WO2024217209A1 - Long-acting method for coronavirus polypeptide fusion inhibitor - Google Patents

Abstract

Provided is a long-acting method for a coronavirus polypeptide fusion inhibitor. The method comprises coupling an IgG Fc binding peptide to a coronavirus polypeptide therapeutic agent, wherein the coronavirus polypeptide therapeutic agent comprises an amino acid sequence selected from SEQ ID NOs: 1-3, or a variant thereof having substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4, or a variant thereof having substitution, insertion, deletion or addition of one or more amino acids. By means of the method, the coronavirus polypeptide therapeutic agent can be long-acting or the effect of the coronavirus polypeptide therapeutic agent is improved.

Description

Method for prolonging the effect of coronavirus polypeptide fusion inhibitors

This application claims priority to a Chinese invention patent application filed on April 19, 2023, with application number: 202310423264.7, and entitled “Method for Prolonging the Effect of Coronavirus Peptide Fusion Inhibitors”. This priority application is incorporated herein by reference.

Technical Field

The present invention belongs to the field of biomedicine, and relates to a method for long-acting a coronavirus polypeptide fusion inhibitor, and specifically relates to a long-acting broad-spectrum coronavirus polypeptide fusion inhibitor. The method can be used to prepare a long-acting broad-spectrum anti-coronavirus drug.

Background Art

Coronaviruses (CoVs) pose a huge threat to human health and public health. Currently, there are seven confirmed coronaviruses (HCoVs) that can infect humans, including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as the new coronavirus). SARS-CoV, MERS-CoV and SARS-CoV-2 are highly pathogenic HCoVs. SARS-CoV-2 has been prevalent in more than 200 countries and regions around the world, with a cumulative death toll of more than 7 million. The SARS-CoV-2 genome mutation rate is very high, and variants of concern (VOCs) such as Alpha, Beta, Gamma, Delta and Omicron have appeared so far. Compared with previous VOCs, the Omicron mutant strain that is currently prevalent in the world has lower pathogenicity, but its infectivity is significantly enhanced. Moreover, Omicron has a strong ability to escape immune responses triggered by infection or vaccination, as well as the monoclonal neutralizing antibodies that have been developed. In addition, small molecule inhibitors of SARS-CoV-2 have been reported to be prone to drug-resistant strains and viral rebound. Small molecule inhibitors can also be toxic to the liver and kidneys.

Coronavirus peptide fusion inhibitors, such as EK1, inhibit the formation of the six-helix bundle (6-HB) during viral membrane fusion by targeting the highly conserved region on the coronavirus spike protein S2 subunit, thereby preventing coronaviruses from entering cells. Therefore, compared with monoclonal antibody drugs, they have a better broad spectrum, higher specificity and lower toxicity than small molecule inhibitors. However, unmodified peptide drugs generally have a small molecular weight and a short half-life in the body.

Therefore, there is a need in the art for a method for prolonging the effectiveness of coronavirus polypeptide fusion inhibitors.

Summary of the invention

At present, there are strategies for the long-acting peptides that have been marketed, including coupling with polymers (such as polyethylene glycol), fusion with long-acting fragments (IgG Fc domain, human serum albumin), coupling with albumin binding ligands (such as fatty acid chains), etc. However, the inventors have found that these long-acting strategies have their own limitations when applied to peptide drugs. For example, there are problems such as immunogenicity and safety in peptide PEGylation, which leads to reduced therapeutic effects and adverse events. After the peptide is fused with the Fc domain of IgG or albumin, the molecular weight increases more than ten times, which will lead to increased steric hindrance and easy loss of drug activity.

Therefore, the inventors have developed a new long-acting method to extend the half-life of anti-coronavirus peptide drugs in vivo. The inventors of the present application proposed to couple an IgG Fc binding peptide that can bind to the human IgG Fc domain with a coronavirus peptide therapeutic agent to achieve the purpose of extending the half-life of the peptide drug in vivo. Thus, a new long-acting method is provided for extending the half-life of coronavirus peptide therapeutic agents in vivo, and a long-acting, broad-spectrum coronavirus peptide therapeutic agent is provided, which can be used to combat the current and possible future coronavirus outbreaks.

In one aspect, the present invention provides a method for prolonging the effect of a coronavirus polypeptide therapeutic agent or a method for extending the in vivo half-life of a coronavirus polypeptide therapeutic agent. In one aspect, the present invention provides a method for increasing the efficacy of a coronavirus polypeptide therapeutic agent against coronavirus or a method for increasing the binding ability of an IgG Fc binding peptide to an IgG protein.

Each method may include coupling an IgG Fc binding peptide to a polypeptide (e.g., a coronavirus polypeptide therapeutic agent, such as a coronavirus polypeptide fusion inhibitor). The coronavirus polypeptide fusion inhibitor may comprise an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof that substitutes, inserts, deletes, or adds one or more amino acids. The IgG Fc binding peptide may comprise an amino acid sequence of SEQ ID NO: 4 or a variant thereof that substitutes, inserts, deletes, or adds one or more amino acids.

In one embodiment, the IgG Fc binding peptide is coupled to the N-terminus or C-terminus of the polypeptide therapeutic.

In one embodiment, the IgG Fc binding peptide is coupled to a coronavirus polypeptide fusion inhibitor directly or via a linker.

In one embodiment, the linker is a flexible linker or a rigid linker.

In one embodiment, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, and n is an integer from 1 to 20. For example, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

In one aspect, the present invention provides a fusion polypeptide comprising a coronavirus polypeptide therapeutic agent (e.g. The coronavirus polypeptide therapeutic agent may comprise an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids. The IgG Fc binding peptide may comprise an amino acid sequence of SEQ ID NO: 4 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids.

In one embodiment, the IgG Fc binding peptide is coupled to the N-terminus or C-terminus of the coronavirus polypeptide fusion inhibitor.

In one embodiment, the IgG Fc binding peptide is coupled to a coronavirus polypeptide fusion inhibitor directly or through a linker.

In one embodiment, the linker is a flexible linker or a rigid linker.

In one embodiment, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is an integer from 1-20.

In one embodiment, the fusion polypeptide comprises the amino acids shown in SEQ ID NO:5 or a variant thereof that substitutes, inserts, deletes or adds one or more amino acids.

In one aspect, the invention provides nucleic acids encoding the fusion polypeptides described herein.

In one aspect, the invention provides a vector comprising a nucleic acid described herein.

In one embodiment, the vector comprises nucleotides encoding a purification tag, nucleotides encoding a leader and/or regulatory elements.

In one embodiment, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag.

In one embodiment, the regulatory element is selected from a promoter, a terminator and/or an enhancer.

In one aspect, the invention provides a host cell comprising a nucleic acid described herein or a vector described herein.

In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell.

In one embodiment, the eukaryotic cell is a yeast cell, an animal cell and/or an insect cell. In one embodiment, the prokaryotic cell is an E. coli cell.

In one aspect, the present invention provides a composition comprising a fusion polypeptide, a nucleic acid, a vector and/or a host cell described herein and a pharmaceutically acceptable carrier.

In one embodiment, the composition comprises an additional anti-coronavirus drug.

