WO2023179543A1

WO2023179543A1 - Nano-antibody r14 construct and application thereof

Info

Publication number: WO2023179543A1
Application number: PCT/CN2023/082523
Authority: WO
Inventors: 王奇慧; 高福; 刘红辉; 刘博�; 仵丽丽; 韩鹏程
Original assignee: 中国科学院微生物研究所
Priority date: 2022-03-21
Filing date: 2023-03-20
Publication date: 2023-09-28
Also published as: WO2023179545A1; CN114805560B; CN116813759A; CN116813757A; CN114805560A

Abstract

Provided are a construct (comprising a multivalent nano-antibody and a nano-antibody fusion protein) based on a nano-antibody R14 specifically binding to SARS-CoV-2 RBD, a related product thereof, and a use thereof. The construct (comprising a multivalent nano-antibody and a nano-antibody fusion protein) based on the nano-antibody R14 specifically binding to SARS-CoV -2 RBD can effectively inhibit SARS-CoV -2 infection and variant strain infection thereof, be administered as a nebulized drug, can directly reach the lungs, takes effect quickly, has a long half-life period, and provides a more effective treatment strategy for clinically preventing or treating novel coronavirus and variant strain infection thereof.

Description

The construct of Nanobody R14 and its application

cross reference

This application claims priority to the Chinese patent application with application number 202210278872.9 and the invention title "Construct of Nanobody R14 and its Application", submitted on March 21, 2022, the entire content of which is incorporated herein by reference.

Technical field

This application relates to the field of biomedicine, specifically to the construct of Nanobody R14 and its application, and more specifically to multivalent Nanobodies, Nanobody fusion proteins, and coding based on Nanobody R14 that specifically binds SARS-CoV-2 RBD. Polynucleotides thereof, nucleic acid constructs containing the polynucleotides, expression vectors containing the nucleic acid constructs, transformed cells containing the above polynucleotides, nucleic acid constructs or expression vectors, and medicines containing any of the above products The composition and its application in the preparation of drugs for preventing or treating the new coronavirus, and its application in the preparation of reagents or kits for detecting the new coronavirus or diagnosing the new coronavirus infection.

Background technique

The epidemic caused by the new coronavirus (SARS-CoV-2) of the Coronaviridae family continues to spread around the world. In addition, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), both belonging to the Coronaviridae family, are also major pathogens targeting the human respiratory system, mainly through droplets and air It is transmitted through aerosols and contact and is highly contagious. Therefore, this type of virus that causes respiratory diseases seriously endangers public health safety.

The current COVID-19 outbreak has promoted the development of various vaccines and antiviral drugs. Vaccination can effectively prevent the occurrence of serious infectious diseases. However, vaccines are only suitable for uninfected people, and the development cycle is long and the clinical research process is complex. For confirmed patients, they can only be treated with antiviral drugs. One of the antiviral drugs is therapeutic antibody drugs, which mainly refer to neutralizing antibodies. Neutralizing antibody drugs mainly prevent the infection by binding to antigens on the surface of pathogenic microorganisms. Specific molecules expressed by pathogenic microorganisms bind to cell surface receptors to achieve a "neutralization" effect. The SARS-CoV-2 virus has a glycosylated spike protein (S) on its surface. The S protein can interact with the host cell receptor protein ACE2 and trigger membrane fusion. Therefore, blocking the binding of the S protein to ACE2 is An effective way to treat COVID-19 infection.

Conventional monoclonal antibodies are generally administered through intravenous injection. However, the drug concentration of monoclonal antibodies administered through intravenous injection from the systemic circulation into the lungs is very low, which greatly reduces the antiviral effect of the neutralizing antibody itself, resulting in inability to Effectively reduces viral load in the lungs. The new coronavirus initially infects the upper respiratory tract, and its first interaction with the immune system mainly occurs on the surface of the respiratory mucosa. In view of this, for the new coronavirus infected through the respiratory tract, while paying attention to serum antibodies, it is also necessary to detect the mucosal Think, design and develop appropriate Antibody drugs. For example, aerosol administration can enable antibody drugs to reach higher local concentrations in the respiratory tract, which can more effectively block viral infection when the virus invades.

Nanobodies have attracted much attention as therapeutic drugs. For example, the nanobody drug Caplacizumab (Cablivi ^TM ) developed by Ablynx is used to treat acquired thrombotic thrombocytopenic purpura and is the first nanobody drug approved for marketing; For example, the nanobody drug candidate ALX-0171 is a trivalent form of nanobody used to treat respiratory syncytial virus (RSV) infection in children. It is administered by aerosol and has entered the clinical phase II stage (https://clinicaltrials .gov), these all suggest that nanobody drugs are safe and feasible.

Therefore, the development of nanobody drugs targeting the new coronavirus and suitable for respiratory mucosal immunity has potential clinical application value and prospects.

