Anti-sars-cov-2 humanized nanobodies and uses thereof

Abstract

The application relates to the technical field of antibodies, in particular to an anti-novel coronavirus humanized nanobody, the humanized nanobody has low immunogenicity risk, can effectively block the combination of wild type, Delta mutant and Omicron mutant novel coronavirus SARS-COV-2 RBD protein and human ACE2 receptor protein, and has significant novel coronavirus neutralization activity; therefore, the humanized nanobody, nucleic acid, expression vector and host cell provided by the application have good application prospects in the preparation of drugs for treating and / or preventing novel coronavirus.

Description

[0001] This application is a divisional application of Chinese invention patent application 202210493578.X (application number: 202210493578.X, application date: 2022-05-07, invention title: humanized nanobody against novel coronavirus and its application). Technical Field

[0002] This invention belongs to the field of antibody technology. More specifically, it relates to humanized nanobodies against the novel coronavirus and their applications. Background Technology

[0003] The successful application of monoclonal antibodies in cancer detection and targeted therapy has revolutionized tumor treatment. However, 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, limiting their widespread clinical application.

[0004] Nanobodies are currently the smallest antibody molecules, with a molecular weight only 1 / 10 that of ordinary antibodies. In addition to possessing the antigenic reactivity of monoclonal antibodies, nanobodies also have some unique functional characteristics, such as small molecular weight, high stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, simple humanization, and low preparation cost. They almost perfectly overcome the shortcomings of traditional antibodies, such as long development cycle, low stability, and harsh storage conditions.

[0005] Multiple variants of the novel coronavirus (SARS-CoV-2) have been discovered. These variants are those resulting from mutations in a single gene base or deletions of certain bases within the original viral genome. These mutations or deletions alter the properties of the virus, potentially leading to changes in 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 impact immunogenicity and preventative immunity.

[0006] While existing broad-spectrum small-molecule antiviral drugs such as Remdesivir have shown some efficacy against wild-type novel coronaviruses, they lack efficacy and specificity against wild-type, Delta variant, and Omicron variants. Clinically, there is a lack of specific drugs for novel coronaviruses. Therefore, there is an urgent need to develop drugs that have specific efficacy against both wild-type and mutant strains of the novel coronavirus. Summary of the Invention

[0007] The purpose of this invention is to provide a single-domain antibody against the novel coronavirus, wherein the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO:5 to 14.

[0008] Another object of the present invention is to provide a humanized nanobody against the novel coronavirus, the antibody comprising the single-domain antibody. This humanized nanobody has a low immunogenicity risk and can effectively block the binding of the wild-type, Delta variant, and Omicron variant SARS-CoV-2 RBD protein to the human ACE2 receptor protein, exhibiting significant neutralizing activity against the novel coronavirus.

[0009] Another object of the present invention is to provide a nucleic acid that encodes the single-domain antibody or the humanized nanobody.

[0010] The present invention also provides an expression vector carrying the nucleic acid.

[0011] The present invention also provides a host cell that carries the nucleic acid, contains the expression vector, or is capable of expressing the single-domain antibody, or is capable of expressing the humanized nanobody.

[0012] The present invention also provides a pharmaceutical composition comprising the single-domain antibody, the humanized nanobody, the nucleic acid, the expression vector, or the host cell.

[0013] The present invention also provides the use of the single-domain antibody, the humanized nanobody, the nucleic acid, the expression vector, and the host cell in the preparation of drugs for treating and / or preventing the novel coronavirus.

[0014] The present invention also provides a method for treating and / or preventing the novel coronavirus, comprising administering an effective dose of the pharmaceutical composition to a novel coronavirus infected subject. Attached Figure Description

[0015] Figure 1 It is a plasmid image containing the target gene.

[0016] Figure 2 This is a graph showing the results of the neutralizing activity of humanized nanobodies against the wild-type SARS-CoV-2 RBD protein.

[0017] Figure 3 This is a graph showing the results of the neutralizing activity of humanized nanobodies against the Delta variant of the novel coronavirus SARS-CoV-2 RBD protein.

[0018] Figure 4This is a graph showing the neutralizing activity of humanized nanobodies against the Omicron mutant SARS-CoV-2 RBD protein. Detailed Implementation

[0019] 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.

[0020] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0021] The present invention provides a single-domain antibody against the novel coronavirus, wherein the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO: 5 to 14.

