 UK; SARS-CoV-2 Describe a method for selecting from the group consisting of India; SARS-CoV-2 India (uncleaved); HCoV-OC43; MERS-CoV; MERS-CoV (uncleaved); herpes simplex virus (HSV) 1; HSV 1 (uncleaved); influenza A H5N1 virus (IAV H5N1); human papillomavirus (HPV); human metapneumovirus; and human immunodeficiency virus (HIV).

[0031] Furthermore, the present disclosure provides a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2) or fragments thereof, and (b) a recombinant polypeptide comprising an Fc domain, wherein one or more b1, b2 or fragments thereof are derived from the NRP1 or NRP2 protein; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids; and the virus is:GTCTQSGERRREKR;KNTNVTLSKKRKRR;LTHKMIEESHRLRR;VSFKPPPPPSRRRR;VSFKPPPPPSRRRRGACVVY;CASYQTQ A method is described that has a CendR motif selected from the group consisting of TNSPRRAR;CASYQTQTNSPRRARSVASQSIIAYTMSLG;ASYQTQTNSHRRAR;ASYQTQTNSRRRAR;ASYQTQTNSRRRARSVASQSIIAY;GSGYCVDYSKNRRSR;LLEPVSISTGSRSAR;LLEPVSISTGSRSARSAIEDLLFDK;ERPRAPARSASRPRR;ERPRAPARSASRPRRPV;VLATGLRNVPQRKKR;PTTSSTSTTAKRKKR;IDMLKARVKNRVAR;AKRRVVQREKR; and AKRRVVQREKRAVGIGALFLG.

[0032] Furthermore, the disclosure describes a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of a recombinant polypeptide having an amino acid sequence that is at least 90% identical to one of the amino acid sequences shown in SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162, and 193-201, or a pharmaceutically acceptable salt thereof.

[0033] Furthermore, the disclosure describes a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of a recombinant polypeptide having an amino acid sequence that is at least 90% identical to one of the amino acid sequences shown in SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162, and 193-201, or a pharmaceutically acceptable salt thereof.

[0034] Furthermore, the disclosure describes a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of a recombinant polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162, and 193-201, or a pharmaceutically acceptable salt thereof.

[0035] Furthermore, this disclosure describes recombinant polypeptides having an amino acid sequence that is at least 90% identical to any one of the amino acid sequences shown in SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162 and 193-201, or pharmaceutically acceptable salts thereof, further comprising excipients.

[0036] Furthermore, the Disclosure describes a method for limiting, reducing the risk of, or reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of the recombinant polypeptide of the present invention, wherein the virus is a virus belonging to one of the following families: Astroviridae, Bunyaviridae, Bornaviridae, Chuviridae, Flaviviridae, Filoviridae, Hantaviridae, Hepeviridae, Herpesviridae, Nairoviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Peribunyaviridae, Fenuiviridae, Pneumoviridae, Poxviridae, Retroviridae, Raptoviridae, or Togaviridae.

[0037] Furthermore, the Disclosure describes a method for limiting, reducing the risk of, or reducing the severity of a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of the recombinant polypeptide of the present invention, wherein the virus is selected from the group consisting of: dengue fever; respiratory syncytial virus (RSV); hantavirus; Epstein-Barr virus (EBV); EBV (uncleaved); HCoV-OC43; MERS-CoV; MERS-CoV (uncleaved); herpes simplex virus (HSV) 1; HSV 1 (uncleaved); influenza A H5N1 virus (IAV H5N1); human papillomavirus (HPV); human metapneumovirus; and human immunodeficiency virus (HIV).

[0038] Furthermore, the Disclosure describes a method for limiting, reducing the risk of, or reducing the severity of a viral infection in a subject requiring it, comprising administering to the subject a composition comprising a therapeutically effective amount of the recombinant polypeptide of the present invention, wherein the virus has a CendR motif selected from the group consisting of:GTCTQSGERRREKR;KNTNVTLSKKRKRR;LTHKMIEESHRLRR;VSFKPPPPPSRRRR;VSFKPPPPPSRRRRGACVVY;GSGYCVDYSKNRRSR;LLEPVSISTGSRSAR;LLEPVSISTGSRSARSAIEDLLFDK;ERPRAPARSASRPRR;ERPRAPARSASRPRRPV;VLATGLRNVPQRKKR;PTTSSTSTTAKRKKR;IDMLKARVKNRVAR;AKRRVVQREKR; andAKRRVVQREKRAVGIGALFLG.

[0039] Furthermore, this disclosure relates to a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject that requires it, wherein the subject is provided with (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP The administration of a composition comprising a therapeutically effective amount of a recombinant polypeptide or a pharmaceutically acceptable salt thereof comprising the b2 domain (b2) or a fragment thereof, and (b)Fc domain, wherein one or more b1, b2 or fragment thereof are derived from the NRP1 or NRP2 protein; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, X1 and X2 are any amino acids, Z is arginine or lysine, and X is any amino acid; the viruses include: dengue fever; respiratory syncytial virus (RSV); hantavirus; Epstein-Barr virus (EBV); EBV (uncleaved); HCoV-OC43; MERS-CoV; MERS-CoV (uncleaved); herpes simplex virus (HSV) 1; HSV 1 (uncleaved); influenza A H5N1 virus (IAV Describe a method selected from the group consisting of H5N1, human papillomavirus (HPV), human metapneumovirus, and human immunodeficiency virus (HIV).

[0040] Furthermore, the present disclosure relates to a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2) or fragments thereof, and (b) a recombinant polypeptide or a pharmaceutically acceptable salt thereof comprising an Fc domain, wherein one or more b1, b2 or fragments thereof are derived from the NRP1 or NRP2 protein; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids; and the virus is:GTCTQSGERRREKR;KNTNVTLSKKRKRR A method is described that has a CendR motif selected from the group consisting of ;LTHKMIEESHRLRR;VSFKPPPPPSRRRR;VSFKPPPPPSRRRRGACVVY;GSGYCVDYSKNRRSR;LLEPVSISTGSRSAR;LLEPVSISTGSRSARSAIEDLLFDK;ERPRAPARSASRPRR;ERPRAPARSASRPRRPV;VLATGLRNVPQRKKR;PTTSSTSTTAKRKKR;IDMLKARVKNRVAR;AKRRVVQREKR; and AKRRVVQREKRAVGIGALFLG.

[0041] Furthermore, this disclosure describes a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of the recombinant polypeptide of the present invention or a pharmaceutically acceptable salt thereof, wherein the virus is not SARS-CoV-2.

[0042] Furthermore, the Disclosure describes a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of the recombinant polypeptide of the present invention or a pharmaceutically acceptable salt thereof, wherein the virus is neither SARS-CoV-2 Wuhan; SARS-CoV-2 Wuhan (uncleaved); SARS-CoV-2 UK; SARS-CoV-2 India; or SARS-CoV-2 India (uncleaved).

[0043] Furthermore, this disclosure describes a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of the recombinant polypeptide of the present invention or a pharmaceutically acceptable salt thereof, wherein the virus is a virus not belonging to the Coronaviridae family.

[0044] Furthermore, this disclosure describes a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of the recombinant polypeptide of the present invention or a pharmaceutically acceptable salt thereof, wherein the virus is a virus not belonging to the genus Betacoronavirus.

[0045] Furthermore, the Disclosure describes a method for limiting, reducing the risk of, reducing the severity of, or treating a viral infection in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of the recombinant polypeptide of the present invention or a pharmaceutically acceptable salt thereof, wherein the virus is a virus not belonging to the subgenus Salvecovirus.

[0046] The following drawings are provided as examples and are not intended to limit the scope of the invention. [Brief explanation of the drawing]

[0047] [Figure 1A] This is a schematic diagram of the ACE2 receptor and its function, in addition to the mechanism by which the SARS-CoV-2 spike protein binds and enables viral entry. [Figure 1B] This is a schematic diagram of antibody neutralization of SARS-CoV-2 virus particles. [Figure 1C] This is a comparative chart of SARS-CoV-1 and SARS-CoV-2 entry sites, corresponding symptoms, and infected tissues. [Figure 2A] This is an illustration of polypeptides used to neutralize the SARS-CoV-2 virus. [Figure 2B] This is a schematic diagram of a polypeptide having sNRP1(b1), sACE2(ACE2), a linker, and an immunoglobulin (Fc) domain that can effectively bind to one or more SARS-CoV-2 virus particles. [Figure 2C] b1 is a diagram showing the furin cleavage sites on the SARS-CoV-2 virus spike protein that bind to the NRP1 binding region. [Figure 3A] This is a schematic diagram of SARS-CoV-2 pinocytotic infection using NRP1 and / or ACE2 receptors. [Figure 3B] This is a diagram illustrating how antibody constructs can neutralize and opsonize the SARS-CoV-2 virus. [Figure 3C] This is a diagram illustrating the proposed mechanism of current vaccines and / or therapeutic polypeptide therapy for SARS-CoV-2 treatment. [Figure 3D] This is a diagram illustrating the proposed mechanism of the disclosed polypeptide therapeutics for SARS-CoV-2 treatment. [Figure 4] This is a schematic diagram of an exemplary N1-Fc polypeptide construct disclosed in the present invention. [Figure 5]This is a schematic diagram of an exemplary N1-Fc-ACE2 polypeptide construct disclosed in the present invention. [Figure 6] This is a plot of the relative fluorescence units (RFUs) in the wells corresponding to the ACE2 peptide (A) bound to the S1 / S2 spike protein and the S protein CendR (B) bound to hu-b1b2-His. [Figure 7] This is a plot of HuN1abHuIgG bound to the SARS-CoV-2 spike protein, determined using relative fluorescence units (RFU). [Figure 8] This graph shows the mean daily weight of hamsters inoculated with 51 plaque-forming units (PFUs) of SARS-CoV-2. The three groups include virus-free controls; hamsters inoculated with 51 PFUs; and hamsters inoculated with 51 PFUs and treated with compound 1 (SEQ ID NO: 122) at a dose of 15 mg / kg intraperitoneally. Error bars indicate the standard deviation (SD). [Figure 9] This graph shows the mean daily weight of hamsters inoculated with 510 plaque-forming units (PFUs) of SARS-CoV-2. The three groups include virus-free controls; hamsters inoculated with 510 PFUs; and hamsters inoculated with 510 PFUs and treated with compound 1 (SEQ ID NO: 122) or SAD-S35 at 15 mg / kg intraperitoneal injection. Error bars indicate the standard deviation (SD). [Figure 10] This is a graphical representation of the respiratory cycle, showing the various measurements used to calculate respiratory parameters for comparison between groups in this study. (Enhanced Pause) Penh shows a single respiratory cycle, showing the various measurements used to calculate respiratory parameters. Here, PEF is the expiratory flow peak of respiration; PIF is the inspiratory flow peak of respiration; Te is the time of the expiratory portion of respiration; and Tr is the time required to exhale 65% of the respiratory volume. [Figure 11] This graph shows a single expiratory portion of the respiratory cycle, representing the total expiratory time and the time to the expiratory flow peak relative to Te. [Figure 12]This graph shows a single exhalation portion of the respiratory cycle, indicating the time it takes to exhale 50% of the total expiratory volume. [Figure 13] This graph shows the logarithm of Penh results for plethysmography data from hamsters treated with 51 PFU. Data are expressed as mean + 1 standard error. [Figure 14] This graph shows the logarithm of the Penh results for plethysmography data from hamsters treated with 510 PFU. The data is expressed as mean + 1 standard error. [Figure 15] This graph shows the natural logarithm (ln) of the Penh results for plethysmography data from hamsters treated with 51 PFU. The data is expressed as mean + 1 standard error. [Figure 16] This graph shows the ln of the Penh results for plethysmography data from hamsters treated with 510 PFU. The data is expressed as mean + 1 standard error. [Figure 17] This graph shows the square root of the EF50 results of plethysmography data from hamsters treated with 51 PFU. The data is expressed as mean + 1 standard error. [Figure 18] This graph shows the square root of the EF50 results of plethysmography data from hamsters treated with 510 PFU. The data is expressed as mean + 1 standard error. [Figure 19] This is a 4x magnified micrograph of a paraffin histological thin section stained with H&E, obtained from a hamster belonging to the control group inoculated with culture medium. No inflammation is present. Red size bar = 150 μM. [Figure 20] Micrograph at 4x magnification of paraffin histological thin sections stained with H&E, obtained from hamsters treated with 51 PFU. Several areas of chronic active inflammation are present, consisting of macrophages, lymphocytes, plasma cells, and neutrophils; see, for example, inset (A) at 40x magnification. Red size bars = 150 μM, 40x magnification; green size bars = 50 μM. [Figure 21]This is a 4x magnification micrograph of a paraffin histological thin section stained with H&E, obtained from a hamster belonging to the control group inoculated with 510 PFU SARS-CoV-2. Several areas of chronic active inflammation are present, consisting of macrophages, lymphocytes, plasma cells, and neutrophils; see, for example, inset (A) at 40x magnification. Red size bars = 150 μM, 40x magnification; green size bars = 50 μM. [Figure 22] This is a 4x magnification micrograph of paraffin histological thin sections stained with H&E, obtained from hamsters treated with 51 PFU and SEQ ID NO: 122 (15 mg / kg) by intraperitoneal injection. The red size bars represent 150 μM and 40x magnification, while the green size bars represent 50 μM. [Figure 23] This is a 4x magnification micrograph of paraffin histological thin sections stained with H&E, obtained from hamsters treated with 510 PFU and SEQ ID NO: 122 (15 mg / kg) by intraperitoneal injection. The red size bar represents 150 μM at 40x magnification, and the green size bar represents 50 μM. [Figure 24] This is a 4x magnification micrograph of paraffin histological thin sections stained with H&E, obtained from hamsters treated with 510 PFU and SAD35 (15 mg / kg) by intraperitoneal injection. The red size bar represents 150 μM at 40x magnification, and the green size bar represents 50 μM. [Figure 25] This graph shows the weight in grams over time for hamsters treated with a construct and loaded with 1500 PFU of SARS-CoV-2. Here, 1 = Sequence ID 122; 2 = Sequence ID 154; and 3 = Sequence ID 192. Error bars indicate the standard deviation (SD). [Figure 26] This graph shows the nucleocapsid gene copy number divided by the viral titer detected as 1 μL of RNA extracted from a pharyngeal swab or BAL. Here, 1 = SEQ ID NO: 122; 2 = SEQ ID NO: 154; and 3 = SEQ ID NO: 192. Error bars indicate the standard deviation (SD). [Figure 27]This graph shows the SARS-CoV-2 copy number per 1 μL of RNA extracted from the olfactory bulb. Here, 1 = SEQ ID NO: 122; 2 = SEQ ID NO: 154; and 3 = SEQ ID NO: 192. Error bars indicate the standard deviation (SD). [Figure 28] This graph shows the EF50 (mL / sec) for each group over time. Here, 1 = Sequence ID 122; 2 = Sequence ID 154; and 3 = Sequence ID 192. Error bars indicate the standard deviation (SD). [Figure 29] This graph shows the Penh sequence over time for each group. Here, 1 = sequence number 122; 2 = sequence number 154; and 3 = sequence number 192. Error bars indicate the standard deviation (SD). [Figure 30] This graph shows the Rpef (Rate of Performance) for each group over time. Here, 1 = Sequence ID 122; 2 = Sequence ID 154; and 3 = Sequence ID 192. Error bars indicate the standard deviation (SD). [Figure 31] This graph shows the interferon-gamma (IFNγ) levels (pg / mL) in each group over time. Here, 1 = SEQ ID NO: 122; 2 = SEQ ID NO: 154; and 3 = SEQ ID NO: 192. Error bars indicate the standard deviation (SD). [Figure 32] This graph shows the results of cytokine analysis of angiotensin 1-7 (Ang1-7) levels during SARS-CoV-2 infection in hamsters over time. Here, 1 = SEQ ID NO: 122; 2 = SEQ ID NO: 154; and 3 = SEQ ID NO: 192. Error bars indicate standard deviation (SD). [Figure 33] This graph shows the results of cytokine analysis of angiotensin II levels during SARS-CoV-2 infection in hamsters over time. Here, 1 = SEQ ID NO: 122; 2 = SEQ ID NO: 154; and 3 = SEQ ID NO: 192. Error bars indicate standard deviation (SD). [Figure 34] This graph shows the ratios of angiotensin II levels Ang1-7 in hamsters over time during SARS-CoV-2 infection. Here, 1 = SEQ ID NO: 122; 2 = SEQ ID NO: 154; and 3 = SEQ ID NO: 192. Error bars indicate standard deviation (SD). [Figure 35] Micrograph of a formalin-fixed, H&E-stained hamster lung at 4x magnification. Evidence of bronchopneumonia is shown on day 7, the end of the experiment. Panel A = medium control; Panel B = virus control; Panel C = SEQ ID NO: 122; Panel D = SEQ ID NO: 154; and Panel E = SEQ ID NO: 192. Note the areas of inflammation, which are strongly blue and contain macrophages, neutrophils, and lymphocytes. Size bar = 1 mm. [Figure 36] The following histogram shows the scoring of inflammation in histological sections provided in Figure 35, scored using the scheme below: (a) Lesion distribution: none=0; localized=1; multifocal=2; diffuse=3; (b) Inflammation intensity: none=0; mild (thickness of 2-3 inflammatory cells)=1; moderate (thickness of 3-20 inflammatory cells), severe (thickness of more than 20 inflammatory cells); (c) Small vessel thrombosis: none=0; present=1. There was no significant difference between the treatment group and the control (Wilcoxon rank-sum test). [Figure 37] This graph shows the body weight of all K18ACE2 mice measured daily during the progression of SARS-CoV-2 B1.351 infection. Body weight of K18-ACE2 mice loaded with SARS-CoV-2 B1.351 in the following four groups: Group (1): N=13 each for viral inoculation and administration of SEQ ID NO: 113 (15 mg / kg); Group (2): N=13 each for viral inoculation and administration of an antibody that binds to the SARS-CoV-2 spike protein, having a heavy chain containing the amino acid sequence shown in SEQ ID NO: 189 and a light chain containing the amino acid sequence shown in SEQ ID NO: 190 (anti-SARS-CoV-2 spike protein antibody), 1.2 mg / kg; Group (3): N=6 intranasal control in cell culture medium with IP saline administration; Group (4): N=6 viral inoculation with IP saline administration. Viral inoculated mice in Groups 1 and 4 began showing clinical signs of disease by day 4, a small number died from viral infection, and the rest were all diseased by day 7, the end of the study. Here, 1 = Sequence ID 113; 2 = Anti-SARS-CoV-2 spike protein antibody. [Figure 38]This graph shows the clinical scores of all K18ACE2 mice measured daily during the progression of SARS-CoV-2 infection of strain B1.351. Here, 1 = SEQ ID NO: 113; 2 = Anti-SARS-CoV-2 spike protein antibody. [Figure 39] This graph shows the serum fibrin degradation products of all K18ACE2 mice, measured daily during the progression of SARS-CoV-2 infection with strain B1.351. Here, 1 = SEQ ID NO: 113; 2 = Anti-SARS-CoV-2 spike protein antibody. [Figure 40] This graph shows D-dimer levels in K18ACE2 mice measured at the end of a study following infection with strain B1.351. SARS-CoV-2. A decrease in mean serum D-dimer levels appears to be present in the groups treated with SEQ ID NO: 113 and anti-SARS-CoV-2 spike protein antibody, but the mean was not statistically different from placebo and viral controls (one-way ANOVA with Tukey's multiple comparisons). Here, 1 = SEQ ID NO: 113; 2 = anti-SARS-CoV-2 spike protein antibody (heavy chain containing the amino acid sequence shown in SEQ ID NO: 189 and light chain containing the amino acid sequence shown in SEQ ID NO: 190). [Figure 41] This graph shows the heparin binding affinity profile for the construct molecule of the present invention. (A) is SEQ ID NO: 113; (B) is SEQ ID NO: 121; (C) is SEQ ID NO: 122; (N) is SEQ ID NO: 191; (O) is SEQ ID NO: 121; (G) is SEQ ID NO: 128; (L) is SEQ ID NO: 129; (M) is SEQ ID NO: 154; (D) is SEQ ID NO: 135; (E) is SEQ ID NO: 136; (F) is SEQ ID NO: 137; (H) is SEQ ID NO: 114; (I) is SEQ ID NO: 115; (J) is SEQ ID NO: 116; (K) is SEQ ID NO: 133. [Figure 42] This figure illustrates the construct of the present invention. The upper gray portion shows the neuropilin b1 domain linked to a further b1 domain (shown in double tandem) by a short-chain peptide sequence known as a G4S linker, and the black portion shows the stem of IgG Fc consisting of constant heavy chains (CH2 and CH3). [Figure 43] This figure shows the cumulative distribution of PiTou scores in human peptides. [Figure 44] This figure shows the cumulative distribution of PiTou scores for viral peptides. [Figure 45] This figure shows the cumulative distribution of PiTou scores in bacterial peptides. [Figure 46] This figure shows the PiTou score at known virus cleavage sites. [Figure 47] This figure shows the prioritized PiTou score distribution. [Modes for carrying out the invention]

[0048] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by a person skilled in the art to which this disclosure belongs. Therefore, the following terms have the following meanings:

[0049] As used herein and in the claims, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include the plural forms.

[0050] As used herein, “administration” of the disclosed polypeptides includes the delivery of the polypeptides or compositions of the present invention as described herein, or prodrugs or other pharmaceutically acceptable derivatives thereof, to a subject using any preferred formulation or route of administration, such as those described herein.

[0051] As used herein, the term "and / or" when used in a list of two or more items means that any one of the listed items may be used alone, or any combination of two or more of the listed items may be used. For example, if a composition is described as containing components A, B and / or C, the composition may contain only A; only B; only C; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B and C.

[0052] As used herein, “treatment” and “to treat” are used interchangeably herein and refer to an approach to obtain a beneficial or desired outcome, including but not limited to therapeutic effects. A therapeutic benefit means the eradication or improvement of the underlying disease being treated. Furthermore, a therapeutic benefit is achieved by the eradication or improvement of one or more physiological symptoms associated with the underlying disease, such that improvement is observed in the patient, but nevertheless, the patient may still suffer from the underlying disease. The term “to treat” is used herein in all its verbal forms to mean reducing, alleviating, preventing and / or managing at least one symptom of a disorder in a subject.

[0053] As used herein, “subject” can refer to any animal susceptible to viral infections, such as mammals, including laboratory animals, farm animals, and pets. In some embodiments, the animal is a primate, preferably a human. As used herein, the terms “subject” and “patient” are used interchangeably. The terms “subject” and “patient” refer to animals (e.g., birds, e.g., chickens, quail, or turkeys, or mammals), specifically “mammals” including non-primates (e.g., cattle, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs, and mice) and primates (e.g., monkeys, chimpanzees, and humans), more specifically humans. In one embodiment, the subject is a non-human animal, such as a farm animal (e.g., a horse, cattle, pig, or sheep) or a pet (e.g., a dog, cat, guinea pig, or rabbit). In a preferred embodiment, the subject is a “human.”

[0054] As used herein, the term “fusion” refers to the integration of two molecules having the same or different function or structure, and the method of fusion may include any physical, chemical or biological method capable of binding a peptide to a protein, small molecule drug, nanoparticle or liposome. Preferably, the fusion may be mediated by a linker peptide, for example, a linker peptide may be fused to the C-terminus of a fragment of the antibody light chain variable region (Fc).

[0055] The terms “disease,” “disorder,” and “condition” can be used interchangeably here to refer to a medical or pathological condition transmitted by a virus.

[0056] As used herein, the term “biological sample” includes, but is not limited to, cell cultures or extracts thereof; biopsy material obtained from mammals or extracts thereof; and blood, saliva, urine, feces, semen, tears or other bodily fluids or extracts thereof.

[0057] As used herein, “multiplicity of infection” or “MOI” is the ratio of an infectious agent (e.g., a phage or virus) to an infectious target (e.g., a cell). For example, when referring to a population of cells inoculated with infectious viral particles, the multiplicity of infection or MOI is the ratio determined by dividing the number of infectious viral particles placed in a well by the number of target cells present in that well.

[0058] As used herein, the term “inhibition of SARS-CoV-2 virus replication” includes both a reduction in the amount of viral replication (e.g., a reduction of at least 10%) and a complete cessation of viral replication (i.e., a 100% reduction in the amount of viral replication). In some embodiments, SARS-CoV-2 replication is inhibited by at least 50%, at least 65%, at least 75%, at least 85%, at least 90%, or at least 95%.

[0059] As used herein, “viral titer (or titer)” is a measure of viral concentration. Titer testing can utilize serial dilutions to obtain approximate quantitative information from analytical procedures that would otherwise only be evaluated as positive or negative. The titer corresponds to the highest dilution factor that still yields a positive reading; for example, a positive reading in the first eight consecutive 2-fold dilutions is interpreted as a titer of 1:256. A specific example is viral titer. To determine titer, several dilutions such as 10⁻¹, 10⁻², 10⁻³, ... 10⁻⁸ can be prepared. The lowest concentration of virus that still infects cells is the viral titer.

[0060] As used herein, the terms “to treat,” “treatment,” and “doing to treat” are used herein in general to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely stabilizes or cures a disease and / or adverse effects resulting from the disease. “Treatment” as used herein encompasses any treatment of a disease in a subject, particularly in humans, and includes (a) preventing the occurrence of a disease or symptoms in a subject who may be predisposed to the disease or symptoms but has not yet been diagnosed with it; (b) suppressing disease symptoms, i.e., preventing their occurrence; or (c) reducing disease symptoms, i.e., causing regression of the disease or symptoms. Persons requiring treatment include individuals already diagnosed with a disease, such as a viral infection, as well as persons who should be prevented from developing a disease. Thus, the terms “to treat,” “treatment,” and “doing to treat” refer to both therapeutic and prophylactic treatments. For example, therapeutic treatments include reducing or improving the progression, severity, and / or duration of a disease-mediated condition, or improving one or more symptoms of a disease-mediated condition (specifically, one or more identifiable symptoms), resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as polypeptides or compositions of the present invention).