Contents of the invention

Purpose of invention

The purpose of this application is to provide constructs (including multivalent Nanobodies and Nanobody fusion proteins) based on Nanobodies R14 that specifically bind SARS-CoV-2 RBD, polynucleotides encoding them, and nucleic acids containing the polynucleotides. Constructs, expression vectors containing the nucleic acid constructs, transformed cells containing the above-mentioned polynucleotides, nucleic acid constructs or expression vectors, and pharmaceutical compositions containing any of the above products and their preparation for preventing or treating the new coronavirus Application in virus medicines, application in preparing reagents or kits for detecting novel coronavirus or diagnosing novel coronavirus infection.

The construct of the present application based on the Nanobody R14 that specifically binds SARS-CoV-2 RBD (including multivalent Nanobodies and Nanobody fusion proteins) can effectively inhibit SARS-CoV-2 infection and its variant strain infection, and can Aerosol administration can directly reach the lungs, has a rapid onset of action and a long half-life, providing a more effective treatment strategy for infections caused by the new coronavirus and its mutant strains.

solution

In order to achieve the above purpose, this application provides the following technical solutions:

In the first aspect, the present application provides a multivalent Nanobody, which includes more than two VHH chains of Nanobodies that specifically bind SARS-CoV-2 RBD, wherein the specific binding is SARS-CoV-2 RBD The VHH chain of the Nanobody includes the following CDRs:

The amino acid sequence is CDR1 shown in SEQ ID NO:1 (i.e., GFTLDYYAIG),

CDR2 with the amino acid sequence shown in SEQ ID NO:2 (i.e., CISSSDGSTSYADSVKG), and

The amino acid sequence is CDR3 shown in SEQ ID NO:3 (i.e., TPATYYSGRYYYQCPAGGMDY).

In a specific embodiment, the VHH chain of the Nanobody that specifically binds SARS-CoV-2 RBD also includes 4 framework regions FR1-4, which are staggered with the CDR1, CDR2 and CDR3 in order ;

Preferably, the amino acid sequences of FR1-4 are as follows: SEQ ID NO: 4 (i.e., QVQLQESGGGLVQPGGSLRLSCAVS), SEQ ID NO:5 (i.e., WFRQAPGKEREGVS), SEQ ID NO:6 (i.e., RFTISRDNAKNTVYLQMNSLKPEDTALYYCAA), and SEQ ID NO:7 (i.e., WGQGTQVTVSS).

In a preferred embodiment, the VHH chain of the Nanobody that specifically binds SARS-CoV-2 RBD has an amino acid sequence as shown in SEQ ID NO: 8, or is identical to the amino acid sequence shown in SEQ ID NO: 8 An amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity; preferably, the amino acid sequence of the VHH chain is as shown in the following SEQ ID NO: 8:

Among them, the underlined parts are the framework regions FR1-4 respectively, and the black parts are CDR1, CDR2 and CDR3 of the heavy chain variable region respectively.

In a preferred embodiment 1, the multivalent Nanobody is composed of two or more, preferably three, VHH chains of the Nanobody that specifically binds SARS-CoV-2 RBD, connected through a Linker;

Wherein, the Linker is (GGGGS)n, where n=1, 2, 3, or 4, preferably, n=2 or 3.

As a further preference of specific embodiment 1, the multivalent Nanobody is a trivalent Nanobody and has the amino acid sequence shown in SEQ ID NO: 9:

In a preferred embodiment II, the multivalent Nanobody is an IgM pentamer formed from the following fusion protein, and the structure of the fusion protein from the N-terminus to the C-terminus is shown in formula (I):
ALB(I)

in,

A is a single VHH chain of the Nanobody that specifically binds to the SARS-CoV-2 RBD, or a multivalent Nanobody as described in the preferred embodiment I above;

B is the Fc fragment of human IgM; Preferably, the Fc fragment of human IgM has a structure such as SEQ ID NO: 10 (i.e., VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQD TAIRVFAIPPSFASIFLTKSTKLTCLLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLY NVSLVMSDTAGTCY), or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10;

L is (GGGGS)m, where m=0, 1, 2, 3, or 4.

As a preferred embodiment of the above fusion protein, it has the amino acid sequence shown in SEQ ID NO: 11:

In the second aspect, the present application provides a Nanobody fusion protein. The structure of the Nanobody fusion protein from the N-terminus to the C-terminus is as shown in Formula (I):
ALB(I)

in,

A is a single VHH chain of a Nanobody that specifically binds to SARS-CoV-2 RBD, and the VHH chain of a Nanobody that specifically binds to SARS-CoV-2 RBD is as defined in the first aspect above; or, A is According to the multivalent Nanobody described in the preferred specific embodiment I of the first aspect above, that is, the VHH chain of more than two, preferably three, Nanobodies that specifically bind SARS-CoV-2 RBD is passed through the Linker Multivalent Nanobodies composed of linkers, wherein the Linker is (GGGGS)n, where n=1, 2, 3, or 4, preferably, n=2 or 3;

B is the Fc fragment of human IgM; preferably, the Fc fragment of human IgM has the amino acid sequence shown in SEQ ID NO: 10, or has at least 95%, 96% of the amino acid sequence shown in SEQ ID NO: 10 An amino acid sequence with %, 97%, 98% or 99% sequence identity;

L is (GGGGS)m, where m=0, 1, 2, 3, or 4.

