WO2021258218A1 - Utilisation de polypeptides nrp1 solubles pour le traitement d'infections à coronavirus - Google Patents

Utilisation de polypeptides nrp1 solubles pour le traitement d'infections à coronavirus Download PDF

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WO2021258218A1
WO2021258218A1 PCT/CA2021/050878 CA2021050878W WO2021258218A1 WO 2021258218 A1 WO2021258218 A1 WO 2021258218A1 CA 2021050878 W CA2021050878 W CA 2021050878W WO 2021258218 A1 WO2021258218 A1 WO 2021258218A1
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seq
residues
nrp1 polypeptide
soluble nrp1
amino acid
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PCT/CA2021/050878
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English (en)
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Przemyslaw SAPIEHA
Normand BEAULIEU
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Semathera Inc.
Rsem, Limited Partnership
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Publication of WO2021258218A1 publication Critical patent/WO2021258218A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • Asequence listing is submitted herewith as an ASCII compliant text file named 16761_43 - Seq listing_ST25.txt, that was created on June 16, 2021 and having a size of ⁇ 102 kilobytes.
  • the content of the aforementioned file hereby incorporated by reference in its entirety.
  • the present invention generally relates to viral infection, and more particularly to the treatment of coronavirus infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
  • coronavirus infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • Coronaviruses are large, roughly spherical, RNA viruses with bulbous surface projections that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19). Coronaviruses have four structural proteins, namely the spike (S), envelope (E), and membrane (M) proteins, forming the viral envelope, as well as the nucleocapsid (N) protein, holding the viral RNA genome.
  • S spike
  • E envelope
  • M membrane proteins
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the strain of coronavirus that causes COVID-19, the respiratory illness responsible for the COVID-19 pandemic.
  • the spike protein SARS-CoV-2 is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its S1 subunit catalyzes attachment, the S2 subunit fusion.
  • the main receptor involved in SARS-CoV-2 entry into human cells is the angiotensin converting enzyme 2 (ACE2).
  • TMPRSS2 protease transmembrane protease, serine 2
  • the B.1.1.7 variant (23 mutations with 17 amino acid changes) was first described in the United Kingdom in December 2020; the 501Y.V2 variant (23 mutations with 17 amino acid changes) was initially reported in South Africa in December 2020; and the P.1 variant (approximately 35 mutations with 17 amino acid changes) was reported in Brazil in January 2021.
  • the B.1.1.7 variant had been reported in 93 countries, the 501Y.V2 variant in 45, and the P.1 variant in 21. All three variants have the N501Y mutation, which changes the amino acid asparagine (N) to tyrosine (Y) at position 501 in the receptor-binding domain of the spike protein.
  • the 501Y.V2 and P.1 variants both have two additional receptor-binding-domain mutations, K417N/T and E484K. These mutations increase the binding affinity of the receptor binding domain to the angiotensin-converting enzyme 2 (ACE2) receptor.
  • ACE2 angiotensin-converting enzyme 2
  • SARS-CoV-2 variants B.1.427 and B.1.429, which were first detected in California, have been shown to be approximately 20% more transmissible than pre-existing variants and have been classified by the CDC as variants of concern. Studies on these variants have provided compelling evidence that they have the potential to escape naturally-induced immunity as well as the immunity induced by currently approved vaccines.
  • SARS-CoV-2 the etiologic agent of COVID-19
  • the current pandemic is aggravated by the apparition of variants of concern that are feared to result in an antigenic drift that could evade vaccine-elicited immune responses.
  • the present disclosure provides the following items 1 to 81 :
  • a method for treating an infection by a coronavirus, or for reducing the risk and/or severity of a coronavirus-related disease, in a subject comprising administering to said subject an effective amount of a soluble NRP1 polypeptide comprising a sequence having at least 70% identity with residues 276 to 428 of the amino acid sequence of SEQ ID NO:1.
  • a soluble NRP1 polypeptide comprising a sequence having at least 70% identity with residues 276 to 428 of the amino acid sequence of SEQ ID NO:1.
  • the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 590 to 859 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 148 to 275 of the amino acid sequence of SEQ ID NO:1.
  • the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 148 to 275 of the amino acid sequence of SEQ ID NO:1.
  • the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 22 to 147 of the amino acid sequence of SEQ ID NO:1.
  • the soluble NRP1 polypeptide comprises residues 22 to 147 of the amino acid sequence of SEQ ID NO:1, or a variant thereof in which the isoleucine residue at position 140 is replaced by a leucine residue.
  • soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 22 to 859 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence corresponding to residues 22-653 of SEQ ID NO: 7, residues 22-812 of SEQ ID NO: 8, residues 22-658 of SEQ ID NO: 12, residues 22-696 of SEQ ID NO:14, residues 22-1085 of SEQ ID NO: 15 or residues 22-708 of SEQ ID NO: 16.
  • coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • a soluble NRP1 polypeptide comprising a sequence having at least 70% identity with residues 276 to 428 of the amino acid sequence of SEQ ID NO:1 for use in the treatment of an infection by a coronavirus in a subject.
  • soluble NRP1 polypeptide for use according to item 28, wherein the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 276 to 428 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 31 , wherein the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 429 to 589 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 32, wherein the soluble NRP1 polypeptide comprises residues 429 to 589 of the amino acid sequence of SEQ ID NO: 1.
  • soluble NRP1 polypeptide for use according to any one of items 28 to 33, wherein the soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 590 to 859 of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 34, wherein the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 590 to 859 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to any one of items 28 to 36, wherein the soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 148 to 275 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 37, wherein the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 148 to 275 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 38, wherein the soluble NRP1 polypeptide comprises residues 148 to 275 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to any one of items 28 to 39, wherein the soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 22 to 147 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 40, wherein the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 22 to 147 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 40, wherein the soluble NRP1 polypeptide comprises residues 22 to 147 of the amino acid sequence of SEQ ID NO:1, or a variant thereof in which the isoleucine residue at position 140 is replaced by a leucine residue.
