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WO2021213437A1 - Protéine de fusion ace2-fc et son utilisation - Google Patents

Protéine de fusion ace2-fc et son utilisation Download PDF

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Publication number
WO2021213437A1
WO2021213437A1 PCT/CN2021/088681 CN2021088681W WO2021213437A1 WO 2021213437 A1 WO2021213437 A1 WO 2021213437A1 CN 2021088681 W CN2021088681 W CN 2021088681W WO 2021213437 A1 WO2021213437 A1 WO 2021213437A1
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Prior art keywords
ace2
fusion protein
antibody
protein
seq
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Chinese (zh)
Inventor
姜伟东
刘俊利
刘光
宋戈
李超群
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Shanghai Henlius Biotech Inc
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Shanghai Henlius Biotech Inc
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Priority claimed from CN202010325666.XA external-priority patent/CN113549154A/zh
Priority claimed from CN202010528750.1A external-priority patent/CN113801865A/zh
Priority claimed from CN202010745204.3A external-priority patent/CN114057887A/zh
Priority claimed from CN202110193511.XA external-priority patent/CN114958811A/zh
Application filed by Shanghai Henlius Biotech Inc filed Critical Shanghai Henlius Biotech Inc
Publication of WO2021213437A1 publication Critical patent/WO2021213437A1/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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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)

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a fusion protein of angiotensin converting enzyme 2 (ACE2) protein and an Fc fragment of an antibody and therapeutic applications thereof.
  • ACE2 angiotensin converting enzyme 2
  • ACE2 is a member of the angiotensin converting enzyme family. As a carboxypeptidase, ACE2 is mainly involved in catalyzing the hydrolysis between proline and hydrophobic amino acids or basic C-terminal amino acids. The most important function of ACE2 in the human body is to catalyze the hydrolysis of angiotensin 1-8 (Ang1-8) to form Ang1-7. Studies have shown that Ang1-7 has the effects of promoting vasodilation, inhibiting malignant cell proliferation, inhibiting angiogenesis, and inhibiting inflammatory reactions. Therefore, ACE2 recombinant protein may be used in the treatment of diseases related to the pathological increase of Ang1-8, such as acute lung injury, pulmonary hypertension, acute respiratory distress, diabetic nephropathy and other diseases.
  • diseases related to the pathological increase of Ang1-8 such as acute lung injury, pulmonary hypertension, acute respiratory distress, diabetic nephropathy and other diseases.
  • ACE2 recombinant protein has been used in clinical studies of pulmonary hypertension, acute lung injury and other diseases, and has shown good safety. In vivo pharmacokinetic studies have shown that the in vivo half-life of ACE2 is only 10 hours, so it needs to be administered daily in the actual medication process.
  • ACE2 is also a key receptor for some viruses to invade the body.
  • SARS-CoV-2 novel coronavirus that broke out in 2019 invaded the body through ACE2.
  • the Spike protein (S protein) on the surface of the virus binds to the ACE2 on the surface of the host cell through its receptor binding domain (RBD), thereby mediating the invasion of the virus.
  • RBD receptor binding domain
  • COVID-19 new coronavirus pneumonia
  • the present invention provides an ACE2 fusion protein, which comprises an extracellular region of the ACE2 protein and a polypeptide that can promote the dimerization of the fusion protein.
  • the present invention provides the ACE2 fusion protein as described above, wherein the polypeptide that can promote the dimerization of the fusion protein is an Fc fragment of an antibody, preferably a human IgG antibody Fc fragment, more preferably a human IgG1 antibody Fc fragment.
  • the present invention provides the aforementioned ACE2 fusion protein, wherein the amino acid sequence of the extracellular region of the ACE2 protein is shown in SEQ ID No. 1.
  • the present invention provides the aforementioned ACE2 fusion protein, wherein the amino acid sequence of the Fc fragment of the human IgG1 antibody is shown in SEQ ID No.2.
  • the present invention provides the aforementioned ACE2 fusion protein, wherein the amino acid sequence of the ACE2 fusion protein is shown in SEQ ID No. 4.
  • the present invention also provides a nucleic acid molecule, which encodes the ACE2 fusion protein as described above.
  • the present invention also provides an expression vector comprising the nucleic acid molecule as described above.
  • the present invention also provides a host cell, which contains the expression vector as described above and can express the fusion protein as described above.
  • the present invention also provides a pharmaceutical composition, which comprises the aforementioned ACE2 fusion protein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition as described above further comprises a neutralizing antibody against the S protein of the novel coronavirus (SARS-CoV-2).
  • SARS-CoV-2 novel coronavirus
  • the pharmaceutical composition as described above, wherein the anti-new coronavirus (SARS-CoV-2) S protein antibody comprises an antibody heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region and the heavy chain variable region shown in SEQ ID NO. 8 have the same HCDR1, HCDR2, and HCDR3, and the light chain variable region and the light chain variable region shown in SEQ ID NO. 7 can be The variable regions have the same LCDR1, LCDR2 and LCDR3; or
  • the heavy chain variable region has the same HCDR1, HCDR2, and HCDR3 as the heavy chain variable region shown in SEQ ID NO. 10, and the light chain variable region has the same HCDR1, HCDR2, and HCDR3 as the light chain variable region shown in SEQ ID NO.
