WO2021213437A1 - Ace2-fc fusion protein and use thereof - Google Patents

Abstract

Provided is a fusion protein formed by means of connecting an ACE2 extracellular region with a polypeptide fragment capable of dimerizing ACE2, preferably by means of connecting an Fc fragment of a human IgG1 antibody with a human ACE2 extracellular region. The protein can be combined with RBD, and can simultaneously inhibit the combination between the molecular and cellular levels of RBD and an ACE2 protein. The fusion protein can be used for preventing and treating infection with the novel coronavirus (SARS-CoV-2).

Description

ACE2-Fc fusion protein and its application Technical field

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.

Background technique

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.

At present, 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.

In addition to performing its enzymatic function as an enzyme in the body, ACE2 is also a key receptor for some viruses to invade the body. Studies have shown that the 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. However, there are currently no specific drugs to treat the new coronavirus pneumonia (COVID-19). It is necessary to find drugs that can effectively treat the new coronavirus (SARS-CoV-2) as soon as possible.

Summary of the invention

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.

In some aspects, 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.

In some aspects, 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.

In some aspects, 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.

In some aspects, 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.

In some aspects, the pharmaceutical composition as described above further comprises a neutralizing antibody against the S protein of the novel coronavirus (SARS-CoV-2).

In some aspects, 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:

a) 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

b) 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.

In some aspects, 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:

c) 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

d) 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.

In some aspects, 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:

e) 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

f) 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.

In some aspects, 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.

In some aspects, 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:

g) 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

h) 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). 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.

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.

Generally, 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.

hACE2-Fc is used for the prevention of close contacts of patients with new coronavirus infection or people with a history of virus exposure.

Description of the drawings

Figure 1. Binding affinity results of hACE2-Fc and RBD-His

Figure 2. The blocking effect of hACE2-Fc on the binding of RBD-Fc to ACE2-His

Figure 3. The blocking effect of hACE2-Fc on the binding of RBD-Fc to the full-length ACE2 on the cell membrane

Figure 4. Figure 4A: 0.5μg/ml ACE2-Fc combined with different concentrations of antibody P17-A11 to block the detection results of virus infection; Figure 4B: 0.05μg/ml antibody P17-A11 respectively to block with different concentrations of ACE2-Fc Virus infection test results.

Figure 5. 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 6. Survival curve of SARS-CoV-2 infected mice treated with ACE2-Fc fusion protein.

Figure 7A-7B. In vivo efficacy of antibody P17-A11+ACE2-Fc fusion protein combination.

Detailed ways

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.

definition

The term "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. The term "antibody portion" refers to a full-length antibody or antigen-binding fragment thereof.

As used herein, "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). 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. 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 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain) or lgA2 (α2 heavy chain) chain).

The term "antigen-binding fragment" as used herein 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. In some embodiments, 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 . However, even a single variable domain (or a half Fv containing only three CDRs against the original specificity) has the ability to recognize and bind to an antigen, although usually with a lower affinity than the complete binding site.

"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. In some embodiments, 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. For a review of scFv, see Plückthun's The Pharmacology of Monoclonal Antibodies, Volume 113, Rosenburg and Moore eds Springer-Verlag [Springer Press], New York, pp. 269-315 ( 1994).

As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen binding sites found in the variable regions of heavy and light chain polypeptides. These specific regions have been described in: Kabat et al., J. Biol. Chem. [Biochemistry Journal], 252: 6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest [Protein sequence of immunological significance]” (1991); Chothia et al., J.Mol. Biol. [Journal of Molecular Biology] 196:901-917 (1987); Al-Lazikani B. et al., J. Mol .Biol. [Journal of Molecular Biology], 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. [Journal of Molecular Biology] 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol. [Molecular Immunology], 45: 3832-3839 (2008); Lefranc MP et al., Dev. Comp. Immunol. [Development and Comparative Immunology], 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol. [Journal of Molecular Biology], 309: 657-670 (2001), where the definition includes the overlap or subset of amino acid residues when compared to each other. However, application of either definition to refer to the CDR of an antibody or a grafted antibody or variant thereof is intended to fall within the scope of the term as defined and used herein. The amino acid residues covering the CDRs as defined in each of the above references are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol. [Molecular Immunology], 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res. [Nucleic Acid Research] ], 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res. [Nucleic Acid Research], 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more of the claims herein.