[0061] In specific embodiments, therapeutic treatments include improvement of at least one measurable physical parameter of the virus-borne condition. In other embodiments, therapeutic treatments include inhibition of the progression of the virus-borne condition, for example, by stabilization of identifiable symptoms, by physiological stabilization of physical parameters, or both. In other embodiments, therapeutic treatments include reduction or stabilization of the virus-borne infection. Antiviral drugs can be used in social settings to treat people who already have COVID-19 in order to reduce the severity of symptoms and the number of days people are ill.

[0062] As used herein, the terms “prevention,” “preventive use,” and “preventive action” refer to any medical or public health procedure whose purpose is to prevent a disease rather than to treat or cure it. As used herein, the terms “prevent,” “prevent,” and “preventing” refer to reducing the risk of obtaining or causing a given condition, or reducing or inhibiting the recurrence of such condition, in a subject that is not diseased but has been in close contact with or potentially been in close contact with a diseased person. The term “preventive chemotherapy” refers to the use of a drug, such as a small molecule drug (rather than a “vaccine”), for the prevention of a disorder or disease.

[0063] As used herein, prophylactic use includes use to prevent the transmission or spread of an infectious disease in situations where an outbreak has been detected, in places where many people at high risk of serious viral (e.g., COVID-19) complications live in close proximity to one another (e.g., hospital wards, daycare centers, prisons, nursing homes, etc.). It also includes use in populations where protection from SARS-CoV-2 is needed but protection is not obtained after vaccination (e.g., due to a weakened immune system), or when the vaccine is unavailable or they are unable to receive the vaccine due to side effects. It also includes use in the two weeks following vaccination, as the vaccine is not yet effective during this period. Prophylactic use may also include treating individuals who have not developed the disease from SARS-CoV-2 and are not considered to be at high risk of complications, in order to reduce the likelihood of infection with SARS-CoV-2 and the likelihood of transmitting it to high-risk individuals with whom they have had close contact (e.g., healthcare workers, ward workers, etc.).

[0064] As used herein, “effective dose” refers to an amount sufficient to produce a desired biological response. In the present invention, a desired biological response is to inhibit the replication of a virus (e.g., SARS-CoV-2), reduce the amount of virus, reduce or improve the severity, duration, progression or onset of a viral infection, prevent the progression of a viral infection, prevent the recurrence, occurrence, onset or progression of symptoms associated with a viral infection, or enhance or improve the prophylactic or therapeutic effect (may be one) of another therapy used against a viral infection. The exact amount of compound administered to a subject depends on the mode of administration, the type and severity of the infection, and the subject’s characteristics, such as general health status, age, sex, weight, and tolerance to the drug. Those skilled in the art can determine an appropriate dosage based on these and other factors. When administered concurrently with other antiviral agents, for example, concurrently with antiviral pharmaceuticals, the “effective dose” of the second agent depends on the type of drug used. Preferred dosages are known for approved agents and can be adjusted by those skilled in the art depending on the condition of the subject, the type of condition being treated, and the amount of polypeptide described herein used. Where the amount is not explicitly stated, the effective dose should be assumed. For example, the compounds described herein can be administered to subjects in a dosage range of approximately 0.01 to 100 mg / kg body weight / day for therapeutic or prophylactic treatment.

[0065] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues.

[0066] The term “reduce” or other forms of the word, e.g., “reduce” or “to reduce,” generally refers to reducing an event or characteristic (e.g., one or more symptoms, or the binding of one protein to another). This is generally understood to be relative to some standard or expected value, in other words, it is understood not necessarily to be relating to the standard or relative value being referenced. In some embodiments, when the term “reduce” is used in the context of “reduce risk” or “reduce severity,” it means reducing the risk of infection with a given disease or virus compared to a subject not treated according to the compositions and / or methods of the present invention; or reducing the severity and / or frequency of symptoms, and / or eliminating the symptoms, of a given disease or virus.

[0067] In the context of "reduced binding," the term "reduced" refers to a decrease in the affinity of one molecule to another. For example, in some embodiments, a protein, domain, or motif can specifically bind to a particular target, such as a peptide, polypeptide, protein, carbohydrate, saccharide, polysaccharide, glycosaminoglycan, or any of its epitopes, with a given affinity; said reduction in binding refers to a decrease in the affinity of said protein, domain, or motif to its target. Measuring binding affinity is well known in the art. In some embodiments, the affinity of one molecule to another to which it specifically binds is characterized by a dissociation constant (KD or Kd).

[0068] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule and its binding target or partner (e.g., an antigen). The affinity of a molecule to its target can generally be expressed by the dissociation constant (KD), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). In short, the strength or affinity of a binding interaction can be expressed by the dissociation constant (KD) of the interaction, with smaller KDs indicating greater affinity. The binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, whose rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally influence the rates in both directions. Thus, both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculating the concentrations as well as the actual rates of association and dissociation. (See Nature 361: pp. 186-87 (1993)). The Koff / Kon ratio allows for the dissociation of all parameters unrelated to affinity and is equal to the dissociation constant KD. (Generally, refer to Davies et al. (1990) Annual Rev Biochem 59: pp. 439-473).

[0069] Therefore, equivalent affinities can include different rate constants, as long as the ratio of rate constants is the same. Affinity can be measured by established methods known in the art, including those described herein. Accordingly, in some embodiments, “reduced binding” refers to a decrease in affinity to each interaction. Conversely, “increased binding” refers to an increase in binding affinity to each interaction.

[0070] In some embodiments, the recombinant polypeptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is ≤1 μM. In some embodiments, the recombinant polypeptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is ≤100 nM. In some embodiments, the recombinant polypeptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is ≤10 nM. In some embodiments, the recombinant polypeptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is ≤100 pM to about 1 pM, as measured by assays such as surface plasmon resonance (SPR), Octet assay, or similar assays known to those skilled in the art. In some embodiments, KD may be 10⁻⁵M or less (for example, 10⁻⁶M or less, 10⁻⁷M or less, 10⁻⁸M or less, 10⁻⁹M or less, 10⁻⁰ or 10⁻⁶M or less).

[0071] In some embodiments, the recombinant polypeptide may have a binding reduction of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the control. For example, in recombinant polypeptides having reduced heparin or heparan sulfate binding, the recombinant polypeptide may have a binding reduction of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the control.

[0072] "Derived from" or "derived from" means obtaining a peptide, polypeptide, protein, or polynucleotide from a known and / or original peptide, polypeptide, protein, or polynucleotide. Therefore, as used herein, the term "derived from" includes, but is not limited to: a protein or polynucleotide isolated or obtained directly from an original source (e.g., an organism); a protein or polynucleotide that is identical, substantially related to, modified from, synthesized, or produced by recombinant means of a protein or polynucleotide from a known / original source (e.g., NRP1 or NRP2); or a protein or polynucleotide made from a protein or polynucleotide or a fragment of a known / original source. As used herein, the term "substantially related" means that the protein may have been modified by chemical, physical or other means (e.g., sequence modification).

[0073] Therefore, “derived” can mean obtaining a protein or polynucleotide directly or indirectly from a known and / or original protein or polynucleotide. For example, in some embodiments, “derived” can mean obtaining a protein or polynucleotide from a known and / or original protein or polynucleotide by looking at the sequence of the known / original protein or polynucleotide and preparing a protein or polynucleotide having a sequence that is at least partially similar to the sequence of the known and / or original protein or polynucleotide. In yet another embodiment, “derived” can mean obtaining a protein or polynucleotide from a known and / or original protein or polynucleotide by isolating the protein or polynucleotide from the organism to which the known protein or polynucleotide is related. Other methods of “deriving” a protein or polynucleotide from a known protein or polynucleotide are known to those skilled in the art.

[0074] In some embodiments, "derived from" in relation to a protein (e.g., "protein derived from a living organism") describes a state in which the protein is originally identified in a living organism and is regenerated therefrom through isolation from the organism or through synthesis or recombinant means.

[0075] "Excipients" refers to any pharmaceutically inactive, natural or synthetic component or substance formulated alongside (e.g., simultaneously with) or after the active ingredient of the present invention. In some embodiments, excipients may be any additives, adjuvants, binders, expanders, carriers, coatings, diluents, disintegrants, fillers, flow enhancers, lubricants, preservatives, vehicles or combinations thereof, which the recombinant polypeptide of the present invention may administer together with and / or which may be beneficial in preparing the compositions of the present invention. Excipients include any such substance known in the art that is non-toxic and does not interact with other components of the composition. In some embodiments, when preparing a composition for the purpose of increasing the volume of the composition, excipients may be formulated together with the recombinant polypeptide (and thus often referred to as expanders, fillers, or diluents). In other embodiments, excipients may be used to impart enhancement of the active ingredient in the final dosage form, for example, to promote absorption and / or solubility. In yet other embodiments, excipients may be used to provide stability or to prevent contamination (e.g., microbial contamination). In other embodiments, excipients can be used to impart physical properties to a composition (e.g., a composition in the physical form of dry granules or a dry, flowable powder). References to excipients include one and more such excipients. Preferred pharmaceutical excipients are described in Remington's Pharmaceutical Sciences by E.W. Martin, and their disclosure is fully incorporated herein by reference.

[0076] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. When both positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, then these molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. Therefore, in some embodiments, the term "homonymous" refers to sequence similarity between two polypeptide molecules or two nucleic acid molecules. When both positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, then these molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, if 6 out of 10 positions of two sequences are identical or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology.

[0077] When used in relation to nucleic acids, the term "homology" refers to the degree of complementarity. There may be partial homology or complete homology, and therefore identity. "Sequence identity" refers to a measure of the relevance between two or more nucleic acids, given as a percentage of the total length being compared. The calculation of identity considers nucleotide residues that are identical and occupy the same relative positions in their respective larger sequences.

[0078] "Identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by the comparison of the sequences. The term "identity" also means the degree of sequence relationship between polypeptide or polynucleotide sequences, which may be determined by the matching of strings of such sequences. "Identity" and "similarity" can be readily calculated by any one of the countless methods known to those skilled in the art, including, but not limited to, those described below: Computational Molecular Biology, Lesk, AM ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988), these disclosures are fully incorporated herein by reference. Furthermore, methods for determining identity and similarity have been codified into publicly available computer programs. For example, in some embodiments, methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol. 215:403-410 (1990)).The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894; Altschul, S., et al., J.Mol.Biol, 215:403-410 (1990), its disclosure is fully incorporated herein by reference).

[0079] "Mutant" refers to an organism, DNA sequence, polynucleotide, amino acid sequence, peptide, polypeptide, or protein that has alterations, mutations, or modifications (e.g., in a nucleotide or amino acid sequence) that cause the organism and / or sequence to differ from a naturally occurring or wild-type organism, wild-type sequence, and / or reference sequence to which the mutant is being compared. In some embodiments, this alteration, mutation, or modification may be a substitution or modification (e.g., deletion or addition) of one or more nucleotides and / or amino acids. In some embodiments, one or more amino acid substitutions or modifications may be conserved; here, such conserved amino acid substitutions and / or modifications in the "mutant" do not substantially reduce the activity of the mutant compared to its non-mutant form. For example, in some embodiments, the "mutant" has one or more conserved amino acid substitutions when compared to a peptide having the disclosed and / or claimed sequence as indicated by the sequence number.

[0080] "Operable" refers to usability, the ability to perform something and / or achieve a certain function or result. For example, in some embodiments, "operable" refers to the ability of a polynucleotide, DNA sequence, RNA sequence or other nucleotide sequence or gene to code for peptides, polypeptides and / or proteins. For example, in some embodiments, a polynucleotide may be operable to code for a protein, meaning that the polynucleotide contains information that imparts to it the ability to make a protein (for example, by transcribing mRNA which will then be translated into a protein).

[0081] "Wild type" or "WT" refers to the phenotype and / or genotype (i.e., appearance or sequence) of an organism, polynucleotide sequence, and / or polypeptide sequence as it is found and / or observed in the state or conditions in which it naturally exists.

[0082] Throughout this specification, unless otherwise required by the context, variations of the word such as "comprise," "comprises," or "comprising" imply that they include the explicitly stated step, element, or integer, or group of steps, elements, or integers, but not that any other steps, elements, integers, or groups of elements or integers are excluded.

[0083] All patent applications, patents, and printed publications referenced herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is specifically and individually shown to be incorporated by reference in full. And all patent applications, patents, and printed publications cited herein are incorporated by reference in full, except for any definitions, abandonment or denial of the subject invention, and unless the incorporated material is inconsistent with the express disclosure herein, as governed by the language of this disclosure.

[0084] SARS-CoV-2 virion particle model background and mechanism Severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2) stimulates an immune response in the body that leads to increased vascular permeability, increased endothelial inflammatory response, decreased nitric oxide (NO) levels, and impaired angiogenesis. Endothelial dysfunction due to a cytokine storm from SARS-CoV-2 can lead to multi-organ failure, including heart and kidney failure. Co-existing factors such as age, hypertension, and / or obesity can worsen the effects of SARS-CoV-2. In many severe cases of SARS-CoV-2, severe acute respiratory syndrome occurs in the alveoli and endothelium of the lungs, where COVID-19 involves vascular leakage, coagulation, and inflammation. Unlike the original SARS virus, H1N1, or other types of viruses such as Ebola or dengue fever, which damage endothelial cells but do not infect the lungs, SARS-CoV-2 is a respiratory virus that has the ability to infect vascular cells and circulate throughout the body.

[0085] As illustrated in Figure 1A, the SARS-CoV-2 virus can enter and infect human cells by binding its spike protein (SARS-S) to the angiotensin-converting enzyme 2 (ACE2) cell receptor present on the cell surface. Once the spike protein binds to the ACE2 receptor, the SARS-CoV-2 virus can enter the cell, where its viral sheath is broken down, releasing RNA into the host cell, where it replicates and produces more viral particles. The ACE2 receptor's ability to break down angiotensin II, regulate blood pressure, and block organ damage can be severely inhibited during SARS-CoV-2 infection.

[0086] Referring now to Figure 1B, one known mechanism that can be used to help neutralize the spread of SARS-CoV-2 infection is antibody neutralization. As illustrated in Figure 1B, antibodies or immunoglobulin constructs can bind to SARS-CoV-2 particles and block their attachment to the ACE2 cell receptor on cells by conferring steric hindrance, capsid stabilization, and / or structural changes. In some cases, antibodies, polypeptides, or immunoglobulin constructs can aggregate onto multiple SARS-CoV-2 particles to further inhibit the internal migration of the virus into cells. In these cases, where SARS-CoV-2 particles bind to one or more antibodies, the corresponding larger particles can enter the cell through phagocytosis, which neutralizes the conjugated SARS-CoV-2 particles.

[0087] Based on the pathogenesis of coronaviruses, which allows for the discovery of blood clotting in almost all organs during autopsies of COVID-19 patients, scientists are beginning to consider coronaviruses as a vascular disease. If COVID-19 is indeed a vascular disease, then the best antiviral therapy may not actually be conventional antiviral therapy. As explained above, both SARS-CoV-1 and SARS-CoV-2 are understood to enter cells through the ACE2 cell receptor. Based on the increased number of additional symptoms and infected tissues in patients infected with SARS-CoV-2, it is thought that one or more additional sites of entry may explain the prevalence of microthrombosis and the amount of angiogenesis in the lungs compared to influenza A and SARS-CoV-1. Referring to Figure 1C, a flowchart is provided outlining the additional symptoms observed, such as vascular damage, blood clotting, angiogenesis, AKI, and diabetes, as well as further infected tissues such as the heart, kidneys, and vascular endothelium, with SARS-CoV-2 using neuropilin-1 (NRP-1) as a means of entering cells in addition to the ACE2 receptor. Loss of taste / smell is also frequently observed.

[0088] Recent studies have confirmed that neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway to the central nervous system. It has further been noted that neuropilin-1 is a host factor for SARS-CoV-2 infection. Referring here to Figure 2A, it has been shown that the soluble domain of the SARS-CoV-2 virus utilizes the binding ability of the b1b2 domains of the neuropilin-1 receptor and the soluble ACE2 receptor. By designing the Fc domain of the immunoglobulin to provide a double decoy soluble protein, this corresponding fusion polypeptide can effectively bind to at least one, at least two, at least three, at least four, at least five, or at least six different spike proteins of one or more SARS-CoV-2 virus particles. As illustrated in Figure 2B and described in further detail herein, the exemplary fusion polypeptide may include sNRP1(b1), sACE2(ACE2), linker, and immunoglobulin (Fc) domains that can effectively bind to at least one, at least two, at least three, at least four, at least five, or at least six different spike proteins of one or more SARS-CoV-2 virus particles.

[0089] Referring next to Figure 2C, SARS-CoV-2 has a quaternary (4) amino acid insertion (PRRA) between Ser680 and ARG685 that generates a furin cleavage site. The furin cleavage of SARS-CoV-2 exposes the C-terminal motif RXXR-OH (C-terminal R rule), which is known to bind to the neuropilin-1 (NRP1) and / or neuropilin-2 (NRP2)b1 binding site.

[0090] Therefore, in designing therapeutic agents effective in reducing and treating viral infections such as the SARS-CoV-2 virus, fusion peptides comprising a neuropilin-1 (NRP1) domain, a neuropilin-2 (NRP2) domain, angiotensin-converting enzyme 2 (ACE2) domain, or a combination thereof are designed herein to specifically bind to the coat protein of viral particles, particularly the S protein of COVID-19 particles. In some embodiments, these fusion peptides further comprise, for example, an immunoglobulin domain and an Fc domain derived from human immunoglobulin.

[0091] Referring to Figure 3A, pinocytosis infection by the SARS-CoV-2 virus using the NRP1 receptor and / or ACE2 receptor is illustrated. In both cases, the SARS-CoV-2 virus is introduced into the cell by budding of vesicles from the cell membrane, where the viral shell is broken down in an acidic environment, allowing the RNA to be released into the host cell, where it replicates and generates more viral particles.

[0092] Referring next to Figure 3B, an illustrative schematic diagram is provided demonstrating how the disclosed polypeptide can neutralize and opsonize SAR-CoV-2 virus particles. In step 1, the polypeptide (A) and pathogen (B) circulate and move freely in the blood. In step 2, the disclosed polypeptide can bind to the pathogen, and can do so in different configurations such as opsonization (2a), neutralization (2b), and agglutination (2c). In step 3, a phagocyte (C) approaches the pathogen, where the b1 and / or ACE2 domains, which are optionally bound to the Fc region (D) of the disclosed polypeptide, bind to one of the receptors (E) on the phagocyte. Finally, in step 4, the pathogen is ingested and phagocytosis occurs.

[0093] A currently proposed mechanism for reducing and treating SAR-CoV-2 virus infection is illustrated in Figure 3C. In this mechanism, a therapeutic antibody and / or vaccine would be provided, which would produce an antibody that can bind to the SAR-CoV-2 virus and induce phagocytosis. The problem with this approach is that the SAR-CoV-2 virus particle may instead bind to the NRP1 receptor and infect cells through CendR-NRP1-mediated pinocytosis. The SAR-CoV-2 virus, which can maintain its activity in the body through the CendR-NRP1-mediated pinocytosis mechanism, could lead to incomplete treatment and / or difficulty in effectively treating patients.

[0094] Alternatively, as illustrated in Figure 3D, proposed treatments using the polypeptides disclosed herein may improve upon the shortcomings of the mechanism outlined in Figure 3C. For example, polypeptides comprising both the b1 domain of neuropyrin or its derivatives or fragments; and both the ACE2 domain of angiotensin-converting enzyme 2 or its derivatives or fragments; target both the CendR-NRP1 and ACE2-mediated pinocytosis mechanisms by which the SAR-CoV-2 virus enters cells. By eliminating the mechanism that the SAR-CoV-2 virus uses to enter cells and effectively neutralizing the viral action through phagocytosis, a complete and effective treatment can be provided to the patient.

[0095] Neutralization of SARS-CoV-2 with neuropilin Neuropyrin consists of five main domains. Starting from the N-terminus, the a1 and a2 domains are classified as CUB domains, to which Ig-like C2 type semaphorins bind. In particular, this site forms a complex with plexin, playing a role in increasing semaphorin-plexin binding affinity. The b1 and b2 domains are classified as FV / VIII domains, to which the C-terminus of VEGF family ligands or class 3 semaphorin ligands binds. In particular, this region contains a site to which heparin can bind, making it easier for ligands with many (+) charged residues to bind. Furthermore, the MAM induces oligomerization, the transmembrane domain (TM) allows neuropyrin to be fixed to the cell surface, and the cytosolic domain contains a site that can bind to the postsynaptic density 95, is dispersive, and binds to the Zona occludens 1 (PDZ) domain.

[0096] The four extracellular domains (a1, a2, b1, and b2) above determine the binding specificity of multiple ligands to NRP1 / 2, and the final extracellular c domain, along with the transmembrane domain, is linked to dimerization or oligomerization between NRP and its coreceptors. Both VEGF and Sema3 family ligands specifically bind to the VEGF-binding region of the b1 domain of NRP1 / 2 via a C-terminal R / KxxR / K sequence motif, where x represents any amino acid. In fact, all known proteins and peptides that bind to the ligand-binding pocket in the NRP1-b1 domain share this sequence motif. This fundamental sequence motif of NRP1-binding ligands and peptides has the strict requirement that it must be exposed at the C-terminus for binding to NRP1, and that the last C-terminal residue requires Arg (or rarely Lys); this requirement is called the "C-terminal rule" (CendR).

[0097] Multiple viruses possess a CendR motif within their capsid proteins and can undergo proteolytic cleavage to expose the CendR motif in order to become infectious. Most viral envelope glycoproteins need to be proteolytically cleaved before they can mediate viral entry into host cells. Often, viruses utilize the cell's trypsin- or subtilisin-like endoproteases for this purpose. Subtilisin-like proteases such as furin require a multinucleotide cleavage site, while trypsin-like proteases also recognize single-nucleotide motifs and cleave after a single arginine or lysine residue. Furin-mediated cleavage has been described for envelope glycoproteins encoded by numerous evolutionarily diverse families of viruses, including herpesviridae, coronavirusidae, flaviviridae, togaviridae, bornaviridae, bunyaviridae, filoviridae, orthomyxoviridae, paramyxoviridae, pneumoviridae, and retroviridae.

[0098] In recent years, researchers have observed in cell culture experiments that NRP1 enhances the ability of SARS-CoV-2 to infect cells. They also discovered that the S1 polypeptide is responsible for binding to NRP1. The S1 polypeptide is a one- or two-nucleotide molecule formed when the SARS-CoV-2 spike protein is cleaved and activated. It contains a sequence that conforms to the C-terminal rule (CendR).

[0099] Furthermore, the b1b2 domains of Nrp1 and Nrp2 contain C-terminal Sema3F and VEGF-binding-capable structure-determining factors. The intact b1b2 domains in NRP1 serve as VEGF165-, P1GF-2-, and heparin-binding sites, and this heparin is a crucial component for regulating the interaction of VEGF165 and P1GF-2 with NRP1 by physically interacting with both the receptor and ligand.

[0100] Mutations in the VEGF-binding pocket within the b1 domain altered the binding affinity to VEGF165A. Y297A / S346A / Y353A mutations in the b1 domain prevented binding to VEGF. Furthermore, the E319A mutation in the b1 domain resulted in stronger binding affinity to VEGFA with heparin, while the T349A and K351A mutations in the b1 domain resulted in weaker binding affinity to VEGFA with or without heparin.

[0101] Neuropilins, which are transmembrane glycoproteins, are classified into two types: neuropilin-1 (NRP1; the human NRP1 amino acid sequence is provided as SEQ ID NO: 1) and neuropilin-1 (NRP2; the human NRP2 amino acid sequence is provided as SEQ ID NO: 1) (Kolodkin et al. 1997). Neuropilins-1 and-2 consist of 923 and 931 amino acids, respectively, exhibiting approximately 44% amino acid sequence homology and sharing several structural characteristics and biological activities. Neuropilins-1 and-2 generally consist of extracellular a1, a2, b1, b2, and MAM domains as well as an intracellular PDZ-binding domain (Appleton et al. 2007). Neuropilins are expressed very weakly in normal cells but are overexpressed in most tumor-associated endothelial cells, solid tumor cells, and hematological malignancies (Grandclement, C. and C. Borg 2011). Neuropilins act as co-receptors of the VEGF receptor (VEGFR) by binding to VEGF25 family ligands. In particular, NRP1 acts as a co-receptor for VEGFR1, VEGFR2, and VEGFR3, binding to various VEGF ligands and thereby contributing to angiogenesis, cell migration and adhesion, and invasion. On the other hand, NRP2 acts as a co-receptor for VEGFR2 and VEGFR3, thereby contributing to lymphangiogenesis and cell adhesion. Furthermore, neuropilin 1 and 2 act as co-receptors for plexin family receptors, binding to secreted class 3 semaphorin ligands (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F, Sema3G). Neuropilin does not have a domain in functional cells, so even if a ligand is bound to it, it does not have activity on its own. Neuropilin signaling is known to occur through the co-receptors, VEGF receptors or plexin co-receptors. Sema3 binds to neuropilin and plexin receptors in a 2:2:2 ratio and acts accordingly. However, many studies have shown that neuropilin proteins alone can carry out signal transduction without their interaction with VEGF receptors or plexin coreceptors. The precise molecular mechanism of this signal transduction remains unclear, however.

