WO2022253272A1 - Ac2 recombinant multivalent et ses utilisations - Google Patents

Ac2 recombinant multivalent et ses utilisations Download PDF

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WO2022253272A1
WO2022253272A1 PCT/CN2022/096610 CN2022096610W WO2022253272A1 WO 2022253272 A1 WO2022253272 A1 WO 2022253272A1 CN 2022096610 W CN2022096610 W CN 2022096610W WO 2022253272 A1 WO2022253272 A1 WO 2022253272A1
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ace2
fragments
protein
domain
recombinant
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PCT/CN2022/096610
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Xiaohu Liu
Yanal M. Murad
Kuan Zhang
William Wei-Guo JIA
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Virogin Biotech Canada Ltd.
Virogin Biotech (Shanghai) Ltd.
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Publication of WO2022253272A1 publication Critical patent/WO2022253272A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the present invention relates generally to blocking of SARS-CoV-2 infection with multivalent recombinant ACE2.
  • the present invention provides compositions and methods for treating and/or preventing coronavirus infections, such as SARS-CoV-1 or CoV-2 infections.
  • soluble ACE2 receptor trap constructs are provided that have high binding affinities to the SARS-CoV-2 spike protein.
  • recombinant multivalent proteins comprisingtwo or more angiotensin-converting enzyme 2 (ACE2) fragments (e.g., SEQ ID NO: 1) and an antibody (e.g., a humanized immunoglobin Fc domain, SEQ ID NO: 2) , wherein the ACE2 fragments are capable of binding to a coronavirus.
  • ACE2 angiotensin-converting enzyme 2
  • the human immunoglobin Fc domain comprises an N-terminal flexible hinge region, and wherein the C-terminal ends of a first and a second of the two or more ACE2 fragments are joined to the N-terminal flexible hinge region of the human immunoglobin Fc domain.
  • the N-terminal ends of a first and a second of the two or more ACE2 fragments are joined to the C-terminal of the human immunoglobin Fc domain.
  • the recombinant multivalent protein comprises two or more ACE2 fragments with different amino acid sequences.
  • the antibody of the recombinant multivalent proteins is a Fc domain derived from IgG4.
  • expression vectors which comprise a nucleic acid sequence encoding the recombinant multivalent protein as disclosed herein, operably linked to a suitable promoter, as well as host cells which can contain the expression vector (and from which the recombinant multivalent protein can be expressed) .
  • compositions comprising the recombinant multivalent protein provided herein, as well as methods of neutralizing a coronavirus, blocking the ability of a coronavirus to infect a target cell, and for treating or preventing coronavirus infections.
  • FIG. 1A is a schematic illustration of two exemplary multivalent recombinant ACE2 protein constructs VG3.1 and VG3.2.
  • FIG. 1B and 1C are gels showing restriction digest patterns of the expression vectors encoding two exemplary multivalent recombinant ACE2 protein constructs.
  • FIG. 2A and 2B are Western blots showing expression of two exemplary multivalent recombinant ACE2 protein constructs.
  • FIG. 3 is a graph showing neutralizing efficacy of two exemplary multivalent recombinant ACE2 protein constructs against SARS-CoV-2 pseudoparticles in a cell culture system.
  • FIG. 4A and 4B present data and a graph, respectively, showing the efficacy of one exemplary multivalent recombinant ACE2 protein construct in inhibiting SARS-CoV-2 infection of cells in culture.
  • FIG. 5A and 5B present data and a graph, respectively, showing the effects of one exemplary multivalent recombinant ACE2 protein construct on inhibiting cell death caused by SARS-Co-2 infection in vitro.
  • FIG. 6A and 6B present data and a graph, respectively, showing the toxicity of one exemplary multivalent recombinant ACE2 protein construct to mammalian cells in vitro.
  • FIG. 7 provides the amino acid sequence of one exemplary construct, VG3.1 (ACE2-Fc4, SEQ ID NO: 8) .
  • FIG. 8 provides the amino acid sequence of one exemplary construct, VG3.2 (ACE2-Fc4-ACE2, SEQ ID NO: 9) .