As a preferred embodiment of the above-mentioned fusion protein, it has the amino acid sequence shown in SEQ ID NO: 11.

In a third aspect, the present application provides a polynucleotide encoding a multivalent Nanobody as described in the above first aspect, or encoding a Nanobody fusion protein as described in the above second aspect.

In specific embodiments, the polynucleotide is DNA or mRNA;

Preferably, the polynucleotide encodes a multivalent Nanobody according to the preferred specific embodiment 1 of the above-mentioned first aspect. Further preferably, the polynucleotide includes as shown in SEQ ID NO: 12 (i.e., ) Nucleotide sequence;

Preferably, the polynucleotide encodes a Nanobody fusion protein as described in the second aspect above, further preferably, the polynucleotide includes a protein such as SEQ ID NO: 13 (i.e., CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGTCTCTGGATTTACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTAGTAGTGATGGTAGCACATC GTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAAAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCC ) the nucleotide sequence shown.

In a fourth aspect, the present application provides a nucleic acid construct comprising the polynucleotide as described in the third aspect above, and optionally, at least one expression control element operably linked to the polynucleotide. For example, histidine tag, stop codon, etc.

In a fifth aspect, the present application provides an expression vector comprising the nucleic acid construct described in the fourth aspect.

In the sixth aspect, the present application provides a transformed cell, which includes the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, or the expression vector as described in the fifth aspect. .

In the seventh aspect, the present application provides a pharmaceutical composition, which includes the multivalent Nanobody as described in the first aspect, the Nanobody fusion protein as described in the second aspect, and the nanobody fusion protein as described in the third aspect. Polynucleotides, nucleic acid constructs as described in the fourth aspect, expression vectors as described in the fifth aspect, or transformed cells as described in the sixth aspect, and pharmaceutically acceptable vectors and/or vectors. form agent.

In specific embodiments, the pharmaceutical composition may be in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated agents, pellets, sublingual tablets and ointments;

Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, a topical liquid, an injectable or a pushable preparation.

The dosage of the active ingredient of the pharmaceutical composition of the present application varies depending on the administration target, the target organ, symptoms, administration method, etc., and may take into consideration the type of dosage form, administration method, age and weight of the patient, The patient's symptoms, etc., are determined based on the doctor's judgment.

In the eighth aspect, the present application provides a multivalent Nanobody as described in the first aspect, a Nanobody fusion protein as described in the second aspect, a polynucleotide as described in the third aspect, The nucleic acid construct described in the fourth aspect, the expression vector described in the fifth aspect, the transformed cell described in the sixth aspect or the pharmaceutical composition described in the seventh aspect are used for the prevention and treatment of /or application in drugs to treat novel coronavirus infection.

In specific embodiments, the new coronavirus can be the original strain of SARS-CoV-2 and/or the mutant strain of SARS-CoV-2;

Preferably, the SARS-CoV-2 variant strains are Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B. 1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain, further preferably Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain.

In the ninth aspect, the present application provides a multivalent Nanobody as described in the first aspect, a Nanobody fusion protein as described in the second aspect, a polynucleotide as described in the third aspect, The nucleic acid construct described in the fourth aspect, the expression vector as described in the fifth aspect, or the transformed cells as described in the sixth aspect are used in preparing reagents for detecting a new coronavirus or for diagnosing a new coronavirus infection. or application in a kit.

In the tenth aspect, the present application provides a novel coronavirus detection kit, which includes the multivalent Nanobody as described in the above first aspect, the Nanobody fusion protein as described in the above second aspect, and the above third aspect. The polynucleotide, the nucleic acid construct as described in the fourth aspect, the expression vector as described in the fifth aspect, or the above The transformed cells described in the sixth aspect.

beneficial effects

This application is for the drug development of Nanobody constructs for the new coronavirus. The constructs based on Nanobody R14 of this application can bind to SARS-CoV-2 RBD with high affinity and can neutralize SARS-CoV-2 with high neutralizing activity. Prototype strains and a series of mutant strains of pseudoviruses and live viruses, all of which show that the construct based on Nanobody R14 is a novel coronavirus that can bind to the SARS-CoV-2 RBD with high affinity and has high neutralizing activity. Virus (SARS-CoV-2) Nanobodies.

