Anti-EpCAM nano antibody and application thereof

Abstract

The invention discloses an anti-EpCAM nano antibody and application thereof, belongs to the technical field of biology, is used for solving the problem of accurate recognition of EpCAM, and is characterized in that a complementary determining region CDR of the nano antibody comprises CDR1, CDR2 and CDR3 sequences: (I): (1) the amino acid sequence of the CDR2 is shown as a formula (1-1), and (2) the amino acid sequence of the CDR3 is shown as a formula (1-2); (1-1), SRSX21RX22X23 (1-1); x < 31 > X < 32 > X < 33 > N < Y > X < 34 > X < 35 > X < 36 > N < 37 > X < 38 > RX < 39 > (1-2); in the formula, X represents a site which can be modified, substituted or deleted in an amino acid sequence, can be used for capturing and detecting EpCAM positive cells, and can be applied to immunofluorescence analysis or immunohistochemical analysis and the like through a proper antibody labeling technology.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-EpCAM nano antibody, a polypeptide containing the nano antibody, and preparation and application thereof. Background Art

[0002] Epithelial cell adhesion molecule (EpCAM) is a single-pass transmembrane glycoprotein with a molecular weight of about 40 kDa and composed of 314 amino acids. Its molecular structure consists of three parts: the extracellular domain, the single-pass transmembrane domain, and the intracellular domain. It participates in regulating cell adhesion, proliferation, differentiation, migration, and signal transduction. It interacts with a variety of cell adhesion molecules and participates in cell signal transduction through its intracellular domain (EpICD). Abnormal expression of EpCAM is closely related to the occurrence, development, and prognosis of tumors, especially in the process of epithelial-mesenchymal transition (EMT), which is one of the important molecular mechanisms of tumor invasion and metastasis. EpCAM is highly expressed in a variety of human malignant tumors, especially in epithelial tumors, such as colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer, and ovarian cancer, making it a hot target for cancer treatment. After circulating tumor cells (CTCs) break away from solid tumors and enter the blood circulation system, they can spread to other parts of the body through the blood, potentially causing distant metastasis and spread of the tumor.

[0003] Therefore, the capture and detection of EpCAM-positive cells (such as CTCs) are of great significance in cancer research, especially in tumor biology and new drug development.

[0004] The capture methods of EpCAM-positive cells can be roughly divided into two categories: one is antibody-based affinity enrichment method, and the other is physical enrichment method based on cell characteristics. Its detection methods can also be divided into two categories: one is based on antibody technology, and the other is based on nucleic acid detection. The selection and application of these methods depend on the specific needs of the experiment, including the required sensitivity, specificity, throughput, and whether the captured cells need to be subsequently cultured or molecularly analyzed.

[0005] Antibodies can capture and detect EpCAM-positive cells with high sensitivity and specificity, and do not damage cells or affect the subsequent culture of captured cells. However, traditional antibodies are large in size and expensive to prepare, which limits the practical application of affinity enrichment and detection. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problem of accurate recognition of EpCAM. In the first aspect, according to the anti-EpCAM nanobody in some embodiments of the present application, the complementary determining region CDR of the nanobody includes CDR1, CDR2 and CDR3 sequences:

[0007] (I): (1) the amino acid sequence of the CDR2 is as shown in formula (1-1), (2) the amino acid sequence of the CDR3 is as shown in formula (1-2);

[0008] SRSX 21 RX 22 X 23 (1-1);

[0009] X 31 X 32 X 33 NYX 34 X 35 X 36 NX 37 X 38 RX 39 (1-2);

[0010] In the formula, X represents a site in the amino acid sequence that can be modified, substituted or deleted.

[0011] According to the anti-EpCAM nanobody in some embodiments of the present application, wherein:

[0012] (II): (1) the amino acid sequence of CDR2 as described in (1) of (I) is SRSGRDI, and the amino acid sequence of CDR3 as described in (2) of (I) is RWTNYLGANPGRY; (SEQ ID.1, SEQ ID.2, SEQ ID.3, SEQ ID.4, SEQ ID.5, SEQID.6, SEQ ID.12, SEQ ID.15, SEQ ID.16, SEQ ID.17, SEQ ID.23, SEQ ID.24, SEQ ID.27, SEQ ID.28, SEQ ID.31, SEQ ID.32, SEQ ID.35, SEQID.36) or

[0013] (2) the amino acid sequence of CDR2 as described in (1) of (I) is SRSDRGI, and the amino acid sequence of CDR3 as described in (2) of (I) is SGTNYLGANPGRY; (SEQ ID.7, SEQ ID.14, SEQ ID.25, SEQ ID.29, SEQ ID.33, SEQID.37) or

[0014] (3) the amino acid sequence of CDR2 in (1) of (I) is SRSDRGL, and the amino acid sequence of CDR3 in (2) of (I) is SGTNYLGANPGRY; (SEQ ID.8, SEQ ID.9, SEQ ID.21) or

[0015] (4) The amino acid sequence of CDR2 described in (1) of (I) is SRSDRGT, and the amino acid sequence of CDR3 described in (2) of (I) is SGTNYLGANPGRY; (SEQ ID.10, SEQ ID.11, SEQ ID.13, SEQ ID.18, SEQ ID.19, SEQ ID.20, SEQ ID.26, SEQ ID.30, SEQ ID.34, SEQ ID.38) or

[0016] (5) The amino acid sequence of CDR2 described in (1) of (I) is SRSDRGT, and the amino acid sequence of CDR3 described in (2) of (I) is RWTNYLGANPGRY.