  • the variable zones have the same LCDR1, LCDR2 and LCDR3;
  • the HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 are defined according to the rules of Kabat, Chothia, MacCallum, IMGT, AHo or ABM.
  • the pharmaceutical composition as described above, wherein the neutralizing antibody against the S protein of the novel coronavirus (SARS-CoV-2) comprises an antibody heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region has HCDR1 shown in SEQ ID NO.14, HCDR2 shown in SEQ ID NO.15 and HCDR3 shown in SEQ ID NO.16, and the light chain variable region has The same LCDR1 shown in SEQ ID NO.11, LCDR2 shown in SEQ ID NO.12 and LCDR3 shown in SEQ ID NO.13; or
  • the heavy chain variable region has the HCDR1 shown in SEQ ID NO. 20, the HCDR2 shown in SEQ ID NO. 21, and the HCDR3 shown in SEQ ID NO. 22, and the light chain variable region has The same LCDR1 shown in SEQ ID NO.17, LCDR2 shown in SEQ ID NO.18 and LCDR3 shown in SEQ ID NO.19.
  • the pharmaceutical composition as described above, wherein the neutralizing antibody against the S protein of the novel coronavirus (SARS-CoV-2) comprises an antibody heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region is the heavy chain variable region shown in SEQ ID NO. 8 and the light chain variable region is the light chain variable region shown in SEQ ID NO. 7; or
  • the heavy chain variable region is the heavy chain variable region shown in SEQ ID NO. 10 and the light chain variable region is the light chain variable region shown in SEQ ID NO. 9.
  • the pharmaceutical composition as described above wherein the full-length antibody of the neutralizing antibody against the S protein of the novel coronavirus (SARS-CoV-2) is composed of an antibody heavy chain and a light chain, wherein the The antibody heavy chain constant region is selected from the constant regions of human IgG1, IgG2 or IgG4, and the antibody light chain constant region is selected from the human antibody lambda chain or kappa chain.
  • SARS-CoV-2 novel coronavirus
  • the pharmaceutical composition as described above wherein the full-length antibody of the neutralizing antibody against the S protein of the novel coronavirus (SARS-CoV-2) is composed of an antibody heavy chain and a light chain, wherein:
  • the antibody heavy chain amino acid sequence is shown in SEQ ID NO. 23, and the antibody light chain amino acid sequence is shown in SEQ ID NO. 24; or
  • the antibody heavy chain amino acid sequence is shown in SEQ ID NO. 25, and the antibody light chain amino acid sequence is shown in SEQ ID NO. 26.
  • the present invention also provides a method for preventing or treating novel coronavirus pneumonia (COVID-19), which is to administer an effective amount of susceptible people or infected patients to novel coronavirus pneumonia (COVID-19).
  • COVID-19 novel coronavirus pneumonia
  • ACE2 fusion protein or pharmaceutical composition as described above.
  • the present invention also provides a method for blocking infection of a new type of coronavirus, which is to administer an effective amount of ACE2 fusion protein as described above or as described above to susceptible people or infected patients with new type of coronavirus pneumonia (COVID-19)
  • the pharmaceutical composition is to administer an effective amount of ACE2 fusion protein as described above or as described above to susceptible people or infected patients with new type of coronavirus pneumonia (COVID-19)
  • COVID-19 coronavirus pneumonia
  • the present invention also provides the use of the aforementioned ACE2 fusion protein or the aforementioned pharmaceutical composition in the preparation of drugs for the prevention or treatment of new coronavirus pneumonia (COVID-19), and the use is to give new coronavirus pneumonia (COVID-19) -19) Susceptible people or infected patients administer an effective amount of the ACE2 fusion protein as described above or the pharmaceutical composition as described above.
  • the present invention also provides the use of the ACE2 fusion protein as described above or the pharmaceutical composition as described above in the preparation of drugs for blocking new coronavirus infections, and the use is to give new coronavirus pneumonia (COVID-19) susceptibility
  • the population or the infected patient is administered an effective amount of the ACE2-Fc fusion protein as described above or the pharmaceutical composition as described above.
  • the half-life of ACE2 recombinant protein is relatively short, and the half-life of hACE2-Fc of the present invention will be significantly prolonged.
  • the Fc fragment can make hACE2-Fc form a dimer through disulfide bonds, which is closer to the conformation of the native ACE2 protein.
  • the hACE2-Fc of the present invention can be combined with the Spike protein on the surface of the novel coronavirus (SARS-CoV-2) so as to inhibit the virus from invading the host cell, and finally achieve the effect of anti-viral infection.
  • SARS-CoV-2 novel coronavirus
  • hACE2-Fc is used for the prevention of close contacts of patients with new coronavirus infection or people with a history of virus exposure.
  • Figure 4A 0.5 ⁇ g/ml ACE2-Fc combined with different concentrations of antibody P17-A11 to block the detection results of virus infection
  • Figure 4B 0.05 ⁇ g/ml antibody P17-A11 respectively to block with different concentrations of ACE2-Fc Virus infection test results.
  • Figure 5A Neutralizing effect of the combined treatment of P17-A11 and ACE2-Fc fusion protein at normal viral load
  • Figure 5B Neutralizing medicine of combined treatment of P17-A11 and ACE2-Fc fusion protein at 5 times the viral load effect.