  • FIG. 9 illustrates neutralizing efficacy of VG3.1 and VG3.2 against SARS-CoV-2 pseudoparticles.
  • FIG. 10 illustrates IC50 value of VG3.1 and VG3.2 neutralizing against SARS-CoV-2 pseudoparticles.
  • FIG. 11 illustrates neutralizing efficacy of VG3.1 and VG3.2 against SARS-CoV-2 pseudoparticles.
  • FIG. 12 is a schematic illustration of two exemplary multivalent recombinant ACE2 protein constructs VG4.1 and VG4.1.1.
  • FIG. 13A-13D presents binding ability of VG4.1 and VG4.1.1 to spike protein.
  • FIG. 14 is a schematic illustration of eight exemplary multivalent recombinant ACE2 protein constructs VG3.2.1 to VG3.2.8.
  • FIG. 15A-15F present binding ability of exemplary multivalent recombinant ACE2 protein constructs to spike protein.
  • the present invention provides, a mongst other things, compositions and methods for treating and/or preventing coronavirus infections, such as SARS-CoV-1 or CoV-2 infections, with ACE2 receptor trap constructs which have a high binding affinity to the SARS-CoV-2 spike protein.
  • ACE2 Angiotensin-converting enzyme 2
  • a coding sequence for ACE2 can comprise the entire extracellular domain of ACE2, or it may be a fragment thereof.
  • the selected coding sequence of ACE2 can also comprise the natural sequence of ACE2, or it may be modified to contain mutations that increase affinity to SARS-CoV-2 spike protein. A list of such mutations can be found at https: //www. uniprot. org/uniprot/Q9BYF1 under the subheading “Mutagenesis” , and are listed as below.
  • a "vector” refers to a nucleic acid construct for introducing a nucleic acid sequence into a cell.
  • the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polypeptide encoded in a DNA sequence.
  • an "expression vector” has a promoter sequence operably linked to a DNA sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.
  • the term "expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.
  • the term “produces” refers to the production of proteins and/or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.
  • the terms "host cell” and "host strain” refer to suitable hosts for expression vectors comprising nucleic acid sequences provided herein (e.g., the polynucleotides encoding the recombinant multivalent proteins) .
  • the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant techniques as known in the art.
  • RNA viruses are a group of related RNA viruses in the Orthornavirae Kingdom. They are enveloped viruses that have a positive-sense single-stranded RNA genome of approximately 26-32kb. They also have characteristic club-shaped spikes that project from their surface. In humans and other animals such as birds, they cause respiratory tract infections. Mild illnesses in humans include some cases which mimic the of the common cold, while more lethal varieties can cause SARS, MERS, and COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes COVID-19 (coronavirus disease 2019) , the respiratory illness responsible for the COVID-19 pandemic.
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
  • Antibody refers to any protein or protein fragment derived, designed or constructed (naturally or by synthetic or recombinant means) which is based upon an immunoglobulin (e.g., IgA, IgD, IgE, IgM, or any of the various forms of IgG) . Chimeric constructs including a portion of an immunoglobulin (e.g. an IgG binding domain) fused to another polypeptide are also included within the meaning of the term “antibody” as used herein.
  • a “humanized antibody” refers to an antibody which has had its respective fragment having amino acid residues that are substantially from a human antibody (as opposed to, for example, a mouse antibody) . Humanized antibodies can have human antibody sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. Within certain embodiments of the invention the antibody is a humanized Fc fragment.
  • Treat” or “treating” or “treatment, ” as used herein, means an approach for obtaining beneficial or desired results, including clinical results.
  • Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total) , whether detectable or undetectable.
  • the terms “treating” and “treatment” can also mean prolonging survival or reducingthe infectious of an infected subject, as compared to an individual not receiving treatment.
  • prevent means precluding or prohibiting a disease state from developing to the point of a subject exhibiting clinical symptoms, or, from being detectable using clinical assays.