[0022] In some embodiments, the amino acid sequence of the single-domain antibody is shown in SEQ ID NO:10.

[0023] The present invention provides a humanized nanobody against the novel coronavirus, wherein the antibody comprises the single-domain antibody.

[0024] In this invention, the "nanobody" refers to a heavy chain antibody lacking the light chain (e.g., derived from camel cells), and a single-domain antibody obtained by cloning its variable region. It is the smallest functional antigen-binding fragment with a relative molecular mass (Mr) of only about 15,000. Nanobodies are characterized by their small molecular weight, high stability, good solubility, ease of expression, and low immunogenicity.

[0025] This invention also includes variants, derivatives, and analogs of the nanobodies described herein. As used herein, the terms “variant,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the nanobodies of this invention. The polypeptide variants, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify this polypeptide or the original polypeptide sequence, or a fusion polypeptide). These variants, derivatives, and analogs, as defined herein, are within the scope well known to those skilled in the art.

[0026] Furthermore, other amino acid sequences that do not substantially affect the activity, expression level, and stability of the nanobody described in this invention may be added to the amino or carboxyl terminus of the nanobody. These added amino acid sequences are beneficial for expression (e.g., signal peptides), for purification (e.g., 6×His sequences), or other sequences that can promote the activity, expression level, or stability of the nanobody.

[0027] In some embodiments, the humanized nanobody further includes a half-life extension domain.

[0028] In a preferred embodiment, the half-life extension domain is selected from the immunoglobulin Fc region or the serum albumin binding domain.

[0029] In a preferred embodiment, the half-life extension domain is the immunoglobulin Fc region.

[0030] In a preferred embodiment, the amino acid sequence of the Fc region of the immunoglobulin is shown in SEQ ID NO:15.

[0031] In some embodiments, the amino acid sequence of the humanized nanobody is as shown in any one of SEQ ID NO:16 to 25.

[0032] In a preferred embodiment, the amino acid sequence of the humanized nanobody is shown in SEQ ID NO:21.

[0033] The present invention also provides a nucleic acid that encodes the single-domain antibody or the humanized nanobody.

[0034] Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. 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 ligated into a vector.

[0035] The present invention also provides an expression vector carrying the nucleic acid.

[0036] 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, an expression vector can be formed by selecting a commercially available vector and then operatively linking the nucleotide sequence encoding the nanobody of this invention to an expression regulatory sequence.

[0037] The present invention also provides a host cell that carries the nucleic acid, contains the expression vector, or is capable of expressing the single-domain antibody, or is capable of expressing the humanized nanobody.

[0038] 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 nanobodies include *Escherichia coli*, yeast cells, insect cells, COS cells, and CHO cells. After obtaining the transformed host cells, the cells can be cultured under conditions suitable for expressing the nanobodies of this invention to express the nanobodies; the expressed nanobodies are then isolated.

[0039] The present invention also provides a pharmaceutical composition comprising the single-domain antibody, the humanized nanobody, the nucleic acid, the expression vector, or the host cell.

[0040] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0041] In this invention, a "pharmaceutically acceptable carrier" can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents. Specifically, it can be any one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and combinations thereof. Of course, a "pharmaceutically acceptable carrier" may also include trace amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, to extend the shelf life or potency of the antibody.

[0042] Furthermore, the use of the single-domain antibody, the humanized nanobody, the nucleic acid, the expression vector, and the host cell in the preparation of drugs for treating and / or preventing the novel coronavirus is also within the scope of protection of this invention.

[0043] The present invention also provides a method for treating and / or preventing the novel coronavirus, comprising administering an effective dose of the pharmaceutical composition to a novel coronavirus infected subject.

[0044] The present invention has the following beneficial effects:

[0045] This invention provides a humanized nanobody against the novel coronavirus. This humanized nanobody has a low risk of immunogenicity and can effectively block the binding of the wild-type, Delta variant, and Omicron variant SARS-CoV-2 RBD protein to the human ACE2 receptor protein, exhibiting significant neutralizing activity against the novel coronavirus. Therefore, the humanized nanobody, nucleic acid, expression vector, and host cell provided by this invention have great application prospects in the preparation of drugs for the treatment and / or prevention of the novel coronavirus.

[0046] The embodiments of the present invention will now be described in detail with reference to examples.