In addition to the CDRs determined by the ABM rules of antibodies, there are also a variety of conventional CDR determination methods for antibodies, as shown in the following table.

Table 1: CDR definition

	Kabat 1	Chothia 2	MacCallum 3	IMGT 4	AHo 5
V H CDR1	31-35	26-32	30-35	27-38	25-40
V H CDR2	50-65	53-55	47-58	56-65	58-77
V H CDR3	95-102	96-101	93-101	105-117	109-137
V L CDR1	24-34	26-32	30-36	27-38	25-40
V L CDR2	50-56	50-52	46-55	56-65	58-77
V L CDR3	89-97	91-96	89-96	105-117	109-137

¹ Residue numbering follows the nomenclature of Kabat et al. (ibid.).

² residue numbering follows the nomenclature of Chothia et al (supra).

³ Residue numbering follows the nomenclature of MacCallum et al. (ibid.).

⁴ Residue numbering follows the nomenclature of Lefranc et al. (ibid.).

⁵ Residue numbering follows Honegger and Plückthun's nomenclature (ibid.).

The expression "number of 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. Using this numbering system, 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. For example, 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.

Unless otherwise stated herein, the amino acid residues encompassing the CDRs of full-length antibodies (e.g., anti-S protein antibodies disclosed herein) 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. In most cases, 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. In some embodiments, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, 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. For further details, see Jones et al., Nature [Nature], 321:522-525, (1986); Riechmann et al., Nature [Nature], 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. [State of Biology] 2:593-596 (1992).

"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.

"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.

"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). Unless otherwise indicated, as used herein, "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.

The non-limiting embodiment of the 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" or "homology" 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. For the purpose of determining the percentage of amino acid sequence identity, 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. However, for the purpose of this article, the 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.

The term "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.

The term "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. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG 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. Therefore, the 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.

A non-limiting example of the 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. In addition, a preferred FcR is an FcR that binds IgG antibodies (γ receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and spliced forms of these receptors. FcγRII receptors include FcγRIIA (" Activating receptor") and FcγRIIB ("inhibiting receptor"), they have similar amino acid sequences, the main difference lies in their cytoplasmic domains. The activated receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M.

Annu. Rev. Immunol. [Annual Review of Immunology] 15:203-234 (1997). FcR is reviewed in Ravetch and Kinet, Annu. Rev. Immunol. [Annual Review of Immunology], 9:457-92 (1991); Capel et al., Immunomethods [Immunomethods] 4:25-34 (1994); and de Haas Et al., J.Lab.Clin.Med. [Journal of Experimental and Clinical Medicine] 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including FcRs that will be identified in the future.

An "isolated" antibody (or construct) is a fusion protein that has been identified, isolated, and/or recovered from a component (eg, natural or recombinant) of its production environment. In certain embodiments, 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.

The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a specific host organism. For example, 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. For example, if 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. Generally, "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. However, 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.

The terms "subject", "individual" and "patient" are used interchangeably herein and refer 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.

As used herein, "treatment or treating" is a method used to obtain beneficial or desired results (including clinical results). For the purposes of this application, 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.

It should be understood that the examples of the application described herein include "consisting of" and/or "essentially consisting of".

Substitution, insertion and deletion variants

In certain embodiments, 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).

Table 2. Amino acid substitutions

Original residue	Exemplary substitution	Preferred substitution
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; Ser	Ser
Trp(W)	Tyr; Phe	Tyr
Tyr(Y)	Trp; Phe; Thr; Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala; Norleucine	Leu

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.

Fc region variants

In certain embodiments, 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.

