Anti-VIM Nanobody and Its Applications

Abstract

VIM-specific nanobody and its applications belong to the field of biotechnology. To solve the problem of detecting vimentin (VIM), the key point is that the complementarity-determining regions (CDRs) of the nanobody include CDR1, CDR2, and CDR3 sequences: (I): (1) The amino acid sequence of CDR1 is shown in SEQ ID NO.27; (2) The amino acid sequence of CDR2 is shown in SEQ ID NO.33; and (3) The amino acid sequence of CDR3 is shown in SEQ ID NO.39. The effect is that the nanobody and its polypeptide of the present invention have high affinity and activity, can specifically recognize and bind to VIM, can be used for the capture and detection of VIM-positive cells, and can be applied to immunofluorescence analysis or immunohistochemical analysis through appropriate antibody labeling techniques.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an anti-VIM nanobody, a polypeptide comprising the nanobody, and their preparation and applications. Background Art

[0002] Vimentin (VIM) is a type III intermediate filament protein with a molecular weight of about 54 kDa, mainly present in cells derived from the mesoderm. Vimentin plays a key role in numerous biological processes, including constructing the cytoskeleton, participating in signal transduction pathways, and a significant upregulation in its expression during epithelial-mesenchymal transition (EMT), making it a marker of EMT. Therefore, the detection of vimentin not only has profound significance in the study of VIM-related pathways but also plays a crucial role in the field of oncology.

[0003] Specifically, as a marker molecule of EMT, vimentin not only participates in regulating the EMT process but also is involved in the formation of mesenchymal phenotype cancer stem cells, being closely related to tumor invasiveness, metastatic ability, and drug resistance; when vimentin is used in combination with other tumor markers (such as alpha-fetoprotein), it can significantly improve the specificity and sensitivity of diagnosis; the change in the expression level of vimentin is also regarded as an important indicator for evaluating the therapeutic effect of tumors; vimentin plays a key role in the occurrence, development, invasion, and metastasis of tumors, becoming a key target for studying the biological behavior of tumors; the dynamic changes of CTCs are closely related to the EMT process, and detecting the phenotypic changes of CTCs through VIM reflects the heterogeneity of tumors and different EMT states.

[0004] These findings further emphasize the importance of vimentin detection and the detection and capture of VIM-positive CTCs in oncology research and clinical applications.

[0005] Currently, the detection of VIM and its positive CTCs is achieved by monoclonal antibodies (EPR3776, RV202) or polyclonal antibodies (10366-1-AP) to recognize the corresponding antigens, and these antibodies can be used for different experimental applications, including Western Blot (WB), immunohistochemistry (IHC), immunocytochemistry (ICC / IF), flow cytometry (Flow Cyt), and enzyme-linked immunosorbent assay (ELISA), etc., to meet the needs of VIM detection.

[0006] However, traditional antibodies are large in size and high in preparation cost, thus limiting the practical applications of affinity enrichment methods and detection. Summary of the Invention

[0007] To solve the problem of vimentin (VIM) detection, in a first aspect, according to some embodiments of the present application, an anti-VIM nanobody, the complementarity-determining region CDR of the nanobody includes CDR1, CDR2 and CDR3 sequences:

[0008] (I): (1) The amino acid sequence of CDR1 is shown in SEQ ID NO.27, (2) The amino acid sequence of CDR2 is shown in SEQ ID NO.33; and (3) The amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II): An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequences described in (1), (2), (3) of (I), and having the same function as the amino acid sequence described in (I).

[0009] According to some embodiments of the present application, an anti-VIM nanobody, wherein: the amino acid sequence obtained by substituting one or more amino acids in (II) for the amino acid sequences described in (1), (2), (3) of (I) includes (II-1): The amino acid sequence of CDR1 is shown in SEQ ID NO.28, the amino acid sequence of CDR2 is shown in SEQ ID NO.33, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-2): The amino acid sequence of CDR1 is shown in SEQ ID NO.29, the amino acid sequence of CDR2 is shown in SEQ ID NO.34, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-3): The amino acid sequence of CDR1 is shown in SEQ ID NO.30, the amino acid sequence of CDR2 is shown in SEQ ID NO.35, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-4): The amino acid sequence of CDR1 is shown in SEQ ID NO.31, the amino acid sequence of CDR2 is shown in SEQ ID NO.36, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-5): The amino acid sequence of CDR1 is shown in SEQ ID NO.32, the amino acid sequence of CDR2 is shown in SEQ ID NO.37, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-6): The amino acid sequence of CDR1 is shown in SEQ ID NO.32, the amino acid sequence of CDR2 is shown in SEQ ID NO.38, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39.

[0010] According to the anti-VIM nanobody in some embodiments of the present application, wherein: (III) the framework regions FR of the nanobody include FR1, FR2, FR3, and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO. 49, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO. 57, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO. 65, (4) the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV) an amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), and (4) of (III).

[0011] According to the anti-VIM nanobody in some embodiments of the present application, the amino acid sequence having more than 50% homology with the amino acid sequence described in (1), (2), (3), and (4) of (III) in (IV) includes (IV-1): the amino acid sequence of FR1 is as shown in SEQ ID NO. 40, the amino acid sequence of FR2 is as shown in SEQ ID NO. 57, the amino acid sequence of FR3 is as shown in SEQ ID NO. 65, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV-2): the amino acid sequence of FR1 is as shown in SEQ ID NO. 40, the amino acid sequence of FR2 is as shown in SEQ ID NO. 53, the amino acid sequence of FR3 is as shown in SEQ ID NO. 65, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV-3): the amino acid sequence of FR1 is as shown in SEQ ID NO. 40, the amino acid sequence of FR2 is as shown in SEQ ID NO. 59, the amino acid sequence of FR3 is as shown in SEQ ID NO. 64, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV-4): the amino acid sequence of FR1 is as shown in SEQ ID NO. 43, the amino acid sequence of FR2 is as shown in SEQ ID NO. 56, the amino acid sequence of FR3 is as shown in SEQ ID NO. 64, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV-5): the amino acid sequence of FR1 is as shown in SEQ ID NO. 45, the amino acid sequence of FR2 is as shown in SEQ ID NO. 58, 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. 72; or (IV-6): the amino acid sequence of FR1 is as shown in SEQ ID NO. 45, the amino acid sequence of FR2 is as shown in SEQ ID NO. 58, the amino acid sequence of FR3 is as shown in SEQ ID NO. 63, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV-7): the amino acid sequence of FR1 is as shown in SEQ ID NO. 47, the amino acid sequence of FR2 is as shown in SEQ ID NO. 60, the amino acid sequence of FR3 is as shown in SEQ ID NO. 62, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV-8): the amino acid sequence of FR1 is as shown in SEQ ID NO. 47, the amino acid sequence of FR2 is as shown in SEQ ID NO. 54, the amino acid sequence of FR3 is as shown in SEQ ID NO. 62, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV-9): the amino acid sequence of FR1 is as shown in SEQ ID NO. 46, the amino acid sequence of FR2 is as shown in SEQ ID NO.As shown in 53, the amino acid sequence of FR3 is as shown in SEQ ID NO.69, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-10): the amino acid sequence of FR1 is as shown in SEQ ID NO.43, the amino acid sequence of FR2 is as shown in SEQ ID NO.56, the amino acid sequence of FR3 is as shown in SEQ ID NO.71, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-11): the amino acid sequence of FR1 is as shown in SEQ ID NO.45, the amino acid sequence of FR2 is as shown in SEQ ID NO.52, the amino acid sequence of FR3 is as shown in SEQ ID NO.63, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-12): the amino acid sequence of FR1 is as shown in SEQ ID NO.46, the amino acid sequence of FR2 is as shown in SEQ ID NO.54, the amino acid sequence of FR3 is as shown in SEQ ID NO.62, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-13): the amino acid sequence of FR1 is as shown in SEQ ID NO.40, the amino acid sequence of FR2 is as shown in SEQ ID NO.53, the amino acid sequence of FR3 is as shown in SEQ ID NO.70, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-14): the amino acid sequence of FR1 is as shown in SEQ ID NO.43, the amino acid sequence of FR2 is as shown in SEQ ID NO.51, the amino acid sequence of FR3 is as shown in SEQ ID NO.64, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-15): the amino acid sequence of FR1 is as shown in SEQ ID NO.44, the amino acid sequence of FR2 is as shown in SEQ ID NO.50, the amino acid sequence of FR3 is as shown in SEQ ID NO.63, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-16): the amino acid sequence of FR1 is as shown in SEQ ID NO.48, the amino acid sequence of FR2 is as shown in SEQ ID NO.54, the amino acid sequence of FR3 is as shown in SEQ ID NO.62, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-17): the amino acid sequence of FR1 is as shown in SEQ ID NO.41, the amino acid sequence of FR2 is as shown in SEQ ID NO.55, the amino acid sequence of FR3 is as shown in SEQ ID NO.61, and the amino acid sequence of FR4 is as shown in SEQ ID NO.73; or (IV-18): the amino acid sequence of FR1 is as shown in SEQ ID NO.44, the amino acid sequence of FR2 is as shown in SEQ ID NO.51, the amino acid sequence of FR3 is as shown in SEQ ID NO.As shown in Figure 67, the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-19): the amino acid sequence of FR1 is as shown in SEQ ID NO.41, the amino acid sequence of FR2 is as shown in SEQ ID NO.52, the amino acid sequence of FR3 is as shown in SEQ ID NO.61, and the amino acid sequence of FR4 is as shown in SEQ ID NO.73; or (IV-20): the amino acid sequence of FR1 is as shown in SEQ ID NO.42, the amino acid sequence of FR2 is as shown in SEQ ID NO.50, 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.72.