  • Figure 7A-7B In vivo efficacy of antibody P17-A11+ACE2-Fc fusion protein combination.
  • the present invention provides an ACE2 fusion protein in the form of a dimer, which includes a fusion protein (hACE2-Fc) formed by connecting the extracellular region of the ACE2 protein and the Fc fragment of a human antibody IgG1 antibody.
  • the soluble hACE2-Fc recombinant protein can competitively bind to the Spike protein on the surface of the virus, so that the virus cannot bind to the ACE2 on the cell surface, and ultimately inhibits the virus's invasion of the body. Since it takes a certain incubation period after the virus invades the body, a certain dose of hACE2-Fc may have the effect of suppressing the disease if given to people exposed to coronavirus or high-risk groups in advance. For infected patients, the virus will continue to replicate and expand in the body to infect more cells. After hACE2-Fc treatment, the newly amplified virus will not be able to further infect new cells, thereby inhibiting the deterioration of the disease.
  • antibody is used in its broadest meaning herein, and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), whole Long antibodies and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity.
  • antibody portion refers to a full-length antibody or antigen-binding fragment thereof.
  • neutralizing antibody refers to an antibody capable of blocking the recognition and binding between human ACE2 and the S protein of SARS-CoV-2 virus.
  • a full-length antibody contains two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable domain of the heavy chain and light chain may be referred to "V H" and "V L".
  • the variable region in the two chains usually contains three highly variable loops, called complementarity determining regions (CDR) (including LC-CDR1, LC-CDR2 and LC-CDR3 light chain (LC) CDR (or LCDR) ), including the heavy chain (HC) CDR (or HCDR) of HC-CDR1, HC-CDR2 and HC-CDR3).
  • CDR complementarity determining regions
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the following conventions: Kabat, Chothia or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDRs of the heavy or light chain are inserted between flanking sections called framework regions (FR), which are more highly conserved than the CDRs and form a scaffold that supports the hypervariable loop.
  • FR flanking sections
  • the constant regions of the heavy and light chains do not participate in antigen binding, but exhibit multiple effector functions.
  • Antibodies are classified based on the amino acid sequence of the constant region of the antibody heavy chain.
  • the five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
  • Several major antibody categories are divided into subcategories, such as lgG1 ( ⁇ 1 heavy chain), lgG2 ( ⁇ 2 heavy chain), lgG3 ( ⁇ 3 heavy chain), lgG4 ( ⁇ 4 heavy chain), lgA1 ( ⁇ 1 heavy chain) or lgA2 ( ⁇ 2 heavy chain) chain).
  • antigen-binding fragment refers to antibody fragments, which include, for example, bispecific antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide bond stabilized Fv fragments (dsFv), (dsFv) 2.
  • Multispecific dsFv (dsFv-dsFv'), disulfide bond-stabilized bispecific antibodies (ds bispecific antibodies), single chain Fv (scFv), scFv dimers (bivalent diabodies), the inclusion of antibodies
  • a multispecific antibody a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure formed by part of one or more CDRs .
  • the antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or parent antibody fragment (e.g., parent scFv) binds.
  • the antigen-binding fragment may comprise one or more CDRs from a particular human antibody that are grafted to framework regions from one or more different human antibodies.
  • Fv is the smallest antibody fragment, which contains a complete antigen recognition site and an antigen binding site.
  • the fragment is composed of a dimer of a heavy chain variable region domain and a light chain variable region domain that are tightly non-covalently associated. From the folding of these two domains, six hypervariable loops (each from the 3 loops of the heavy chain and the light chain) are emitted, which contribute amino acid residues for antigen binding and give the antibody specificity for binding to the antigen .
  • six hypervariable loops are emitted, which contribute amino acid residues for antigen binding and give the antibody specificity for binding to the antigen .
  • a single variable domain or a half Fv containing only three CDRs against the original specificity
  • Single-chain an Fv (also abbreviated as “sFv” or “the scFv”) is an antibody fragment and V L, V H antibody domains connected into a single polypeptide chain comprising.
  • the scFv polypeptide further comprises a polypeptide linker between the V H and V L, domain, the polypeptide linker which enables the scFv to form the desired structure for antigen binding.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • variable domain residues as in Kabat or “number of amino acid positions as in Kabat” and variants thereof refer to the heavy chain variable domain or light chain used in the antibody compilation of Kabat et al. above
  • the numbering system for variable domains the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or hypervariable region (HVR) of the variable domain.
  • the heavy chain variable domain may comprise a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (for example, residue according to Kabat 82a, 82b, 82c, etc.).
  • the Kabat numbering of residues in a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbering sequence in the region of homology.
  • amino acid residues encompassing the CDRs of full-length antibodies are defined according to the Kabat nomenclature of Kabat et al. above, and immunoglobulin heavy chains such as Fc
  • the residue numbering in the region is the numbering of the EU index as described by Kabat et al. above, except that the amino acid residues of the CDRs covering any consensus sequence are defined according to the Kabat nomenclature, where the modifications are based on experimental conditions.
  • EU index as described by Kabat refers to the residue numbering of the human IgG1 EU antibody.
  • Framework or "FR” residues are those variable domain residues other than the CDR residues defined herein.
