A llama-derived nanobody targeting monkeypox virus e8 protein, detection reagent comprising same and application thereof

Abstract

The present application relates to a llama-derived nanobody targeting monkeypox virus E8 protein, a detection reagent comprising the same and application thereof. It comprises a heavy chain variable region, which comprises a complementarity determining region CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a complementarity determining region CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and three complementarity determining regions CDR3 with an amino acid sequence as shown in SEQ ID NO: 3. The E8-17-mFc nanobody of the present application has a binding constant of 3.25 pM to the monkeypox virus E8 antigen, and has clinical application value for detecting monkeypox virus infection and laboratory application value for detecting E8 antigen.

Description

[0001] Cross-referencing This application claims priority to Chinese Patent Application No. 202610483478.7, filed on April 13, 2026, entitled "A nanobody of alpaca origin targeting monkeypox virus E8 protein, a detection reagent containing the same, and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of immunology, specifically to an alpaca-derived nanobody targeting the E8 protein of monkeypox virus, a detection reagent containing the nanobody, and its application; more specifically, to an alpaca-derived nanobody targeting the E8 protein of monkeypox virus or its antigen-binding fragment, a polynucleotide encoding the nanobody, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a method for its preparation, transformed cells, and a pharmaceutical composition containing the above, and their application in the preparation of medicaments for the prevention, treatment, or detection of poxvirus and / or related poxvirus infections. Background Technology

[0003] To address the public health threat posed by monkeypox virus, especially when the virus has spread and clinical symptoms have appeared, the development of novel monkeypox virus drugs is of great significance. Nanobodies, due to their structural specificity, possess unique advantages and are particularly suitable for antiviral drug development. Recent studies have shown that nanobodies exhibit great potential in the field of antiviral therapy, effectively targeting cryptic epitopes of viral proteins and possessing stronger tissue penetration capabilities. Drug research based on nanobodies shows great development potential and application prospects for the treatment of monkeypox virus.

[0004] Monkeypox virus has six important neutralizing antigens, which are key targets for antibodies to exert their neutralizing effects. Among them, antigen E8 is an important antigen on the surface of the viral particle IMV. The N-terminal domain of antigen E8 contains a carbonic anhydrase folding region (CAH), which can bind to GAGs and chondroitin sulfate on the surface of host cells, promoting the initial contact between the virus and the cell. Therefore, it plays a key role in the process of viral attachment and entry into the host cell. This characteristic makes E8 an ideal target for antiviral drugs.

[0005] However, there are currently few reports on antibodies against the E8 antigen, making it difficult to provide a good application for the treatment and detection of monkeypox virus. Summary of the Invention

[0006] Purpose of the invention To address the problems and needs existing in the prior art, the present invention aims to provide an alpaca-derived nanobody targeting the E8 protein of monkeypox virus with high affinity, a detection reagent containing the nanobody, and its applications. The nanobody E8-17-mFc (also referred to as E8 antibody) screened by this invention exhibits extremely high affinity (the binding constant between the E8-17-mFc nanobody and the monkeypox virus E8 antigen is 3.25 pM), which can significantly improve the detection sensitivity of monkeypox virus and has extremely high clinical application value.

[0007] Solution To achieve the above objectives, the present invention has obtained an alpaca-derived nanobody targeting monkeypox virus E8 protein or its antigen-binding fragment through extensive screening. The nanobody contains a heavy chain variable region, which includes: a complementarity-determining region CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a complementarity-determining region CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and three complementarity-determining regions CDR3 with amino acid sequences as shown in SEQ ID NO: 3.

[0008] As a possible embodiment, the heavy chain variable region further includes four frame regions FR1. 4 (i.e., FR1, FR2, FR3, FR4), wherein FR1 4 is arranged alternately with CDR1, CDR2 and CDR3 in sequence; FR1 (optional) The amino acid sequences of 4 are as shown in SEQ ID NO:9, 10, 11, and 12, respectively.

[0009] As a possible embodiment, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

[0010] And / or, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, human antibody, chimeric antibody, or bispecific or multispecific antibody.

[0011] As a feasible embodiment, an mFc tag is also attached to the C-terminus of the variable region of the heavy chain. Optionally, the amino acid sequence of the mFc tag is shown in SEQ ID NO: 13, and the nucleotide sequence encoding it is shown in SEQ ID NO: 14.

[0012] In a second aspect, a polynucleotide is provided that encodes a nanobody or an antigen-binding fragment thereof as described in the first aspect.