[0012] According to the anti-VIM nanobody in some embodiments of the present application, the nanobody has (V) an amino acid sequence as shown in SEQ ID NO.1; or (VI) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence as described in (V), and having the same function as the amino acid sequence described in (I).

[0013] According to the anti-VIM nanobody in some embodiments of the present application, wherein the amino acid sequence of the nanobody of (VI) is any one of SEQ ID NO.2 to SEQ ID NO.26.

[0014] In a second aspect, according to the polypeptide in some embodiments of the present application, including any one of the above-mentioned nanobodies.

[0015] In a third aspect, according to the nucleic acid molecule encoding any one of the above-mentioned nanobodies in some embodiments of the present application.

[0016] In a fourth aspect, according to the expression vector in some embodiments of the present application, including the nucleic acid molecule described above.

[0017] In a fifth aspect, according to the host cell transformed or transfected with the above-mentioned expression vector in some embodiments of the present application.

[0018] In a sixth aspect, according to the conjugate or coupling agent in some embodiments of the present application, including any one of the above-mentioned nanobodies chemically labeled or biologically labeled.

[0019] In a seventh aspect, according to the adsorbent in some embodiments of the present application, including any one of the above-mentioned nanobodies; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the coupling agent, and a carrier.

[0020] In an eighth aspect, a kit according to some embodiments of the present application is characterized by comprising any one of the nanobodies described above; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent, and an auxiliary agent acceptable in detection.

[0021] In a ninth aspect, a device according to some embodiments of the present application for capturing, adsorbing, and / or detecting VIM comprises any one of the nanobodies described above; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit.

[0022] In a tenth aspect, the use of any one of the nanobodies described above; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit according to some embodiments of the present application in the preparation of a preparation for specifically capturing, adsorbing, and / or detecting VIM.

[0023] The use of any one of the nanobodies described above; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit according to some embodiments of the present application in the preparation of a tumor detection preparation for specifically capturing, adsorbing, and / or detecting VIM.

[0024] The use of any one of the nanobodies described above; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit according to some embodiments of the present application in the preparation of a cell preparation for specifically capturing, adsorbing, and / or detecting VIM for enrichment and / or purification.

[0025] The use of any one of the nanobodies described above; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit according to some embodiments of the present application in the preparation of a reagent for immunofluorescence analysis or immunohistochemical analysis for specifically capturing, adsorbing, and / or detecting VIM.

[0026] Beneficial effects: The nanobody of the present invention is a nanobody against VIM screened and discovered with a new amino acid sequence. This nanobody and its polypeptides, etc., have high affinity and activity, can specifically recognize and bind to VIM, and can be used for VIM detection and purification, as well as the capture and detection of VIM-positive cells. Through appropriate antibody labeling techniques, it can be applied to immunofluorescence analysis or immunohistochemical analysis, etc. Brief Description of the Drawings

[0027] Figure 1 It is the kinetic curve of the nanobody in Example 5 of the present invention. Among them, V1. Response curve of nanobody V1, V2. Response curve of nanobody V2, V3. Response curve of nanobody V3, V4. Response curve of nanobody V4, V5. Response curve of nanobody V5, V6. Response curve of nanobody V6, V7. Response curve of nanobody V7, V8. Response curve of nanobody V8, V9. Response curve of nanobody V9.

[0028] Figure 2 It is the labeling result after V1-biotin capture in Example 8 of the present invention. (a). Magnetic beads capturing cells, (b). Magnetic beads, (c). Cells.

[0029] Figure 3 It is the fluorescence staining result of V1-FITC in Example 9 of the present invention. A. Human cell MCF-7, B. African green monkey kidney cell Vero E6, C. Mouse cell l929.

[0030] Figure 4 It is the flow cytometry graph of the fluorescence staining of V1-FITC in Example 9 of the present invention.

[0031] Figure 5 It is the immunohistochemical staining result of V1-HRP in Example 10 of the present invention. Detailed Embodiments

[0032] The above content and other aspects of the present invention will be further described clearly below, where:

[0033] (1) Unless otherwise specified, the term "sequence" as used herein (such as in terms like "antibody sequence", "variable region sequence", "V HH sequence" or "protein sequence") should generally be understood to include the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the amino acid sequence, unless the context requires a more narrow interpretation.

[0034] (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, reference is still made to the comprehensive background art cited above and other references cited therein.

[0035] (3) The term "specificity" refers to the ability of a specific antigen-binding molecule (such as the nanobody or polypeptide 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 (K D ) of the antigen and the antigen-binding molecule, which is a measure of the binding strength between the antigen and the antigen-binding molecule. The smaller the K D value, the stronger the binding strength between the antigen and the antigen-binding molecule. Conversely, the larger the K D value, the weaker the binding strength between the antigen and the antigen-binding molecule. K a represents the binding constant. The larger the K a , the faster the binding. The smaller the K a , the slower the binding. K d represents the dissociation constant. The larger the K d , the faster the dissociation. The smaller the K d , the slower the dissociation. And K D = K d / K a .

[0036] (4) The term "cognate" refers to a family of nanobody sequences that bind the same antigen, have the same number of amino acids, and have an amino acid sequence identity greater than 75%.

[0037] (5) Amino acid substitutions can generally be described as where 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 there is 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.

[0038] For the above substitution methods, the situations disclosed in the documents WO04 / 037999, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300 can be cited, but are not limited thereto. In addition, the (preferred) types and / or combinations of the substitutions selected based on the relevant information of other references cited in WO 04 / 037999 and WO 06 / 122786 can also be cited.

[0039] The amino acid substitutions of the present invention include, but are not limited to, substitution methods such as substituting one amino acid within the following groups (a) to (e) with another amino acid within 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.

[0040] Preferred amino acid substitutions 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.

[0041] The framework region is more conserved compared to the complementarity-determining region. Those skilled in the art will reasonably screen the sequence structure of the framework region according to the actual use and function of the nanobody. As the amino acid sequence of the framework region, an amino acid sequence with a homology of more than 50% is preferred, and further preferably an amino acid sequence with a homology of more than 70%, and still further preferably an amino acid sequence with a homology of more than 95%.