[0047] Example 1: Humanization Design of Humanized Nanobodies Against Novel Coronavirus

[0048] Camel-derived nanobody against novel coronavirus (see CN202210089563.7, the amino acid sequences of its CDR1, CDR2, and CDR3 are shown in SEQ ID NO:1-3, and the amino acid sequence of VHH is shown in SEQ ID NO:1-3). As shown in IDNO:4, the camel-derived nanobody (VHH number RX011) was designed for humanization using the CDR-grafting method. First, MOE software was used to model the camel-derived nanobody for homology. Based on the model structure and the inventors' experience, key amino acid residues affecting the conformational stability of the antigen-binding region were analyzed. Then, the human immunoglobulin database was searched, and human IGVH sequences with high homology to the camel-derived nanobody were used as templates for humanization. The camel-derived nanobody was compared with the matching human IGVH sequences. Based on the inventors' experience, the sites on the camel-derived nanobody that were inconsistent with the human IGVH sequence, affecting the conformational stability of the antigen-binding region, were analyzed, and whether humanization replacement could be performed in the model were observed. Based on different degrees of humanization replacement, a total of 10 VHH amino acid sequences for anti-novel coronavirus humanized nanobodies were designed, numbered RX012-RX021. Their specific amino acid sequences are shown in Table 1.

[0049] SEQ ID NO:1:RCTFNWDG

[0050] SEQ ID NO:2: ISSSGQEP

[0051] SEQ ID NO:3:AAAQYTGASYSILRDQVGYDY

[0052] SEQ ID NO:4:

[0053] QVQLVESGGGPVQAGGSLRLSCTCSRCTFNWDGMGWFRQAPGKEREFVATISWSGQEPAYADSVKGRFTISRDKPKNTVYLQMTSLKSEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTRVTVSA

[0054] Table 1

[0055]

[0056]

[0057] For ease of verification, the huFc constant region was chosen as the expression tag, and its specific amino acid sequence is shown in Table 2:

[0058] Table 2

[0059]

[0060] Example 2: Preparation of humanized nanobodies against novel coronavirus

[0061] Transient protein expression:

[0062] Image of plasmid containing the target gene as shown below Figure 1 As shown, a 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.

[0063] 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.

[0064] Purification of the complex sample:

[0065] 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.

[0066] Antibodies were 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.

[0067] Sample concentration was determined using the Biotek-Epoch-Take-3 assay. Antibody concentration was detected using the A280 method, 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 antibody was calculated according to the Lambert-Beer law. If the sample concentration was too low, ultrafiltration concentration was required using an ultrafiltration concentrator (…). Following the general operating procedure provided in the instruction manual, concentrate the sample concentration to >0.5 mg / mL using the Ultra-15 Centrifugal Filter Devices (30 kDa). Collect the concentrated sample, sterilize it using a 0.22 μm sterile syringe filter (Cobbat, PES, 0.22 μm, 13 mm diameter), and then aliquot and freeze for later use.

[0068] Ten humanized nanobodies against the novel coronavirus were directly linked with hIgG1-Fc (SEQ ID NO:15) via VHH amino acid sequences RX012-RX021 to obtain humanized nanobodies against the novel coronavirus, numbered R1383-R1392 respectively; RX011 was directly linked with hIgG1-Fc (SEQ ID NO:15) to obtain camel-derived nanobodies, numbered R1382.

[0069] Table 3

[0070]

[0071]

[0072]

[0073]

[0074] The expression level and purity of the humanized nanobody against the novel coronavirus are shown in Table 4. The results show that the expression level and purity of the humanized antibody are both ideal.

[0075] Table 4

[0076]

[0077]

[0078] Example 3: Neutralizing Activity of Humanized Nanobody Against Wild-type SARS-CoV-2

[0079] The neutralizing activity of humanized nanobodies against the wild-type SARS-CoV-2 RBD protein was detected using a competitive assay, with R1382 from Example 2 serving as the control group. The specific experimental steps are as follows:

[0080] Envelope conditions:

[0081] Viral protein: Wild-type SARS-CoV-2 RBD protein, 2 μg / mL;

[0082] Coating solution: 50mm pH 9.51 CB;

[0083] Encapsulation volume: 100ul / well;

[0084] Coating temperature: 2℃-8℃;

[0085] Wrapping time: 18 hours;

[0086] Blocking solution: containing 1% BSA + 1×PBS;

[0087] Sealing temperature: 37℃;

[0088] Closure time: 3 hours;

[0089] Sample loading: Add 50 μL of the antibody to be tested (all antibodies are initially diluted at a concentration of 5 μg / mL and serially diluted 5-fold), incubate for 30 min, wash 5 times with washing buffer (1×PBS), add 50 μL of ACE2 protein, wash 3 times with washing buffer (1×PBS), and then perform colorimetric detection.