In particular, the inventors have demonstrated through a series of experiments that the trivalent Nanobody (TR14) and IgM pentameric form (MR14) based on Nanobody R14 of the present application have significantly improved performance compared to its monomer (i.e., Nanobody R14). With its neutralizing activity and significantly extended half-life, it achieves mucosal immunity, can limit the reproduction of the virus and further cross the mucosal barrier, control the mucosal spread of the virus, and provides a potential aerosolized solution for the clinical prevention and treatment of the new coronavirus. The new antibody drug administered can achieve sensitive and reliable detection of the new coronavirus.

Description of the drawings

One or more embodiments are exemplified by the pictures in the corresponding drawings, and these exemplary illustrations do not constitute limitations to the embodiments. The word "exemplary" as used herein means "serving as an example, example, or illustrative." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or superior to other embodiments.

Figure 1 is a schematic structural diagram of the Nanobody constructs TR14 and MR14 constructed in Example 1 of the present application;

Figure 2 is a diagram of the R14 protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of the present application;

Figure 3 is a diagram of the TR14 protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of the present application;

Figure 4 is a diagram of the MR14 protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of the present application;

Figure 5 is the SARS-CoV-2 RBD-his protein (A) described in Example 2 of the present application, the RBD-his protein (B) of the mutant strain Omicron (B.1.1.529) subtype BA.1 and SDS-PAGE identification results of RBD-his protein (C) of Omicron (B.1.1.529) subtype BA.2.

Figure 6 is a schematic diagram of the effect of the three antibodies measured in Example 5 of the present application on neutralizing SARS-CoV-2 prototype strain pseudovirus infection.

Figure 7 is a schematic diagram of the effect of the three antibodies measured in Example 5 of the present application on neutralizing SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus infection.

Figure 8 is a schematic diagram of the effect of the three antibodies measured in Example 5 of the present application on neutralizing SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 pseudovirus infection.

Figure 9 is a schematic diagram of the effect of the three antibodies measured in Example 5 of the present application on neutralizing SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.2 pseudovirus infection.

Figure 10 is the neutralizing activity of the three antibodies measured in Example 7 of the present application against the pseudovirus before and after atomization; where A is the neutralizing activity of Nanobody R14 against the SARS-CoV-2 prototype virus before and after atomization. Neutralizing activity results of pseudovirus strains, B is the neutralizing activity result of Nanobody construct TR14 against SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus, C is the result of Nanobody construct MR14 against SARS-CoV-2 variant strain Delta ( B.1.617.2) Neutralizing activity results of pseudoviruses.

Figure 11 is the half-life of the three antibodies in blood measured in Example 8 of the present application.

Figure 12 is the atomization system used in Embodiment 8 of the present application.

Figure 13 is the half-life of the three antibodies in the lungs measured in Example 8 of the present application.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present application, not all of them. Embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of this application. Unless expressly stated otherwise, throughout the specification and claims, the term "comprises" or its variations such as "comprises" or "comprising" will be understood to include the stated elements or components, and to Other elements or other components are not excluded.

In addition, in order to better explain the present application, numerous specific details are given in the following detailed description. It will be understood by those skilled in the art that the present application may be practiced without certain specific details. In some embodiments, raw materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail in order to highlight the gist of the present application.

The present application is described in detail below.

definition

"Nanobody" is a "heavy chain single domain antibody". This type of antibody only contains a heavy chain variable region (VHH, variable domain of heavy chain of heavy-chain antibody). Compared with other antibodies, the light chain is naturally missing. .

Due to their biophysical advantages, nanobodies can be easily atomized and delivered directly to the lungs through inhalers to treat infections caused by respiratory viruses and are considered to be very promising antibody-based drugs.

When referring to a ligand/receptor, antibody/antigen or other binding pair, "specific" binding refers to the determination of the presence or absence of said protein, such as the Nanobodies of the present application, in a heterogeneous population of proteins and/or other biological agents. Binding reaction with SARS-CoV-2 RBD protein. Thus, under specified conditions, a specific ligand/antigen binds to a specific receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The reagents, enzymes, culture media, antibiotics, milk and other chemical materials used in the following examples of this application are all commercially available products. For example, TRIzol is purchased from Invitrogen, and Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.

Some commonly used biological materials, such as competent cells, vectors, helper phages, cells to be transformed, etc. are also commercially available products. For example, pCAGGS vector was purchased from MiaoLingPlasmid; 293F cells, HEK293T cells, etc. were purchased from ATCC; Series Sensor Chip SA chip was purchased from GE Healthcare; Vero cells were purchased from ATCC CCL81.

Some synthetic biological materials, such as primers, sequences and other materials that require artificial synthesis, are entrusted to synthesis companies. For example, the coding sequence of TR14 in this application was synthesized by Nanjing Genscript Biotechnology Co., Ltd.