  • soluble NRP1 polypeptide for use according to item 44, wherein the soluble NRP1 polypeptide comprises residues 22 to 589 of the amino acid sequence of SEQ ID NO:1, or a variant thereof in which the isoleucine residue at position 140 is replaced by a leucine residue.
  • soluble NRP1 polypeptide for use according to any one of items 28 to 45, wherein the soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 22 to 859 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 46 wherein the soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 22 to 859 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide for use according to item 47, wherein the soluble NRP1 polypeptide comprises residues 22 to 859 of the amino acid sequence of SEQ ID NO:1 , or a variant thereof in which the isoleucine residue at position 140 is replaced by a leucine residue.
  • soluble NRP1 polypeptide for use according to any one of items 28 to 48, wherein the soluble NRP1 polypeptide is fused to one or more peptides or polypeptides.
  • soluble NRP1 polypeptide for use according to item 49, wherein the soluble NRP1 polypeptide is fused to a purification or affinity tag.
  • soluble NRP1 polypeptide for use according to item 49, wherein the soluble NRP1 polypeptide is fused to fragment crystallizable (Fc) domain of an antibody, for example a variant of an IgG 1 or lgG4 Fc fragment having reduced effector activity.
  • Fc fragment crystallizable
  • soluble NRP1 polypeptide for use according to any one of items 28 to 53, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • soluble NRP1 polypeptide comprising a sequence having at least 70% identity with residues 276 to 428 of the amino acid sequence of SEQ ID NO:1 for the manufacture of a medicament for the treatment of an infection by a coronavirus in a subject.
  • soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 429 to 589 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 429 to 589 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 590 to 859 of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 590 to 859 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 148 to 275 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 148 to 275 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 22 to 147 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 22 to 147 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises residues 22 to 147 of the amino acid sequence of SEQ ID NO:1 , or a variant thereof in which the isoleucine residue at position 140 is replaced by a leucine residue.
  • 70. The use according to any one of items 55 to 69, wherein the soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 22 to 589 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 22 to 589 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises residues 22 to 589 of the amino acid sequence of SEQ ID NO:1 , or a variant thereof in which the isoleucine residue at position 140 is replaced by a leucine residue.
  • soluble NRP1 polypeptide comprises a sequence having at least 70% identity with residues 22 to 859 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises a sequence having at least 90% identity with residues 22 to 859 of the amino acid sequence of SEQ ID NO:1.
  • soluble NRP1 polypeptide comprises residues 22 to 859 of the amino acid sequence of SEQ ID NO:1 , or a variant thereof in which the isoleucine residue at position 140 is replaced by a leucine residue.
  • soluble NRP1 polypeptide comprises a sequence corresponding to residues 22-653 of SEQ ID NO: 7, residues 22-812 of SEQ ID NO: 8, residues 22-658 of SEQ ID NO: 12, residues 22-696 of SEQ ID NO: 14, residues 22-1085 of SEQ ID NO: 15 or residues 22-708 of SEQ ID NO:16.
  • coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • FIG. 1A depicts the amino acid sequence of membrane-bound NRP1 (isoform 1 , canonical sequence, SEQ ID NO:1).
  • FIG. 1B depicts the amino acid sequence of soluble NRP1 (isoform 2, SEQ ID NO:2).
  • FIG. 1C depicts the amino acid sequence of isoform 3 of NRP1 (SEQ ID NO:3).
  • FIGs. 1D-E depict an alignment of the amino acid sequence of rat (SEQ ID NO:4), human (SEQ ID NO:1) and mouse (SEQ ID NO:5) NRP1 with the predicted signal peptide and different domains identified (a1 , a2, b1 , b2, C, transmembrane (TM) and cytoplasmic).
  • FIG. 1F depicts the amino acid sequence of the Fc domain of IgG 1 incorporated into some of the soluble NRP1 polypeptide traps described herein (SEQ ID NO:6).
  • FIG. 2A shows the structure of SARS-CoV-2 S protein with the soluble receptor binding domain (RBD, residues 319-541) used in the binding studies highlighted.
  • FIGs. 2B-K are graphs showing the results of binding studies on SARS-CoV-2 S protein RBD using a control (vehicle) and the various NRP1 polypeptide traps depicted
  • FIG. 2L is a representation of output models from ZDOCK (http://zdock.umassmed.edu/: Pierce BG et al. (2014) ZDOCK Server: Interactive Docking Prediction of Protein-Protein Complexes and Symmetric Multimers. Bioinformatics 30(12): 1771-3) depicting the binding of the S protein ectodomain to NRP1.
  • FIG. 3A is a graph showing the results of cell fusion studies between hACE2-expressing cells and cell expressing SARS-CoV-2 S protein in the presence of vehicle (control), recombinant human ACE2 (rhACE2) and the various soluble NRP1 polypeptide traps depicted.
  • FIG. 3B is a graph showing the results of infectivity studies in the presence of vehicle (control) and the various NRP1 polypeptide traps depicted.
  • FIG. 4 is a graph showing the results of a competitive ELISA assay between soluble ACE2 and soluble NRP1 polypeptide traps #2, #4 and #8 for binding to the SARS-CoV-2 S protein RBD. The reduction of the binding of ACE2 to SARS-CoV-2 S protein RBD in the presence of increasing concentrations of the soluble NRP1 polypeptide traps is depicted.