  • the present invention provides recombinant multivalent proteins comprised of two or more angiotensin-converting enzyme 2 (ACE2) fragments and a humanized antibody (e.g., a humanized immunoglobin Fc domain) , wherein the ACE2 fragments are capable of binding to a coronavirus.
  • ACE2 angiotensin-converting enzyme 2
  • SARS-CoV-2 entry of SARS-CoV-2 into target cells is mediated by binding of viral spike (S) protein to angiotensin-converting enzyme 2 (ACE2) on the host cell surface.
  • ACE2 angiotensin-converting enzyme 2
  • the present invention provides novel soluble ACE2 receptor trap construct with binding affinity to SARS-CoV-2 spike protein that is multiple orders of magnitude greater than the current state of the art.
  • This recombinant multivalent protein can act as a decoy and thereby neutralizes infection by a coronavirus.
  • This receptor trap construct can also be used to neutralize SARS-CoV-1 which likewise relies on ACE2 binding.
  • One benefit of this strategy is the use of natural ACE2 fragments or engineered ACE2 with high similarity to the natural ACE2 receptor, thereby limiting the possibility of viral escape and enabling inhibition of infection of SARS-CoV-2 variants that can escape vaccine-induced immunity or impair therapeutic antibody targeting.
  • a recombinant multivalent protein is a Fc4-fused bivalent ACE2 construct as depicted in Figure 1A (VG3.1) , and which has an exposed ACE2 N-termini to facilitate S binding.
  • VG3.1 is designed to interact with the S protein (as is confirmed by the binding and virus inhibition assays shown in Figures 3-5.
  • VG3.2 tetravalent molecule
  • Figure 1A the two ACE2 attached to the N-terminal region of Fc4 in the tetravalent molecule depicted in Figure 1A have their N-terminal regions exposed and available for binding to S protein.
  • the two additional ACE2 molecules that are attached to the C-terminal end of Fc4 via the (GS) 3 linker (SEQ ID NO: 4) do not have their N-terminal regions readily accessible for S protein binding, thus reducing their predicted contribution to the overall interaction with SARS-CoV-2 S protein.
  • Figure 3 shows that VG3.2 possesses a much higher ability to inhibit SARS-CoV-2 pseudovirus infection compared to VG3.1.
  • recombinant multivalent proteins which have a significantly increased affinity to the WT receptor binding domain (RBD) of the S protein.
  • RBD WT receptor binding domain
  • a dimeric ACE2-Fc that was engineered with mutations to enhance binding to spike protein can interact with the WT receptor binding domain (RBD) of S protein with an affinity of 22 nM (see Chan et al., “Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2” Science. 2020 Sep 4; 369 (6508) : 1261-1265. doi: 10.1126/science. abc0870. Epub 2020 Aug 4.
  • VG3.2 binds to WT RBD of the S protein with more than 20,000 times greater affinity despite lacking any mutations in ACE2 that increase binding affinity.
  • all four ACE2 in VG3.2 contribute to binding SARS-CoV-2 S protein, and that such binding is facilitated by the unusual configuration of VG3.2. Additional tetravalent and hexavalent constructs can also be produced.
  • Constructs for expression of recombinant ACE2 can be generated by linking the selected coding sequence of ACE2 to that of an antibody such as human Fc4 (human Fc1 may be used as an alternative option) .
  • the selected coding sequence of ACE2 may comprise the entire extracellular domain of ACE2, or it may be a fragment thereof.
  • the selected coding sequence of ACE2 may comprise the natural sequence of ACE2, or it may be modified to contain mutations that increase affinity to SARS-CoV-2 spike protein. A list of such mutations can be found at https: //www. uniprot. org/uniprot/Q9BYF1 under the subheading “Mutagenesis” .
  • the Fc4 hinge region used in VG3.1 and VG3.2 contains 3 mutations (S228P, F234A, and L235A) when compared to the wild-type human Fc4 hinge region (see Dumet et al., “Insights into the IgG heavy chain engineering patent landscape as applied to IgG4 antibody development” .
  • PMID 31556789; PMCID: PMC6816381.
