Anti-sars-cov humanized multivalent binding proteins and uses thereof

Abstract

The present application relates to the technical field of antibodies, in particular to an anti-novel coronavirus humanized multivalent binding protein and application thereof. The multivalent binding protein provided by the present application comprises at least two antigen epitope binding domains, wherein the antigen epitope binding domain is a VHH; the amino acid sequence of the VHH is shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence with at least 95% similarity to SEQ ID NO: 1 or SEQ ID NO: 2. The multivalent binding protein can effectively block the binding of wild-type strains, Delta mutant strains and Omicron mutant strains of SARS-COV-2 RBD proteins to human ACE2 receptor proteins, and has more significant neutralization activity of the novel coronavirus than a monovalent binding protein, and can be widely used for the prevention and treatment of the novel coronavirus.

Description

[0001] This application is a divisional application of Chinese invention patent application 202210724686.3 (application number: 202210724686.3, application date: 2022-06-24, invention title: anti-novel coronavirus humanized multivalent binding protein and its application). Technical Field

[0002] This invention belongs to the field of antibody technology. More specifically, it relates to a humanized multivalent binding protein against the novel coronavirus and its applications. Background Technology

[0003] Traditional monoclonal antibodies have a large molecular weight (150kD), making it difficult to penetrate tissues and resulting in low effective concentrations in tumor areas and insufficient therapeutic effects. In addition, traditional antibodies have high immunogenicity, while modified antibodies are difficult to achieve the original affinity, which limits their widespread clinical application.

[0004] Since its outbreak in 2019, the novel coronavirus (SARS-CoV-2) has spread widely throughout the world. Furthermore, various variants of the novel coronavirus have been discovered, such as the Delta variant first discovered in India in October 2020, and the Omicron variant first discovered in South Africa on November 24, 2021. Variant strains refer to those that have undergone mutations in a single gene base or deletions of certain bases in the original viral genome. Mutations or deletions lead to changes in the properties of the virus, potentially altering its infectivity, host range, transmissibility, virulence, pathogenicity, disease severity, prognosis, and immunogenicity. This can alter the entire clinical course of the disease and its epidemiology, and may even affect immunogenicity and immune prophylaxis, causing severe economic losses, social burden, and other negative impacts. Therefore, there is an urgent need to develop drugs with specific therapeutic effects against wild-type and mutant strains of the novel coronavirus. Summary of the Invention

[0005] The purpose of this invention is to provide a humanized multivalent binding protein against the novel coronavirus and its applications. The multivalent binding protein provided by this invention can effectively block the binding of wild-type, Delta variant, and Omicron variant SARS-CoV-2 RBD proteins to human ACE2 receptor proteins, and compared with monovalent binding proteins, the humanized multivalent binding protein of this invention has more significant neutralizing activity against the novel coronavirus.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a humanized multivalent binding protein against the novel coronavirus, comprising at least two antigen epitope binding domains, wherein the antigen epitope binding domain is VHH; the amino acid sequence of the VHH is as shown in SEQ ID NO:1 or SEQ ID NO:2, or as shown in an amino acid sequence having at least 95% similarity to SEQ ID NO:1 or SEQ ID NO:2.

[0008] In a second aspect, the present invention provides a fusion protein comprising the multivalent binding protein.

[0009] Thirdly, the present invention provides a conjugate comprising the multivalent binding protein.

[0010] Fourthly, the present invention provides a nucleic acid that encodes the multivalent binding protein, or the fusion protein, or the conjugate.

[0011] Fifthly, the present invention provides a recombinant vector carrying the nucleic acid.

[0012] In a sixth aspect, the present invention provides a host cell that carries the nucleic acid or contains the recombinant vector.

[0013] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the multivalent binding protein, the fusion protein, the conjugate, the nucleic acid, the recombinant vector, or the host cell.