Example 1: Construction, expression and purification of antibodies based on the trivalent form of Nanobody R14 (TR14) and the IgM pentameric form (MR14)

The schematic structural diagram of the single-chain Nanobody and its trivalent form and IgM pentameric form in this embodiment is shown in Figure 1.

The basic nanobody R14 used in this laboratory was obtained by simultaneously immunizing alpacas with SARS-CoV-2 RBD protein and SARS-CoV-2NTD protein, constructing an antibody library, and screening using phage display technology; the single-chain nanobody R14 The amino acid sequence of the VHH chain is shown in SEQ ID NO:8. It can bind SARS-CoV-2 RBD with high affinity and specificity (binding constant is less than 1E-10M), and in pseudovirus neutralization experiments, it can bind to SARS-CoV-2 RBD with high affinity and specificity. High neutralizing activity and neutralizing SARS-CoV-2 pseudovirus, all of which indicate that R14 nanobody is a novel coronavirus (SARS-CoV-2) that can bind to SARS-CoV-2 RBD with high affinity and has high neutralizing activity. ) Alpaca-derived Nanobodies.

A signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) is connected to the 5' end of the coding sequence of the R14VHH chain of the above single-chain Nanobody (as shown in SEQ ID NO: 14), and 6 histidine tags are connected to the 3' end ( hexa-His-tag) and the translation stop codon TGA, it was constructed into the pCAGGS vector (purchased from Invitrogen) through the restriction endonuclease sites EcoRI and XhoI, and then the resulting recombinant vector was transfected into 293F In cells (purchased from Invitrogen), R14-his protein was expressed. After the cell culture medium containing the target protein is purified by nickel ion affinity chromatography (HisTrap ^TM excel ((GE Healthcare))) and gel filtration chromatography (Superdex ^TM 75Increase 10/300GL column (GE Healthcare)), a relatively pure product can be obtained The target protein. The size of R14-his protein identified by SDS-PAGE is about 15KD. The results are shown in Figure 2.

Through two (GGGGS) ₃ sequences, the coding sequences of three Nanobody R14VHH chains shown in SEQ ID NO: 14 are connected in a head-to-tail format (directly synthesized by Nanjing Genscript Biotechnology Co., Ltd.), in which The 5' end is connected to the signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15), the 3' end is connected to the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon TGA, and the restriction endonuclease is used. EcoRI and XhoI sites were constructed into the pCAGGS vector (purchased from Invitrogen), and then the resulting recombinant vector was transfected into 293F cells (purchased from Invitrogen) to express TR14-his protein. After the cell culture medium containing the target protein is purified by nickel ion affinity chromatography (HisTrap ^TM excel ((GE Healthcare))) and gel filtration chromatography (Superdex ^TM 200Increase 10/300GL column (GE Healthcare)), a relatively pure product can be obtained The target protein. The size of TR14-his protein identified by SDS-PAGE is about 50KD. The results are shown in Figure 3.

Through homologous recombination, the coding sequence of the Nanobody R14VHH chain (shown in SEQ ID NO:14) Connect to the Fc coding sequence of the human IgM antibody (as shown in SEQ ID NO: 16), connect the signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) to its 5' end, and connect the translation termination codon TGA to its 3' end , construct it into the pCAGGS vector (purchased from Invitrogen) through the restriction endonuclease sites EcoRI and XhoI, and obtain the pCAGGS-R14-IgM Fc recombinant expression vector. Connect the translation stop codon TGA to the 3' end of the coding sequence of the J chain (Joining chain) (as shown in SEQ ID NO: 17), and construct it into the pCAGGS vector through the restriction endonuclease sites EcoRI and XhoI. (purchased from Invitrogen), the pCAGGS-J chain recombinant expression vector was obtained. The above two recombinant expression vectors pCAGGS-R14-IgM Fc and pCAGGS-J chain were co-transfected into 293F cells (purchased from Invitrogen) to express the R14-IgM Fc fusion protein and J chain, and the two were then self-assembled. , forming the IgM form of the MR14 protein. The obtained MR14 protein was purified by HiTrap ^™ IgM Purification HP (GE Healthcare) and Superose ^™ 6increase 10/300GL (GE Healthcare), and then identified by SDS-PAGE. The size of the MR14 protein identified by SDS-PAGE is about 70KD, and the results are shown in Figure 4.

Example 2: Expression and purification of RBD of SARS-CoV-2 and its mutant strains

Connect the coding sequence of 6 histidine tags (hexa-His-tag) to the 3' end of the coding sequence of the RBD protein of the original strain of SARS-CoV-2 (its amino acid sequence is shown in SEQ ID NO: 18) and the translation stop codon TGA, which was constructed into the pCAGGS vector (purchased from Invitrogen) through the restriction endonuclease sites EcoRI and XhoI, and then the resulting recombinant vector was transfected into 293F cells (purchased from Invitrogen) for Expression of SARS-CoV-2 RBD-his protein.