In one embodiment, the composition is in the form of a tablet, capsule, pellet, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or lyophilized powder.

In one embodiment, the anti-coronavirus drug is an antibody or a small molecule drug.

In one embodiment, the coronavirus is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2.

In one embodiment, the antibody is ambavirimab, romidesevimab, sotrovimab (ie, S309 mAb), imdevimab, casirivimab (ie, 10933 mAb), tixagevimab, cilgavimab, bamlanivimab, etesevimab, amubarvimab, and/or romlusevimab.

In one embodiment, the small molecule drug is mononavir, namatevir, ritonavir, and/or azithromycin.

In one aspect, the present invention provides the use of an IgG Fc binding peptide for increasing the efficacy of a coronavirus polypeptide drug (fusion inhibitor) against a coronavirus, wherein the IgG Fc binding peptide is coupled to a coronavirus polypeptide therapeutic agent, wherein the coronavirus polypeptide therapeutic agent comprises an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids.

In one embodiment, the IgG Fc binding peptide is coupled to the N-terminus or C-terminus of the coronavirus polypeptide therapeutic.

In one embodiment, the IgG Fc binding peptide is coupled to the coronavirus polypeptide therapeutic agent directly or via a linker.

In one embodiment, the linker is a flexible linker or a rigid linker.

In one embodiment, the linker is (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, where n is an integer from 1-20.

In one embodiment, the coronavirus is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2.

In one aspect, the invention provides an in vitro method for inhibiting coronavirus proliferation in a cell, comprising contacting the cell with a fusion polypeptide or composition described herein.

In one embodiment, the coronavirus is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2.

In one aspect, the present invention provides the use of the fusion polypeptide or composition herein in the preparation of a medicament or a kit for inhibiting the proliferation of coronavirus, treating and/or preventing the occurrence of coronavirus infection. Diseases or symptoms caused by viruses.

In one embodiment, the coronavirus is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2.

In one embodiment, the drug is in the form of a tablet, capsule, pellet, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or lyophilized powder.

On the one hand, the present invention provides a method/use of the fusion polypeptide of the present invention for increasing the efficacy of coronavirus polypeptide therapeutic agents against coronaviruses or for increasing the binding ability of IgG Fc binding peptides to IgG proteins.

Advantages of the present invention include:

1. The present invention provides a method for extending the in vivo half-life of a coronavirus polypeptide therapeutic agent or making the coronavirus polypeptide therapeutic agent long-acting. Compared with the half-life of an unmodified polypeptide, the method can extend the in vivo half-life of a coronavirus polypeptide therapeutic agent to about 40 hours (Figure 2).

2. IgG Fc binding peptides can enhance the ability of coronavirus peptide drugs (coronavirus peptide fusion inhibitors) to inhibit coronavirus-mediated cell-cell fusion (Figure 3A).

3. IgG Fc binding peptide can enhance the ability of coronavirus peptide drugs to inhibit infection.

4. The fusion polypeptide of the present invention can synergistically exert a therapeutic effect with coronavirus antibodies (such as S309 and/or 10933 monoclonal antibodies).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the binding of IBP-EK1 to monkey IgG.

Figure 2 shows the curve of IBP-EK1 concentration in plasma over time. Based on the in vitro IC50 value of IBP-EK1 inhibiting SARS-CoV pseudovirus and the measured DF-IC ₅₀ of serum samples (left figure), the concentration of active peptide in rhesus monkey serum samples at different time points after administration was estimated (right figure).

FIG3 shows the inhibitory activity of IBP-EK1 on SARS-CoV-2S protein-mediated cell-cell fusion (A) and SARS-CoV-2 pseudovirus infection (B).

Figure 4 shows the inhibitory activity of IBP-EK1 against Omicron BQ.1 mutant pseudovirus infection.

FIG5 shows the cytotoxicity of IBP-EK1 to target cells Caco-2 (A) and Huh-7 (B).

FIG6 shows the circular dichroism spectrum of IBP-EK1 (A), the circular dichroism spectrum of the IBP-EK1/HR1P complex (B), and the Tm value of the IBP-EK1/HR1P complex (C).

Figure 7 shows the dose reduction of IBP-EK1 peptide combined with S309 monoclonal antibody to inhibit Delta pseudovirus infection. Low situation.

DETAILED DESCRIPTION

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Although it is believed that those of ordinary skill in the art fully understand the following terms, the following definitions are still stated to help illustrate the subject matter disclosed by the present invention.

As used in this article, "coronavirus" is an enveloped, linear single-stranded positive-strand RNA virus with spikes on the envelope. The entire virus resembles a solar corona and is a large class of viruses that are widely found in nature. SARS-CoV-2 is the seventh known coronavirus that can infect humans. The other six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (which causes severe acute respiratory syndrome), and MERS-CoV (which causes Middle East respiratory syndrome).

As used herein, "long-acting" refers to making a drug (e.g., a polypeptide) effective over an extended period of time. Long-acting methods include chemical modification, polyethylene glycol (PEG) modification, glycosyl modification, fusion with human serum albumin, fusion with antibody Fc fragment, microencapsulation, etc. In this article, the inventors use IBP polypeptides, which are fused or conjugated to the end of the polypeptide to achieve long-acting polypeptides.

As used herein, "coronavirus polypeptide therapeutic agent" refers to a polypeptide drug that can be used to inhibit the growth of coronavirus, prevent or treat coronavirus-related diseases or symptoms. In this article, the coronavirus polypeptide therapeutic agent is a coronavirus polypeptide fusion inhibitor, which is an inhibitor that inhibits coronavirus-mediated cell fusion. This inhibitor can be a polypeptide inhibitor, which has been widely reported in the art (such as Chinese patent application 202180001804.1). For example, the coronavirus polypeptide therapeutic agent comprises an amino acid sequence selected from the following SEQ ID NO: 1-3 or a variant thereof that replaces, inserts, deletes or adds one or more amino acids.
SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL(SEQ ID NO:1)
DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL(SEQ ID NO:2)
ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQ(SEQ
ID NO:3)

As used herein, "IgG Fc binding peptide" refers to a polypeptide having IgG Fc binding ability. The IgG Fc binding peptide can be the amino acid sequence of SEQ ID NO:4 or a variant thereof that replaces, inserts, deletes or adds one or more amino acids. The sequence of SEQ ID NO:4 is DCAWHLGELVWCT.

As used herein, "fusion polypeptide" is used interchangeably with fusion or fusion protein, and refers to two or more polypeptides with the same or different functions linked together. In one embodiment, the fusion polypeptide has the following amino acid sequence:
DCAWHLGELVWCTSLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL
(SEQ ID NO:5).

As used herein, amino acid addition refers to adding amino acids to the C-terminus or N-terminus of an amino acid sequence, such as any one of SEQ ID NO: 1-5, as long as the polypeptide has inhibitory activity against coronavirus.

As used herein, amino acid substitution refers to the replacement of an amino acid residue at a certain position in an amino acid sequence, such as any one of SEQ ID NO: 1-5, by another amino acid residue, as long as the polypeptide has inhibitory activity against coronavirus.