[0017] (SEQ ID.22)

[0018] (6) An amino acid sequence having 75% or more homology with any one of the amino acid sequences described in (1) to (5) of (II).

[0019] According to the anti-EpCAM nanobody in some embodiments of the present application, wherein:

[0020] (III): (1) the amino acid sequence of the CDR1 is ESNH--A, the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences of (1) of (II); (SEQ ID.1, SEQ ID.2, SEQ ID.3, SEQ ID.15, SEQ ID.23, SEQ ID.27, SEQ ID.31, SEQ ID.35) or

[0021] (2) the amino acid sequence of the CDR1 is RTFSLYA, the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences of (II) (1); (SEQ ID.4, SEQ ID.5, SEQ ID.6, SEQ ID.17, SEQ ID.24, SEQ ID.28, SEQ ID.32, SEQ ID.36) or

[0022] (3) the amino acid sequence of the CDR1 is RTVDIYG, the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (1); (SEQ ID.12, SEQ ID.16) or

[0023] (4) the amino acid sequence of the CDR1 is RTVDIYG, the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (2); (SEQ ID.7, SEQ ID.25, SEQ ID.29, SEQ ID.33, SEQ ID.37) or

[0024] (5) The amino acid sequence of the CDR1 is RTSSSNA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (2). (SEQ ID.14) or

[0025] (6) the amino acid sequence of the CDR1 is RTFSLYA, the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (3); (SEQ ID. 21) or

[0026] (7) The amino acid sequence of the CDR1 is RTVDIYG, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (3); (SEQ ID.8, SEQ ID.9) or

[0027] (8) The amino acid sequence of the CDR1 is ESNH--A, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (4); (SEQ ID.18) or

[0028] (9) the amino acid sequence of the CDR1 is RTFSLYA, the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (4); (SEQ ID.13, SEQ ID.19) or

[0029] (10) The amino acid sequence of the CDR1 is RTSSSNA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (4); (SEQ ID.1SEQ ID., SEQ ID.11, SEQ ID.2SEQ ID., SEQ ID.26, SEQ ID.3SEQ ID., SEQ ID.34, SEQ ID.38) or

[0030] (11) The amino acid sequence of the CDR1 is RTSSSNA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (5) of (II); (SEQ ID.22)

[0031] According to the anti-EpCAM nanobody in some embodiments of the present application, the framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, wherein:

[0032] (IV) (1) the amino acid sequence of FR1 is as shown in formula (4-1), (2) the amino acid sequence of FR2 is as shown in formula (4-2), (3) the amino acid sequence of FR3 is as shown in formula (4-3), (4) the amino acid sequence of FR4 is as shown in formula (4-4);

[0033] QX 41 X 42 SGGGX 43 VxD 44 GX 45 SLRLSCX 46 ASX 47 (4-1);

[0034] X 51 QXd 52 PGX 53 X 54 X 55 EX 56 VX 57 X 58 X 59 (4-2);

[0035] X 61 YX 62 X 63 SVKGRFTIX 64 X 65 DNX 66 X 67 NX 68 X 69 YQ 610 X 611 LX 612 X 613 EDTAX 614 YX 615 CX 616 A(4-3);

[0036] DYWGQG 71 VTVSS(4-4);

[0037] In the formula, X represents a site in the amino acid sequence that can be modified, substituted or deleted.

[0038] According to the anti-EpCAM nanobody in some embodiments of the present application, wherein:

[0039] (V): (1) X in the amino acid sequence of FR1 as described in (1) of (IV) 41 Selected from Q or V; X 42 Selected from E or A alone; X 43 Selected from L or F alone; X 44 Individually selected from A or P or I; X 45 Selected from D or G alone; X 46 Selected from A or V; X 47 selected from G alone or missing;

[0040] (2) In the amino acid sequence of FR2 as described in (IV) (2), X 51 Selected alone from M or L; X 52 Selected from A or V; X 53 Selected from K or Q alone; X 54 Individually selected from E or Q or G; X 55 Selected alone from R or L; X 56 Selected from F or A alone; X 57 Selected alone from A or S; X 58 Selected from A or S alone; X 59 Individually selected from V or I;

[0041] (3) In the amino acid sequence of FR3 described in (IV) (3), X 61 Individually selected from Y or N or T; X 62 Individually selected from A or G; X 63 Selected from D or G alone; X 64 Individually selected from S or V; X 65 Selected alone from R or K; X 66 Selected from A or S alone; X 67 Selected alone from K or A; X 68 Selected alone from T or R; X 69 Individually selected from V or M or A; X 610 Individually selected from N or S; X 611 Selected from S or T alone; X 612 Selected alone from K or R; X 613 Selected from A or P alone; X 614 Selected from V or T alone; X 615 Selected from Y or F alone; X 616 Selected from A or V alone;

[0042] (4) In the amino acid sequence of FR4 as described in (IV) (4), X 61 Selected from Q or L alone.

[0043] According to the anti-EpCAM nanobody in some embodiments of the present application, wherein:

[0044] (VI): (1) the amino acid sequence of FR1 is shown in any one of SEQ ID.68 to SEQ ID.76, (2) the amino acid sequence of FR2 is shown in any one of SEQ ID.77 to SEQ ID.85, (1) the amino acid sequence of FR3 is shown in any one of SEQ ID.86 to SEQ ID.96, (4) the amino acid sequence of FR4 is shown in any one of SEQ ID.97 to SEQ ID.98;

[0045] (2) or an amino acid sequence having 50% or more homology with the amino acid sequence described in (1), (2), (3) or (4).