The coding sequence of the RBD protein (the amino acid sequence of which is shown in SEQ ID NO: 19) of the SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529 ) The 3' end of the coding sequence of the RBD protein of subtype BA.2 (the amino acid sequence of which is shown in SEQ ID NO: 20) is connected to the coding sequence and translation of 6 histidine tags (hexa-His-tag). Stop codon TGA, expressed by bac-to-bac baculovirus expression system (Invitrogen). Transform the pFastbac1 plasmid containing the target gene into DH10Bac competent cells to generate recombinant bacmids (bacmids). The recombinant bacmid bacmids were transfected into Sf9 cells for virus amplification, and protein expression was performed in Hi5 cells. After 48 h of expression, the Hi5 cell supernatant was collected, and the soluble protein was purified by nickel affinity chromatography using HisTrap ^TM excel (GE Healthcare).

After the cell culture medium containing the target protein is purified by the nickel ion affinity chromatography column HisTrap ^TM excel (GE Healthcare) and the gel filtration chromatography Superdex ^TM 200Increase 10/300GL column (GE Healthcare), a relatively pure target protein can be obtained. SARS-CoV-2 RBD-his protein, RBD-his protein of mutant strain Omicron (B.1.1.529) subtype BA.1, and RBD-his protein of Omicron (B.1.1.529) subtype BA.2 The SDS-PAGE identification size is about 30KD, as shown in Figures 5A to C respectively.

Example 3: Surface plasmon resonance technology detects the binding ability of each antibody to the RBD protein of the original strain of SARS-CoV-2 and its mutant strains

Surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). Specific steps are as follows:

The single-chain Nanobody R14 and its constructs TR14 and MR14 prepared in the above examples were biotinylated respectively, and then immobilized on the Series Sensor Chip SA chip (Cytiva Life Sciences); PBST buffer (2.7mM KCl, 137mM NaCl, 4.3mM Na ₂ HPO ₄ , 1.4mM KH ₂ PO ₄ , 0.05% Tween) were used to dilute the RBD proteins of the original SARS-CoV-2 strain and its mutant strains prepared in the above examples, from Load samples from low to high concentrations onto the chip one by one. Calculation of binding kinetic constants was performed using BIAevaluation software 8K (Biacore, Inc.). The equilibrium dissociation constant (K _D ) between each antibody and each RBD is shown in Table 1. The results in Table 1 show that the single-chain nanobody R14 and its constructs TR14 and MR14 can all interact with the SARS-CoV-2 prototype strain. And the RBD proteins of mutant strains Omicron subtype BA.1 and Omicron subtype BA.2 bind with higher affinity.

Table 1. Affinity results between single-chain Nanobody R14 and its constructs TR14 and MR14 and the RBD of the original and mutant strains of SARS-CoV-2

Example 4: Packaging of SARS-CoV-2 original strain and mutant strain pseudovirus

1) Separately encode the SARS-CoV-2 original strain (WT) and variant strains Delta (B.1.617.2), Omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529) ) The gene for the last 18 amino acids of the S protein of subtype BA.2 was removed, and the remaining sequences of the S protein were synthesized (synthesis services provided by Suzhou Genewise), and SARS-CoV-2-WT-S-del18 and B.1.617 were obtained. The nucleotide sequences of 2-S-del18, B.1.1.529-BA.1-S-del18 and B.1.1.529-BA.2-S-del18 genes are as follows: SEQ ID NO: 21～24 shown.

2) Clone the protein gene obtained in 1) into the pCAGGS vector to obtain expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18, pCAGGS-B.1.617.2-S-del18, and pCAGGS-B .1.1.529-BA.1-S-del18 and pCAGGS-B.1.1.529-BA.2-S-del18.

The packaging steps for SARS-CoV-2 original strain and mutant strain pseudovirus are as follows:

a. Cell preparation: Plate HEK293T cells (purchased from ATCC CRL-3216) in a 10cm cell culture dish, so that the cell confluence density reaches about 80% the next day. The culture medium was DMEM medium containing 10% FBS.

b. Transfection: Take the expression plasmid of each S protein in the above step 2), and use PEI to transfect 30 μg plasmid/10cm cell culture dish. Mix the target plasmid and PEI in a ratio of 1:3 before transfection, and transfect after 4-6 hours. Culture medium (DMEM medium containing 10% FBS), culture at 37°C for 24 hours.

c. Toxin addition: Add the pseudovirus packaging skeleton virus G*VSV-delG (purchased from Wuhan Shumi Brain Science and Technology Co., Ltd.) to the above-transfected HEK293T cells, incubate at 37°C for 2 hours, and change the culture medium (containing 10% FBS) DMEM medium), And add VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank), and continue to culture in the incubator for 30 hours.

d. Collect the virus: Collect the supernatant and centrifuge it at 3000 rpm for 10 minutes, filter it through a 0.45 μm sterile filter in a clean workbench, remove cell debris, aliquot, and freeze in a -80°C refrigerator.