As used in this article, amino acid insertion refers to inserting an amino acid residue at an appropriate position in an amino acid sequence, such as any one of SEQ ID NO: 1-5. The inserted amino acid residues may also be completely or partially adjacent to each other, or the inserted amino acids may not be adjacent to each other, as long as the polypeptide has inhibitory activity against coronavirus.

As used in this article, amino acid deletion means that 1, 2 or more amino acids can be deleted from an amino acid sequence, such as any one of SEQ ID NO: 1-5, as long as the polypeptide has inhibitory activity against coronavirus.

In the present invention, the substitution may be a conservative amino acid substitution, which means that compared with the amino acid sequence of any one of SEQ ID NO: 1-5, 3, preferably 2, or 1 amino acid is replaced by an amino acid with similar or similar properties to form a peptide. These conservative variant peptides can be generated by amino acid substitution according to Table 1.

In the context of the present invention, conservative substitutions may be defined according to substitutions within the amino acid classes reflected in one or more of the following three tables:

Table 1: Categories of conservatively substituted amino acid residues

Table 2: Alternative conservative amino acid residue substitution categories

Table 3: Alternative physical and functional classifications of amino acid residues

As used herein, coronavirus can be any kind of coronavirus. For example, coronavirus is HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome).

The polypeptide or composition of the present invention can be used alone or in combination, or in combination with other agents having anti-coronavirus activity or coronavirus inhibitory activity. For example, these agents can be agents that are currently reported to have inhibitory activity against coronaviruses or have therapeutic effects on coronavirus diseases, such as coronavirus pneumonia, such as favipiravir, nelfinavir, arbidol, lopinavir, ritonavir, chloroquine phosphate, darunavir or remdesivir, etc. The anti-coronavirus drug can be an antibody or a small molecule drug. The antibody can be ambavir monoclonal antibody, romisvir monoclonal antibody, sotrovimab, imdevimab, The small molecule drug may be mononavir, namatevir, ritonavir and/or azithromycin.

Herein, the polypeptide of the present invention can be prepared into a composition for application. The composition may include a suitable carrier, such as a pharmaceutically acceptable carrier. Such compositions may be external, for example, used as external preparations, external smear preparations, such as external gels or external infiltration preparations. Such compositions can be applied to articles that need to inhibit viruses, such as, but not limited to, masks, paper towels, gloves, clothing, such as protective clothing, etc. Alternatively, it can be added as an active ingredient to hand washing products, such as hand sanitizers, shower gels, etc. Such polypeptides or polypeptide derivatives can be used to inhibit coronaviruses in vitro to prevent and/or reduce viral infection. Such polypeptides or polypeptide derivatives can be used to prevent or treat coronavirus infections in subjects or diseases caused by coronaviruses.

The polypeptide or composition of the present invention can also be prepared into a medicine or pharmaceutical composition. Such medicines can be tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories or freeze-dried powder injections. These medicines or pharmaceutical compositions can be applied by various administration methods, such as injection, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracisternal injection or infusion, etc., cavity administration, such as rectal, vaginal and sublingual, respiratory administration, such as nasal; mucosal administration, or surface administration, etc.

The present invention provides a method for preventing or treating coronavirus infection in a subject or a disease caused by a coronavirus, comprising administering a polypeptide or composition of the present invention. The present invention also provides a polypeptide or polypeptide derivative used in preventing or treating coronavirus infection in a subject or a disease caused by a coronavirus.

The present invention also relates to a method for preventing or treating coronavirus infection or a disease caused by coronavirus in a subject, which comprises administering the polypeptide or composition of the present invention to the subject/patient.

The present invention also relates to polypeptides, nucleic acids, vectors, host cells and/or compositions for preventing or treating coronavirus infection or a disease caused by coronavirus in a subject.

The present invention also relates to polypeptides, nucleic acids, vectors, host cells, polypeptides or compositions, such as pharmaceutical compositions and kits, for preventing or treating coronavirus infection or diseases caused by coronavirus in subjects/patients.

Conventional pharmaceutical practice can be used to provide suitable formulations or compositions for administration to subjects suffering from coronavirus infection. Any suitable route of administration can be used, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ocular, intraventricular, intracapsular, Intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, topical or oral administration. The therapeutic formulation may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; for intranasal formulations, it may be in the form of a powder, nasal drops or aerosol. Preferably, in this context, the polypeptide or composition of the invention is used for intravenous administration.

Methods for preparing formulations known in the art can be found, for example, in "Remington's Pharmaceutical Sciences" (19th edition), edited by A. Gennaro, 1995, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water or saline, poly(alkylene) glycols such as polyethylene glycol, vegetable oils or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compound. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Pseudovirus preparation

Pseudoviruses can be prepared using methods known in the art herein. For example, as previously described (L. Lu, Q. Liu, Y. Zhu, K. -H. Chan, L. Qin, Y. Li, Q. Wang, J. F-W. Chan, L. Du, F. Yu, C. Ma, S. Ye, K. -Y. Yuen, R. Zhang, S. Jiang, Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nat. Commun. 5, 3067 (2014)), 293T cells were transfected with plasmids, and the plasmids included HIV backbone plasmids pNL4-3. Luc. R ^- . E ^- and different SARS-CoV-2 mutant strains S protein expression plasmids. The amino acid sequences of different SARS-CoV-2 mutant strains S proteins are known. Various SARS-CoV-2 variant strains S protein expression plasmids are provided in Table 4. The amino acid sequences of one or more SARS-CoV-2 mutant strains S proteins listed in Table 4 can be used herein.

Table 4:

Preparation of effector cells

Effector cells can be prepared herein using methods known in the art, i.e., 293T/coronavirus S protein/GFP cells required for cell-cell fusion experiments. For example, as described in the prior art literature (Xia, S. et al. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv 5, eaav4580, doi: 10.1126/sciadv.aav4580 (2019)), the gene sequences of different coronavirus S proteins can be cut from the pcDNA3.1 vector using endonucleases. , connected to the pAAV-IRES-GFP vector, and the following fusion plasmids of different coronaviruses were obtained (encoding GFP and coronavirus S protein, expressed in the cytoplasm and cell membrane surface, respectively): pAAV-SARS-2-IRES-GFP; pAAV-SARS-IRES-GFP; pAAV-MERS-IRES-GFP; pAAV-WIV1-IRES-GFP; pAAV-OC43-IRES-GFP; pAAV-NL63-IRES-GFP; pAAV-229E-IRES-GFP. The amino acid sequence of the S protein is shown in Table 5. Then, the constructed vector can be used to transfect 293T cells.

Table 5

method

The present invention provides a method for prolonging the effect of a coronavirus polypeptide therapeutic agent, which comprises coupling an IgG Fc binding peptide with a coronavirus polypeptide therapeutic agent, wherein the coronavirus polypeptide therapeutic agent comprises an amino acid sequence selected from SEQ ID NOs: 1-3 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids.

The present invention provides a method for increasing the efficacy of a coronavirus polypeptide therapeutic against a coronavirus, the method comprising coupling an IgG Fc binding peptide to a coronavirus polypeptide therapeutic, wherein the coronavirus polypeptide therapeutic comprises an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids.