[0042] Nanobody Serial Number and FR Sequence Information Comparison Table - 1

[0043] Antibody Name Antibody Serial Number FR1 Sequence SEQ ID FR2 Sequence SEQ ID V1 SEQ ID No.1 QLQESGGGLVQPGGSLRLSCVTS No.49 MGWYRQAPGKERELVAHI No.57 V2 SEQ ID No.2 QLQESGGGLVQPGGSLRLSCVAS No.40 MGWYRQAPGKERELVAHI No.57 V3 SEQ ID No.3 QLQESGGGLVQPGGSLRLSCVAS No.40 MGWYRQAPGKERELVATI No.53 V4 SEQ ID No.4 QLQESGGGLVQPGGSLRLSCVAS No.40 MGWYRQAPGKEREFVATI No.59 V5 SEQ ID No.5 QLQESGGGLAQPGGSLRLSCAAS No.43 MGWYRQAPGEQREFVATI No.56 V6 SEQ ID No.6 QLQESGGGLVQAGGSLRLSCATS No.45 MGWYRQAPGKQRELVASI No.58 V7 SEQ ID No.7 QLQESGGGLVQAGGSLRLSCATS No.45 MGWYRQAPGKQRELVASI No.58 V8 SEQ ID No.8 QLQESGGGLVQPGGSLRLSCVVS No.47 MGWYRRAPGKERELVAVI No.60 V9 SEQ ID No.9 QLQESGGGLVQPGGSLRLSCVVS No.47 MGWYRQGPGKERELVAVI No.54 V11 SEQ ID No.10 QLQESGGGLVQPGGSLRLSCAAS No.46 MGWYRQAPGKERELVATI No.53 V12 SEQ ID No.11 QLQESGGGLAQPGGSLRLSCAAS No.43 MGWYRQAPGEQREFVATI No.56 V13 SEQ ID No.12 QLQESGGGLVQAGGSLRLSCATS No.45 MGWFRQAPGQGLEAVAAI No.52 V14 SEQ ID No.13 QLQESGGGLVQPGGSLRLSCAAS No.46 MGWYRQGPGKERELVAVI No.54 V15 SEQ ID No.14 QLQESGGGLVQPGGSLRLSCVAS No.40 MGWYRQAPGKERELVATI No.53 V16 SEQ ID No.15 QLQESGGGLAQPGGSLRLSCAAS No.43 MGWFRQAPGKEREFVAAI No.51 V17 SEQ ID No.16 QLQESGGGLVQAGGSLRLSCAAS No.44 MGWFRQAPGKEREFVSAI No.50 V18 SEQ ID No.17 QLQESGGGFVQPGGSLRLSCAAS No.48 MGWYRQGPGKERELVAVI No.54 V1-ah SEQ ID No.18 QLVESGGGLVQPGGSLRLSCAAS No.41 MGWFRQAPGKGLEAVAAI No.55 V2-ah SEQ ID No.19 QLVESGGGLVQPGGSLRLSCAAS No.41 MGWFRQAPGKGLEAVAAI No.55 V3-com SEQ ID No.20 QLQESGGGLVQAGGSLRLSCAAS No.44 MGWFRQAPGKEREFVAAI No.51 V4-com SEQ ID No.21 QLQESGGGLVQAGGSLRLSCAAS No.44 MGWFRQAPGKEREFVAAI No.51 V5-FGLA SEQ ID No.22 QLVESGGGLVQPGGSLRLSCAAS No.41 MGWFRQAPGQGLEAVAAI No.52 V6-FGLA SEQ ID No.23 QLVESGGGLVQPGGSLRLSCAAS No.41 MGWFRQAPGQGLEAVAAI No.52 V7-hs SEQ ID No.24 QLQASGGGFVQPGGSLRLSCAAS No.42 MGWFRQAPGKEREFVSAI No.50 V8-hs SEQ ID No.25 QLQASGGGFVQPGGSLRLSCAAS No.42 MGWFRQAPGKEREFVSAI No.50 V9-hs SEQ ID No.26 QLQASGGGFVQPGGSLRLSCAAS No.42 MGWFRQAPGKEREFVSAI No.50

[0044] Nanobody Serial Number and FR Sequence Information Comparison Table - 2

[0045]

[0046]

[0047] The framework region contributes less to the affinity. Therefore, amino acid substitutions in the framework region generally do not affect the affinity of the nanobody, as long as it can exist in a soluble form. The amino acid substitutions described above are also applicable to the amino acids in the framework region. Among them, humanization is a typical example of amino acid substitution in the framework region. In the present invention, SEQ ID No: 10 to SEQ ID No: 26 are humanized forms of SEQ ID No: 1 to SEQ ID No: 9, and none of them affect the affinity of the original sequence.

[0048] In addition, the total number of residues of the nanobody can be in the range of 110 - 120. However, parts, fragments or analogs of the nanobody are not particularly limited by their length and / or size, as long as such parts, fragments or analogs meet the further requirements listed below and are also suitable for the purposes described herein.

[0049] The nanobodies in the present invention belong to homologous nanobodies, with the same total length of amino acid sequences, the same lengths of each framework region FR and antigen-binding region CDR, high sequence identity, similar structures, and substantially equivalent antigen-binding capabilities.

[0050] As a method for preparing "nanobodies", in its broadest sense, it is not limited to specific biological resources or specific preparation methods. For example, the nanobodies of the present invention can be obtained as follows: (1) by isolating the V HH domain of naturally occurring heavy chain antibodies; (2) by expressing the nucleotide sequence encoding the naturally occurring V HH domain; (3) by "humanizing" the naturally occurring V HH domain (as described below) or by expressing the nucleic acid encoding the humanized V HH domain; (4) applying synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (5) by applying nucleic acid synthesis techniques to prepare the nucleic acid encoding the nanobody, and then expressing the nucleic acid thus obtained; and / or (6) by any combination of the foregoing.

[0051] In addition, based on a variant of the nanobody of the present invention, it also includes nanobodies having an amino acid sequence corresponding to the naturally occurring V HH domain but having been humanized. Humanization means replacing one or more amino acid residues in the sequence of the naturally occurring V H domain with one or more amino acid residues present at the corresponding positions in the V HH domain of a conventional 4-chain antibody from humans.

[0052] According to a non - limiting embodiment of the present invention, the above - mentioned polypeptide consists essentially of nanobodies. "Consisting essentially of" means that the amino acid sequence of the polypeptide of the present invention is identical or corresponding to the amino acid sequence of the nanobody, with a limited number of amino acid residues, such as 1 - 10 amino acid residues, and preferably 1 - 6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added to the amino - terminal (N - terminal) and / or carboxyl - terminal (C - terminal) of the said nanobody or polypeptide.

[0053] The above - mentioned amino acid residues may not change the biological properties of the nanobody and may add other functionality to the said nanobody. For example, the amino acid residues may:

[0054] a is a purification tag, that is, an amino acid sequence or residue that facilitates the purification of the said nanobody. For example, purification is carried out using affinity techniques directed against the said sequence or residue. Some preferred but non - limiting examples of such residues are multiple His - tags (His6 or His8), GST - tag, MBP - tag, Myc - tag, Strep - tag, Flag - tag, HA - tag, V5 - tag, S - tag, E - tag;

[0055] b is a solubility tag, that is, a tag that facilitates the increase in the solubility of the nanobody, such as SUMO;

[0056] c is an N - terminal amino acid residue, such as Met, Ala, Gln or MetAlaGln, AlaGln, by means of which expression can be carried out in a heterologous host cell or host organism;

[0057] d is a C - terminal Cys residue, for example, by means of which reaction with - SH on a ligand or reaction with the Au surface can be carried out;

[0058] e is a hinge to provide a link or spacer between the nanobody and other groups, such as a combination of GlySer, IgG hinge, IgA hinge, or other synthetic hinges;

[0059] f is one or more amino acid residues that can be provided with functional groups and / or have been functionalized in a known manner. For example, as is known in the art, amino acid residues such as lysine or cysteine allow the attachment of PEG groups.

[0060] The polypeptide of the present invention may also include 2 or more of the said nanobodies, also known as multivalent polypeptides.

[0061] Bivalent polypeptides include 2 nanobodies, optionally linked by a hinge sequence; trivalent polypeptides include 3 nanobodies, optionally linked by two hinge sequences; tetravalent polypeptides include 4 nanobodies, optionally linked by three hinge sequences. Multivalent polypeptides can bind to the same epitope or to different antigen-binding epitopes, the latter also being referred to as multispecific polypeptides.

[0062] Regarding the multivalent and multispecific polypeptides containing one or more V HH domains and their preparation, reference can be made to the description in EP0822985.

[0063] The hinges for multivalent and multispecific polypeptides should be well-known to those skilled in the art and include, for example, Gly-Ser, such as (Gly4Ser)3 or (Gly3Ser2)3 as described in WO 99 / 42077; or the naturally occurring heavy-chain antibody hinge region or a partial region thereof. For other suitable hinges, reference can also be made to the comprehensive background art cited above.

[0064] In addition, in addition to the one or more nanobodies described above, the polypeptides of the present invention may further contain functional groups, moieties or residues, such as therapeutically active substances, and / or tags, such as fluorescein labeling, isotope labeling, biotin labeling and enzyme-catalyzed tags, etc.

[0065] In addition, the dissociation equilibrium constant (K D ) of the nanobody or polypeptide of the present invention binding to VIM is 10 -11 mol / L (M). The dissociation equilibrium constant of the present invention was measured by surface plasmon resonance technology.