[0046] According to the anti-EpCAM nanobody in some embodiments of the present application, wherein:

[0047] (VII): (1) the amino acid sequence of the nanobody is as shown in any one of SEQ ID.1, SEQ ID.2, SEQ ID.3, SEQ ID.4, SEQ ID.5, SEQ ID.6, SEQ ID.12, SEQ ID.15, SEQ ID.16, SEQ ID.17, SEQ ID.23, SEQ ID.24, SEQ ID.27, SEQ ID.28, SEQ ID.31, SEQ ID.32, SEQ ID.35, and SEQ ID.36; or

[0048] (2) the amino acid sequence of the Nanobody is shown in any one of SEQ ID.7, SEQ ID.14, SEQ ID.25, SEQ ID.29, SEQ ID.33, and SEQ ID.37; or

[0049] (3) the amino acid sequence of the Nanobody is as shown in any one of SEQ ID.8, SEQ ID.9, and SEQ ID.21; or

[0050] (4) the amino acid sequence of the Nanobody is as shown in any one of SEQ ID.10, SEQ ID.11, SEQ ID.13, SEQ ID.18, SEQ ID.19, SEQ ID.20, SEQ ID.26, SEQ ID.30, SEQ ID.34, and SEQ ID.38; or

[0051] (5) The amino acid sequence of the Nanobody is shown in SEQ ID.22.

[0052] In a second aspect, the polypeptide according to some embodiments of the present application comprises any one of the Nanobodies described.

[0053] On the third aspect, according to the nucleic acid molecule encoding any one of the Nanobodies in some embodiments of the present application.

[0054] In a fourth aspect, the expression vector according to some embodiments of the present application comprises the nucleic acid molecule.

[0055] In a fifth aspect, a host cell is transformed or transfected with the expression vector according to some embodiments of the present application.

[0056] In a sixth aspect, the binding substance or conjugate according to some embodiments of the present application comprises the nanobody which is chemically labeled or biologically labeled.

[0057] In a seventh aspect, the adsorbent according to some embodiments of the present application comprises any one of the Nanobodies; or the polypeptide; or the nucleic acid molecule;

[0058] or the expression vector; or the host cell;

[0059] Or the combination; or the conjugate, and a carrier.

[0060] According to the kit in some embodiments of the present application, comprising any one of the Nanobodies; or the polypeptide; or the nucleic acid molecule;

[0061] or the expression vector; or the host cell;

[0062] or the combination described; or the conjugate described;

[0063] Or the adsorbent described above, and an auxiliary agent acceptable in the detection.

[0064] In an eighth aspect, the device according to some embodiments of the present application is characterized in that it is used for capturing, adsorbing and / or detecting EpCAM, comprising any one of the nanobodies; or the polypeptide; or the nucleic acid molecule;

[0065] or the expression vector; or the host cell;

[0066] or the combination described; or the conjugate described;

[0067] Or the adsorbent; or the kit.

[0068] In the ninth aspect, the use of the nanobody described in any one of some embodiments of the present application; 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 in the preparation of a specific capture, adsorption and / or detection EpCAM preparation.

[0069] In the tenth aspect, the use of the nanobody described in any one of some embodiments of the present application; 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 in the preparation of a tumor detection preparation that specifically captures, adsorbs and / or detects EpCAM.

[0070] In the eleventh aspect, the use of the Nanobody described in any one of some embodiments of the present application; 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 in the preparation of enriched and / or purified cell preparations for specific capture, adsorption and / or detection of EpCAM.

[0071] In the twelfth aspect, the use of the nanobody described in any one of some embodiments of the present application; 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 in the preparation of immunofluorescence analysis or immunohistochemistry analysis reagents for heterosexual capture, adsorption and / or detection of EpCAM.

[0072] Beneficial effects: The nanoantibody of the present invention is an anti-EpCAM nanoantibody with a new amino acid sequence discovered through screening. The nanoantibody and its polypeptide have high affinity and activity, can specifically recognize and bind to EpCAM, and can be used for EpCAM detection and purification, as well as capture and detection of EpCAM-positive cells, solving the problems of complex antibody preparation process, high cost, and poor antibody affinity and stability faced by existing EpCAM enrichment, purification, and detection, and can be applied to immunofluorescence analysis or immunohistochemistry analysis, etc. through appropriate antibody labeling technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is the overall effect of the docking diagram of E4 and EpCAM in Example 4 of the present invention;

[0074] Figure 2 It is the CDR2 portion of the E4 and EpCAM docking diagram in Example 4 of the present invention;

[0075] Figure 3 It is the CDR3 portion of the E4 and EpCAM docking diagram in Example 4 of the present invention;

[0076] Figure 4 is a schematic diagram of the interaction between E4 and EpCAM in Example 4 of the present invention;

[0077] Figure 5 It is the kinetic curve of the Nanobody in Example 6 of the present invention, wherein, E1. the response curve of Nanobody E1, E2. the response curve of Nanobody E2, E3. the response curve of Nanobody E3, E4. the response curve of Nanobody E4, E5. the response curve of Nanobody E5, E6. the response curve of Nanobody E6, E7. the response curve of Nanobody E7, E8. the response curve of Nanobody E8, E9. the response curve of Nanobody E9, E10. the response curve of Nanobody E10, and E11. the response curve of Nanobody E11.