The present invention provides a method for increasing the binding ability of an IgG Fc binding peptide to an IgG protein, the method comprising coupling the IgG Fc binding peptide with a coronavirus polypeptide therapeutic agent, wherein the coronavirus polypeptide therapeutic agent comprises an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids.

Herein, the IgG protein may be an IgG1 protein, an IgG2 protein, an IgG3 protein or an IgG4 protein.

The IgG Fc binding peptide can be coupled to the N-terminus or C-terminus of the coronavirus polypeptide therapeutic agent. The IgG Fc binding peptide can be coupled to the coronavirus polypeptide therapeutic agent directly or via a linker. The type of linker may not be particularly limited and may be a flexible linker or a rigid linker. For example, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is an integer from 1 to 20. The coronavirus polypeptide therapeutic agent may be a coronavirus polypeptide fusion inhibitor.

Fusion peptide

The present invention provides a fusion polypeptide, which comprises a coronavirus polypeptide therapeutic agent and an IgG Fc binding peptide, wherein the coronavirus polypeptide therapeutic agent comprises an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids. The fusion polypeptide can be used to prolong the effect of coronavirus polypeptide therapeutic agents, to increase the binding ability of IgG Fc binding peptides to IgG proteins and/or to increase the efficacy of coronavirus polypeptide therapeutic agents against coronaviruses.

The IgG Fc binding peptide can be coupled to the N-terminus or C-terminus of the coronavirus polypeptide therapeutic. The IgG Fc binding peptide is coupled to the coronavirus polypeptide therapeutic directly or through a linker. The linker is not particularly limited and can be a flexible linker or a rigid linker. The linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is an integer from 1 to 20. The fusion polypeptide may comprise the amino acids shown in SEQ ID NO:5 or a variant thereof that substitutes, inserts, deletes, or adds one or more amino acids. The coronavirus polypeptide therapeutic may be a coronavirus polypeptide fusion inhibitor.

Composition

Each component of the present invention, such as fusion polypeptide, nucleic acid, vector and/or host cell can be prepared into a composition or included in a kit. The composition or kit may include a pharmaceutically acceptable carrier to facilitate the application of each component. The composition also includes one or more additional anti-coronavirus drugs. The anti-coronavirus drug can be an antibody or a small molecule drug, such as a drug that is currently or will be commercialized in the future. For example, the antibody is 10933 monoclonal antibody, S309 monoclonal antibody, ambavir monoclonal antibody, romisvir monoclonal antibody, sotrovimab, imdevimab, casirivimab, tixagevimab, cilgavimab, bamlanivimab, etesevimab, amubarvimab and/or romlusevimab. The small molecule drug can be monovir, namatevir, ritonavir and/or azithromycin.

The fusion polypeptide or composition of the present invention can be formulated into a suitable dosage form, such as tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories or lyophilized powders. The fusion polypeptide or composition of the present invention can be used to treat various coronaviruses or related diseases. The coronavirus can be selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2.

Treatment

The present invention provides an in vitro method for inhibiting coronavirus proliferation in cells, which comprises contacting the cells with the fusion polypeptide or composition described herein.

The present invention provides a method for inhibiting coronavirus proliferation, treating and/or preventing diseases or symptoms caused by coronavirus, which comprises administering the fusion polypeptide or composition described herein to a subject (eg, a human or a mammal).

The coronavirus can be selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2.

The present invention also provides the use of the fusion polypeptide or composition herein in the preparation of a drug or kit for inhibiting coronavirus proliferation, treating and/or preventing diseases or symptoms caused by coronavirus. The coronavirus can be selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2.

The present invention also provides the fusion polypeptide or composition of the present invention for inhibiting the proliferation of coronavirus, treating and/or preventing diseases or symptoms caused by coronavirus. The coronavirus can be selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2.

In these methods and uses, the drug can be in the form of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories or lyophilized powders.

Example

The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In combination with this specification and common knowledge in the art, a person of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, a person of ordinary skill in the art can make any modification or change to the technical solution of the present invention, and such modification and change are also included in the scope of the present invention.

Example 1: Preparation of polypeptides, packaging of pseudoviruses and preparation of effector cells

1. Preparation of long-acting coronavirus fusion inhibitor peptide IBP-EK1

A short peptide that can specifically bind to the CH2 and CH3 interfaces of the human IgG Fc domain was selected and named IBP. IBP consists of 13 amino acids and the sequence is N-DCAWHLGELVWCT-C (SEQ ID NO: 4). The sequence of the coronavirus fusion inhibitor peptide EK1 is SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL (SEQ ID NO: 1). The long-acting coronavirus fusion inhibitor peptide IBP-EK1 consists of 49 amino acids, and the specific amino acid sequence is: N-DCAWHLGELVWCTSLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-C (SEQ ID NO: 5); where N- represents the N-terminus and C- represents the C-terminus. The above peptides were commissioned to be synthesized by an outsourcing company (Nanjing Jiepeptide Biotechnology Co., Ltd.).

2. Packaging of the Coronavirus Fake Virus

The pseudovirus was obtained by transfecting 293T cells with plasmids, including HIV backbone plasmid pNL4-3.Luc.R ^- .E ^- and different SARS-CoV-2 mutant strain S protein expression plasmids, see Table 4; the literature report on the construction method is: L.Lu, Q.Liu, Y.Zhu, K.-H.Chan, L.Qin, Y.Li, Q.Wang, JF-W.Chan, L.Du, F.Yu, C.Ma, S.Ye, K.-Y.Yuen, R.Zhang, S.Jiang, Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor.Nat.Commun.5,3067(2014). Specifically, the steps of packaging all pseudoviruses in this patent are as follows:

1) Digest 293T cells 24 hours before transfection, adjust to an appropriate concentration with DMEM medium containing 10% FBS, spread 12 mL/dish into a 100 mm cell culture dish, and culture in a 5% CO2, 37°C cell culture incubator until the cell conjugation rate is about 60-70% after 24 hours.

2) The total amount of plasmid added to each 100mm culture dish is 25μg, and the amount of EZ trans transfection reagent is 55μL. The transfection ratio of the vector plasmid pNL4-3.Luc.R-.E- and the coronavirus S protein expression plasmid is 4:1, that is, 20μg vector plasmid and 5μg expression plasmid/dish. Add the two plasmids to 330μL of serum-free DMEM medium and mix well, and dilute EZ trans to 330μL with serum-free DMEM medium and mix well. Let the plasmid dilution and transfection reagent dilution stand at room temperature for 5 minutes.

3) Add the transfection reagent dilution dropwise into the plasmid dilution, mix gently, and let stand at room temperature for 15 minutes.

4) Slowly and evenly drip the above mixture onto the surface of 293T cells in the cell culture dish, gently shake it, and place it in an incubator for culture.

5) About 9-10 hours after transfection, gently aspirate the culture medium in the dish and replace it with fresh DMEM medium containing 10% FBS. Continue culturing for about 48 hours to allow the pseudovirus to be packaged and released into the supernatant.

6) Aspirate the supernatant from the culture dish, filter it through a 0.45 μm sterile filter into a 50 mL sterile centrifuge tube, divide it into EP tubes or 15 mL centrifuge tubes (avoid multiple freeze-thaw cycles), and store it in a -80°C refrigerator for later use.