[0102] The activity of neuropilin and its co-receptors has been reported to be inhibited even when neuropilin alone is targeted. For example, anti-neuropilin-1 antibodies have been reported to competitively bind only to neuropilin-1, against VEGF-A, which is known to bind to both VEGFR2 and neuropilin-1, and to inhibit the functions of VEGFR2, namely angiogenesis, cell survival, migration and adhesion, and invasion (Pan Q et al. 2007). Anti-neuropilin-2 antibodies have been reported to competitively bind to neuropilin-2, against VEGF-C, which is known to bind to both VEGFR3 and neuropilin-2, and to inhibit the functions of VEGFR3, namely lymphangiogenesis and cell adhesion (Caunt M et al. 2008).

[0103] The C-terminal regions of the VEGF ligand family and Sema3 ligands that bind to neuropilin 1 and 2 bind to the VEGF binding site (the so-called arginine-binding pocket) in the b1 domain commonly present in neuropilin 1 and 2 (MW Parker et al. 2012). Here, binding to the arginine-binding pocket occurs through the R / KxxR / K motif (R=arginine, K=lysine, and x=any amino acid) commonly present in the C-terminal region of neuropilin-binding ligands. When mutations are induced in amino acid sequences that deviate from this motif, the ligands have reduced binding affinity to neuropilin or do not bind to neuropilin at all, and therefore lose their biological activity. In particular, cationic arginine (Arg) or lysine (Lys) in the C-terminal region is essential for binding; therefore, when it is substituted with another amino acid residue, the ligand loses its binding affinity to neuropilin and loses its biological activity. Therefore, the need for an R / KxxR / K motif in the C-terminal region of such neuropilin-binding ligands is called "C-terminal rule" (CendR) (Teesalu et al., 2009). Proteins or peptides containing a C-terminal rule sequence can bind to neuropilin via a C-terminal arginine (Arg) or lysine (Lys) residue (Zanuy et al., 2013).

[0104] The C-terminal regions of VEGF ligands and Sema3 ligands generally contain an R / KxxR / K motif; therefore, most ligands have the property of binding to both neuropilin 1 and 2 rather than selectively binding to one of them.

[0105] In addition to ligands that bind to neuropilin 1 and 2, many peptides that bind to neuropilin have been selected or designed and reported. All of these peptides possess an R / KxxR / K motif and are therefore thought to bind to the arginine-binding pocket in the b1 domain of neuropilin 1 and 2. Furthermore, the iRGD peptide (Sugahara et al. 2010), which binds to neuropilin 1 and 2 to increase the penetration of co-administered drugs into tumor tissue, and the A22p peptide (Shin et al. 2014), which fuses to the heavy chain terminus of antibodies to increase the penetration of antibodies into tumor tissue, also have amino acid sequences that follow the CendR rule.

[0106] The neuropilin domains or their derivatives or fragments used herein include the b1 domain or its derivatives or fragments. Table 1 provides some exemplary neuropilin domains, but is not limited to the full-length NRP1 and NRP2 portions, in addition to some smaller domains.

[0107] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0108] Immunoglobulins In some embodiments, the polypeptide of the present invention comprises an immunoglobulin domain. The immunoglobulin domain may be derived from an immunoglobulin molecule from any mammal. In one embodiment, the immunoglobulin domain is derived from human immunoglobulin and may be derived from an immunoglobulin isotype selected from the group consisting of IgG, IgA, and IgD antibody isotypes. In a particular embodiment, the immunoglobulin domain is derived from an IgG isotype. In yet another embodiment, the immunoglobulin domain is derived from a subclass of IgG selected from the group consisting of IgG1, IgG2, and IgG4.

[0109] The IgG Fc region, including the hinge region, was selected as a biocompatible material capable of increasing the half-life of the physiologically active polypeptide linked to it.

[0110] To reduce this unexpected effector function, human IgG4 mutants L235E or F234A / L235A, and human IgG1 mutants L234A / L235A were created, all of which reduced inflammatory cytokine release. Another early approach intended to reduce effector function was to mutate the glycosylation site of N297 with mutations such as N297A, N297Q, and N297G. This glycosylation approach proved favorable in suppressing effector functions such as Fc interaction with low affinity FcγR and CDC and ADCC. Among the four IgG subclasses, each has a different ability to induce immune effector function. For example, IgG1 and IgG3 are recognized to recruit complement more effectively than IgG2 and IgG4. Furthermore, IgG2 and IgG4 have a very limited ability to induce ADCC. Therefore, several researchers have used cross-subclass approaches to reduce effector function.

[0111] FcRn is known to prolong the half-life of IgG, and the obvious strategy has been to modulate the FcRn-IgG interaction to prolong or shorten the antibody half-life. Prolonging the half-life of a therapeutic antibody would help maintain drug therapeutic levels and reduce administration frequency, while shortening the half-life is ideal for diagnostic testing or toxicity management.

[0112] Attempts to extend antibody half-life by mutations in the Fc region crucial for FcRn binding have been relatively successful. However, increasing Fc for FcRn binding does not necessarily extend serum half-life. In fact, IgG1 mutants designed to significantly increase binding at pH 6.0 and pH 7.4 did not contribute to extending serum half-life; instead, at pH 6.0 alone, they offset the benefit of enhanced binding. FcRn-IgG binding at pH 7.4 is thought to prevent IgG release into circulation and instead redirect it to the degradation pathway. Furthermore, the dissociation rate at pH 7.4 has been suggested to be equally, or perhaps even more, important in determining serum half-life.

[0113] In previous comprehensive screening of Fc mutations in FcRn binding assays, N434A and T307A / E380A / N434A(AAA) were shown to have 3.4-fold and 11.8-fold increases in binding to human FcRn, respectively. Trastuzumab substitution resulted in 1.3-fold and 3.3-fold increases in binding to human FcRn using cell-based assays, and in mice expressing the human FcRn transgene and lacking endogenous FcRn (hFcRn-Tg), resulted in 2.2-fold and 2.5-fold increases in serum half-life.

[0114] The fusion protein that binds to the viral spike protein comprises an IgG Fc region including a hinge region, an ACE2 (angiotensin-converting enzyme 2) fragment including a spike protein binding region, and an NRP (neuropilin) ​​fragment including a CendR binding region, either by linker or direct fusion.

[0115] In some embodiments, the polypeptide of the present invention comprises an immunoglobulin domain including a heavy chain portion. In one embodiment, the immunoglobulin domain comprises a fragmentary crystalline (Fc) domain containing the antibody hinge-CH2-CH3. In specific embodiments, the polypeptide comprises an immunoglobulin domain comprising an Fc domain selected from IgG1 and IgG2 subclasses.

[0116] In some embodiments, the immunoglobulin domain includes a modified Fc domain.

[0117] For example, in certain embodiments, it may be preferable to have increased affinity for the Fc fragment (FcRn) of the IgG receptor and transporter. In adults, FcRn is expressed in epithelial tissue. FcRn can perform diverse roles through the transport and recirculation of bound IgG within and beyond cells. Antibodies and other Fc-containing peptides are internalized through binding to FcRn and directed toward the acidic endosomes and lysosomes of cells for degradation. Therefore, in certain embodiments, the polypeptide comprises an immunoglobulin domain containing an Fc domain having increased binding affinity to FcRn. In one embodiment, the immunoglobulin domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 27, and 29.

[0118] In other embodiments, it may be desirable to have reduced affinity for one or more Fcγ receptors. In some cases, for example, with HIV and dengue virus, a virus-antibody complex that binds to the Fcγ receptor (FcγR) allows the virus to approach the virus-specific receptor, leading to infection. Thus, a virus conjugated with an antibody can result in a phenomenon called antibody-dependent enhancement (ADE) of infection. Therefore, in certain embodiments, the polypeptide comprises an immunoglobulin domain containing an Fc domain having reduced affinity for one or more Fcγ receptors. In one embodiment, the immunoglobulin domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0119] In further embodiments, it may be desirable to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the immunoglobulin domain. In some cases, hyperactivation of ADCC leads to uncontrolled high release of cytokines, causing a "cytokine storm." In other cases, ADCC may lead to the internal transfer of viral particles into susceptible target cells. Thus, in one embodiment, the polypeptide comprises an immunoglobulin domain that is defective in producing ADCC. In one embodiment, the polypeptide comprises an immunoglobulin domain comprising an Fc domain having reduced ADCC, for example, containing the N297A mutation. In one embodiment, the immunoglobulin domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 25 and 27.

[0120] As used herein, the term "heavy chain" can be interpreted to include a full-length heavy chain comprising a heavy chain variable region domain VH containing an amino acid sequence having a variable region sequence sufficient to confer antigen specificity, as well as three heavy chain constant region domains, CH1, CH2, and CH3, and fragments thereof. Furthermore, as used herein, the term "light chain" can be interpreted to include a full-length light chain comprising a light chain variable region domain VL containing an amino acid sequence having a variable region sequence sufficient to confer antigen specificity, as well as a light chain constant region domain CL, and fragments thereof.

[0121] Furthermore, the antibody fragment may be a monomer, dimer, or polymer.

[0122] Antibodies include, without limitation, monoclonal antibodies, nonspecific antibodies, non-human antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv(scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(sdFV), and anti-idiotype (anti-Id) antibodies, as well as epitope-linked fragments of these antibodies.

[0123] The monoclonal antibody may be IgG, IgM, IgA, IgD, or IgE. For example, the monoclonal antibody may be of type IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD, or it may be of type IgG1. Furthermore, the constant region of the antibody's light chain may be of type λ or κ.

[0124] The peptide may be bound to the heavy chain constant region (Fc) fragment of the antibody, preferably to the C-terminus of the heavy chain constant region (Fc) fragment. This binding can be performed by a linker peptide.

[0125] In some embodiments, the polypeptide of the present invention includes an Fc domain.

[0126] Table 2 provides some exemplary immunoglobulin domains used in the therapeutic polypeptides described herein.

[0127] [Table 2-1] [Table 2-2]

[0128] Angiotensin-converting enzyme 2 neutralization effect of SARS-CoV-2 ACE2, an angiotensin-converting enzyme (ACE)-related carboxypeptidase, is a type I intrinsic membrane protein of approximately 805 amino acids containing a single HEXXH+E zinc-binding consensus sequence. ACE2 is a close homolog of somatic angiotensin-converting enzyme (ACE; EC3.4.15.1), a peptidyl dipeptidase that plays a crucial role in the renin-angiotensin system. The ACE2 sequence includes an N-terminal signal sequence (amino acids 1-18), a latent transmembrane domain (amino acids 740-763), and a latent metalloproteinase zinc-binding site (amino acids 374-378, HEMGH).

[0129] The crystal structure of ACE2 bound to an S protein fragment containing RBDs (residues 306-527) has been published. ACE2 residues in direct contact with the RBDs included Q24, T27, K31, H34, E37, D38, Y41, Q42, L45, L79, M82, Y83, N90, Q325, E329, N330, K353, and G354. Comparative structural analysis suggests that most of the key residues of ACE2 involved in S protein binding are found on back-to-back alpha helices 1 and 2 at the N-terminus. The question remains whether the two helices actually remain stable in the complex with the S protein.

[0130] In some embodiments of the present invention, polypeptides comprising an ACE2 domain are disclosed. In some embodiments, the ACE2 domain is derived from a mammalian ACE2 sequence. In one embodiment, the ACE2 domain is a human ACE2 sequence (the full-length amino acid sequence including the signal sequence is provided in SEQ ID NO: 32), a derivative thereof comprising amino acids 22-44 of SEQ ID NO: 32, or a fragment thereof. For example, in one particular embodiment, the ACE2 domain comprises an ACE2 domain comprising an amino acid sequence selected from: EEQAKTFLDKFNHEAEDLFYQSS (SEQ ID NO: 34) and IEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEI (SEQ ID NO: 35). In another embodiment, the ACE2 domain further comprises amino acids 351-357 of SEQ ID NO: 32. For example, in another embodiment, the ACE2 domain is selected from the group consisting of: EEQAKTFLDKFNHEAEDLFYQSS(X)nLGKGDFR (Sequence ID 36) and IEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEI(X)nWDLGKGDFR (Sequence ID 37) During the ceremony: n=0~30 X = any amino acid selected from Gly, Ala, Ser, Ile, Leu, and Val, or any combination thereof.

[0131] In certain embodiments, the ACE2 domain can be modified to include amino acid substitutions, for example, to alter its affinity for viral particles. In one embodiment, the ACE2 domain includes amino acid substitutions at positions selected from the group consisting of F28, D30, and L79 (amino acid numbering based on the full-length human ACE2 sequence). In one embodiment, the ACE2 domain includes an F28W substitution. In another embodiment, the ACE2 domain includes a D30A substitution. In yet another embodiment, the ACE2 domain includes an L79T substitution. Non-limiting examples of various ACE2 domains include, but are not limited to, ACE2-1, ACE2-2, ACE2-3, ACE2-4, ACE2-5, and ACE2-6. The ACE2-1 domain contains α-helix 1 + β-sheet)-(G4S)*2-(α-helix 1 + β-sheet). The ACE2-2 domain contains α-helix 1 + α-helix 2 + β-sheet)-(G4S)*2-(α-helix 1 + α-helix 2 + β-sheet). ACE2-3 contains ACE2-1 with F28W. ACE2-4 contains ACE2-2 with F28W. ACE2-5 contains ACE2-2 with D30A. ACE2-6 contains ACE2-2 with L79T.

[0132] To induce decoy proteins that specifically bind to spike proteins, the sequences of angiotensin-converting enzyme 2 (ACE2), neuropilin-1 (NRP-1), and neuropilin-2 (NRP-2) were analyzed. As a representative example, the complete sequences of angiotensin-converting enzyme 2, neuropilin-1, and neuropilin-2 were selected from the PubMed Entrez protein database.

[0133] Table 3 provides some exemplary ACE2 domains used in the therapeutic polypeptides described herein.

[0134] [Table 3-1] [Table 3-2] [Table 3-3]

[0135] Linker section The peptides that specifically bind to NRP1 according to embodiments of this disclosure may further comprise a linker peptide. The linker peptide may comprise or consist of 1 to 50 amino acids, 4 to 20 amino acids, or 4 to 10 amino acids. Furthermore, the linker peptide may comprise or consist of glycine or serine, and may comprise or consist of an amino acid sequence of (GGGGS)n (wherein n is an integer between 1 and 20 independently) or (GGGGS)2.

[0136] In some embodiments of this disclosure, the peptide having a linker peptide bound thereto may include any one of the amino acid sequences of SEQ ID NOs. 46-52 provided in Table 4 below.

[0137] [Table 4-1] [Table 4-2]

[0138] signal sequence Signal sequences can be located at the N-terminus of a peptide, allowing those proteins to find their correct positions outside the cell membrane. Signal sequences can tag proteins so that they can pass through the cell membrane and be removed from the cell. Signal peptides can play a role in prompting cells to move proteins to the cell membrane as usual. In prokaryotes, signal peptides can guide newly synthesized proteins to SecYEG protein conduction channels present in the plasma membrane.

[0139] In some embodiments of this disclosure, the signal sequence may include the amino acid sequence of SEQ ID NO: 53, provided in Table 5 below.

[0140] [Table 5]

[0141] Immunoglobulin-ACE2 construct In one aspect of the present invention, the polypeptide may include the ACE domain or derivatives or fragments of angiotensin-converting enzyme 2, and an immunoglobulin domain. The ACE2 domain of these polypeptides can bind to the coat protein of viruses selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Retroviridae. Table 6 provides an exemplary but non-limiting list of immunoglobulin-ACE2 constructs.

[0142] [Table 6]

[0143] Immunoglobulin-neuropilin construct In another aspect of the present invention, the polypeptide may include the b1 domain or derivatives or fragments thereof of neuropyrin; and an immunoglobulin domain. The bl domains of these polypeptides may be able to bind to the coat proteins of viruses selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Retroviridae. In some embodiments, the b1 domain may include the full-length NRP1 or NRP2 peptide. In other embodiments, the b1 domain or derivatives or fragments thereof may further include one or more additional b1 domains or derivatives or fragments thereof; one or more b2 domains or derivatives or fragments thereof of neuropyrin; or a combination thereof. Referring to Figure 4, various exemplary schematic designs are provided for these polypeptides in which the b1 domain or derivatives or fragments thereof are bound to an immunoglobulin domain. Table 7A outlines, without limitation, the general structure and connectivity of several exemplary constructs, neuropilin-immunoglobulin constructs (construction numbers 12–36), or polypeptides containing b1, a linker, and an immunoglobulin domain. In some embodiments, the polypeptide has a configuration selected from the group of construction numbers 12–36. Table 7B provides a list of these same exemplary constructs or neuropilin-immunoglobulin polypeptides, including b1, a linker, and an immunoglobulin domain, and their corresponding peptide sequences.

[0144] [Table 7-1] [Table 7-2]

[0145] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9]

[0146] Neuropilin-immunoglobulin-ACE2 construct In yet another aspect of the present invention, the polypeptide may include the neuropyrin b1 domain or a derivative or fragment thereof; an immunoglobulin domain; and the angiotensin-converting enzyme 2 ACE2 domain or a derivative or fragment thereof. Both the b1 and ACE2 domains of these polypeptides may bind to the coat proteins of viruses selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Retroviridae. Referring to Figure 5, various exemplary schematic designs are provided for polypeptides having a b1 domain or a derivative or fragment thereof that binds to the ACE2 domain or a derivative or fragment thereof and further binds to an immunoglobulin domain. Table 8A outlines, without limitation, the general structure and connectivity of several exemplary neuropyrin-immunoglobulin-ACE2 constructs (construct numbers 4-11 and 37-59), or polypeptides containing the b1, ACE2, linker, and immunoglobulin domains. In some embodiments, the polypeptide has a configuration selected from the groups of construct numbers 4-11 and 37-59. Table 8B provides a list of these same exemplary constructs from Table 8A, including neuropilin-immunoglobulin-ACE2 polypeptides, which include b1, ACE2, linker, and immunoglobulin domains, and their corresponding peptide sequences.

[0147] [Table 9-1] [Table 9-2] [Table 9-3]

[0148] [Table 10-1] Table 10-2 Table 10-3 Table 10-4 Table 10-5 Table 10-6 Table 10-7 Table 10-8 Table 10-9 Table 10-10 Table 10-11 Table 10-12 Table 10-13

[0149] ニューロピリン-ACE2 structure In yet another aspect of the present invention, the polypeptide may include the bl domain or a derivative or fragment thereof of neuropyrin; and the ACE2 domain or a derivative or fragment thereof of angiotensin-converting enzyme 2. Each of the bl domain and ACE2 domain in these embodiments is capable of binding to the coat protein of a virus selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Togaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Retroviridae.

[0150] In some embodiments, the b1 domain or derivative or fragment of neuropyrin can be selected from the group including one or more of SEQ ID NOs: 1-22, which is bound to the ACE2 domain or derivative or fragment of angiotensin-converting enzyme 2, including one or more of SEQ ID NOs: 32-43. In some embodiments, the b1 domain is bound to the C-terminus of the ACE2 domain. In other embodiments, the b1 domain is bound to the N-terminus of the ACE2 domain. In yet another embodiment, a linker selected from the group including one or more of SEQ ID NOs: 44-50 can be linked between any combination of one or more b1 domains fused to one or more ACE2 domains.

[0151] In some embodiments, neuropilin-ACE2 polypeptides comprising the b1 domain or its derivatives or fragments, and the ACE2 domain or its derivatives or fragments, can be used in intranasal spray compositions.

[0152] Pharmaceutical composition The polypeptides described herein may be formulated into pharmaceutical compositions further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In one embodiment, the present invention relates to a pharmaceutical composition comprising the polypeptide of the present invention as described above, and a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In one embodiment, the present invention is a pharmaceutical composition comprising an effective amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. The pharmaceutically acceptable carrier includes, for example, a pharmaceutically acceptable diluent, excipient, or carrier that is appropriately selected with respect to the intended dosage form and conforms to conventional pharmaceutical practices.

[0153] The term "effective dose" includes "therapeutic effective dose" and "preventive effective dose." The term "therapeutic effective dose" refers to the amount effective in treating and / or mitigating a viral infection in a patient infected with a viral infection, such as SARS-CoV-2. The term "preventive effective dose" refers to the amount effective in preventing an outbreak of a viral infection and / or substantially reducing the likelihood or scale of an outbreak of a viral infection.

[0154] A pharmaceutically acceptable carrier may contain inactive components that do not excessively inhibit the biological activity of the polypeptide. A pharmaceutically acceptable carrier must be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or free from other undesirable reactions or side effects upon administration to the subject. Standard pharmaceutical formulation techniques can be employed.

[0155] When used herein, pharmaceutically acceptable carriers, adjuvants, or vehicles include all solvents, diluents, or other liquid vehicles, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., depending on the desired specific dosage form. Remingson's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa, 1980) discloses various carriers used in the formulation of pharmaceutically acceptable compositions and known techniques for their preparation. Unless any conventional carrier medium is incompatible with the polypeptides described herein, such as producing any undesirable biological effects or adversely interacting with any other component(s) of a pharmaceutically acceptable composition, its use is intended to be within the scope of the invention. When used herein, the term “adverse effects” includes unwanted and adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Adverse effects are not always undesirable, but undesirable effects are not necessarily harmful. Adverse effects of therapy (e.g., prophylactic or therapeutic agents) may be harmful, unpleasant, or dangerous. Side effects include, but are not limited to, fever, chills, lethargy, gastrointestinal toxicity (including gastric and intestinal ulceration and erosion), nausea, vomiting, neurotoxicity, nephrotoxicity, renal toxicity (including conditions such as papillary necrosis and chronic interstitial nephritis), hepatotoxicity (including elevated serum liver enzyme levels), myelotoxicity (including leukopenia, myelosuppression, thrombocytopenia, and anemia), dry mouth, metallic taste, prolonged pregnancy, weakness, somnolence, pain (including muscle pain, bone pain, and headache), alopecia, asthenia, dizziness, extrapyramidal symptoms, akathisia, cardiovascular disorders, and sexual dysfunction.

[0156] Some examples of materials that can act as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as Twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, methylcellulose, hydroxypropyl methylcellulose, lanolin, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; cocoa butter, and suppository waxes. Excipients such as wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol and phosphate buffer; and other non-toxic, suitable lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coatings, sweeteners, flavors, fragrances, preservatives, and antioxidants may also be present in the composition at the discretion of the compounder.

[0157] In some embodiments, the compositions of the present invention include pharmaceutically acceptable salts.

[0158] The term “pharmaceutically acceptable salt” is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found in the compounds described herein. If the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting the neutral form of such compound with a sufficient amount of the desired base, either neatly or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. If the compounds of the present invention contain relatively basic functional groups, acid addition salts may be obtained by contacting the neutral form of such compound with a sufficient amount of the desired acid, either neatly or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, oxalic acid, and methanesulfonic acid. This also includes salts of amino acids such as alginates, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present invention contain both basic and acidic functional groups, which allow the compound to be converted into either a base-addition salt or an acid-addition salt.

[0159] Therefore, the compounds of the present invention may exist as salts, for example, salts with pharmaceutically acceptable acids. The present invention includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobroms, phosphates, sulfates, methanesulfons, nitrates, maleates, acetates, citrates, fumarates, propions, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts of amino acids such as glutamic acid, as well as quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, etc.). These salts can be prepared by methods known to those skilled in the art.

[0160] The neutral form of the compound is preferably regenerated by conventional methods, such as contacting the salt with a base or acid and isolating the parent compound. The original compound may have various salt forms and differ in certain physical properties, such as solubility in polar solvents.

[0161] In addition to salt forms, the present invention provides compounds in prodrug form. The prodrugs of the compounds described herein are compounds that readily undergo chemical transformation under physiological conditions to provide the compounds of the present invention. The prodrugs of the compounds described herein may be converted in vivo after administration. Furthermore, the prodrugs may be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment, for example, by contact with a suitable enzyme or chemical reagent.

[0162] Certain compounds of the present invention may exist in an unsolvated form, or in a solvated form, including a hydrated form. In general, the solvated form is equivalent to the unsolvated form and is included within the scope of the present invention. Certain compounds of the present invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent for the intended use of the present invention and are intended to be within the scope of the present invention.

[0163] Method of administration In some embodiments, the compositions of the present invention may be administered to subjects requiring them. In some embodiments, the compositions of the present invention may be administered concurrently with one or more further therapies.

[0164] The term “administer” or “to administer” refers to the act of providing a composition of the present invention, such as a polypeptide or a pharmaceutically acceptable salt thereof, to a subject requiring treatment.

[0165] In some embodiments, the compositions of the present invention may be administered as follows: orally, as suppositories, by topical contact, intravenously, parenterally, intraperitoneally, intramuscularly, intrafocally, intraspinally, intranasally or subcutaneously to a subject, or by implantation of a slow-release device, such as a mini osmotic pump. Thus, administration may be obtained by any route, including parenterally and transmucosal (e.g., buccal, sublingual, palatal, gingival, transnasal, intravaginal, intrarectally, or transdermally). Parenteral administration may include, for example, intravenously, intramuscularly, intraarteriolely, intradermally, subcutaneously, intraperitoneally, or intraventricularly. Other modes of delivery, but not limited to, include the use of liposomal formulations, intravenous injections, transdermal patches, etc.

[0166] "Concurrent administration" means that the composition described herein is administered simultaneously with, immediately before, or immediately after the administration of an additional therapeutic agent. The therapeutic agents may be administered to the patient alone or concurrently. Concurrent administration is intended to include the simultaneous or sequential administration of separate or combined components. Thus, the preparation may also be combined with other active substances, if desired. As used herein, "sequential administration" includes the fact that the administration of two drugs (e.g., the drugs described herein) does not occur on the same day.

[0167] As used herein, “concurrent administration” includes at least some overlap in duration. For example, when two compositions (e.g., any of the compositions described herein) are administered concurrently, their administrations occur within a specific desired time period. The administrations of the compositions may begin and end on the same day. The administration of one composition may also precede the administration of the second composition by more than one day, as long as both compositions are taken at least once on the same day. Similarly, the administration of one composition may extend beyond the administration of the second composition, as long as both drugs are taken at least once on the same day. The compositions do not have to be taken simultaneously on the same day in order to include concurrent administration.