In certain embodiments, Fc fragments with some (but not all) effector functions, such 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. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody has no FcγR binding ability (and therefore may lack ADCC activity), but can retain FcRn binding ability. 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. Proceedings of the National Academy of Sciences], 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences], 82:1499-1502 (1985); 5,821,337 (See Bruggemann, M. et al., J. Exp. Med. [Journal of Experimental Medicine], 166: 1351-1361 (1987)). Alternatively, 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. Alternatively or additionally, 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. For example, see C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, 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) )).

Method for producing fusion protein containing antibody Fc region

Any available or known technique in the art can be used to produce the antibody Fc region-containing fusion proteins disclosed herein. For example, but not limited to, 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.

In certain embodiments, the nucleic acid may be present in one or more vectors (e.g., expression vectors). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be joined. Another type of vector is a 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). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell's genome after being introduced into the host cell, and thus replicate along with the host's genome. In addition, certain vector expression vectors can direct the expression of genes to which they are operably linked. Generally speaking, expression vectors used in recombinant DNA technology are often in the form of plasmids (vectors). However, 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.

In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, the host cell is eukaryotic, such as Chinese Hamster Ovary (CHO) cells or lymphoid cells (eg, YO, NSO, Sp20 cells).

In certain embodiments, 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. In certain embodiments, the fusion protein is recovered from the host cell by chromatographic techniques.

In order to recombinantly produce the fusion protein of the present invention, 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. For example, fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expressing fusion proteins in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Volume 248 (BKCLo, editor, Human Press, Totowa, New Jersey (NJ), 2003) , Page 245-254, describes the expression of the fusion protein fragment in E. coli) after expression, the soluble fraction of the fusion protein can be separated from the bacterial cell paste and can be further purified.

In certain embodiments, vertebrate cells can also be used as hosts. For example, but not limited to, 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. [Reproductive Biology] 23 : 243-251 (described in 1980)); monkey kidney cells (CV 1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo rat) Hepatocytes (BRL 3A); Human lung cells (W138); Human hepatocytes (Hep 02); Mouse breast tumors (MMT 060562); TRI cells, such as in Mather et al., Annals NYAcad. Sci. [Annual of the New York Academy of Sciences ] 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc. Natl. Acad.Sci.USA [Proceedings of the National Academy of Sciences] 77:42I6 (1980)); and myeloma cell lines (such as YO, NSO, and Sp2/0). A review of some mammalian host cell lines suitable for antibody production , See, for example, Yazaki and Wu, Methods in Molecular Biology, Volume 248 (Edited by BKCLo, Human Press, Totowa, New Jersey (NJ)) , Pages 255-268 (2003).

The subject of the present invention further provides a method of using the disclosed fusion protein. In certain embodiments, these methods involve the therapeutic use of the currently disclosed fusion protein.

treatment method

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. In certain embodiments, diseases and conditions that can be treated by the fusion protein disclosed herein include, but are not limited to, novel coronavirus pneumonia (COVID-19).

Depending on the indication to be treated and factors related to administration familiar to those skilled in the art, the fusion protein provided herein will be administered at a dose effective to treat the indication while minimizing toxicity and side effects. For the treatment of novel coronavirus pneumonia (COVID-19), a typical dosage may be, for example, in the range of 0.001 to 1000 μg; 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 μg/kg to about 100 mg/kg of total body weight, about 0.1 μg/kg to about 100 μg/kg of total body weight, or about 1 μg/kg to about 100 μg/kg of total body weight. As mentioned above, 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. However, other dosage regimens may be useful and within the scope of the invention. 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.

In certain embodiments, 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. In certain embodiments, the article of manufacture may further comprise a package insert, which indicates that the composition can be used to treat a particular condition.

Alternatively or in addition, 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. The product may include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

Purification of the antibody part

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 (e.g., anion exchange chromatography and/or cation 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.) can also be applied to purify certain polypeptides, such as antibodies. Many methods of purifying polypeptides are known in the art.

In addition to the disclosure of a new ACE2-Fc fusion protein capable of inhibiting the binding of the new coronavirus to human ACE-2 and viral infection, 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 following embodiments are only illustrations of the currently disclosed subject matter, and should not be regarded as limiting in any way.