[0078] Figure 6 It is the standard curve of sandwich ELISA detection in Example 11 of the present invention.

[0079] Figure 7 This is the labeling result after E1-biotin capture in Example 12 of the present invention, wherein: (a) there are no cells with HER2 fluorescent signals on the magnetic beads, and (b) all cells on the magnetic beads have HER2 fluorescent signals.

[0080] Figure 8 This is the fluorescence staining result of E1-FITC in Example 13 of the present invention.

[0081] Fig. 9 This is the immunohistochemical staining result of E1-HRP in Example 14 of the present invention. DETAILED DESCRIPTION

[0082] The above contents and other aspects of the present invention will be further described below, wherein:

[0083] (1) Unless otherwise indicated, the term "sequence" as used herein (e.g., in phrases such as "antibody sequence," "variable region sequence," "V HH The term "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 narrower interpretation.

[0084] (2) Unless otherwise indicated, 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 technology cited above and other references cited therein.

[0085] (3) The term "specificity" refers to the ability of a particular antigen-binding molecule (such as a Nanobody or polypeptide of the 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) between the antigen and the antigen-binding molecule. D ), is a measure of the binding strength between antigen and antigen-binding molecule, K D The smaller the value, the stronger the binding strength between the antigen and the antigen-binding molecule. D The larger the value, the weaker the binding strength between the antigen and the antigen-binding molecule. a represents the binding constant, K a The larger the value, the faster the binding. a The smaller the K, the slower the binding; d represents the dissociation constant, K d The larger the K, the faster the dissociation. d The smaller the K, the slower the dissociation; and D =K d / K a .

[0086] (4) The term "homologous" refers to a family of Nanobody sequences that bind to the same antigen, have the same number of amino acids in CDR3, and have an amino acid sequence identity greater than 75%.

[0087] (5) Amino acid substitution can generally be described as a substitution in which an amino acid residue can be substituted with an amino acid having a similar chemical structure or with an amino acid having 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 substituted with an amino acid having a similar chemical structure.

[0088] For the above-mentioned substitution modes, the situations disclosed in documents WO04 / 037999, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300 can be cited, but they are not limited thereto. In addition, the (preferred) types and / or combinations of the substitutions can be selected based on the relevant information of other references cited in WO 04 / 037999 and WO 06 / 122786.

[0089] The amino acid substitutions of the present invention may include, but are not limited to, substitutions 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.

[0090] Preferred amino acid substitutions can be listed 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.

[0091] For the framework region, it is more conservative than the complementary 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 an amino acid sequence with a homology of more than 70% is further preferred, and an amino acid sequence with a homology of more than 95% is further preferred.

[0092] The contribution of the framework region to affinity is small, so amino acid substitutions in the framework region generally do not affect the affinity of the nanobody, and as long as they can exist in a soluble form, the amino acids in the framework region are also suitable for the above amino acid substitutions. Among them, humanization is a typical example of amino acid substitutions in the framework region. In the present invention, SEQ ID No: 23 to SEQ ID No: 38 are four humanized forms of SEQ ID No: 1 to SEQ ID No: 11, and none of them affects the affinity of the original sequence.

[0093] Furthermore, the total number of residues in a Nanobody may be in the interval 110 - 120. However, parts, fragments or analogs of Nanobodies are not particularly limited in 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.

[0094] The nanobodies of the present invention belong to the same family of nanobodies, whose amino acid sequences have the same total length, and whose framework regions FR and antigen-binding regions CDR have the same length and high sequence identity, similar structures, and substantially equivalent antigen-binding abilities.

[0095] The structure of the nanoantibody of the present invention can be determined by any appropriate method known, including Alohafold2mutimer v3 (a protein structure prediction tool used to predict the three-dimensional structure of protein complexes, which can help researchers understand how different proteins interact with each other to form complexes), Amber (a widely used molecular dynamics simulation software package that can be used to simulate the behavior of proteins and other biological molecules. In structural biology, Amber is often used to perform energy minimization, side chain optimization and molecular dynamics simulation), Relax Process (in molecular dynamics simulations, Relax Process usually refers to the process of energy minimization of protein structures to optimize the geometric configuration of side chains and reduce conflicts and poor geometric configurations within molecules), ADT (AutoDockTools, a software tool set for molecular docking and molecular dynamics simulations that can help researchers predict the binding mode and affinity between ligands and receptors) and / or Ligplot+ (a tool for analyzing the interface of protein-ligand complexes that can identify and visualize hydrogen bonds, hydrophobic interactions and other non-covalent interactions).

[0096] As a method for preparing "nanoantibodies", in its broadest sense, it is not limited to a specific biological resource or a specific preparation method. For example, the nanoantibodies of the present invention can be obtained by: (1) isolating the V chain of a naturally occurring heavy chain antibody; HH domain; (2) by expressing a naturally occurring V HH The nucleotide sequence of the domain; (3) by HH domains are "humanized" (as described below) or by expressing a polypeptide encoding the humanized V HH (4) using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (5) preparing nucleic acids encoding Nanobodies by applying nucleic acid synthesis techniques and then expressing the nucleic acids thus obtained; and / or (6) by any combination of the foregoing.