[0014] Eighthly, the present invention provides the use of the multivalent binding protein, the fusion protein, the conjugate, the nucleic acid, the recombinant vector, or the host cell in the preparation of a medicament for treating and / or preventing the novel coronavirus.

[0015] In a ninth aspect, the present invention provides a method for preparing the multivalent binding protein, comprising: culturing the host cells and isolating and purifying the multivalent binding protein from the culture product. Attached Figure Description

[0016] Figure 1 This is a structural diagram of the multivalent binding proteins R1406, R1407, R1475, and R1464 of this invention.

[0017] Figure 2 This is a graph showing the neutralizing activity of R1382, R1406, and R1407 against the wild-type novel coronavirus.

[0018] Figure 3 This is a graph showing the neutralizing activity of R1463, R1475, and R1464 against the wild-type novel coronavirus.

[0019] Figure 4 This is a graph showing the neutralizing activity of R1382, R1406, and R1407 against the Delta variant of the novel coronavirus.

[0020] Figure 5 This is a graph showing the neutralizing activity of R1463, R1475, and R1464 against the Delta variant of the novel coronavirus.

[0021] Figure 6 This is a graph showing the neutralizing activity of R1382, R1406, and R1407 against the Omicron mutant strain of the novel coronavirus.

[0022] Figure 7 This is a graph showing the neutralizing activity of R1463, R1475, and R1464 against the Omicron mutant strain of the novel coronavirus. Detailed Implementation

[0023] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0024] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0025] In this invention, the term "amino acid" refers to a naturally occurring or non-naturally occurring carboxyl α-amino acid. The term "amino acid" as used in this application can include both naturally occurring and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three-letter code: Ala, single-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Non-naturally occurring amino acids include, but are not limited to, α-aminoadipic acid, GABA, citrulline, high-citrulline, high-leucine, high-arginine, hydroxyproline, ortholeucine, pyridylalanine, and sarcosine.

[0026] In this invention, the term "amino acid sequence" refers to the order in which amino acids are linked together to form a peptide chain (or polypeptide). The amino acid sequence can only be read in one direction. There are over 100 different types of amino acids, of which 20 are commonly used. This invention does not exclude modifications to the amino acid chain by other substances, such as carbohydrates or lipids, nor is it limited to the 20 commonly used amino acids.

[0027] In this invention, the term "Fc region" refers to the C-terminal region of an immunoglobulin, which is a functional structural unit composed only of CH2 and CH3 in the heavy chain constant domain. The Fc region does not have the ability to bind antigens; however, it has a prolonged half-life and a constant amino acid sequence.

[0028] The present invention provides a humanized multivalent binding protein against the novel coronavirus, characterized in that it comprises at least two antigen epitope binding domains, wherein the antigen epitope binding domain is VHH.

[0029] In some embodiments, the amino acid sequence of the VHH is as shown in SEQ ID NO:1 or SEQ ID NO:2, or as shown in an amino acid sequence having at least 95% similarity to SEQ ID NO:1 or SEQ ID NO:2.

[0030] In some embodiments, the number of antigen epitope binding domains is 2-6.

[0031] In some embodiments, the antigen epitope binding domain is linked by a linker peptide. Commonly used linker peptides in the art can be used in this invention.

[0032] In a preferred embodiment, the amino acid sequence of the linker peptide is: GGGGSGGGGSGGGGS (SEQ ID NO:11).

[0033] In a preferred embodiment, the connection is made in series.

[0034] In some embodiments, the multivalent binding protein further includes a half-life extension domain.

[0035] In a preferred embodiment, the half-life extension domain is selected from the immunoglobulin Fc region or the serum albumin binding domain.

[0036] In a preferred embodiment, the immunoglobulin is selected from IgA, IgD, IgE, IgG, and IgM.

[0037] In some embodiments, the half-life extension domain is an immunoglobulin Fc region, and the antigen epitope binding domain is connected to the N-terminus or C-terminus of the Fc region.

