A nanobody targeting NS3a and its application

Abstract

The present invention discloses a nanobody targeting NS3a and its use in a chemically induced disaggregation system. The nanobody is composed of a heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein the amino acid sequence of the CDR1 is as shown in SEQ ID NO:5, the amino acid sequence of the CDR2 is as shown in SEQ ID NO:6, and the amino acid sequence of the CDR3 is as shown in SEQ ID NO:7. The nanobody of the present invention can specifically recognize and bind to the NS3a protein, and its interaction with NS3a can be controllably regulated by small molecule inducers, thereby constructing a chemically induced disaggregation system. The system can be applied to synthetic biology fields such as artificial regulation of protein interactions, cell engineering, and gene expression regulation.

Description

Technical Field

[0001] The present invention relates to the fields of antibody engineering and synthetic biology, and in particular to a method for screening and preparing a nano antibody targeting NS3a and its application. Background Art

[0002] In biological and medical research, regulating the interaction between proteins is an important means of studying cell functions and developing new therapies. Currently, there are relatively mature chemically induced dimerization systems, but there are few easy-to-use chemically induced disaggregation systems. NS3a is a fusion protein of hepatitis C virus NS3-NS4A, which combines the catalytic domain of NS3 and 11 amino acids on NS4A that help stabilize it, and is orthogonal to human proteins. Nanobodies, as the smallest known antibodies, have the advantages of high stability, small molecular weight, easy production and low cost while retaining the high affinity of traditional antibodies, making them an important choice for regulatory elements. The small molecule drug grazoprevir is an FDA-approved NS3a inhibitor with a high safety profile. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to prepare a nanobody that can specifically recognize NS3a and whose binding activity can be controllably regulated by a small molecule inducer (for example, Grazoprevir), thereby providing a new tool molecule for constructing a chemically induced disaggregation system (CIDiss).

[0004] The present invention relates to a method for screening nanobodies. NS3a protein is used as an antigen and recombinantly expressed to possess a unique biotinylation tag. In the first step, based on a synthetic humanized nanobody library, three rounds of enrichment are performed, combined with magnetic bead separation and a gradient reduction of antigen concentration, to obtain nanobodies that bind to the antigen. In the second step, a small molecule competitive elution method is used. Once the nanobodies bind, they are eluted with the small molecule grazoprevir, thereby collecting nanobodies that can be activated by the small molecule to interact with NS3a. In the third step, a flow-through method is used to first bind NS3a with the small molecule grazoprevir, and then collect nanobodies that cannot bind. After a total of five rounds of screening, the nanobodies obtained in each round are tested using enzyme-linked immunosorbent assay (ELISA) to assess the screening efficacy. Once the efficacy reaches a certain level, a phage monoclonal ELISA assay is performed to identify phage expressing nanobodies that bind to the antigen without the addition of a small molecule, but do not bind with the addition of a small molecule. The identified nanobody nucleic acid sequence was transferred to a new expression vector and expressed periplasmically to maintain the disulfide bonds within the nanobody, thus maintaining its stability. Affinity chromatography was then used to purify the nanobody to obtain a high-purity yield of 10-20 mg / L. Biochemical characterization was performed using bio-layer interferometry (BLI) to determine the binding kinetics of the bound nanobody by performing a concentration gradient assay. Finally, a molecular sieving co-migration assay was performed to determine the binding of the nanobody to NS3a in the presence and absence of the small molecule grazoprevir.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0006] In a first aspect, the present invention provides a nanobody targeting NS3a, wherein the nanobody is composed of a heavy chain variable region comprising CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is as shown in SEQ ID NO: 5, the amino acid sequence of the CDR2 is as shown in SEQ ID NO: 6, and the amino acid sequence of the CDR3 is as shown in SEQ ID NO: 7.

[0007] In some embodiments of the invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4, or has a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 4.

[0008] The second aspect of the present invention provides a binding molecule targeting NS3a, which comprises the Nanobody as described in the first aspect of the present invention; preferably, the binding molecule is a monovalent or multivalent Nanobody or fusion protein comprising one or more of the Nanobodies; the fusion protein is preferably an Fc fusion protein or a multispecific antibody.

