Anti-ck8 nanobody, polypeptide comprising the same and use thereof

Abstract

The application provides an anti-CK8 nanobody, a polypeptide containing the nanobody and application thereof. A variable region in the amino acid sequence of the nanobody comprises three complementarity determining regions CDR and a framework region FR, the complementarity determining regions CDR comprising a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein the most important sites participating in antigen recognition and combination are RXXRXYX on the CDR1 and AASPAVSPPRDGRAFTY on the CDR3. The nanobody and the polypeptide thereof have high affinity and activity, can specifically recognize and combine CK8, and can be used for enrichment, removal and detection of CK8 and CK8 positive cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-CK8 nanobody, a polypeptide containing the nanobody, and their preparation and application. Background Technology

[0002] Cytokeratin 8 (CK8) belongs to the type II cytokeratin family, with a molecular weight of approximately 53.5 kDa and composed of 485 amino acids. Its molecular structure consists of three main parts: an N-terminal head domain, a central α-helical rod domain, and a C-terminal tail domain.

[0003] CK8 is an important intermediate filament protein in epithelial cells, widely involved in key biological processes such as maintaining cell structural integrity, signal transduction, and stress response. CK8, together with cytokeratin 18 (CK18), forms an insoluble matrix extending from the nucleus to the inner layer of the cell membrane, providing mechanical support and helping cells resist mechanical stress. CK8 plays a crucial role in various cellular processes, including cell adhesion, proliferation, differentiation, and migration. Through its interaction with the extracellular matrix, it participates in cell morphology maintenance and signal transduction.

[0004] Meanwhile, studies have shown that CK8 is abnormally highly expressed in various epithelial tumors (such as breast cancer, colorectal cancer, and liver cancer) and liver diseases (such as non-alcoholic steatohepatitis and fibrosis). Its expression level is closely related to disease progression, metastatic potential, and prognosis, making it an important diagnostic biomarker and therapeutic target. In some cancers, such as esophageal squamous cell carcinoma, CK8 expression is associated with tumor invasiveness and poor prognosis. Abnormal CK8 expression may also be related to the invasive and metastatic capabilities of tumor cells; for example, in breast cancer, increased CK8 expression is associated with tumor invasiveness and metastatic potential. Circulating tumor cells (CTCs), after detaching from solid tumors and entering the bloodstream, can spread to other parts of the body via the bloodstream, potentially leading to distant metastasis and spread of tumors.

[0005] CK8-positive cells are a type of cancer cell that highly expresses CK8 on its surface, including circulating tumor cells (CTCs). Therefore, the detection of CK8 and CK8-positive cells is of great significance in cancer research, especially in the fields of tumor biology and new drug development.

[0006] Currently, the detection of intracellular and extracellular CK8 and CK8-positive cells relies on traditional monoclonal antibodies (mAbs) or polyclonal antibodies. These antibodies are widely used in various experimental scenarios, including Western blotting (WB), immunohistochemistry (IHC), flow cytometry (FCM), and enzyme-linked immunosorbent assay (ELISA), to detect CK8 expression levels. The detection of intracellular CK8 is particularly complex, involving the following steps: 1. Cell fixation and permeabilization, 2. Blocking, 3. Primary antibody incubation, 4. Washing, 5. Secondary antibody incubation, and 6. Counterstaining and observation. By utilizing fluorescently labeled antibodies to specifically bind to the CK8 protein, the fluorescence signal can be observed using a fluorescence microscope to determine the intracellular localization and expression level of CK8.

[0007] Traditional antibodies are large in size and expensive to prepare, which limits their practical application in fields such as capture and detection, especially in the field of blood purification.

[0008] Nanobodies are single-domain antibodies derived from animals such as camels and sharks. Composed solely of the heavy chain variable region, they have a molecular weight of approximately 12-15 kDa and a size only about 1 / 10 that of ordinary antibodies. This allows them to penetrate cells and tissues more quickly and deeply, reaching areas inaccessible to ordinary antibodies, such as the interior of dense solid tumors and fine intracellular structures, resulting in more efficient and comprehensive intracellular protein staining. Furthermore, the genes of nanobodies are easily manipulated and modified using genetic engineering techniques, such as fusing fluorescent proteins or adding tags, to meet diverse experimental needs. Bispecific or multispecific nanobodies can also be constructed to achieve simultaneous detection of multiple intracellular proteins. Nanobodies can be mass-produced using microbial fermentation systems (such as E. coli and yeast), a simple and efficient process with a significantly lower cost than traditional antibody preparation methods. Summary of the Invention

[0009] The purpose of this invention is to solve the above-mentioned problems by providing nanobodies with amino acid sequences having specific structures, peptides containing such nanobodies, and their applications, in order to solve the problems of complex antibody preparation processes, high costs, poor antibody affinity and stability in existing intracellular and extracellular CK8 detection, CK8 positive cell detection and capture, and CK8 enrichment, purification, detection and removal.

[0010] To achieve the above technical objectives, the technical solution adopted in this application is as follows:

[0011] In a first aspect, according to the anti-CK8 nanobody in some embodiments of this application, the complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2 and CDR3 sequences: (I): (1) the amino acid sequence of CDR1 is shown in SEQ ID NO.47, (2) the amino acid sequence of CDR2 is shown in SEQ ID NO.51; and (3) the amino acid sequence of CDR3 is shown in SEQ ID NO.59; or (II): an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequences (1), (2) and (3) of (I), and having the same function as the amino acid sequence of (I).

[0012] According to some embodiments of the anti-CK8 nanobody of this application, wherein: (II) the amino acid sequence obtained by substituting one or more amino acids of the amino acid sequence described in (1), (2), (3) of (I) includes (II1): the amino acid sequence of CDR1 as shown in SEQ ID NO.47, the amino acid sequence of CDR2 as shown in SEQ ID NO.52, and the amino acid sequence of CDR3 as shown in SEQ ID NO.59; or (II2): the amino acid sequence of CDR1 as shown in SEQ ID NO.48, the amino acid sequence of CDR2 as shown in SEQ ID NO.53, and the amino acid sequence of CDR3 as shown in SEQ ID NO.59; or (II3): the amino acid sequence of CDR1 as shown in SEQ ID NO.48, the amino acid sequence of CDR2 as shown in SEQ ID NO.54, and the amino acid sequence of CDR3 as shown in SEQ ID NO.59; or (II4): the amino acid sequence of CDR1 as shown in SEQ ID NO.49, the amino acid sequence of CDR2 as shown in SEQ ID NO.55, and the amino acid sequence of CDR3 as shown in SEQ ID NO.59. (II) 5: The amino acid sequence of CDR1 is shown in SEQ ID NO. 49, the amino acid sequence of CDR2 is shown in SEQ ID NO. 56, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 59; or (II) 6: The amino acid sequence of CDR1 is shown in SEQ ID NO. 50, the amino acid sequence of CDR2 is shown in SEQ ID NO. 57, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 59; or (II) 7: The amino acid sequence of CDR1 is shown in SEQ ID NO. 50, the amino acid sequence of CDR2 is shown in SEQ ID NO. 58, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 59.

[0013] According to some embodiments of the present application, the anti-CK8 nanobody includes: (III) the framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO. 60, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO. 64, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO. 66, and (4) the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV) an amino acid sequence having more than 50% homology with the amino acid sequences of (1), (2), (3) and (4) of (III).

[0014] According to the anti-CK8 nanobody in some embodiments of this application, (IV) of the amino acid sequences described in (1), (2), (3), and (4) of (III) have more than 50% homology, including (IV-1): the amino acid sequence of FR1 is as shown in SEQ ID NO. 61, the amino acid sequence of FR2 is as shown in SEQ ID NO. 64, the amino acid sequence of FR3 is as shown in SEQ ID NO. 66, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-2): the amino acid sequence of FR1 is as shown in SEQ ID NO. 62, the amino acid sequence of FR2 is as shown in SEQ ID NO. 64, the amino acid sequence of FR3 is as shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-3): the amino acid sequence of FR1 is as shown in SEQ ID NO. 60, the amino acid sequence of FR2 is as shown in SEQ ID NO. 64, the amino acid sequence of FR3 is as shown in SEQ ID NO. 66, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 64. As shown in NO. 70; or (IV-4): the amino acid sequence of FR1 is as shown in SEQ ID NO. 60, the amino acid sequence of FR2 is as shown in SEQ ID NO. 64, the amino acid sequence of FR3 is as shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-5): the amino acid sequence of FR1 is as shown in SEQ ID NO. 60, the amino acid sequence of FR2 is as shown in SEQ ID NO. 64, the amino acid sequence of FR3 is as shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-6): the amino acid sequence of FR1 is as shown in SEQ ID NO. 60, the amino acid sequence of FR2 is as shown in SEQ ID NO. 65, the amino acid sequence of FR3 is as shown in SEQ ID NO. 68, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-7): the amino acid sequence of FR1 is as shown in SEQ ID NO. 63, the amino acid sequence of FR2 is as shown in SEQ ID NO. 65, and the amino acid sequence of FR3 is as shown in SEQ ID NO. 70. As shown in NO. 69, the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-8): the amino acid sequence of FR1 is as shown in SEQ ID NO. 60, the amino acid sequence of FR2 is as shown in SEQ ID NO. 65, the amino acid sequence of FR3 is as shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-9): the amino acid sequence of FR1 is as shown in SEQ ID NO. 60, and the amino acid sequence of FR2 is as shown in SEQ ID NO.As shown in SEQ ID NO. 65, the amino acid sequence of FR3 is shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70.