[0168] As used herein, “intermittent administration” includes a period of administration of the drug (which may be considered a “first administration period”), a period during which the composition is not taken or is taken at a lower maintenance dose (which may be considered an “off period”), and a subsequent period during which the composition is taken again (which may be considered a “second administration period”). Generally, during the second administration period, the drug dosage level is the same as that administered during the first administration period, but may be increased or decreased if medically necessary.

[0169] The polypeptides and pharmaceutically acceptable compositions described above may be administered to humans or other animals orally, rectally, parenterally, intracisional, vaginally, intraperitoneally, topically (as a powder, ointment, or infusion), buccally, as an oral spray or nasal drop, etc., depending on the severity of the infection being treated.

[0170] In some embodiments of the present invention, when treating a target, the inhibitor is administered by systemic intravenous (IV) or by a local intranasal route such as an intranasal spray, metered-dose inhaler, nebulizer, or dry powder inhaler. Formulations for specific delivery methods (e.g., solutions, buffers, and preservatives, as well as droplet or particle sizes for intranasal administration) can be optimized by conventional methods well known in the art. For inhibitors in the form of aerosol formulations administered by inhalation, the aerosol formulations can be placed in a pressurized, acceptable spray such as dichlorodifluoromethane, propane, or nitrogen.

[0171] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsifies, solutions, suspensions, syrups, and elixirs. In addition to the active polypeptide, the liquid dosage forms may also include, for example, water, or other solvents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, as well as inert diluents commonly used in the art, such as solubilizers and emulsifiers, and mixtures thereof. In addition to inert diluents, the oral composition may also include adjuvants such as wetting agents, emulsifiers, and suspending agents, sweeteners, flavorings, and fragrances.

[0172] Injectable preparations, such as sterile injectable aqueous or oily suspensions, may be formulated according to known techniques using appropriate dispersants or wetting and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenterally acceptable diluents or solvents, such as a solution of 1,3-butanediol. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, USP, and isotonic salines. Sterile non-volatile oils have also been conventionally employed as solvents or suspensions. For this purpose, any non-irritating non-volatile oil, including synthetic monoglycerides or diglycerides, may be employed. Fatty acids, such as oleic acid, are also used in the preparation of injectable preparations.

[0173] The injectable formulation may be sterilized, for example, by filtration using a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition which may be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0174] To extend the effects of the polypeptides described herein, it is often desirable to delay the absorption of the polypeptides from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. In this case, the absorption rate of the polypeptide may depend on its dissolution rate, and further, on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered polypeptide forms can be achieved by dissolving or suspending the polypeptide in an oil vehicle. Injectable depot formulations are produced by forming a microencapsulation matrix of polypeptides in biodegradable polymers such as polylactic acid-polyglycerides. The polypeptide release rate can be controlled depending on the ratio of polypeptide to polymer and the properties of the specific polymer employed. Examples of other bioactive polymers include poly(orthoesters) and poly(acid anhydrides). Depot injectable formulations are also prepared by encapsulating polypeptides in liposomes or microemulsifies that are compatible with body tissues.

[0175] Compositions for rectal or vaginal administration are suppositories that can be prepared by mixing the polypeptides described herein with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or suppository wax, which is solid at ambient temperature but liquid at body temperature, and therefore melts in the rectum or vaginal cavity to release the active polypeptide.

[0176] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the polypeptide (i.e., the active polypeptide) is provided with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) hydrating agents such as glycerin; d) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; and e) solution retarding agents such as paraffin. f) an absorption enhancer such as a quaternary ammonium compound, g) a wetting agent such as cetyl alcohol and glycerol monostearate, h) an absorbent such as kaolin and bentonite clay, and i) a lubricant such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer.

[0177] Similar solid compositions may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or lactose and high-molecular-weight polyethylene glycol. Solid dosage forms of tablets, sugars, capsules, pills, and granules may be prepared using coatings and shells, such as enteric coatings and other coatings known in pharmaceutical formulation technology. These dosage forms may optionally contain opacifiers and may be compositions that release only the active ingredient(s) or preferentially release the active ingredient(s) in a specific portion of the intestinal tract, with a delay as needed. Examples of embedding compositions that can be used include polymer substances and waxes. Similar solid compositions may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or lactose and high-molecular-weight polyethylene glycol.

[0178] The active polypeptide may also be in microencapsulated form with one or more of the aforementioned excipients. Solid dosage forms of tablets, sugars, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings, controlled-release coatings, and other coatings known in pharmaceutical formulation technology. In such solid dosage forms, the active polypeptide may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms may also contain further substances other than the inert diluent, as is common practice, such as a tableting lubricant and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain a buffer. These dosage forms may optionally contain an opacifier and may be compositions that release only the active ingredient(s) or preferentially release the active ingredient(s) in a specific portion of the intestinal tract, with a delay as necessary. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0179] Dosage forms for topical or transdermal administration of polypeptides described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed, if necessary, under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers. Ophthalmic formulations, ear drops, and eye drops are also intended to be within the scope of the present invention. Furthermore, the present invention intends for the use of transdermal absorption patches, which have the additional advantage of providing control over the delivery of polypeptides to the body. Such dosage forms may be produced by dissolving or compounding polypeptides in a suitable medium. Absorption enhancers may also be used to increase the flow of polypeptides through the skin. The rate can be controlled either by providing a rate-controlling membrane or by dispersing polypeptides in a polymer matrix or gel.

[0180] The compositions described herein may be administered orally, parenterally, or by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term “parenteral” as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-arterial, intra-arterial, intra-articular, intra-sternal, intrathecal, intrahepatic, intrafocal, and intracranial injection or infusion techniques. In particular, the compositions are administered orally, intraperitoneally, or intravenously.

[0181] The sterile injectable forms of the compositions described herein may be aqueous or oily suspensions. These suspensions may be formulated according to art-known techniques using appropriate dispersants or wetting and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, for example, as a solution in 1,3-butanediol. Water, Ringer's solution, and isotonic salines are among the acceptable vehicles and solvents that may be employed. Sterile non-volatile oils have also been conventionally employed as solvents or suspensions. For this purpose, conventionally non-irritating non-volatile oils, including synthetic monoglycerides or diglycerides, have been employed. Fatty acids such as oleic acid and their glyceride derivatives are useful in the preparation of injectables, as are naturally pharmaceutically acceptable oils such as olive oil or castor oil, particularly their polyoxyethylated varieties. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants such as carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tween and Span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms may also be used for formulation purposes.

[0182] The pharmaceutical compositions described herein may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. For tablets for oral use, commonly used carriers include, but are not limited to, lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. If an aqueous suspension is required for oral use, the active ingredient is mixed with emulsifiers and suspending agents. Certain sweeteners, flavorings, or colorants may also be added, if desired.

[0183] Alternatively, the pharmaceutical compositions described herein may be administered in the form of suppositories for rectal administration. These dosage forms can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0184] The pharmaceutical compositions described herein may also be administered topically if the target of treatment includes, in particular, diseases of the eyes, skin, or lower intestines, and includes areas or organs that are readily accessible by topical application. Suitable topical formulations are readily prepared for each of these areas or organs.

[0185] Topical application for the lower intestine can be achieved with anal suppositories (see above) or with appropriate enema formulations. Topical transdermal patches may also be used.

[0186] For topical application, the pharmaceutical composition may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the polypeptide of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutical composition may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0187] For ophthalmic use, the pharmaceutical composition may be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, or, in particular, as a solution in isotonic, pH-adjusted sterile saline, with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical composition may be formulated in an ointment such as petrolatum.

[0188] The pharmaceutical composition may also be administered by nasal aerosol or inhalation. Such compositions may be prepared according to well-known techniques in the field of pharmaceutical formulations and may be prepared as a solution in saline solution employing benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, fluorocarbons, and / or other conventional solubilizers or dispersants.

[0189] Polypeptides for use in the methods of the present invention may be formulated in unit dosage forms. The term "unit dosage form" means a physically separate unit appropriate as a unit dose for the subject to be treated, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in conjunction with an appropriate drug carrier as needed. A unit dosage form may be for a single daily dose or for a single dose of a plurality of daily doses (e.g., about 1 to 4 times or more per day). If a plurality of daily doses are used, the unit dosage forms may be the same or different for each dose.

[0190] Intrapulmonary / nasal administration For intrapulmonary administration, preferably, at least one polypeptide composition is delivered in a particle size effective for reaching the lungs or the lower airways of the sinuses. According to the present invention, at least one polypeptide disclosed herein can be delivered by any of the various inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, capable of depositing aerosolized formulations into the patient's sinus cavities or alveoli, include metered-dose inhalers, nebulizers, dry powder generators, nebulizers, and the like. Other devices suitable for directing intrapulmonary or nasal administration of polypeptides are also known in the art. Many of these devices can use formulations suitable for administration to disperse polypeptides in an aerosol. Such aerosols may consist of either solutions (both aqueous and non-aqueous) or solid particles.

[0191] Metered dose inhalers, such as Ventolin® metered dose inhalers, typically use a propellant gas and require actuation during inspiration (see, for example, International Publication No. WO 94 / 16970, International Publication No. WO 98 / 35888). Dry powder inhalers such as TURBUHALER™ (Astra), ROTAHALER® (Glaxo), DISKUS® (Glaxo), SPIROS™ inhaler (Dura), devices marketed by Inhale Therapeutics, and the SPINHALER® powder inhaler (Fisons) use breath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 to Astra, European Patent No. 237507 to Astra, International Publication No. WO 97 / 25086 to Glaxo, International Publication No. WO 94 / 08552 to Dura, U.S. Pat. No. 5,458,135 to Inhale, International Publication No. WO 94 / 06498 to Fisons, each of which is incorporated herein by reference in its entirety). Nebulizers such as AERX™ from Aradigm, ULTRAVENT® nebulizer (Mallinckrodt), and ACORN II® nebulizer (Marquest Medical Products) (U.S. Pat. No. 5,404,871 to Aradigm, International Publication No. WO 97 / 22376, all of the above references are incorporated herein by reference in their entireties) produce aerosols from solutions, while metered dose inhalers, dry powder inhalers and the like generate small particle aerosols. These specific examples of commercially available inhalation devices are intended to be representative of particular devices suitable for the practice of the present invention, and are not intended to limit the scope of the present invention.

[0192] In some embodiments, a composition comprising at least one polypeptide disclosed herein is delivered by a dry powder inhaler or nebulizer. There are several desirable features of an inhalation device for administering at least one polypeptide of the present invention. For example, delivery by an inhalation device is advantageously reliable, reproducible, and accurate. For good breathability, the inhalation device may deliver small dry particles, for example, less than about 10 μm, preferably about 1 to 5 μm, as needed.

[0193] A spray containing a polypeptide composition can be produced by extruding a suspension or solution of at least one polypeptide through a nozzle under pressure. The size and configuration of the nozzle, the pressure applied, and the liquid supply rate can be selected to achieve the desired output and particle size. Electrospray can be produced, for example, by an electric field connected to the supply of a capillary tube or nozzle. Advantageously, the particles of at least one polypeptide delivered by the sprayer have a particle size of less than about 10 μm, and in some embodiments, have particle sizes in the range of about 1 μm to about 5 μm, and about 2 μm to about 3 μm.

[0194] A formulation having at least one polypeptide suitable for use with a nebulizer typically comprises the polypeptide composition in an aqueous solution at a concentration of the at least one polypeptide of from about 0.1 mg to about 100 mg per ml of solution or mg / gm, or any range, value, or fraction therein. The formulation may comprise excipients, buffers, isotonic agents, preservatives, surfactants, and preferably agents such as zinc. The formulation may also comprise excipients or agents for stabilizing the polypeptide composition, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful for formulating the polypeptide composition include albumin, protamine, and the like. Typical carbohydrates useful for formulating the polypeptide composition include sucrose, mannitol, lactose, trehalose, glucose, and the like. The polypeptide composition formulation may also comprise a surfactant that can reduce or prevent surface-induced aggregation of the polypeptide composition caused by atomization of the solution during aerosol formation. Various conventional surfactants such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters can be employed. The amount generally ranges between 0.001% to 14% of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Additional agents known in the art for formulating polypeptides, such as IL-23p19 antibodies, or specific portions or variants thereof, can also be included in the formulation.

[0195] Administration of Polypeptide Composition by Nebulizer The polypeptide composition of the present invention can be administered by a nebulizer such as a jet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source is used to generate a high-speed air jet through an orifice. As the gas spreads beyond the nozzle, a low-pressure region is created, which draws the polypeptide composition solution through a capillary tube connected to a liquid reservoir. The liquid stream from the capillary tube is sheared into unstable filaments and droplets, and when the droplets exit the tube, an aerosol is generated. By employing a range of configurations, flow rates, and baffling types, desired performance characteristics can be achieved from a given jet nebulizer. In ultrasonic nebulizers, a piezoelectric transducer is typically used to generate vibration or mechanical energy using high-frequency electrical energy. This energy is transmitted either directly to the polypeptide composition formulation or through a binding fluid, generating an aerosol containing the polypeptide composition. Advantageously, the particles of the polypeptide composition delivered by the nebulizer have a particle size of less than about 10 μm, and in some embodiments, have a particle size in the range of about 1 μm to about 5 μm, or about 2 μm to about 3 μm.

[0196] A formulation of at least one polypeptide, suitable for use with either a jet or ultrasonic nebulizer, typically contains a concentration of at least one polypeptide of about 0.1 mg to about 100 mg per ml of solution. The formulation may also contain excipients, buffers, isotonic agents, preservatives, surfactants, and preferably agents such as zinc. The formulation may also contain excipients or agents for stabilizing the at least one polypeptide composition, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful for formulating the at least one polypeptide composition include albumin, protamine, etc. Typical carbohydrates useful for formulating the at least one polypeptide include sucrose, mannitol, lactose, trehalose, glucose, etc. The at least one polypeptide formulation may also contain a surfactant that can reduce or prevent surface-induced aggregation of the at least one polypeptide caused by atomization of the solution in aerosol formation. Various conventional surfactants such as polyoxyethylene fatty acid esters and alcohols, as well as polyoxyethylene sorbitol fatty acid esters, may be employed. The amount will generally be in the range of about 0.001 to 4% of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, etc. Further agents known in the field for the formulation of polypeptides, such as antibody proteins, may also be included in the formulation.

[0197] Administration of polypeptide compositions using a metered-dose inhaler. In a metered-dose inhaler (MDI), the spray, at least one polypeptide disclosed herein, and any excipients or other additives are contained in a canister as a mixture containing liquefied compressed gas. By operating a throttle valve, a mixture containing particles, preferably less than about 10 μm, and in some embodiments, about 1 μm to about 5 μm and about 2 μm to about 3 μm in size range, is released as an aerosol. Desired aerosol particle sizes can be obtained by employing formulations of polypeptide compositions produced by various methods known to those skilled in the art, including jet milling, spray drying, critical point condensation, etc. Preferred metered-dose inhalers include those employing hydrofluorocarbon sprays manufactured by 3M or Glaxo. Formulations of at least one polypeptide for use with a metered-dose inhaler device generally include a suspension in a non-aqueous medium in which a fine powder containing at least one polypeptide is suspended in the spray, for example, with the help of a surfactant. The spray may be any conventional material employed for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), etc. Preferably, the spray is a hydrofluorocarbon. The surfactant may be selected to stabilize at least one polypeptide as a suspension in the spray, to protect the activator from chemical decomposition, etc. Suitable surfactants include sorbitan trioleate, soy lecithin, oleic acid, etc. In some cases, a solution aerosol using a solvent such as ethanol is preferred. Further agents known in the art for the formulation of polypeptides may also be included in the formulation. Those skilled in the art will recognize that the method of the present invention can be achieved by intrapulmonary administration of at least one polypeptide composition by a device not described herein.

[0198] Combination therapy An effective dose can be achieved in the methods or pharmaceutical compositions of the present invention, employing a polypeptide or a pharmaceutically acceptable salt or solvate thereof (e.g., a hydrate) alone or in combination with a further suitable therapeutic agent, such as an antiviral agent or a vaccine. When "combination therapy" is employed, an effective dose can be achieved using a first amount of polypeptide, or a pharmaceutically acceptable salt or solvate thereof (e.g., a hydrate), and a second amount of a further suitable therapeutic agent (e.g., an antiviral agent or a vaccine).

[0199] In other embodiments of the present invention, the polypeptide and the further therapeutic agent are administered in an effective amount (i.e., an amount that would be therapeutically effective if administered individually). In yet another embodiment, the polypeptide and the further therapeutic agent are administered in an amount that does not provide a therapeutic effect individually (a dose below the therapeutic dose). In yet another embodiment, the polypeptide may be administered in an effective amount, but the further therapeutic agent may be administered at or below the therapeutic dose. In yet another embodiment, the polypeptide may be administered at or below the therapeutic dose, but the further therapeutic agent, such as a suitable antiviral therapeutic agent, may be administered in an effective amount.

[0200] As used herein, the terms “combined” or “concurrent administration” may be used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). The use of the terms does not restrict the order in which the therapies (e.g., prophylactic and / or therapeutic agents) are administered to the subject.

[0201] Concurrent administration includes the administration of polypeptides in substantially simultaneous amounts, such as in a single pharmaceutical composition containing first and second amounts in fixed ratios of first and second amounts, or in multiple separate capsules or tablets for each polypeptide. Such concurrent administration also includes the sequential use of each polypeptide in any order.

[0202] In some embodiments, the present invention relates to combination therapy methods for treating viral infections (e.g., COVID-19) by inhibiting viral replication in biological samples or patients, or for treating or preventing viral infections in patients using polypeptides or pharmaceutical compositions of the present invention. Therefore, the pharmaceutical compositions of the present invention also include those comprising an antiviral polypeptide exhibiting antiviral activity in combination with an inhibitor of viral replication of the present invention.

[0203] If the co-administration involves separate administrations of a first dose of polypeptide and a second dose of an additional therapeutic agent, the polypeptides are administered over sufficiently close intervals to produce the desired therapeutic effect. For example, the interval between each administration that can produce the desired therapeutic effect may range from a few minutes to several hours and can be determined by considering the properties of each polypeptide, such as its efficacy, solubility, bioavailability, plasma half-life, and kinetic profile. For example, the polypeptide and the second therapeutic agent can be administered in any order within approximately 24 hours, 16 hours, 8 hours, 4 hours, 1 hour, or 30 minutes from each other.

[0204] Furthermore, the first therapy (e.g., a prophylactic or therapeutic agent such as any one of the polypeptides of the present invention) may be administered to the subject before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after after) the administration of the second therapy (e.g., a prophylactic or therapeutic agent such as an antiviral agent) to the subject.

[0205] The method of co-administering a first amount of polypeptide and a second amount of further therapeutic agent may result in an enhanced or synergistic therapeutic effect, and the combined effect may be greater than the additive effect produced by the separate administration of the first amount of polypeptide and the second amount of further therapeutic agent.

[0206] As used herein, the term “synergistic” means a combination of the polypeptide of the present invention and another therapy (e.g., a prophylactic or therapeutic agent) that is more effective than the additive effect of the therapies. The synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) enables the use of one or more therapies in lower doses and / or less frequent administration of such therapies to a subject. The ability to utilize lower doses of therapies (e.g., prophylactic or therapeutic agents) and / or administer such therapies in a lower frequency can reduce the toxicity to a subject associated with the administration of such therapies without reducing the effectiveness of such therapies in preventing, managing, or treating the disorder. Furthermore, the synergistic effect may result in improved efficacy of the agents in preventing, managing, or treating the disorder. Finally, the synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) can avoid or reduce adverse or undesirable side effects associated with the use of any of therapies alone.

[0207] When the combination therapy using the polypeptide of the present invention is combined with a vaccine, both therapeutic agents may be administered with longer intervals between each dose (e.g., several days, several weeks, or several months).

[0208] The presence or absence of synergistic effects can be determined using appropriate methods for evaluating drug interactions. Appropriate methods include, for example, the sigmoid-emax equation (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6:429-453 (1981)), the Loewe additive equation (Loewe, S. and Muischnek, H., Arch. Exp. Pethol Pharmacol. 114:313-326 (1926)), and the median-effect equation (Chou, TC and Talalay, P., Adv. Enzyme Regul. 22:27-55 (1984)). Each of the equations mentioned above can be applied to experimental data to create corresponding graphs to help evaluate the effects of drug combinations. The corresponding graphs associated with the equations mentioned above are the concentration-effect curve, the isobologram curve, and the combined exponential curve, respectively.

[0209] Specific examples of polypeptides that can be co-administered with the polypeptides described herein include neuraminidase inhibitors such as remdesivir, oseltamivir (Tamiflu®), and zanamivir (RLENZA®), viral ion channel (M2 protein) blockers such as amantadine (SYMMETREL®) and rimantadine (FLUMADINE®), and antiviral agents described in International Publication No. 2003 / 015798, including T-705 (Ruruta et al., Antiviral Research, 82:95-102 (2009), “T-705 (flavipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections.”), currently under development by Toyama Chemical of Japan. In some embodiments, the polypeptides described herein may be co-administered with a conventional influenza vaccine.

[0210] Exemplary Embodiments In some embodiments, the present invention comprises, consists essentially of, or consists of a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) domains or fragments thereof and (b) an immunoglobulin domain, wherein the one or more mutant NRP domains result in reduced binding of the recombinant polypeptide to heparin or heparan sulfate compared to a wild-type NRP domain.

[0211] In some embodiments, the recombinant polypeptide comprises a recombinant polypeptide comprising one or more mutant NRP domains derived from NRP1 or NRP2 protein.

[0212] In some embodiments, the one or more mutant NRP domains are one or more mutant b1 domains or one or more mutant b2 domains.

[0213] In some embodiments, the one or more mutant NRP domains have one or more amino acid substitutions selected from the group consisting of K373E, K351A, E319A, K358E, R513E, K514E, K516E, R513A, K514A, K516A, Y297A, S345A and Y353A, as compared to the wild-type amino acid sequence set forth in SEQ ID NO: 1.

[0214] In some embodiments, the one or more mutant NRP domains result in reduced binding of the recombinant polypeptide to heparin.

[0215] In some embodiments, the one or more mutant NRP domains result in reduced binding of the recombinant polypeptide to heparan sulfate.

[0216] In some embodiments, the immunoglobulin domain is an Fc domain.

[0217] In some embodiments, the present invention comprises, essentially comprises, or consists of a recombinant polypeptide, wherein (a) one or more mutant neuropilin (NRP) b1 domains, NRP b2 domains or fragments thereof, and (b) an Fc domain, wherein one or more mutant NRP b1 domains, NRP b2 domains or fragments thereof are derived from an NRP1 or NRP2 protein; one or more mutant NRP b1 domains, NRP b2 domains or fragments thereof have one or more amino substitutions selected from the group consisting of K373E, K351A, E319A, K358E, R513E, K514E, K516E, R513A, K514A, K516A, Y297A, S345A and Y353A, compared to the wild-type amino acid sequence shown in SEQ ID NO: 1; and one or more amino substitutions result in a reduction in the binding of the recombinant polypeptide to heparin or heparan sulfate.

[0218] In some embodiments, the present invention relates to a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2), or fragments thereof, and (b) an Fc domain, wherein (a) and (b) are b1-Fc;b1b1-Fc;b1b1b1-Fc;b1-Fc;b1b1b1-Fc;b1b2-Fc;b1b2-Fc;b1b2-Fc;b1b2-Fc;Fc-b1b2;Fc-b1b2 Includes a structure having the orientation of ;b1-Fc-b1;b1b1-Fc-b1;b1-Fc;b1b1-Fc;b1b1b1-Fc;b1-Fc;b1b1b1-Fc;b1b2-Fc;b1b2-Fc;Fc-b1b2;Fc-b1b2;b1-Fc-b1;b1b1-Fc-b1;one or The molecule comprises multiple b1, b2 or fragments thereof derived from the NRP1 or NRP2 protein; one or more b1, b2 or fragments thereof have one or more amino substitutions selected from the group consisting of K373E, K351A, E319A, K358E, R513E, K514E, K516E, R513A, K514A, K516A, Y297A, S345A and Y353A, compared to the wild-type amino acid sequence shown in SEQ ID NO: 1; and the molecule comprises, essentially consists of, or is composed of a recombinant polypeptide, in which one or more amino substitutions result in reduced binding of the recombinant polypeptide to heparin or heparan sulfate.

[0219] In some embodiments, the present invention comprises, essentially consists of, or comprises a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) domains or fragments thereof, and (b) an immunoglobulin domain, wherein the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids.

[0220] In some embodiments, the recombinant polypeptide has one or more mutant NRP domains or fragments derived from the NRP1 or NRP2 protein.

[0221] In some embodiments, one or more mutant NRP domains or fragments thereof are one or more mutant b1 domains or one or more mutant b2 domains.

[0222] In some embodiments, the present invention comprises a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) domains or fragments thereof, and (b) an immunoglobulin domain, wherein the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids, comprising, essentially comprising, or comprising a recombinant polypeptide, wherein the virus is a virus belonging to one of the following regions: duplodonnavirus region, monodonnavirus region, ribovirus region, or validnavirus region.

[0223] In some embodiments, the virus is a virus belonging to one of the following kingdoms: Banfoldvirus, Heunggongvirae, Orthorunavirus, Paralunavirus, or Shoutokuvirus.

[0224] In some embodiments, the virus is a virus belonging to one of the following phyla: Altwell's virus, Kossavirus, Citrinovirus, Negarnavirus, Nucleocytovirus, Peplovirus, or Pisvirus.

[0225] In some embodiments, the virus is a virus belonging to one of the following classes: Arsvirus, Heliovirus, Frasvirus, Helwivirus, Instvirus, Monivirus, Papovavirus, Pisonivirus, Pockesvirus, Leutravirus, or Sterpavirus.