  • Particularly preferred mutations include S228P, F234A and L235A. While unmodified Fc hinge region may be used as an alternative, the S228P mutation is particularly preferred when using Fc4.
  • the expression construct can also be comprised of any secretorysignal peptide known to those skilled in the art, although the specific examples used herein utilize a secretion signal derived from human immunoglobulin heavy chain.
  • the IS peptide (IEEQAKTFLDKFNHEAEDLFYQS) used for a hexavalent construct is a natural part of the ACE2 extracellular domain. Therefore, the IS peptide is also present in the portion of ACE2 used in the bivalent and tetravalent constructs, as shown in the annotated sequence provided in the Figures.
  • Linkage between ACE2 and Fc is mediated by a sequence encoding the flexible (GS) n peptide linker GGGGSGGGGSGGGGS (3GS, SEQ ID NO: 4) .
  • GSAGSAAGSGEF SEQ ID NO: 5
  • GGSGGGSGG SEQ ID NO: 6
  • KRVAPELLGGPS SEQ ID NO: 7
  • GGGGSGGGGS 2GS, SEQ ID NO: 13
  • GGGGSGGGGSGGGGSGGGGS 4GS, SEQ ID NO: 14
  • Chen et al. “Fusion protein linkers: property, design and functionality” Adv Drug Deliv Rev. 2013 Oct; 65 (10) : 1357-69. doi: 10.1016/j. addr. 2012.09.039. Epub 2012 Sep 29. PMID: 23026637; PMCID: PMC3726540
  • the four or more ACE2 fragment can be the same or different.
  • the recombinant multivalent protein comprises four ACE2 fragment, wherein two ACE2 fragments are fused to the N-terminal of Fc domain and two ACE2 fragments are fused to the C-terminal of Fc domain, with or without linker.
  • the recombinant multivalent protein comprises four ACE2 fragment, and four ACE2 fragments are fused to the C-terminal of Fc domain, with or without linker.
  • the recombinant multivalent protein is a homodimer comprise an ectodomain ofACE2, Fc domain and a peptide derived from ACE2, e.g., a peptide with 23 amino acids (SEQ ID NO: 12) .
  • linkers do not have to be identical to each other.
  • the choice of peptide linker will depend upon the ability of the linker to maximize the structural flexibility of the ACE2 construct necessary for binding the SARS-CoV-2 spike protein, while maintaining a functional conformation.
  • the recombinant ACE2 construct sequences are cloned into a suitable vector such as pcDNA3.1 or pET22b for expression and purification.
  • suitable vectors are available and known to those skilled in the art.
  • compositions are provided that may be used to prevent, treat, or ameliorate the effects of a coronavirus infection. More particularly, therapeutic compositions are provided comprising recombinant multivalent protein as described herein. In certain embodiments, the compositions will further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is meant to encompass any carrier, diluent or excipient that does not interfere with the effectiveness of the biological activity of the oncolytic virus and that is not toxic to the subject to whom it is administered (see generally Remington: The Science and Practice of Pharmacy, Lippincott Williams&Wilkins; 21st ed. (May 1, 2005 and in The United States PharmacopE1A: The National Formulary (USP 40–NF 35 and Supplements) .
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions (such as oil/water emulsions) , various types of wetting agents, sterile solutions, and others.
  • Such carriers can be formulated by conventional methods and can be administered to the subject at an effective dose.
  • Additional pharmaceutically acceptable excipients include, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid and ethanol.
  • salts can also be included therein, e.g., mineral acid salts (such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like) and the salts of organic acids (such as acetates, propionates, malonates, benzoates, and the like) .
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like
  • the therapeutic composition is provided in a soluble form suitable for parenteral administration (e.g., intravenously, intramuscularly, or by a nasal spray) , or, enterally (e.g., orally) .
  • compositions may be stored at a temperature conducive to stable shelf-life and includes room temperature (a bout 20°C) , 4°C, -20°C, -80°C, and in liquid N2. Because compositions intended for use in vivo generally don’t have preservatives, storage will generally be at colder temperatures. Compositions may be stored dry (e.g., lyophilized) or in liquid form.