[0038] In some embodiments, the multivalent binding protein forms a dimer structure through interchain disulfide bonds in the Fc region.

[0039] In a preferred embodiment, the amino acid sequence of the Fc region is shown in SEQ ID NO:3.

[0040] In some embodiments, the amino acid sequence of the multivalent binding protein is as shown in any one of SEQ ID NO: 6 to 9.

[0041] In some embodiments, the amino acid sequence of the multivalent binding protein may or may not contain a His tag.

[0042] The present invention also provides a fusion protein comprising the multivalent binding protein.

[0043] In this invention, the term "fusion protein" refers to a fusion protein obtained by fusing the multivalent binding protein of this invention with other functional protein fragments.

[0044] The present invention also provides conjugates comprising the multivalent binding protein.

[0045] In this invention, the term "conjugate" refers to a conjugate obtained by coupling the multivalent binding protein of this invention with one or more of the following phases: enzyme phase (such as horseradish peroxidase, alkaline phosphatase, etc.), radioisotope, fluorescent compound or chemiluminescent compound, therapeutic agent, etc. These conjugates can be used to detect the novel coronavirus, prepare drugs for treating and / or preventing the novel coronavirus, or treat the disease caused by the novel coronavirus infection.

[0046] The present invention also provides a nucleic acid that encodes the multivalent binding protein, or the fusion protein, or the conjugate.

[0047] In this invention, nucleic acids are typically RNA or DNA, and the nucleic acid molecules can be single-stranded or double-stranded. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence. DNA nucleic acids are used when they are incorporated into a vector.

[0048] Currently, the nucleic acid molecule sequence encoding the protein of this invention can be obtained entirely through chemical synthesis.

[0049] The present invention also provides a recombinant vector carrying the nucleic acid.

[0050] In this invention, the term "vector" includes plasmids, expression vectors, cloning vectors, viral vectors, etc. Various vectors known in the art can be used. For example, a commercially available vector can be selected, and then the nucleic acid sequence encoding the multivalent binding protein of this invention can be operatively linked to the expression regulatory sequence to form a recombinant vector.

[0051] The present invention also provides a host cell that carries the nucleic acid or contains the recombinant vector.

[0052] In this invention, the term "host cell" includes both prokaryotic and eukaryotic cells. Common examples of prokaryotic host cells include *Escherichia coli* and *Bacillus subtilis*. Host cells used for expressing the multivalent binding protein include *Escherichia coli*, yeast cells, insect cells, COS cells, and CHO cells. After obtaining the transformed host cell, the cell can be cultured under conditions suitable for expressing the multivalent binding protein of this invention to express the multivalent binding protein; then the expressed multivalent binding protein can be isolated.

[0053] The present invention also provides a pharmaceutical composition comprising the multivalent binding protein, the fusion protein, the conjugate, the nucleic acid, the recombinant vector, or the host cell.

[0054] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, binders, flow aids, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavoring agents, etc., can be used for oral administration; buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., can be used for injectable mixtures; and matrices, excipients, lubricants, preservatives, etc., can be used for surface application.

[0055] In some embodiments, the pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0056] The present invention also provides the use of the multivalent binding protein, the fusion protein, the conjugate, the nucleic acid, the recombinant vector, or the host cell in the preparation of medicaments for treating and / or preventing the novel coronavirus.

[0057] The present invention also provides a method for preparing the multivalent binding protein, comprising: culturing the host cells and isolating and purifying the multivalent binding protein from the culture product.