  • the term “about” has its ordinary meaning.
  • the term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% or 5% of the recited values (or range of values).
  • soluble NRP1 polypeptides have the ability to bind to the RBD of SARS-CoV-2 S protein and compete with the binding to ACE2, to reduce the fusion between cells expressing ACE2 and cells expressing the SARS-CoV-2 S protein, and to reduce the infectivity of recombinant lentiviral viruses expressing the SARS-CoV-2 S protein on ACE2-expressing cells.
  • soluble NRP1 polypeptide traps have the ability to reduce infection of human cells by SARS- CoV-2, and thus may be used for the treatment of patients infected by SARS-CoV-2 and suffering from COVID-19.
  • the present disclosure provides a method for treating an infection by a virus, preferably a coronavirus, in a subject comprising administering to said subject an effective amount of a soluble NRP1 polypeptide.
  • the present disclosure also provides the use of a soluble NRP1 polypeptide for treating an infection by a virus, for example a coronavirus, in a subject.
  • the present disclosure also provides the use of a soluble NRP1 polypeptide for the manufacture of a medicament for treating an infection by a virus, preferably a coronavirus, in a subject.
  • the present disclosure also provides a soluble NRP1 polypeptide for use in treating an infection by a virus, preferably a coronavirus, in a subject.
  • the present disclosure provides a method for blocking the entry of a virus, for example a coronavirus, in a cell, such as an ACE2- and/or NRP1 -expressing cell, comprising contacting the cell and/or virus with an effective amount of a soluble NRP1 polypeptide.
  • the present disclosure also provides the use of a soluble NRP1 polypeptide for blocking the entry of a virus, for example a coronavirus, in a cell, such as an ACE2- and/or NRP1- expressing cell.
  • the present disclosure also provides the use of a soluble NRP1 polypeptide for the manufacture of a medicament for blocking the entry of a virus, for example a coronavirus, in a cell, such as an ACE2- and/or NRP1 -expressing cell.
  • a soluble NRP1 polypeptide for use in blocking the entry of a virus, for example a coronavirus, in a cell, such as an ACE2- and/or NRP1 -expressing cell.
  • the present disclosure provides a method for preventing a coronavirus infection (e.g., SARS-CoV-2 infection) or a related disease (Coronavirus disease 2019, COVID- 19), in a subject in need thereof, the method comprising administering to the subject an effective amount of with an effective amount of a soluble NRP1 polypeptide.
  • the present disclosure also provides the use of a soluble NRP1 polypeptide for preventing a coronavirus infection (e.g., SARS-CoV-2 infection) or a related disease (e.g., COVID-19), in a subject.
  • the present disclosure also provides the use of a soluble NRP1 polypeptide for the manufacture of a medicament for preventing a coronavirus infection (e.g., SARS-CoV-2 infection) or a related disease (e.g., COVID-19), in a subject.
  • a coronavirus infection e.g., SARS-CoV-2 infection
  • a related disease e.g., COVID-19
  • the present disclosure also provides a soluble NRP1 polypeptide for the manufacture of a medicament for use in preventing a coronavirus infection (e.g., SARS-CoV- 2 infection) or a related disease (e.g., COVID-19), in a subject.
  • the present disclosure provides a method for reducing the risk of developing a coronavirus-related disease (e.g., COVID-19), or the severity of a coronavirus- related disease (e.g., COVID-19) in a subject in need thereof, the method comprising administering to the subject an effective amount of with an effective amount of a soluble NRP1 polypeptide.
  • the present disclosure also provides the use of a soluble NRP1 polypeptide for reducing the risk of developing a coronavirus-related disease (e.g., COVID-19), or the severity of a coronavirus-related disease (e.g., COVID-19), in a subject.
  • the present disclosure also provides the use of a soluble NRP1 polypeptide for the manufacture of a medicament for reducing the risk of developing a coronavirus-related disease (e.g., COVID-19), or the severity of a coronavirus- related disease (e.g., COVID-19), in a subject.
  • a soluble NRP1 polypeptide for the manufacture of a medicament for use in reducing the risk of developing a corona virus- related disease (e.g ., COVID-19), or the severity of a coronavirus-related disease (e.g., COVID-19), in a subject.
  • soluble NRP1 polypeptide or “NRP1 trap” or “NRP1 polypeptide trap” refers to the naturally occurring soluble NRP1 polypeptide (such as isoform 1, 2 or 3 of human NRP1 depicted in FIG. 1A, FIG. 1B and 1C), or a synthetic soluble NRP1 polypeptide “derived” from naturally occurring NRP1 polypeptides, for example variants or fragments of naturally occurring NRP1 proteins which has the ability to compete with endogenous NRP1 for ligand binding, and more particularly to compete with endogenous NRP1 for binding to a coronavirus envelope protein (e.g., the spike protein of SARS-CoV-2).
  • a coronavirus envelope protein e.g., the spike protein of SARS-CoV-2
  • the soluble NRP1 polypeptide is a synthetic soluble NRP1 polypeptide “derived” from naturally occurring NRP1 polypeptides, for example variants or fragments of naturally occurring NRP1 proteins.
  • Soluble NRP1 polypeptides according to the present disclosure preferably lack amino acids 1-21 of native NRP1 that correspond to the signal peptide that is cleaved upon secretion of native NRP1 by cells.
  • the soluble NRP1 polypeptide comprises a fragment of at least 100, 150, 200, 250, 300, 350 or 400 amino acids from native NRP1 , or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the fragment, wherein the fragment or variant thereof has the ability to compete with endogenous NRP1 for binding to a coronavirus envelope protein (e.g., the spike protein of SARS-CoV-2).