  • the therapeutic composition are used to treat or prevent a coronavirus infection, comprising the step of administering to a subject in need thereof an effective dose of a recombinant multivalent protein as described herein.
  • effective dose and “effective amount” refers to amounts of the recombinant multivalent protein as described herein that is sufficient to effect treatment or prevention of a coronavirus infection. Effective amounts may vary according to factors such as the subject’s disease state, age, gender, and weight, as well as the pharmaceutical formulation, the route of administration, and the like, but can n evertheless be routinely determined by one skilled in the art.
  • the recombinant multivalent proteins described herein may be given by a route that is parenteral (e.g., intravenously, intramuscularly, or by a nasal spray) , or, enterally (e.g., orally) .
  • parenteral e.g., intravenously, intramuscularly, or by a nasal spray
  • enterally e.g., orally
  • the optimal or appropriate dosage regimen of the recombinant multivalent protein is readily determinable within the skill of the art, by the attending physician based on patient data, patient observations, and various clinical factors, including for example a subject’s size, body surface area, age, gender, degree of severity of the illness, the general health of the patient, and other drug therapies to which the patient is being subjected.
  • a therapeutically effective amount is preferably administered. This is an amount that is sufficientto show benefit to the subject.
  • the actual amount administered and time-course of administration will depend at least in part on the nature of the corornavirus infection, the condition of the subject, site of delivery, and other factors.
  • the therapeutic compositions provided herein can be delivered by inhalation, e.g., by use of a device that can create or deliver small particles that can get into the lung.
  • Larger particle sizes e.g., greater than about 10 um
  • smaller particles e.g., less than about 0.5 um tend to be exhaled.
  • the particles are less than about 10 um in average size, but greater than about 0.5um in average size.
  • the particles can range in average size from about 0.5, to 5 um in size (including subsets of this range, e.g., from about 3 to 5 um in size.
  • inhalation therapy as well as the pharmacodynamics, pharmacokinetics, manufacture and formulation, and other methods and devices related to the delivery of therapeutic compositions to the respiratory tract are described in: “Pharmaceutical Inhalation Aerosol Technology” , Hickey and R. P. da Rocha (eds. ) , Third Ed., CRC Press, Boca Raton, Florida, 2019, ISBN 9780429055201; Gardenhire et al., “A Guide to Aerosol Delivery Devices for Respiratory Therapists” 3rd Edition, American Association for Respiratory Care, 2013; “Optimization of Aerosol Drug Delivery” , Grado ⁇ and Marijnissen (eds.
  • nebulizers e.g., small-volume nebulizers, jet nebulizers, pneumatic compressor nebulizers, ultrasonic nebulizers, vibrating mesh/horn nebulizers and microprocessor-controlled breath-actuated nebulizers
  • pressurized metered-dose inhalers e.g., pressurized metered-dose inhalers, and dry-powder inhalers.
  • This example describes the design and expression cloning of two exemplary multivalent protein constructs engineered to target the SARS-CoV-2 coronavirus and inhibit binding of the virus to target cells.
  • VG3.1 and VG3.2 The structures of two exemplary multivalent recombinant ACE2 protein constructs, VG3.1 and VG3.2, are depicted in Fig. 1A. These constructs are generated by linking two or more fragments of ACE2 to a human Fc domain.
  • the fragment of ACE2 may include the entire extracellular domain (e.g., the n-terminal domain) of ACE2, or a fragment thereof.
  • the VG3.1 construct is bivalent for ACE2 (e.g., includes two ACE2 fragments)
  • the VG3.2 construct is tetravalent for ACE2 (e.g., includes four ACE2 fragments) .
  • the human Fc domain used in this Example is the human Fc4 domain with mutations in the hinge region that enhance stability and reduce aggregation (S228P) and eliminate Fc effector function (F234A and L235A) .