[0090] The results of the neutralizing activity of humanized nanobodies against the wild-type SARS-CoV-2 RBD protein are shown in Table 5. Figure 2 As shown, the results indicate that the humanized nanobodies prepared in Example 2 can effectively block the binding of wild-type SARS-CoV-2RBD protein to human ACE2 receptor protein, and all have neutralizing activity against the novel coronavirus; among them, R1383, R1384, R1385, R1386, R1387, and R1388 showed significant neutralizing activity against the wild-type novel coronavirus.

[0091] Table 5

[0092]

[0093]

[0094] Example 4: Neutralizing activity of humanized nanobody against Delta variant of novel coronavirus

[0095] The neutralizing activity of humanized nanobodies R1383-R1392 against the Delta variant of the novel coronavirus SARS-CoV-2 RBD protein was detected using a competitive assay, with R1382 from Example 2 serving as the control group. The specific experimental steps were the same as in Example 3.

[0096] The results of the neutralizing activity of humanized nanobodies against the Delta variant of the novel coronavirus SARS-CoV-2 RBD protein are shown in Table 6 and... Figure 3 As shown, the results indicate that the humanized nanobodies prepared in Example 2 can effectively block the binding of the Delta variant of the novel coronavirus SARS-CoV-2 RBD protein to the human ACE2 receptor protein, and all have novel coronavirus neutralizing activity; among them, R1385, R1386, R1387, R1388, and R1389 showed significant Delta variant novel coronavirus neutralizing activity.

[0097] Table 6

[0098]

[0099]

[0100] Example 5: Neutralizing activity of humanized nanobody against Omicron mutant strain of novel coronavirus

[0101] The neutralizing activity of humanized nanobodies R1383-R1392 against the Omicron mutant SARS-CoV-2 RBD protein was detected using a competitive assay, with R1382 from Example 2 serving as the control group. The specific experimental steps were the same as in Example 3.

[0102] The results of the neutralizing activity of humanized nanobodies against the Omicron mutant SARS-CoV-2 RBD protein are shown in Table 7 and Figure 4 As shown, the results indicate that the humanized nanobodies prepared in Example 2 can effectively block the binding of the Omicron mutant SARS-CoV-2 RBD protein to the human ACE2 receptor protein, and all have SARS-CoV-2 neutralizing activity; among them, R1385, R1386, R1387, R1388, and R1389 showed significant Omicron mutant SARS-CoV-2 neutralizing activity.

[0103] Table 7

[0104] serial number Omicron variant of SARS-CoV-2 IC50 R1382 0.17nM R1383 0.7067nM R1384 0.8007nM R1385 0.1458nM R1386 0.08825nM R1387 0.1157nM R1388 0.1174nM R1389 0.122nM R1390 0.2232nM R1391 0.2169nM R1392 0.2369nM

[0105] 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 nanobody against the novel coronavirus, characterized in that, The amino acid sequence of the VHH of the antibody is shown in SEQ ID NO:

7.

2. The humanized nanobody according to claim 1, characterized in that, The humanized nanobody has a half-life extended domain.

3. The humanized nanobody according to claim 2, characterized in that, The extended half-life domain is selected from the Fc region of immunoglobulin or the serum albumin-binding domain.

4. A nucleic acid, characterized in that, The nucleic acid encodes the humanized nanobody according to any one of claims 1 to 3.

5. An expression carrier, characterized in that, The expression vector carries the nucleic acid described in claim 4.

6. A host cell, characterized in that, The host cell carries the nucleic acid of claim 4, includes the expression vector of claim 5, or is capable of expressing the humanized nanobody of any one of claims 1 to 3.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the humanized nanobody of any one of claims 1 to 3, the nucleic acid of claim 4, the expression vector of claim 5, or the host cell of claim 6.

8. The use of the humanized nanobody according to any one of claims 1 to 3, the nucleic acid according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating the novel coronavirus.

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