3. Preparation of 293T/coronavirus S protein/GFP cells (effector cells) required for cell-cell fusion experiments

The gene sequences of different coronavirus S proteins were cut out from the pcDNA3.1 vector using endonucleases and ligated into the pAAV-IRES-GFP vector to obtain the following fusion plasmids of different coronaviruses (encoding GFP and coronavirus S proteins, expressed in the cytoplasm and cell membrane surface, respectively): pAAV-SARS-2-IRES-GFP; pAAV-SARS-IRES-GFP; pAAV-MERS-IRES-GFP; pAAV-WIV1-IRES-GFP; pAAV-OC43-IRES-GFP; pAAV-NL63-IRES-GFP; pAAV-229E-IRES-GFP (see, for example, Xia, S. et al. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv 5, eaav4580, doi: 10.1126/sciadv.aav4580 (2019)). The amino acid sequence of S protein is shown in Table 5.

1) Digest 293T cells 24 hours before transfection, spread them into 6-well plates at an appropriate density, add 2 mL of cell suspension to each well, shake thoroughly, and place in a 5% CO2, 37°C cell culture incubator to culture the cells so that the cell conjugation rate is about 60% after 24 hours.

2) 2 hours before transfection, the cell supernatant in the six-well plate was gently aspirated and the medium was replaced with 2 mL/well of fresh DMEM medium (containing 10% FBS).

3) The amount of plasmid added to each well of the six-well plate is 5μg, and the amount of Vigofect transfection reagent is 2μL. 5μg pAAV-SARS-2-IRES-GFP (or other coronavirus fusion plasmid) plasmid and 2μL transfection reagent are added dropwise to 100μL serum-free DMEM medium, and stand at room temperature for 5 minutes. Add the transfection reagent diluent dropwise to the plasmid diluent, mix gently, and stand at room temperature for 15 minutes.

4) About 200 μL of the above-mentioned mixed solution was evenly added to the surface of the 293T cells in the six-well plate, gently shaken, and cultured in an incubator to express green fluorescent protein (GFP) in the cytoplasm of the 293T cells and SARS-CoV-2S protein on the cell membrane surface.

5) 6 to 8 hours after transfection, replace the 293T cell culture medium with 2 mL of fresh DMEM culture medium (containing 10% FBS) per well.

6) Continue to culture in the incubator for 24 to 36 hours, observe and confirm the expression of GFP in the cells under a fluorescence microscope, and then conduct subsequent cell-cell fusion experiments.

Example 2: Detection of the ability of the polypeptide to bind to monkey IgG by ELISA experiment

The ability of peptides IBP, EK1 and IBP-EK1 to bind to monkey IgG was detected by ELISA experiments as described previously (reference: Du L, Kou Z, Ma C, Tao X, Wang L, Zhao G, Chen Y, Yu F, Tseng CT, Zhou Y, Jiang S. A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implications for developing therapeutics and vaccines. PLoS One. 2013 Dec 4; 8(12): e81587).

The specific experimental process is as follows.

(1) _The corresponding peptides (peptides IBP, EK1 and IBP-EK1) were diluted to 5 μg/mL with 0.05 M NaHCO3 coating buffer solution at pH 9.6, and coated into a 96-well plate at 50 μL/well at 4°C overnight.

(2) Add 150 μL of 2% milk to each well and block at 37°C for 2 h, then wash the plate three times.

(3) Rhesus monkey IgG (prepared in-house in this laboratory and purified from rhesus monkey serum using rProtein G column material from Tiandirenhe Biotechnology Co., Ltd. according to the product instructions) was diluted to a concentration of 100 μg/mL, then diluted in multiples, incubated at 37°C for 1 h, and then washed three times.

(4) Add 50 μL of 1:4000 diluted goat anti-monkey IgG-Fc-HRP (purchased from Abcam) to each well, incubate at 37 degrees for 1 hour, and wash the plate 5 times.

(5) 50 μL of TMB color development solution was added to each well, and the color was developed for 3-10 min. Subsequently, 50 μL of 1M H ₂ SO ₄ was added to each well to terminate the reaction. The absorbance at 450 nm (OD450) was then detected using Ultra386 (Tecan).

Figure 1 shows that the absorbance of IBP-EK1 binding to monkey IgG increases with the concentration of monkey IgG. The coupling of IBP and EK1 retains the IgG binding ability of IBP. The absorbance of IBP-EK1 at 450nm (OD450) is positively correlated with the concentration of monkey IgG, indicating that IBP-EK1 can indeed bind to monkey IgG, and the binding is significantly stronger than that of IBP alone.

Example 3: Pharmacokinetics of Peptide in Rhesus Monkeys

The pharmacokinetics of peptides IBP, EK1 and IBP-EK1 in rhesus monkeys were determined as described previously (Reference: Su S, Rasquinha G, Du L, Wang Q, Xu W, Li W, Lu L, Jiang S. A Peptide-Based HIV-1 Fusion Inhibitor with Two Tail-Anchors and Palmitic Acid Exhibits Substantially Improved In Vitro and Ex Vivo Anti-HIV-1 Activity and Prolonged In Vivo Half-Life. Molecules. 2019 Mar 21; 24(6): 1134.).

The specific experimental process is as follows.

(1) Before intravenous injection of rhesus monkeys, serum was collected as a negative control.

(2) Rhesus monkeys were given 10 mg/kg IBP-EK1 by intravenous injection (single dose).

(3) Blood samples were collected at 2h, 4h, 6h, 24h, 72h, and 144h after the intravenous injection.

(4) After the sample has been allowed to stand at room temperature, the serum is separated and stored in a -80°C refrigerator.

(5) The serum at each time point was diluted in multiples to detect the inhibition of SARS-CoV pseudovirus (prepared in-house in our laboratory, obtained by transfecting 293T cells with HIV backbone plasmid pNL4-3.Luc.R ^{- .E-} and SARS-CoV S protein expression plasmid, the specific construction method is as described above), and the serum dilution factor that inhibits 50% of the pseudovirus (DF- _IC50 ) was calculated.

(6) The peptide concentration in serum was estimated based on the in vitro IC ₅₀ , and the half-life (t _1/2 ) was calculated using MODFIT software.

Figure 2 shows the curve of IBP-EK1 concentration in plasma over time. Based on the in vitro IC50 value of IBP-EK1 inhibiting SARS-CoV pseudovirus and the measured DF-IC ₅₀ of serum samples (left figure), the concentration of active peptide in rhesus monkey serum samples at different time points after administration was estimated (right figure). Based on the concentration-administration time curve, the serum half-life (t _1/2 ) of IBP-EK1 was fitted by MODFIT software, which was 39.72h, which was about 21 times longer than the previously reported half-life of EK1.

Example 4: Peptide inhibition of SARS-CoV-2 pseudovirus infection experiment

Peptide inhibition experiments of SARS-CoV-2 pseudovirus infection were performed as previously described (reference: Xia S, Yan L, Xu W, Agrawal AS, Algaissi A, Tseng CK, Wang Q, Du L, Tan W, Wilson IA, Jiang S, Yang B, Lu LA pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike.Sci Adv.2019Apr10;5(4):eaav4580).