  • the VG3.1 construct is designed such that the N-terminal ends of both ACE2 fragments are free and exposed to solution, while the C-terminal ends are joined to the N-terminus of the Fc4 fragment.
  • the VG3.2 construct is constructed such that the additional third and fourth ACE2 fragments arejoined to the Fc4 fragments at their N-terminal ends, and are therefore in opposite orientations relative to the first and second ACE2 fragments.
  • the VG3.2 construct further includes a flexible peptide linker joining the third and fourth ACE2 fragments to the Fc4 domain.
  • telomere sequences encoding VG3.1 and VG3.2 protein were individually cloned into a CHO expression vector pCHO (e.g. catalog no. HG-VPI0983, available from Invitrogen) vectors using restriction enzymes, AvrII and BstZ17I, resulting in plasmids pCHO-VG3.1 and pCHO-VG3.2.
  • the 5’ end of the expression constructs also included DNA fragments encoding the secretory signals derived from the human immunoglobin heavy chain (SEQ ID NO: 3) .
  • Figs. 1B and 1C show the results of agarose gel electrophoresis using plasmid DNA digested with BstZ17I or double-digested with AvrII-BstZ17I. All DNA fragments displayed the expected molecular weight.
  • VG3.1 and VG3.2 protein constructs were transfected to HEK293T cells with lipofectamine 3000.
  • Cell lysates were prepared and analyzed by Western Blotting assay to detect VG3.1 and VG3.2 proteins using antibodies against ACE-2 and hFc. Results are presented in Fig. 2 which shows that both VG3.1 and VG3.2 were detected by antibodies against ACE-2 (Fig. 2A) and hFc (Fig. 2B) . Both ACE-2 multivalent protein constructs migrated in the gel at the expected molecular weight.
  • the ExpiCHO TM Expression System was used to express VG3.1 and VG3.2 proteins in CHO cells. Briefly, pCHO-VG3.1 and pCHO-VG3.2 were transfected to cells with ExpiFectamine TM CHO Reagent. The transfected cells were cultured for 2-4 days at 32°C, 120 rpm, with 5%CO 2 . Cell culture medium was collected and clarified by centrifugation. After passing through a 0.22 um filter, culture medium was used for VG3.1 and VG3.2 protein purification. Protein A was used in the initial capture step, followed by washing and elution. The eluted protein was maintained in PBS buffer at 4°C.
  • VG3.1 and VG3.2 protein completely neutralize the SARS-CoV pseudoparticle at 316 ⁇ g/ml and 1 ug/ml, respectively.
  • IC 50 half maximal inhibitory concentration
  • the efficacy of the VG3.2 protein construct in inhibiting SARS-CoV infection was analyzed in a cell culture system with the SARS-CoV-2 virus and Vero cells.
  • the virus was pre-incubated with a gradient concentration of purified VG3.2 protein.
  • Vero cells were plated in 96-well plates and then infected with virus at a MOI of 0.1.
  • the viral content of cellular supernatant was determined by RT-PCR at 48 hr post infection. Results indicated thatthe IC 50 of VG3.2 protein in inhibition of SARS-CoV-2 infection was 6.1ng/mL (see Fig. 4A and 4B) .
  • the viability of Vero cells was determined using a commercially available Cell Counting Kit-8 (CCK8) . Results of this experiment are shown in Figs. 5A and 5B, which indicate that the IC 50 of VG3.2 in inhibiting SARS-CoV-2-induced cell death was 0.95 ⁇ g/mL.
  • VG3.2 protein was analyzed for the toxicity of the VG3.2 protein on mammalian cells.
  • Vero cells were plated in a 96-well plate. Cells were then treated with a gradient concentration of purified VG3.2 protein. At 3 days post-treatment, cell viability was determined with a CCK8 Kit. The half cytotoxic concentration (CC 50 ) of VG3.2 protein was calculated and determined to be over 500 ⁇ g/mL (see Figs. 6A and 6B) .
  • the avidity between the VG3.1 or VG3.2 protein constructs and ligand proteins may be analyzed by co-crystallization followed by X-ray crystal analysis.