  • a coronavirus envelope protein e.g., the spike protein of SARS-CoV-2
  • the soluble NRP1 polypeptide comprises a sequence derived from the sequence of domain b1 of NRP1 , for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 276 to 424, 428 or 430 of FIG. 1A.
  • the soluble NRP1 polypeptide comprises residues corresponding to the serine at position 346, glutamic acid at position 348, and threonine at position 349, i.e. the residues at positions 346, 348 and 349 are the amino acids present in native NRP1.
  • the soluble NRP1 polypeptide further comprises a sequence derived from one or more of the other extracellular domains of NRP1 , for example domain a1 , a2, b2 and/or c.
  • the soluble NRP1 polypeptide further comprises a sequence derived from the sequence of domain a2 of NRP1, for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 148 to 275 of FIG.
  • the soluble NRP1 polypeptide further comprises a sequence derived from the sequence of domain a1 of NRP1, for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 22 to 147 of FIG. 1A.
  • the residue corresponding to the residue at position 140 of the sequence depicted in FIG. 1A is a leucine (I) residue in the soluble NRP1 polypeptide.
  • the soluble NRP1 polypeptide further comprises a sequence derived from the sequence of domain b2 of NRP1, for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 425, 429 or 431 to 589 of FIG. 1A.
  • the soluble NRP1 polypeptide further comprises a sequence derived from the sequence of domain c of NRP1 , for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 590 to 859 of FIG.
  • the soluble NRP1 polypeptide comprises a sequence derived from the sequence of domains a1 , a2 and b1 of NRP1 , for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 22 to 424, 428 or 430 of FIG. 1A.
  • the soluble NRP1 polypeptide comprises a sequence derived from the sequence of domains a2, b1 and b2 of NRP1 , for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 148 to 589 of FIG. 1A.
  • the soluble NRP1 polypeptide comprises a sequence derived from the sequence of domains a2, b1 , b2 and C of NRP1 , for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 148 to 859 of FIG. 1A.
  • the soluble NRP1 polypeptide comprises a sequence derived from the sequence of domains a1 , a2, b1 and b2 of NRP1 , for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 22 to 589 of FIG. 1A.
  • the soluble NRP1 polypeptide comprises a sequence derived from the sequence of domains a1 , a2, b1 , b2 and c domains of NRP1 , for example a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with residues 22 to 859 of FIG. 1A.
  • the soluble NRP1 polypeptide does not comprise the full domain a1 of NRP1 (e.g., residues 22 to 147 of FIG. 1A). In an embodiment, the soluble NRP1 polypeptide does not comprise the full domain a2 of NRP1 (e.g., residues 148 to 275 of FIG. 1A). In an embodiment, the soluble NRP1 polypeptide does not comprise the full domains a1 and a2 of NRP1 (e.g., residues 22 to 275 of FIG. 1A). In an embodiment, the soluble NRP1 polypeptide does not comprise the full domain C of NRP1 (e.g., residues 590 to 859 of FIG. 1A).
  • the soluble NRP1 polypeptides encompassed by the present disclosure share the ability to interfere with the binding of a viral envelope protein to endogenous NRP1 and/or to ACE2, thereby reducing the ability of viruses to infect cells such as NRP1 -expressing cells and ACE2- expressing cells.
  • the soluble NRP1 polypeptides may comprise amino acid variations (deletions, additions and/or substitutions) relative to the sequence of the native NRP1 protein as long as the above-noted function is maintained.
  • the substitutions are preferably conservative, i.e., an amino acid is replaced by another amino acid having similar physico-chemical properties (size, hydrophobicity, charge/polarity, etc.) as well known by those of ordinary skill in the art.
  • the soluble NRP1 polypeptide may include one or more amino acid mutations relative to the native NRP1 sequence, for example mutations to reduce the binding to endogenous NRP1 ligands such as SEMA3A, VEGF and/or TGF-beta.
  • endogenous NRP1 ligands such as SEMA3A, VEGF and/or TGF-beta.
  • mutation of the tyrosine residue at position 297, the glutamic acid residue at position 319 and the aspartic acid residue at position 320 reduce binding to VEGF.
  • Non-limiting examples of substitutions that may be introduced the soluble NRP1 polypeptide/NRP1 traps of the present disclosure are depicted in Table 1:
  • Identity refers to sequence identity between two polypeptides or peptides. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences. As used herein, a given percentage of identity between sequences denotes the degree of sequence identity in optimally aligned sequences. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl.
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software and tools for performing BLAST analysis may be available through the National Center for Biotechnology Information (NCBI) or other bioinformatic platforms such as EMBL-EBI and ExPASy.
  • NCBI National Center for Biotechnology Information
  • the soluble NRP1 polypeptide may include one or more linkers, such as peptide/polypeptide linkers.
  • linkers may be within the sequence, for example between two domains of the soluble NRP1 polypeptide, or may be incorporated at the amino and/or carboxy-terminal end(s) of the soluble NRP1 polypeptide.
  • Such linkers may be used, for example, to attach one or more additional polypeptide domain(s) to the soluble NRP1 polypeptide.
  • Such polypeptide domain may for example increases the stability of the polypeptide in vivo and/or a domain which facilitates purification of the polypeptide.
  • Linker sequence may optionally include peptidase or protease cleavage sites which may be used to remove one or more polypeptide fragments or domains (e.g., removal of purification tag prior to in vivo administration of the soluble NRP1).
  • a protease cleavage site is a Tobacco Etch Virus (TEV) protease cleavage site (e.g., GSKENLYFQ’G).
  • TSV Tobacco Etch Virus
  • GSKENLYFQ’G GSKENLYFQ’G
  • Many other protease/peptidase cleavage sites are known to the skilled person and may be introduced in the soluble NRP1 polypeptides to optionally remove one or more polypeptide domains or fragments.