The specific experimental process is as follows.

(1) Digest and count target cells (Caco-2 cells preserved in our laboratory: 6000 cells/well; Caco-2 (human colon adenocarcinoma cell line) was preserved in liquid nitrogen in our laboratory, and after recovery, placed in a 5% CO ₂ , 37°C incubator, and cultured in DMEM medium containing 10% FBS), add 100 μL per well to a 96-well cell culture plate. Incubate in a 37°C incubator for 24 hours to allow cells to grow and attach completely.

(2) The stock solutions of IBP, EK1 and IBP-EK1 were diluted with serum-free DMEM and vortexed to mix, and then added to the first row of a 96-well round-bottom dilution plate. Three replicate wells were set for each peptide. In addition, three virus control wells without drug and three cell control wells without virus were set.

(3) Dilute the drug in multiples and add prepackaged coronavirus pseudovirus (prepared in-house in this laboratory. Pseudoviruses are obtained by transfecting 293T cells with plasmids, including HIV backbone plasmid pNL4-3.Luc.R ^{- .E-} and S protein expression plasmids of different SARS-CoV-2 mutants; the construction method is as described above) to each well except the cell well, and incubate in a 37°C incubator for 45 min.

(4) Discard the supernatant in the cell culture plate, mix the peptide-pseudovirus mixture in the 96-well round-bottom plate by pipetting, transfer 100 μl of the mixture to the 96-well cell plate, and incubate it in a 37°C incubator.

(5) After incubation for 10 to 12 hours, the medium was replaced with DMEM medium containing serum.

(6) After incubation for 48 h, discard the cell supernatant, add 50 μL of lysis buffer to each well, and place on a shaker for 45 min at room temperature for lysis.

(7) Pipette 30 μL of lysate from each well of the 96-well plate and transfer to the corresponding 96-well opaque white plate.

(8) Add 30 μL luciferase substrate from the first row to the last row, place the white plate into the ELISA reader, and read the value of each well.

(9) The inhibition rate of the peptide on pseudovirus infection at different concentrations was calculated based on the values of the virus pores and cell pores.

Figure 3 B shows the percentage of polypeptides IBP, EK1 and IBP-EK1 inhibiting SARS-CoV-2 pseudovirus infection. Fusion of IBP with EK1 to form IBP-EK1 did not affect the ability of EK1 to inhibit SARS-CoV-2 pseudovirus infection. Table 6 shows the inhibitory activity of IBP-EK1 against pseudovirus infection of SARS-CoV-2 mutant strains (especially Omicron sublineage strains).

Table 6 shows the inhibitory activity of IBP-EK1 against pseudovirus infection of SARS-CoV-2 mutant strains (especially Omicron sublineage strains).

Table 6

The inhibitory activity of the long-acting coronavirus polypeptide fusion inhibitor IBP-EK1 prepared according to the method of the present invention on SARS-CoV-2 is not affected by the mutation site of the variant strain (as shown in Table 6 and Figure 4). Compared with EK1, the IC50 of IBP-EK1 is further reduced, which is unexpected.

Example 5: Peptide inhibition experiment on SARS-CoV-2S protein-mediated cell-cell fusion

The inhibition experiments of peptides IBP, EK1 and IBP-EK1 on SARS-CoV-2S protein-mediated cell-cell fusion were performed as described previously (reference: Xia S, Yan L, Xu W, Agrawal AS, Algaissi A, Tseng CK, Wang Q, Du L, Tan W, Wilson IA, Jiang S, Yang B, Lu L. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv. 2019 Apr 10; 5(4):eaav4580).

The specific experimental process is as follows:

(1) 293T cells (293T (human embryonic kidney cell line) were stored in liquid nitrogen in our laboratory and cultured in a 5% CO ₂ , 37°C incubator after recovery, using DMEM medium containing 15% FBS) were digested and plated into 6-well plates at an appropriate density and incubated in a 37°C incubator to allow the cells to adhere to the wall.

(2) 24 hours later, 293T cells were transfected with the SARS-CoV-2 fusion plasmid pAAV-SARS-2-IRES-GFP (preserved in this laboratory, the construction method can be found in: Xia, S. et al. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv 5, eaav4580, doi: 10.1126/sciadv.aav4580 (2019)). 6 hours after transfection, the medium was replaced with DMEM containing 10% serum, and the cells were incubated at 37 degrees Celsius for 36 to 48 hours to observe the expression of green fluorescence.

(3) 12 h before the inhibition experiment, the target cells were digested and 25,000 cells/well were added to a 96-well cell culture plate with 100 μL per well. The plate was placed in a 37°C incubator to allow the cells to adhere to the wall.

(4) Dilute the peptide stock solution with serum-free DMEM and vortex to mix, then add it to the first row of a 96-well round-bottom dilution plate. Set up three replicate wells for each peptide. In addition, set up three virus (293T) control wells without drug and three cell control wells without virus (293T).

(5) Digest 293T cells, resuspend in serum-free DMEM, and dilute to an appropriate density. Dilute the drug in multiples in a 96-well round-bottom plate, add 293T cells expressing the S protein of each coronavirus (see Table 4) to each well except the cell well, and incubate in a 37°C incubator for 45 min.

(6) Discard the supernatant in the cell culture plate, mix the polypeptide-cell mixture in the 96-well round-bottom plate by pipetting, transfer 100 μL to the corresponding 96-well cell plate, and incubate in a 37°C incubator.

(7) Observe every two hours until obvious signs of cell-cell fusion appear in the virus control wells without peptide addition.

(8) Add 100 μL of 4% paraformaldehyde solution to each well of the 96-well plate to terminate fusion and fix the cells.

(9) Adjust the fluorescence microscope to the GFP channel and randomly select 4 fields of view in each well to take pictures of cell-cell fusion.

(10) ImageJ software was used to count the number of fused cells and the total number of cells in each image, and the fusion rate of each image was calculated as: number of fused cells/total number of cells × 100%. The average fusion rate of each well was then calculated as the average fusion rate of the four fields of view in each well.

(11) The inhibition rate of cell-cell fusion of the polypeptide at different concentrations was calculated based on the fusion rates of the virus wells and the cell wells: (fusion rate of the virus control wells - fusion rate of the polypeptide wells)/(fusion rate of the virus control wells - fusion rate of the cell control wells) × 100%.

Figure 3, Panel A shows the inhibition of SARS-CoV-2 S protein-mediated cell-cell fusion by peptides IBP, EK1, and IBP-EK1. Compared with EK1, IBP-EK1 had an improved inhibitory effect on SARS-CoV-2 S protein-mediated cell-cell fusion, which was unexpected.

Table 7 shows the inhibitory activity of IBP-EK1 on cell-cell fusion and pseudovirus infection mediated by different coronavirus S proteins. IC50, or half inhibitory concentration, refers to the drug concentration that can inhibit 50% pseudovirus infection/cell-cell fusion.

Table 7.