  • a list of SARS-CoV-2 spike protein ligands is listed in table 1. Briefly, purified VG3.1 or VG3.2 protein is mixed with each of the tested spike proteins and subjected to standard crystallization procedures. The final structures are imaged by X-ray analysis.
  • VG3.1 or VG3.2 protein constructs VG3.1 or VG3.2 protein constructs, and recombinant anti-ACE2 antibodies REGN10933 and REGN10987 (Regeneron Pharmaceuticals Inc. ) .
  • VG3.1, VG3.2, REGN10933 and REGN10987 were diluted to a concentration of 1 ⁇ g/ml in serum-free medium.
  • 10 ⁇ l diluted VG3.1, VG3.2, REGN10933 and REGN10987 solutions were mixed with 10 ⁇ l 10 7 TU/ml pseudovirus in a 96-well plate and incubated at 37°C for 1 hour.
  • 100 ⁇ l of 5 ⁇ 10 5 /ml ACE2-293T cells Novoprotein, XCC14 were added to the 96-well plate to incubate for 48h.
  • luciferase detection kit Bolysis Buffer (Promega, E2661) and recombinant anti-ACE2 antibody (Sino Biological, 10108-R003) were also used.
  • VG3.1 and VG3.2 are capable of blocking four SARS-CoV-2 variants pseudoparticles bind to ACE, with a comparable inhibitory activity comparing to REGN10933 and REGN10987.
  • VG3.2 has the strongest inhibitory activity, followed by VG3.1.
  • VG4.1 and VG4.1.1 were constructed, expressed and purified as described in examples 1-3.
  • the VG4.1 is a homodimer containing ACE2 (Q18–S740, SEQ ID NO: 10) , and the Fc domain of human IgG4.
  • VG4.1.1 is a homodimer containing a mutant ACE2 (SEQ ID NO: 11) and the Fc domain of human IgG4.
  • the structures of VG4.1 and VG4.1.1, are depicted in Fig. 12.
  • the binding activity of VG4.1 or VG4.1.1 with Spike protein were tested. Briefly, Sspike protein was added to a 96-well plate, and the VG4.1 or VG4.1.1 was add to the 96-well plate and the mixture were incubated. Then anti-IgG antibody was added after removing unbinding VG4.1 or VG4.1.1. OD value was read by a microplate reader.
  • VG4.1 is ACE2-740-Fc4, VG4.1.1 is ACE2-740-H378A-Fc4. VG4.1 and VG4.1.1 have a higher binding ability to Spike protein than VG3.1 at different concentrations of Spike protein.
  • VG3.2.1, VG3.2.2, VG3.2.3, VG3.2.4, VG3.2.5, VG3.2.6, VG3.2.7 and VG3.2.8 were constructed, expressed and purified as described in examples 1-3.
  • VG3.2.1 is a homodimer containing the Fc domain of human IgG4 and ACE2 (Q18–D614, SEQ ID NO: 1) from N-terminal and C-terminal.
  • the linker between Fc domain and ACE2 is GGGGSGGGGS (2GS) .
  • VG3.2.5, VG3.2.6, VG3.2.7, and VG3.2.8 the linker between Fc domain and ACE2 is GGGGSGGGGSGGGGS (3GS) .
  • VG3.2.2, VG3.2.3, VG3.2.4, VG3.2.6, VG3.2.7 and VG3.2.8 a 23-mer spike binding peptide (PI, SEQ ID NO: 12) is fused to the C-terminal of IgG4 Fc, with a linker of 2GS, 3GS or 4GS.
  • PI 23-mer spike binding peptide
  • Fig. 14 The structures of VG3.2.1 to VG3.2.8, are depicted in Fig. 14. Then the binding ability of these ACE2-Fc proteins to Spike protein was tested as described in example 11.