The SARS-CoV-2 prototype strain (SARS-CoV-2WT) and variant strains Delta (B.1.617.2) and Omicron (B.1.1.529) subtypes BA.1 and Omicron (B.1.1. 529) Pseudovirus of subtype BA.2.

Example 5: Detection of Antibody Neutralizing Pseudovirus Infection

The purified single-chain Nanobody R14 and its constructs TR14 and MR14 (prepared from Example 1) were diluted 5-fold to the ninth gradient (2.56pg/mL), and the dilution was mixed with 1.6×10 ⁴ TCID ₅₀ A series of pseudoviruses of the original SARS-CoV-2 strain and variant strains obtained in Example 4 were mixed separately, mixed and incubated at 37°C for 1 hour, and then added to 96 cells pre-inoculated with Vero cells (purchased from ATCC CCL81) in the orifice plate. After incubation for 18 to 20 hours, it was detected by CQ1 Confocal Quantitative Image Cytometer (Yokogawa). Based on the number of cells with GFP fluorescence, the neutralizing ability of the antibody against the pseudoviruses of the above-mentioned series of SARS-CoV-2 prototype strains and variant strains Delta, BA.1 and BA.2 was calculated. The results are shown in Figure 6~ 9, and the result statistics are shown in Table 2; the results show that compared with the single-chain Nanobody R14, the pseudovirus neutralizing effects of its constructs TR14 and MR14 are both improved.

Table 2. Neutralizing ability of single-chain nanobody R14 and its constructs TR14 and MR14 against pseudoviruses of the original strain and mutant strains of SARS-CoV-2

Note: IC ₅₀ (μg/mL) is the half inhibitory concentration of the antibody.

Example 6: Detection of Antibody Neutralizing Live Virus Infection

In this example, the neutralizing effect of each antibody on the live virus of the new coronavirus was measured through a live virus neutralization test based on cytopathic effect (CPE). Specific steps are as follows:

The purified single-chain Nanobody R14 and its TR14 and MR14 (prepared in Example 1) were diluted 2-fold to the 11th gradient, with 4 replicate wells for each gradient, 50 μL per well, and the diluent was Incubate with an equal volume of 100 TCID ₅₀ of the original SARS-CoV-2 strain or its variant strain Delta and Omicron subtype BA.1 at 37°C. After 1 hour, the mixture was added to the suspended Vero cells and incubation was continued at 37°C for 3 days. Observe and record cell lesions. _IC50 of Nanobodies and their constructs were calculated using GraphPad Prism 7.0. The experiments were all conducted in the biosafety level three laboratory (BSL3) of the Chinese Center for Disease Control and Prevention.

Single-chain nanobody R14 and its TR14 and MR14 are effective against live viruses of the original strain of the new coronavirus and its mutant strains The neutralizing effects are shown in Table 3. The results in Table 3 show that TR14 and MR14 have good inhibitory effects on both the original strain and the mutated live virus of the new coronavirus.

Table 3. Neutralizing ability of single-chain nanobody R14 and its constructs TR14 and MR14 against live viruses of original and mutant strains of SARS-CoV-2

Note: IC ₅₀ (μg/mL) is the half inhibitory concentration of the antibody. # indicates that at the lowest measured concentration of 0.001 μg/mL, it still has 100% inhibitory effect on live viruses.

Example 7: Detection of antibody stability before and after atomization

The single-chain Nanobody R14 and its constructs TR14 and MR14 were atomized using an Aerogen Solo (Aerogen Inc., Chicago, USA) nebulizer, respectively, and then used in an all-glass SKC (Eighty Four, PA, USA) containing 20 mL of PBS. ) Collect the aerosolized antibodies and conduct a pseudovirus neutralization test as described in Example 5. The results are shown in Figure 10, where A is the neutralizing activity result of Nanobody R14 against SARS-CoV-2 prototype strain pseudovirus before and after aerosolization, and B is the effect of Nanobody construct TR14 on SARS-CoV-2 Neutralizing activity results of mutant strain Delta (B.1.617.2) pseudovirus. C is the neutralizing activity result of Nanobody construct MR14 against SARS-CoV-2 mutant strain Delta (B.1.617.2) pseudovirus. . The results of Figure 10 show that the neutralizing activity of the nanobody constructs TR14 and MR14 of the present application against the pseudovirus of the new coronavirus prototype strain or its mutant strain remains stable before and after atomization, suggesting that they are both suitable for aerosolization. route of administration.