[0015] According to some embodiments of the anti-CK8 nanobody of this application, the nanobody has (V) an amino acid sequence as shown in SEQ ID NO. 5; or (VI) an amino acid sequence obtained by modifying, substituting, deleting, or adding one or more amino acids to the amino acid sequence of (V), and having the same function as the amino acid sequence of (I). According to some embodiments of the anti-CK8 nanobody of this application, wherein the amino acid sequence of the nanobody of (VI) is as shown in any one of SEQ ID NO. 6 to SEQ ID NO. 14.

[0016] In a second aspect, according to some embodiments of the present application, the humanized nanobody (VII): the amino acid sequence of the humanized nanobody is shown in any one of SEQ ID NO.15 to SEQ ID NO.46.

[0017] On a third-party level, the polypeptides in some embodiments of this application include any of the nanobodies described herein.

[0018] In a fourth aspect, nucleic acid molecules encoding any one of the nanobodies according to some embodiments of this application.

[0019] In a fifth aspect, the expression vector according to some embodiments of this application includes the nucleic acid molecule.

[0020] In a sixth aspect, the host cells of the expression vector described herein are transformed or transfected according to some embodiments of this application.

[0021] In a seventh aspect, the conjugates or conjugates according to some embodiments of this application include nanobodies described in any one of the chemically labeled or biologically labeled methods.

[0022] In an eighth aspect, the adsorbents according to some embodiments of this application include any of the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the couplings, and the carriers.

[0023] In a ninth aspect, the kit according to some embodiments of this application includes any of the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the conjugates; or the adsorbents, as well as adjuvants acceptable for detection.

[0024] In a tenth aspect, the apparatus according to some embodiments of this application is used to capture, adsorb, and / or detect CK8, including any of the nanobodies described herein; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the coupling agents; or the adsorbents; or the kits described herein.

[0025] In the eleventh aspect, the use of the nanobody; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the coupling compound; or the adsorbent; or the kit; or the device according to some embodiments of this application in the preparation of preparations for the specific capture, adsorption, and / or detection of CK8; in the preparation of cell preparations for the specific capture, adsorption, and / or detection of CK8, for enrichment and / or purification; or in the preparation of immunofluorescence or immunohistochemical assay reagents for the specific capture, adsorption, and / or detection of CK8.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The nanobody of the present invention is an anti-CK8 nanobody with a novel amino acid sequence discovered through screening. This nanobody and its polypeptide have high affinity and activity, and can specifically recognize and bind to CK8. It can be used for CK8 capture, detection and purification, as well as the capture and detection of CK8 positive cells. With appropriate antibody labeling technology, it can be applied to immunofluorescence analysis or immunohistochemical analysis, etc. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the interaction between K8-A0 and CK8 in Embodiment 4 of the present invention.

[0029] Figure 2 This is a diagram of the docking of K8-A0 and CK8 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0030] Figure 3 This is a diagram of the docking of K8-A1 and CK9 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0031] Figure 4 This is a diagram of the docking of K8-A2 and CK10 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0032] Figure 5This is a diagram of the docking of K8-A3 and CK11 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0033] Figure 6 This is a diagram of the docking of K8-A4 and CK12 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0034] Figure 7 This is a diagram of the docking of K8-A5 and CK13 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0035] Figure 8 This is a diagram of the docking of K8-A6 and CK14 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0036] Figure 9 This is a diagram of the docking of K8-A7 and CK15 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0037] Figure 10 This is a diagram of the docking of K8-A8 and CK16 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0038] Figure 11 This is a diagram of the docking of K8-A9 and CK17 in Embodiment 4 of the present invention, where a is the overall effect, b is a partial magnification from the first angle, and c is a partial magnification from the second angle.

[0039] Figure 12 This is a kinetic curve of the nanobody in Example 6 of the present invention.

[0040] Figure 13 This is a fluorescence immunoassay result of the nanobody in Example 10 of the present invention, where a is MCF-7 cells and b is primary mouse hepatocytes.

[0041] Figure 14 This is an immunohistochemical image of the nanobody in Example 11 of the present invention. Detailed Implementation

[0042] The foregoing and other aspects of the present invention will be further described below, wherein:

[0043] (1) Unless otherwise specified, the term “sequence” is used herein (as in similar terms such as “antibody sequence”, “variable region sequence”, “V”). HHIn the term "sequence" or "protein sequence," it should generally be understood to include the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said amino acid sequence, unless the context requires a narrower interpretation.

[0044] (2) Unless otherwise specified, all methods, steps, techniques and operations not specifically described are known and well known to those skilled in the art. For example, references are still made to the general background art cited above and other references cited therein.

[0045] (3) The term "specificity" refers to the ability of a specific antigen-binding molecule (such as the nanobodies or peptides of the present invention) to bind to different types of antigens or antigenic determinants. The specificity of an antigen-binding molecule can be determined based on its affinity and / or activity. Affinity is expressed as the dissociation equilibrium constant (K0) between the antigen and the antigen-binding molecule. D K is a measure of the binding strength between antigens and antigen-binding molecules. D The smaller the K value, the stronger the binding strength between the antigen and the antigen-binding molecule; conversely, the larger the K value, the stronger the binding strength between the antigen and the antigen-binding molecule. D The higher the value, the weaker the binding strength between the antigen and the antigen-binding molecule. a K represents the associative constant. a The larger the value, the faster the binding. a The smaller the value, the slower the binding; K d K represents the dissociation constant. d The larger the value, the faster the dissociation. d The smaller the value of K, the slower the dissociation; and K D = K d / K a .

[0046] (4) The term “family” refers to a group of nanobodies with highly similar CDR3 sequences, where highly similar means that the CDR3 length is the same and the sequence similarity is greater than 80%. These family nanobodies are derived from the same B cell line and bind to the same epitope of the same target (antigen) (Pardon E, et.al, A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 2014 Mar;9(3):674-93).

[0047] (5) Amino acid substitution can generally be described as follows: an amino acid residue can be replaced by an amino acid with a similar chemical structure or by an amino acid with a dissimilar chemical structure, as long as it has little or no effect on the function, activity or other biological properties of the polypeptide. Preferably, the amino acid residue can be replaced by an amino acid with a similar chemical structure.

[0048] For the above-mentioned substitution methods, examples disclosed in documents WO04 / 037999, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300 can be listed, but are not limited to these. In addition, (preferred) types and / or combinations of the substitution can be selected based on relevant information from other references cited in WO 04 / 037999 and WO 06 / 122786.

[0049] The amino acid substitutions of the present invention can be listed, but are not limited to, the following substitution methods, in which one amino acid in the following groups (a) to (e) is replaced by another amino acid in the same group: (a) Ala, Ser, Thr, Pro and Gly; (b) Asp, Asn, Glu and Gln; (c) His, Lys and Arg; (d) Met, Leu, Ile, Val and Cys; (e) Phe, Tyr and Trp.

[0050] Preferred amino acid substitutions may include, but are not limited to, the following: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser or Thr; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted with Ile or Val; Lys is substituted with Arg, Glu, or Gln; Met is substituted with Leu, Tyr, or Ile; Phe is substituted with Met, Tyr, or Leu; Ser is substituted with Thr; Thr is substituted with Ser; Tyr is substituted with Trp; Trp is substituted with Tyr.

[0051] The framework region is more conserved compared to the complementarity-determining region. Those skilled in the art will rationally screen the sequence structure of the framework region based on the actual application and function of the nanobody. For the amino acid sequence of the framework region, an amino acid sequence with homology of 50% or more is preferred, further preferred is an amino acid sequence with homology of 75% or more, and even more preferred is an amino acid sequence with homology of 95% or more. Tables 1 and 2 show the framework region sequence information of each antibody in Example 3:

[0052] Table 1. Comparison of Nanobody Serial Numbers and FR Sequence Information - 1

[0053]

[0054] Table 2 Comparison of Nanobody Serial Numbers and FR Sequence Information - 2

[0055]

[0056] The framework region contributes relatively little to affinity; therefore, amino acid substitutions in the framework region generally do not affect the affinity of nanobodies. As long as they can exist in a soluble form, the amino acid substitution methods described above also apply. Humanization is a typical example of amino acid substitution in the framework region. In this invention, SEQ ID No. 15 to SEQ ID No. 46 represent four humanized forms of SEQ ID No. 5 to SEQ ID No. 14, none of which affect the affinity of the original sequence.