[0009] The third aspect of the present invention provides a chemically induced disaggregation system comprising an NS3a protein and a Nanobody as described in the first aspect of the present invention, or a binding molecule as described in the second aspect of the present invention;

[0010] In some embodiments of the present invention, the chemically induced disaggregation system further comprises a small molecule drug; preferably, the small molecule drug comprises grazoprevir; and / or the NS3a protein has a tag such as biotin.

[0011] The fourth aspect of the present invention provides an isolated nucleic acid encoding the Nanobody as described in the first aspect of the present invention, or the binding molecule as described in the second aspect of the present invention, or the chemically induced disaggregation system as described in the third aspect of the present invention.

[0012] The fifth aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid as described in the fourth aspect of the present invention; preferably, the backbone of the recombinant expression vector is pSB.

[0013] The sixth aspect of the present invention provides a transformant, which comprises the isolated nucleic acid as described in the fourth aspect of the present invention, or the recombinant expression vector as described in the fifth aspect of the present invention; preferably, the host of the transformant is a prokaryotic cell or a eukaryotic cell; more preferably, the prokaryotic cell is E. coli MC1061 strain.

[0014] The seventh aspect of the present invention provides a method for preparing the Nanobody as described in the first aspect of the present invention or the binding molecule as described in the second aspect of the present invention or the chemically induced disaggregation system as described in the third aspect of the present invention, the method comprising culturing the transformant as described in the sixth aspect of the present invention, thereby obtaining the Nanobody or the binding molecule or the chemically induced disaggregation system.

[0015] An eighth aspect of the present invention provides a pharmaceutical composition comprising the Nanobody as described in the first aspect of the invention or the binding molecule as described in the second aspect of the invention, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0016] The ninth aspect of the present invention provides a kit comprising the nanobody as described in the first aspect of the present invention, the binding molecule as described in the second aspect of the present invention, the chemically induced disaggregation system as described in the third aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention.

[0017] In a tenth aspect, the present invention provides the use of the Nanobody as described in the first aspect of the present invention, the binding molecule as described in the second aspect of the present invention, the chemically induced disaggregation system as described in the third aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention in the preparation of a kit or drug for regulating cell signal transduction, gene editing, protein colocalization or cell therapy;

[0018] Preferably, the cell therapy is CAR-T cell therapy.

[0019] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0020] The reagents and raw materials used in the present invention are commercially available.

[0021] The positive advances of this invention include the development of a novel chemically induced disaggregation system that combines the advantageous properties of NS3a nanobodies with the regulatory effects of small molecule inducers to achieve controlled regulation of protein interactions. The nanobodies screened and obtained by this invention exhibit high specificity, excellent stability, low molecular weight, and ease of production, making them ideal tool molecules for constructing chemically induced disaggregation systems. Furthermore, the chemically induced disaggregation system developed by this invention leverages the molecular properties of nanobodies to enhance the system's flexibility and applicability, while enabling precise control of protein interactions through regulation by small molecule inducers. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This graph shows the results of a poly-ELISA targeting NS3a. The vertical axis represents absorbance. In the first phase, three rounds of screening (R1: 700nM, R2: 700nM, R3: 200nM) were used, with the antigen concentration gradually decreasing and the screening conditions becoming increasingly stringent to obtain nanobodies with higher affinity.

[0023] Figure 2 This graph shows the Mono-ELISA results for the nanobody Nb0G8. The vertical axis represents absorbance. In the absence of the grazoprevir small molecule, Nb0G8 binds to NS3a, resulting in a high absorbance. In the presence of grazoprevir, Nb0G8 does not bind to NS3a, resulting in a low absorbance, demonstrating that Nb0G8 is a suitable nanobody for CIDiss applications.

[0024] Figure 3Figures 1 and 2 show the molecular sieving profile of the nanobody Nb0G8 and its binding kinetics for NS3a. (A) Molecular sieving peak profile (Superdex 75 Increase 10 / 300 GL) and purification results for the nanobody Nb0G8, demonstrating its purity. (B) Binding kinetics of the nanobody to NS3a.