[0226] In some embodiments, the virus is a virus belonging to one of the following orders: Amarilloviruses, Articulaviruses, Bunyaviroides, Cytoviruses, Heperiviruses, Herpesviruses, Jingchuvirales, Martelliviruses, Mononegaviruses, Nidoviruses, Orterviruses, Stellaviruses, or Zurhausenviruses.

[0227] In some embodiments, the virus is a virus belonging to one of the following families: Astroviridae, Bunyaviridae, Bornaviridae, Chuviridae, Coronaviridae, Flaviviridae, Filoviridae, Hantaviridae, Hepeviridae, Herpesviridae, Nairoviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Peribunyaviridae, Fenuiviridae, Pneumoviridae, Poxviridae, Retroviridae, Raptoviridae, or Togaviridae.

[0228] In some embodiments, the virus is selected from the group consisting of dengue fever; respiratory syncytial virus (RSV); hantavirus; Epstein-Barr virus (EBV); EBV (uncleaved); SARS-CoV-2 Wuhan; SARS-CoV-2 Wuhan (uncleaved); SARS-CoV-2 UK; SARS-CoV-2 India; SARS-CoV-2 India (uncleaved); HCoV-OC43; MERS-CoV; MERS-CoV (uncleaved); herpes simplex virus (HSV) 1; HSV 1 (uncleaved); influenza A H5N1 virus (IAV H5N1); human papillomavirus (HPV); human metapneumovirus; and human immunodeficiency virus (HIV).

[0229] In some embodiments, the virus has a CendR motif selected from the group consisting of GTCTQSGERRREKR;KNTNVTLSKKRKRR;LTHKMIEESHRLRR;VSFKPPPPPSRRRR;VSFKPPPPPSRRRRGACVVY;CASYQTQTNSPRRAR;CASYQTQTNSPRRARSVASQSIIAYTMSLG;ASYQTQTNSHRRAR;ASYQTQTNSRRRAR;ASYQTQTNSRRRARSVASQSIIAY;GSGYCVDYSKNRRSR;LLEPVSISTGSRSAR;LLEPVSISTGSRSARSAIEDLLFDK;ERPRAPARSASRPRR;ERPRAPARSASRPRRPV;VLATGLRNVPQRKKR;PTTSSTSTTAKRKKR;IDMLKARVKNRVAR;AKRRVVQREKR; and AKRRVVQREKRAVGIGALFLG.

[0230] In some embodiments, the present invention relates to a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2), or fragments thereof, and (b) an Fc domain, wherein one or more b1, b2, or fragments thereof are derived from an NRP1 or NRP2 protein; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids, and the virus is dengue fever; respiratory syncytial virus (RSV); hantavirus; Epstein-Barr virus (EBV); EBV (uncleaved); SARS-CoV-2 Wuhan; SARS-CoV-2 Wuhan (uncleaved); SARS-CoV-2 UK; SARS-CoV-2 India; SARS-CoV-2 The product contains, essentially consists of, or comprises recombinant polypeptides selected from the group consisting of India (uncleaved); HCoV-OC43; MERS-CoV; MERS-CoV (uncleaved); herpes simplex virus (HSV) 1; HSV1 (uncleaved); influenza A H5N1 virus (IAV H5N1); human papillomavirus (HPV); human metapneumovirus; and human immunodeficiency virus (HIV).

[0231] In some embodiments, the present invention relates to a recombinant polypeptide comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2), or fragments thereof, and (b) an Fc domain, wherein one or more b1, b2 or fragments thereof are derived from NRP1 or NRP2 proteins; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids; and the virus is GTCTQSGERRREKR;KNTNVTLSKKRKRR;LTHKMIEESHRLRR;VSFKPPPPPSRRRR;VSFKPPPPPSRRRRGACVVY;CASYQTQTNSPRRAR;CASYQTQTNSPRRARSVASQ The invention comprises, essentially consists of, or comprises recombinant polypeptides having a CendR motif selected from the group consisting of SIIAYTMSLG;ASYQTQTNSHRRAR;ASYQTQTNSRRRAR;ASYQTQTNSRRRARSVASQSIIAY;GSGYCVDYSKNRRSR;LLEPVSISTGSRSAR;LLEPVSISTGSRSARSAIEDLLFDK;ERPRAPARSASRPRR;ERPRAPARSASRPRRPV;VLATGLRNVPQRKKR;PTTSSTSTTAKRKKR;IDMLKARVKNRVAR;AKRRVVQREKR; and AKRRVVQREKRAVGIGALFLG.

[0232] In part by implementation, the present invention is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, and at least 92% identical to the amino acid sequence shown in any one of the sequence numbers listed in Table 21. or comprising a recombinant polypeptide or a pharmaceutically acceptable salt thereof containing an amino acid sequence that is at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, or consisting of a recombinant polypeptide or a pharmaceutically acceptable salt thereof.

[0233] In some implementations, the present invention is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, and at least A recombinant polypeptide or a pharmaceutically acceptable salt thereof containing an amino acid sequence that is 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, comprising, essentially consisting of, a recombinant polypeptide or a pharmaceutically acceptable salt thereof, or comprising a recombinant polypeptide or a salt thereof, further comprising an excipient.

[0234] In some embodiments, the present invention is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, and at least... A recombinant polypeptide or a pharmaceutically acceptable salt thereof containing an amino acid sequence that is at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, contains, essentially consists of, a recombinant polypeptide or a pharmaceutically acceptable salt thereof, or consists of a recombinant polypeptide or a salt thereof.

[0235] In some embodiments, the present invention is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, and at least 91% identical. A recombinant polypeptide or a pharmaceutically acceptable salt thereof containing an amino acid sequence that is 100% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, comprising, essentially, a recombinant polypeptide or a pharmaceutically acceptable salt thereof, or comprising a recombinant polypeptide or a salt thereof, further comprising an excipient.

[0236] In some embodiments, the present invention includes a recombinant polypeptide having an amino acid sequence that is at least 90% identical to any one of the amino acid sequences shown in SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162 and 193-201, or a pharmaceutically acceptable salt thereof, or essentially consists of a recombinant polypeptide or a pharmaceutically acceptable salt thereof, or consists of a recombinant polypeptide or a pharmaceutically acceptable salt thereof.

[0237] In some embodiments, the present invention comprises a recombinant polypeptide having an amino acid sequence that is at least 90% identical to any one of the amino acid sequences shown in SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162 and 193-201, or a pharmaceutically acceptable salt thereof, or essentially consists of a recombinant polypeptide or a pharmaceutically acceptable salt thereof, or consists of a recombinant polypeptide or a pharmaceutically acceptable salt thereof.

[0238] In some embodiments, the present invention comprises a recombinant polypeptide having an amino acid sequence shown in any one of SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162, and 193-201, or a pharmaceutically acceptable salt thereof, or essentially consists of a recombinant polypeptide or a pharmaceutically acceptable salt thereof, or consists of a recombinant polypeptide or a pharmaceutically acceptable salt thereof.

[0239] In some embodiments, the present invention comprises, essentially comprises, or consists of a method for limiting the occurrence of a viral infection, reducing the risk, reducing the severity, or treating a subject in need thereof, comprising administering to a subject a composition comprising (a) one or more mutant neuropilin (NRP) domains or fragments thereof, and (b) a recombinant polypeptide comprising an immunoglobulin domain, wherein the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids.

[0240] In some embodiments of the method, one or more mutant NRP domains or fragments thereof are derived from the NRP1 or NRP2 protein.

[0241] In some embodiments of the method, one or more mutant NRP domains or fragments thereof are one or more mutant b1 domains or one or more mutant b2 domains.

[0242] In some embodiments of the method, the virus is a virus belonging to one of the following regions: the duplodnavirus region, the monodnavirus region, the ribovirus region, or the validnavirus region.

[0243] In some embodiments, the virus is a virus belonging to one of the following kingdoms: Banfoldvirus, Heunggongvirae, Orthorunavirus, Paralunavirus, or Shoutokuvirus.

[0244] In some embodiments of the method, the virus is a virus belonging to one of the following phyla: Altwell's virus, Kossavirus, Chitorinovirus, Negarnavirus, Nucleocytovirus, Peplovirus, or Pisvirus.

[0245] In some embodiments of the method, the virus is a virus belonging to one of the following classes: Arsvirus, Heliovirus, Frasvirus, Helwivirus, Instvirus, Monivirus, Papovavirus, Pisonivirus, Pockesvirus, Leutravirus, or Sterpavirus.

[0246] In some embodiments of the method, the virus is a virus belonging to one of the following orders: Amarillovirales, Articulavirales, Bunyaviroides, Cytovirales, Heperivirales, Herpesvirales, Jingchuvirales, Martellivirales, Mononegavirales, Nidovirales, Ortervirales, Stellavirales, or Zurhausenvirales.

[0247] In some embodiments of the method, the virus is a virus belonging to one of the following families: Astroviridae, Bunyaviridae, Bornaviridae, Chuviridae, Coronaviruses, Flaviviridae, Filoviridae, Hantaviridae, Hepeviridae, Herpesviridae, Nairoviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Peribunyaviridae, Fenuiviridae, Pneumoviridae, Poxviridae, Retroviridae, Raptoviridae, or Togaviridae.

[0248] In some embodiments of the method, the virus is selected from the group consisting of dengue fever; respiratory syncytial virus (RSV); hantavirus; Epstein-Barr virus (EBV); EBV (uncleaved); SARS-CoV-2 Wuhan; SARS-CoV-2 Wuhan (uncleaved); SARS-CoV-2 UK; SARS-CoV-2 India; SARS-CoV-2 India (uncleaved); HCoV-OC43; MERS-CoV; MERS-CoV (uncleaved); herpes simplex virus (HSV) 1; HSV 1 (uncleaved); influenza A H5N1 virus (IAV H5N1); human papillomavirus (HPV); human metapneumovirus; and human immunodeficiency virus (HIV).

[0249] In some embodiments of the method, the virus has a CendR motif selected from the group consisting of GTCTQSGERRREKR;KNTNVTLSKKRKRR;LTHKMIEESHRLRR;VSFKPPPPPSRRRR;VSFKPPPPPSRRRRGACVVY;CASYQTQTNSPRRAR;CASYQTQTNSPRRARSVASQSIIAYTMSLG;ASYQTQTNSHRRAR;ASYQTQTNSRRRAR;ASYQTQTNSRRRARSVASQSIIAY;GSGYCVDYSKNRRSR;LLEPVSISTGSRSAR;LLEPVSISTGSRSARSAIEDLLFDK;ERPRAPARSASRPRR;ERPRAPARSASRPRRPV;VLATGLRNVPQRKKR;PTTSSTSTTAKRKKR;IDMLKARVKNRVAR;AKRRVVQREKR; and AKRRVVQREKRAVGIGALFLG.

[0250] In some embodiments, the present invention relates to a method for limiting the occurrence of a viral infection, reducing the risk, reducing the severity, or treating a subject in need thereof, comprising administering to the subject a composition comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2) or fragments thereof, and (b) a recombinant polypeptide comprising an Fc domain, wherein one or more b1, b2 or fragments thereof are derived from the NRP1 or NRP2 protein; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids; and the virus is dengue fever; respiratory syncytial virus (RSV); hantavirus; Epstein-Barr virus (EBV); EBV (uncleaved); SARS-CoV-2 Wuhan; SARS-CoV-2 Wuhan (uncleaved); SARS-CoV-2 UK; SARS-CoV-2 A method comprising, essentially comprising, or comprising a method selected from the group consisting of India; SARS-CoV-2 India (uncleaved); HCoV-OC43; MERS-CoV; MERS-CoV (uncleaved); herpes simplex virus (HSV) 1; HSV1 (uncleaved); influenza A H5N1 virus (IAV H5N1); human papillomavirus (HPV); human metapneumovirus; and human immunodeficiency virus (HIV).

[0251] In some embodiments, the present invention comprises, essentially comprises, or comprises a method for limiting the occurrence of a viral infection, reducing the risk, reducing the severity, or treating a subject in need thereof, comprising administering to a subject a composition comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2) or fragments thereof, and (b) a recombinant polypeptide comprising an Fc domain, wherein one or more b1, b2 or fragments thereof are derived from the NRP1 or NRP2 protein; the recombinant polypeptide is operable to bind to a virus having a -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids; and the virus has a CendR motif selected from the group comprising any motif disclosed in Table 36 or 37.

[0252] In some embodiments, the present invention relates to a method for limiting the occurrence of a viral infection, reducing the risk, reducing the severity, or treating a subject in need thereof, comprising administering to the subject a composition comprising (a) one or more mutant neuropilin (NRP) b1 domains (b1), NRP b2 domains (b2) or fragments thereof, and (b) a therapeutically effective amount of a recombinant polypeptide comprising an Fc domain, wherein one or more b1, b2 or fragments thereof are derived from the NRP1 or NRP2 protein; the recombinant polypeptide is operable to bind to a virus having the -Z1-X1-X2-Z2-(CendR) motif, where Z1 and Z2 are arginine or lysine, and X1 and X2 are any amino acids; and the virus is GTCTQSGERRREKR;KNTNVTLSKKRKRR;LTHKMIEESHRLRR;VSFKPPPPPSRRRR;VSFKPPPPPSRRRRGACVVY;CASYQTQTNSPRRAR;C A method comprising, essentially consisting of, or consisting of, a CendR motif selected from the group consisting of ASYQTQTNSPRRARSVASQSIIAYTMSLG;ASYQTQTNSHRRAR;ASYQTQTNSRRRAR;ASYQTQTNSRRRARSVASQSIIAY;GSGYCVDYSKNRRSR;LLEPVSISTGSRSAR;LLEPVSISTGSRSARSAIEDLLFDK;ERPRAPARSASRPRR;ERPRAPARSASRPRRPV;VLATGLRNVPQRKKR;PTTSSTSTTAKRKKR;IDMLKARVKNRVAR;AKRRVVQREKR; and AKRRVVQREKRAVGIGALFLG.

[0253] In some embodiments, the present invention includes, essentially, or consists of, a method for limiting the occurrence of a viral infection, reducing the risk, reducing the severity, or treating a subject in need thereof, comprising administering to the subject a composition comprising a recombinant polypeptide having an amino acid sequence that is at least 90% identical to one of the amino acid sequences shown in SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162, and 193-201, or a therapeutically effective amount of a pharmaceutically acceptable salt thereof.

[0254] In some embodiments, the present invention includes, essentially, or constitutes a method for limiting the occurrence of a viral infection, reducing the risk, reducing the severity, or treating a subject in need thereof, comprising administering to the subject a composition comprising a recombinant polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162, and 193-201, or a therapeutically effective amount of a pharmaceutically acceptable salt thereof.

[0255] In some embodiments, the present invention includes, essentially, or consists of, a method for limiting the occurrence of a viral infection, reducing the risk, reducing the severity, or treating a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of a recombinant polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 113-116, 121-122, 133-137, 148-149, 154, 162, and 193-201, or a pharmaceutically acceptable salt thereof.

[0256] Examples The examples provided herein are not intended to limit the present invention and should not be used to limit it. They are provided solely to illustrate the present invention.

[0257] Example 1. Design and generation of a structure Several types of expression vectors have been designed in this specification. The first type of expression vector comprises the b1 domain of neuropyrin and an immunoglobulin domain (e.g., Fc domain), as described in Tables 7A and 7B. The second type of expression vector comprises the ACE2 domain of angiotensin-converting enzyme 2 and an immunoglobulin domain (e.g., Fc domain), as described in Table 6. The third type of expression vector comprises the b1 domain of neuropyrin, the ACE2 domain of angiotensin-converting enzyme 2 and an immunoglobulin domain (e.g., Fc domain), as described in Tables 8A and 8B.

[0258] To increase the binding affinity to the spike protein of a specific virus, such as SARS-CoV-2, the polypeptide, also referred to herein as a fusion protein, may be designed to comprise one or more b1 or neuropilin domains and / or one or more ACE2 or angiotensin-converting enzyme 2 domains.

[0259] To increase the binding affinity to the spike protein of a specific virus, such as SARS-CoV-2, polypeptides, also referred to herein as fusion proteins, may be designed with mutations in the b1 or neuropilin domain and / or in the ACE2 or angiotensin-converting enzyme 2 domain. In some embodiments, the mutation in the b1 or neuropilin domain may include E319A. In some embodiments, the mutation in the ACE2 or angiotensin-converting enzyme 2 domain may include F28W, D30A, or L79T mutations.

[0260] To reduce the binding affinity of the virus to the spike protein, the fusion protein may be designed with mutations in the b1 or neuropilin domain and / or in the angiotensin-converting enzyme 2 domain. In some embodiments, the neuropilin fragment mutations may include Y297A / S346A / Y353A, T349A, and K351A mutations in the b1 domain.

[0261] To increase binding affinity to FcRn, the fusion protein may be designed with mutations in the immunoglobulin Fc domain. In some embodiments, the immunoglobulin domain mutations may include N434A and T307A / E380A / N434A(AAA) mutations in the Fc domain.

[0262] To reduce Fc-mediated effector function, polypeptides or fusion proteins may be designed with mutations in the immunoglobulin Fc domain. In some embodiments, these mutations in the immunoglobulin domain may include L235E, F234A / L235A, N297A, N297Q, or N297G mutations in the Fc domain. In other embodiments, the immunoglobulin Fc domains of IgG2 and IgG4 may be utilized to reduce the Fc-mediated effector function of the fusion protein.

[0263] To reduce antibody-dependent enhancement (ADE), some decoys were designed with mutations in the immunoglobulin Fc domain. In some embodiments, the mutations in the immunoglobulin domain may include L235E, F234A / L235A, N297A, N297Q, or N297G mutations in the Fc domain. In other embodiments, the immunoglobulin Fc domains of IgG2 and IgG4 may be used to reduce the Fc-mediated effector function of the fusion protein.

[0264] Human neuropilin-1 (UniProt number: 014786, SEQ ID NO: 1) and human neuropilin-2 (UniProt number: 060462, SEQ ID NO: 2) were used as sources for neuropilin fragments. Human angiotensin-converting enzyme 2 (UniProt number: Q9BYF1, SEQ ID NO: 32) was used as a source for angiotensin-converting enzyme 2 fragments. Human IgG variants were used as sources for the immunoglobulin Fc region. Derivatives of these sequences, including amino acid substitutions, are shown in Tables 1-3.

[0265] Generally, protein constructs are synthesized as follows: The expression construct is generated by codon-optimized gene synthesis and inserted into pcDNA3.4 as an expression vector using NotI and HindIII restriction enzymes. The constructed expression vector contains a signal peptide, and a Kozak sequence may be included in the 5' untranslated region for optimized transcription. One Shot® Top10 E. coli competent cells are transformed with the resulting plasmid containing the gene encoding the protein construct, and the transformed cells are cultured overnight. The constructed plasmid is obtained using the PureLink® HiPure Expi Plasmid Megaprep Kit (ThermoFisher Scientific, Waltham, Mass.).

[0266] The fusion protein was transiently expressed in the CHO-S system (ThermoFisher Scientific). The proteins were expressed individually according to the manufacturer's recommended conditions. Briefly, plasmid DNA with a total of 0.8 μg per 1 mL of CHO-S culture, with a 1:1 light-to-heavy chain ratio, was prepared using OPTIPRO® SFM and EXPIFECTAMINE®. The mixture was added to CHO-S cells at a viable cell density of 6 × 10⁶ cells / mL and a viability of over 98%. The cell culture was incubated overnight at 37°C, 80% humidity, and 8% CO₂ in a Nalgene® Single-Use PETG Erlenmeyer flask with shaking at 125 RPM in a 19 mm orbit. The following day, the culture is augmented (EXPICHO® enhancer; ThermoFisher Scientific), nutrient supplied (EXPICHO® feed; ThermoFisher Scientific), transferred to 32°C, 80% humidity, 5% CO2, and shaken at 125 RPM in a 19 mm orbit. A second nutrient supply is given on day 5, and the culture is returned to 32°C until collection on day 12. Collection is achieved by centrifugation at 4000 × g for 20 minutes. The purified supernatant is sterilized using an asymmetric polyethersulfone (PES) 0.22 μM filter assembly (Nalgene). The filtrate is stored at 4°C until purified.

[0267] All sterile supernatant containing antibodies is purified using MabSelect PRISMA® resin (GE Healthcare Life Sciences) in AKTApure (GE Healthcare Life Sciences). The resin is equilibrated with a buffer of 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. The supernatant containing antibodies is then loaded onto the column. The resin is washed with a buffer of 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 until the chromatography baseline returns to the column equilibration level. Elution is then performed using a buffer of 100 mM sodium acetate, 20% glycerol, pH 3.0, and the fraction is collected. The fraction is immediately neutralized with 1 M Tris, pH 9. The fraction containing the dominant absorbance at a wavelength of 280 nm is pooled and placed in an Amicon 10 kDa ultrafiltration device for buffer exchange. The elution buffer is removed by centrifugation in seven half-dilutions in an Amicon concentrator using storage buffer (phosphate-buffered saline). This substance will be submitted to the SEC and then stored at 4°C.

[0268] Size exclusion chromatography (SEC) analysis was performed using an Agilent Infinity 1260 II Quatenary Pump high-performance liquid chromatography (HPLC) system with a diode array UV detector (WR). 20(10) μg of antibody material was injected into a TSKgel G3000SWXL, 5 μm, 7.8 mm ID × 30 cm column. The mobile phase was phosphate-buffered saline at a flow rate of 1 mL / min. The antibody material was detected at wavelengths of 220, 280, and 330 nm during a 15-minute acquisition at a sampling rate of 1 Hz.

[0269] Example 2. In vitro test In vitro studies, using fluorescence-based enzyme-linked immunosorbent assay (ELISA), identified the binding properties of human neuropilin and angiotensin-converting enzyme 2 (ACE2)-based human IgG Fc constructs to the SARS-CoV-2 spike protein. The ELISA method used herein was used to rank the binding affinities of the disclosed neuropilin-1 / 2 (NP1 or NP2) and ACE2 human IgG Fc fusion constructs. Furthermore, the binding of two peptides identified from the SAR-CoV-2 S1 / S2 junction of the spike protein, enabling viral entry into cells via N1 and N2 receptors, is described, along with a peptide derived from the ACE2 amino-terminal alpha-1 domain that has been demonstrated to bind to the SAR-CoV-2 spike protein.

[0270] The reagents and materials used in the fluorescence-conjugated assay are shown in Table 9 below. The measurement methods used are shown in Table 10.

[0271] [Table 11]

[0272] [Table 12]

[0273] Example 3. Sample Preparation All protein constructs and peptides were diluted with PBS to the concentrations listed in Table 11. BSA was diluted to 5% in 1×PBS.

[0274] [Table 13]

[0275] Example 4. Plate preparation and sample addition

[0276] In Experiment 1, wells A1-B10 (darkly shaded) of Plate 1 (see Table 12) were coated with 200 μL of SARS-CoV-2 spike S1+S2 ECD-His protein to a final coating amount of 0.66 μg per well, and wells C1-D10 were coated with hub1b2-His to a final coating amount of 0.06 μg per well. The plate was incubated overnight in a refrigerator at 5°C. The following day, all assay preparations were performed at room temperature, except for incubation at 37°C. Plate 1 was removed from the refrigerator and the liquid was removed by gently tapping the plate in a sink. All wells were rinsed three times with 250 μL of 1×PBS. After the final rinse, the plate was inverted and tapped firmly on a paper towel to remove any remaining excess liquid. Then, all wells were blocked with 200 μL of 5% BSA, and the plate was incubated at 37°C for 1 hour. After incubation, the liquid was removed and the wells were cleaned as described above.

[0277] The FITC-labeled ACE2 a1 peptide was further diluted to 1.15 μg / mL in 800 μL of PBS, while the FITC-labeled CendR peptide was also diluted to 1.54 μg / mL in 800 μL of PBS. 190 μL of 1×PBS was added to wells A2-A10, B2-B10, C2-C10, and D2-D10. 380 μL of the ACE2 a1 peptide dilution was added to wells A1 and B1, while the same amount of CendR peptide was added to wells C1 and D1. Then, using a multichannel pipette, 190 μL was taken from each of the first wells (A1, B1, C1, and D1) and transferred to the wells in the next row. The samples were carefully mixed to avoid mixing with adjacent wells. This procedure was completed for all subsequent wells until wells A10, B10, C10, and D10 were completed. This process resulted in a 2-fold dilution of each well in columns 1 through 10 of rows A, B, C, and D (see Table 12). The remaining 190 μL of excess from the final dilution in column 10 was discarded.

[0278] [Table 14]

[0279] The plate was placed in a 37°C incubator for 1 hour. After incubation, the liquid was removed, and the plate was washed and dried as described above.

[0280] Relative fluorescence units (RFUs) from each well were measured immediately using a Varioskan Lux multimode fluorescence microplate reader under manufacturer-suggested conditions, with excitation at 495 nm and emission at 519 nm. The resulting graph is shown in Figure 6.

[0281] As shown in Figure 6, Graph A shows the 1:1 binding relationship of the ACE2 peptide to the SAR-CoV-2 viral spike protein across the doses used in this study, with an R² value of 0.9865 for curve fitting. Furthermore, referring to Figure 6, Graph B shows the 1:1 binding relationship of the CendR peptide to the SARS-CoV-2 viral spike protein across the doses used in this study, with an R² value of 0.9870 for curve fitting.

[0282] In Experiment 2, wells A1-F10 of Plate 2 (see Table 13) were coated with 200 μL of SARS-CoV-2 spike S1+S2 ECD-His protein to a final coating amount of 0.66 μg per well. The plate was incubated overnight in a refrigerator at 5°C. The following day, all assay preparations were performed at room temperature, except for incubation at 37°C. Plate 2 was removed from the refrigerator and the liquid was removed by gently tapping the plate in a sink. All wells were rinsed three times with 250 μL of 1×PBS. After the final rinse, any remaining excess liquid was removed by inverting the plate on a paper towel and tapping it firmly. Then, all wells were blocked with 200 μL of 5% BSA and the plate was incubated at 37°C for 1 hour. After incubation, the liquid was removed and the wells were washed as described above.