[0057] Furthermore, the total number of residues in nanobodies can be in the range of 110-120. However, the portions, fragments, or analogs of nanobodies are not particularly limited in their length and / or size, provided that such portions, fragments, or analogs meet the further requirements listed below and are also suitable for the purposes described herein.

[0058] The nanobodies in this invention belong to the same family of nanobodies, with the same total length of amino acid sequences, the same length of each frame region (FR) and antigen-binding region (CDR), high sequence identity, similar structure, and basically equivalent antigen-binding capacity.

[0059] The core of nanobody encoding lies in the standardized numbering of the amino acid sequence of its VHH domain, thereby distinguishing between the framework region (FR) responsible for maintaining spatial structural stability and the complementarity-determining region (CDR) mediating antigen-specific binding. Commonly used encoding methods in this field include IMGT, Kabat, and Chothia. This application adopts the IMGT encoding method to define the FR and CDR. IMGT coding is a universal standard recognized by the International Federation of Immunological Societies (IFIS) of the World Health Organization. It is based on sequence alignment using a complete reference gene database of the entire immunoglobulin superfamily and serves as a consensus coding system in the global research field of immunoglobulins and single-domain antibodies. It ensures the comparability and interoperability of different research data and cross-species single-domain antibody sequences. For nanobodies, residues are counted consecutively from 1 to 128. If a residue is missing, the corresponding number is left blank without complex additional annotations, avoiding problems such as numbering confusion and inconsistent labeling of inserted residues that are prone to occur in other coding methods. IMGT coding has exclusive FR-IMGT and CDR-IMGT definitions, with clear boundaries and a high degree of fit with the structure and function of single-domain antibodies. It can accurately correspond to the conserved regions of its backbone and the antigen-binding variable regions, providing reliable support for subsequent sequence analysis and epitope identification.

[0060] The preparation method of "nanobody" is, in its broadest sense, not limited to specific biological resources or specific preparation methods. For example, the nanobody of the present invention can be obtained as follows: (1) by isolating V of naturally occurring heavy chain antibodies. HH Structural domain; (2) Encoding the naturally occurring V through expression HH The nucleotide sequence of the domain; (3) by using naturally occurring V HH The structural domain is "humanized" (as described below) or the humanized V is expressed by encoding the expression. HH (4) preparing a protein, polypeptide or other amino acid sequence by means of synthetic or semi-synthetic techniques; (5) preparing a nucleic acid encoding a nanobody by means of nucleic acid synthesis techniques and then expressing the nucleic acid thus obtained; and / or (6) by any of the foregoing combinations.

[0061] Furthermore, a variant of the nanobody based on the present invention also includes having a similarity to naturally occurring V. HH Nanobodies with corresponding but humanized amino acid sequences for their structural domains. Humanization refers to the use of V-chain antibodies derived from conventional 4-chain antibodies from humans. H One or more amino acid residues at the corresponding positions in the domain replace the naturally occurring V. HH One or more amino acid residues in a domain sequence.

[0062] According to a non-limiting embodiment of the present invention, the above-mentioned polypeptide is substantially composed of nanobodies. "Substantially composed of" means that the amino acid sequence of the polypeptide of the present invention is exactly the same as or corresponds to the amino acid sequence of the nanobodies, wherein a limited number of amino acid residues, such as 1 to 10 amino acid residues, and preferably 1 to 6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, are added to the amino terminus (N-terminus) and / or carboxyl terminus (C-terminus) of the nanobodies or polypeptides.

[0063] The aforementioned amino acid residues may not alter the biological properties of the nanobody, and may even impart other functionalities to the nanobody. For example, the amino acid residues may:

[0064] 'a' is a purification tag, i.e., an amino acid sequence or residue that facilitates the purification of the nanobody, for example, by using affinity techniques targeting the sequence or residue for purification. Some preferred, but not limited, examples of such residues are multiple sets of His-tags (His6 or His8), GST-tags, MBP-tags, Myc-tags, Strep-tags, Flag-tags, HA-tags, V5-tags, S-tags, and E-tags;

[0065] b is a soluble tag, which is a tag that increases the solubility of nanobodies, such as SUMO;

[0066] c is an N-terminal amino acid residue, such as Met, Ala, Gln or MetAlaGln, AlaGln, which can be expressed in a heterologous host cell or in a host organism.

[0067] d is a C-terminal Cys residue, which can, for example, react with -SH on a ligand or with the Au surface;

[0068] e is a hinge to provide a link or spacer between the nanobody and other groups, such as a combination of GlySer, an IgG hinge, an IgA hinge, or other synthetic hinges.

[0069] f is provided in a known manner with one or more amino acid residues that have functional groups and / or have been functionalized, for example, as is known in the art, amino acid residues such as lysine or cysteine ​​allow PEG groups to attach.

[0070] The polypeptide of the present invention may also include two or more of the nanobodies, also known as multivalent polypeptides.

[0071] A bivalent polypeptide comprises two nanobodies, optionally linked by one hinge sequence; a trivalent polypeptide comprises three nanobodies, optionally linked by two hinge sequences; and a tetravalent polypeptide comprises four nanobodies, optionally linked by three hinge sequences. Multivalent polypeptides can bind to the same antigenic epitope or different antigen-binding epitopes; the latter are also called multispecific polypeptides.

[0072] Regarding containing one or more V HH For information on multivalent and multispecific polypeptides with structural domains and their preparation, please refer to EP 0822985.

[0073] Hinges for multivalent and multispecific peptides should be well known to those skilled in the art, including, for example, Gly-Ser, such as (Gly4Ser)3 or (Gly3Ser2)3 as described in WO 99 / 42077; or naturally occurring heavy chain antibody hinge regions or portions thereof. For other suitable hinges, reference may also be made to the comprehensive background art cited above.

[0074] In addition to the one or more nanobodies mentioned above, the polypeptides of the present invention may also contain functional groups, portions or residues, such as therapeutically active substances, and / or tags, such as fluorescent labels, isotope labels, biotin labels and enzyme catalytic tags.

[0075] Furthermore, the dissociation equilibrium constant (K) of the nanobody or peptide of the present invention binding with CK8 D ) is 10 -8~10 -10 Moles per liter (M). The dissociation equilibrium constant of this invention was determined using plasmon resonance technology.

[0076] The specific binding between the aforementioned antigen and antigen-binding molecules can be determined by any suitable known method, including Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), as well as other novel methods known in the art, such as plasma resonance (SPR) and / or biomembrane interference (BLI) techniques. Furthermore, those skilled in the art should recognize that affinity parameters measured using different methods can vary considerably, even by two to three orders of magnitude.

[0077] The nanobody structure of this invention can be determined using any suitable known method, including Alohafold2multimer v3 (a protein structure prediction tool used to predict the three-dimensional structure of protein complexes, helping researchers understand how different proteins interact to form complexes), Amber (a widely used molecular dynamics simulation software package used to simulate the behavior of proteins and other biomolecules. In structural biology, Amber is often used to perform energy minimization, side-chain optimization, and molecular dynamics simulations), Relax Process (in molecular dynamics simulations, Relax Process typically refers to the process of minimizing the energy of protein structures to optimize the geometry of side chains and reduce intramolecular conflicts and undesirable geometries), ADT (AutoDock Tools, a software toolset for molecular docking and molecular dynamics simulations that helps researchers predict binding patterns and affinities between ligands and receptors), and / or Ligplot+ (a tool for analyzing protein-ligand complex interfaces, identifying and visualizing hydrogen bonds, hydrophobic interactions, and other non-covalent interactions).

[0078] The nanobodies, peptides, and nucleic acids encoding the present invention can be prepared in known ways, as will become clear to those skilled in the art from the further description herein. A particularly useful method for preparing the nanobodies, peptides, and nucleic acids typically includes the following steps:

[0079] (1) Expressing nucleic acid encoding the nanobody or polypeptide of the present invention in a suitable host cell or host organism or in another suitable expression system, optionally followed by;

[0080] (2) Isolate and / or purify the nanobodies or polypeptides of the present invention thus obtained.

[0081] Alternatively, other methods can be used, including the following steps:

[0082] (3) Cultivate and / or maintain the host of the present invention under certain conditions, so that the host of the present invention expresses and / or produces a nanobody and / or polypeptide of the present invention; optionally, proceed thereafter;

[0083] (4) Isolate and / or purify the nanobodies or polypeptides of the present invention thus obtained.