[0025] Figure 4 Figures show the results of a co-migration experiment between the nanobody Nb0G8 and NS3a. (A) shows the molecular sieve peak patterns of the complex formed between Nb0G8 and NS3a in the presence and absence of the small molecule grazoprevir. (B) shows an SDS-PAGE analysis of the corresponding fractions. It can be seen that in the presence of the small molecule, Nb0G8 and NS3a do not form a stable complex. In the absence of the small molecule, Nb0G8 and NS3a form a co-migrating complex. DETAILED DESCRIPTION

[0026] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the corresponding fields. In addition, for a better understanding of the present invention, the following definitions and explanations of relevant terms are provided:

[0027] In the present invention, the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0028] In the present invention, the amino acid sequences of the complementarity determining regions (CDRs) listed are shown according to the definition of the IMGT numbering rules. However, it is well known to those skilled in the art that antibody CDRs can be defined by a variety of methods in the art, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international ImMunoGeneTicsdatabase (IMGT, World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0029] Therefore, when referring to antibodies defined by specific CDR sequences defined herein, the scope of such antibodies also encompasses antibodies whose variable region sequences comprise such specific CDR sequences, but whose claimed CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination). Although the scope of protection claimed in the present invention is based on the sequences defined according to the IMGT numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0030] In the present invention, "NS3a binding molecules" are proteins that have the function of recognizing and binding to NS3a, including but not limited to antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines and chemokines.

[0031] In the present invention, the terms "single-domain antibody," "anti-NS3a single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," "VHH," and "nanobody" are used interchangeably to refer to nanobodies that specifically recognize and bind to NS3A. A nanobody is the variable region of a heavy chain antibody. Typically, a nanobody contains three CDRs and four FRs. Nanobodies are the smallest functional antigen-binding fragments. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody consisting solely of a single heavy chain variable region.

[0032] In the present invention, a binding molecule comprising two or more Nanobodies is a multivalent Nanobody; a binding molecule comprising two or more Nanobodies with different specificities is a multispecific Nanobody. A multivalent Nanobody or a multispecific Nanobody is connected to multiple Nanobodies via a linker. The linker is typically composed of 1-15 amino acids selected from G and S, for example (G4S)3.

[0033] In the present invention, "multispecific antibody" refers to a type of antibody that can simultaneously recognize and bind to at least two different antigenic epitopes, such as a bispecific antibody.

[0034] In the present invention, a "fusion protein" refers to a protein formed by genetically linking two or more distinct protein or polypeptide sequences. The Fc fusion protein structure consists of two components: the Fc region of an immunoglobulin and the Nanobody described herein. These components possess relatively independent structural domains and functions, each capable of influencing its physicochemical properties and biological activity in distinct ways. Fc fusion proteins can dimerize to form heavy-chain antibodies.

[0035] For the purposes of this invention, "heavy chain antibodies" are antibodies derived from camelids or elasmobranchs. Compared to the aforementioned full-length antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), consisting of only two heavy chains consisting of a variable region (VHH) and other constant regions. The variable regions are connected to the constant regions by a hinge-like structure. Each heavy chain of a camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of an elasmobranch heavy chain antibody contains one variable region and five constant regions (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. By fusion with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 of human IgG Fc.

[0036] In the present invention, heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies.

[0037] Without materially affecting the activity of the antibody, those skilled in the art may modify one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletion, insertion, and / or substitution of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids having similar or similar properties generally do not alter protein function. For example, amino acids with similar properties may be substituted in the FR and / or CDR regions of the variable region. Amino acid residues suitable for conservative substitutions are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For another example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein and is considered to be included in the scope of protection of the present invention.

[0038] Variants of the various antibodies of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the DNA encoding the various antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera against the various antibodies of the present invention.

[0039] In some embodiments, the sequence of the variants described herein can be at least 95%, 96%, 97%, 98%, or 99% identical to the sequence from which it is derived. Sequence identity as described herein can be measured using sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have greater than 90% (preferably greater than 95%, and most preferably greater than 98%) homology to the CDRs identified herein.

[0040] Nanobodies and heavy-chain antibodies of the invention can be prepared using conventional methods in the art, such as phage display techniques well known in the art. Alternatively, the various antibodies of the invention can be expressed in other cell lines. Sequences encoding the various antibodies of the invention can be used to transform suitable mammalian host cells. Transformation can be carried out using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial NS3a binding properties.