  • Non-human (e.g., rodent) antibodies in "humanized” forms are chimeric antibodies that contain minimal sequences derived from non-human antibodies.
  • humanized antibodies are human immunoglobulins (receptor antibodies), in which residues from the receptor hypervariable region (HVR) are derived from non-human species (e.g., mouse, rat, rabbit, or non-human). (Primates) with the desired antigen specificity, affinity and capacity of the hypervariable region (donor antibody) residue replacement.
  • the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues.
  • humanized antibodies may include residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance.
  • a humanized antibody will include at least one, and typically substantially all of the two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all All the FRs above are those of the human immunoglobulin sequence.
  • the humanized antibody will also optionally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • S protein and “Spike protein” refer to the structural protein "spindle protein” of the novel coronavirus (SARS-CoV-2), which binds to the human cell surface receptor-ACE2 protein to fuse the virus envelope with the cell membrane. Infect cells.
  • SARS-CoV-2 novel coronavirus
  • HACE2-Fc fusion protein refers to a fusion protein formed by linking the extracellular region of human angiotensin converting enzyme 2 (ACE2) with the Fc region of a human IgG antibody.
  • ACE2 angiotensin converting enzyme 2
  • Binding affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (for example, ACE2) and its binding partner (for example, the Spike protein of the SARS-CoV-2 virus).
  • binding affinity refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, receptor and ligand).
  • the affinity of a molecule X to its partner Y can usually be represented by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
  • amino acid sequence of the extracellular region of human ACE2 of the present invention is as follows [SEQ ID NO:1]:
  • the ACE2 protein can also be a natural or functional variant of the entire human ACE2 protein or the extracellular region of the human ACE2 protein.
  • the functional variants of the ACE2 protein may include conservative mutations in the extracellular region of ACE2 shown in SEQ ID NO. 1, and do not lose or weaken the S protein of ACE2 and the new coronavirus (SARS-CoV-2) Affinity.
  • Percentage (%) of amino acid sequence identity with respect to the polypeptide and fusion protein sequences identified herein is defined as the sequence aligned with the candidate sequence (considering any conservative substitutions as part of the sequence identity) The percentage of amino acid residues with the same amino acid residues in the compared polypeptides.
  • the alignment can be achieved in a variety of ways in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithm that needs to achieve the maximum alignment over the full length of the sequence being compared.
  • sequence comparison computer program MUSCLE is used to generate the% value of amino acid sequence identity (Edgar, RC, Nucleic Acids Research [Nucleic Acid Research] 32(5): 1792-1797, 2004; Edgar, RC, BMC Bioinformatics [BMC Bioinformatics] 5(1): 113, 2004).
  • “Homologous” refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When two positions in two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecule is in that position Are homologous.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, comparisons are made when two sequences are aligned to give maximum homology.
  • constant domain refers to a part of an immunoglobulin molecule that has a more conservative amino acid sequence relative to another part of an immunoglobulin, that is, a variable domain, which contains an antigen binding site.
  • C H constant domain of the heavy chain comprises 1, C L domain, and C H C H 2 domain. 3 (collectively referred to as C H) and light chains.
  • Fc region or "Fc fragment” herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc region.
  • Fc region of an immunoglobulin heavy chain is generally defined as an amino acid residue at position Cys226 or extending from Pro230 to its carboxyl terminus.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain.
  • composition of intact antibodies may include an antibody population with all K447 residues removed, an antibody population without K447 residues removed, and an antibody population with a mixture of antibodies with and without K447 residues.
  • Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.
  • amino acid sequence of the Fc fragment is shown in the following amino acid sequence [SEQ ID NO.2]:
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • a preferred FcR is a natural human FcR.
  • a preferred FcR is an FcR that binds IgG antibodies ( ⁇ receptors) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (" Activating receptor") and Fc ⁇ RIIB ("inhibiting receptor”), they have similar amino acid sequences, the main difference lies in their cytoplasmic domains.
  • the activated receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • an “isolated” antibody is a fusion protein that has been identified, isolated, and/or recovered from a component (eg, natural or recombinant) of its production environment.
  • the isolated polypeptide has no or substantially no association with all other components in its production environment.
  • the "isolated" nucleic acid molecule encoding the construct or fusion protein described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule normally associated with it in its production environment. In certain embodiments, the isolated nucleic acid has no or substantially no association with all components related to the production environment.
  • the form of the isolated nucleic acid molecules encoding the polypeptides and fusion proteins described herein is different from the naturally occurring form or background. Therefore, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and fusion proteins described herein that are naturally present in the cell.
  • An isolated nucleic acid includes the nucleic acid molecule contained in a cell that usually contains the nucleic acid molecule, but the nucleic acid molecule exists outside the chromosome or at a chromosomal location different from its natural chromosomal location.
  • control sequence refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a specific host organism.
  • suitable control sequences for prokaryotes include promoters, optional operator sequences, and ribosome binding sites. It is known that eukaryotic cells utilize promoters, polyadenylation signals and enhancers.
  • a nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence.