[0084] The nucleic acid of the present invention can be in the form of single-stranded or double-stranded DNA or RNA, and is preferably in the form of double-stranded DNA. For example, the nucleic acid sequence of the present invention can be genomic DNA, cDNA or synthetic DNA (such as DNA having codons particularly suitable for expression in the host cell or host organism to which it is intended, i.e., codon optimized).

[0085] The nucleic acids of this invention can be prepared or obtained by methods that are essentially known, based on the amino acid sequence information of the nanobodies or polypeptides of this invention given herein, and / or can be isolated from suitable natural sources. For example, for naturally occurring V... HH The nucleic acid sequence of the structural domain is subjected to site-directed gene mutation to provide the nucleic acid of the present invention encoding the analogue.

[0086] The nucleic acids of the present invention can also be present in and / or as part of a genetic construct, as is well known to those skilled in the art. Such genetic constructs typically include at least one nucleic acid of the present invention, which can be in vector form, such as a plasmid, YAC, viral vector, or transposon. In particular, the vector can be an expression vector, i.e., a vector that can provide in vitro and in vivo expression (e.g., in a suitable host cell, host organism, and / or expression system).

[0087] The nucleic acids and / or genetic constructs of the present invention can be used to transform host cells or host organisms, i.e., for expressing and / or producing the nanobodies or peptides of the present invention. Suitable hosts or host cells are well known to those skilled in the art, and may be, for example, any suitable fungus, prokaryotic or eukaryotic cell or organelle or organism, as well as all other hosts or host cells known essentially for expressing and producing antibodies and antibody fragments (including, but not limited to, single-domain antibodies and ScFv fragments), are well known to those skilled in the art.

[0088] For production, the nanobodies and peptides of the present invention can be produced in the milk of transgenic mammals, such as rabbits, cows, goats or sheep, or in plants or parts of plants, including but not limited to their leaves, flowers, fruits, roots or seeds.

[0089] As mentioned above, one advantage of using nanobodies is that the peptides based on them can be expressed and prepared in prokaryotic systems, and suitable prokaryotic expression systems, vectors, and host cells are well known to those skilled in the art, as cited in the references above. However, it should be noted that this invention is not limited, in its broadest sense, to expression in bacterial systems.

[0090] Preferably, in this invention, the nanobodies or peptides are produced in bacterial cells, particularly in bacterial cells suitable for large-scale drug production, as described above.

[0091] When the nanobodies or peptides of the present invention are expressed in cells, the nanobodies or peptides of the present invention may be generated intracellularly (e.g., in the cytoplasm or periplasmic space), then isolated from the host cell, and optionally further purified; or they may be generated extracellularly (i.e., secreted expression), then isolated from the culture medium, and optionally further purified.

[0092] Some preferred but non-limiting vectors for use with these host cells include vectors for expression in mammalian cells—pMANneo (Clonetech), pUCTtag (ATCC37460), and pMClneo (Stratagene); vectors for expression in bacterial cells—pET vector (Novagen) and pQE vector (Qiagen); expression vectors for expression in yeast or other fungal cells—pYES2 (Invitrogen) and Pichia pastoris expression vector (Invitrogen); expression vectors for expression in insect cells—pBlueBacⅡ (Invitrogen) and other baculovirus vectors; and so on.

[0093] The corresponding techniques used to transform the host or host cells of the present invention are well known to those skilled in the art.

[0094] After transformation, it is possible to detect and select those hosts that have been successfully transformed with the nucleotide sequence / genetic construct of the present invention. The transformed host cells (which may be in the form of stable cell lines) or host organisms (which may be in the form of stable mutant lines or strains) form another aspect of the present invention.

[0095] The amino acid sequence of the present invention can then be isolated from the host cell / host organism and / or from the culture medium in which the host cell or host organism is cultured, using essentially known protein separation and / or purification techniques, such as (preparative) chromatography and / or electrophoresis, differential precipitation, affinity techniques (e.g., using a specific / cleavable amino acid sequence fused with the amino acid sequence of the present invention) and / or preparative immunological techniques (i.e., using antibodies against the amino acid sequence to be isolated).

[0096] The nanobodies of this invention can be used to specifically recognize CK8 and CK8-positive cells.

[0097] The nanobodies or peptides of the present invention can be used for enrichment, purification, removal and detection of CK8.

[0098] This invention provides nanobodies with specific amino acid sequences, peptides containing these nanobodies, and their applications, to address the problems of complex and costly antibody preparation processes, as well as poor antibody affinity and stability, in existing methods for intracellular and extracellular CK8 detection, CK8-positive cell detection and capture, and CK8 enrichment, purification, detection, and removal. Specifically, in a first aspect, this invention provides a CK8-binding nanobody, wherein the variable region in the amino acid sequence of the nanobody includes a complementarity-determining region (CDR) and a framework region (FR). The CDR includes CDR1, CDR2, and CDR3, wherein the most important sites involved in antigen recognition and binding are R25, R28, and Y30 on CDR1 and S101, R104, R107, and Y111 on CDR3.

[0099] Preferably, the amino acid sequence of the complementarity-determining region CDR1 includes SEQ ID No. 47 to SEQ ID No. 50, the amino acid sequence of the complementarity-determining region CDR2 includes SEQ ID No. 51 to SEQ ID No. 58, and the amino acid sequence of the complementarity-determining region CDR3 includes SEQ ID No. 59 and a sequence with more than 75% homology to it.

[0100] Preferably, the amino acid sequence of the nanobody includes: SEQ ID No. 5 ~ SEQ ID No. 14.

[0101] Preferably, the nanobody is a humanized nanobody, and more preferably, the humanized nanobody includes: SEQ ID No. 15 ~ SEQ ID No. 46.

[0102] In a second aspect, the present invention provides a polypeptide obtained by modifying the amino acids at the N-terminus and / or C-terminus of the aforementioned nanobody.

[0103] Preferably, the N-terminal and / or C-terminal amino acid modification of the nanobody includes:

[0104] Method 1: Tagging the N-terminal and / or C-terminal amino acids of the nanobody;

[0105] Method 2: After tagging the N-terminal and / or C-terminal amino acids of the nanobody, the tag is further connected to protect the amino acids via a hinge;

[0106] Preferably, the tag includes at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag, and Flag-tag; the hinge includes at least one of GS hinge, IgG hinge, IgA hinge, and PEG; and the protected amino acid includes Ala, Gln, Glu, Met, or any combination of two or more of the aforementioned amino acids.

[0107] Thirdly, the present invention provides a polypeptide obtained by multivalent synthesis of the aforementioned nanobody.

[0108] Fourthly, the present invention provides a nucleic acid that encodes the aforementioned nanobody or the aforementioned polypeptide.

[0109] Fifthly, the present invention provides an expression vector comprising the expression frame of the nucleic acid described above.

[0110] In a sixth aspect, the present invention provides a host cell containing the expression vector described above.

[0111] In a seventh aspect, the present invention provides the application of the nanobodies and / or the peptides described herein in immunoassay, enrichment and / or purification.

[0112] Preferably, the nanobody and / or the polypeptide are used in the preparation of CK8 adsorbents, CK8 purification kits, and CK8 detection kits.

[0113] Preferably, the nanobody and / or the polypeptide are used in the capture and detection of CK8 positive cells.

[0114] Example

[0115] The following examples illustrate specific implementations of the present invention. However, the implementation of the present invention is not limited to these examples, and any selections and modifications can be made within the scope of the technical effects to be achieved by the present invention.

[0116] Example 1: Construction of an anti-CK8 nanobody library.

[0117] The phage display library used in this invention is an immune library based on T7 phage, and the establishment steps are as follows:

[0118] (1) Alpacas (numbered 2305-1 and 2305-2) were immunized with CK8. After four immunizations, jugular vein blood was collected from the two alpacas, peripheral blood lymphocytes were isolated, and total RNA (PuerLink) was extracted. TM RNA Mini Kit, Life Technologies: 12183018A);

[0119] (3) Total RNA was reverse transcribed into cDNA, and V was amplified by two rounds of nested PCR. HH Gene;

[0120] The first round of PCR used cDNA as a template, with UP primer1 and DOWN primer1 as upstream and downstream primers, respectively. After amplification, a band of 650-750 bp was recovered. This band was then used as the template for the second round of PCR, with UP primer2 and DOWN primer2 as upstream and downstream primers, respectively. A PCR product of 450-500 bp was recovered.

[0121] UP primer1:CTTGGTGGTCCTGGCTGCTCT,

[0122] DOWN primer1:GGTACGTGCTGTTGAACTGTTCC,

[0123] UP primer2:TATCTAGTCGAATTCCGCCCAGGTGCAGCTC,

[0124] DOWN primer2: AGCGACTAAGCTTTGAGGAGACGGTGAC;

[0125] (3) The PCR product was digested with EcoRI and HindIII and subjected to agarose gel electrophoresis. The gene band of 350-500 bp was recovered, which is V. HH Gene fragments;

[0126] (4) Ligate the T7 vector (T7Serelect® 10-3 Cloning Kit, Merck Millipore Novagen®: 70550-3) and V using T4 ligase. HH Gene fragments;

[0127] (5) The ligation product is mixed with the packaging protein to form a complete T7 phage. The mixture is then amplified to obtain the original phage library.