[0058] Example 1: Design and Construction of Humanized Multivalent Binding Protein Against Novel Coronavirus

[0059] Humanized multivalent binding proteins with different structures were designed by tandemly incorporating the Fc region or His tag and the VHH domain. The structures of the modified humanized multivalent binding proteins are shown below. Figure 1As shown, the specific amino acid sequences are shown in Table 1:

[0060] Among them, the parent sequence of R1406 and R1407 is RX011 (amino acid sequence as shown in SEQ ID NO:1), and the sequence structure is as follows: R1406: VHH-Linker-VHH-Linker-VHH-Fc region, R1407: Fc region-VHH-Linker-VHH-Linker-VHH; the parent sequence of R1475 and R1464 is RX017 (amino acid sequence as shown in SEQ ID NO:2), and the sequence structure is as follows: R1475: VHH-Linker-VHH-His tag, R1464: His tag-VHH-Linker-VHH-Linker-VHH; the amino acid sequence of the Fc region is shown in SEQ ID NO:3, and the amino acid sequence of the His tag is HHHHHH (SEQ ID NO:10).

[0061] Using R1382 (amino acid sequence as shown in SEQ ID NO:4) and R1463 (amino acid sequence as shown in SEQ ID NO:5) as controls, the sequence structures are as follows: R1382: RX011 VHH-Fc region, R1463: RX017 VHH-His tag.

[0062] SEQ ID NO:1:

[0063] QVQLVESGGGPVQAGGSLRLSCTCSRCTFNWDGMGWFRQAPGKEREFVATISWSGQEPAYADSVKGRFTISRDKPKNTVYLQMTSLKSEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTRVTVSA

[0064] SEQ ID NO:2:

[0065] QVQLVESGGGVVQPGGSLRLSCTCSRCTFNWDGMGWFRQAPGKGLEFVATISWSGQEPAYADSVKGRFTISRDNSKNTLYLQMTSLRAEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTLVTVSS

[0066] SEQ ID NO:3:

[0067] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0068] SEQ ID NO: 4:

[0069] QVQLVESGGGPVQAGGSLRLSCTCSRCTFNWDGMGWFRQAPGKEREFVATISWSGQEPAYADSVKGRFTISRDKPKNTVYLQMTSLKSEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTRVTVSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0070] SEQ ID NO: 5:

[0071] QVQLVESGGGVVQPGGSLRLSCTCSRCTFNWDGMGWFRQAPGKGLEFVATISWSGQEPAYADSVKGRFTISRDNSKNTLYLQMTSLRAEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTLVTVSSHHHHHH

[0072] Table 1. Amino acid sequences of humanized multivalent binding proteins

[0073]

[0074]

[0075] Example 2: Preparation of humanized multivalent binding protein against novel coronavirus

[0076] Transient protein expression:

[0077] The plasmid containing the target gene is introduced into the host cell Expi293 after forming a cationic complex with the transfection reagent PEI. During the time the plasmid is in the cell, the foreign gene on the plasmid is transcribed and translated into the target protein.

[0078] Expi293 cells were cultured at 37°C, 8% CO2, and 130 rpm. Before transfection, 2E6 cells were seeded into 1L shake flasks (approximately 300 ml) by cell counting. Transfection complex preparation: 750 μg of the target plasmid was added to a 50 ml centrifuge tube containing 15 ml of Opti-MEM reagent, gently mixed, and labeled tube A. 1.5 mg of PEI transfection reagent was added to a 50 ml centrifuge tube containing 15 ml of Opti-MEM reagent, gently mixed, and incubated at room temperature for 5 min; this tube was labeled tube B. The PEI dilution in tube B was added dropwise to the DNA dilution in tube A, gently mixed, and incubated at room temperature for 15 min. After incubation, the PEI-target plasmid complex was added to the Expi293 cells, and the cells were cultured in a shaker at 37°C. Samples were collected after days 7-10.