[0097] In addition, based on a variation of the Nanobodies of the invention, also includes a V HH The amino acid sequence of the nanobody corresponds to the structural domain but has been humanized. Humanization is to use the V of a conventional 4-chain antibody from humans. H One or more amino acid residues existing at corresponding positions in the structural domain replace the naturally occurring V HH One or more amino acid residues of a domain sequence.

[0098] According to one non-limiting embodiment of the invention, the above-mentioned polypeptide consists essentially of a Nanobody. "Essentially consisting of" means that the amino acid sequence of the polypeptide of the invention is identical or corresponds to the amino acid sequence of a Nanobody, 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 Nanobody or polypeptide.

[0099] The above amino acid residues may not change the biological properties of the Nanobody, and may add other functionalities to the Nanobody. For example, the amino acid residues may:

[0100] a is a purification tag, i.e. an amino acid sequence or residue that facilitates the purification of the Nanobody, for example, using an affinity technique for the sequence or residue for purification. Some preferred but non-limiting examples of such residues are groups of His-tags (His6 or His8), GST-tag, MBP-tag, Myc-tag, Strep-tag, Flag-tag, HA-tag, V5-tag, S-tag, E-tag;

[0101] b is a soluble tag, i.e., a tag that helps increase the solubility of the nanobody, such as SUMO;

[0102] c is the N-terminal amino acid residue, for example, Met, Ala, Gln or MetAlaGln, AlaGln, whereby it can be expressed in a heterologous host cell or host organism;

[0103] d is a C-terminal Cys residue, which can react with -SH on the ligand or with the Au surface, for example;

[0104] e is a hinge to provide a link or spacer between the nanobody and other groups, for example, a combination of GlySer, IgG hinge, IgA hinge, or other artificially synthesized hinges;

[0105] f is one or more amino acid residues which may be provided with a functional group and / or have been functionalized in a known manner, for example amino acid residues such as lysine or cysteine ​​allow for the attachment of a PEG group as is known in the art.

[0106] The polypeptide of the present invention may also include two or more of the above-described Nanobodies, also known as multivalent polypeptides.

[0107] 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 antigen epitope or to different antigen binding epitopes, the latter also being referred to as multispecific polypeptides.

[0108] About containing one or more V HH For multivalent and multispecific polypeptides of the structural domain and their preparation, reference may be made to the description in EP0822985.

[0109] The hinges used for multivalent and multispecific polypeptides should be well known to those skilled in the art, for example, including Gly-Ser, such as (Gly4Ser)3 or (Gly3Ser2)3 described in WO 99 / 42077; or naturally occurring heavy chain antibody hinge regions or partial regions thereof. For other suitable hinges, reference can also be made to the comprehensive background technology cited above.

[0110] Furthermore, in addition to the one or more Nanobodies, the polypeptides of the invention may also comprise functional groups, parts or residues, such as therapeutically active substances, and / or labels, such as fluorescent labels, isotope labels, biotin labels and enzyme catalytic labels, and the like.

[0111] In addition, the dissociation equilibrium constant (K) of the Nanobodies or polypeptides of the present invention for binding to EpCAM D ) is 10 -8 ~10 -9 Mole / liter (M). The dissociation equilibrium constant of the present invention is measured using the plasma resonance technique.

[0112] The specific binding between the above antigen and the antigen binding molecule can be determined by any known appropriate 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 plasma resonance technology (SPR) and / or biomembrane interferometry (BLI) technology, etc. It should be known to those skilled in the art that the affinity parameters measured by different methods vary greatly, and may even differ by 2 to 3 orders of magnitude.

[0113] The nanobodies, polypeptides, and nucleic acids encoding the same 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 comprises the following steps:

[0114] (1) expressing a nucleic acid encoding a Nanobody or polypeptide of the invention in a suitable host cell or host organism or in another suitable expression system, optionally followed by;

[0115] (2) Isolating and / or purifying the Nanobodies or polypeptides of the invention thus obtained.

[0116] Alternatively, other methods may include the following steps:

[0117] (3) culturing and / or maintaining the host of the invention under conditions such that the host of the invention expresses and / or produces a Nanobody and / or polypeptide of the invention; optionally followed by;

[0118] (4) Isolating and / or purifying the Nanobody or polypeptide of the invention thus obtained.

[0119] Nucleic acid of the present invention can be the form of single-stranded or double-stranded DNA or RNA, and preferably double-stranded DNA form.For example, nucleotide sequence of the present invention can be genomic DNA, cDNA or synthetic DNA (as DNA with codon usage that is particularly suitable for expressing in the host cell or host organism to be used, that is, codon optimized).

[0120] The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information given herein on the amino acid sequences of the Nanobodies or polypeptides of the invention, and / or can be isolated from suitable natural sources. HH The nucleic acid sequence of the structural domain is subjected to site-directed mutagenesis to provide the nucleic acid of the present invention encoding the analog.

[0121] Nucleic acid of the present invention can also be in such a form, is present in and / or is the part of a genetic construct, which is well known to those skilled in the art. Such a genetic construct generally comprises at least one nucleic acid of the present invention, can be in the form of a vector, such as a plasmid, YAC, viral vector or transposon. Especially, the vector can be an expression vector, that is, a vector (such as in a suitable host cell, host organism and / or expression system) for external and in vivo expression can be provided.

[0122] The nucleic acids of the invention and / or the genetic constructs of the invention can be used to transform host cells or host organisms, i.e. for expressing and / or producing the Nanobodies or polypeptides of the invention. Suitable hosts or host cells are well known to those skilled in the art and may for example be any suitable fungal, prokaryotic or eukaryotic cell or organelle or organism, as well as all other hosts or host cells known per se for the expression and production of 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.