[0066] The specific binding between the above-mentioned antigen and the antigen-binding molecule 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 new methods known in the art, such as surface plasmon resonance technology (SPR) and / or biolayer interferometry (BLI) technology, etc. And those skilled in the art should know that the affinity parameters measured by different methods vary greatly, even by 2-3 orders of magnitude.

[0067] The nanobodies, polypeptides and nucleic acids encoding them of the present invention can be prepared in a known manner, which will be clear to those skilled in the art from the further description herein. A particularly useful method for preparing the nanobodies, polypeptides and nucleic acids generally includes the following steps:

[0068] (1) Express the 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;

[0069] (2) Isolate and / or purify the nanobody or polypeptide of the present invention thus obtained.

[0070] Alternatively, other methods may include the following steps:

[0071] (3) Culture and / or maintain the host of the present invention under certain conditions such that the host of the present invention expresses and / or produces a nanobody and / or polypeptide of the present invention; optionally followed by;

[0072] (4) Isolate and / or purify the nanobody or polypeptide of the present invention thus obtained.

[0073] The nucleic acid of the present invention may be in the form of single-stranded or double-stranded DNA or RNA, and preferably in the form of double-stranded DNA. For example, the nucleic acid sequence of the present invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with codon usage specifically adapted for expression in the host cell or host organism to be used, i.e., codon-optimized).

[0074] The nucleic acid of the present invention may be prepared or obtained by essentially known methods, based on the information on the amino acid sequence of the nanobody or polypeptide of the present invention given herein, and / or may be isolated from appropriate natural sources. For example, site-directed mutagenesis is performed on the nucleic acid sequence of the naturally occurring V HH structural domain to provide the nucleic acid of the present invention encoding the analogue.

[0075] The nucleic acid of the present invention may also be in the form of, present in and / or be part of a genetic construct, which is well known to those skilled in the art. Such genetic constructs generally include at least one nucleic acid of the present invention and may be in the form of a vector, such as a plasmid, YAC, viral vector or transposon. In particular, the vector may be an expression vector, i.e., a vector that can provide in vitro and in vivo expression (such as in a suitable host cell, host organism and / or expression system).

[0076] The nucleic acid of the present invention and / or the genetic construct of the present invention can be used to transform a host cell or host organism, i.e., for expressing and / or producing the nanobody or polypeptide 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 fungal, prokaryotic or eukaryotic cell or organelle or organism, as well as all other hosts or host cells essentially known for expressing and producing antibodies and antibody fragments (including but not limited to single-domain antibodies and ScFv fragments), which are well known to those skilled in the art.

[0077] For production, the nanobodies and polypeptides of the present invention can be produced in the milk of transgenic mammals, such as in the milk of rabbits, cows, goats or sheep, or can also be produced in plants or parts of plants, which plant parts include but are not limited to their leaves, flowers, fruits, roots or seeds.

[0078] As mentioned above, one advantage of applying nanobodies is that the polypeptides based thereon can be expressed and prepared in a prokaryotic system, and suitable prokaryotic expression systems, vectors, host cells, etc. are well-known to those skilled in the art, as cited in the references above. However, it should be noted that the present invention is not limited to expression in a bacterial system in its broadest sense.

[0079] Preferably, in the present invention, the nanobody or polypeptide is produced in bacterial cells, especially in bacterial cells suitable for large-scale drug production, as described above.

[0080] When the nanobodies or polypeptides of the present invention for production are expressed in cells, the nanobodies or polypeptides of the present invention can be produced intracellularly (e.g., in the cytoplasm or periplasmic space), then separated from the host cells, and optionally further purified; or can be produced extracellularly (i.e., secreted expression), then separated from the culture medium, and optionally further purified.

[0081] Some preferred but non-limiting vectors used 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 vectors (Novagen) and pQE vectors (Qiagen); expression vectors for use in yeast or other fungal cells - pYES2 (Invitrogen) and Pichia expression vector (Invitrogen); expression vectors for use in insect cells - pBlueBacⅡ (Invitrogen) and other baculovirus vectors; and so on.

[0082] The corresponding techniques for transforming the hosts or host cells of the present invention are well-known to those skilled in the art.

[0083] After transformation, detection can be carried out and those hosts that have successfully transformed the nucleotide sequence / genetic construct of the present invention can be selected. The transformed host cells (which can be in the form of a stable cell line) or host organisms (which can be in the form of a stable mutant line or strain) form another aspect of the present invention.

[0084] Then the amino acid sequence of the present invention can be isolated from the host cell / host organism and / or from the culture medium for culturing the host cell or host organism, and can be obtained by protein separation and / or purification techniques essentially known in the art, such as (preparative) chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g., using specific / cleavable amino acid sequences fused to 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).

[0085] The adsorbent of the present invention can be used to specifically recognize VIM.

[0086] The nanobody or polypeptide or adsorbent of the present invention can be used for purifying and detecting VIM, and can also be used for capturing and detecting VIM-positive cells.

[0087] The object of the present invention is to solve the above problems and provide a nanobody with an amino acid sequence of a specific structure, a polypeptide containing the nanobody and its applications, so as to solve the problems of complex antibody preparation process, high cost, poor antibody affinity and stability in the aspects of VIM enrichment, purification, detection, etc.

[0088] To achieve the above technical object, the technical solution adopted in the present application is as follows:

[0089] In the first aspect, the present invention provides a nanobody that binds to VIM. The variable region in the amino acid sequence of the nanobody includes complementarity-determining regions CDR and framework regions FR. The complementarity-determining regions CDR include complementarity-determining region CDR1, complementarity-determining region CDR2 and complementarity-determining region CDR3. Among them, CDR1 is the sequence of SEQ ID No. 27 to SEQ ID No. 32, CDR2 is the sequence of SEQ ID No. 33 to SEQ ID No. 38, CDR3 is the sequence of SEQ ID No. 39, and sequences with a homology of more than 75% thereto.

[0090] Preferably, the amino acid sequence of the nanobody includes: SEQ ID No: 1 to SEQ ID No: 9.

[0091] Preferably, the nanobody is a humanized nanobody. Preferably, the humanized nanobody includes: SEQ ID No: 10 to SEQ ID No: 26.

[0092] In the second aspect, the present invention provides a polypeptide obtained by modifying the amino acids at the N-terminus and / or C-terminus of the above-mentioned nanobody.

[0093] Preferably, the ways of modifying the amino acids at the N-terminus and / or C-terminus of the nanobody include:

[0094] Method 1: Add a tag to the N-terminal and / or C-terminal amino acids of the nanobody;

[0095] Method 2: After adding a tag to the N-terminal and / or C-terminal amino acids of the nanobody, the tag is further connected to a protective amino acid through a hinge;

[0096] Preferably, the tag includes at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag, Flag-tag, the hinge includes at least one of GS hinge, IgG hinge, IgA hinge, PEG, and the protective amino acid includes Ala, Gln, Glu, Met or any combination of two or more of the foregoing amino acids.

[0097] In a third aspect, the present invention provides a polypeptide obtained by multivalent synthesis of the above-mentioned nanobody.

[0098] In a fourth aspect, the present invention provides a nucleic acid encoding the above-mentioned nanobody or the above-mentioned polypeptide.

[0099] In a fifth aspect, the present invention provides an expression vector containing an expression cassette of the above-mentioned nucleic acid.

[0100] In a sixth aspect, the present invention provides a host cell containing the above-mentioned expression vector.

[0101] In a seventh aspect, the present invention provides the use of the above-mentioned nanobody and / or the above-mentioned polypeptide in immunoassay, enrichment and / or purification.

[0102] Preferably, the use of the above-mentioned nanobody and / or the above-mentioned polypeptide in the preparation of a VIM adsorbent, a VIM purification kit, and a VIM detection kit.

[0103] Preferably, the use of the above-mentioned nanobody and / or the above-mentioned polypeptide in capturing and detecting VIM-positive cells.

[0104] Example

[0105] Hereinafter, examples are given to illustrate the specific embodiments of the present invention. However, the embodiments of the present invention are not limited by these examples, and any selection and modification can be made within the scope not affecting the technical effects to be achieved by the present invention.

[0106] Example 1

[0107] Construction of an anti-VIM nanobody library.

