Nanometer antibody targeting NS3a and application thereof

Abstract

The invention discloses an NS3a-targeted nano antibody and an application of the NS3a-targeted nano antibody in a chemical induction depolymerization system. The nano antibody is composed of a heavy chain variable region containing 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. The nano antibody can specifically recognize and bind to NS3a protein, and the interaction between the nano antibody and NS3a can be controllably adjusted through a small molecule inducer, so that a chemical induction depolymerization system is constructed. The system can be applied to the synthetic biology fields of artificial regulation of protein interaction, cell engineering, gene expression regulation and the like.

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 an application thereof. Background Art

[0002] In biological and medical research, regulating the interaction between proteins is an important means to study cell functions and develop new therapies. At present, 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 can help stabilize it, and is orthogonal to human proteins. Nanobodies, as the smallest known molecular weight antibodies, have the advantages of high stability, small molecular weight, easy production and low cost while retaining the high affinity of traditional antibodies. They are an important choice for regulatory elements. The small molecule drug grazoprevir is an FDA-approved NS3a inhibitor with high safety. 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 (e.g., Grazoprevir), thereby providing a new type of tool molecule for constructing a chemically induced disaggregation system (CIDiss).

[0004] The present invention relates to a method for screening nano antibodies. NS3a protein is used as an antigen and recombinantly expressed so that it has a unique biotinylation tag. In the first step, based on an artificially synthesized humanized nano antibody library, three rounds of enrichment are adopted, combined with magnetic bead sorting and a scheme of decreasing antigen concentration gradient, to obtain a nano antibody that binds to the antigen. In the second step, a small molecule competitive elution method is used. After the nano antibody binds, the small molecule grazoprevir is used for elution, so as to collect the nano antibody that can be opened 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 the nano antibody that cannot be bound is collected. After a total of 5 rounds of screening, the nano antibody obtained in each round of screening is tested by enzyme-linked immunosorbent assay (ELISA) to evaluate the screening effect of each round. After the effect reaches a certain level, a phage monoclonal ELISA experiment is continued to identify phages expressing nano antibodies that bind to the antigen without adding small molecules and do not bind with small molecules. The identified nanobody nucleic acid sequence was transferred to a new expression vector, and periplasmic expression was used to maintain the disulfide bonds inside the nanobody and maintain its stability. Afterwards, affinity chromatography was used for purification to obtain nanobodies with good purity, with a yield of 10-20 mg / L. Biochemical identification used the Bio-Layer Interferometry (BLI) method to conduct a concentration gradient experiment on the bound nanobody to determine its binding kinetic parameters. Finally, a molecular sieve co-migration experiment was performed to determine the binding of the nanobody to NS3a in the presence and absence of 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 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% sequence identity with 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, which comprises 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] Preferably, NS3b protein is also included.

[0011] 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 label such as biotin.

[0012] The fourth aspect of the invention provides an isolated nucleic acid, which encodes the Nanobody as described in the first aspect of the invention, or the binding molecule as described in the second aspect of the invention, or the chemically induced disaggregation system as described in the third aspect of the invention.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] The eighth aspect of the present invention provides a pharmaceutical composition, which comprises 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, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0017] The ninth aspect of the present invention provides a kit, which comprises the nanoantibody 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.

[0018] The tenth aspect of 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, and 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;

[0019] Preferably, the cell therapy is CAR-T cell therapy.

[0020] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0021] The reagents and raw materials used in the present invention are commercially available.

[0022] The positive and progressive effects of the present invention are: a new type of chemically induced disaggregation system has been developed, which realizes controllable regulation of protein interactions by combining the advantageous characteristics of NS3a nano antibodies and the regulatory effects of small molecule inducers. The nano antibodies screened and obtained by the present invention have the characteristics of high specificity, good stability, small molecular weight, and are easy to produce, making them ideal tool molecules for constructing chemically induced disaggregation systems. In addition, the chemically induced disaggregation system developed by the present invention improves the flexibility and applicability of the system based on the molecular characteristics of nano antibodies, and realizes precise control of protein interactions through the regulation of small molecule inducers. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The result of poly-ELISA targeting NS3a is shown in the figure. The vertical axis is the absorbance value. In the first stage, three rounds of screening (R1: 700nM, R2: 700nM, R3: 200nM) were used to continuously reduce the concentration of the antigen and increase the severity of the screening conditions to obtain nanobodies with higher affinity.

[0024] Figure 2 The result of Mono-ELISA of nanoantibody Nb0G8 is shown in the figure. The vertical axis is the absorbance value. When there is no Grazoprevir small molecule, Nb0G8 binds to NS3a and the absorbance value is high; when there is Grazoprevir small molecule, Nb0G8 does not bind to NS3a and the absorbance value is low, which proves that Nb0G8 is a suitable CIDiss nanoantibody.