[0079] Purification of the complex sample:

[0080] The transiently transfected cell expression solution was centrifuged at 9000 rpm for 20 min, and the supernatant was collected and then sterilized by filtration through a 0.22 μm filter membrane. Purification was performed using ProA affinity chromatography. The procedure was as follows: using an AKTA Avant 150 chromatography system, the chromatography column (e.g., MabSelect SuRe LX, GE) was equilibrated with at least 5 CV equilibration buffer (10 mM PBS). The sample was loaded onto the column, allowing the target protein to adsorb onto the column while other impurities permeated and separated. After loading, the column was washed again with at least 5 CV equilibration buffer (10 mM PBS), followed by elution with elution buffer (20 mM NaAc, pH 3.4). Neutralization buffer (1 M Tris, pH 8.0) was pre-added to the collection tube. The volume of neutralization buffer added depended on the estimated concentration of the eluted sample, generally 10% of the elution volume.

[0081] The binding protein was prepared using standard methods, and the expression supernatant was purified by ProA affinity chromatography. The procedure was as follows: using an AKTA Avant 150 chromatography system, the column (e.g., MabSelect SuRe LX, GE) was equilibrated with at least 5 CV equilibration buffer (10 mM PBS). The sample was loaded onto the column, allowing the target protein to adsorb onto the column while other impurities permeated and separated. After loading, the column was washed again with at least 5 CV equilibration buffer (10 mM PBS), followed by elution with elution buffer (20 mM NaAc, pH 3.4). Neutralization buffer (1 M Tris, pH 8.0) was pre-added to the collection tube; the volume of neutralization buffer added depended on the estimated concentration of the eluted sample, generally 10% of the elution volume.

[0082] Sample concentration was determined using the Biotek-Epoch-Take-3 assay. The A280 method was used to detect the binding protein concentration, with an extinction coefficient EC = 1.37 (predicted based on amino acid sequence) and a path length of 0.05 mm (slight differences in path length between wells of the Take-3 plate are automatically corrected). The absorbance of the sample was measured using the instrument, and the concentration of the binding protein was calculated according to the Lambert-Beer law. If the sample concentration was too low, ultrafiltration concentration was required using ultrafiltration concentrators (…). Using the Ultra-15 Centrifugal Filter Devices (30kD), concentrate the sample to >0.5mg / ml according to the general operating procedure provided in the instruction manual; collect the concentrated sample, sterilize it with a 0.22um sterile needle filter (Cobbat, PES, 0.22um, 13mm diameter), and then aliquot and freeze for later use.

[0083] The titer and purity results of the humanized multivalent binding protein against the novel coronavirus are shown in Table 2. The results show that the titer and purity data of the humanized multivalent binding protein are both ideal.

[0084] Table 2. Titer and purity results of humanized multivalent binding protein against novel coronavirus.

[0085]

[0086]

[0087] Example 3: Neutralizing activity of humanized multivalent binding protein against wild-type novel coronavirus

[0088] The neutralizing activity of the humanized multivalent binding protein prepared in Example 2 against the wild-type SARS-CoV-2 RBD protein was detected using a competitive assay, with R1382 and R1463 as controls. The specific experimental steps are as follows:

[0089] Envelope conditions:

[0090] Viral protein: wild-type SARS-CoV-2 RBD protein (Ag27), 2ug / ml;

[0091] Coating solution: 50mm pH 9.51CB;

[0092] Encapsulation volume: 100ul / well;

[0093] Coating temperature: 2℃-8℃;

[0094] Wrapping time: 18 hours;

[0095] Blocking solution: containing 1% BSA + 1×PBS;

[0096] Sealing temperature: 37℃;

[0097] Closure time: 3 hours;

[0098] Sample loading: Add 50 μL of the target binding protein (all binding proteins start at a concentration of 5 μg / mL and are serially diluted 5-fold), incubate for 30 min, wash 5 times with wash buffer (1x PBS), add 50 μL of ACE2 protein, wash 3 times with wash buffer (1x PBS), and then perform colorimetric detection.

[0099] The results of the neutralizing activity of humanized multivalent binding proteins against wild-type SARS-CoV-2 are shown in Table 3. Figure 2 and Figure 3 As shown in Table 3, Figure 2 and Figure 3 The results showed that the humanized multivalent binding protein prepared in Example 2 had neutralizing activity against the novel coronavirus and could effectively block the binding of wild-type SARS-CoV-2 RBD protein to human ACE2 receptor protein. Among them, R1406, R1407, R1475, and R1464 showed significant neutralizing activity against the wild-type novel coronavirus compared with the control group.