[0108] The phage display library used in the present invention is an immune library with T7 phage as the vector, and the establishment steps are as follows:

[0109] (1) Immunize alpacas (numbered 2209-1 and 2209-2) with human VIM. After four immunizations, collect jugular vein blood from the two alpacas, isolate peripheral blood lymphocytes, and extract total RNA (PuerLink TM RNA Mini Kit, Life Technologies: 12183018A);

[0110] (3) Reverse transcribe the total RNA into cDNA, and amplify the V HH gene using two rounds of nested PCR;

[0111] For the first round of PCR, use cDNA as the template, and UP primer1 and DOWN primer1 as the upstream and downstream primers respectively. After amplification, recover the band with a size of 650-750 bp, and use this as the template for the second round of PCR. The upstream and downstream primers are UP primer2 and DOWN primer2 respectively, and recover the PCR product of 450-500 bp;

[0112] UP primer1: CTTGGTGGTCCTGGCTGCTCT,

[0113] DOWN primer1: GGTACGTGCTGTTGAACTGTTCC,

[0114] UP primer2: TATCTAGTC GAATTC CGCCCAGGTGCAGCTC,

[0115] DOWN primer2: AGCGACTAAGCTTTGAGGAGACGGTGAC;

[0116] (3) Double digest the PCR product with EcoRΙ and HindⅢ, and perform agarose gel electrophoresis. Recover the gene band of 350-500 bp, which is the V HH gene fragment;

[0117] (4) Use T4 ligase to ligate the T7 vector ( 10-3 Cloning Kit, MeterckMetillipore : 70550-3) and the V HH gene fragment;

[0118] (5) Mix the ligation product with packaging proteins to form a complete T7 phage, and amplify the mixture to obtain the original phage library;

[0119] (6) After detection, the titer of the original library is 5.32×10 9pfu / mL, with a diversity of 7.5×10 6 .

[0120] Example 2

[0121] Screening of nanobodies.

[0122] First, dilute the antigen VIM to 10 μg / mL with TBS. Take 100 μL and add it to a 96-well plate, and incubate at 4 °C for 12 h. Aspirate the antigen dilution in the wells, wash the plate 3 times with TBS, pat dry, add 1% protein-free blocking solution (purchased from Sangon Biotech Co., Ltd.), 300 μL / well, and incubate at room temperature for 2 h (alternately use 1% protein-free blocking solution and 1% BSA during screening). Aspirate the blocking agent in the wells, wash the plate 6 times with TBST, pat dry, add the amplified phage, 100 μL / well, and incubate at room temperature for 30 min. Wash the plate 10 times with TBST, add T7 elution buffer (1% SDS) to elute the phage, incubate at room temperature for 30 min, and amplify the eluate for the next round of screening.

[0123] Example 3

[0124] Construction of genetically engineered bacteria

[0125] (1) After four rounds of screening, perform solid amplification on the screening eluate, pick phage plaques, use the phage plaque amplification solution as a template, and use UP primer3 and DOWN primer3 as upstream and downstream primers for PCR amplification;

[0126] UP primer3: TTCCTTAA CATATG GCCCAGGTGCAGCTCGT,

[0127] DOWN primer3: TTAAGGAA CTCGAG CACGGTGACCAGGGTC;

[0128] (2) Send a part of the PCR product for external sequencing to obtain the nanobody sequence information. According to the CDR region length and homology, select 9 monoclonal sequences belonging to the same family. The nanobody naming and serial number information are shown in Table 1.

[0129] (3) Double-digest the other part of the PCR product with NdeΙ and XhoΙ, and recover the digested product. At the same time, perform digestion and recovery of the vector in the same way, use T4 ligase to ligate the digested product and the vector, and transfer the ligation product into Escherichia coli to obtain genetically engineered bacteria expressing VIM-specific nanobodies.

[0130] Table 1 Comparison table of nanobody serial numbers and CDR sequence information

[0131] Antibody Name Antibody Serial Number CDR1 Sequence SEQ ID CDR2 Sequence SEQ ID CDR3 Sequence SEQ ID V1 SEQ ID No.1 GNAFSDNA No.27 SKNSNT No.33 AIGPRYG No.39 V2 SEQ ID No.2 GNAGSDNA No.28 SKNSNT No.33 AIGPRYG No.39 V3 SEQ ID No.3 GSTSSENA No.29 SRNTNT No.34 AIGPRYG No.39 V4 SEQ ID No.4 GSTSSDNA No.30 SWDSNT No.35 AIGPRYG No.39 V5 SEQ ID No.5 GSTSSDNA No.30 SWDSNT No.35 AIGPRYG No.39 V6 SEQ ID No.6 GFAFSVNA No.31 SNGGNT No.36 AIGPRYG No.39 V7 SEQ ID No.7 GFAFSVNA No.31 SNGGNT No.36 AIGPRYG No.39 V8 SEQ ID No.8 GFPSSVNA No.32 SWDENT No.37 AIGPRYG No.39 V9 SEQ ID No.9 GFPSSVNA No.32 SWDGNT No.38 AIGPRYG No.39

[0132] SEQ ID No.1: QLQESGGGLVQPGGSLRLSCVTS - GNAFSDNA - MGWYRQAPGKERELVAHI - SKNSNT - NYPDS VKGRFIISRNNAQNTVYLQMNSLNPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0133] SEQ ID No.2: QLQESGGGLVQPGGSLRLSCVAS - GNAGSDNA - MGWYRQAPGKERELVAHI - SKNSNT - NYPDS VKGRFIISRNNAQNTVYLQMNSLNPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0134] SEQ ID No.3: QLQESGGGLVQPGGSLRLSCVAS - GSTSSENA - MGWYRQAPGKERELVATI - SRNTNT - NYPDSV KGRFIISRNNAQNTVYLQMNSLNPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0135] SEQ ID No.4: QLQESGGGLVQPGGSLRLSCVAS - GSTSSDNA - MGWYRQAPGKEREFVATI - SWDSNT - NYPDSV KGRFIISRDNAQNTVYLQMNSLKPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0136] SEQ ID No.5: QLQESGGGLAQPGGSLRLSCAAS - GSTSSDNA - MGWYRQAPGEQREFVATI - SWDSNT - NYPDSV KGRFIISRDNAQNTVYLQMNSLKPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0137] SEQ ID No.6: QLQESGGGLVQAGGSLRLSCATS - GFAFSVNA - MGWYRQAPGKQRELVASI - SNGGNT - NYKDS VKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0138] SEQ ID No.7: QLQESGGGLVQAGGSLRLSCATS-GFAFSVNA-MGWYRQAPGKQRELVASI-SNGGNT-NYKDSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0139] SEQ ID No.8: QLQESGGGLVQPGGSLRLSCVVS-GFPSSVNA-MGWYRRAPGKERELVAVI-SWDENT-YYKDLAKGRFIISRDNAQNTVFLQMNSLTPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0140] SEQ ID No.9: QLQESGGGLVQPGGSLRLSCVVS-GFPSSVNA-MGWYRQGPGKERELVAVI-SWDGNT-YYKDLAKGRFIISRDNAQNTVFLQMNSLTPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0141] Example 4

[0142] Preparation of VIM Nanobody

[0143] (1) The basal medium for the nanobody is TB medium. Inoculate at an inoculation amount of 5%, culture at 37 °C for 3 - 5 h, and add the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) (final concentration 0.25 mM, the same below) for overnight induction;

[0144] (2) After the induction is completed, centrifuge at 4000 rpm for 20 min to obtain the wet bacteria containing the nanobody.

[0145] (3) Add the lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.4 0.02 M PB) to the obtained wet bacteria at a ratio of 1:10, and use a high-pressure homogenizer at 700 bar to disrupt the cells;

[0146] (4) Centrifuge at 4 °C and 10000 rpm for 20 min, and take the supernatant;

[0147] (5) Filter the supernatant through a 0.45 μm filter, and then separate and purify the VIM nanobody through an affinity chromatography column (GE Healthcare, US), where the packing material of the affinity chromatography column is Ni Sepharose High Perfomance;

[0148] (6) The nanobody after affinity chromatography purification was subjected to SDS-PAGE electrophoresis to determine the purity, and the protein solution with higher purity was selected to measure the protein concentration using the BCA method.