Example

Example 1 Preparation of ACE2-Fc fusion protein and ACE2 protein

Plasmid construction and protein expression

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.

Purification of fusion protein

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. After loading, wash the affinity column with pH 7.4 PBS until The UV absorption fell back to the baseline level. 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.

The 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:

>Signal peptide sequence [SEQ ID NO.3]: MGWSLILLFLVAVATRVHS

>hACE2-Fc fusion protein amino acid sequence (fusion protein of human ACE2 extracellular domain (ECD) and human IgG1 Fc fragment) [SEQ ID NO.4]:

Note: 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.

>ACE2-His[SEQ ID NO.5]:

Note: The body part is the extracellular region of human ACE2, and the underlined part is the His tag

>ACE2 full-length protein amino acid sequence [SEQ ID NO.6]

Example 2 Detection of binding affinity between hACE2-Fc and RBD

100μl 2μg/ml RBD-His (Nearshore Biologics Catalog Number: DRA42) was added to 96-well plate and coated overnight at 4℃, washed with PBST three times and then washed with 5% bovine serum albumin (BSA) in 1×PBS (pH 7.4) Seal at room temperature for 1h. Wash with PBST three times, add 100 μl of hACE2-Fc recombinant protein with an initial concentration of 20 μg/ml and incubate at room temperature for 1 hour. Wash with PBST three times, add Anti-hFc-HRP and incubate at room temperature for 30 min, wash with PBST five times and start to develop color. After adding stop solution, use a microplate reader (TECAN Spark) to detect the absorbance value.

After testing, it was determined that the affinity of hACE2-Fc to RBD-His was 0.54 nM, which showed a strong affinity to RBD-Fc (Figure 1).

Example 3 Detection of the blocking effect of hACE2-Fc on the binding of RBD and ACE2-His

Method: 100μl 2μg/ml 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).

After testing, it was determined that the IC50 of hACE2-Fc blocking the binding of ACE2-His to RBD-Fc was 5.02 nM, showing that hACE2-Fc can block the binding of ACE2-His to RBD-Fc very well (Figure 2).

Example 4 Detection of the blocking effect of hACE2-Fc on the binding of RBD to ACE2 on the cell membrane

Method: According to the manufacturer's instructions, use PEI to transfer a plasmid containing the full length of hACE2 (vector is pAS-Puro) into HEK-293T cells for use. Specific method: Inoculate HEK-293T into a 6-well plate, when the cell density grows to about 70%, according to the instructions of polyethyleneimine (PEI), the ratio of DNA:PEI is 1:3, and the amount of plasmid per well is 4μg Transfect the cells and change the medium 8h after transfection. After culturing in a 37°C, 5% carbon dioxide incubator for 24 hours, 4μg/ml puromycin was added for resistance screening, and HEK-293T (hACE2-293T) overexpressing hACE2 was obtained for use. Trypsin digest hACE2-293T, wash with PBS for 2 times, centrifuge to remove the supernatant, mix 0.1μg/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).

After testing, it was determined that the IC50 of hACE2-Fc blocking the binding of ACE2 expressed on the cell membrane to RBD-Fc was 7.6nM, showing that hACE2-Fc can block the binding of ACE2 expressed on the cell membrane to RBD-Fc very well. (image 3).

Example 5 Anti-SARS-CoV-2 S protein neutralizing antibody

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.

The relevant amino acid sequences of antibodies P17-A11 and P16-A3 are shown below:

>P16-A3 VL[SEQ ID NO.7]:

>P16-A3 VH[SEQ ID NO.8]:

>P17-A11 VL[SEQ ID NO.9]:

>P17-A11 VH[SEQ ID NO.10]:

Table 3 CDR sequences of anti-S protein antibodies determined according to ABM rules

>P16-A3 heavy chain [SEQ ID NO.23]:

>P16-A3 light chain [SEQ ID NO.24]:

>P17-A11 heavy chain [SEQ ID NO.25]:

>P17-A11 light chain [SEQ ID NO.26]:

Example 6 Study on the cytotoxicity of anti-SARS-CoV-2 S protein antibody and ACE2-Fc fusion protein