[0123] For production, the Nanobodies and polypeptides of the invention can be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep, but also in plants or parts of plants, including but not limited to their leaves, flowers, fruits, roots or seeds.

[0124] As mentioned above, one advantage of using nanobodies is that polypeptides based thereon can be expressed and prepared in prokaryotic systems, and suitable prokaryotic expression systems, vectors, host cells, etc. are well known to those skilled in the art, such as the references cited above. However, it should be noted that the present invention in its broadest sense is not limited to expression in bacterial systems.

[0125] Preferably, in the present invention, the Nanobodies or polypeptides are produced in bacterial cells, in particular in bacterial cells suitable for large-scale drug production, as described above.

[0126] When the Nanobodies or polypeptides of the invention are expressed for production in cells, the Nanobodies or polypeptides of the invention may be produced intracellularly (e.g. in the cytoplasm or periplasmic space), then isolated from the host cell, and optionally further purified; or may be produced extracellularly (i.e. secretory expression), then isolated from the culture medium, and optionally further purified.

[0127] 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 vectors (Novagen) and pQE vectors (Qiagen); vectors for expression in yeast or other fungal cells - pYES2 (Invitrogen) and Picha expression vector (Picha expression vector) (Invitrogen); vectors for expression in insect cells - pBlueBacⅡ (Invitrogen) and other baculovirus vectors; and the like.

[0128] The corresponding techniques for transforming the host or host cell of the present invention are well known to those skilled in the art.

[0129] After transformation, those hosts that can detect and select to have transformed nucleotide sequence / genetic construct of the present invention successfully.Converted host cell (it can be in the form of stable cell line) or host organism (it can be in the form of stable mutant line or strain) form another aspect of the present invention.

[0130] The amino acid sequence of the invention can then be isolated from the host cells / host organisms and / or from the culture medium in which the host cells or host organisms are cultivated, by means of protein separation and / or purification techniques known per se, 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 invention) and / or preparative immunological techniques (i.e. using antibodies directed against the amino acid sequence to be isolated).

[0131] The adsorbent of the present invention can be used to specifically recognize EpCAM.

[0132] The nanobodies, polypeptides or adsorbents of the present invention can be used to purify and detect EpCAM, and can also be used to capture and detect EpCAM-positive cells.

[0133] In the first aspect, the present invention provides a nanobody that binds to EpCAM, wherein the variable region in the amino acid sequence of the nanobody includes a complementary determining region CDR and a framework region FR, wherein the complementary determining region CDR includes a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein the most important site involved in antigen recognition and binding is the CDR2 region SRSX 21 RX 22 X 23 and CDR3 region X 31 X 32 X 33 NYX 34 X 35 X 36 NX 37 X 38 RX 39 .

[0134] Preferably, the amino acid sequence of the complementary determining region CDR3 includes SEQ ID No: 58 or SEQ ID No: 59, and the amino acid sequence of the complementary determining region CDR2 includes SEQ ID No: 60 to SEQ ID No: 63, and sequences with a homology of more than 75% thereto.

[0135] Preferably, the framework region FR includes FR1, FR2, FR3 and FR4 sequences, (1) the amino acid sequence of FR1 is shown in any one of SEQ ID.68 to SEQ ID.76, (2) the amino acid sequence of FR2 is shown in any one of SEQ ID.77 to SEQ ID.85, (1) the amino acid sequence of FR3 is shown in any one of SEQ ID.86 to SEQ ID.96, (4) the amino acid sequence of FR4 is shown in any one of SEQ ID.97 to SEQ ID.98:

[0136] SEQ ID No.68:QLQESGGGLVQAGGSLRLSCAASG

[0137] SEQ ID No.69:QLVESGGGLVQPGGSLRLSCAASG

[0138] SEQ ID No.70:QLQESGGGLVQAGDSLRLSCAASG

[0139] SEQ ID No.71:QLQASGGGFVQPGGSLRLSCAASG

[0140] SEQ ID No.72:QLQESGGGLVQPGGSLRLSCAASG

[0141] SEQ ID No.73:QLQESGGGLVQAGDSLRLSCVASG

[0142] SEQ ID No.74:QLQESGGGLVQIGDSLRLSCAASG

[0143] SEQ ID No.75:QLQESGGGLVQAGGSLRLSCVAS

[0144] SEQ ID No.76:QLQESGGGLVQPGDSLRLSCAASG

[0145] SEQ ID No.77:MGWFRQAPGKEREFVAAV

[0146] SEQ ID No.78:MGWFRQVPGKQREFVASV

[0147] SEQ ID No.79:LGWFRQAPGQGLEAVAAV

[0148] SEQ ID No.80:MGWFRQAPGKEREFVAAI

[0149] SEQ ID No.81:MGWFRQAPGKEREFVSAV

[0150] SEQ ID No.82:MGWFRQAPGKGLEAVAAI

[0151] SEQ ID No.83:MGWFRQVPGKQREFVAAV

[0152] SEQ ID No.84:VGWFRQAPGKEREFVAAV

[0153] SEQ ID No.85:MGWFRQVPGKEREFVAAV

[0154] SEQ ID No.86:YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

[0155] SEQ ID No.87:TYGDSVKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA

[0156] SEQ ID No.88:YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA

[0157] SEQ ID No.89:YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAA

[0158] SEQ ID No.90:NYADSVKGRFTISRDNAANTVYLQMNSLKPEDTAVYYCVA

[0159] SEQ ID No.91:NYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVA

[0160] SEQ ID No.92:YYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAA

[0161] SEQ ID No.93:TYAGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAA

[0162] SEQ ID No.94:TYAGSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYFCAA

[0163] SEQ ID No.95:TYAGSVKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA

[0164] SEQ ID No.96:TYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAA

[0165] SEQ ID No.97:DYWGQGTQVTVSS

[0166] SEQ ID No.98: DYWGQGTLVTVSS

[0167] Preferably, the amino acid sequence of the nanobody includes: SEQ ID No: 1 to SEQ ID No: 22.