[0283] The NRP1ab-huIgG Fc(D12A N1E) protein was further diluted to 64 μg / mL in 1200 μL of PBS. 190 μL of 1×PBS was added to wells A2-A10, B2-B10, and C2-C10. 380 μL of the NRP1ab-huIgG Fc(D12A N1E) protein dilution was added to wells A1, B1, and C1. Then, using a multichannel pipette, 190 μL was taken from each of the first wells (A1, B1, and C1) and transferred to the wells in the next column. The samples were carefully mixed to avoid mixing with adjacent wells. This procedure was completed for all subsequent wells until wells A10, B10, and C10 were completed. This process resulted in a 2-fold dilution of each well in columns 1-10 of rows A, B, and C. The remaining 190 μL of excess from the final dilution in row 10 was discarded.

[0284] [Table 15]

[0285] The plate was placed in a 37°C incubator for 1 hour. After incubation, the liquid was removed, and the plate was washed and dried as described above.

[0286] FITC-labeled anti-human IgG was diluted in PBS to 3.1 μg / mL, and 200 μL of this dilution was added to all wells on the plate except for rows D-H and columns 11 and 12. The plate was placed in a 37°C incubator for 1 hour. After incubation, the liquid was removed, and the plate was washed and dried as described above.

[0287] Relative fluorescence units (RFUs) from each well were measured using a Varioskan Lux multimode fluorescence microplate reader under manufacturer-suggested conditions, with excitation at 495 nm and emission at 519 nm. The resulting graph is shown in Figure 7.

[0288] Referring to Figure 7, the graph shows the 1:1 binding of the huN1ab-huIgG Fc construct to the SARS-CoV-2 viral spike protein across dose usage in this study, with an R² value of 0.9966 for curve fitting. It is important to note that the points 41.5 and 0.7 ng are considered outliers that deviate significantly from the other data points on the curve, and have therefore been omitted from the graph.

[0289] Furthermore, the affinity of the various polypeptide constructs described herein to the SARS-CoV-2 spike S1+S2 ECD-His is determined using the methods described above.

[0290] Example 5. Cell-based assay Cell line development - Vector cloning and manipulation of transgene expression cell lines In cell-based assay systems, HEK-293T and Vero E6 cells are manipulated with lentiviruses (LV) to stably overexpress ACE2, NRP1, TMPRSS2, or any combination of these three, in order to test various therapeutic agents that may interfere with SARS-CoV-2 infection. Table 14 lists the plasmids developed in-house for lentiviral transduction of cells.

[0291] [Table 16]

[0292] Plasmid development

[0293] The full-length ACE2 gene was amplified by PCR using the hACE2 plasmid (Addgene 1786) as a DNA template and cloned into the Pinetree lentivirus plasmid LV-IRES-Zeo using NheI and XhoI. The full-length NRP1 gene was amplified by PCR using pcDNA3.4-NRP1 synthesized by Genewiz Inc. (Cambridge, MA) and cloned into the Pinetree lentivirus plasmid LV-2A-Puro using NheI and XhoI. The full-length TMPRSS2 gene was amplified by PCR using the TMPRSS2 plasmid (Addgene 53887) as a DNA template and cloned into the Pinetree lentivirus plasmid LV-2A-Blast. The DNA sequences of ACE2, NRP1, and TMPRSS2 in the lentivirus plasmids were confirmed by Sanger sequencing at Genewiz Inc. (Cambridge, MA).

[0294] Lentiviral packaging of LV-ACE2, LV-NRP1, and LV-TMPRSS2 in HEK-293T cells

[0295] For lentiviral packaging, HEK-293T cells were transfected at 90% confluence in a single well of a 6-well plate. Transfection was performed using LIPOFECTAMINE® 3000 (ThermoFisher Scientific) according to the manufacturer's instructions, with 3.6 μg of pCMV delta plasmid DNA (Pinetree) and 2.4 μg of pVSV-G plasmid DNA (Pinetree) per well, along with 5 μg of either LV-ACE2, LV-NRP1, or LV-TMPRSS2 plasmid DNA. 24 hours after transfection, the medium was replaced with 1.5 ml of OPTIMEM® medium (GIBCO®), and the supernatant containing the lentivirus was collected at 48 and 72 hours after transfection.

[0296] Lentiviral packaging of LV-ACE2, LV-NRP1, and LV-TMPRSS2 in HEK-293T cells

[0297] For lentiviral packaging, HEK-293T cells were transfected at 90% confluence in a single well of a 6-well plate. Transfection was performed using LIPOFECTAMINE® 3000 (ThermoFisher Scientific) according to the manufacturer's instructions, with 3.6 μg of pCMV delta plasmid DNA (Pinetree) and 2.4 μg of pVSV-G plasmid DNA (Pinetree) per well, along with 5 μg of either LV-ACE2, LV-NRP1, or LV-TMPRSS2 plasmid DNA. 24 hours after transfection, the medium was replaced with 1.5 ml of OPTIMEM® medium (GIBCO®). The supernatant containing the lentivirus was collected at 48 and 72 hours after transfection, filtered using a 45 μm filter, and stored at -80°C until further use.

[0298] Stable cell line generation To generate HEK-293T, Vero E6, or other cell lines that stably express ACE2, NRP1, TMPRSS2, or a combination of these three receptors, seed cells in their respective full growth media in 6-well plates 24 hours before viral infection. On the day of infection, cells should reach 70–80% confluence. The LV used for transduction is thawed on ice, and serial dilutions (1:10, 1:50, 1:100, 1:500) containing either a single LV or multiple LVs are prepared in full growth media supplemented with polyblen or another transduction-enhancing additive. The dilutions are then added to the seeded cells (1.5 ml total volume per well). After 48–72 hours of incubation, the medium is removed and replaced with a selective medium containing the antibiotic blastosidine, zeosin, puromycin, or hygromycin. The optimal concentration of each selection marker varies depending on the cell line and culture conditions and is determined before selection begins by treating untransduced parental cells (death curve). Cells are selected for 6–14 days, or until the parental cells (untransduced) are completely dead, if the control is adopted. During selection, the culture medium containing each selection agent is changed every 72 hours. Once selection is complete, the antibiotic concentration may be reduced or removed completely.

[0299] After stable pool formation, further serial dilutions and subsequent single-cell clone expansion may be performed. To confirm sufficient transgene expression, the stable pool or single-cell clones are analyzed using flow cytometry or Western blotting.

[0300] Packaging of lentiviruses pseudotyped with either SARS-CoV-2 spike protein (S) or VSV spike G (control). To investigate the effectiveness of various therapeutic agents that interfere with SARS-CoV-2 infection, we first generated replication-deficient lentiviruses pseudotyped with either the SARS-CoV-2 spike protein (S) or a variant version thereof, lacking 19 amino acids in the C-terminus predicted to function as an endoplasmic reticulum retention signal. For use as a control, lentivirus pseudotyped with VSV spike G was packaged in parallel. Both the SARS-CoV-2 and VSV-G pseudotyped lentiviruses contain firefly luciferase and an eGFP cassette, which are co-expressed under the CMV promoter in transduced cells. Therefore, even when both the control and the SARS-CoV-2 spiked virus are replication-deficient, viral entry into target cells can be microscopically monitored by detection of eGFP fluorescence and by luminescence measurement after incubation of infected cells in luciferin-containing medium. The plasmids used for pseudovirus packaging are listed in Table 15.

[0301] [Table 17]

[0302] To generate a sufficient quantity of SARS-CoV-2 lentivirus pseudovirus, we used a large-scale packaging protocol with PEI as the transfection agent, as described below.

[0303] Day 1: HEK-293T cells are seeded onto a 15 cm plate at a rate of approximately 1.4 × 10⁶ cells.

[0304] Day 2: HEK-293T cells in a 15cm plate should reach 70-80% confluence, and the medium should be replaced with 15ml of pre-warmed complete growth medium 2 hours before transfection. Add the plasmid DNA shown in Table 16 or Table 17 to 5ml of OptiMEM. In parallel, mix 1μl of 10mM PEI (Sigma Aldrich 408727) with 5ml of OptiMEM and filter sterilize through a 0.22μm filter. Then, add the PEI / OptiMEM solution drop by drop to 5ml of OptiMEM / DNA mix and incubate at room temperature for 20 minutes. After incubation, add 10ml of the PEI / OptiMEM / DNA mixture to each 15cm plate, taking care not to disturb the adherent HEK-293T cells, and incubate overnight at 37°C and 5% CO2.

[0305] [Table 18]

[0306] [Table 19]

[0307] Day 3: Morning: Replace the culture medium with 20 mL of pre-warmed complete medium. Evening (approximately 24 hours after transfection): Replace the culture medium with 11 mL of OptiMEM (collection medium).

[0308] Day 4: Evening: Collect the culture medium containing the pseudovirus and add 11 mL of fresh, pre-warmed culture medium. The collected virus-containing culture medium should be stored at 4°C.

[0309] Day 5: Evening: Collect the culture medium and pool it with the first collection. Bleach and discard the plate. Filter the collected medium containing the virus using a syringe filter with a 0.22 μm filter and a 60 mL syringe.

[0310] Enrichment and titration of lentiviral vectors After collection and filtration, the lentiviruses pseudotyped with SARS-CoV-2 spike protein (S) or VSV spike G (control) are concentrated using ultracentrifugation or using a viral precipitation solution according to the manufacturer's instructions for use (e.g., PEG-it, System Biosciences No. LV825A-1). The concentrated lentiviruses may be titrated using quantitative PCR, flow cytometry (the lentiviral vector used in this protocol expresses GFP), or by determining the relative vector particle count based on virion RNA as previously described.

[0311] Example 6. Cell assay for quantitative measurement of pseudoviral transduction in the presence or absence of a therapeutic agent. Day 1: Cell seeding Target cells to be investigated for SARS-CoV-2 lentivirus pseudoviral infectivity, such as 293T, 293T-ACE2, 293T-TMPRSS2, 293T-NRP1, 293T-ACE2 / TMPRSS2, 293T-ACE2 / NRP1, and 293T-NRP1 / TMPRSS2, are seeded in 50 μl of complete medium at a density of 5,000-10,000 cells per well in an opaque black, transparent-bottomed 96-well microplate (ThermoFisher, Nunc 165305) and incubated overnight at 37°C in 5% CO2.

[0312] Day 2: Introduction of viral phenotype Viral transduction in the absence or presence of antiviral drugs: Prepare serial dilutions of the antiviral agent to be tested in complete culture medium. • To test drugs that bind to the SARS-CoV-2 spike protein S: 5–25 μL of concentrated SARS-CoV-2 pseudotyped lentivirus is pre-incubated for 30 minutes with serially diluted anti-spike therapeutics (e.g., anti-spike monoclonal antibody, ACE2, or NRP1 decoy receptor construct) in equal volumes. After incubation, 10–40 μL of virus / antibody mixtures containing various dilutions of anti-spike therapeutics are added to each well. To test drugs that bind to ACE2, NRP1, TMPRSS2, or other receptors expressed on target cells and predicted to be involved in viral transduction: 5-25 μL of serially diluted antireceptor therapy (e.g., anti-ACE2 mAb) is added to each well containing target cells and incubated for 30 minutes. After incubation, an equal volume of concentrated SARS-CoV-2 pseudotyped lentivirus is added to each well. • Control wells containing the same amount of target cells should not be treated with therapeutic agents or pseudoviruses. The VSV-G pseudotyped lentivirus may contain firefly luciferase and be used as another control by adding an eGFP cassette in a similar manner.

[0313] After the processing outlined above, the plates are incubated at 37°C in 5% CO2. 48–72 hours after transduction, eGFP expression may be observed and quantified using a fluorescence microscope. To read the luminescence, a luciferin solution is prepared in complete medium and added to each well (final luciferin concentration 0.4 mg / mL), followed by incubation for 20–30 minutes. Transduction efficacy is determined by measuring the luciferase activity of the transduced cells using a luminescence microplate reader (e.g., ThermoFisher Scientific Varioskan Lux multimode microplate reader). Alternatively, transduction efficacy may be further measured by flow cytometry and gating for GFP-positive cells.

[0314] Example 7. In vitro assay of SARS-CoV-2 based on Vero E6 for infectivity and cytopathic effects. To test and analyze the antiviral activity of the investigational drug against replicated SARS-CoV-2 virus in Vero E6 cells, an in vitro inhibitory assay may be performed to test and analyze the antiviral activity of the compound of the present invention against replicated SARS-CoV-2 virus in Vero E6 cells.

[0315] method For testing the antiviral compounds of the present invention, a modified version of the bioassay protocol from the Biological Research Information Center (BRIC), for example, may be used. For example, the compounds may be those described in Samples 1-4 (described below), and all samples may be liquid or water-soluble. The solvent for diluting Samples 1-3 may be PBS, and for Sample 4, it may be DMSO. The cell line may be Vero E6 cells (ATCC). The virus strain may be SARS-CoV-2 (e.g., NCCP43326; supplied by the CDC of the Republic of Korea).

[0316] The cell culture conditions may be as follows: Cells are cultured in an incubator at 37°C, 95% humidity, and 5% CO2, and their condition is monitored every 8 hours. Culture medium and reagents: DMEM, 10% FBS, 1% Pen / Strep, 1% L-glutamine 200mM, 1% sodium pyruvate 100mM, non-essential amino acids. Flask and cell density: Cells may be cultured in a 96-well plate at 1 × 10⁴ cells / well, but the cell density may be changed if stable virus is not detected.

[0317] Antiviral assays using the compounds of the present invention can be performed with the following in mind: Since the mechanism of action differs for each antiviral compound, it may not be feasible to find standard assay conditions. Therefore, assay conditions may be adjusted on a case-by-case basis (e.g., entry blockers, replication blockers).

[0318] The viral replication assay can use active virus at a TCiD50 / mL concentration greater than 102.

[0319] The compound to be tested may be serially diluted in DMEM in a 2-1 to 2-6 ratio. The virus may be diluted as follows: The titer of SARS-CoV-2 may be measured before use, or 1.0 mL of the virus may be used after being diluted 10-fold in PBS at 4°C.

[0320] The processing conditions may be as follows: The culture medium of Vero E6 cells in the 96-well plate may be removed and washed twice with 100 μL of PBS. Then, 50 μL of serially diluted test drug may be added, and 50 μL of virus (102 TCID50 / ml) may be added for infection.

[0321] The antiviral activity of the compounds of the present invention may be evaluated as follows: Cytopathic effects were monitored 48 to 72 hours after infection. RNA may be isolated from infected cells, and the residual viral load was estimated using qPCR analysis.

[0322] Example 8. Infectivity assay The infectivity assay may be performed as follows. First, the COVID virus used for efficacy evaluation may be a virus adapted by three passages of SARS-CoV-2 (e.g., supplied by the CDC of the Republic of Korea) in Vero E6 cells. The virus (minimum 103 TCID50 / mL) may be diluted 10-fold and inoculated into Vero E6 cells, which may be simultaneously treated with various concentrations of the compounds listed below.

[0323] Sample 1: hNRP1ab-hFc (human NRP1 fragment containing a and b domains, fused with human IgG1 Fc); Sample 2: hNRP1b1b2-his (human NRP1 fragment containing the b1b2 domain, fused to a 6× histidine tag at its C-terminus); Sample 3: hNRP1b1b2-aaa-his (a human NRP1 fragment containing a b1b2 domain with Y297A / S346A / Y353A mutations that reduce VEGF binding, fused to a 6× histidine tag at its C-terminus); and Sample 4: Remdesivir (control).

[0324] Remdesivir is well known to those skilled in the art and is available under the trade name "VEKLURY®" (Gilead Sciences; CAS number 1809249-37-3).

[0325] Cell viability and morphology may be observed 48 to 96 hours after sample processing.

[0326] Cytotoxicity results: No cytotoxicity may be observed at the highest concentration of any of the test substances (data not shown).

[0327] Infectivity results: In experiments where the virus culture solution and test diluents were treated simultaneously, cytopathic effects (CPE) could be observed from 48 hours in the virus-treated group. No CPE was observed in any of the diluents of samples 1-3, but CPE was observed in sample 4 72 hours after inoculation at a concentration of 1.11 μM.

[0328] Each sample may be subjected to two freeze-thaw cycles over 96 hours, and the degree of viral replication may be quantitatively evaluated. For samples 1, 2, and 3, the lowest dilution factor may be 0.00003 μM, and for sample 4, RNA may be extracted from the 1.11 μM well where CPE was observed, followed by real-time PCR. These results can be interpreted as a significant inhibition of viral infection and replication of viruses that have infected cells without toxicity, and it may be necessary to clarify the therapeutic effect in infected animals through animal experiments.

[0329] Example 9. Animal Testing The infectivity assay may be performed as follows: SARS-CoV-2 isolates may be grown in Vero E6 cells in OptiMEM containing 0.3% bovine serum albumin (BSA) and trypsin treated with 1 μg of L-1-tosylamido-2-phenylethylchloromethyl ketone per mL, or in Vero 76 cells in minimal essential medium (MEM) supplemented with 2% fetal bovine serum, at 37°C.

[0330] All experiments involving SARS-CoV-2 may be conducted in a high biosafety level 3 (BSL3) containment laboratory or a high BSL3 containment laboratory.

[0331] Experimental infection Female Syrian hamsters aged 1 month and female Syrian hamsters aged 7-8 months may be used in this study. Baseline body weight may be measured before infection. Under ketamine-xylazine anesthesia, 4 hamsters per group may be inoculated with 105.6 PFU (in 110 μL) or 103 PFU (in 110 μL) of SARS-CoV-2 isolate via a combination of intranasal (100 μL) and ocular (10 μL) routes. Body weight may be monitored daily for 14 days.

[0332] For virological and pathological experiments, 2, 4, or 5 hamsters per group may be infected with 105.6 PFU (in 110 μL) or 103 PFU (in 110 μL) of the virus via a combination of intranasal and ocular routes. Three, six, and ten days after infection, the animals may be killed and their organs (e.g., nasal turbinates, trachea, lungs, eyelids, brain, heart, liver, spleen, kidneys, jejunum, colon, and blood) may be collected.

[0333] For reinfection experiments, three hamsters per group may be infected with 105.6 PFU (in 110 μL) or 103 PFU (in 110 μL) of SARS-CoV-2 or PBS (simulation) via a combination of intranasal and ocular routes. Twenty days after infection, these animals may be reinfected with 105.6 PFU of the virus via a combination of intranasal and ocular routes. Four days after reinfection, the animals may be killed, and viral titers in the nasal turbinates, trachea, and lungs may be determined by a plaque assay in VeroE6 / TMPRSS2 cells.

[0334] For passive transport experiments, eight hamsters may be infected with 105.6 PFU (in 110 μL) or 103 PFU (in 110 μL) of SARS-CoV-2 via a combination of intranasal and ocular routes. Serum samples may be collected or pooled from these infected hamsters 38 or 39 days post-infection. Control serum can be obtained from uninfected hamsters of appropriate age. Three hamsters per group may be intranasally inoculated with 103 PFU of SARS-CoV-2. On 1 or 2 days post-infection, the hamsters may be intraperitoneally injected with post-infection serum or control serum (2 mL per hamster). On 4 days post-infection, the animals may be killed, and viral titers in the nasal turbinates and lungs may be determined by plaque assay in VeroE6 / TMPRSS2 cells. All experiments with hamsters may be conducted in accordance with approved protocols, as well as the Proper Conduct of Animal Experiments and corresponding guidelines.

[0335] Example 10. In vivo efficacy of Sequence ID No. 122 against SARS-CoV-2 in hamsters. method The in vivo efficacy of sequence number 122 against SARS-CoV-2 in hamsters was evaluated.

[0336] This example included six arms, each containing four hamsters. All animals were male Syrian golden hamsters from Charles River Laboratories, weighing approximately 60–70 grams. The hamsters were weighed daily throughout the first four days after inoculation, and again on day seven when they were euthanized. On days 0, 2, 4, and 7, after acclimatization, plethysmography (PFT) recordings were taken for 20 minutes, followed by the collection of 100 μL of blood from either the jugular vein or the anterior vena cava (under isoflurane anesthesia).

[0337] On day 0, blood was collected and plethysmography (PFT) was performed, followed by intratracheal viral inoculation with a given exposure. Animals were anesthetized with ketamine / xylazine at doses of 133 mg / kg and 13.3 mg / kg, respectively, followed by viral exposure. Viral exposure was administered by intratracheal viral inoculation of the isolate USA-WA1 / 2020, which is severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), consisting of either 51 plaque-forming units (PFUs) or 510 PFUs in 50 μL of EMEM. Controls included a virus-free control consisting of 50 μL of EMEM, and a positive control consisting of SAD-S35 (anti-SARS-CoV-2 RBD neutralizing antibody, human IgG1) (see information on SAD-S35 below).

[0338] The six arms evaluated were as follows: Arm (1): Virus-free EMEM control, followed by 0.5 mL of PBS and 250 mM NaCl given 12 and 24 hours post-inoculation (PI); Arm (2): 51 PFU, followed by 0.5 mL of PBS and 250 mM NaCl given over 12 and 24 hours; Arm (3): 510 PFU, followed by 0.5 mL of PBS and 250 mM NaCl given over 12 and 24 hours; Arm (4): 51 PFU, followed by PI SEQ ID NO: 122 intraperitoneal injection (15 m / kg) administered at 12 and 24 hours; Arm (5): 510 PFU, followed by PI SEQ ID NO: 122 intraperitoneal injection (15 m / kg) given at 12 and 24 hours; and Arm (6): 510 PFU, followed by intraperitoneal injection of SAD-S35 (15 m / kg) administered at 12 and 24 hours.

[0339] SAD-S35 (anti-SARS-CoV-2 RBD neutralizing antibody, human IgG1) was used as a positive control. SAD-S35 is a specific antibody against the SARS-CoV-2 spike protein RBD domain. SAD-S35 is isolated from patients infected with SARS-CoV-2 and recombinantly generated from human 293 cells (HEK293). SAD-S35 is available from ACROBiosystems (1 Innovation Way, Newark, DE 19711; catalog number SAD-S35).

[0340] On day 7, after plethysmography recording, the hamsters were euthanized using 0.1 mL of IP Euthasol® (an anesthetic solution containing pentobarbital sodium and phenytoin sodium), blood was collected, bronchoalveolar lavage was performed with 1.5 mL of PBS, the lungs were inflated with 1.5 mL of 10% neutral buffered formalin, then fixed, and stained with hematoxylin and eosin (H&E).

[0341] The animal weights were compiled into a table in Excel. Plethysmography data are expressed as the square roots of log Penh, ln Rpef, and EF50 in the standard format published in rodent virus vital capacity measurement literature known to those skilled in the art.

[0342] RT-qPCR was performed on RNA isolated from blood samples and bronchoalveolar lavage (BAL) using the Qiagen viral RNA isolation kit. CDC TaqMan® assay primer / probe sequences and conditions targeting the SARS-CoV-2 viral nucleocapsid protein were used in an ABI QuantStudio 3 thermal cycler. Promega GoTaq® RT-PCR and standards for cloned viral nucleocapsids were used to quantify genome copies.

[0343] result During virus inoculation, hamster #8 in the 51 PFU saline treatment group died, and at the end of the experiment, data for hamster #4 in the virus-free group (vegetation inoculation only, PBS injection) was digitally corrupted and unusable. Each of these control groups remained with 3 animals. All other groups contained 4 hamsters.

[0344] No significant weight changes were observed between groups (Figures 8 and 9). Initially, poor weight gain during days 1-2 of PI was observed in all groups, and weight loss occurred on day 1 of PI in the virus-free control group. However, all animals continued to gain weight throughout the remainder of the study. Since the virus-free control group experienced only significant changes in weight gain during days 0-1, no adverse effects for treatment or viral inoculation were detected between groups.

[0345] Plethysmography The effects of viral infection and treatment on respiratory physiology were characterized using analysis of three parameters calculated from whole-body plethysmography. The three parameters were enhanced pause (Penh), Rpef, and EF50.

[0346] Enhanced Pause (Penh) is a unitless calculated airway function index. Penh calculation was performed according to the following formula.

number

[0347] Here, PEF is the expiratory flow peak of respiration; PIF is the inspiratory flow peak of respiration; Te is the expiratory portion time of respiration; and Tr is the time required to exhale 65% of the respiratory volume. Penh is used as an indirect measure of airway resistance and provides a nonspecific assessment of the breathing pattern.

[0348] The Penh formula considers four respiratory parameters, including the peak expiratory flow (PEF), peak inspiratory flow (PIF), expiratory portion time (Te), and the time required to exhale 65% of the respiratory volume (Tr). Penh is used as an indirect measure of airway resistance and provides a nonspecific assessment of the breathing pattern.

[0349] Figure 10 is a graphical representation of the respiratory cycle, showing various measurements used to calculate respiratory parameters for comparison between groups in this study.

[0350] "Rpef" measures the ratio of the time to the peak expiratory follow (PEF) to the total exhalation time. Rpef is calculated according to the following formula.

number

[0351] Here, PEF is the expiratory flow peak; Te is the total expiratory time.

[0352] This calculation parameter is lower in chronic obstructive pulmonary disease and SARS-CoV-1 infection, when volume reduction in the final part of respiration is hindered due to airway obstruction. Figure 11.

[0353] EF50 is the flow rate at 50% volume (mL / sec). EF50 is similar to Rpef and is sensitive to airway obstruction during exhalation. However, changes in exhalation are detected in the later part of the exhalation cycle. This calculation parameter is shorter in SARS-CoV-1 and longer in asthma. Figure 12.