[0149] Example 5 Affinity Analysis

[0150] The binding ability of the nanobody to human VIM was analyzed using SPR technology. VIM was amino-coupled to the CM5 sensor chip at a density of 500 - 800 RU, and the nanobody was injected at 7 different concentrations in the range of 1 - 100 nM. The flow rate was 45 μL / min in all experiments. The chip regeneration condition was glycine-HCl pH 1.5. The kinetic parameters K a , K d and K D . Figure 1 Shown in the figure are the 9 response curves of nanobodies V1 - V9. Each response curve from top to bottom is the response curve of the nanobody at 7 concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, etc. The kinetic parameters shown in Table 2 were calculated by equation fitting. The nanobodies all have relatively high affinity for VIM, and the range of K D is 10 -11 (M).

[0151] Table 2 Affinity of Nanobodies for VIM

[0152] Nanobody ka (1 / Ms) kd (1 / s) KD (M) V1 2.07E+04 4.05E-07 1.96E-11 V2 2.31E+04 3.05E-07 1.32E-11 V3 3.07E+04 4.88E-07 1.59E-11 V4 2.49E+04 3.08E-07 1.24E-11 V5 3.08E+04 4.27E-07 1.39E-11 V6 6.67E+03 5.78E-07 8.67E-11 V7 8.50E+03 5.27E-07 6.20E-11 V8 9.90E+03 3.22E-07 3.25E-11 V9 1.06E+04 6.14E-07 5.79E-11

[0153] Example 6: Humanization of Nanobodies.

[0154] Humanization Method 1

[0155] The protein sequences of nanobodies V3 - V9 were aligned with the human germline, and the different amino acids were marked in the framework region. Then, some of the different amino acids were replaced with human amino acids by site-directed mutagenesis to generate partially humanized nanobodies V11 - V18, and the corresponding sequence numbers are SEQ ID No.10 - SEQ ID No.17. Subsequently, the expression and purification of the humanized nanobodies were carried out.

[0156] Humanization Method 2

[0157] CDR region transplantation based on common humanized or highly stable nanobody scaffolds: The complementary determining region sequences of the nanobodies were transplanted onto the humanized scaffolds reported in the literature, and humanized nanobody sequences V1-ah, V2-ah, V3-com, V4-com, V5-FGLA, V6-FGLA, V7-hs, V8-hs, and V9-hs were generated by gene synthesis, with corresponding sequence numbers SEQ ID No.18 to SEQ ID No.26. Subsequently, the humanized nanobodies were expressed and purified.

[0158] Four nanobody scaffolds selected in this invention are respectively:

[0159] The ah scaffold is a general-purpose fully humanized scaffold, taken from the literature (Chi, X.J. 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).).

[0160] The FGLA is a general-purpose fully humanized scaffold, taken from the literature (Vincke, C. et al. General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold. J. Biol. Chem. 284, 3273-3284, doi:10.1074 / jbc.M806889200 (2009).).

[0161] The hs is a highly stable, highly expressed, and partially humanized scaffold, from 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).).

[0162] com is a general scaffold with high stability and high expression, which is 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).).

[0163] The expression and purification process is briefly described as follows:

[0164] (1) The basal medium for the humanized nanobody is TB medium. Inoculate according to an inoculation amount of 5%, culture at 37 °C for 3 - 5 h, and add the inducer isopropyl β-D-thiogalactoside (IPTG) for overnight induction;

[0165] (2) After the induction is completed, centrifuge at 4000 rpm for 20 min to obtain the wet bacteria containing the humanized nanobody;

[0166] (3) Add the lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.4 0.02 M PB) to the obtained wet bacteria at a ratio of 1:10, and use a high-pressure homogenizer at 700 bar to disrupt the cells;

[0167] (4) Centrifuge at 4 °C and 10000 rpm for 20 min, and take the supernatant;

[0168] (5) Filter the supernatant through a 0.45 μm filter, and then separate and purify the humanized nanobody through an affinity chromatography column (GE Healthcare, US), where the packing material of the affinity chromatography column is Ni Sepharose High Performance;

[0169] (6) Perform SDS-PAGE electrophoresis on the nanobody purified by affinity chromatography to judge the purity, and select the protein solution with higher purity to measure the protein concentration by the BCA method.

[0170] Apply SPR technology to analyze the binding ability of the humanized nanobody to VIM. The analysis process is as described in Example 5, and the affinity results are shown in Table 3.

[0171] Table 3 Affinity of the humanized antibody to VIM

[0172] Sequence number Nanobody ka (1 / Ms) kd (1 / s) KD (M) SEQ ID No.10 V11 1.84E+04 6.69E-07 3.64E-11 SEQ ID No.11 V12 3.36E+04 5.59E-07 1.66E-11 SEQ ID No.12 V13 1.07E+04 8.18E-07 7.64E-11 SEQ ID No.13 V14 1.30E+04 4.28E-07 3.31E-11 SEQ ID No.14 V15 2.32E+04 5.77E-07 2.49E-11 SEQ ID No.15 V16 5.62E+04 7.97E-07 1.42E-11 SEQ ID No.16 V17 1.67E+04 6.86E-07 4.11E-11 SEQ ID No.17 V18 2.11E+04 7.86E-07 3.72E-11 SEQ ID No.18 V1-ah 8.73E+03 4.16E-07 4.76E-11 SEQ ID No.19 V2-ah 3.31E+04 5.05E-07 1.53E-11 SEQ ID No.20 V3-com 1.26E+04 1.17E-06 9.28E-11 SEQ ID No.21 V4-com 4.28E+04 7.72E-07 1.80E-11 SEQ ID No.22 V5-FGLA 3.81E+04 2.24E-06 5.87E-11 SEQ ID No.23 V6-FGLA 7.75E+03 5.55E-07 7.16E-11 SEQ ID No.24 V7-hs 4.49E+04 1.97E-06 4.39E-11 SEQ ID No.25 V8-hs 8.70E+03 4.58E-07 5.26E-11 SEQ ID No.26 V9-hs 2.17E+04 8.88E-07 4.09E-11

[0173] SEQ ID No.10: QLQESGGGLVQPGGSLRLSCAAS - GSTSSENA - MGWYRQAPGKERELVATI - SRNTNT - NYKDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0174] SEQ ID No.11: QLQESGGGLAQPGGSLRLSCAAS - GSTSSDNA - MGWYRQAPGEQREFVATI - SWDSNT - NYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0175] SEQ ID No.12: QLQESGGGLVQAGGSLRLSCATS - GFAFSVNA - MGWFRQAPGQGLEAVAAI - SNGGNT - NYKDSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0176] SEQ ID No.13: QLQESGGGLVQPGGSLRLSCAAS - GFPSSVNA - MGWYRQGPGKERELVAVI - SWDGNT - YYKDLAKGRFIISRDNAQNTVFLQMNSLTPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0177] SEQ ID No.14: QLQESGGGLVQPGGSLRLSCVAS - GSTSSENA - MGWYRQAPGKERELVATI - SRNTNT - NYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0178] SEQ ID No.15: QLQESGGGLAQPGGSLRLSCAAS - GSTSSDNA - MGWFRQAPGKEREFVAAI - SWDSNT - NYPDSVKGRFIISRDNAQNTVYLQMNSLKPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0179] SEQ ID No.16: QLQESGGGLVQAGGSLRLSCAAS - GFAFSVNA - MGWFRQAPGKEREFVSAI - SNGGNT - NYKDSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0180] SEQ ID No.17: QLQESGGGFVQPGGSLRLSCAAS - GFPSSVNA - MGWYRQGPGKERELVAVI - SWDGNT - YYKDLAKGRFIISRDNAQNTVFLQMNSLTPEDTAVYYCT - AIGPRYG - WGQGTQVTVSS

[0181] SEQ ID No.18: QLVESGGGLVQPGGSLRLSCAAS - GNAFSDNA - MGWFRQAPGKGLEAVAAI - SKNSNT - YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA - AIGPRYG - WGQGTLVTVSS

[0182] SEQ ID No.19: QLVESGGGLVQPGGSLRLSCAAS - GNAGSDNA - MGWFRQAPGKGLEAVAAI - SKNSNT - YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA - AIGPRYG - WGQGTLVTVSS

[0183] SEQ ID No.20: QLQESGGGLVQAGGSLRLSCAAS - GSTSSENA - MGWFRQAPGKEREFVAAI - SRNTNT - YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA - AIGPRYG - WGQGTQVTVSS

[0184] SEQ ID No.21: QLQESGGGLVQAGGSLRLSCAAS - GSTSSDNA - MGWFRQAPGKEREFVAAI - SWDSNT - YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA - AIGPRYG - WGQGTQVTVSS