  • Fc-2GS-ACE2 refers to VG3.2.5
  • Fc4-2GS-ACE2-2GS-PI refers to VG3.2.6
  • Fc4-2GS-ACE2-3GS-PI refers to VG3.2.7
  • Fc4-2GS-ACE2-4GS-PI refers to VG3.2.8
  • Fc4-3GS-ACE2 refers to VG3.2.1
  • Fc4-3GS-ACE2-2GS-PI refers to VG3.2.2
  • Fc4-3GS-ACE2-3GS-PI refers to VG3.2.3
  • Fc4-3GS-ACE2-4GS-PI refers to VG3.2.4.
  • All recombinant proteins with ACE2 fused to C-terminal of Fc domain are capable of binding Spike protein and recombinant proteins with PI have higher binding ability than those without PI.
  • a recombinant multivalent protein comprising two or more angiotensin-converting enzyme 2 (ACE2) fragments and a linker such as an antibody (e.g., a human immunoglobin Fc domain) , wherein said ACE2 fragments are capable of binding to a coronavirus.
  • ACE2 angiotensin-converting enzyme 2
  • the recombinant multivalent protein of embodiment 1 comprising four or more ACE2 fragments, wherein the N-terminal ends of a third and a fourth of the four or more ACE2 fragments are joined to the C-terminal end of the Fc domain.
  • a recombinant multivalent protein is provided with the amino acid sequence comprising the sequence set forth in FIG. 7.
  • a recombinant multivalent protein is provided with the amino acid sequence comprising the sequence set forth in FIG. 8.
  • a recombinant multivalent protein is provided which is soluble and/or sterile.
  • An expression vector comprising a nucleic acid sequence encoding the recombinant multivalent protein of any of embodiments 1–8 operably linked to a suitable promoter.
  • nucleic acid sequence encoding the recombinant multivalent protein construct of any of embodiments 1–8 comprises a nucleic acid sequence encoding a secretory signal, wherein the secretory signal is operably linked to the soluble recombinant multivalent protein construct.
  • inventions are provided which contain an expression vector according to any one of embodiments 9-11, as well as methods for producing the recombinant multivalent protein constructs utilizing the expression vector containing host cells.
  • a pharmaceutical composition comprising the recombinant multivalent protein of any of embodiments 1–8 and an a pharmaceutically acceptable excipient or buffer.
  • a method of neutralizing a coronavirus comprising contacting the coronavirus with an effective dose of the composition of embodiment 12.
  • a method of blocking the ability of a coronavirus to infect a target cell comprising administering to the target cell an effective dose of the composition of embodiment 12, wherein the effective dose is capable of neutralizing the coronavirus.
  • a method of treating or preventing a coronavirus infection in a patient comprising administering to the patient a therapeutically effective dose of the composition of embodiment 12.
  • the therapeutically effective dose is administered parenterally (e.g., intravenously, intramuscularly, or by a nasal spray) .
  • the therapeutically effective dose is administered enterally (e.g., orally) .
  • the coronavirus infection can be caused by SARS-CoV-1, or, SARS-CoV-2 virus.
  • a method for detecting the presence of coronavirus comprising the step admixing a composition according to any one of embodiments 1-8 with a sample to be tested, and determining whether said composition had bound a coronavirus.
  • any concentration range, percentage range, ratio range, or integer range provided herein is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer) , unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term "about” means ⁇ 20%of the indicated range, value, or structure, unless otherwise indicated.

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Abstract

L'invention concerne des protéines multivalentes recombinantes ayant au moins deux fragments d'enzyme de conversion de l'angiotensine 2 (ACE2) et un domaine Fc d'immunoglobuline humaine, les fragments ACE2 étant capables de se lier à un coronavirus. Dans un mode de réalisation, le domaine Fc d'immunoglobuline humaine a une région charnière flexible N-terminale qui est liée aux extrémités C-terminales d'un premier et d'un second des deux fragments ACE2 ou plus. L'invention concerne également des vecteurs d'expression et des cellules hôtes pour produire les protéines multivalentes recombinantes, ainsi que des compositions pharmaceutiques comprenant des protéines multivalentes recombinantes, et des procédés d'utilisation de celles-ci (par exemple, pour le traitement et/ou la prévention d'infections à coronavirus).
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