[0025] Figure 3The molecular sieve diagram of the nanobody Nb0G8 and the results of the determination of the binding kinetic parameters with NS3a. (A) The molecular sieve peak diagram (Superdex 75 Increase 10 / 300 GL) and purification results of the nanobody Nb0G8 show its purity. (B) The binding kinetic curve of the nanobody with NS3a.

[0026] Figure 4 The results of the co-migration experiment of the nanoantibody Nb0G8 and NS3a are shown. (A) shows the molecular sieve peak diagram of the complex formed by Nb0G8 and NS3a in the presence or absence of Grazoprevir small molecules. (B) The figure is the SDS-PAGE analysis gel diagram of the corresponding components. It can be seen that in the presence of small molecules, Nb0G8 and NS3a did not form a stable complex. In the absence of small molecules, Nb0G8 and NS3a formed a co-migrating complex. DETAILED DESCRIPTION

[0027] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are all conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below:

[0028] 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.

[0029] In the present invention, the amino acid sequences of complementarity determining regions (CDRs) listed are shown according to the definition of IMGT numbering rules. However, it is well known to those skilled in the art that the CDR of an antibody 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 loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), 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 appreciated 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 the complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0030] Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of the present invention is based on the sequences shown in the definition according to the IMGT numbering rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of the present invention.

[0031] 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.

[0032] In the present invention, "single domain antibody", "anti-NS3a single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", and "nanoantibody" are used interchangeably, all referring to nanoantibodies that specifically recognize and bind to NS3A. Nanoantibodies are the variable regions of heavy chain antibodies. Generally, nanoantibodies contain three CDRs and four FRs. Nanoantibodies are the smallest functional antigen-binding fragments. Usually, antibodies that naturally lack light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanoantibody consisting of only one heavy chain variable region.

[0033] 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 usually composed of 1-15 amino acids selected from G and S, for example (G4S) 3 .

[0034] 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.

[0035] In the present invention, "fusion protein" refers to a protein formed by connecting two or more different protein or polypeptide sequences through genetic engineering. Among them, the structure of Fc fusion protein consists of two parts, namely the Fc segment of immunoglobulin and the nanobody described in the present invention. Both have relatively independent domains and functions, and can affect their own physicochemical properties and biological activities from different aspects. Fc fusion protein dimerization can form heavy chain antibodies.

[0036] In the present invention, "heavy chain antibodies" are antibodies derived from camelids or cartilaginous fish. Compared with the above-mentioned full-length antibodies, heavy chain antibodies lack light chains and heavy chain constant region 1 (CH1), and only contain two heavy chains composed of variable regions (VHH) and other constant regions, and the variable regions are connected to the constant regions through a hinge region-like structure. Each heavy chain of the camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3), and each heavy chain of the cartilaginous fish heavy chain antibody contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can have CH2 and CH3 of human IgG Fc.

[0037] In the present invention, heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies.

[0038] Without substantially affecting the activity of the antibody, a person skilled in the art may change one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence 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 (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, conservative substitution with amino acids with similar or similar properties usually does not change the function of the protein. For example, amino acids with similar properties are substituted in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted 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 will generally not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.

[0039] Variant forms of the various antibodies described in the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of 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.

[0040] In some embodiments, the sequence of the variant of the present invention can have at least 95%, 96%, 97%, 98% or 99% identity with its source sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters is used. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0041] Nanobodies and heavy chain antibodies of the present invention can be prepared by conventional methods in the art, such as phage display technology well known in the art. Alternatively, various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding various antibodies of the present invention. Transformation can be carried out by any known method, for example, including packaging polynucleotides in viruses (or viral vectors) and transducing host cells with viruses (or vectors). 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, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, polynucleotide encapsulation in liposomes and direct microinjection of DNA into the nucleus, etc. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but 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), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial NS3a binding properties.

[0042] 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.

[0043] In the present invention, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of mRNA, protein, polypeptide or peptide by a host cell when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector contacts the cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector of the present invention is generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vector 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 a natural source, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring nucleotides, or both types of connections. In exemplary aspects, altered nucleotides or non-naturally occurring nucleotides are connected without hindering transcription or replication of the vector.

[0044] The recombinant expression vector of the present invention can be any suitable recombinant expression vector, which can be used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably express the gene or sequence 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.

[0045] 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, an animal, a fungus, or an alga; or it can be a prokaryotic cell, such as a bacterium or a protozoan.

[0046] In the present invention, "pharmaceutically acceptable carrier" refers to a carrier substance that can be used for pharmaceutical preparation, is non-toxic, and does not interfere with the active ingredients. For example, aqueous carriers (such as water for injection, physiological saline), non-aqueous carriers (such as vegetable oil, glycerol), organic solvents (such as ethanol, propylene glycol), etc. "Pharmaceutically acceptable excipients" refer to auxiliary substances added to the formulation to improve the performance of the formulation, including but not limited to: stabilizers (such as mannitol, sucrose), isotonicity regulators (such as sodium chloride), pH regulators (such as citrate, phosphate), preservatives (such as benzyl alcohol), surfactants (such as tween-80).