[0041] In the present invention, "nucleic acid" refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0042] In the present invention, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of the mRNA, protein, polypeptide, or peptide by the host cell when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is in contact with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors of the present invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and which may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In exemplary aspects, the altered nucleotides or non-naturally occurring internucleotide linkages do not hinder transcription or replication of the vector.

[0043] The recombinant expression vector of the present invention can be any suitable recombinant expression vector that can be used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0044] In the present invention, the term "host cell" refers to any type of cell that can contain the nucleic acid or vector described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it can be a prokaryotic cell, such as a bacterium or protozoa.

[0045] In this invention, "pharmaceutically acceptable carrier" refers to a non-toxic carrier substance that can be used for pharmaceutical preparations and does not interfere with the active ingredients. Examples include aqueous carriers (e.g., water for injection, physiological saline), non-aqueous carriers (e.g., vegetable oils, glycerol), and organic solvents (e.g., ethanol, propylene glycol). "Pharmaceutically acceptable excipients" refer to auxiliary substances added to a formulation to improve its performance, including but not limited to stabilizers (e.g., mannitol, sucrose), isotonicity regulators (e.g., sodium chloride), pH regulators (e.g., citrate, phosphate), preservatives (e.g., benzyl alcohol), and surfactants (e.g., tween-80).

[0046] The "Chemically Induced Disaggregation System (CIDiss)" described in this invention is a controllable protein disaggregation technology based on small molecule compound regulation. By adding specific small molecule compounds, this system enables precise regulation of the disaggregation of target protein complexes. This system primarily consists of two components: a ligand that specifically binds to the target protein and a domain that responds to the small molecule compound. When specific small molecule compounds are added to the system, they trigger the dissociation of protein-protein interactions, leading to disaggregation of the protein complex. This technology has important applications in studying protein-protein interactions, signaling pathway regulation, and the dynamics of functional proteins.

[0047] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0048] Example 1: Construction, expression and purification of NS3a protein

[0049] Construction of the NS3a expression plasmid: From the N-terminus to the C-terminus, the plasmid contains a glutathione S-transferase (GST), a tobacco etch virus (TEV) cleavage site, NS3a, and an Avi tag (GLNDIFEAQKIEWHE, SEQ ID NO: 3). NS3a was expressed in Escherichia coli. Expression was induced with 1 mM IPTG at an OD of 0.6 at 37°C for 18 hours. The culture was harvested by centrifugation at 5000 g for 15 minutes. The cells were disrupted by sonication and centrifuged, followed by the addition of GST magnetic beads and incubation for 2 hours. After washing on the column, the beads were resuspended and digested with TEV enzyme overnight at 4°C. The next day, the flow-through was collected, concentrated, and further purified by size-exclusion chromatography (Superdex Increase 75 10 / 300 GL). The resulting NS3a protein, with a uniform molecular weight and greater than 95% purity, contained only the Avi tag at the C-terminus.

[0050] Glutathione S-transferase (GST):

[0051] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAY SKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK (SEQ ID NO: 1)

[0052] NS3a full length:

[0053] MKKKGSVVIVGRINLSGDTAYSQQTRGLEGCQETSQTGRDKNQVEGEVQVVSTATQSFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSP (SEQ ID NO: 2)

[0054] Example 2: Biotinylation

[0055] Biotinylation of NS3a: Purified MBP-BirA (maltose binding protein tag, MBP, UniProtKB / Swiss-Prot database, accession number P0AEX9.1) (a fusion protein of biotin ligase and MBP, at a molar concentration of 1 / 25 that of NS3a) was added to 1 mg / mL NS3a protein. 5 mM ATP, 10 mM magnesium acetate, and a biotin concentration 3 times that of NS3a were added. The mixture was incubated at 4°C for 12 h. The protein was then further purified by size exclusion chromatography (Superdex Increase 75 10 / 300 GL). Fractions containing the protein were collected, aliquoted, snap-frozen in nitrogen, and stored at -80°C for phage display screening.