  • the polypeptide linker comprises at least 2, 3, 4 or 5 amino acids.
  • the polypeptide linker comprises about 100, 90, 80, 70, 60 or 50 amino acids or less. In a further embodiment, the polypeptide linker comprises about 5 to about 60 amino acids, about 5 to about 50 amino acids, for example about 5 to about 40 amino acids or about 5 to about 30 amino acids, for example about 7 to about 20 or 25 amino acids.
  • the linker is a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc. Such linkers may be attached to one or more of the amino acids of the soluble NRP1 polypeptide ( e.g ., to the side chain) to conjugate molecules of interest to the soluble NRP1 polypeptide.
  • the linker is a polypeptide linker comprising a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence
  • the soluble NRP1 polypeptide is conjugated with a molecule, e.g., is fused to one or more additional polypeptide domains.
  • additional polypeptide domain(s) may be directly attached to the amino and/or carboxy-terminal end(s) of the soluble NRP1 polypeptide (NRP1 domains) or through a linker, as defined above.
  • the one or more additional polypeptide domains comprise a purification or affinity tag, and/or a molecule that increases the stability of the soluble NRP1 polypeptide, for example the stability in vivo.
  • Example of purification or affinity tags include, for example, polyhistidine tags (His-tags), polyarginine tags, polyaspartate tags, polycysteine tags, polyphenylalanine tags, glutathione S-transferase (GST) tags, Maltose binding protein (MBP) tags, calmodulin binding peptide (CBP) tags, Streptavidin/Biotin-based tags, HaloTag ® , Profinity eXact ® tags, epitope tags (such as FLAG, hemagglutinin (HA), HSV, S/S1 , c-myc, KT3, T7, V5, E2, and Glu-Glu epitope tags) (see, e.g., Kimple et al., Curr Protoc Protein Sci.
  • His-tags polyhistidine tags
  • polyarginine tags polyaspartate tags
  • polycysteine tags polyphenylalanine tags
  • GST glutathione S-transfer
  • the soluble NRP1 polypeptide is conjugated with a molecule that increases the stability of the soluble NRP1 polypeptide, for example the stability in vivo.
  • a molecule that increases the stability of the soluble NRP1 polypeptide for example the stability in vivo.
  • examples of such molecule include fatty acids (e.g., C 6 -Cis), sugars/polysaccharides, polyethylene glycol (PEG) or polypeptides that have a long half- life in vivo such as a Fragment crystallizable (Fc) fragment of the constant heavy chain of an antibody, preferably a human Fc fragment (e.g., an Fc fragment comprising the sequence depicted in FIG. 1F, SEQ ID NO:6), albumin or transferrin.
  • Fc Fragment crystallizable fragment of the constant heavy chain of an antibody
  • the Fc fragment is a variant of a human Fc fragment, for example a variant of an IgG 1 or lgG4 Fc fragment having reduced effector activity, such as variants comprising a P329G substitution and/or the “LALA” double mutation (Leu234Ala and Leu235Ala) as disclosed in Schlothauer et al., Protein Eng Des Sel. 2016 Oct;29(10):457-466).
  • the additional polypeptide domain e.g., Fc fragment
  • the additional polypeptide domain is fused to the amino-terminal end of the soluble NRP1 polypeptide.
  • the additional polypeptide domain is fused to the carboxy-terminal end of the soluble NRP1 polypeptide.
  • the soluble NRP1 polypeptide is conjugated at its amino- terminal end with an Fc fragment.
  • the conjugate comprises a linker (e.g., a peptide or polypeptide linker as described above) between the soluble NRP1 polypeptide and the Fc fragment.
  • the soluble NRP1 polypeptides may be conjugated to oligomerization domains to obtain multimeric forms of the soluble NRP1 polypeptides, e.g., dimeric, trimeric, tetrameric, pentameric heptameric, or decameric forms of soluble NRP1 polypeptides.
  • Such oligomeric forms may be obtained through methods well known in the art, for example by adding oligomerization domains or scaffolds to the soluble NRP1 polypeptides (e.g., IgM scaffold, Fc domain, biotin/avidin complexes), by favoring the formation of disulfide bonds (e.g., via engineered cysteine residues), cofactor bridging or metal ion bridging (see, e.g., Gwyther et al., Biochem Soc Trans (2019) 47(6): 1773-1780).
  • the soluble NRP1 polypeptide is in an oligomerized form.
  • the soluble NRP1 polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 7 (preferably residues 22-653), SEQ ID NO: 8 (preferably residues 22-812), SEQ ID NO: 12 (preferably residues 22-658), SEQ ID NO: 14 (preferably residues 22-696), 15 (preferably residues 22-1085) or SEQ ID NO: 16 (preferably residues 22-708).
  • SEQ ID NO: 7 preferably residues 22-653
  • SEQ ID NO: 8 preferably residues 22-812
  • SEQ ID NO: 12 preferably residues 22-658
  • SEQ ID NO: 14 preferably residues 22-696
  • 15 preferably residues 22-1085
  • SEQ ID NO: 16 preferably residues 22-708
  • the soluble NRP1 polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 7, preferably with residues 22-653 of SEQ ID NO: 7.
  • the soluble NRP1 polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8, preferably with residues 22-812 of SEQ ID NO: 8.
  • the soluble NRP1 polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 12, preferably with residues 22-658 of SEQ ID NO: 12.
  • the soluble NRP1 polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 14, preferably with residues 22-696 of SEQ ID NO: 14.
  • the soluble NRP1 polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 15, preferably with residues 22-1085 of SEQ ID NO: 15.