[0185] SEQ ID No.22: QLVESGGGLVQPGGSLRLSCAAS-GSTSSDNA-MGWFRQAPGQGLEAVAAI-SWDSNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA-AIGPRYG-WGQGTLVTVSS

[0186] SEQ ID No.23: QLVESGGGLVQPGGSLRLSCAAS-GFAFSVNA-MGWFRQAPGQGLEAVAAI-SNGGNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA-AIGPRYG-WGQGTLVTVSS

[0187] SEQ ID No.24: QLQASGGGFVQPGGSLRLSCAAS-GFAFSVNA-MGWFRQAPGKEREFVSAI-SNGGNT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA-AIGPRYG-WGQGTQVTVSS

[0188] SEQ ID No.25: QLQASGGGFVQPGGSLRLSCAAS-GFPSSVNA-MGWFRQAPGKEREFVSAI-SWDENT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA-AIGPRYG-WGQGTQVTVSS

[0189] SEQ ID No.26: QLQASGGGFVQPGGSLRLSCAAS-GFPSSVNA-MGWFRQAPGKEREFVSAI-SWDGNT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA-AIGPRYG-WGQGTQVTVSS

[0190] Note: 1. The "-" between FR and CDR represents a linker used to distinguish FR from CDR, and there are no missing sites.

[0191] Compared with the original sequence, the affinity of the humanized nanobody for the antigen VIM has not decreased significantly, and the affinity for related antigens remains at the same order of magnitude. The affinity level of the above humanized antibody is still 10 -11 M. This indicates that the humanization of the nanobody of the present invention is successful.

[0192] Example 7: Antibody Labeling

[0193] Antibody labeling techniques well-known to those skilled in the art, such as: enzyme labeling method, biotin labeling method, fluorescein labeling method, colloidal gold labeling method, and radioisotope labeling method, etc. Each labeling technique has its specific application scenarios and advantages. Selecting the appropriate labeling technique can improve the sensitivity and specificity of the experiment.

[0194] The present invention uses two common antibody labeling methods as follows:

[0195] a) Fluorescent labeling: 1 mg / mL of V1 is dissolved in PBS, and the FITC fluorescent coupling kit (Fast)-Lightning- (ab188285) from Abcam is used. According to the instructions, the nanobody is labeled with FITC. It is denoted as V1-FITC.

[0196] b) HRP labeling: The nanobody V1 is exchanged into PBS (pH 7.4) at a concentration of about 10 mg / mL, and it is labeled using the HRP coupling kit (abcam, Lightning- ab102890), and then thoroughly dialyzed and exchanged with liquid. It is denoted as V1-HRP.

[0197] c) Biotinylation: 1 mg / mL of V1 is dissolved in PBS, 10-fold molar amount of NHS-biotin is added, and after mixing, it is incubated overnight at 4 °C in the dark, and then ultrafiltered and exchanged with liquid. It is denoted as V1-biotin.

[0198] Example 8 Capture of VIM-positive cells

[0199] Streptavidin-coated magnetic beads are mixed and incubated in V1-biotin (0.1 mg / mL) for 30 min, and then thoroughly washed with PBST buffer for standby; MCF-7 cells (VIM-positive cells) are used as model cells. The experimental group is 5 mL of fresh healthy human whole blood added with 100 MCF-7 cells, and the control group is 5 mL of fresh healthy human whole blood without adding MCF-7 cells.

[0200] 20 μL of the above-mentioned magnetic beads are added to the experimental group and the control group respectively, and incubated at 37 °C for 30 min. The magnetic beads are thoroughly washed with PBS buffer, and then DAPI labeling is carried out by the conventional test method and observed under a fluorescence microscope. As Figure 2 shown, Figure 2 (a). Pictures taken under bright field and fluorescence channels, showing the MCF-7 cells captured by the magnetic beads; Figure 2 (b). The magnetic beads taken under bright field; Figure 2 (c). The cells taken under fluorescence channel. It is proved that the nanobody of the present invention can be used for the capture of VIM-positive cells.

[0201] Example 9: Fluorescence Immunoassay

[0202] Application (1): Verification of Antibody Cross-Reactivity

[0203] Antibody cross-species reactivity refers to the property of the same antibody to react with the same or similar antigens from different species. This property is very important in research and clinical applications, especially when using animal models for disease research and drug development. For example, during drug development, researchers often need to evaluate the activity of antibodies in humans and animal models, and cross-reactivity determines the applicability of antibodies in these models.

[0204] In this invention, after culturing human cells MCF-7 ( Figure 3 (A)), African green monkey kidney cells Vero E6 ( Figure 3 (B)), and mouse cells l929 ( Figure 3 (C)) separately, fluorescence labeling was carried out, and confocal photos were taken using a fluorescence microscope. The results are as Figure 3 shown. The green fluorescence shows the cytoskeleton labeled by V1-FITC, and the blue shows the DAPI-stained cell nucleus. It shows that the nanobody of this invention can effectively label the vimentin of the cytoskeleton of human and common experimental animals (monkey, mouse) cells, has cross-reactivity, and strong applicability.

[0205] Application (2): Cell Sorting Using Flow Cytometry

[0206] Take 2 mL of human peripheral whole blood, add 6 mL of red blood cell lysate to remove red blood cells, and then centrifuge at 450×g for 10 min to obtain white blood cells. Add ≈1×10 6 A431 cells to the above cell mixture.

[0207] Directly perform immunofluorescence labeling on the cells. The amount of antibody (V1-FITC) added is 200 μL / 2 mL cell suspension, and the working concentration of the antibody is 4 μg / mL. Incubate at 4°C for 30 - 60 min. After centrifuging and washing clean with cold PBS, add 500 μL of cold PBS to the cells, pipette and mix well, and place in a flow tube. At this time, the cell concentration of the test sample is 1×10 6 cells / mL.

[0208] The results are as Figure 4 shown. The horizontal axis of the scatter plot represents FITC fluorescence, and the vertical axis represents side scatter (SSC), which is usually related to the size and internal complexity of the cells. The horizontal axis of the histogram represents FITC fluorescence, and the vertical axis represents the number of cells detected at that fluorescence intensity. From Figure 4As can be seen, most cells (blue dots) have low VIM expression, while cells in the red region show high VIM expression. This indicates the existence of a specific cell population with significantly higher VIM expression than other cells, and this cell population is A431.

[0209] This shows that the nanobody of the present invention can effectively label cells with high VIM expression, supporting the application of flow cytometry for cell sorting.

[0210] Example 10: Immunohistochemical analysis

[0211] MCF-7 cells (VIM-positive cells) were inoculated into the tail vein of mice and normally fed until tumor tissue could be observed. The tumor tissue was excised and paraffin sections were prepared. The paraffin sections were labeled with V1-HRP and developed by conventional experimental methods. As Figure 5 shown, in the tumor tissue sections, the staining intensity of VIM protein was strongly positive, and the positively stained cells were diffusely distributed in the tumor tissue, covering most of the tumor cells. This proves that the nanobody of the present invention can be used for the labeling and immunohistochemical analysis of VIM-positive cells.

[0212] Industrial applicability

[0213] The nanobody of the present invention is an anti-VIM nanobody with a new amino acid sequence discovered through screening of a phage library. This nanobody and its polypeptide have high affinity and activity, can specifically recognize and bind to VIM. The nanobody prepared by the present invention can be used for the capture and detection of VIM-positive cells, and through appropriate antibody labeling techniques, can be applied to immunofluorescence analysis or immunohistochemical analysis, etc.

[0214] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. An anti-VIM nanobody, characterized in that, The complementarity-determining regions CDR of the nanobody include CDR1, CDR2, and CDR3 sequences: The amino acid sequence of CDR1 is shown in SEQ ID NO.27, the amino acid sequence of CDR2 is shown in SEQ ID NO.33, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

39.

2. The nanobody according to claim 1, wherein The framework regions FR of the nanobody include FR1, FR2, FR3, and FR4 sequences, where: (1) the amino acid sequence of FR1 is shown in SEQ ID NO.49, (2) the amino acid sequence of FR2 is shown in SEQ ID NO.57, (3) the amino acid sequence of FR3 is shown in SEQ ID NO.65, and (4) the amino acid sequence of FR4 is shown in SEQ ID NO.

72.

3. The nanobody according to claim 2, wherein Wherein: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), and (4).

4. An anti-VIM nanobody, characterized in that, The nanobody has the amino acid sequence shown in SEQ ID NO.

1.