[0013] As a possible embodiment, the polynucleotide is a polynucleotide set, which includes nucleotide sequences encoding complementarity-determining regions CDR1, CDR2, and CDR3 respectively; Optionally, the nucleotide sequence encoding CDR1 is a DNA molecule having the nucleotide sequence shown in SEQ ID NO: 5 or its corresponding mRNA molecule; Optionally, the nucleotide sequence encoding CDR2 is a DNA molecule having the nucleotide sequence shown in SEQ ID NO: 6 or its corresponding mRNA molecule; Optionally, the nucleotide sequence encoding CDR3 is a DNA molecule or its corresponding mRNA molecule having the nucleotide sequence shown in SEQ ID NO: 7.

[0014] As one possible embodiment, it comprises a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO: 8.

[0015] Thirdly, a nucleic acid construct or recombinant vector is provided, comprising a polynucleotide as described in the first aspect, and optionally, at least one expression regulatory element operatively linked to the polynucleotide.

[0016] Fourthly, a transformed host cell is provided, wherein the transformation comprises a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect, or a recombinant vector.

[0017] Fifthly, a pharmaceutical composition is provided comprising the nanobody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct or expression vector as described in the third aspect, or the transformed cells as described in the fourth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0018] As one possible embodiment, the pharmaceutical composition is in the form of a parenteral preparation, nasal spray, suppository, or oral preparation; Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation. Preferably, the nasal spray is selected from aerosols, sprays, and powders; Preferably, the oral formulation is selected from powders, pills, tablets, granules, soft / hard capsules, film-coated agents, and ointments; More preferably, the powder is a granule; More preferably, the pills are small pills; More preferably, the tablet is a sublingual tablet; More preferably, the granules are fine granules.

[0019] A sixth aspect provides a method for preparing nanobodies or antigen-binding fragments thereof as described in the first aspect, the method comprising: expressing the nanobodies or antigen-binding fragments thereof in host cells transformed as described in the fourth aspect under conditions suitable for expression of the nanobodies or antigen-binding fragments thereof, and recovering the expressed nanobodies or antigen-binding fragments thereof from a culture of the host cells.

[0020] In a seventh aspect, the use of the nanobody or antigen-binding fragment thereof described in the first aspect, the nucleotide described in the second aspect, the nucleic acid construct or expression vector described in the third aspect, the transformed cells described in the fourth aspect, the pharmaceutical composition described in the fifth aspect, or the nanobody or antigen-binding fragment thereof prepared by the method described in the sixth aspect in the preparation of a medicament for the prevention, treatment, or detection of poxvirus and / or related poxvirus infections is provided, wherein the poxvirus is optionally orthopoxvirus or optionally monkeypoxvirus.

[0021] Eighthly, the present invention provides a method for preventing or treating poxviruses or related poxviruses, comprising: administering to a subject in need a preventive or therapeutically effective amount of a nanobody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct or expression vector as described in the third aspect above, or transformed cells as described in the fourth aspect above, or a pharmaceutical composition as described in the sixth aspect above.

[0022] Preferably, the poxvirus is an orthopoxvirus, and optionally a monkeypoxvirus.

[0023] In a ninth aspect, the present invention provides a method for detecting poxviruses or related poxviruses, comprising using nanobodies or antigen-binding fragments thereof as described in the first aspect above.

[0024] Preferably, the poxvirus is an orthopoxvirus, and optionally a monkeypoxvirus.

[0025] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0026] Beneficial effects This invention establishes a nanobody affinity library by immunizing alpacas with the E8 antigen; then, using phage display technology, candidate nanobodies E8-17-mFc are screened from it. The nanobodies E8-17-mFc (also referred to as E8 antibodies) screened by this invention have extremely high affinity (the binding constant between E8-17-mFc nanobodies and monkeypox virus E8 antigen is 3.25 pM), which can greatly improve the detection sensitivity of monkeypox virus, has extremely high clinical application value, and can be stored stably, suitable for large-scale production, and has multiple potential uses (detection reagent development, diagnosis, vaccine development, conjugation of targeted drugs (strong tissue penetration ability), analysis of virus models, and tracking of virus life cycle).

[0027] This invention provides potential nanobody drugs for the clinical prevention, treatment and detection of monkeypox virus and related monkeypox virus infections. Attached Figure Description

[0028] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0029] Figure 1 The images show molecular sieve chromatography and SDS-PAGE identification results of the purified E8 antigen expressed by Escherichia coli BL-21 in Example 1.

[0030] Figure 2 In Example 1, the serum antibody titers were determined by ELISA for different numbers of immunizations, where no immunization and four to eight immunizations represent the serum antibody titers when no immunization was performed and when immunizations were performed 4 to 8 times, respectively.