Example 6: Detection of polypeptide cytotoxicity

For the peptides IBP, EK1 and IBP-EK1, the peptide cytotoxicity was tested as described previously (reference: Xia S, Yan L, Xu W, Agrawal AS, Algaissi A, Tseng CK, Wang Q, Du L, Tan W, Wilson IA, Jiang S, Yang B, Lu L. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv. 2019 Apr 10; 5(4):eaav4580).

The specific experimental process is as follows:

(1) Digest and count target cells (Caco-2 cells: 7000 cells/well; Huh7 cells: 10000 cells/well; Huh-7 (human liver cancer cell line) was stored in liquid nitrogen in our laboratory and placed in a 5% CO ₂ , 37°C incubator after thawing, cultured in DMEM medium containing 15% FBS), add 100 μL per well to a 96-well cell culture plate, and incubate in a 37°C incubator for 24 hours to allow cells to grow and adhere completely.

(2) Dilute the peptide stock solution to the target concentration and vortex to mix, then add to the first row of a 96-well round-bottom dilution plate, with three replicate wells for each peptide. In addition, set up three cell control wells without drug.

(3) Dilution of the drug in multiple ratios. Transfer 100 μL of the corresponding volume into a 96-well cell plate from which the supernatant has been discarded and incubate in a 37°C incubator.

(4) After 48 hours, discard the cell supernatant, add 100 μL CCK8 diluent to each well, and incubate in a 37°C incubator for 2-3 hours.

(5) Measure the absorbance of each well at 450 nm (OD ₄₅₀ ) using an ELISA reader.

The calculation method of cell viability = OD ₄₅₀ of polypeptide wells / OD ₄₅₀ of cell control wells × 100%.

The long-acting coronavirus polypeptide fusion inhibitor IBP-EK1 prepared according to the method of the present invention has no obvious cytotoxicity to target cells (as shown in FIG. 5 ).

Example 7: Determination of secondary structure and melting temperature of polypeptide complex by circular dichroism

The secondary structure and melting temperature of the peptide complex were determined by circular dichroism spectroscopy as described previously (reference: Bi W, Xu W, Cheng L, Xue J, Wang Q, Yu F, Xia S, Wang Q, Li G, Qin C, Lu L, Su L, Jiang S. IgG Fc-binding motif-conjugated HIV-1 fusion inhibitor exhibits improved potency and in vivo half-life: Potential application in combination with broad neutralizing antibodies. PLoS Pathog. 2019 Dec 5; 15(12): e1008082).

The specific experimental process is as follows:

The SARS-CoV-2 HR1P and HR2P peptides used in this part of the study were synthesized by Shanghai Jier Biochemical Co., Ltd. and tested by HPLC with a purity of over 95%. The sequences are as follows:
HR1P: ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQ;
HR2P: DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL.

(1) Dilute the peptides (HR1P, EK1 and IBP-EK1) with phosphate buffer at pH 7.2 to a final concentration of 10 μM for each peptide and incubate at 4°C overnight.

(2) Add blank phosphate buffer to a 0.1 cm quartz cuvette and place it in the machine slot as a blank control calibration. Then add the mixture (HR1P+IBP, HR1P+HR2P, HR1P+EK1, or HR1P+IBPEK1) to the quartz cuvette. Detect the change in CD value from 260 nm to 200 nm.

(3) In the instrument's own software, the CD results were converted to molar ellipticity based on the peptide concentration and the number of amino acids.

(4) At 222 nm, the temperature was raised from 20 to 98 degrees Celsius (5°C/min), and the change in the CD value of the mixture (HR1P+EK1, or HR1P+IBPEK1) was measured during this process to obtain the corresponding melting curve. The midpoint temperature of the melting curve, i.e., the Tm value, was obtained using the instrument's own software.

Figure 6 shows the molar ellipticity. Figure 6A: IBP-EK1 presents a double negative peak spectrum at 208 and 222 nm similar to EK1, indicating that the two polypeptides have comparable α-helical structures. IBP itself presents a random structure. This result shows that coupling with IBP does not change the original secondary structure of EK1. Figure 6B: Compared with EK1 or IBP-EK1 alone, EK1 or IBP-EK1 mixed with HR1P both form significantly deepened double negative peaks at 208 and 222 nm, indicating that the two interact to form a complex with a high α-helix content, indicating that IBP-EK1 can combine with HR1P to form a heterologous six-helix bundle (6-HB). This result suggests that IBP-EK1 uses the same inhibitory mechanism as EK1 to fight coronavirus infection. Figure 6C: As the temperature gradually increases, the CD signal weakens, that is, the six-helix structure of the complex gradually denatures and disintegrates, and eventually tends to a relatively stable value. The melting point is obtained through the instrument's built-in software. The midpoint temperature of the curve is the melting temperature (Tm value). The Tm value of the complex formed by IBP-EK1 and HR1P is higher than that of the complex formed by EK1 and HR1P, indicating that the complex formed by IBP-EK1 and HR1P has better thermal stability.

Example 8: Peptide and antibody combined to inhibit Delta variant pseudovirus infection experiment

An experiment was conducted to use peptides and antibodies in combination to inhibit Delta variant pseudovirus infection (Reference: Pan C, Cai L, Lu H, Qi Z, Jiang S. Combinations of the first and next generations of human immunodeficiency virus (HIV) fusion inhibitors exhibit a highly potent synergistic effect against enfuvirtide-sensitive and -resistant HIV type 1 strains. J Virol. 2009 Aug; 83(16): 7862-72).

The specific experimental process is as follows:

(1) Expi293 cells were used to express the S309 monoclonal antibody (PDB ID: 6WS6) and purified using a protein G column. The buffer system was then replaced with PBS using a 50 kD ultrafiltration tube.

S309 Fab Heavy Chain:

S309 Fab light chain:

10933 Fab heavy chain:

10933 Fab light chain:

(2) Digest the target cells and count them (Caco-2: 6000 cells/well), add 100 μL per well into a 96-well cell culture plate, and incubate in a 37°C incubator for 24 h to allow the cells to grow and attach completely.

(3) Based on the previously measured IC50 of IBP-EK1 peptide and S309 monoclonal antibody for inhibiting Delta pseudovirus, calculate the appropriate starting concentrations of peptide and antibody.

(4) Dilute the peptide sample tube, antibody sample tube, and peptide/antibody combination sample tube to the appropriate starting concentration and immediately vortex to mix. Add to the first row of a 96-well round-bottom dilution plate, with three replicate wells for each sample. In addition, set up three virus control wells without drug and three cell control wells without virus.

(5) Dilute the drug in multiple ratios and add prepackaged Delta pseudovirus to each well except the cell well, and incubate in a 37°C incubator for 45 min.

(6) Discard the supernatant in the cell culture plate, mix the drug-pseudovirus mixture in the 96-well round-bottom plate by pipetting, transfer 100 μl of the mixture to the 96-well cell plate, and incubate in a 37°C incubator.

(7) After incubation for 10 to 12 hours, the medium was replaced with DMEM medium containing serum.

(8) After incubation for 48 h, discard the cell supernatant, add 50 μL of lysis buffer to each well, and place on a shaker for 45 min at room temperature for lysis.

(9) Pipette 30 μL of lysate from each well of the 96-well plate and transfer to the corresponding 96-well opaque white plate.