1) Inoculate 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);

2) Observe the cell status. When the cell confluence reaches about 50%, dilute ACE2-Fc and P17-A11 with 2% FBS-containing DMEM medium (ACE2-Fc concentration is set to: 16, 8, 4). , 2, 1, 0.5, 0.25, 0.125, 0.0625μg/ml; P17-A11 concentration is set to: 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125μg/ml);

3) Add 100μl/well to the cell plate with 4 replicates for each concentration; set the control group (without drug group) and the blank group (without cell group) at the same time, and place them at 37°C, 5% CO ₂ Cultivation in an incubator;

4) 48h after adding the drug, directly add 1/10 volume of Cell Counting Kit-8 (CCK-8) to the cell culture medium, mix well, but avoid air bubbles. Incubate in a 37°C incubator for 1 hour until the color turns orange. Set the blank group to zero, measure the absorbance at 450nm with a multifunctional microplate reader, and calculate it according to the following formula: survival rate (%) = OD450 of the drug-added group/OD450 of the control group×100%. At the same time, calculate the half-toxic concentration (CC50) of the drug.

The half-toxic concentration of protein to cells is shown in the table below.

Table 4. The half toxic concentration of ACE2-Fc and P17-A11 on Vero-E6 cells

The results showed (see Table 4) that the ACE2-Fc fusion protein had very low toxicity to Vero-E6 cells, and the antibody P17-A11 and the Vero-E6 cell model did not detect cytotoxicity.

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.

^{1) Inoculate 8×10 4} Vero-E6 cells in each well of a 24-well cell culture plate. Under 37℃, 5% CO ₂ culture conditions, when the confluence reaches 70%～80%, use ACE2-Fc DMEM medium containing 2% FBS was diluted by a factor of two, discarding the medium in the wells, and adding 1 ml of SARS-CoV-2 virus liquid (according to the multiplicity of infection MOI as 0.005) and the corresponding concentration of drug DMEM medium to each well. (ACE2-Fc concentration is set to: 1.0, 0.5, 0.25, 0.125, 0.0625, 0.03125μg/ml; P17-A11 concentration is set to: 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.0015625μg/ml), and the control group is set at the same time (No drug group), the supernatant virus liquid was collected 24h after infection.

2) Use real-time RT-PCR (qRT-PCR) to quantify RNA of the collected virus:

Take 200 μl of the collected supernatant virus solution, and perform RNA extraction according to the QIAamp viral RNA minikit kit instructions. QRT-PCR detection (Taqman probe method) was performed with the new coronavirus nucleic acid detection (fluorescence quantitative PCR) kit.

3) Calculate the drug inhibition rate at each concentration. 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%. At the same time, 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.

Table 5. Different concentrations of ACE2-Fc fusion protein and P17-A11 inhibit SARS-CoV-2 virus

_{Table 6. The half effective concentration (EC 50} ) and 90% effective concentration (EC ₉₀ ) of ACE2-Fc and P17-A11 to inhibit SARS-CoV-2 virus replication in the Vero-E6 cell model

The results show that P17-A11 and ACE2-Fc have strong inhibitory activity against the new coronavirus.

Example 8 Evaluation of the effect of anti-SARS-CoV-2 S protein antibody and ACE2-Fc fusion protein in inhibiting 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.

A. 0.5μg/ml ACE2-Fc was mixed with different concentrations of antibody P17-A11, and then 0.1μg/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.

B. The 0.05μg/ml antibody P17-A11 was mixed with different concentrations of ACE2-Fc, and 0.1μg/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 results show that when the antibody P17-A11 against the S protein of SARS-CoV-2 and the ACE2-Fc fusion protein are used in combination, they can synergistically block the infection of cells by the virus more effectively.