[0056] Example 3: Phage display and screening of nanobodies

[0057] The present invention carried out a total of five rounds of phage display. The first round used 700 nM antigen. The purified phage was first incubated with streptavidin-coupled magnetic beads to remove phages that had the ability to bind to the magnetic beads. The phage was then incubated with 700 nM biotinylated NS3a protein and then transferred to another portion of magnetic beads. After binding at room temperature, the non-specifically bound phage was washed to remove the non-specifically bound phage, and then the bound phage was released with 0.2 M glycine solution (pH 3), and 1 M Tris·HCl pH 8.0 was added in time to neutralize. The screened phages were amplified in vivo and purified in vitro, and then the second and third rounds of phage display were carried out with antigen concentrations of 700 nM and 200 nM, respectively. The screened phages were then subjected to Poly-ELISA experiments to obtain the enrichment after each round of screening (see Figure 1 ). After the screening phages obtained in the third round were bound to the magnetic beads, the non-specifically bound phages were washed away, and the fourth round phages were eluted with 5 μM small molecule Grazoprevir. In the fifth round of screening, small molecules were first used to bind to NS3a, and then the fourth round phages were added to collect the flow-through phages, that is, the phages that did not interact with NS3a bound to Grazoprevir. The results of the fifth round of screening were used to select single clones for ELISA experiments and sequencing to obtain the nano antibody nucleic acid sequence (Nb) that can bind to NS3a. This nano antibody cannot bind to NS3a in the presence of small molecule Grazoprevir (see Figure 2 The screened Nb gene was cloned into the expression vector pSB (Addgene Plasmid #110100) and transformed into E. coli MC1061 for expression.

[0058] Example 4: Expression and purification of nanobodies

[0059] After the nanobody expression plasmid was transformed into E. coli MC1061, a single colony was selected and cultured overnight in 1 mL of LB medium containing 50 μg / mL chloramphenicol (37°C, 220 rpm). The colony was then transferred to fresh TB medium (containing chloramphenicol) at a ratio of 1:100. When the OD600 reached 0.5, the temperature was lowered to 22°C and cultured for 1.5-2 hours. The culture was then induced with 0.02% (w / v) arabinose for 18 hours. The culture was harvested by centrifugation at 5000 g for 15 minutes and resuspended in 5 mL of TES (0.5 M sucrose, 0.5 mM EDTA, 0.2 M Tris-HCl, pH 8.0). After rotation for 30 minutes, 10 mL of milliQ H2O was added. After rotation for 1 hour, the supernatant was collected by centrifugation and applied to a 2 mL Ni-NTA affinity column. After incubation for 1 hour, the column was passed through by gravity, washed with 30 mM imidazole in a buffer solution (250 mM NaCl, 20 mMTris pH 8.0), and the protein was eluted with a buffer solution containing 300 mM imidazole. The purified nanobody was subjected to SDS-PAGE gel and SEC analysis (Superdex Increase 75 10 / 300 GL) (see Figure 3 The purified Nb monomeric protein (as shown below, where the variable region sequences and CDR regions are defined according to the IMGT numbering system) can be directly used for BLI binding and co-migration assays.

[0060] >Nb0G8 amino acid sequence

[0061] AVQLQASGGGFVQPGGSLRLSCAASGLTSPVVDMGWFRQAPGKEREFVSAIENRFQNRYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAVVAWNDHDMTGYWRYWGQGTQVTVSS (SEQ ID NO: 4)

[0062] CDR1:GLTSPVVD (SEQ ID NO: 5)

[0063] CDR2:IENRFQNR (SEQ ID NO: 6)

[0064] CDR3:AVVAWNDHDMTGYWRY (SEQ ID NO: 7)

[0065] >Nb0G8 nucleotide sequence

[0066] GCCGTGCAGCTGCAGGCCAGCGGCGGCGGCTTCGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCCTGACTTCTCCGGTTGTTGACATGGGCTGGTTCAGGCAGGCTCCCGGCAAGGAGAGGGAGTTCGTGGCCGCCATCGAAAACCGTTTCCAGAACCGTTACTACGCCGACA GCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCACCTACTACTGCGCCGTTGTTGCTTGGAACGACCATGACATGACTGGTTACTGGCGTTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC (SEQID NO: 8)

[0067] Example 5: BLI method to determine the affinity between antigen NS3a and nanobody