(10) Add 30 μL luciferase substrate from the first row to the last row, place the white plate into the ELISA reader, and read the value of each well.

(11) The inhibition rate of the peptide/antibody/peptide-antibody combination on pseudovirus infection was calculated based on the values of the virus pores and cell pores.

(12) Based on CalcuSyn, the CI values and dose reduction values of peptide-antibody combination at different inhibition rates were obtained (reference Chou, T.C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58, 621-681, doi:10.1124/pr.58.3.10 (2006)).

The long-acting coronavirus polypeptide fusion inhibitor IBP-EK1 prepared according to the method of the present invention can be used in combination with antibodies targeting different epitopes of SARS-CoV-2. The combination of IBP-EK1 and S309 monoclonal antibody inhibits SARS-CoV-2 and its VOC mutants, with a synergistic index as low as 0.1; after the combination, the dosage of S309 monoclonal antibody is generally reduced by at least 10 times (as shown in Table 3 and Figure 7). This synergistic effect is unexpected. The class 3 antibody S309 targeting RBD was selected in this paper, and the class 1 antibody 10933 (PDB ID: 6XDG) targeting RBM was also selected; the monoclonal antibody heavy chain and light chain expression plasmids (sequences see above) preserved in our laboratory were used to transfect Expi293 cells and purified using rProtein G column materials.

Table 8 shows the synergistic coefficient and the dose reduction value of the combined use of IBP-EK1 polypeptide and 10933 monoclonal antibody or S309 monoclonal antibody at 50% inhibition of pseudovirus infection. CI, namely synergistic coefficient.

Table 8

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (12)

A method for prolonging the effect of a coronavirus polypeptide therapeutic agent, the method comprising coupling an IgG Fc binding peptide to a coronavirus polypeptide therapeutic agent, wherein the coronavirus polypeptide therapeutic agent comprises an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids; Preferably, the IgG Fc binding peptide is coupled to the N-terminus or C-terminus of the coronavirus polypeptide therapeutic agent; Preferably, the IgG Fc binding peptide is coupled to the coronavirus polypeptide therapeutic agent directly or via a linker; Preferably, the joint is a flexible joint or a rigid joint; Preferably, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, and n is an integer from 1 to 20; Preferably, the coronavirus polypeptide therapeutic agent is a coronavirus polypeptide fusion inhibitor. A fusion polypeptide comprising a coronavirus polypeptide therapeutic agent and an IgG Fc binding peptide, wherein the coronavirus polypeptide therapeutic agent comprises an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4 or a variant thereof with substitution, insertion, deletion or addition of one or more amino acids; Preferably, the IgG Fc binding peptide is coupled to the N-terminus or C-terminus of the coronavirus polypeptide therapeutic agent; Preferably, the IgG Fc binding peptide is coupled to the coronavirus polypeptide therapeutic agent directly or through a linker; Preferably, the joint is a flexible joint or a rigid joint; Preferably, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is an integer from 1 to 20. Preferably, the fusion polypeptide comprises the amino acids shown in SEQ ID NO:5 or a variant thereof which substitutes, inserts, deletes or adds one or more amino acids; Preferably, the coronavirus polypeptide therapeutic agent is a coronavirus polypeptide fusion inhibitor. A nucleic acid encoding the fusion polypeptide according to claim 2. A vector comprising the nucleic acid according to claim 3, Optionally, wherein the vector comprises nucleotides encoding a purification tag, nucleotides encoding a leader and/or a regulatory element; Preferably, the purification tag is selected from the group consisting of His tag, GST tag, MBP tag, SUMO tag or NusA tag; Preferably, the regulatory element is selected from a promoter, a terminator and/or an enhancer. A host cell comprising the nucleic acid according to claim 3 or the vector according to claim 4; Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cell is a yeast cell, an animal cell and/or an insect cell, and/or the prokaryotic cell is an Escherichia coli cell. A composition comprising the fusion polypeptide according to claim 2, the nucleic acid according to claim 3, the vector according to claim 4 and/or the host cell according to claim 5 and a pharmaceutically acceptable carrier; Preferably, the composition comprises an additional anti-coronavirus drug; Preferably, the composition is in the form of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories or lyophilized powders; Preferably, the anti-coronavirus drug is an antibody or a small molecule drug; Preferably, the coronavirus is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2; Preferably, the antibody is ambavirimab, romisvir, sotrovimab, imdevimab, casirivimab, tixagevimab, cilgavimab, bamlanivimab, etesevimab, amubarvimab and/or romlusevimab; Preferably, the small molecule drug is mononavir, namatevir, ritonavir and/or azithromycin. Use of an IgG Fc binding peptide for increasing the efficacy of a coronavirus polypeptide therapeutic against a coronavirus, wherein the IgG Fc binding peptide is coupled to a coronavirus polypeptide therapeutic, wherein the coronavirus polypeptide therapeutic comprises an amino acid sequence selected from SEQ ID NO:1-3 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO:4 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids; Preferably, the IgG Fc binding peptide is coupled to the N-terminus or C-terminus of the coronavirus polypeptide therapeutic agent; Preferably, the IgG Fc binding peptide is coupled to the coronavirus polypeptide therapeutic agent directly or via a linker; Preferably, the joint is a flexible joint or a rigid joint; Preferably, the linker is (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, and n is an integer from 1 to 20; Preferably, the coronavirus is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2; Preferably, the coronavirus polypeptide therapeutic agent is a coronavirus polypeptide fusion inhibitor. An in vitro method for inhibiting the proliferation of coronavirus in a cell, comprising contacting the cell with the fusion polypeptide of claim 2 or the composition of claim 6; Preferably, the coronavirus is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2. Use of the fusion polypeptide of claim 2 or the composition of claim 6 in the preparation of a medicament or a kit for inhibiting the proliferation of coronaviruses, treating and/or preventing diseases or symptoms caused by coronaviruses; Preferably, the coronavirus is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2. The use according to claim 9, wherein the drug is in the form of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories or lyophilized powders. A method for increasing the efficacy of a coronavirus polypeptide therapeutic against a coronavirus or a method for increasing the binding ability of an IgG Fc binding peptide to an IgG protein, the method comprising coupling an IgG Fc binding peptide to a coronavirus polypeptide therapeutic, wherein the coronavirus polypeptide therapeutic comprises an amino acid sequence selected from SEQ ID NO: 1-3 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids, and the IgG Fc binding peptide comprises an amino acid sequence of SEQ ID NO: 4 or a variant thereof with a substitution, insertion, deletion or addition of one or more amino acids; Preferably, the IgG Fc binding peptide is coupled to the N-terminus or C-terminus of the coronavirus polypeptide therapeutic agent; Preferably, the IgG Fc binding peptide is coupled to the coronavirus polypeptide therapeutic agent directly or via a linker; Preferably, the joint is a flexible joint or a rigid joint; Preferably, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, and n is 1-20. integer; Preferably, the coronavirus polypeptide therapeutic agent is a coronavirus polypeptide fusion inhibitor. The fusion polypeptide of claim 2 is used to increase the efficacy of coronavirus polypeptide therapeutic agents against coronaviruses or to increase the binding ability of IgG Fc binding peptides to IgG proteins.