[0128] (6) The titer of the original library was found to be 8.74 × 10⁻⁶. 9 pfu / mL, diversity was 8.9 × 10 6 .

[0129] Example 2: Screening of nanobodies.

[0130] First, the antigen CK8 was diluted to 10 μg / mL with TBS, and 100 μL was added to a 96-well plate and incubated at 4°C for 12 h. The antigen dilution was aspirated from the wells, the plate was washed 3 times with TBS, blotted dry, and 300 μL of 1% protein-free blocking buffer (purchased from Sangon Biotech Co., Ltd.) was added to each well. The plate was incubated at room temperature for 2 h (1% protein-free blocking buffer and 1% BSA were used alternately during screening). The blocking agent was aspirated from the wells, the plate was washed 6 times with TBST, blotted dry, and 100 μL of amplified phage was added to each well. The plate was incubated at room temperature for 30 min. The plate was washed 10 times with TBST, and the phage was eluted with T7 elution buffer (1% SDS). The plate was incubated at room temperature for 30 min, and the elution buffer was amplified for the next round of screening.

[0131] Example 3: Construction of genetically engineered bacteria.

[0132] (1) After four rounds of screening, the screening eluent was amplified on solid, plaques were picked, and PCR amplification was performed using plaque amplification solution as template and UP primer3 and DOWN primer3 as upstream and downstream primers.

[0133] UP primer3:TTCCTTAACATATGGCCCAGGTGCAGCTCGT,

[0134] DOWN primer3: TTAAGGAACTCGAGCACGGTGACCAGGGTC;

[0135] (2) A portion of the PCR products were sequenced externally to obtain the nanobody sequence information. Based on the CDR region length and homology, 10 monoclonal sequences belonging to the same family were selected. The nanobody naming and sequence number information is shown in Table 1.

[0136] (3) The other part of the PCR product was double-digested with NdeI and XhoI, and the digested products were recovered. At the same time, the same method was used to digest and recover the vector. The digested products and the vector were ligated with T4 ligase, and the ligation product was transformed into Escherichia coli to obtain the genetically engineered bacteria expressing CK8 specific nanobodies.

[0137] Table 3. Comparison of Nanobody Serial Numbers and CDR Sequence Information

[0138]

[0139] SEQ ID No.5:

[0140] QLQESGGGLVQAGGSLRLSCAASG-RSFRNYV-LGWFRQAPGKEREFVTSI-SSAGGTQ-DYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0141] SEQ ID No.6:

[0142] QLQESGGGLVQAGDSLRLSCAASG-RSFRNYV-LGWFRQAPGKEREFVTSI-SWAGGTQ-DYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0143] SEQ ID No.7:

[0144] QLQESGGGLVQPGGSLRLSCAASG-RSFRNYV-LGWFRQAPGKEREFVTSI-SWAGGTQ-DYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0145] SEQ ID No.8:

[0146] QLQESGGGLVQAGGSLRLSCAASG-RSFRNYV-LGWFRQAPGKEREFVTSI-SWAGGTQ-DYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0147] SEQ ID No.9:

[0148] QLQESGGGLVQAGGSLRLSCAASG-RPFRIYA-LGWFRQAPGKEREFVTSI-SWSGGNT-DYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0149] SEQ ID No.10:

[0150] QLQESGGGLVQAGGSLRLSCAASG-RPFRIYA-LGWFRQAPGKEREFVTSI-NWSGGNT-DYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0151] SEQ ID No.11:

[0152] QLQESGGGLVQAGGSLRLSCAASG-RPFRLYA-MGWFRQAPGKEREFVTSI-NWTGTST-DYADSVKGRFTISRNNAKNTLYLQMSSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0153] SEQ ID No.12:

[0154] QLQESGGGLVQAGGSLRLSCAVSG-RPFRLYA-MGWFRQAPGKEREFVTSI-NKTGTST-DYADSVKGRFTVSRNNAKNTLYLQMSSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0155] SEQ ID No.13:

[0156] QLQESGGGLVQAGGSLRLSCAASG-RGFRWYA-MGWFRQAPGKEREFVTSI-TRGGTST-DYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0157] SEQ ID No.14:

[0158] QLQESGGGLVQAGGSLRLSCAASG-RGFRWYA-MGWFRQAPGKEREFVTSI-TRGGSST-DYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGPGTQVTVSS

[0159] Among them, the CDR sequences of K8-A1, K8-A2 and K8-A3 are the same, and only the FR sequences are different

[0160] Example 4: Calculation of key amino acids.

[0161] Using sequences K8-A0 and CK8 (205~483aa) as model proteins, molecular docking was performed to predict the antigen-binding epitope of VHH on CK8 and the key amino acids in the interaction.

[0162] First, the CK8 structure was obtained from the database. The CK8 sequence and the VHH antibody sequence were combined, and Alohafold2multimer v3 was used for complex structure prediction and search. After generating the complex structure, Amber was used to perform a Relax Process to optimize the side chain structure. ADT (AutoDock Tools) was used to check hydrogen atoms and calculate potentials. Ligplot+ was used to analyze the hydrogen bonding and hydrophobic interaction network at the interface between the VHH CDR region amino acids and CK8, and to check the atomic contacts between structural domains. A reasonable conformation that met the above conditions was selected as the structural model of the VHH antibody-CK8 protein complex.

[0163] Through molecular docking, the binding epitopes of VHH on CK8 were calculated: K8-A0 is involved in antigen recognition and binding via R25, R28, and Y30 on CDR1, and S101, R104, R107, and Y111 on CDR3. The interaction between VHH and CK8 is as follows: Figure 1 As shown, R25 on CDR1 of VHH identifies and combines with E234 on CK8, R28 identifies and combines with E230 on CK8, and Y30 identifies and combines with E231 on CK8. S101 and R104 on CDR3 of VHH jointly identify and combine with E288 and S291 on CK8, R107 identifies and combines with E287 on CK8, and Y111 identifies and combines with E277 on CK8.

[0164] The molecular docking results of K8-A0 and CK8 protein are as follows: Figure 2 As shown, 'a' represents the molecular docking diagram and a magnified view of the nanobody K8-A0 and the CK8 protein. To further clarify each point and the docking relationship, Figure 2 Figures b and c show magnified views of the molecular docking diagram from different angles. It can be seen that the main amino acid residues involved in antigen recognition and binding on VHH are R25, R28, and Y30 (blue) on CDR1, and S101, R104, R107, and Y111 (red) on CDR3. They bind to CK8 through electrostatic interactions. The binding epitopes are two antiparallel α-helices, specifically at sites E230, E231, E234, E277, E287, E288, and S291.

[0165] The molecular docking results of K8-A1 and CK8 protein are as follows: Figure 3As shown, the molecular docking results of K8-A2 and CK8 proteins are as follows: Figure 4 As shown, the molecular docking results of K8-A3 and CK8 protein are as follows: Figure 5 As shown, the molecular docking results of K8-A4 and CK8 proteins are as follows: Figure 6 As shown, the molecular docking results of K8-A5 and CK8 proteins are as follows: Figure 7 As shown, the molecular docking results of K8-A6 and CK8 proteins are as follows: Figure 8 As shown, the molecular docking results of K8-A7 and CK8 proteins are as follows: Figure 9 As shown, the molecular docking results of K8-A8 and CK8 proteins are as follows: Figure 10 As shown, the molecular docking results of K8-A9 and CK8 proteins are as follows: Figure 11 As shown, the main amino acid residues involved in antigen recognition and binding on VHH are R25, R28, and Y30 (blue) on CDR1, and S101, R104, R107, and Y111 (red) on CDR3. They bind to CK8 via electrostatic interactions, with the binding epitopes being two antiparallel α-helices, specifically at sites E230, E231, E234, E277, E287, E288, and S291. This is consistent with the molecular docking results of K8-A0 with the CK8 protein.

[0166] Sequence analysis revealed that in the homologous nanobody sequences described in Example 3, these two regions (RXXRXYX on CDR1 and ASPAVSPPRDGRAFTY on CDR3) are strictly conserved. That is, the most important sites for antigen recognition and binding of the nanobodies K8-A0~K8-A9 of the present invention are R25, R28, and Y30 on CDR1 of VHH, and S101, R104, R107, and Y111 on CDR3 of VHH.

[0167] Example 5: Preparation of CK8 nanobody.

[0168] (1) The basic culture medium for nanobodies is TB medium. The inoculum is 5% and cultured at 37°C for 3-5 hours. The inducing agent galactoside (IPTG) (final concentration 0.25 mM, the same below) is added for overnight induction.