[0068] The Octet RED96 instrument was used to detect the interaction between the Nb0G8 nanobody and the NS3a protein using biolayer optical interferometry (BLI) technology. The nanobody to be tested was diluted to different concentration gradients with buffer (PBS, 10 mg / mL BSA). The biotinylated NS3a protein was immobilized on a streptavidin probe, and then the probe was inserted into a gradient dilution solution (1200 nM, 600 nM, 300 nM, 150 nM, 75 nM) containing the Nb0G8 nanobody to be tested to measure the affinity between the NS3a protein and the Nb0G8 nanobody. The experimental results can be used to obtain the kinetic parameters of the NS3a protein and the test protein. Among them, kon refers to the association rate constant, koff refers to the dissociation rate constant, and KD (KD = koff / kon) refers to the equilibrium dissociation constant, which is used to characterize the affinity between the antibody and the antigen. The smaller the KD value, the lower the concentration of the drug required to cause the maximum effect and the higher the affinity. The experimental results show that Nb0G8 can bind to NS3a (see Figure 3 Part B, Table 1).

[0069] Table 1. Summary of kinetic parameters for the binding of nanobodies to NS3a

[0070]

[0071] Example 6: Co-migration method to identify the interaction between nanobodies and NS3a with and without small molecules

[0072] 50 μg Nb0G8 and 50 μg NS3a were incubated in a buffer solution (20 mM Tris, 100 mM NaCl). 20 μM of the small molecule Grazoprevir was added to the experimental group, and the corresponding concentration of DMSO was added to the control group. After incubation, the binding between Nb0G8 and NS3a was observed by size exclusion chromatography (Superdex Increase 75 10 / 300 GL) and SDS-PAGE gel. The experimental results showed that Nb0G8 and NS3a interacted in the absence of the small molecule Grazoprevir, but could not interact in the presence of the small molecule Grazoprevir, demonstrating that Nb0G8 is a feasible CIDiss nanobody (see Figure 4 ).

Claims

1. A nanobody targeting NS3a derived from hepatitis C virus, characterized in that The Nanobody is composed of a heavy chain variable region comprising CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is shown in SEQ ID NO: 5, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:

7.

2. The Nanobody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4, or has a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO:

4.

3. A binding molecule targeting NS3a, characterized in that The binding molecule comprises the Nanobody according to claim 1 or 2.

4. The binding molecule according to claim 3, wherein The binding molecule is a monovalent or multivalent Nanobody or Fc fusion protein comprising one or more Nanobodies.

5. A chemically induced depolymerization system, characterized in that: It comprises NS3a protein and the Nanobody as claimed in claim 1 or 2, or the binding molecule as claimed in claim 3 or 4.

6. The chemically induced depolymerization system according to claim 5, characterized in that It also includes small molecule drugs.

7. The chemically induced depolymerization system according to claim 6, wherein The small molecule drug includes grazoprevir; and / or the NS3a protein has a tag.

8. The chemically induced depolymerization system according to claim 6 or 7, characterized in that The NS3a protein has a biotin tag.

9. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the Nanobody according to claim 1 or 2, or the binding molecule according to claim 3 or 4, or the chemically induced disaggregation system according to claim 5.

10. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 9.

11. The recombinant expression vector according to claim 10, wherein The backbone of the recombinant expression vector is pSB.

12. A transformant, characterized in that: The transformant comprises the isolated nucleic acid according to claim 9, or the recombinant expression vector according to claim 10 or 11.

13. The transformant according to claim 12, wherein The host of the transformant is a prokaryotic cell or a eukaryotic cell.

14. The transformant according to claim 13, wherein The prokaryotic cell is E. coli MC1061 strain.

15. A method for preparing the Nanobody according to claim 1 or 2, the binding molecule according to claim 3 or 4, or the chemically induced disaggregation system according to claim 5, characterized in that: The method comprises culturing the transformant according to any one of claims 12 to 14, thereby obtaining the nanobody or the binding molecule or the chemically induced disaggregation system.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody as claimed in claim 1 or 2 or the binding molecule as claimed in claim 3 or 4, and optionally a pharmaceutically acceptable carrier and / or excipient.

17. A kit, characterized in that The kit comprises the Nanobody according to claim 1 or 2, the binding molecule according to claim 3 or 4, the chemically induced disaggregation system according to any one of claims 5 to 8, or the pharmaceutical composition according to claim 16.

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