Example 8: Determination of antibody half-life

The half-life of nanobody R14 and its constructs TR14 and MR14 in the blood was studied by intraperitoneal injection. The specific procedures were as follows: 6-8 week old female SPF grade BALB/c mice (purchased from Vital River )) were anesthetized with isoflurane, and 200 μl of 2 mg/mL (20 mg/kg body weight) of R14, TR14, and MR14 were slowly injected via intraperitoneal injection. Blood samples were collected via orbital bleeding at 1 hour, 6 hours, 10 hours, 24 hours, 48 hours, 72 hours and 96 hours after injection. After centrifugation, the supernatant was taken and the concentration of antibodies in the serum was determined by ELISA. The half-life (t _1/2 ) of Nanobody R14 and its constructs TR14 and MR14 was calculated using the software PKSolver. The results of the half-life of R14, TR14 and MR14 in the blood are shown in Figure 11. The results of Figure 11 show that compared to the monovalent antibody R14, the constructs TR14 and MR14 show a significantly longer half-life, which is beneficial to improving the resistance of the antibody in the body. Viral effect.

Next, the residence time of Nanobody R14 and its constructs TR14 and MR14 in the lungs was studied through aerosol administration. Use the atomization system (as shown in Figure 12) to atomize the three antibodies R14, TR14 and MR14 at 20 mg/ml respectively. After the mice were continuously exposed to the atomization chamber for 10 minutes, they were injected intraperitoneally with three antibodies. bromine Mice were anesthetized with ethanol, and bronchoalveolar lavage fluid was collected at 0, 1, 6, and 24 hours. The antibody concentration in the lavage fluid was determined by ELISA, and PKSolver was used to calculate the half-life of the nanobody in the lungs (t _{1 /2} ). The results of the half-life of R14, TR14 and MR14 in the lungs are shown in Figure 13. The results of Figure 13 show that compared to the monovalent antibody R14, the constructs TR14 and MR14 show a significantly longer half-life, which is beneficial to improving the delivery of antibodies through the aerosol route. The antiviral effect of the drug in the body.

The above results show that the nanobody R14-based constructs TR14 and MR14 of the present application have the potential to be developed into highly neutralizing active antibody drugs, especially aerosolized drugs, for the treatment of infections caused by the new coronavirus and its mutant strains.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent substitutions are made to some of the technical features; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions in the embodiments of the present application.

Industrial applicability

The construct of the present application based on the nanobody R14 that specifically binds to the SARS-CoV-2 RBD can effectively inhibit SARS-CoV-2 infection and its mutant strain infection. It can be administered by aerosol, can directly reach the lungs, and has a rapid onset of effect. , and has a long half-life, providing a more effective treatment option for the clinical prevention or treatment of infections caused by the new coronavirus and its mutant strains.

Claims

An application of an IgM pentamer formed by the following Nanobody fusion protein in the preparation of drugs for preventing or treating new coronavirus infection, characterized in that the structure of the Nanobody fusion protein from the N-terminus to the C-terminus is as follows: I) as shown:

A-L-B (I)

in,

A is a Nanobody that specifically binds SARS-CoV-2 RBD;

B is the Fc fragment of human IgM;

L is (GGGGS)m, where m=0, 1, 2, 3, or 4.
The application according to claim 1, characterized in that the Nanobody that specifically binds SARS-CoV-2 RBD includes the following CDR: CDR1 with an amino acid sequence such as SEQ ID NO:1, and CDR1 with an amino acid sequence such as SEQ ID NO CDR2 shown in :2, and CDR3 whose amino acid sequence is shown in SEQ ID NO:3.
The application according to claim 2, characterized in that the Nanobody that specifically binds SARS-CoV-2 RBD also includes 4 framework regions FR1-4, and the FR1-4 and the CDR1, CDR2 and CDR3 staggered in sequence;

Preferably, the FR1-4 are shown in SEQ ID NO: 4, 5, 6, and 7 respectively.
The application according to any one of claims 1-3, characterized in that the Nanobody that specifically binds SARS-CoV-2 RBD has an amino acid sequence as shown in SEQ ID NO: 8.
The application according to any one of claims 1-4, characterized in that the Fc fragment of the human IgM has the amino acid sequence shown in SEQ ID NO: 10.
The application according to any one of claims 1-5, characterized in that the Nanobody fusion protein has an amino acid sequence as shown in SEQ ID NO: 11.
The application according to any one of claims 1 to 6, characterized in that the new coronavirus is an original strain of SARS-CoV-2 and/or a mutant strain of SARS-CoV-2;

Preferably, the SARS-CoV-2 variant strains are Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B. 1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain and/or Omicron (B.1.1.529) subtype BA.2 strain.
The application according to any one of claims 1 to 7, characterized in that the drug is in the form of a nasal spray, oral preparation, suppository or parenteral preparation;

Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft/hard gums sachets, film-coated tablets, pellets, sublingual tablets and ointments;

Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, a topical liquid, an injectable or a pushable preparation.