[0354] The three calculation parameters were mathematically transformed according to conventional methods known to those skilled in the art when presenting this type of data, namely, Penh was transformed to log Penh, Rpef to ln Rpef, and EF50 to the square root of EF50.

[0355] The plethysmography data results for the hamster groups treated with 51 PFU and 510 PFU are shown in Figures 13 and 14.

[0356] Log Penh data for the 51 PFU group show that both the SEQ ID NO: 122 and untreated arms returned to baseline values ​​at 7 days, with no significant residual changes in respiratory parameters (Figure 13). Compared to the untreated group, which showed the largest deviation on day 4, the SEQ ID NO: 122 group showed a larger deviation from baseline on day 2, however, this was not statistically significant. A significant difference was observed between the 51 PFU-treated and untreated groups at day 7 (T-test, T=3.998, p<0.01), indicating that the SEQ ID NO: 122-treated group continued to return to baseline, while the untreated group gradually deviated from its baseline values ​​at the end of the study.

[0357] A significant difference (T-test, T=6.058, P<0.001) was observed on day 7 between the SEQ ID NO: 122-treated group and the untreated control group. The log Penh parameter remained relatively unchanged throughout the experimental period in the untreated control group. In contrast, 510 PFU exposure resulted in a persistent deviation of log Penh from baseline, with the deviation being largest in the untreated arm, smaller in the SEQ ID NO: 122-treated arm, and even smaller in the SAD-S35 antibody-treated arm. A significant difference (T-test, T=2.544, P<0.05) was observed between the SAD-S35-treated group and the untreated virus group in log Penh on day 4, and no other statistical associations were found between the 510 PFU-exposed arms.

[0358] In the ln Rpef data, 510 PFU exposure was found to exaggerate the effect of the treatment more than it was measured in the 51 PFU exposure group (Figures 15 and 16). Compared to the SEQ ID NO: 122 treated arm, which returned to baseline values, the untreated 51 PFU exposure showed a sustained change in the ln Rpef parameter. On day 4, there was a statistically significant difference (T-test, T=2.75, P<0.05) between the virus-free control arm and the 51 PFU arm treated with SEQ ID NO: 122. On day 7, there was a statistically significant difference (T-test, T=2.775, P<0.05) between the virus-free control and the untreated 51 PFU exposed arm in ln Rpef. 510 PFU exposure indicates that the deviation from baseline values ​​was smaller in the treated groups (SEQ ID NO: 122 and SAD-S35) compared to the untreated arm. Rpef largely stems from changes in the final part of exhalation when airway obstruction delays the time to final exhalation.

[0359] The last parameter investigated, the square root of EF50, showed little to no change in both the treated and untreated exposure groups. There were no statistically significant differences between the 51 PFU and 510 PFU exposure arms on any day. Figures 17 and 18. This parameter is sensitive to the initial portion of exhaled breath, which showed little change even during SARS-CoV-2 infection.

[0360] RT-qPCR Heparinized blood was diluted 1:10 in PBS and then stored at -80°C. Total RNA was isolated from 140 μL each of the virus-free control, 51 PFU PBS-treated, and 510 PFU PBS-treated groups. One microliter of the isolated RNA was subjected to CDC RT-qPCR for viral nucleocapsids, and the results were compared to the cloned standard. No SARS-CoV-2 genome copies were detected in any of the samples, while the control functioned as expected; therefore, the remaining samples were not tested.

[0361] histology Tissue slices obtained from representative hamster lungs selected from each study arm are shown in Figures 19–24. Seven days after viral or vector exposure, bronchoalveolar lavage was performed, followed by fixation and staining of the tissue sections. No inflammation was observed in the vector-inoculated control arm compared to several areas with mild to moderate chronic active inflammation in the 51 PFU arm (Figure 20) (Figure 19). In the 510 PFU arm, several areas with more severe chronic active inflammation were observed (Figure 21).

[0362] In tissues treated with 51 PFU and 510 PFU, inflammatory regions primarily contained lymphocytes and plasma cells, along with macrophages and neutrophils, which included fewer fragments of these cells. (Figures 20-21). Syncytia of terminal bronchiolar epithelial cells were rarely observed.

[0363] In the 51 PFU or 510 PFU arms, treatment with SEQ ID NO: 122 did not reduce the area of ​​inflammation. Figures 22-23. Similarly, in the 510 PFU arms, treatment with the antibody SAD-S35 did not reduce the size or severity of inflammation. Figure 24.

[0364] Quantitative RT-PCR of bronchoalveolar lavage (BAL) fluid After euthanasia on day 7, the lungs were lavaged with 1 mL of PBS. Total RNA was isolated from 140 μL of BAL and eluted into 50 μL; from this, 1 μL was subjected to RT-qPCR for the SARS-CoV-2 nucleocapsid gene using the US CDC real-time reverse transcription PCR panel for the detection of severe acute respiratory syndrome coronavirus 2 (https: / / www.cdc.gov / coronavirus / 2019-ncov / lab / rt-pcr-panel-primer-probes.html).

[0365] The results of the RT-qPCR assay are shown in the table below.

[0366] [Table 20-1] [Table 20-2]

[0367] No virus was detected in the EMEM-vaccinated control group (Group 1). One-factor analysis of variance did not reveal any significant differences (F=0.788, F critical=3.326, within-group df=5, between-group df=10). The absence of statistical differences is a result of the substantial standard deviation in the copy number of the nucleocapsid gene. Generally, the standard errors for each group were greater than or equal to the group mean due to a single outlier in the copy number in the BAL. Individual one-sided t-test comparisons between exposure and exposure + treatment did not show significant differences in the amount of nucleocapsid copies per microliter of RNA isolated from the BAL.

[0368] Consideration The beneficial effects of treatment with Sequence ID No. 122 and SAD35, as shown in plethysmography data, do not appear to correlate with the degree of inflammation observed histologically. Several reasons may explain this, including: the drugs attenuated the inflammatory response in the early stages of infection before histological evaluation was performed; the drug treatment reduced exhaled airway resistance and alleviated airway obstruction to some extent, but had no effect on inflammation; and / or the treatment altered the progression of early fibrosis initiated during bronchopneumonia, allowing for improved lung compliance.

[0369] Since the pharmacokinetics of SEQ ID NO: 122 and SAD35 in hamsters are unknown, the small but significant beneficial effect of the treatment may have been attenuated. If the half-lives of these treatments are short in hamsters, or if the minimum inhibitory concentration of the compound for viral replication is higher than the concentration achieved, SARS-CoV-2 viral replication may have been transiently slowed or impaired in a way that was insufficient to halt the subsequent inflammatory response. Since the SAD35 IgG antibody that interfered with SARS-CoV-2 viral replication in vitro was not a secreted antibody, it appears that it did not reach a level sufficient to completely inhibit viral replication in the alveolar space.

[0370] Analysis of BAL solutions for SARS-CoV-2 nucleocapsid gene target per microliter of isolated RNA by RT-qPCR showed no significant difference between titers in viral exposure and PBS and titers in viral exposure and SEQ ID NO: 122 or SAD35 treatment. Generally, one hamster in each group showed a higher copy number, which was most significant in animals treated with SEQ ID NO: 122.

[0371] Example 11. In vivo efficacy of VT116, VT114, and VT130 against SARS-CoV-2 in hamsters. In this example, young male hamsters weighing 95-100 grams were purchased and given intratracheal exposure to 1500 PFU of SARS-CoV-2, followed by intravenous (IV) administration of SEQ ID NO: 122, SEQ ID NO: 154, and SEQ ID NO: 192 via jugular vein catheter.

[0372] The exposure virus was diluted to a concentration of 100 PFU / μL, and 15 μL was administered orally intratracheally (IT). This example had five arms (three drug administration groups, each with n=5, and two control groups, each with n=4). Hamsters were monitored daily, their body weight was measured, and clinical disease scores were determined. Whole-body plethysmography (PFT) was recorded for each hamster on days 0, 2, 4, and 7. Serum levels of interferon-gamma (IFNγ) were determined from blood collected on days 0, 2, 4, and 7. RT-qPCR targeting the nucleocapsid gene was performed on the following samples: oropharyngeal swabs collected on days 2 and 4, bronchoalveolar lavage (BAL) from one hamster in each group on day 4, and from the remaining hamsters on day 7. RT-qPCR was also performed on RNA extracted from olfactory bulb samples.

[0373] All hamsters were euthanized on day 7. Bronchoalveolar lavage was performed using 1.5 mL of PBS, followed by fixation of the lungs for histological examination and excision of the olfactory bulbs. The viral control group was administered the virus via intracellular therapy (IT) and given intravenous saline after SARS-CoV-2 exposure. The sham infection control group was administered 15 μL of DMEM via IT, followed by intravenous saline treatment.

[0374] Each compound was administered at a dose of 15 mg / kg, which was less than 0.7 mL in volume. Control animals were given 0.5 mL of 250 mM saline intravenously. The compounds or saline were administered intravenously 12, 24, and 48 hours after IT virus exposure.

[0375] method Throughout the study, all hamsters were weighed and observed at least once daily. On days 0, 2, 4, and 7, PFT was performed after weighing the animals. On the morning of day 0, after weighing and obtaining PFT, the hamsters were anesthetized with intraperitoneal ketamine / xylazine anesthesia (133 mg / kg and 13.3 mg / kg, respectively). Once anesthetized, the hamsters were suspended by their incisors on an inclined board, and the glottis was made visible using transtracheal illumination. Next, 5 μL of 2% lidocaine solution was infused into the glottis for 30 seconds, followed by the placement of an 18-gauge catheter in the IT. The position of the intratracheal catheter was confirmed by observing the exhaled condensate on a cooled dental mirror held at the catheter opening. After confirming the catheter position, 15 μL of a viral inoculum containing 1500 PFU, or 15 μL of DMEM for a virus-free control, was administered through the IT catheter using a 100 μL gel-loading pipette tip. After removing the gel-loading chip, 1 milliliter of air was forced into the lungs through the IT catheter to help disperse the inoculation source. The catheter was then removed, and the hamster recovered from anesthesia.

[0376] The source of viral inoculation was the severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), isolate USA-WA1 / 2020, obtained from BEI Resources (www.beiresources.org).

[0377] The hamsters had jugular vein catheters, which were maintained by removing the anticoagulant solution (25% dextrose containing 500 U / mL of heparin) at each procedure (catheter volume 41 μL); thereafter, after blood collection or administration of the procedure, 250 μL of saline was flushed through the catheter to replace the heparin-dextrose solution.

[0378] Each time a hamster was manipulated for blood sampling or substance injection, it was anesthetized with isoflurane. At 12, 24, and 48 hours after viral inoculation, the hamster was weighed and administered the given treatment (15 mg / kg) or saline via jugular vein catheter. After collecting body weight on days 0, 2, 4, and 7, PFT was recorded for a 20-minute period; this was followed by isoflurane anesthesia and collection of 500 μL of blood for analysis of selected cytokines. Finally, pharyngeal swabs (days 2 and 4) or BAL after euthanasia (day 7) were collected. The five study arms were as follows:

[0379] Arm (1): A virus-free DMEM control was administered via intravenous injection, followed by 0.5 mL of saline intravenously. Arm (2): Untreated control, 1500 PFU SARS-CoV-2 (WA-1) administered via intravenous injection (IT), followed by sterile 250 mM NaCl administered via intravenous injection (IV). Arm (3): 1500 PFU, followed by IV administration of sequence number 122 at 15 mg / kg. Arm (4): 1500 PFU, followed by IV administration of SEQ ID NO: 154 at 15 mg / kg. Arm (5): 1500 PFU, followed by IV administration of SEQ ID NO: 192 at 15 mg / kg.

[0380] Hamsters were euthanized on days 4 and 7, and samples were collected. On day 4, after recording plethysmography and body weight, one hamster from each group was euthanized using 0.1 mL IP Euthasol®; the remaining hamsters were euthanized on day 7 after inoculation. After euthanasia, blood was collected for IFNγ determination, tracheotomy and cannulation were performed, followed by BAL collection and lung fixation. BAL collection consisted of injecting 1.5 mL of saline into the lung and then withdrawing it. RNA extracted from the BAL was used for RT-qPCR to determine the relative amount of SARS-CoV-2 in the lung. After BAL collection, the lung was inflated with 3 mL of 10% neutral buffered formalin via tracheal cannula and submitted for paraffin embedding and H&E staining. In addition, the olfactory bulb was dissected from the brain, and RNA extraction and SARS-CoV-2 detection were performed.

[0381] PFT is shown as Penh, Rpef, and EF50 in the publicly available standard format for rodent virus vital capacity measurement, and as calculated in the above example. A pharyngeal swab (tip of a cotton swab) was placed in 175 μL of PBS and vortexed for 15 seconds; then 140 μL was used for RNA extraction. RNA was extracted from the pharyngeal swabs and BAL using the Qiagen viral RNA isolation kit. RNA was extracted from the olfactory bulb using Qiagen RNeasy and Qia-shredder columns.

[0382] The TaqMan® assay primer / probe sequences and reaction conditions used in this study to target the SARS-CoV-2 virus nucleocapsid were published by the CDC (see above). The TaqMan® assay was performed using Promega GoTaq® RT-PCR on an ABI QuantStudio 3 thermal cycler. The amplicons generated by the primers were cloned into plasmid pCR4 and used as a quantitative standard to determine the genomic copy number in 1 microliter of RNA extracted from swabs, BALs, or tissues. Cytokine analysis for IFNγ, tumor necrosis factor alpha (TNFα), angiotensin II, and angiotensin I-7 was performed by antigen capture ELISA using kits from MyBioSource and Genorise Scientific.

[0383] result No significant weight loss occurred in any of the experimental groups (or individuals). Figure 25.

[0384] Viral titer The viral titer, detected as nucleocapsid gene copy number / 1 μL of RNA extracted from pharyngeal swabs or BALs, was highest on day 4 after IT inoculation (Figure 26). The viral control group had a large standard deviation of titers, ranging from 807 to 17,158 PFUs eluted from pharyngeal swabs collected on day 4. RT-qPCR was performed on 140 μL of BALs on day 7; however, by this time, the copy number had been minimized in all groups (<17 PFU / μL). Each compound-treated group had the highest viral copy number in samples collected post-inoculation and on day 4 post-inoculation, and these copy numbers were lower than those detected in the untreated viral exposure control (Figure 26).

[0385] In addition, the presence of the SARS-CoV-2 genome in the olfactory bulb of the brain was detected by RT-qPCR in all groups except the medium control group. Treatment with the compound did not result in a decrease in the viral nucleocapsid gene copy number detected by RT-qPCR in RNA extracted from the olfactory bulb compared to the untreated viral control. Figure 27. The highest copy number was found in RNA extracted from hamsters treated with VT116, followed by VT130 treatment, SEQ ID NO: 122, and the untreated viral exposure control. It was not determined whether this represents infectious, reproducible SARS-CoV-2 in the brain, but the higher copy number in the treated groups is noteworthy.

[0386] On day 7, when SARS-CoV-2 could no longer be detected in BALs collected from mice, the genome was present in the olfactory bulb of the brain (see below).

[0387] Whole-body plethysmography A significant difference in PFT test results was observed between the compound-treated group and the virus control group on day 2 after inoculation (T-test, P<0.01 for SEQ ID NO: 122 and SEQ ID NO: 154, p<0.05 for VT130). Figures 28-30. This difference was not retained on days 4 and 7 when comparing the groups. Since a single adverse reaction to SEQ ID NO: 192 was observed in a single animal, the significant difference detected on day 2 may be related to the administration of the test compound. The decrease in EF50 values ​​in the treatment group compared to the control group is related to a shorter duration to exhalation of 50% of the breath in the treatment group. Figure 28. This shorter duration is physiologically related to reduced resistance to respiratory movement and absence of bronchoconstriction, which may be a direct effect on airway smooth muscle or an indirect effect in reducing lung inflammation.

[0388] Plasma cytokine levels Interferon-gamma (IFNγ) levels increased in all virus-vaccinated groups, peaking on day 2 in the virus control group, as well as in the groups treated with SEQ ID NO: 122 and SEQ ID NO: 192. Figure 31.

[0389] IFNγ plasma levels peaked on day 4 in the SEQ ID NO: 154 treatment group (500 pg / mL plasma). These levels decreased to near baseline by day 7 in the SEQ ID NO: 122 group and by day 4 in the SEQ ID NO: 192 group; however, there was an unusual increase in IFNγ levels on day 7 in the SEQ ID NO: 192 and SEQ ID NO: 154 groups. This may be related to the viral genome copies detected in the hamster brains on day 7 in the SEQ ID NO: 154 and SEQ ID NO: 192 treatment groups. The viral control group never returned to baseline IFNγ levels until the end of the study. Tumor necrosis factor alpha (TNFα) levels were also measured, but the results were inconclusive (data not shown).

[0390] Angiotensin I-VII levels decreased in all groups throughout the study, and this decrease was significant between the virus control group and the mediation control group (T-test, two-sided, p<0.05). Figure 32. On day 2 post-vaccination, the decrease was statistically significant between the mediation control group and all other groups, but on day 7, the decrease was statistically significant only between the determined levels in the two control groups. In comparison, angiotensin II levels decreased in a similar manner in all groups throughout the study, but these decreases were not statistically significant. Figure 33.

[0391] Angiotensin II (AngII) is involved in blood pressure regulation and is converted to angiotensin I-7 by angiotensin-converting enzyme type 2 (ACE2), which is the receptor that SARS-CoV-2 uses to bind to cells. Since people with hypertension are more susceptible to the harmful consequences of coronavirus, we thought it prudent to investigate these metabolites. The ratios of these metabolites (AngII / Ang1-7) are shown in Figure 34 and differed significantly between the control groups, with this ratio becoming closer to the media control ratio in the treatment group throughout the experiment.

[0392] histopathology Excluding the sham-inoculated control, all hamster groups most frequently presented with pulmonary lesions described as chronic active multifocal bronchopneumonia with perivascular edema. Substantial type 2 lung cell proliferation was present in the affected areas of the lungs. Arteriosclerotic thrombosis was very rarely found in areas where blood vessels were surrounded by localized, severe inflammation. Histopathological lesions were scored using the following scheme: (a) Lesion distribution: none=0, localized=1, multifocal=2, diffuse=3; (b) Inflammation intensity: none=0, mild (thickness of 2-3 inflammatory cells)=1, moderate (thickness of 3-20 inflammatory cells), severe (thickness of more than 20 inflammatory cells); (c) Small vessel thrombosis: absent=0, present=1. Figures 35-36.

[0393] conclusion Several points are noteworthy in this study. First, there was an initial spike in IFNγ two days after viral exposure, which decreased before the viral load peak detected four days post-inoculation. Typically, IFNγ levels continue to rise during viral replication. Second, in BAL, the viral copy number per microliter decreased to near zero by day seven, but lung inflammation continued to a disease-vigorous level in the absence of the virus. Third, although the viral copy number decreased to near zero by day seven in BAL, viral copies were detected in the olfactory bulbs of the animals; however, we did not test whether these copies were associated with infectious virus or simply detected viral nucleic acid.

[0394] Treatment of hamsters with any of the three compounds reduced IFNγ levels compared to the viral control (however, statistical significance could not be determined due to the small group size and variability in measurements between individuals). Plethysmography data showed that SEQ ID NOs. 122 and 192 helped hamsters achieve near-normal respiration compared to the media control and viral control groups. Finally, viral titers in the olfactory bulb persisted at higher levels on day 7 in hamsters treated with SEQ ID NO. 192 compared to the viral control and SEQ ID NOs. 122 or 154. Viral titers on day 4 were reduced in the treatment group compared to the viral control (corrected by removing a single outlier in the SEQ ID NO. 192 treatment group). Histologically, there was no clear difference in the distribution or severity of pneumonia between the treatment group and the viral control group, which is likely due to the persistence of the inflammatory response even in the absence of the virus in the BAL. Since the administered treatments focus on interfering with the entry of the virus into cells, the inflammatory response, once initiated, appears to be separate from the ongoing presence of the virus, and therefore, once inflammation has started, it is not affected by the treatment.

[0395] Finally, the severity of the angiotensin II-to-angiotensin I-7 conversion imbalance as a potential cause of residual lung inflammation when the virus is minimal or no longer present was assessed by the AngII / Ang1-7 ratio. Compared to viral controls, treatment with SEQ ID NO: 122 and SEQ ID NO: 192 kept the ratio normal or improved to a greater extent. However, this was not accompanied by a significant reduction in lung inflammation.

[0396] Example 12. K18-ACE2 mouse study Introduction Young female K18-ACE2(JAX) mice weighing 20-25 grams were treated with 1,250 PFU of SARS-CoV-2 (strain B1.351) administered via intranasal exposure, followed by intraperitoneal (IP) administration of either an antibody (anti-SARS-CoV-2 spike protein antibody) containing a heavy chain with the amino acid sequence shown in SEQ ID NO: 189 and a light chain with the amino acid sequence shown in SEQ ID NO: 190, or saline. Intraperitoneal (IP) drug administration was initiated 12 hours before viral exposure, with subsequent doses continued 12 hours and 24 hours after exposure.

[0397] Upon exposure to the virus, the compound was mixed with the virus in a volume of 50 μL, incubated at 37°C for 15 minutes, and then intranasally administered to mice under isoflurane anesthesia. For SEQ ID NO: 113, due to the concentration of the compound, the intranasal administration volume was 83 μL to achieve a concentration of 15 mg / kg.

[0398] The following four arms were evaluated.

[0399] Arm (1): Groups inoculated with the virus and those administered with SEQ ID NO: 113 (15 mg / kg), n=13 for each group. Arm (2): Groups administered with viral inoculation and anti-SARS-CoV-2 spike protein antibody (1.2 mg / kg), n=13 each. Arm (3): Cell culture medium, intranasal control, and IP saline administration, n=6. Arm (4): Viral inoculation and IP saline administration, n=6.

[0400] Mice were monitored daily, their weight was measured, and objective clinical disease scores were evaluated.

[0401] The mice were euthanized according to the following schedule: 2 hours after drug administration on day 0; on days 1, 2, and 4: arm 1 n=2; arm 2 n=2, arm 3 n=1, and arm 4 n=1. The remaining mice were euthanized on day 7.

[0402] Prior to euthanasia, the mice were weighed and assigned a clinical score. Blood was collected under anesthesia. After euthanasia, bronchoalveolar lavage (BAL) was performed using 1 mL of sterile phosphate-buffered saline, and the lungs were fixed by blowing 1 mL of formalin into them.

[0403] RNA was isolated and quantified from all BAL samples using Qiagen RNeasy for viral RNA, and the samples were heated to 60°C for 20 minutes. Serum was tested for mouse fibrin degradation products and D-dimers using a MyBiosource kit.

[0404] result Figure 37 shows the body weight of mice in the four arms. The virus-inoculated mice in arms 1 and 4 began showing clinical signs of disease by day 4, several died from the viral infection, and the rest remained ill throughout the study until day 7, the end of the study.

[0405] Clinical scores paralleled changes in body weight as the mice began to show signs of disease on day 5 (Figure 38). This slow progression to clinical disease and weight loss is due to the slower nature of the South African variant of SARS-CoV-2, both in vitro and in vivo, compared to the WA-1 virus strain.

[0406] Clinical scores paralleled changes in body weight as the mice began to show signs of illness on day 5. This slow progression to clinical disease and weight loss is due to the slower nature of the South African variant of SARS-CoV-2, both in vitro and in vivo, compared to the WA-1 virus strain.

[0407] Mixed-model statistical analysis of body weight data showed significant differences between placebo-controlled and viral-controlled mice (p<0.001), and between placebo-controlled and SEQ ID NO: 113-treated mice (p<0.05). While there was no statistically significant difference between placebo-controlled and anti-SARS-CoV-2 spike protein antibody-treated mice, the data tended to move towards a difference if the study had been continued for a longer period.

[0408] Using mixed-model statistical analysis of clinical score data, significant differences were observed on day 6 between placebo control and viral control (p<0.01), and between placebo control and SEQ ID NO: 113 treated mice (p<0.05). On day 7, significant differences were observed among the following groups: placebo vs viral control (p<0.01), placebo vs SEQ ID NO: 113 (p<0.01), SEQ ID NO: 113 vs viral control (p<0.05), and SEQ ID NO: 113 vs anti-SARS-CoV-2 spike protein antibody (p<0.05). No statistically significant differences were observed on any day between SEQ ID NO: 113 vs viral control, or between placebo vs anti-SARS-CoV-2 spike protein antibody treated mice.

[0409] [Table 21-1] [Table 21-2]

[0410] microthrombosis Fibrin degradation products (FDPs) and D-dimers were used as indicators of microthrombosis. Fibrin degradation products and D-dimers were measured in the serum of all mice (excluding those that died). Figures 39-40.

[0411] [Table 22-1] [Table 22-2]

[0412] Using mixed-model statistical analysis for FDP data and separately for D-dimer data, no significant differences were found between any group in the measurements of either of these serum components.

[0413] conclusion Anti-SARS-CoV-2 spike protein antibodies provided some protection against weight loss and clinical signs of the disease. Weight and clinical scores in SEQ ID NO: 113 were not significantly different from those in the viral infection control group.

[0414] Serum levels of D-dimer showed no statistically significant differences between placebo and the viral control or SEQ ID NO: 113 and anti-SARS-CoV-2 spike protein antibody treatment groups when all comparisons between these groups were analyzed. However, the D-dimer graph gives the impression that the mean values ​​of SEQ ID NO: 113 and anti-SARS-CoV-2 spike protein antibodies for animals euthanized on day 8 were closer to the mean values ​​for the placebo control, suggesting that the number of mice in each group may have been insufficient to demonstrate statistical significance.