[0169] (2) After induction, centrifuge at 4000 rpm for 20 min to obtain wet bacteria containing nanobodies.

[0170] (3) Add lysis buffer (10mM imidazole, 500mM NaCl, pH7.4 0.02M PB) to the obtained wet bacteria at a ratio of 1:10, and use a 700bar high-pressure homogenizer to disrupt the cells;

[0171] (4) Centrifuge at 4℃ and 10000 rpm for 20 min and collect the supernatant;

[0172] (5) The supernatant was filtered through a 0.45 μm filter and then purified by affinity chromatography column (GE Healthcare, US) for separation of CK8 nanoantibody. The packing material of the affinity chromatography column was Ni Sepharose High Performance.

[0173] (6) The nanobody purified by affinity chromatography was subjected to SDS-PAGE electrophoresis to determine its purity. The protein solution with high purity was selected and the protein concentration was determined by BCA method.

[0174] Example 6: Affinity analysis.

[0175] The binding ability of nanobodies to human CK8 was analyzed using SPR technology. In this study, CK8 (205-483 aa) can be used as an antigen to prepare antibodies for detecting CK8 expression or conducting related research. Because this fragment contains part of the central "bar" domain and the C-terminal tail, it still possesses certain immunogenicity and can specifically bind to the corresponding antibodies, thus helping researchers detect and locate the presence of CK8 protein in cells or tissues. CK8 (1-483 aa) is the complete CK8 protein, containing its entire amino acid sequence. The nanobodies K8-A0 to K8-A9 of this invention have the ability to bind to both forms of CK8, as demonstrated by the following two methods:

[0176] In this invention, CK8 (205~483 aa) was amino-coupled onto a CM5 sensor chip at a density of 500~800 RU. Seven different concentrations of nanobodies were injected within the range of 1~100 nM, with a flow rate of 45 μL / min in all experiments. The chip regeneration conditions were glycine-HCl pH 1.5. The kinetic parameter K was calculated using binding curves obtained at different nanobodies concentrations. a K d and K D . Figure 12 The curves, from top to bottom, represent the response curves of nanobodies K8-A0 to K8-A9 to CK8 at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM. The kinetic parameters, as shown in Table 4, were calculated through equation fitting. All tested nanobodies exhibited high affinity for CK8 (205–483 aa). D The range is consistent at 10 -9 (M).

[0177] Table 4. Affinity of nanobodies to CK8 (205~483 aa)

[0178]

[0179] ELISA confirmed the binding ability of nanobodies K8-A0 to K8-A9 for both CK8 (205–483 aa) and CK8 (1–483 aa). Both forms of CK8 were coated onto 96-well plates, with blank wells as controls. The binding ability of nanobodies K8-A0 to K8-A9 for both forms was tested, and the absorbance values ​​are shown in Table 5. Comparing the two forms of CK8 protein, the absorbance values ​​of nanobodies K8-A0 to K8-A9 for both forms were significantly higher than those of the blank wells, demonstrating high binding ability. Furthermore, the absorbance values ​​were essentially equivalent, with minimal differences. This indicates that nanobodies K8-A0 to K8-A9 possess strong and comparable binding abilities for both CK8 proteins.

[0180] Table 5. Binding ability of nanobodies to CK8

[0181]

[0182] Example 7: Stability, humanization, and affinity determination of nanobodies.

[0183] To reduce the immunogenicity of nanobodies while maintaining or improving their stability and bioactivity, this invention utilizes a commonly used humanized or highly stable nanobodies backbone for CDR region transplantation, thereby achieving the humanization and stability modification of the nanobodies.

[0184] (1) Sequence design

[0185] This invention selects four nanobody backbones for modification, namely ah, YW-sdAb (hereinafter referred to as sdAb), hs2dAb (hereinafter referred to as hs) and com backbone, the sequence information of which are shown in SEQ ID No.1 to SEQ ID No.4, wherein the CDR region is occupied by X, and X represents any amino acid.

[0186] SEQ ID No. 1:

[0187] QLVESGGGLVQPGGSLRLSCAASGGWFRQAPGKGLEAVAAIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYWGQGTLVTVSS

[0188] SEQ ID No. 2:

[0189] QLVESGGGLVQPGGSLRLSCAASGYLGWFRQAPGQGLEAVAAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAYWGQGTLVTVSS

[0190] SEQ ID No. 3:

[0191] QLQESGGGLVQAGGSLRLSCAASGGWFRQAPGKEREFVAAIYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAYWGQGTQVTVSS

[0192] SEQ ID No.4:

[0193] QLQASGGGFVQPGGSLRLSCAASMGGWFRQAPGKEREFVSAISYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAYWGQGTQVTVSS

[0194] The ah and sdAb backbones are universal fully humanized backbones, taken from the literature (Chi, XJ et al. Humanized single domain antibodies neutralize SARS-CoV-2 by targeting the spike receptor binding domain. Nature Communications 11, doi:10.1038 / s41467-020-18387-8 (2020).).

[0195] hs is a highly stable, highly expressed, partially humanized backbone, as described in the literature (Moutel, S. et al. NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife 5, doi:10.7554 / eLife.16228 (2016).).

[0196] com is a highly stable and highly expressive general-purpose scaffold, derived from the literature (Ferrari, D., Garrapa, V., Locatelli, M. & Bolchi, A. A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries. Molecular Biotechnology 62, 43-55, doi:10.1007 / s12033-019-00224-z (2020).).

[0197] The sequence names and sequence numbers of the modified humanized sequences are shown in Table 6.

[0198] Table 6. Names and Serial Numbers of Humanized Antibodies

[0199]

[0200] SEQ ID No. 15:

[0201] QLVESGGGLVQPGGSLRLSCAASG-RSFRNYV-LGWFRQAPGKGLEAVAAI-SSAGGTQ-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPRDGRAFTY-WGQGTLVTVSS

[0202] SEQ ID No. 16:

[0203] QLVESGGGLVQPGGSLRLSCAASG-RSFRNYV-LGWFRQAPGKGLEAVAAI-SWAGGTQ-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPRDGRAFTY-WGQGTLVTVSS

[0204] SEQ ID No. 17:

[0205] QLVESGGGLVQPGGSLRLSCAASG-RPFRIYA-LGWFRQAPGKGLEAVAAI-SWSGGNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPRDGRAFTY-WGQGTLVTVSS

[0206] SEQ ID No.18:

[0207] QLVESGGGLVQPGGSLRLSCAASG-RPFRIYA-LGWFRQAPGKGLEAVAAI-NWSGGNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0208] SEQ ID No.19:

[0209] QLVESGGGLVQPGGSLRLSCAASG-RPFRLYA-MGWFRQAPGKGLEAVAAI-NWTGTST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0210] SEQ ID No.20:

[0211] QLVESGGGLVQPGGSLRLSCAASG-RPFRLYA-MGWFRQAPGKGLEAVAAI-NKTGTST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0212] SEQ ID No.21:

[0213] QLVESGGGLVQPGGSLRLSCAASG-RGFRWYA-MGWFRQAPGKGLEAVAAI-TRGGTST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0214] SEQ ID No.22:

[0215] QLVESGGGLVQPGGSLRLSCAASG-RGFRWYA-MGWFRQAPGKGLEAVAAI-TRGGSST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0216] SEQ ID No.23:

[0217] QLVESGGGLVQPGGSLRLSCAASG-RSFRNYV-LGWFRQAPGQGLEAVAAI-SSAGGTQ-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0218] SEQ ID No.24:

[0219] QLVESGGGLVQPGGSLRLSCAASG-RSFRNYV-LGWFRQAPGQGLEAVAAI-SWAGGTQ-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0220] SEQ ID No.25:

[0221] QLVESGGGLVQPGGSLRLSCAASG-RPFRIYA-LGWFRQAPGQGLEAVAAI-SWSGGNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0222] SEQ ID No.26:

[0223] QLVESGGGLVQPGGSLRLSCAASG-RPFRIYA-LGWFRQAPGQGLEAVAAI-NWSGGNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0224] SEQ ID No.27:

[0225] QLVESGGGLVQPGGSLRLSCAASG-RPFRLYA-LGWFRQAPGQGLEAVAAI-NWTGTST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0226] SEQ ID No.28:

[0227] QLVESGGGLVQPGGSLRLSCAASG-RPFRLYA-LGWFRQAPGQGLEAVAAI-NKTGTST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0228] SEQ ID No.29:

[0229] QLVESGGGLVQPGGSLRLSCAASG-RGFRWYA-LGWFRQAPGQGLEAVAAI-TRGGTST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0230] SEQ ID No.30:

[0231] QLVESGGGLVQPGGSLRLSCAASG-RGFRWYA-LGWFRQAPGQGLEAVAAI-TRGGSST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTLVTVSS

[0232] SEQ ID No.31:

[0233] QLQESGGGLVQAGGSLRLSCAASG-RSFRNYV-LGWFRQAPGKEREFVAAI-SSAGGTQ-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0234] SEQ ID No.32:

[0235] QLQESGGGLVQAGGSLRLSCAASG-RSFRNYV-LGWFRQAPGKEREFVAAI-SWAGGTQ-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0236] SEQ ID No.33:

[0237] QLQESGGGLVQAGGSLRLSCAASG-RPFRIYA-LGWFRQAPGKEREFVAAI-SWSGGNT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0238] SEQ ID No.34:

[0239] QLQESGGGLVQAGGSLRLSCAASG-RPFRIYA-LGWFRQAPGKEREFVAAI-NWSGGNT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0240] SEQ ID No.35:

[0241] QLQESGGGLVQAGGSLRLSCAASG-RPFRLYA-LGWFRQAPGKEREFVAAI-NWTGTST-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0242] SEQ ID No.36:

[0243] QLQESGGGLVQAGGSLRLSCAASG-RPFRLYA-LGWFRQAPGKEREFVAAI-NKTGTST-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0244] SEQ ID No.37:

[0245] QLQESGGGLVQAGGSLRLSCAASG-RGFRWYA-LGWFRQAPGKEREFVAAI-TRGGTST-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0246] SEQ ID No.38:

[0247] QLQESGGGLVQAGGSLRLSCAASG-RGFRWYA-LGWFRQAPGKEREFVAAI-TRGGSST-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0248] SEQ ID No.39:

[0249] QLQASGGGFVQPGGSLRLSCAASG-RSFRNYV-MGWFRQAPGKEREFVSAI-SSAGGTQ-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0250] SEQ ID No.40:

[0251] QLQASGGGFVQPGGSLRLSCAASG-RSFRNYV-MGWFRQAPGKEREFVSAI-SWAGGTQ-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0252] SEQ ID No.41:

[0253] QLQASGGGFVQPGGSLRLSCAASG-RPFRIYA-MGWFRQAPGKEREFVSAI-SWSGGNT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0254] SEQ ID No.42:

[0255] QLQASGGGFVQPGGSLRLSCAASG-RPFRIYA-MGWFRQAPGKEREFVSAI-NWSGGNT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-AASPAVSPPRDGRAFTY-WGQGTQVTVSS

[0256] SEQ ID No.43:

[0257] QLQASGGGFVQPGGSLRLSCAASG-RPFRLYA-MGWFRQAPGKEREFVSAI-NWTGTST-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-AASPAVSPRDGRAFTY-WGQGTQVTVSS

[0258] SEQ ID No. 44:

[0259] QLQASGGGFVQPGGSLRLSCAASG-RPFRLYA-MGWFRQAPGKEREFVSAI-NKTGTST-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-AASPAVSPRDGRAFTY-WGQGTQVTVSS

[0260] SEQ ID No. 45:

[0261] QLQASGGGFVQPGGSLRLSCAASG-RGFRWYA-MGWFRQAPGKEREFVSAI-TRGGTST-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-AASPAVSPRDGRAFTY-WGQGTQVTVSS

[0262] SEQ ID No. 46:

[0263] QLQASGGGFVQPGGSLRLSCAASG-RGFRWYA-MGWFRQAPGKEREFVSAI-TRGGSST-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-AASPAVSPRDGRAFTY-WGQGTQVTVSS

[0264] (2) Preparation of nanobodies and determination of their binding affinity

[0265] The carrier construction process was as described in Example 3, the nanobody preparation process was as described in Example 5, and the nanobody binding affinity analysis was as described in Example 6. CK8 (205~483 aa) was amino-coupled to the CM5 sensor chip at a density of 500~800 RU. Humanized nanobodies were injected at seven different concentrations ranging from 1~100 nM. The flow rate was 45 μL / min in all experiments, and the kinetic parameters are shown in Table 7, calculated by equation fitting. All tested nanobodies showed high affinity for CK8 (205~483 aa). D The range is consistent at 10-9 (M).

[0266] Table 7. Binding affinity of humanized nanobodies

[0267]

[0268] Compared with the original sequence (Table 4), the affinity of the humanized nanobodies for antigen CK8 was not significantly reduced, and the affinity for related antigens remained at the original level. The unhumanized nanobodies K8-A0 to K8-A9 all exhibited high affinity for CK8 (205~483 aa), with a KD range consistently within 10⁻⁹ (M). The humanized nanobodies (Table 7) still all showed high affinity for CK8 (205~483 aa), with a KD range consistently within 10⁻⁹ (M), indicating that the humanized nanobodies of this invention were successfully modified.

[0269] Example 8: Cross-reactivity of nanobodies.

[0270] Antibody cross-species reactivity refers to the property of an antibody to react with the same or similar antigens from different species. This property is crucial in research and clinical applications, especially when using animal models for disease research and drug development. For example, in drug development, researchers often need to evaluate the activity of antibodies in human and animal models, and cross-reactivity determines the suitability of antibodies in these models. The docking results in Example 4 show that the nanobody of the present invention interacts with the E230, E231, E234, E277, E287, E288, and S291 side chains on CK8 through hydrogen bonding and electrostatic interactions. Sequences of human and common experimental animal CK8 (205-483 aa) obtained from the library revealed that, except for one weakly interacting (hydrogen bond) residue, S291, which is replaced by the chemically similar T291 in some species of CK8, the remaining six amino acid residues are conserved in human and common experimental animal (macaque, mouse, rat, pig, cow, dog, rabbit) CK8 (205-483 aa). Therefore, theoretically, the nanobody K8-A0 to K8-A9 of the present invention can bind to CK8 (205-483 aa) of the aforementioned different animals.

[0271] The present invention has been verified by ELISA to bind to CK8 (205~483aa) in humans and commonly used experimental animals. The experimental results are shown in Table 8. The nanobodies K8-A0~K8-A9 of the present invention can bind to CK8 (205~483aa) in humans and commonly used experimental animals (macaques, cattle, pigs, dogs, mice, rats, rabbits), exhibiting cross-reactivity, strong applicability, and meeting theoretical expectations.

[0272] Table 8 Cross-reactivity of nanobodies

[0273]

[0274] Example 9: Antibody labeling.

[0275] Antibody labeling techniques well-known to those in the art include enzyme labeling, biotin labeling, fluorescein labeling, colloidal gold labeling, and radioisotope labeling. Each labeling technique has its specific application scenarios and advantages. Choosing the appropriate labeling technique can improve the sensitivity and specificity of the experiment.

[0276] This invention employs four commonly used antibody labeling methods, as follows:

[0277] Biotinylation: Dissolve 1 mg / mL K8-A0 in PBS, add 10 molar amounts of NHS-biotin, mix well, and incubate overnight at 4°C in the dark. Then, ultrafilter and change the medium. This is denoted as K8-A0-biotin.

[0278] Fluorescent labeling: Dissolve 1 mg / mL K8-A0 in PBS, add 10 molar amounts of fluorescein isothiocyanate (FITC), mix well, and incubate overnight at 4°C in the dark. Then, ultrafilter and change the solution. This is labeled K8-A0-FITC.

[0279] HRP labeling: Replace the K8-A0 nanobody with PBS (pH 7.4) at a concentration of approximately 10 mg / mL, and label it using an HRP conjugation kit (abcam, Lightning-Link® ab102890). Dialyze thoroughly and replace the medium. Label it as K8-A0-HRP.

[0280] ALP labeling: Replace the K8-A0 nanobody with PBS (pH 7.4) at a concentration of approximately 1 mg / mL, and label it using an ALP conjugation kit (abcam, Lightning-Link® ab102850). Dialyze thoroughly and replace the medium. Label it as K8-A0-ALP.

[0281] Example 10: Immunofluorescence analysis.

[0282] MCF-7 cells and primary mouse hepatocytes were cultured in 24-well plates, respectively. DAPI and K8-AO-FITC labeling were performed using standard methods. The results were observed under a laser confocal microscope. Figure 13 As shown, DAPI signaling can be observed in the cell nucleus, and a significant FITC signal is visible in the cytoskeleton region, indicating that K8-A0-FITC labels CK8 on the cytoskeleton. These results demonstrate that the nanobody of the present invention can be used for CK8 labeling and fluorescence analysis in cells.

[0283] Example 11: ALP (alkaline phosphatase) labeled nanobodies for immunohistochemistry.