  • the soluble NRP1 polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 16, preferably with residues 22-708 of SEQ ID NO: 16.
  • the soluble NRP1 polypeptide may be formulated in a pharmaceutical composition in admixture with suitable pharmaceutically acceptable carrier(s) or excipient(s).
  • suitable pharmaceutically acceptable carrier or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, physiological media, and the like that are physiologically compatible, i.e. that do not interfere with effectiveness of the biological activity of the active ingredients (soluble NRP1 polypeptides) and that is not toxic to the subject, i.e., is a type of excipient and/or is for use in an amount which is not toxic to the subject.
  • the carrier is suitable for systemic administration, e.g., injection.
  • the carrier is suitable for oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents, such as for systemic or oral application, is well known in the art. Except insofar as any conventional media or agent is incompatible with the soluble NRP1 polypeptides described herein, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Suitable routes of administration may, for example, include systemic, oral, subcutaneous, intramuscular, nasal or pulmonary (e.g., aerosol) administration.
  • the formulations may also be in the form of sustained release formulations.
  • Formulations suitable for oral administration may include (a) liquid solutions, such as an effective amount of active agent(s)/composition(s) suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor e.g., sucrose
  • an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for the soluble NRP1 polypeptides include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, (e.g., lactose) or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • excipients e.g., lactose
  • aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate
  • glycocholate and deoxycholate may be oily solutions for administration in the form of nasal drops, or as a gel.
  • the appropriate dosage of the soluble NRP1 polypeptide will depend on the type of disease or condition to be treated, the severity and course of the disease or condition, whether the soluble NRP1 polypeptide is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the soluble NRP1 polypeptide, and the discretion of the attending physician.
  • the soluble NRP1 polypeptide may be suitably administered to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in useful animal models prior to testing in humans.
  • the present disclosure provides dosages for the soluble NRP1 polypeptides and compositions comprising same.
  • the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/ 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values.
  • a typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the term “treating” or “treatment” in reference to viral disease is meant to refer to a reduction/improvement in one or more symptoms or pathological features associated with said viral disease.
  • Non-limiting examples include a decrease in viral load, reduction of cough, fever, fatigue, shortness of breath, reduction/prevention of acute respiratory distress syndrome (ARDS), reduction/prevention of multi-organ failure, septic shock, and blood clots, etc.
  • ARDS acute respiratory distress syndrome
  • the administration/use of the soluble NRP1 polypeptides, or pharmaceutical composition described herein delays the onset of one or more symptoms of a coronavirus-caused infection, such as a SARS-CoV-2-caused infection (e.g., COVID-19).
  • methods and uses described herein are for the treatment of an infection caused by SARS-CoV-2 or a related disease (COVID-19).
  • the subject is infected by the original Wuhan strain.
  • the subject is infected by a variant of the Wuhan strain, such as the B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.427 (Epsilon), B.1.429 (Epsilon), or B.1.617.2 (Delta) variant strain.
  • the soluble NRP1 polypeptides disclosed herein may be useful for blocking the entry of any virus that has a surface protein with CendR elements that can bind to endogenous NRP1 such as the envelope glycoprotein B (UL55) from human cytomegalovirus, the fusion protein from Measles or Mumps virus, the preM protein from tick-born encephalitis or yellow fever virus, hemagglutinin from Influenza A virus (H5N1), envelope precursor gp160 from HIV, the virion spike glycoprotein precursor from Zaire ebolavirus, the BALF4 (glycoprotein B) from human herpesvirus 4, the fusion glycoprotein precursor from human metapneumo-virus, the env propeptide from Human T-lymphotropic virus-2 or the glycoprotein precursor from Crimean-Congo hemorrhagic fever virus.
  • UL55 envelope glycoprotein B
  • fusion protein from Measles or Mumps virus
  • the preM protein from tick-born encephalitis or yellow fever virus
  • the soluble NRP1 polypeptides may be used for the treatment of any diseases or conditions associated with a protein/peptide having a polybasic furin-type cleavage site leading to the generation of a CendR sequence, i.e. a CendR peptide or protein.
  • a CendR sequence such as LyP-1 and iRGD (Teesalu et al., Front. Oncol., 27 August 2013), and thus the soluble NRP1 polypeptides may be used for the treatment of such tumors.
  • the soluble NRP1 polypeptides or pharmaceutical composition described herein may be used alone or in combination with other prophylactic agents such as anti-virals, anti-inflammatory agents, vaccines, immunotherapies, etc.
  • the combination of active agents and/or compositions comprising same may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form.
  • Co-administration in the context of the present disclosure refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time.
  • a first agent e.g., the soluble NRP1 polypeptides or pharmaceutical composition described herein
  • a second active agent e.g., an antiviral or anti-inflammatory agent
  • the agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time.
  • the soluble NRP1 polypeptides or pharmaceutical composition is used in combination with one or more anti-SARS-CoV-2 antibodies.
  • the soluble NRP1 polypeptides described herein and the one or more anti-SARS-CoV-2 antibodies are present in the same composition.
  • the term "subject” is taken to mean warm blooded animals such as mammals, for example, cats, dogs, mice, guinea pigs, horses, bovine cows, sheep and humans. In an embodiment, the subject is a mammal, and more particularly a human.
  • Plasmids for viral assessment The plasmid expressing the human coronavirus Spike of SARS-CoV-2 was previously reported (Hoffmann, M., et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181 , 271-280 e278 (2020)).
  • the plasmid encoding for SARS-CoV-2 S RBD (residues 319-541) fused with a hexahistidine tag was reported elsewhere (Amanat, F., et al. A serological assay to detect SARS- CoV-2 seroconversion in humans. Nat Med 26, 1033-1036 (2020)).