[0100] Table 3. Neutralizing activity of humanized multivalent binding proteins against wild-type SARS-CoV-2.

[0101]

[0102]

[0103] Example 4: Neutralizing activity of humanized multivalent binding protein against Delta variant of novel coronavirus.

[0104] The neutralizing activity of the humanized multivalent binding protein prepared in Example 2 against the Delta mutant SARS-COV-2 RBD protein was detected using a competitive method, with R1382 and R1463 as controls. The viral protein coated in the experiment was the Delta mutant SARS-COV-2 RBD protein (Ag101), 2 ug / ml. The specific experimental steps were the same as in Example 3.

[0105] The results of the neutralizing activity of the humanized multivalent binding protein against the Delta variant of SARS-CoV-2 are shown in Table 4. Figure 4 and Figure 5 As shown in Table 4, Figure 4 and Figure 5 The results showed that the humanized multivalent binding proteins prepared in Example 2 could effectively block the binding of Delta mutant SARS-CoV-2 RBD protein to human ACE2 receptor protein, and all had neutralizing activity against the novel coronavirus; among them, R1406, R1407, R1475, and R1464 showed significant neutralizing activity against the novel coronavirus from the Delta mutant strain compared with the control group.

[0106] Table 4. Neutralizing activity of humanized multivalent binding proteins against Delta variant SARS-CoV-2.

[0107] R1382 0.01369 R1406 0.01476 R1407 0.01582 R1463 0.01805 R1475 0.003341 R1464 0.004316

[0108] Example 5: Neutralizing activity of humanized multivalent binding protein against Omicron mutant SARS-CoV-2.

[0109] The neutralizing activity of the humanized multivalent binding protein prepared in Example 2 against the Omicron mutant SARS-COV-2 RBD protein was detected using a competitive assay, with R1382 and R1463 as controls. The viral protein coated in the experiment was the Omicron mutant SARS-COV-2 RBD protein (covsK20), 2 ug / ml. The specific experimental steps were the same as in Example 3.

[0110] The results of the neutralizing activity of humanized multivalent binding proteins against the Omicron mutant strain of SARS-CoV-2 are shown in Table 5. Figure 6 and Figure 7 As shown, the results indicate that the humanized multivalent binding proteins prepared in Example 2 can effectively block the binding of Omicron mutant SARS-CoV-2 RBD protein to human ACE2 receptor protein, and all have novel coronavirus neutralizing activity; among them, R1406, R1407, R1475, and R1464 showed significant Omicron mutant novel coronavirus neutralizing activity compared with the control group.

[0111] Table 5. Neutralizing activity of humanized multivalent binding proteins against Omicron mutant SARS-CoV-2.

[0112] R1382 0.17 R1406 0.1277 R1407 0.1591 R1463 11.1 R1475 0.07167 R1464 0.06166

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A humanized multivalent binding protein against the novel coronavirus, wherein the multivalent binding protein comprises two or three epitope binding domains, the two or three epitope binding domains being linked in series by a linker peptide; the epitope binding domain is VHH; the amino acid sequence of the VHH is shown in SEQ ID NO:2; the amino acid sequence of the linker peptide is shown in SEQ ID NO:

11.

2. Nucleic acid, characterized in that, The nucleic acid encodes the multivalent binding protein of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector carries the nucleic acid described in claim 2.

4. A host cell, characterized in that, The host cell carries the nucleic acid described in claim 2.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the multivalent binding protein of claim 1.

6. The use of the multivalent binding protein of claim 1, the nucleic acid of claim 2, the recombinant vector of claim 3, the host cell of claim 4, or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating novel coronavirus infection.

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