  • the DNA of the pre-sequence or the secretory leader sequence is expressed as a pre-protein involved in the secretion of the polypeptide, the DNA of the pre-sequence or the secretory leader sequence is operably linked to the DNA of the polypeptide; if the promoter or enhancer affects For transcription of the coding sequence, the promoter or enhancer is operably linked to the sequence; or if the ribosome binding site is positioned so as to facilitate translation, the ribosome binding side is operably linked to the coding sequence.
  • operably linked means that the linked DNA sequences are continuous and, in the case of a secreted leader sequence, are continuous and in reading frame.
  • the enhancer need not be continuous.
  • the connection is achieved by connecting at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.
  • subject refers to mammals, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the subject is a human.
  • the "effective amount” of an agent refers to an amount effective to achieve the desired therapeutic or preventive result within the necessary dose and time period.
  • the specific dose can be changed according to one or more of the following: the specific agent selected, the subsequent dosing regimen (regardless of whether it is combined with other compounds), the time of administration, the tissue that is imaged, and where it is carried Physical delivery system.
  • the "therapeutically effective amount" of the substance/molecule, agonist or antagonist of the present application can be based on, for example, the disease state, age, sex, and individual weight, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual And other factors.
  • the therapeutically effective amount is also the amount at which any toxic or harmful effects of the substance/molecule, agonist or antagonist are offset by the beneficial effects of the treatment.
  • the therapeutically effective amount can be delivered by one or more administrations.
  • prophylactically effective amount refers to an effective amount in a dose meter and for a required period of time to achieve the desired preventive result. Typically, but not necessarily, because the preventive dose is used in the subject before or early in the disease, the preventive effective amount will be less than the therapeutically effective amount.
  • treatment or treating is a method used to obtain beneficial or desired results (including clinical results).
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, reduction of the degree of the disease, and stabilization of the disease (e.g., Prevent or delay the deterioration of the disease), prevent or delay the spread of the disease (for example, metastasis), prevent or delay the recurrence of the disease, delay or slow the progression of the disease, improve the disease state, provide relief (partial or full), and reduce The dosage of one or more other drugs required to treat the disease, delay the progression of the disease, increase or improve the quality of life, increase weight gain and/or prolong survival.
  • Treatment also encompasses reducing the pathological consequences of cancer (like, for example, tumor volume). The method of the application considers any one or more of these therapeutic aspects. “Treatment” does not necessarily mean that the disease being treated will be cured.
  • fusion protein variants with one or more amino acid substitutions are provided.
  • Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions”. More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions” and are described further below with reference to amino acid side chain classes.
  • Amino acid substitutions can be introduced into the fusion protein of interest, and the product screened for the desired activity (e.g. retained/improved binding of the receptor to the ligand).
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val
  • Amino acids can be grouped according to common side chain characteristics: (1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe. In certain embodiments, non-conservative substitutions will require the exchange of members of one of these categories for another category.
  • one or more amino acid modifications may be introduced into the Fc region (e.g., scFv-Fc) of the fusion protein portion, thereby generating Fc region variants.
  • the Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.
  • Fc fragments with some (but not all) effector functions make the fragments an ideal candidate for applications in which the half-life of the fusion protein in vivo is very important. But some effector functions (for example, complement and ADCC) are unnecessary or harmful.
  • In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity.
  • an Fc receptor (FcR) binding assay can be performed to ensure that the antibody has no Fc ⁇ R binding ability (and therefore may lack ADCC activity), but can retain FcRn binding ability.
  • NK cells The primary cells used to mediate ADCC, NK cells, only express FcyRIII, while monocytes express FcyRI, FcyRII, and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, in Table 2 on page 464 of Annu. Rev. Immunol. [Annual Review of Immunology] 9:457-492 (1991).
  • a non-limiting embodiment of an in vitro assay for assessing the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (for example, see Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA [U.S.
  • a non-radioactive assay method can be used (for example, see ACTI TM Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc.) Mountain View, California; and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells.
  • the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models, such as Clynes et al., Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences] 95:652-656 (1998).
  • a C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity.
  • C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods [Journal of Immunological Methods] 202:163 (1996); Cragg, MS et al., Blood [blood] 101: 1045 -1052 (2003); and Cragg, MS and MJ Glennie, Blood [Blood] 103: 2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life determination can also be performed using methods known in the art (for example, see: Petkova, SB et al., Int'l. Immunol. [International Immunology] 18(12): 1759-1769 (2006) )).
  • any available or known technique in the art can be used to produce the antibody Fc region-containing fusion proteins disclosed herein.
  • recombinant methods and compositions can be used to produce fusion proteins containing the Fc region of an antibody, for example, as described in U.S. Patent No. 4,816,567. The detailed procedures for antibody production are described in the following examples.
  • the subject of the present invention also provides an isolated nucleic acid encoding the antibody Fc region-containing fusion protein disclosed herein.
  • the nucleic acid may be present in one or more vectors (e.g., expression vectors).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vector expression vectors can direct the expression of genes to which they are operably linked.
  • expression vectors used in recombinant DNA technology are often in the form of plasmids (vectors).
  • the disclosed subject matter is intended to include other forms of expression vectors with equivalent functions, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).
  • the different portions of the antibodies disclosed herein can be constructed in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors.