[0168] Preferably, the nanobody is a humanized nanobody, preferably, the humanized nanobody comprises: SEQ ID No: 23 to SEQ ID No: 38.

[0169] In a second aspect, the present invention provides a polypeptide obtained by modifying the N-terminus and / or C-terminus of the above-mentioned Nanobody.

[0170] Preferably, the method of modifying the amino acids at the N-terminus and / or C-terminus of the Nanobody comprises:

[0171] Method 1: adding a tag to the N-terminal and / or C-terminal amino acid of the Nanobody;

[0172] Mode 2, after the N-terminal and / or C-terminal amino acids of the Nanobody are tagged, the tags are further connected to protect the amino acids via hinges;

[0173] 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 a combination of any two or more of the foregoing amino acids.

[0174] In a third aspect, the present invention provides a polypeptide obtained by multivalent synthesis of the above-mentioned nanoantibody.

[0175] In a fourth aspect, the present invention provides a nucleic acid encoding the above-mentioned Nanobody or the above-mentioned polypeptide.

[0176] In a fifth aspect, the present invention provides an expression vector comprising an expression cassette of the nucleic acid described above in claim 1.

[0177] In a sixth aspect, the present invention provides a host cell comprising the expression vector as claimed in claim 1.

[0178] In a seventh aspect, the present invention provides the use of the nanobody and / or the polypeptide in immunodetection, enrichment and / or purification.

[0179] Preferably, the nanobody and / or the polypeptide is used in the preparation of an EpCAM adsorbent, an EpCAM purification kit, or an EpCAM detection kit.

[0180] Preferably, the Nanobody and / or the polypeptide are used for capturing and detecting EpCAM-positive cells.

[0181] Example

[0182] The following examples are given to illustrate the specific implementation of the present invention, but the implementation of the present invention is not limited to the following examples, and any selection and change can be made within the scope that does not affect the technical effect to be achieved by the present invention.

[0183] Example 1: Construction of anti-EpCAM nanobody library.

[0184] The phage display library used in the present invention is an immune library with T7 phage as a carrier, and the establishment steps are as follows:

[0185] (1) Alpacas (numbered 2309-1 and 2309-2) were immunized with EpCAM. Jugular vein blood was collected from two alpacas after four immunizations. Peripheral blood lymphocytes were isolated and total RNA was extracted (PuerLink TM RNAMini Kit, Life Technologies: 12183018A);

[0186] (3) Total RNA was reverse transcribed into cDNA and amplified using two rounds of nested PCR. HH Gene;

[0187] In the first round of PCR, cDNA was used as a template, UP primer1 and DOWN primer1 were used as upstream and downstream primers, respectively. After amplification, a band of 650-750 bp was recovered, which was used as a template for the second round of PCR. The upstream and downstream primers were UPprimer2 and DOWN primer2, respectively, and a PCR product of 450-500 bp was recovered.

[0188] UPprimer1:CTTGGTGGTCCTGGCTGCTCT,

[0189] DOWNprimer1:GGTACGTGCTGTTGAACTGTTCC,

[0190] UPprimer2:TATCTAGTC GAATTC CGCCCAGGTGCAGCTC,

[0191] DOWNprimer2: AGCGACTAAGCTTTGAGGAGACGGTGAC;

[0192] (3) The PCR product was double-digested with EcoR Ι and Hind Ⅲ and subjected to agarose electrophoresis to recover the 350-500 bp gene band, which was V HH Gene fragments;

[0193] (4) Use T4 ligase to connect the T7 vector ( 10-3Cloning Kit,MeterckMetillipore 70550-3) and V HH Gene fragments;

[0194] (5) mixing the ligation product with the packaging protein to form a complete T7 phage, and amplifying the mixture to obtain a phage original library;

[0195] (6) After testing, the titer of the original library was 6.68×10 9 pfu / mL, with a diversity of 5.7×10 6 .

[0196] Example 2: Screening of Nanobodies.

[0197] First, dilute the antigen EpCAM to 10 μg / mL with TBS, take 100 μL and add it to a 96-well plate, and incubate at 4°C for 12 hours. Aspirate the antigen dilution in the well, wash the plate 3 times with TBS, pat dry, add 1% protein-free blocking solution (purchased from Sangon Biotechnology Co., Ltd.), 300 μL / well, and incubate at room temperature for 2 hours (1% protein-free blocking solution and 1% BSA are used alternately during screening). Aspirate the blocking agent in the well, wash the plate 6 times with TBST, pat dry, add the amplified phage, 100 μL / well, and incubate at room temperature for 30 minutes. Wash the plate 10 times with TBST, add T7 elution buffer (1% SDS) to elute the phage, incubate at room temperature for 30 minutes, and amplify the eluate for the next round of screening.