[0031] Figure 3 The image shows the molecular sieve chromatography pattern and SDS-PAGE identification results of the E8-17-mFc nanobody purified and expressed in 293F cells in Example 2.

[0032] Figure 4 Example 3 shows the binding kinetics curve of the nanobody E8-17-mFc to the E8 antigen. Detailed Implementation

[0033] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0034] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0035] To facilitate a better understanding of this invention, certain technical terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For specific definitions and terms in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0036] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0037] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0038] As used herein, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as “only one” or “exactly one” or when “consisting of” is used in the claims, will it refer to only one number or one element of the list.

[0039] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to those in a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0040] The term "nanobody" is equivalent to "single-domain antibody," and these antibodies contain only one variable domain of heavy chain (VHH). Light chain antibody, compared to other antibodies, naturally lacks the light chain.

[0041] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy chain of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy chain of the antibody.

[0042] In this invention, the heavy chain complementarity determination region is represented by CDR, which includes CDR1, CDR2 and CDR3.

[0043] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, for example, the binding reaction of the nanobody of the present invention to the monkeypox virus E8 antigen, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0044] "Antigen-binding fragments" refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Antigen-binding fragments include those selected from Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, biantibodies, peptides containing CDRs, etc.

[0045] The “Fab fragment” consists of a light chain, a heavy chain, CH1, and a variable region.

[0046] The “Fc” region contains two heavy chain segments that contain the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0047] The “Fab′ fragment” contains a light chain and a heavy chain portion that includes the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains. Interchain disulfide bonds are formed between the two heavy chains of the two Fab′ fragments to form the F(ab′)2 molecule.

[0048] The “F(ab′)2 segment” contains two light chains and two heavy chain segments containing the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by disulfide bonds between the two heavy chains.

[0049] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0050] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment containing the VH and VL domains of the antibody, which are contained within a single polypeptide chain. Generally, scFv polypeptides contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.

[0051] A "dual antibody" is a small antigen-binding fragment with two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL or VL-VH). By using a linker so short as to prevent pairing between the two domains on the same chain, the domain pairs with a complementary domain of the other chain to form two antigen-binding sites.

[0052] “Affinity” or “binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the surface plasmon resonance technique described herein. The term “non-binding” protein or cell means not binding to a protein or cell, or not binding to it with a high affinity, i.e., a KD of 1.0 × 10⁻⁶ for binding proteins or cells. -6 M or higher, more preferably 1.0 × 10-5 M or higher, more preferably 1.0 × 10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0 × 10 -2 M or higher.

[0053] For IgG antibodies, the term "high affinity" refers to a KD of 1.0 × 10⁻⁶ for the antigen. -6 M or lower, preferably 5.0 × 10 -8 M or lower, more preferably 1.0 × 10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0 × 10 -11 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype refers to a KD of 10. -6 M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0054] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see U.S. Patent No. 8,278,036, belonging to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0055] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following embodiments are given only for illustrative purposes and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0056] The reagents, culture media, and other chemical materials used in the following embodiments of the present invention are all commercially available products. For example, 293F cell culture medium was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (M293TII-N), and 293F cell feed solution was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (M293-SUPI).

[0057] Some commonly used biological materials, such as competent cells, vectors, helper phages, and cells to be transformed, are also commercially available products. For example, competent cells were purchased from Beijing TransGen Biotech Co., Ltd. (CD501).

[0058] Some synthetic biological materials, such as sequences, which require artificial synthesis, are outsourced to synthetic companies or constructed by other companies. For example, the sequencing primers of this invention and the PCR primers for cloning VHH from the pComb3X vector to the pCAGGS vector were purchased from Ruiboxing Technology Co., Ltd.

[0059] Information on some of the sequences involved in this invention is shown in Table 1 below.

[0060] Table 1. Sequence information involved in this invention

[0061] Example 1: Establishment and screening of an alpaca E8 affinity nanobody library Expression and purification of monkeypox virus E8 antigen A pET28a expression vector containing the E8 antigen sequence (NC_003310.1) was constructed and transformed into E. coli BL-21 competent cells. The cells were cultured in liquid LB medium (kanamycin resistant) until the bacterial culture reached OD500. 600 When the concentration of the protein was approximately 0.4-0.6, IPTG was added to a final concentration of 1 mM to induce expression. After culturing for another 3-5 hours, the bacterial culture was taken and sonicated to rupture the protein. Inclusion bodies were extracted, and the denatured proteins in the renatured inclusion bodies were purified using Hiload 16 / 60 Superdex 200 PG molecular sieve chromatography.