[0415] Example 13. NRP molecule with modified heparin binding. Neuropyrin has an acidic polysaccharide binding site that interacts with heparin or heparin sulfate to enhance the interaction between neuropyrin and VEGF. The acidic polysaccharide binding site is located in both b1 and b2, which define the continuous positively charged region.

[0416] To reduce the binding affinity to acidic polysaccharides such as heparin and heparan sulfate, the fusion protein was designed to have mutations in the neuropilin fragment (e.g., K358E, K373E in the b1 domain, and R513E, K514E, K516E in the b2 domain).

[0417] All designed proteins were generated by codon-optimized gene synthesis and inserted into pcDNA3.4 as an expression vector using NotI and HindIII restriction enzymes. The constructed expression vector contains a signal peptide and may include a Kozak sequence in the 5' untranslated region for optimized transcription.

[0418] To obtain the quantity of the constructed plasmid for transfection, One Shot® Top10 competent E. coli cells were transformed with the constructed plasmid and subsequently cultured overnight. The constructed plasmid was obtained using the PureLink® HiPure Expi Plasmid Megaprep kit.

[0419] Fusion proteins were transiently expressed in the CHO-S system (ThermoFisher Scientific Inc.). Proteins were expressed individually according to the manufacturer's instructions. Briefly, plasmid DNA with a total of 0.8 μg per 1 mL of CHO-S culture, with a 1:1 light-to-heavy chain ratio, was prepared using OPTIPRO® SFM and ExpiFectamine®. The mixture was added to CHO-S cells at a viable cell density of 6 × 10⁶ cells / mL and a viability of over 98%. The cell cultures were incubated overnight at 37°C, 80% humidity, and 8% CO₂ in a Nalgene® Single-Use PETG Erlenmeyer flask with shaking at 125 RPM in a 19 mm orbit. The following day, the culture was augmented (ExpiCHO® enhancer; ThermoFisher Scientific Inc.), nutrient supplied (ExpiCHO® feed; ThermoFisher Scientific Inc.), transferred to 32°C, 80% humidity, and 5% CO2, and shaken at 125 RPM in a 19 mm orbit. A second nutrient supply was given on day 5, and the culture was returned to 32°C until harvest on day 12.

[0420] Recovery was achieved by centrifugation at 4000 × g for 20 minutes. The purified supernatant was sterilized using an asymmetric polyethersulfone (PES) 0.22 μM filter assembly (Nalgene). The filtrate was stored at 4°C until purification the following day.

[0421] All sterile supernatants containing antibodies were purified using MabSelect prismA® resin (GE Healthcare Life Sciences) in an AKTApure (GE Healthcare Life Sciences) system. The resin was equilibrated with a buffer solution of 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. The supernatants containing antibodies were then loaded onto the column.

[0422] Next, the resin was washed with a buffer solution of 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 until the chromatography baseline returned to the column equilibration level. Then, elution was performed using 100 mM sodium acetate, 20% glycerol, pH 3.0, and the fraction was collected. The fraction was then immediately neutralized with 1 M Tris, pH 9.

[0423] After obtaining the fractions, ion exchange chromatography was performed. The cation exchange chromatography (CEX) column (Capto S ImpAct) or the anion exchange chromatography (AEX) column (Capto Q Impres) was disinfected with 1M NaOH and rinsed with MQ. For the CEX column, equilibration was performed using 50mM NaAc pH 5.5 (starting buffer) and 50mM NaAc pH 5.5 and IM NaCl (elution buffer). For the AEX column, equilibration was performed using 50mM Tris pH 8.0 (starting buffer) and 50mM Tris-HCl pH 8.0 and IM NaCl (elution buffer).

[0424] The pH of the starting buffer and elution buffer was sometimes according to the protein pI value, with Na-pi pH 7.0 and bicin pH 8.0 for CEX, and Tris 8.0 and Tri 8.5 for AEX. Antibodies purified with Protein A were loaded at a concentration of 1-2 g antibody / 1 mL resin. The column was then washed with the starting buffer. The antibody product was then eluted using a gradient of 0-60% elution buffer in 25 column volume. Each peak was collected separately and concentrated by centrifugation at 4000 × g using an Amicon® Ultra-15 centrifuge filter unit, followed by buffer exchange to PBS.

[0425] Heparin binding To evaluate heparin binding, a heparin affinity column (HiTrap Heparin HP) was disinfected with 1M NaOH and rinsed with MQ. Equilibration was performed using 50mM sodium phosphate pH 7.0 (starting buffer) and 50mM sodium phosphate pH 7.0, 1M NaCl (elution buffer). The construct of the present invention was loaded at a concentration of 1 mg antibody / 1 mL resin. The column was then washed with the starting buffer. The construct of the present invention was then eluted using a gradient of 100% elution buffer in 15 column volumes. A summary of the constructs is shown in the table below.

[0426] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5] [Table 23-6] [Table 23-7] [Table 23-8] [Table 23-9] [Table 23-10] [Table 23-11] Table 23-12 Table 23-13 Table 23-14 Table 23-15 Table 23-16 Table 23-17 Table 23-18 Table 23-19 Table 23-20 Table 23-21 Table 23-22

[0427] Table 24-1 Table 24-2 Table 24-3 [Table 24-4] [Table 24-5] [Table 24-6] [Table 24-7] [Table 24-8]

[0428] Results of heparin binding The heparin affinity results are summarized in the table below. Some of the tested constructs did not bind to the heparin column. For example, SEQ ID NOs. 113, 128, 129, 114, 115, 116, and 133 did not show heparin binding. Of the above constructs, SEQ ID NOs. 113 has the wild type of the b1 domain, while the other SEQ ID NOs. 128, 129, 114, 115, 116, and 133 contain at least one mutation at the putative heparin binding site.

[0429] With the exception of Sequence ID No. 133, all constructs containing either only the wild-type b1 domain or both the b1 and b2 domains were shown to bind to heparin. Constructs containing wild-type b1 and b2 domains, such as Sequence ID Nos. 122, 124, and 125, showed stronger heparin binding than constructs containing only the b1 domain. Furthermore, a single b1 domain did not result in heparin binding; however, increasing the number of b1 domains did result in heparin binding, thus demonstrating that interaction with heparin is possible due to increased b1 domain binding activity.

[0430] [Table 25-1] [Table 25-2]

[0431] Example 14. CendR decoy array overview To demonstrate and characterize the binding of neuropilin-1 b1-domain-antibody-fusion constructs to the CendR motif, and the binding of b1 to viral proteins and / or CendR peptides, the receptor-binding domain CendR motif was evaluated.

[0432] The constructs tested in this embodiment include one or more recognition-binding domains that target the neuropilin-1 b1 domain in a tandem expressed as a recombinant fusion molecule on the n-terminus of human IgG Fc. Figure 42.

[0433] While not bound by theory, the constructs of the present invention are hypothesized to bind to the CendR motif of glycoproteins of SARS-CoV-2, respiratory syncytial virus (RSV), and other viral pathogens via the neuropilin-1 b1 domain. Virus recognition-binding domain (RBD) targeting is primarily mediated by the binding of the neuropilin b1 domain to the CendR motif (e.g., RXXROH) of the viral construct expressed on the spike protein on the viral particle membrane, which is cleaved by the RBD. Neuropilin-1 is a cell surface receptor involved in numerous developmental processes, including axon guidance, angiogenesis, and heterophilic cell adhesion. The neuropilin-1 b1 domain, together with the b2 domain, forms a portion of the tandem coagulation factor domain, and both domains belong to a domain family called the F5 / 8 type C or discoidin domain family. The neuropilin-1 b1 domain has been identified as a major driver of binding of the viral spike protein to the S1 domain, which may aid in viral infectivity. Recombinant fusion of the RBD-binding b1 domain with human IgG Fc activates the adaptive immune system to bind to viral pathogens and prevent their infectivity. The neuropilin-1 b1 domain, along with other neuropilin ectodomains, mediates the binding of ligands including semaphorins, coagulation factor V and VIII, and VEGF, as part of the developmental process, including initiating axon guidance and angiogenic factor binding, as well as other physiological activities.

[0434] Binding analysis was established using Bio-Layer Interferometry (BLI) of the Octet® Red 96 system (ForteBio). The specific objectives of this example were: (1) to establish a construct molecule that binds to the RBD CendR motif of viral particles; and (2) to characterize the binding parameters of the construct molecule to the RBD CendR motif-targeted recombinant viral protein.

[0435] Binding of the constructor molecule to the recombinant viral protein To evaluate the binding of the construct molecule to viral proteins, we performed BLI binding studies using the Octet red 96 system. In short, the construct was immobilized on an anti-human Fc Capture (AHC) biosensor, and its binding to commercially available recombinant viral proteins was investigated. Using BLI technology, binding to the construct was evaluated across various viruses, and both kinetics and binding affinity (equilibrium binding constant, KD) were assessed in monovalent format. In addition, CendR peptide sequences from various viruses were immobilized on a streptavidin (SA) biosensor. These studies were conducted to determine whether the construct molecule exhibits broad spectral activity and its affinity for target proteins and peptides.

[0436] The tested constructs are shown in the table below.

[0437] [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4] [Table 26-5] [Table 26-6]

[0438] material The materials used in this embodiment are shown in the table below.

[0439] [Table 27-1] [Table 27-2]

[0440] CendR peptide The CendR peptides that were evaluated are shown in the table below.

[0441] [Table 28-1] [Table 28-2]

[0442] The conjugation of the construct molecules to viral proteins and CendR peptides, and KD determination, were performed using the Octet Red 96 system, as described below.

[0443] Binding of constructive molecules to viral proteins: Method The construct molecule was immobilized on an anti-hIgG Fc biosensor at various concentrations (0.55–1.56 μg / mL) in 1× kinetic buffer (1× PBS pH 7.4, 0.1% w / v BSA, 0.002% v / v Tween-20), with a loading time of 180 seconds for each stage. A 60-second baseline was maintained after loading. Next, recombinant viral proteins of 25 nM, 50 nM, and 100 nM were prepared in 1× kinetic buffer. A reference sensor was generated by applying the viral protein to a blank anti-hIgG Fc biosensor (without the construct molecule). The association (Ka) and dissociation (Kd) steps were 300 seconds each. Data were analyzed using Octet analysis software, applying 1:1 or 2:1 model fits to report the dissociation constant KD(M).

[0444] Binding of the constructive molecule to the biotinylated CendR peptide: Method Constructs with the amino acid sequences shown in SEQ ID NOs: 113, 122, 154, and 193 were evaluated. The CendR peptide was immobilized on streptavidin (SA) biosensors at various concentrations (0.05–0.65 μg / mL) in 1× kinetic buffer (shown above), with a loading time of 180 seconds for each stage. A 60-second baseline was maintained after loading. Next, 100 nM construct molecules with the amino acid sequences shown in SEQ ID NOs: 113, 122, 154, and 193 were prepared in 1× kinetic buffer. A reference sensor was generated by applying the construct molecule to the surface of a blank streptavidin biosensor (without the CendR peptide). The association (Ka) and dissociation (Kd) steps were 300 seconds each. The data were analyzed using Octet analysis software with a 1:1 model fit, thereby reporting the dissociation constant KD(M).

[0445] result Binding of the constructor molecule to the recombinant RSV F protein. The effectiveness of constructs that bind to the RSV-F protein with high affinity increases the potential of a platform that can provide a therapeutic mechanism to combat RSV (see table below).

[0446] [Table 29-1] [Table 29-2]

[0447] Binding of the constructor molecule to the CendR peptide. The table below shows the results of studies evaluating the binding of the constructor molecule to the CendR peptide.

[0448] [Table 30]

[0449] conclusion The objective of the research described herein was to establish the binding characteristics of the construct molecule to recombinant RBD viral protein and CendR peptide and to determine its KD. Based on the data presented, the following conclusions can be drawn: (1) The construct molecule binds to the RSV F protein with high affinity in monovalent form, and (2) constructs having the amino acid sequences shown in SEQ ID NOs. 113, 122, and 154 exhibit a broad spectrum of the viral CendR motif in octet assay form. In summary, the data presented herein suggest that the biological effect of the construct molecule of the present invention is that it can bind and prevent infectivity.

[0450] Example 15. ELISA test for RSV (Respiratory Syncytial Virus) The construct molecules were evaluated by testing their binding affinity to the RSV (respiratory syncytial virus) F glycoprotein using ELISA.

[0451] Reagents and materials The reagents used were as follows: 1× DPBS (Corning, Corning, NY; catalog number 21-031-CM); Tween-20 (100%) (Boston BioProducts, Ashland, MA; catalog number P-934); Wash buffer: PBST (0.02% Tween-20 in 1× DPBS); Dried milk powder (Research Products International, Mt. Prospect, IL; catalog number M17200-1000.0); RSV-F (amino acids 1-529), (extracellular domain) protein (His tag), ABIN2006856 (Antibodies-online, Inc., Limerick, PA); Anti-human IgG-HRP conjugate (Abcam, Cambridge; United Kingdom; catalog number ab6759); TMB ELISA substrate (high sensitivity) (Abcam, Cambridge, United Kingdom; catalog number ab171523); ELISA stop solution (ThermoFisher Invitrogen, Waltham, MA; catalog number SS04; and Pierce® 96-well high-binding ELISA plate (ThermoFisher Scientific, Waltham, MA; catalog number 15041).

[0452] Device ELISA tests were performed using the following equipment: 450nm 96-well SpectraMax M2e microplate reader (Molecular Devices, San Jose, CA); Wellwash Versa microplate washer (ThermoFisher Scientific, Waltham, MA); P20, P200, P1000 and multi-channel pipettes (Eppendorf).

[0453] procedure The ELISA test was performed using the following steps:

[0454] Step 1: Using a multichannel pipette, plate culture 100 μL of 3 μg / mL RSV F protein in PBS into all wells of a 96-well high protein binding plate; Step 2: Incubate the plate overnight at 2-8°C; Step 3: Wash the plate three times with 1× PBS (PBST) containing 0.05% Tween-20; Step 4: Add 200 μL of 5% dried milk in 1× PBS to each plate per well; Step 5: Incubate the plate at room temperature for 1 hour; Step 6: Wash the plate three times with 1× PBS (PBST) containing 0.05% Tween-20; Step 7: Add 100 μL of PBS to columns 8-12; Step 8: Add 200 μL of each construct (1 μg / mL in PBS) to wells A1-H1; Step 9: Using a multichannel pipette, transfer 100 μL of each construct sequentially from column 1 to column 11. Discard the extra 100 μL from column 11. Leave column 12 with PBS only; Step 10: Incubate at room temperature for 1 hour; Step 11: Wash the plate three times with 1×PBS (PBST) containing 0.05% Tween-20; Step 12: Add 200 μL of rabbit anti-human IgGHRP to all wells of all plates at a 1:20,000 dilution in PBS; Step 13: Incubate at room temperature for 1 hour; Step 14: Wash the plate three times with 1×PBS (PBST) containing 0.05% Tween-20; Step 15: Add 100 μL of TMB reagent to all wells; Step 16: Incubate at room temperature for 15 minutes; Step 17: Add 100 μL of stop reagent to each well; Step 18: Read the absorption on ABS450.

[0455] The results of the ELISA assay are shown in the table below.

[0456] [Table 31]

[0457] [Table 32]

[0458] Example 16. Binding analysis of RSV (respiratory syncytial virus) The construct molecules were evaluated by testing their binding affinity to the RSV (respiratory syncytial virus) F glycoprotein using the Octet® Red96 system (ForteBio).

[0459] [Table 33]

[0460] Example 17. ELISA test for SARS-CoV-2 The construct molecules were evaluated by testing their binding affinity to SARS-CoV-2 via ELISA. The ELISA was performed by the method described in Example 15 above.

[0461] [Table 34]

[0462] Example 18. Binding analysis of influenza A H5N1 virus (IAV H5N1) The construct molecules were evaluated by testing their binding affinity to IAV H5N1 using the Octet® Red96 system (ForteBio).

[0463] [Table 35]

[0464] Example 19. Bioinformatics approach for identifying CendR Prediction of furin transection site Furins are conserved proteases that cleave the "RX-[KR]-R" motif to yield the CendR peptide. Furins belong to an ancient gene family with orthologs and paralogs in all vertebrates, tracing back to the branching of animals and fungi.

[0465] To identify furin cleavage sites (and subsequent formation of CendR peptides for targeting by the constructs of the present invention) in human, viral, and bacterial proteins, bioinformatics evaluations were performed to identify extended consensus sequences.

[0466] To identify furin cleavage sites, we evaluated RefSeq peptide databases containing the following records: human: 114,963 records; virus: 477,258 records; and bacteria: 161,430,766 records. Furthermore, we used two predictive software programs: ProP predictive software and PiTou furin cleavage site computer prediction tool.

[0467] The ProP tool is a neural network with 94.7% sensitivity and 83.7% specificity. An exemplary description of ProP is provided in Duckert et al., Prediction of proprotein convertase cleavage sites, Protein Eng Des Sel. 2004 January;17(1):107-12, the disclosure of which is fully incorporated herein by reference.

[0468] The PiTou furin cleavage site computer prediction tool (hereinafter "PiTou") is a knowledge-based tool with 96.9% sensitivity and 97.3% specificity. PiTou is based on 131 known furin cleavage sites and 4265 arginine sites that furin does not cleave. A logarithmic (odds) score is provided based on the furin binding site profile Hmm and physical properties such as quantity, charge, and hydrophobicity.

[0469] An exemplary description of the PiTou furin cleavage site computer prediction tool is provided in Tian et al., Computational prediction of furin cleavage sites by a hybrid method and understanding mechanism underlying diseases. Sci Rep. 2012;2:261, the disclosure of which is fully incorporated herein by reference.

[0470] The RefSeq peptide results are shown in the table below.

[0471] [Table 36]

[0472] Figures 43-45 show the cumulative distribution of PiTou scores for human, viral, and bacterial peptides, respectively. Figure 46 shows the PiTou scores for known viral cleavage sites. Based on 30 viruses, the known sites had the following PiTou scores: 90%>5 (99%); 75%>7 (99.9%); and 50%>11 (99.99%). Figure 47 shows the prioritized distribution of PiTou scores.

[0473] Novel predicted targets were prioritized based on known diseases, PiTou scores, secreted peptides, and conservation. Known human diseases were identified based on OMIM, ClinVar, GnomAD, and MGI. Bacteria and viruses were identified based on WHO, RKI, Bode Science Center, and Major Infectious Diseases 3rd Ed.

[0474] [Table 37]

[0475] The secreted peptides were prioritized based on DeepSig, a neural network with separate models for eukaryotes and bacteria; Cell-Ploc 2.0 (pre-calculated organelle localization for different proteins); and Spdb 5.1 (a database of signal peptides in Swiss-Prot&EMBL entries; see Choo et al., 2005).

[0476] An exemplary description of DeepSig is provided in Savojardo et al., DeepSig: deep learning improves signal peptide detection in proteins. Bioinformatics. 2018 May 15;34(10):1690-1696, the disclosure of which is fully incorporated herein by reference.

[0477] An exemplary description of Cell-Ploc 2.0 is provided in Chou and Shen, Cell-PLoc: package of Web servers for predicting subcellular localization of proteins in various organisms, Nat Protoc. 2008;3(2):153-62, the disclosure of which is fully incorporated herein by reference.

[0478] An exemplary description of SPDB 5.1 is provided in Choo et al., 2005, and its disclosure is incorporated herein in full by reference.

[0479] [Table 38-1] [Table 38-2] [Table 38-3] [Table 38-4]

[0480] [Table 39-1] [Table 39-2] [Table 39-3]

[0481] Example 20. VEGF binding assay To investigate the binding of the molecular constructs of the present invention to VEGF165, BLI binding studies were performed using the Octet Red 96 system. Briefly, the constructs were immobilized on anti-human Fc capture (AHC) biosensors, and their binding to commercially available recombinant VEGF165 was examined. Using BLI techniques, binding to VEGF165 was investigated using SEQ ID NOs: 122, 154, and 193 for binding affinity (equilibrium binding constant, KD) in monovalent format.

[0482] [Table 40]

[0483] method The construct molecule of the present invention, which binds to VEGF165, and the KD determination of the interaction during KD determination were performed using the Octet Red 96 system. Briefly, the molecule was immobilized on an anti-hIgG Fc biosensor at 1.5 μg / mL in 1× or 2× dynamic buffer with a loading time of 120 or 180 seconds. A post-baseline loading was performed for 60 seconds. Next, recombinant viral proteins at 50 nM, 25 nM, and 12.5 nM per second were prepared in 1× or 2× dynamic buffer. A reference sensor was prepared by coating VEGF165 onto a blank anti-hIgG Fc biosensor (no molecule). The association and dissociation steps were 300 seconds each. Data were analyzed using Octet analysis software with a 1:1 or 2:1 model fit applied, reporting the dissociation steady-state KD(M).

[0484] To investigate the binding of molecules to VEGF165, we performed BLI binding studies using the Octet Red 96 system. Briefly, constructs were immobilized on anti-human Fc capture (AHC) biosensors, and their binding to commercially available recombinant VEGF165 was examined. Using BLI techniques, binding to VEGF165 was investigated using SEQ ID NOs: 122, 154, and 209 for binding affinity (equilibrium binding constant, KD) in monovalent format.

[0485] result Constructs that bind to VEGF165 at affinity levels ranging from single nanomolar to sub-nanomolar concentrations demonstrate naturally occurring interactions between the neuropilin domain and the native ligand. See the table below.

[0486] [Table 41]

[0487] The objective of this embodiment was to determine whether the natural ligand VEGF165 binds to the normal neuropilin-binding domain present in the construct of the present invention. From the above results, the following conclusions can be drawn: VEGF165 binds to the construct molecule containing the neuropilin domain, as described in the published literature on VEGF165-neuropillin interaction; the affinity (KD) of the construct molecule to VEGF165 is potent, ranging from single-digit nanomolar concentrations to sub-nanomolar concentrations; and from the data presented here, the construct molecule maintains the established interaction between VEGF165 and the neuropilin domain, and can help establish therapeutic potential for a platform to combat viral diseases using the naturally occurring interaction.

[0488] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications fall within the scope of the appended claims. All values ​​are approximate and should be further understood as being provided for illustrative purposes only.

Claims

1. (a) A variant of the b1 domain having a mutation in the amino acid sequence of the b1 domain of neuropyrin; (b) immunoglobulin domains; and (c) ACE2 domain Includes, The variant of the b1 domain is the amino acid sequence of Sequence ID No. 4 (NRP1 b1 E319A), in which the E at position 45 of the amino acid sequence of the b1 domain of neuropilin (NRP1) represented by Sequence ID No. 3 is replaced with A. The variant of the b1 domain specifically binds to the S protein of SARS-CoV-2. The ACE2 domain is the amino acid sequence represented by SEQ ID NO: 41 (ACE2-2), the amino acid sequence represented by SEQ ID NO: 43 (ACE2-4), the amino acid sequence represented by SEQ ID NO: 44 (ACE2-5), or the amino acid sequence represented by SEQ ID NO: 45 (ACE2-6). Polypeptide.

2. The polypeptide according to claim 1, wherein the polypeptide is capable of binding to the S protein of SARS-CoV-2.

3. The polypeptide according to claim 1 or 2, wherein the variant of the b1 domain includes a mutation that enhances the affinity for the SARS-CoV-2 S protein when compared with the non-mutant b1 domain.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide further comprises a linker, a neuropyrin b2 domain, or a combination thereof.

5. The polypeptide according to claim 1, further comprising a linker between the variant of the b1 domain and the ACE2 domain.

6. The polypeptide according to claim 5, wherein the linker is selected from the group consisting of sequence numbers 46 to 52.

7. The polypeptide according to any one of claims 1 to 6, wherein the immunoglobulin domain comprises an Fc domain, and the Fc domain contains a mutation that reduces ADCC when compared with a wild-type Fc domain.

8. The polypeptide according to claim 7, wherein the mutation is located at position N297, determined by Kabat numbering.

9. The polypeptide according to claim 7, wherein the Fc domain contains one or more mutations that enhance affinity to FcRn compared to a wild-type Fc domain.

10. The polypeptide according to claim 9, wherein the mutation is located at a position selected from the group consisting of T307, E380, and N434 as determined by Kabat numbering, or a combination thereof.

11. The polypeptide according to claim 7, wherein the Fc domain contains a mutation that reduces affinity to an Fcγ receptor subtype when compared with the wild-type Fc domain.

12. The polypeptide according to claim 11, wherein the mutation is located at a position selected from at least one of L324 and L325 as determined by Kabat numbering, or a combination thereof.

13. The variant of the b1 domain of neuropilin 1, the immunoglobulin domain, and the ACE2 domain are, The polypeptide according to claim 1, having the configuration of (b1b2)-(G4S)*2-(b1b2), b1(E319A), IgG1(N297A / T307A / E380A / N434A), ACE2-5.

14. The polypeptide according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-97, 107, 108, and 110.

15. The polypeptide according to claim 7, wherein the b1 domain variant is bound to the C-terminus of the Fc domain.

16. The polypeptide according to claim 7, wherein the ACE2 domain is bound to the C-terminus of the Fc domain.

17. The polypeptide according to claim 7, wherein the ACE2 domain is bound to the N-terminus of the Fc domain.

18. A polypeptide according to any one of claims 1 to 17, further comprising a signal peptide.

19. The polypeptide according to claim 18, wherein the signal peptide comprises SEQ ID NO:

53.

20. A pharmaceutical composition for reducing COVID-19 infection, comprising a polypeptide according to any one of claims 1 to 19 as an active ingredient.

21. A pharmaceutical composition for treating COVID-19 infection, comprising a polypeptide according to any one of claims 1 to 19 as an active ingredient.

22. A pharmaceutical composition for preventing COVID-19 infection, comprising a polypeptide according to any one of claims 1 to 19 as an active ingredient.

23. A pharmaceutical composition for reducing symptoms of COVID-19, comprising a polypeptide according to any one of claims 1 to 19 as an active ingredient.

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