  • VSV-G vesicular stomatitis virus G
  • pSVCMV-IN-VSV-G The vesicular stomatitis virus G (VSV-G)-encoding plasmid (pSVCMV-IN-VSV-G) was previously described (Lodge, R., Lalonde, J.P., Lemay, G. & Cohen, E.A.
  • the membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells. EMBO J 16, 695-705 (1997)).
  • the lentiviral packaging plasmids pLP1 and pLP2, coding for HIV-1 gag/pol and rev respectively, were purchased from Invitrogen.
  • the transfer plasmid (pLenti- C-mGFP-P2A-Puro-ACE2) encoding for human angiotensin converting enzyme 2 (ACE2) fused with a mGFP C-terminal tag and a puromycin selection marker was purchased from OriGene.
  • the lentiviral vector to produce pseudoparticles was pNL4.3 R-E- Luc.
  • 293T human embryonic kidney cells obtained from ATCC were maintained at 37°C under 5% C0 2 in Dulbecco's modified Eagle's medium (DMEM) (Wisent) containing 5% fetal bovine serum (VWR) and 100 pg/ml of penicillin-streptomycin (Wisent).
  • DMEM Dulbecco's modified Eagle's medium
  • VWR fetal bovine serum
  • Pstreptomycin penicillin-streptomycin
  • 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).
  • pLP1 and pLP2 an envelope plasmid
  • pSVCMV-IN-VSV-G a lentiviral transfer plasmid coding for human ACE2
  • pSVCMV-IN-VSV-G lentiviral transfer plasmid coding for human ACE2
  • pSVCMV-IN-VSV-G lentiviral transfer plasmid coding for human ACE2
  • OriGene lentiviral transfer plasmid coding for human ACE2
  • supernatant containing lentiviral particles was used to infect more 293T cells in presence of 5 p
  • FreestyleTM 293-F cells (Invitrogen) were grown in FreestyleTM 293-F medium (Invitrogen) to a density of 1 x 10 6 cells/mL at 37°C with 8 % C0 2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD using ExpiFectamineTM 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 pm filter (Thermo Fisher Scientific). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen).
  • RBD preparations were dialyzed against phosphate buffered saline (PBS) and stored in aliquots at -80°C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue. For cell-surface staining, RBD proteins were fluorescently labelled with Alexa Fluor 594 (Invitrogen) according to the manufacturer’s protocol.
  • PBS phosphate buffered saline
  • HEK-293T cells transfected with plasmids expressing hACE2 and a luciferase reporter gene under the control of the HIV-1 LTR (pNL4.3 R- EnvLuc) were plated into 96-well plates and served as target cells 24 hours after transfection.
  • Effector cells were HEK- 293T transfected with plasmids expressing for the HIV-1 Tat transactivator and the SARS-CoV-2 Spike (S) glycoprotein or VSV-G glycoprotein.
  • S SARS-CoV-2 Spike
  • effector cells were incubated with 1 mM of NRP1 polypeptide traps for 1 hour at 37°C and then added to targets cells at a 1 :1 ratio. After 8 hours of incubation at 37°C, mixed cells were lysed and luciferase activity (relative lighting unit, RLU) was measured. Data are expressed with background reading subtracted.
  • Anti-SARS-CoV-2 RBD CR3022 mAb 50 ng/ml. Plates were washed four times with washing buffer followed by incubation with secondary Abs (diluted in blocking buffer) for 1 h at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1 :1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB 941 TriStar luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each serum and were then normalized to the signal obtained with CR3022 mAb present in each plate.
  • Target cells were infected with single-round luciferase- expressing lentiviral particles. Briefly, 293T cells were transfected by the calcium phosphate method with the lentiviral vector pNL4.3 R-E-Luc (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike at a ratio of 5:4. Two days after transfection, cell supernatants were harvested and stored in aliquots at -80°C until use. 293T-ACE2 target cells were seeded at a density of 1 x 10 4 cells/well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer) 24 h before infection.
  • Luciferase-expressing recombinant viruses in a final volume of 100 pi were incubated with the indicated sera dilutions (1/50; 1/250; 1/1250; 1/6250; 1/31250) for 1h at 37°C and were then added to the target cells followed by incubation for 48 h at 37°C; the medium was then removed from each well, and the cells were lysed by the addition of 30 pi of passive lysis buffer (Promega) followed by one freeze-thaw cycle.
  • the neutralization half- maximal inhibitory dilution (ID 5 o) or the neutralization 80% inhibitory dilution (IDso) represents the sera dilution to inhibit 50% or 80% of the infection of 293T-ACE2 cells by recombinant lentiviral viruses bearing the indicated surface glycoproteins.
  • Mouse single-cell RNAseq dataset (242 500 cells, Microwell-Seq) was obtained from GEO: GSE108097 (Mapping the Mouse Cell Atlas by Microwell-Seq, Cell, Volume 173, Issue 5, 17 May 2018).
  • Human healthy airways single-cell RNAseq dataset (78 000 cells, 10X Genomics) was obtained from EGAS00001004082 (Single cell RNA-seq mapping of nasal and tracheobronchial airways in human healthy volunteers, Am J Respir Crit Care Med. 2020 Dec 15;202(12):1636-1645).
  • NRP1 is expressed on cells susceptible to SARS-CoV-2 infection
  • Nrp1 RNAseq datasets were re-analysed in order to determine heterogeneity of Nrp1 in mouse and human tissues. In rodents, Nrp1 was strongly expressed in the liver, pancreas and lung, and principally in endothelial cells. In human airways (lung and nasal cavities), Nrp1 was robustly expressed in endothelial cells as well as in fibroblast and macrophages.