[0047] In the present invention, the "chemically induced disaggregation system (CIDiss)" is a controllable protein disaggregation technology based on the regulation of small molecule compounds. The system can achieve precise disaggregation regulation of the target protein complex by adding specific small molecule compounds. This system is mainly composed of two components: one is a ligand that can specifically bind to the target protein, and the other is a domain that is responsive to small molecule compounds. When specific small molecule compounds are added to the system, the dissociation of protein-protein interactions is triggered, resulting in the disaggregation of protein complexes. This technology has important applications in studying protein-protein interactions, signal pathway regulation, and dynamic studies of functional proteins.

[0048] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0049] Example 1: Construction, expression and purification of NS3a protein

[0050] Construction of NS3a expression plasmid: from N-terminus to C-terminus, there are glutathione S transferase (GST), tobacco etch virus cysteinease (TEV) cleavage site, NS3a and Avi tag (GLNDIFEAQKIEWHE, SEQ ID NO: 3). Expression process of NS3a: Escherichia coli expression was adopted. When the OD was 0.6 at 37℃, 1mM IPTG was used to induce expression for 18h. The cultured bacteria were collected by centrifugation at 5000 g for 15 min. The bacteria were broken by ultrasound, and GST magnetic beads were added after centrifugation and incubated for 2h. After washing on the column, the magnetic beads were resuspended and TEV enzyme was added to the column overnight at 4℃ for enzyme digestion. The protein that flowed through was collected the next day, and the collected target protein was concentrated and further purified by molecular exclusion chromatography (Superdex Increase 75 10 / 300 GL). Finally, the NS3a protein with uniform molecular weight and purity greater than 95% with Avi tag only at the C-terminus was obtained.

[0051] Glutathione S-transferase (GST):

[0052] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAY SKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK (SEQ ID NO: 1)

[0053] NS3a full length:

[0054] MKKKGSVVIVGRINLSGDTAYSQQTRGLEGCQETSQTGRDKNQVEGEVQVVSTATQSFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSP (SEQ ID NO: 2)

[0055] Example 2: Biotinylation

[0056] Biotinylation labeling of NS3a protein: Add purified MBP-BirA (maltose binding protein tag, MBP, UniProtKB / Swiss-Prot database, accession number P0AEX9.1) (a fusion protein of biotin ligase and MBP, 1 / 25 of the molar concentration of NS3a protein) to 1 mg / mL NS3a protein, add 5 mM ATP, 10 mM magnesium acetate and 3 times the molar concentration of biotin of NS3a, mix well, place at 4°C for 12h, and then use molecular exclusion chromatography (Superdex Increase 7510 / 300 GL) to further separate and purify the protein. Collect the protein-containing fractions, package them, snap-frozen them with nitrogen, and store them in a -80°C refrigerator for phage display screening.

[0057] Example 3: Phage display and screening of nanobodies

[0058] The present invention carried out a total of five rounds of phage display. The first round used an antigen of 700 nM. 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 away and then released with 0.2 M glycine solution (pH 3), and 1 M Tris·HCl pH 8.0 was added to neutrality in time. 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 the 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.

[0059] Example 4: Expression and purification of nanobodies

[0060] After the expression plasmid of the nanobody was transferred into E.coli MC1061, a single clone was selected and cultured overnight in 1 mL LB medium containing 50 μg / ml chloramphenicol (37°C, 220 rpm), and then transferred to fresh TB medium (containing chloramphenicol) at a ratio of 1:100. When OD600 grew to 0.5, the temperature was lowered to 22°C and cultured for 1.5~2h, and then 0.02% (w / v) arabinose was added for induction culture for 18h. The cultured bacteria were collected by centrifugation at 5000 g for 15 min, resuspended with 5 mL TES (0.5M sucrose, 0.5mM EDTA, 0.2 M Tris-HCl pH 8.0), and added with 10 mL milliQH after rotating for 30 min. 2O, rotate for 1 hour, collect the supernatant by centrifugation and add 2 ml Ni-NTA affinity column. After incubation for 1 hour, flow through the gravity column, add 30 mM imidazole to the buffer (250 mM NaCl, 20 mMTris pH 8.0) to wash, and elute the protein with a buffer 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 monomer 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.