[0284] MCF-7 human breast cancer cells in logarithmic growth phase with adequate viability were selected, and after adjusting the concentration, they were subcutaneously inoculated into immunodeficient mice. The mice were then routinely fed until the tumors reached a suitable size. Subsequently, the mice were anesthetized by inhalation, and the tumor tissue was completely dissected. After ex vivo removal, the tissue was rapidly fixed in 10% neutral buffered formalin solution, trimmed, and then subjected to graded dehydration, clearing, and paraffin embedding to prepare 4-5 μm thick paraffin sections. Subsequent procedures included dewaxing to water, antigen retrieval, and endogenous ALP blocking. K8-A0-ALP was added for incubation, followed by BCIP / NBT staining in the dark, hematoxylin counterstaining, dehydration, clearing, and mounting. Finally, the ALP immunohistochemical results were observed under a microscope. Results are as follows: Figure 14 As shown, after immunohistochemical staining mediated by ALP-labeled nanobodies, MCF-7 human breast cancer xenograft tissue showed clear and specific blue-purple positive staining. The positive signal was mainly located in the cytoplasm of tumor cells. The staining distribution in the tumor parenchyma was uniform and the staining intensity was moderate. There was no obvious non-specific background staining in the tumor stroma and normal tissue areas. After hematoxylin counterstaining, the cell nuclei appeared light blue. The positive staining contrasted sharply with the cell nuclei. The tumor tissue morphology was intact and the cell structure was clearly distinguishable.

[0285] Industrial availability

[0286] The nanobody of the present invention is an anti-CK8 nanobody with a novel amino acid sequence discovered through screening of a phage library. This nanobody and its polypeptide have high affinity and activity, and can specifically recognize and bind to CK8. It can be applied in the field of CK8 detection and is helpful for the diagnosis and treatment of CK8-related diseases.

[0287] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An anti-CK8 nanobody, characterized in that, The complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2, and CDR3 sequences: (I): (1) The amino acid sequence of CDR1 is shown in SEQ ID NO.47, and (2) The amino acid sequence of CDR2 is shown in SEQ ID NO.51; And (3) the amino acid sequence of CDR3 is shown in SEQ ID NO. 59; or (II): Amino acid sequences obtained by modifying, substituting, deleting or adding one or more amino acids as described in (1), (2) and (3) of (I), and which have the same function as the amino acid sequences described in (I).

2. The nanobody according to claim 1, characterized in that, in: (II1): The amino acid sequence of CDR1 is shown in SEQ ID NO.47, the amino acid sequence of CDR2 is shown in SEQ ID NO.52, and the amino acid sequence of CDR3 is shown in SEQ ID NO.59; or (II2): The amino acid sequence of CDR1 is shown in SEQ ID NO.48, the amino acid sequence of CDR2 is shown in SEQ ID NO.53, and the amino acid sequence of CDR3 is shown in SEQ ID NO.59; or (II3): The amino acid sequence of CDR1 is shown in SEQ ID NO.48, the amino acid sequence of CDR2 is shown in SEQ ID NO.54, and the amino acid sequence of CDR3 is shown in SEQ ID NO.59; or (II4): The amino acid sequence of CDR1 is shown in SEQ ID NO.49, the amino acid sequence of CDR2 is shown in SEQ ID NO.55, and the amino acid sequence of CDR3 is shown in SEQ ID NO.59; or (II5): The amino acid sequence of CDR1 is shown in SEQ ID NO.49, the amino acid sequence of CDR2 is shown in SEQ ID NO.56, and the amino acid sequence of CDR3 is shown in SEQ ID NO.59; or (II6): The amino acid sequence of CDR1 is shown in SEQ ID NO.50, the amino acid sequence of CDR2 is shown in SEQ ID NO.57, and the amino acid sequence of CDR3 is shown in SEQ ID NO.59; or (II7): The amino acid sequence of CDR1 is shown in SEQ ID NO.50, the amino acid sequence of CDR2 is shown in SEQ ID NO.58, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

59.

3. The nanobody according to claim 1, characterized in that, in: (III) The framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO. 60, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO. 64, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO. 66, and (4) the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV) An amino acid sequence that has more than 50% homology with the amino acid sequences described in (1), (2), (3), and (4) of (III).

4. The nanobody according to claim 3, characterized in that, (IV) Amino acid sequences with more than 50% homology as described in (III) (1), (2), (3), (4), including (IV-1): The amino acid sequence of FR1 is shown in SEQ ID NO. 61, the amino acid sequence of FR2 is shown in SEQ ID NO. 64, the amino acid sequence of FR3 is shown in SEQ ID NO. 66, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70; or (IV-2): The amino acid sequence of FR1 is shown in SEQ ID NO. 62, the amino acid sequence of FR2 is shown in SEQ ID NO. 64, the amino acid sequence of FR3 is shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70; or (IV-3): The amino acid sequence of FR1 is shown in SEQ ID NO. 60, the amino acid sequence of FR2 is shown in SEQ ID NO. 64, the amino acid sequence of FR3 is shown in SEQ ID NO. 66, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70; or (IV-4): The amino acid sequence of FR1 is shown in SEQ ID NO. 60, the amino acid sequence of FR2 is shown in SEQ ID NO. 64, the amino acid sequence of FR3 is shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70; or (IV-5): The amino acid sequence of FR1 is shown in SEQ ID NO. 60, the amino acid sequence of FR2 is shown in SEQ ID NO. 64, the amino acid sequence of FR3 is shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70; or (IV-6): The amino acid sequence of FR1 is shown in SEQ ID NO. 60, the amino acid sequence of FR2 is shown in SEQ ID NO. 65, the amino acid sequence of FR3 is shown in SEQ ID NO. 68, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70; or (IV-7): The amino acid sequence of FR1 is shown in SEQ ID NO. 63, the amino acid sequence of FR2 is shown in SEQ ID NO. 65, the amino acid sequence of FR3 is shown in SEQ ID NO. 69, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70; or (IV-8): The amino acid sequence of FR1 is shown in SEQ ID NO. 60, the amino acid sequence of FR2 is shown in SEQ ID NO. 65, the amino acid sequence of FR3 is shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is shown in SEQ ID NO. 70; or (IV-9): The amino acid sequence of FR1 is shown in SEQ ID NO.60, the amino acid sequence of FR2 is shown in SEQ ID NO.65, the amino acid sequence of FR3 is shown in SEQ ID NO.67, and the amino acid sequence of FR4 is shown in SEQ ID NO.

70.

5. The nanobody according to any one of claims 1-4, characterized in that, The nanobody has (V) the amino acid sequence shown in SEQ ID NO. 5; or (VI) An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids as described in (V), and having the same function as the amino acid sequence described in (I).

6. The nanobody according to claim 5, characterized in that, in, The amino acid sequence of the nanobody of (VI) is shown in any one of SEQ ID NO.6 to SEQ ID NO.

14.

7. A humanized nanobody, characterized in that, (VII): The amino acid sequence of the humanized nanobody is shown in any one of SEQ ID NO.15 to SEQ ID NO.

46.

8. A polypeptide, characterized in that, Includes the nanobody described in any one of claims 1 to 7.

9. A nucleic acid molecule encoding the nanobody according to any one of claims 1 to 7.

10. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 9.

11. A host cell that transforms or transfects the expression vector of claim 10.

12. A combination or coupling, characterized in that, Including nanobodies of any one of claims 1 to 7 that have been chemically or biologically labeled.

13. An adsorbent, characterized in that, Includes the nanobody according to any one of claims 1 to 7; or the polypeptide according to claim 8; Or the nucleic acid molecule as described in claim 9; or the expression vector as described in claim 10; or the host cell as described in claim 11; Or the combination as described in claim 12; or the coupling as described in claim 12, and a carrier.

14. A reagent kit, characterized in that, Includes the nanobody according to any one of claims 1 to 7; or the polypeptide according to claim 8; Or the nucleic acid molecule as described in claim 9; or the expression vector as described in claim 10; or the host cell as described in claim 11; Or the combination as described in claim 12; or the coupling as described in claim 12; Or the adsorbent as described in claim 13, and an auxiliary agent acceptable in the detection.

15. A device, characterized in that, For capturing, adsorbing and / or detecting CK8, including the nanobody of any one of claims 1 to 7; or the polypeptide of claim 7; Or the nucleic acid molecule as described in claim 9; or the expression vector as described in claim 10; or the host cell as described in claim 11; Or the combination as described in claim 12; or the coupling as described in claim 12; Or the adsorbent of claim 13, or the kit of claim 14.

16. The nanobody according to any one of claims 1 to 7; or the polypeptide according to claim 8; Or the nucleic acid molecule as described in claim 9; or the expression vector as described in claim 10; or the host cell as described in claim 11; Or the combination as described in claim 12; or the coupling as described in claim 12; Or the adsorbent of claim 13, or the kit of claim 14; Applications in the preparation of formulations for the specific capture, adsorption, and / or detection of CK8; or Application in the preparation of cell preparations for the specific capture, adsorption and / or detection of CK8; or Application in the preparation of immunofluorescence or immunohistochemical reagents for the specific capture, adsorption, and / or detection of CK8.

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