  • Embodiments of the elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, for example, FGF-1 IRES, FGF-2IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1IRES, p53IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornavirus IRES, polio virus IRES and encephalomyocarditis virus IRES) and cleavable linkers ( For example 2A peptides, such as P2A, T2A, E2A and F2A peptides).
  • Combinations of retroviral vectors and suitable packaging lines are also suitable, where the capsid protein will have the function of infecting human cells.
  • a variety of cell lines producing amphoteric viruses are known, including but not limited to PA12 (Miller et al. (1985) Mol. Cell. Biol. [Molecular Cell Biology] 5:431-437); PA317 (Miller et al. (1986) Mol Cell. Biol. [Molecular Cell Biology] 6: 2895-2902); and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 85: 6460-6464).
  • Non-amphiphilic particles are also suitable, such as coating with VSVG, RD114 or GALV and any other pseudotyped particles known in the art.
  • the nucleic acid encoding the antibody of the present invention and/or one or more vectors including the nucleic acid may be introduced into the host cell.
  • nucleic acids can be introduced into cells by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with viruses or phage vectors containing nucleic acid sequences, and cells Fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • the host cell may include, for example, a host cell that has been transformed with a vector comprising a nucleic acid encoding an amino acid sequence comprising a hACE2-Fc fusion protein.
  • the host cell is eukaryotic, such as Chinese Hamster Ovary (CHO) cells or lymphoid cells (eg, YO, NSO, Sp20 cells).
  • the method for preparing the fusion protein disclosed herein may include culturing the host cell into which the nucleic acid encoding the fusion protein has been introduced under conditions suitable for protein expression, and optionally from the host cell and/or host
  • the fusion protein is recovered from the cell culture medium.
  • the fusion protein is recovered from the host cell by chromatographic techniques.
  • the nucleic acid encoding the fusion protein as described above can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • Such nucleic acids can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding genes encoding the heavy and light chains of the fusion protein).
  • Suitable host cells for cloning or expressing the vector encoding the fusion protein include prokaryotic or eukaryotic cells as described herein.
  • fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector functions are not required.
  • vertebrate cells can also be used as hosts.
  • mammalian cell lines suitable for growth in suspension may be useful.
  • a non-limiting embodiment of a useful mammalian host cell line is the monkey kidney CV1 line transformed by SY40 (COS-7); the human embryonic kidney line (293 or 293 cells, as for example in Graham et al., J Gen Viral. [ J. General Virology] 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli (sertoli) cells (TM4 cells, for example in Mather, Biol. Reprod.
  • CHO Chinese Hamster Ovary
  • DHFK CHO cells Urlaub et al., Proc. Natl. Acad.Sci.USA [Proceedings of the National Academy of Sciences] 77:42I6 (1980)
  • myeloma cell lines such as YO, NSO, and Sp2/0.
  • the subject of the present invention further provides a method of using the disclosed fusion protein.
  • these methods involve the therapeutic use of the currently disclosed fusion protein.
  • the present invention provides the use of the hACE2-Fc fusion protein disclosed herein for preventing or treating diseases and disorders or for preparing drugs for preventing or treating diseases.
  • diseases and conditions that can be treated by the fusion protein disclosed herein include, but are not limited to, novel coronavirus pneumonia (COVID-19).
  • the fusion protein provided herein will be administered at a dose effective to treat the indication while minimizing toxicity and side effects.
  • a typical dosage may be, for example, in the range of 0.001 to 1000 ⁇ g; however, dosages lower or higher than this exemplary range are within the scope of the present invention.
  • the daily dose may be about 0.1 ⁇ g/kg to about 100 mg/kg of total body weight, about 0.1 ⁇ g/kg to about 100 ⁇ g/kg of total body weight, or about 1 ⁇ g/kg to about 100 ⁇ g/kg of total body weight.
  • the efficacy of treatment or prevention can be monitored by regularly evaluating treated patients. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs.
  • the desired dose can be delivered by administering the composition by a single bolus injection, by administering the composition by multiple bolus injections, or by administering the composition by continuous infusion.
  • the product may include (a) a first container containing a composition, wherein the composition contains the fusion protein of the present invention; and (b) a second container containing A composition, wherein the composition comprises other cytotoxic or therapeutic agents.
  • the article of manufacture may further comprise a package insert, which indicates that the composition can be used to treat a particular condition.
  • the product may further include another container, such as a second or third container, which includes a pharmaceutically acceptable buffer, such as but not limited to bacteriostatic water for injection (BWFI) or physiological saline.
  • a pharmaceutically acceptable buffer such as but not limited to bacteriostatic water for injection (BWFI) or physiological saline.
  • BWFI bacteriostatic water for injection
  • the product may include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.
  • the anti-S antibody (for example, an anti-S protein full-length antibody or an antigen-binding fragment thereof) can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the constant region of the antibody. For example, protein A, protein G, protein A/G, or antibody affinity columns can be used to bind to the constant region and purify anti-S protein antibodies containing Fc fragments. Hydrophobic interaction chromatography, such as butyl or phenyl columns, can also be used to purify certain polypeptides, such as antibodies.
  • Ion exchange chromatography may also be suitable for purifying certain polypeptides, such as antibodies.
  • Mixed mode chromatography e.g. reverse phase/anion exchange, reverse phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.
  • Many methods of purifying polypeptides are known in the art.