[0061] >Nb0G8 amino acid sequence

[0062] AVQLQASGGGFVQPGGSLRLSCAASGLTSPVVDMGWFRQAPGKEREFVSAIENRFQNRYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAVVAWNDHDMTGYWRYWGQGTQVTVSS (SEQ ID NO: 4)

[0063] CDR1:GLTSPVVD (SEQ ID NO: 5)

[0064] CDR2:IENRFQNR (SEQ ID NO: 6)

[0065] CDR3:AVVAWNDHDMTGYWRY (SEQ ID NO: 7)

[0066] >Nb0G8 nucleotide sequence

[0067] GCCGTGCAGCTGCAGGCCAGCGGCGGCGGCTTCGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCCTGACTTCTCCGGTTGTTGACATGGGCTGGTTCAGGCAGGCTCCCGGCAAGGAGAGGGAGTTCGTGGCCGCCATCGAAAACCGTTTCCAGAACCGTTACTACGCCGACA GCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCACCTACTACTGCGCCGTTGTTGCTTGGAACGACCATGACATGACTGGTTACTGGCGTTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC (SEQID NO: 8)

[0068] Example 5: BLI method to determine the affinity between antigen NS3a and nanobody

[0069] The Octet RED96 instrument was used to detect the interaction between the Nb0G8 nanobody and the NS3a protein using the bio-layer optical interference (BLI) technique. The nanobody to be tested was diluted to different concentration gradients with a buffer solution (PBS, 10 mg / mL BSA). The biotinylated NS3a protein was fixed on the streptavidin probe, and then the probe was inserted into the gradient dilution solution (1200 nM, 600 nM, 300 nM, 150 nM, 75 nM) containing the Nb0G8 nanobody to be tested to determine the affinity between the NS3a protein and the Nb0G8 nanobody. The kinetic parameters of the NS3a protein and the protein to be tested can be obtained from the experimental results. Among them, kon refers to the binding 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).

[0070] Table 1. Summary of kinetic parameters of nanobody binding to NS3a

[0071] <![CDATA[K on (1 / Ms)]]> <![CDATA[K off (1 / s)]]> KD (nM) Nb2G 7.587E+03 1.002E-3 132 nM

[0072] Example 6: Co-migration method to identify the interaction between nanobodies and NS3a with or without small molecules

[0073] 50 μg Nb0G8 and 50 μg NS3a were incubated in a buffer solution (20 mM Tris, 100 mM NaCl). 20 μM small molecule Grazoprevir was added to the experimental group, and the corresponding concentration of DMSO was added to the control group. After incubation, molecular exclusion chromatography (Superdex Increase 75 10 / 300 GL) and SDS-PAGE gel were used to observe the binding between Nb0G8 and NS3a. The experimental results showed that Nb0G8 and NS3a interacted in the absence of small molecule Grazoprevir, but could not interact in the presence of small molecule Grazoprevir, proving that Nb0G8 is a feasible CIDiss nanoantibody (see Figure 4 ).

Claims

1. A nanobody targeting NS3a, 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, characterized in that The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4, or has 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% sequence identity with SEQ ID NO:

4.

3. A binding molecule targeting NS3a, characterized in that The binding molecule comprises the Nanobody as described in claim 1 or 2; 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.

4. A chemically induced depolymerization system, characterized in that: It comprises NS3a protein and a Nanobody as claimed in claim 1 or 2, or a binding molecule as claimed in claim 3; Preferably, NS3b protein is also included.

5. The chemically induced depolymerization system according to claim 4, characterized in that: It also includes small molecule drugs; preferably, the small molecule drugs include grazoprevir; and / or, the NS3a protein has a label such as biotin.

6. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the Nanobody as claimed in claim 1 or 2, or the binding molecule as claimed in claim 3, or the chemically induced disaggregation system as claimed in claim 4.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid as described in claim 6; preferably, the backbone of the recombinant expression vector is pSB.

8. A transformant, characterized in that The transformant comprises the isolated nucleic acid according to claim 6, or the recombinant expression vector according to claim 7; preferably, the host of the transformant is a prokaryotic cell or a eukaryotic cell; more preferably, the prokaryotic cell is E. coli MC1061 strain.

9. A method for preparing the Nanobody according to claim 1 or 2, or the binding molecule according to claim 3, or the chemically induced disaggregation system according to claim 4, characterized in that: The method comprises culturing the transformant as claimed in claim 8, thereby obtaining the nanobody or the binding molecule or the chemically induced disaggregation system.

10. 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, and optionally a pharmaceutically acceptable carrier and / or excipient.

11. A kit, characterized in that: The kit comprises the Nanobody as claimed in claim 1 or 2, the binding molecule as claimed in claim 3, the chemically induced disaggregation system as claimed in claim 4 or 5, or the pharmaceutical composition as claimed in claim 10.

12. Use of the nanobody according to claim 1 or 2, the binding molecule according to claim 3, the chemically induced disaggregation system according to claim 4 or 5, or the pharmaceutical composition according to claim 10 in the preparation of a kit or drug for regulating cell signal transduction, gene editing, protein colocalization or cell therapy; Preferably, the cell therapy is CAR-T cell therapy.

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