  • Nrp1 bronchoalveolar lavage fluid
  • soluble NRP1 polypeptide traps comprising different domains (a1 , a2, b1 , b2, and c) from human NRP1 fused to the Fc domain of human IgG 1 to bind to the RBD of the SARS-CoV-2 S protein (FIG. 2A) was assessed by ELISA.
  • FIGs. 2B-K show that the b1 domain of human NRP1 is primarily involved in the binding to the SARS-CoV-2 S protein as soluble NRP1 polypeptide traps that lack this domain did not bind the RBD of the SARS-CoV-2 S protein.
  • the results suggest that the presence of the C domain in the soluble NRP1 polypeptide reduces the binding to the RBD of the SARS-CoV-2 S protein.
  • FIG. 2L is a representation of output models from ZDOCK (http://zdock.umassmed.edu/1 depicting showing the whole S ectodomain (left panel) where the b1 domain of NRP1 is in its most-preferred pose relative to RBD, directly on the domain.
  • the right panel shows that when an entire S1 subunit is used (extracted from 6vyb with the 682RRAR685 motif reverted to the wild- type sequence), the b1 domain of NRP1 preferentially binds the 682-685 site (white balls).
  • this modeling predicts and confirms RBD ELISA binding data that NRP1 , mostly through its b1 domain, directly binds the RBD of SARS-CoV-2 S protein.
  • soluble NRP1 polypeptide traps may be useful to interfere with SARS-CoV-2 cell entry by blocking the binding of SARS-CoV-2 S protein to the cell membrane and/or by competing with endogenous NRP1 for binding to the SARS-CoV-2 S protein.
  • Example 4 Effect of soluble NRP1 polypeptide traps on the fusion of hACE2-expressing cells with cells expressing the SARS-CoV-2 S protein
  • soluble NRP1 polypeptide traps could block the fusion between HEK-293T cells expressing hACE2 and HEK-293T cells expressing the SARS-CoV-2 S glycoprotein.
  • incubation with soluble NRP1 polypeptide traps comprising the b2 domain of NRP1 significantly reduced cell fusion relative to incubation with vehicle (control).
  • a non-statistically significant trend towards reduced cell fusion (relative to control) was obtained with soluble NRP1 polypeptide traps comprising the b1 domain but lacking the b2 domain, whereas a soluble NRP1 polypeptide trap comprising only the a1 and a2 domains did not reduce cell fusion.
  • Example 5 Effect of soluble NRP1 polypeptide traps on the infectivity of SARS-CoV-2 S- expressing recombinant lentiviral viruses Infection of hACE2-expressing 293T cells by recombinant lentiviral viruses expressing SARS-CoV-2 Spike was used as a model to mimic viral entry of SARS-CoV-2 in human cells. The neutralizing effect of representative soluble NRP1 polypeptide traps on viral infection was assessed in this model. As shown in FIG.
  • Example 6 Effect of soluble NRP1 polypeptide traps on the interaction between ACE2 and SARS-CoV-2 S protein.
  • soluble NRP1 polypeptides comprising different domains (a1 , a2, b1 , b2, and c) from human NRP1 fused to the Fc domain of human IgG 1 to prevent binding of ACE2 to the RBD of the SARS-CoV-2 S protein was assessed by competition ELISA.
  • the results depicted in FIG. 4 show that soluble NRP1 polypeptides comprising the b1 domain of human NRP1 can compete with ACE2 for the RBD of SARS-CoV-2 S protein and hence prevent its binding to this domain of the SARS-CoV-2 S protein.
  • soluble NRP1 polypeptide traps have the ability to interfere with the binding of SARS-CoV-2 S protein to human cells and to reduce infection of human cells by SARS-CoV-2, and thus may be used for the treatment of patients infected by SARS-CoV-2 and suffering from COVID-19.

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Abstract

La présente invention concerne le traitement d'une infection à coronavirus, tel que le coronavirus à syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) et des maladies associées. Le traitement est réalisé par l'administration de polypeptides de neuropiline-1 solubles (NRP1) qui sont en compétition avec la NRP1 endogène et l'ACE2 pour se lier à la glycoprotéine de spicule (S) située sur l'enveloppe du coronavirus.
PCT/CA2021/050878 2020-06-26 2021-06-25 Utilisation de polypeptides nrp1 solubles pour le traitement d'infections à coronavirus WO2021258218A1 (fr)

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Citations (1)

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CA2960054A1 (fr) * 2014-09-05 2016-03-10 Rsem, Limited Partnership Compositions et methodes pour traiter et prevenir l'inflammation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2960054A1 (fr) * 2014-09-05 2016-03-10 Rsem, Limited Partnership Compositions et methodes pour traiter et prevenir l'inflammation

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Title
CANTUTI-CASTELVETRI LUDOVICO, OJHA RAVI, PEDRO LILIANA D., DJANNATIAN MINOU, FRANZ JONAS, KUIVANEN SUVI, KALLIO KATRI, KAYA TUĞBER: "Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system", BIORXIV, 15 July 2020 (2020-07-15), XP055892302, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.06.07.137802v3.full.pdf> *
DALY JAMES L., SIMONETTI BORIS, ANTÓN-PLÁGARO CARLOS, KAVANAGH WILLIAMSON MAIA, SHOEMARK DEBORAH K., SIMÓN-GRACIA LORENA, KLEIN KA: "Neuropilin-1 is a host factor for SARS-CoV-2 infection", BIORXIV, 5 June 2020 (2020-06-05), pages 1 - 28, XP055892934, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.06.05.134114v1.full.pdf> [retrieved on 20220217], DOI: 10.1101/2020.06.05.134114 *

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