  • the present invention also unexpectedly discovered that the ACE2-Fc fusion protein and the anti-S protein neutralizing antibody of the new coronavirus have Synergistically block the combination of new coronavirus and human ACE2, and inhibit viral infection. Provide new and more powerful methods for the prevention and treatment of the new coronavirus.
  • the nucleic acid encoding hACE2-Fc fusion protein and ACE2-His protein were cloned and expressed. According to the amino acid sequence, it was constructed on the pAS-Pruo expression vector after codon optimization according to the expression host CHO-S cells. Construct a stable transgenic CHO-S cell line for protein expression.
  • the culture supernatant expressing the fusion protein is centrifuged at high speed, and the supernatant is collected and filtered with a 0.22um filter membrane for use.
  • Wash the Protein A affinity column (3-5 times the column volume) with 0.1M NaOH, and then wash the affinity column with 1 ⁇ PBS. The washing time is 5 times the column volume.
  • Use the loading balance solution (PBS pH 7.4) to equilibrate the affinity column with 3-5 times the column volume and start loading. Control the flow rate to ensure that the retention time is above 1 min.
  • the sample was eluted with 0.1M glycine hydrochloride pH 3.4 buffer, and the eluted product was collected according to the ultraviolet absorption peak. After the elution is completed, use 1M Tris-HCl (pH 8.0) to quickly adjust the pH of the eluted product to 5.5 for temporary storage, and then replace it with other buffer systems by means of ultrafiltration or dialysis as needed.
  • 1M Tris-HCl pH 8.0
  • amino acid sequences of the prepared hACE2-Fc fusion protein, ACE2-His protein, and the signal peptide sequence used in cloning and expression are as follows:
  • the body part is the extracellular region of human ACE2, and the underlined part is the Fc fragment of human IgG1.
  • the specific molecules of ACE2-Fc used in the examples herein are all ACE2-Fc fusion proteins shown in SEQ ID NO. 4. Except for specific limitations, the examples are not a limitation on the rights of the present disclosure.
  • the body part is the extracellular region of human ACE2, and the underlined part is the His tag
  • RBD-Fc (Yiqiao Shenzhou, 40592-V05H, where RBD is the new coronavirus (SARS-CoV-2) surface spike glycoprotein (Spike protein) receptor-binding domain, RBD)) coated 96-well plate overnight at 4°C, washed with PBST three times and then blocked with PBS containing 5% bovine serum albumin (BSA) for 1 hour at room temperature. Wash with PBST three times, mix 50ng/ml ACE2-His with gradient dilution hACE2-Fc and add to 96-well plate at the same time and incubate at room temperature for 1h. After washing with PBST, add anti-his-HRP and incubate at room temperature for 30 min. After washing with PBST five times, add the substrate for color development. After adding the stop solution, read with a microplate reader (TECAN Spark).
  • SARS-CoV-2 surface spike glycoprotein receptor-binding domain
  • PEI polyethyleneimine
  • HEK-293T HEK-293T overexpressing hACE2 was obtained for use. Trypsin digest hACE2-293T, wash with PBS for 2 times, centrifuge to remove the supernatant, mix 0.1 ⁇ g/ml RBD-Fc with serially diluted hACE2-Fc recombinant protein and add to hACE2-293T, resuspend the cells and incubate on ice After washing with PBS for 2 times, add PE-labeled anti-hFc antibody and incubate on ice for 30 minutes. After washing with PBS, resuspend the cells and measure the fluorescence intensity of the cells with a flow cytometer (Beckman CytoFLEX).
  • Anti-S protein antibodies such as P17-A11 were prepared according to the method described in Chinese patent application CN202010236256.8. All the contents in the above-mentioned patent application are simultaneously incorporated into this application.
  • Vero-E6 one day in advance ( CRL-1586 TM ) cells in a 96-well plate, 1 ⁇ 10 4 cells per well (note that the edge of the 96-well plate is not used as an experimental well, and PBS is added to prevent the medium from other wells from evaporating);
  • the half-toxic concentration of protein to cells is shown in the table below.
  • Example 7 Evaluation of the effect of anti-SARS-CoV-2 S protein antibody and ACE2-Fc fusion protein in inhibiting SARS-CoV-2 virus replication in Vero-E6 cell model
  • the antiviral activity was determined on the Vero-E6 cell model, and each experiment was set up with 4 multiple holes, which were repeated 3 times in total.
  • ACE2-Fc concentration is set to: 1.0, 0.5, 0.25, 0.125, 0.0625, 0.03125 ⁇ g/ml
  • P17-A11 concentration is set to: 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.0015625 ⁇ g/ml
  • the control group is set at the same time (No drug group), the supernatant virus liquid was collected 24h after infection.
  • qRT-PCR real-time RT-PCR
  • Inhibition rate (%) 1-the number of viral RNA copies of the experimental group/the number of viral RNA copies of the drug-free group ⁇ 100%.
  • GraphPad PrisM6.0 software was used to analyze and calculate the half effective concentration (EC50) and 90% effective concentration (EC90) of the drug to inhibit SARS-CoV-2 virus.
  • Flow cytometry was used to detect the effects of ACE2-Fc and anti-S protein alone and in combination to block SARS-CoV-2 infection.