Example 9 Study on the combination of neutralizing antibody P17-A11 and ACE2-Fc fusion protein to inhibit SARS-CoV-2 infection of Vero cells

Mix 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 (200μL/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 agar, wash with fixative once; add an appropriate volume of 1% crystal violet solution, stain at room temperature 1 After hours, discard the crystal violet solution, wash with fixative once, and count the number of spots. And calculate the inhibition rate according to the formula (inhibition rate=(1-antibody group/control group)*100%). The experimental results (see Figure 5A and Table 7) show that the combination of P17-A11 and ACE2-Fc fusion protein in different proportions can significantly improve the antiviral effect and has a significant synergistic effect. When the combination ratio is 1:5, the neutralizing activity is EC50=0.001 nM, which is 7 times higher than that of the P17-A11 single treatment group.

In order to further evaluate the virus neutralization activity of the P17-A11 and ACE2-Fc fusion protein, we increased the amount of virus in the infected cells by 5 times, and then studied the virus neutralization effect of different ratios of P17-A11 and ACE2-Fc combined treatment. The results (see Figure 5B and Table 7) show that P17-A11 and ACE2-Fc fusion protein still have a significant synergistic effect when the virus concentration is increased by 5 times, and the synergy is strongest when the ratio is 1:5, EC50=0.41 nM. Compared with the antibody P17-A11 treatment alone, the neutralizing activity increased about 3 times.

Table 7 The half effective concentration of P17-A11 and ACE2-Fc fusion protein combined treatment under different viral load conditions

Example 10 Study on the neutralization effectiveness of the combination of antibody P17-A11 and ACE2-Fc fusion protein on a wide range of S protein RBD mutants

1) Sample preparation:

1.1) Fake virus packaging:

The ratio of backbone plasmid pNL4-3.Luc/envelope plasmid pV-S(nCoV)=4:1 was transfected with LipofectamineTM 3000 (Thermo Fisher Scientific) into 293T cells with a ^{density of about 1×10 5} /cm ^2. About 8 hours after transfection, fresh culture medium was replaced, 12-16 hours after the medium change, the cell supernatant was collected, and the supernatant was taken as pseudovirus supernatant after centrifugation.

1.2) Determination of pseudovirus titer:

Prepare 54 μL of pseudovirus supernatant and its 10～10 ⁸ gradient dilutions, place them in a 96-well plate, and add 50 μL of 4× ^{10 5} /mL stable cell line 293T-ACE2 to the above virus dilutions at 37°C, 5 Culture in %CO2 for 20-24 hours, add 25μL of DMEM medium containing 10% FBS to each well, and continue to culture until 48h.

Add 100μL of Luc detection reagent to each well. After lysis for 2 minutes at room temperature, transfer 100μL to a 96-well white plate for detection using a microplate reader.

1.3) Dilute the sample concentration to the required initial concentration with DMEM medium without FBS and filter for later use. The required initial concentration is shown in Table 8:

Table 8. Initial concentration of samples

Sample type	Antibody P17-A11	ACE2-Fc fusion protein	Antibody P17-A11+ACE2-Fc fusion protein
Concentration (μg/ml)	100	2000	50+250

2) Take a transparent 96-well plate, add 112 μl of the initial concentration of antibody in row 1, and add 84 μl/well of DMEM medium without FBS (the amount of three multiple wells) in rows 2-7. Pipette and mix the antibody from row 1 for 15 times, then take 28μl of liquid to row 2, gently pipette and mix 15 times, take 28μl to row 3, dilute to row 8 by 4 times, then discard after pipetting and mixing. 28μl of antibody dilution; add 84μl of serum-free DMEM medium to another well as a virus control; then add 84μl of pseudovirus solution with half of the effective infection concentration in the titer determination in the above-mentioned treatment well, gently mix the wells Incubate the transparent plate in a 37°C, 5% CO ₂ incubator for 1 hour;

3) Take 293-ACE2 cells, count and adjust the density to 0.4×10 ⁶ cells/mL, then take enough cells according to the amount of cells required for the experiment; after incubating for 1 hour, divide the mixed solution in the 96-well transparent plate into three turns To a 96-well white transparent plate, 50μl per well. In addition, select three wells and add 50μl of DMEM medium containing 10% FBS as a negative control, and then add 50μl of cells with adjusted density to each well, and finally put them in a 37°C, 5% CO ₂ cell incubator for culture; incubate for 24 hours After rehydration, add 25μl of DMEM medium containing 10% FBS to each well and continue culturing for 24h; after the completion of culture, add 100μl of Bright-Light Luciferase Assay System detection reagent to each well, and use a multifunctional microplate reader to detect the luminescence value (RLU);

4) Data processing: Bring the data into GraphPad Prism software for data analysis, and output IC ₅₀ value and R ² value.