[0062] The results of analyzing the eluent from molecular sieve chromatography are as follows: Figure 1As shown, the peak position of the E8 antigen is around 16.213 mL, and its molecular weight is approximately 35 kDa as determined by SDS-PAGE.

[0063] Establishment of an alpaca immunoaffinity nanobody library for monkeypox virus E8 antigen Alpacas were immunized with the purified E8 antigen protein described above. A total of 8 immunizations were performed. For the initial immunization, Freund's complete adjuvant was mixed with the E8 antigen protein at a 1:1 volume ratio, with a dose of 0.5 mg / alpacia. Each immunization resulted in a final volume of 1.5 mL / alpacia, with immunizations performed two weeks apart. For the 2nd to 5th immunizations, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant (purchased from Beyotime) mixed with the E8 antigen protein (1:1). For the last three immunizations, the adjuvant was replaced with MF59 adjuvant mixed with the E8 antigen protein (1:1), with an immunization dose of 0.5 mg / alpacia for each immunization.

[0064] Blood was collected after each immunization, and serum antibody titers were measured using ELISA. Serum was serially diluted 10-fold starting at 1:100, for a total of five dilutions. A curve was plotted, as shown below. Figure 2 As shown, the results indicate that the serum titer against the E8 antigen reached 10 after the fifth immunization. 4 level.

[0065] Peripheral blood mononuclear cells (PBMCs) were isolated from alpacas two weeks after the eighth immunization, and total RNA was extracted. Two rounds of PCR were performed to amplify the nanobody sequence.

[0066] Phage library screening and sequencing The aforementioned nanobody sequences were constructed into the phage display vector pComb3X to obtain a phage display library. Biotinylated antigen E8 was immobilized with streptavidin magnetic beads, and the phage display library was rotated and incubated. After repeated washing and elution, phages specifically binding to the E8 antigen were obtained. Logarithmically growing *E. coli* TG1 was infected, and helper phage M13K07 was added to obtain a screened and amplified phage sublibrary. A second round of screening was performed using the same method to enrich specific phages.

[0067] The phage titers and enrichment levels after two rounds of screening (enrichment level is calculated by dividing the screening titer by the input titer; a higher value indicates greater antibody enrichment in the input library) are shown in the table below:

[0068] Take 10 μL of phage elution buffer from the second round of screening and serially dilute it 10-fold in 1.5 mL centrifuge tubes, for a total of 12 dilutions. Specifically, take 10 μL of the phage library and dilute it to 100 μL, then take another 10 μL and dilute it to 100 μL, and so on, for a total of 12 dilutions. Add 90 μL of OD to each dilution centrifuge tube. 600The bacterial suspension with a pH of 0.5-0.55 was mixed thoroughly. It was then spread onto solid culture plates, and the phage supernatant from single colonies was subjected to two rounds of ELISA detection. Positive single clones were sequenced to obtain the initial screening nanobody sequence library.

[0069] The results of the two rounds of ELISA initial screening are shown in the table below:

[0070] Example 2: Expression and purification of E8 nanobodies in a eukaryotic expression system Construction of E8 nanobody expression vector The wild-type pCAGGS expression vector was digested with EcoRI and XhoI. The nanobody fragment targeting the E8 antigen was amplified from the above vector pComb3X by PCR and incorporated into the pCAGGS expression vector by homologous recombination to construct the secretory nanobody expression vector pCAGGS-E8-17-mFc, which is fused with the mFc protein tag (coding sequence as shown in SEQ ID NO: 14).

[0071] E8 nanobodies were transfected into eukaryotic cells and purified. The pCAGGS-E8-17-mFc expression vector of the correctly sequenced nanobody was transfected into 293F cells, and the supernatant was collected after 3-5 days. First, the mFc-tagged protein was purified by protein A affinity chromatography, and then the approximately 84 kDa nanobody dimer E8-17-mFc (disulfide bonds will form between Fc fragments) was purified by Hiload 16 / 60superdex 200 PG molecular sieve chromatography.

[0072] Results of detection of eluent in molecular sieve chromatography as follows Figure 3 As shown, the results indicate that the peak position of the nanobody E8-17-mFc is at 14.689 mL, and its molecular weight is approximately 84 kDa as determined by SDS-PAGE electrophoresis.

[0073] Example 3: Determination of the affinity between monkeypox virus E8 antigen and E8 nanobody using surface plasmon resonance technique. On the BIACORE 8K instrument, the affinity between the antigen and antibody was determined by using the CM-5 chip amino-coupled mouse secondary antibody (purchased from Cytiva BR100838), capturing nanobody E8-17--mFc as the stationary phase, and E8 antigen as the mobile phase.