  • ACE2-Fc 0.5 ⁇ g/ml ACE2-Fc was mixed with different concentrations of antibody P17-A11, and then 0.1 ⁇ g/ml RBD-Fc was added to pre-incubate for 20 minutes, and then added to 293-hACE2 cell suspension and incubated at room temperature for 30 minutes. After washing the cells with PBS, PE-labeled anti-Fc antibody was added to detect the content of RBD-Fc on the cell membrane, and the blocking efficiency was calculated. See Figure 4A for the results.
  • the 0.05 ⁇ g/ml antibody P17-A11 was mixed with different concentrations of ACE2-Fc, and 0.1 ⁇ g/ml RBD-Fc was added to pre-incubate for 20min, and then added to 293-hACE2 cell suspension and incubated at room temperature for 30min. After washing the cells with PBS, PE-labeled anti-Fc antibody was added to detect the content of RBD-Fc on the cell membrane, and the blocking efficiency was calculated. See Figure 4B for the results.
  • the P17-A11 and ACE2-Fc fusion protein in a ratio of 1:5, 1:10, and 1:30, then use the cell maintenance solution to make a 5-fold gradient dilution, and then mix with an equal volume of the new coronavirus (virus strain : BetaCoV/Beijing/IMEBJ01/2020, the amount of virus added should ensure that the number of plaques in the positive control group is 50-100/well), incubate at 37°C for 1 hour; add the virus-antibody mixture (200 ⁇ L/well) to the In a 24-well culture plate with a single layer of dense Vero cells, incubate at 37°C for 1 hour, shaking gently several times during this period; discard the virus-antibody mixture, add an appropriate volume of preheated nutrient agar to each well, and incubate at 37°C with 5% CO 2 Continue to incubate in the box, add an appropriate volume of fixative on the second day after infection, fix at room temperature for 1 hour, discard the fixative and nutrient
  • mice 6-8 weeks old female hACE2-IRES-luc transgenic mice (source: Shanghai Southern Model Biotechnology Co., Ltd.) were randomly divided into 3 groups, and they were nasally infected with a lethal dose of SARS-CoV-2 virus (nCoV-SH01). , GenBank: MT121215.1). Two hours later, a single dose of ACE2-Fc fusion protein (15 and 50 mg/kg) or PBS control was injected into the intraperitoneal cavity of virus-infected transgenic mice. The survival rate of the mice was recorded every day for 5 days.

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Abstract

L'invention concerne une protéine de fusion formée au moyen de la liaison d'une région extracellulaire ACE2 avec un fragment polypeptidique capable de dimériser l'ACE2, de préférence par liaison d'un fragment Fc d'un anticorps IgG1 humain avec une région extracellulaire ACE2 humaine. La protéine peut être combinée avec RBD, et peut simultanément inhiber la combinaison entre les niveaux moléculaires et cellulaires de RBD et d'une protéine ACE2. La protéine de fusion peut être utilisée pour prévenir et traiter une infection par le nouveau coronavirus (SRAS-CoV-2).
PCT/CN2021/088681 2020-04-23 2021-04-21 Protéine de fusion ace2-fc et son utilisation Ceased WO2021213437A1 (fr)

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CN202010325666.XA CN113549154A (zh) 2020-04-23 2020-04-23 ACE2-Fc融合蛋白及其应用
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CN202010528750.1A CN113801865A (zh) 2020-06-11 2020-06-11 ACE2-Fc融合蛋白及其应用
CN202010528750.1 2020-06-11
CN202010745204.3 2020-07-29
CN202010745204.3A CN114057887A (zh) 2020-07-29 2020-07-29 ACE2-Fc融合蛋白及其应用
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CN114740199A (zh) * 2022-03-18 2022-07-12 北京安奇生物医药科技有限公司 一种SARS-CoV-2中和抗体试剂盒及其应用
WO2022184854A2 (fr) 2021-03-03 2022-09-09 Formycon Ag Formulations de protéines de fusion ace2 fc
CN115521928A (zh) * 2022-05-20 2022-12-27 中山大学·深圳 Ace2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用
CN115991779A (zh) * 2022-11-18 2023-04-21 四川省医学科学院·四川省人民医院 一种抗人ace2的抗体、检测试剂盒及其应用
WO2023081958A1 (fr) * 2021-11-11 2023-05-19 The Macfarlane Burnet Institute For Medical Research And Public Health Ltd Agent antiviral comprenant un récepteur d'entrée cellulaire et un composant de la région fc
WO2023094507A1 (fr) 2021-11-24 2023-06-01 Formycon Ag Protéines de fusion ace2 améliorées
EP4331571A1 (fr) 2022-09-02 2024-03-06 Formycon AG Formulations de protéines de fusion ace2-igm
EP4386084A1 (fr) 2022-12-14 2024-06-19 Formycon AG Protéines de fusion ace2 améliorées

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EP4331571A1 (fr) 2022-09-02 2024-03-06 Formycon AG Formulations de protéines de fusion ace2-igm
CN115991779A (zh) * 2022-11-18 2023-04-21 四川省医学科学院·四川省人民医院 一种抗人ace2的抗体、检测试剂盒及其应用
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EP4386084A1 (fr) 2022-12-14 2024-06-19 Formycon AG Protéines de fusion ace2 améliorées

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