The results showed that 5 out of 38 naturally-occurring S protein mutants (V483A, E484K, G485D, F490L and F490P) showed resistance to the antibody P17-A11 (Table 9). The ACE2-Fc fusion protein treatment alone and the combined antibody P17-A11 treatment can effectively neutralize all 38 S protein mutants, including the above 5 drug-resistant mutants. This indicates that the ACE2-Fc fusion protein and its combined application with the antibody P17-A11 may provide effective therapies against various SARS-CoV-2 virus mutants.

Table 9. Neutralization effectiveness of antibody P17-A11, ACE2-Fc fusion protein and antibody P17-A11+ACE2-Fc fusion protein combination on SARS-CoV-2 virus S protein RBD mutant

Example 11 In vivo efficacy study of ACE2-Fc fusion protein in hACE2-IRES-luc transgenic mouse model

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.

The results showed that all animals showed infectious clinical symptoms caused by a lethal dose of virus infection. However, in the ACE2-Fc fusion protein treatment group receiving a dose of 50 mg/kg, no mice died before the scheduled euthanasia (Figure 6), indicating that the ACE2-Fc fusion protein has therapeutic potential for SARS-CoV-2 virus infection.

Example 12 In vivo pharmacodynamic study of antibody P17-A11 and ACE2-Fc fusion protein in BALB/c mouse model

In the in vivo study of the combination of antibody P17-A11 and ACE2-Fc fusion protein, 1.6×10 ⁴ PFU of MASCp6 (the 6th generation mouse-adapted strain of SARS-CoV-2 with N501Y mutation) was used, source: Beijing Institute of Microbiology and Epidemiology) infect BALB/c mice. After 2 hours, use antibody P17-A11 (5mg/kg), ACE2-Fc fusion protein (25mg/kg) or antibody P17-A11+ACE2-Fc fusion protein (5+25mg). /kg) Treat infected mice. After 5 days, the mice were sacrificed to analyze the viral load in the lungs and trachea. Virus titer is expressed in RNA copies per gram of tissue. *p<0.05.

The results showed that compared with antibody P17-A11 or ACE2-Fc fusion protein monotherapy, the combination of 5 mg/kg antibody P17-A11 plus 25 mg/kg ACE2-Fc fusion protein can significantly increase the virus clearance rate in vivo (Figure 7A- 7B), which indicates that the combination therapy of antibody P17-A11 and ACE2-Fc fusion protein has potential effectiveness against MASCp6 infection.

Claims (10)

1. 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.
2. The ACE2 fusion protein according to claim 1, 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, most preferably as SEQ ID No Fc fragment shown in .2.
3. The ACE2 fusion protein according to claim 1, wherein the amino acid sequence of the extracellular region of the ACE2 protein is shown in SEQ ID No. 1.
4. The ACE2 fusion protein of claim 1, wherein the amino acid sequence of the ACE2 fusion protein is shown in SEQ ID No. 4.
5. A nucleic acid molecule encoding the ACE2 fusion protein according to any one of claims 1 to 4.
6. An expression vector comprising the nucleic acid molecule according to claim 5.
7. A host cell comprising the expression vector according to claim 6 and capable of expressing the ACE2 fusion protein according to any one of claims 1 to 4.
8. A pharmaceutical composition comprising the ACE2 fusion protein according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.
9. The pharmaceutical composition according to claim 8, further comprising a neutralizing antibody against the S protein of the novel coronavirus (SARS-CoV-2).
10. The pharmaceutical composition according to claim 9, 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:

    a) 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

    b) 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 zone has the same LCDR1, LCDR2 and LCDR3.

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