5. An anti-VIM nanobody, characterized in that, The complementarity-determining regions CDR of the nanobody include CDR1, CDR2, and CDR3 sequences: The amino acid sequence of CDR1 is shown in SEQ ID NO.28, the amino acid sequence of CDR2 is shown in SEQ ID NO.33, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

39.

6. The nanobody according to claim 5, wherein The framework regions FR of the nanobody include FR1, FR2, FR3, and FR4 sequences, where: (1) the amino acid sequence of FR1 is shown in SEQ ID NO.40, (2) the amino acid sequence of FR2 is shown in SEQ ID NO.57, (3) the amino acid sequence of FR3 is shown in SEQ ID NO.65, and (4) the amino acid sequence of FR4 is shown in SEQ ID NO.

72.

7. The nanobody according to claim 6, wherein Wherein: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), and (4).

8. An anti-VIM nanobody, characterized in that, The nanobody has the amino acid sequence shown in SEQ ID NO.

2.

9. An anti-VIM nanobody, characterized in that, The complementarity-determining regions CDR of the nanobody include CDR1, CDR2, and CDR3 sequences: The amino acid sequence of CDR1 is shown in SEQ ID NO.29, the amino acid sequence of CDR2 is shown in SEQ ID NO.34, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

39.

10. The nanobody according to claim 9, wherein The framework regions FR of the nanobody include FR1, FR2, FR3, and FR4 sequences, where: (1) the amino acid sequence of FR1 is shown in SEQ ID NO.40, (2) the amino acid sequence of FR2 is shown in SEQ ID NO.53, (3) the amino acid sequence of FR3 is shown in SEQ ID NO.65, and (4) the amino acid sequence of FR4 is shown in SEQ ID NO.

72.

11. The nanobody according to claim 10, characterized in that, Wherein: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), and (4).

12. An anti-VIM nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

3.

13. An anti-VIM nanobody, characterized in that, The complementarity-determining regions CDR of the nanobody include CDR1, CDR2 and CDR3 sequences: The amino acid sequence of CDR1 is as shown in SEQ ID NO.30, the amino acid sequence of CDR2 is as shown in SEQ ID NO.35, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.

39.

14. The nanobody according to claim 13, wherein, The framework regions FR of the nanobody include FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.40, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO.59, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO.64, (4) the amino acid sequence of FR4 is as shown in SEQ ID NO.

72.

15. The nanobody according to claim 14, wherein Wherein: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), (4).

16. An anti-VIM nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

4.

17. The nanobody according to claim 13, characterized in that, The framework regions FR of the nanobody include FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.43, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO.56, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO.64, (4) the amino acid sequence of FR4 is as shown in SEQ ID NO.

72.

18. The nanobody according to claim 17, wherein Wherein: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), (4).

19. An anti-VIM nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

5.

20. An anti-VIM nanobody, characterized in that, The complementarity-determining regions CDR of the nanobody include CDR1, CDR2 and CDR3 sequences: The amino acid sequence of CDR1 is as shown in SEQ ID NO.31, the amino acid sequence of CDR2 is as shown in SEQ ID NO.36, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.

39.

21. The nanobody according to claim 20, wherein The framework regions FR of the nanobody include FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.45, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO.58, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO.68, (4) the amino acid sequence of FR4 is as shown in SEQ ID NO.

72.

22. The nanobody according to claim 21, wherein Wherein: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), (4).

23. An anti-VIM nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

6.

24. The nanobody according to claim 20, characterized in that, The framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, where: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.45, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO.58, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO.63, (4) the amino acid sequence of FR4 is as shown in SEQ ID NO.

72.

25. The nanobody according to claim 24, wherein Where: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), (4).

26. An anti-VIM nanobody, characterized in that, The nanobody has the amino acid sequence as shown in SEQ ID NO.

7.

27. An anti-VIM nanobody, characterized in that, The complementarity-determining region CDR of the nanobody includes CDR1, CDR2 and CDR3 sequences: The amino acid sequence of CDR1 is as shown in SEQ ID NO.32, the amino acid sequence of CDR2 is as shown in SEQ ID NO.37, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.

39.

28. The nanobody according to claim 27, characterized in that, The framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, where: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.47, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO.60, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO.62, (4) the amino acid sequence of FR4 is as shown in SEQ ID NO.

72.

29. The nanobody according to claim 28, wherein Where: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), (4).

30. An anti-VIM nanobody, characterized in that, The nanobody has the amino acid sequence as shown in SEQ ID NO.

8.

31. An anti-VIM nanobody, characterized in that, The complementarity-determining region CDR of the nanobody includes CDR1, CDR2 and CDR3 sequences: The amino acid sequence of CDR1 is as shown in SEQ ID NO.32, the amino acid sequence of CDR2 is as shown in SEQ ID NO.38, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.

39.

32. The nanobody according to claim 31, wherein The framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, where: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.47, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO.54, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO.62, (4) the amino acid sequence of FR4 is as shown in SEQ ID NO.

72.

33. The nanobody according to claim 32, wherein Where: An amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3), (4).

34. An anti-VIM nanobody, characterized in that, The nanobody has the amino acid sequence as shown in SEQ ID NO.

9.

35. An anti-VIM nanobody, characterized in that, The nanobody has the amino acid sequence shown in any one of SEQ ID NO.10 to SEQ ID NO.

26.

36. A polypeptide, characterized in that, Comprising the nanobody according to any one of claims 1 to 35.

37. A nucleic acid molecule encoding the nanobody according to any one of claims 1 to 35.

38. An expression vector, characterized in that, Comprising the nucleic acid molecule of claim 37.

39. A host cell transformed or transfected with the expression vector of claim 38.

40. A conjugate or a coupling substance, characterized in that: A Nanobody according to any one of claims 1 to 35 comprising a chemically labeled or biologically labeled Nanobody.

41. An adsorbent, characterized in that, Comprising the Nanobody described in any one of claims 1 to 35; or the polypeptide described in claim 36; or the nucleic acid molecule described in claim 37; or the expression vector described in claim 38; or the host cell described in claim 39; or the conjugate or coupling described in claim 40, and a vector.

42. A kit, characterized in that, Comprising the Nanobody described in any one of claims 1 to 35; or the polypeptide described in claim 36; or the nucleic acid molecule described in claim 37; or the expression vector described in claim 38; or the host cell described in claim 39; or the conjugate or coupling described in claim 40; or the adsorbent described in claim 41, and an auxiliary agent acceptable in detection.

43. A device, characterized in that, Used for capturing, adsorbing and / or detecting VIM, comprising the Nanobody described in any one of claims 1 to 35; or the polypeptide described in claim 36; or the nucleic acid molecule described in claim 37; or the expression vector described in claim 38; or the host cell described in claim 39; or the conjugate or coupling described in claim 40; or the adsorbent described in claim 41; or the kit described in claim 42.

44. Use of the Nanobody according to any one of claims 1 to 35, the polypeptide according to claim 36, the nucleic acid molecule according to claim 37, the expression vector according to claim 38, the host cell according to claim 39, the conjugate or coupling according to claim 40, the adsorbent according to claim 41, or the kit according to claim 42 in the preparation of a preparation for specific capture, adsorption and / or detection of VIM.

45. Use of the Nanobody according to any one of claims 1 to 35, the polypeptide according to claim 36, the nucleic acid molecule according to claim 37, the expression vector according to claim 38, the host cell according to claim 39, the conjugate or coupling according to claim 40, the adsorbent according to claim 41, or the kit according to claim 42 in the preparation of a tumor detection preparation for specific capture, adsorption and / or detection of VIM.

46. Use of the Nanobody according to any one of claims 1 to 35, the polypeptide according to claim 36, the nucleic acid molecule according to claim 37, the expression vector according to claim 38, the host cell according to claim 39, the conjugate or coupling according to claim 40, the adsorbent according to claim 41, or the kit according to claim 42 in the preparation of an enriched and / or purified cell preparation for specific capture, adsorption and / or detection of VIM. Use of the nanobody according to any one of claims 1 to 35, the polypeptide according to claim 36, the nucleic acid molecule according to claim 37, the expression vector according to claim 38, the host cell according to claim 39, the conjugate or conjugate according to claim 40, the adsorbent according to claim 41, or the kit according to claim 42 in the preparation of an immunofluorescence assay or immunohistochemical assay reagent for specifically capturing, adsorbing, and / or detecting VIM.

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