[0074] The results are as follows Figure 4 As shown, calculations using the Single-cycle kinetics using capture program show that the affinity between the nanobody and the antigen is approximately 3.25 pM, indicating a higher affinity.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and 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 of the embodiments of the present invention.

Claims

1. A nanobody or antigen-binding fragment of an alpaca-derived nanobody targeting the E8 protein of monkeypox virus, comprising a heavy chain variable region, the heavy chain variable region comprising: The amino acid sequence is shown as complementarity-determining region CDR1 in SEQ ID NO: 1, the amino acid sequence is shown as CDR2 in SEQ ID NO: 2, and the amino acid sequence is shown as the three complementarity-determining regions CDR3 in SEQ ID NO:

3.

2. The nanobody or its antigen-binding fragment as described in claim 1, wherein, The heavy chain variable region also includes four frame regions FR1. 4, the FR1 4 is arranged alternately with CDR1, CDR2 and CDR3 in sequence; FR1 (optional) The amino acid sequences of 4 are as shown in SEQ ID NO:9, 10, 11, and 12, respectively.

3. The nanobody or its antigen-binding fragment as described in claim 1, wherein, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

4. And / or, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, human antibody, chimeric antibody, or bispecific or multispecific antibody.

4. The nanobody or its antigen-binding fragment as described in claim 3, wherein, An mFc tag is also attached to the C-terminus of the heavy chain variable region, and optionally the amino acid sequence of the mFc tag is shown in SEQ ID NO:

13.

5. A polynucleotide encoding a nanobody or an antigen-binding fragment thereof as described in any one of claims 1 to 4.

6. The polynucleotide of claim 5, wherein, The polynucleotide is a polynucleotide set, which includes nucleotide sequences encoding complementarity-determining regions CDR1, CDR2, and CDR3, respectively. Optionally, the nucleotide sequence encoding CDR1 is a DNA molecule having the nucleotide sequence shown in SEQ ID NO: 5 or its corresponding mRNA molecule; Optionally, the nucleotide sequence encoding CDR2 is a DNA molecule having the nucleotide sequence shown in SEQ ID NO: 6 or its corresponding mRNA molecule; Optionally, the nucleotide sequence encoding CDR3 is a DNA molecule or its corresponding mRNA molecule having the nucleotide sequence shown in SEQ ID NO:

7.

7. The polynucleotide of claim 6, wherein, It contains a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:

8.

8. A nucleic acid construct or recombinant vector comprising a polynucleotide as described in any one of claims 5-7, and optionally, at least one expression regulatory element operatively linked to said polynucleotide.

9. A transformed host cell, wherein the transformation comprises a polynucleotide as described in any one of claims 5-7, a nucleic acid construct as described in claim 8, or a recombinant vector.

10. A pharmaceutical composition comprising a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 4, a polynucleotide as described in any one of claims 5 to 7, a nucleic acid construct or expression vector as described in claim 8, or a transformed cell as described in claim 9, and a pharmaceutically acceptable carrier and / or excipient.

11. The pharmaceutical composition according to claim 10, characterized in that, The pharmaceutical composition is in the form of a parenteral preparation, nasal spray, suppository, or oral preparation; Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation. Preferably, the nasal spray is selected from aerosols, sprays, and powders; Preferably, the oral formulation is selected from powders, pills, tablets, granules, soft / hard capsules, film-coated agents, and ointments; More preferably, the powder is a granule; More preferably, the pills are small pills; More preferably, the tablet is a sublingual tablet; More preferably, the granules are fine granules.

12. A method for preparing a nanobody or an antigen-binding fragment thereof as described in any one of claims 1-4, the method comprising: Under conditions suitable for the expression of the nanobody or its antigen-binding fragment, the host cells transformed according to claim 9 are made to express the nanobody or its antigen-binding fragment, and the expressed nanobody or its antigen-binding fragment is recovered from the culture of the host cells.

13. The use of a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, a nucleotide according to any one of claims 5 to 7, a nucleic acid construct or expression vector according to claim 8, a transformed cell according to claim 9, a pharmaceutical composition according to claim 10 or 11, or a nanobody or antigen-binding fragment thereof prepared by the method of claim 12 in the preparation of a medicament for the prevention, treatment, or detection of poxvirus and / or related poxvirus infections, wherein the poxvirus is optionally orthopoxvirus, and optionally monkeypoxvirus.

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