[0198] Example 3: Construction of genetically engineered bacteria

[0199] (1) After four rounds of screening, the screening eluate was subjected to solid amplification, plaques were picked, and PCR amplification was performed using the plaque amplification solution as a template and UP primer 3 and DOWN primer 3 as upstream and downstream primers;

[0200] UPprimer3:TTCCTTAA CATATG GCCCAGGTGCAGCTCGT,

[0201] DOWNprimer3:TTAAGGAA CTCGAG CACGGTGACCAGGGTC;

[0202] (2) A portion of the PCR products were outsourced for sequencing to obtain the sequence information of the nanobody. Based on the length and homology of the CDR region, 11 monoclonal sequences belonging to the same family were selected. The naming and sequence number information of the nanobody are shown in Table 1.

[0203] (3) Another part of the PCR product was double-digested with NdeI and XhoI, and the digestion product was recovered. At the same time, the digestion was performed in the same way and the vector was recovered. The digestion product and the vector were ligated with T4 ligase, and the ligation product was transferred into Escherichia coli to obtain a genetically engineered bacterium expressing EpCAM-specific nanobodies.

[0204] Table 1 Comparison table of nanobody sequence numbers and CDR sequence information

[0205] Antibody Name Antibody serial number CDR1 sequence CDR2 sequence CDR3 sequence E1 SEQ ID No.1 ESNH--A SRSGRDI RWTNYLGANPGRY E2 SEQ ID No.2 ESNH--A SRSGRDI RWTNYLGANPGRY E3 SEQ ID No.3 ESNH--A SRSGRDI RWTNYLGANPGRY E4 SEQ ID No.4 RTFSLYA SRSGRDI RWTNYLGANPGRY E5 SEQ ID No.5 RTFSLYA SRSGRDI RWTNYLGANPGRY E6 SEQ ID No.6 RTFSLYA SRSGRDI RWTNYLGANPGRY E7 SEQ ID No.7 RTVDIY SRSDRGI SGTNYLGANPGRY E8 SEQ ID No.8 RTVDIY SRSDRGL SGTNYLGANPGRY E9 SEQ ID No.9 RTVDIY SRSDRGL SGTNYLGANPGRY E10 SEQ ID No.10 RTSSSNA SRSGRGT SGTNYLGANPGRY E11 SEQ ID No.11 RTSSSNA SRSGRGT SGTNYLGANPGRY

[0206] SEQ ID No.1:QLQESGGGLVQAGDSLRLSCAASG-ESNH--A-MGWFRQAPGKEREFVAAV-SRSGRDI-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0207] SEQ ID No.2:QLQESGGGLVQAGDSLRLSCAASG-ESNH--A-MGWFRQAPGKEREFVAAV-SRSGRDI-YYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0208] SEQ ID No.3:QLQESGGGLVQAGDSLRLSCVASG-ESNH--A-VGWFRQAPGKEREFVAAV-SRSGRDI-YYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0209] SEQ ID No.4:QLQESGGGLVQAGGSLRLSCAASG-RTFSLYA-MGWFRQVPGKQREFVASV-SRSGRDI-NYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0210] <h2 style=";text-align:left;direction:ltr">SEQ ID No. 5: QLQESGGGLVQAGGSLRLSCAASG-RTFSLYA-MGWFRQVPGKQREFVASV-SRSGRDI-NYADSVKGRFTISRDNAANTVYLQMNSLKPEDTAVYYCVA-RWTNYLGANPGRY-DYWGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0211] <h2 style=";text-align:left;direction:ltr"> SEQ ID No. 6: QLQESGGGLVQPGGSLRLSCAASG-RTFSLYA-MGWFRQVPGKQREFVASV-SRSGRDI-NYADSVKGRFTISRDNAANTVYLQMNSLKPEDTAVYYCVA-RWTNYLGANPGRY-DYWGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0212] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.7:QLQESGGGLVQIGDSLRLSCAASG-RTVDIYG-MGWFRQVPGKQREFVASV-SRSDRGI-TYGDS VKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA-SGTNYLGANPGRY-DYWGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0213] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.8:QLQESGGGLVQIGDSLRLSCAASG-RTVDIYG-MGWFRQVPGKQREFVASV-SRSDRGL-TYGDS VKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA-SGTNYLGANPGRY-DYWGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0214] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.9:QLQESGGGLVQPGDSLRLSCAASG-RTVDIYG-MGWFRQVPGKQREFVAAV-SRSDRGL-TYGD SVKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA-SGTNYLGANPGRY-DYWGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0215] <h2 style=";text-align:left;direction:ltr"> SEQ ID No. 10: QLQESGGGLVQAGDSLRLSCAASG-RTSSSNA-MGWFRQVPGKQREFVAAV-SRSGRGT-TYAG SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAA-SGTNYLGANPGRY-DYWGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0216] SEQ ID No.11:QLQESGGGLVQAGDSLRLSCAASG-RTSSSNA-MGWFRQVPGKQREFVAAV-SRSGRGT-TYAGSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYFCAA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0217] Note: 1. The "-" between FR and CDR represents a connector, which is used to distinguish FR from CDR, and there is no vacant site. 2. The "--" in "ESNH--A" in the CDR1 region represents a placeholder, which is used to maintain comparability with other sequences in the same family.

[0218] Example 4: Calculation of key amino acids

[0219] Using sequence E4 and EpCAM as model proteins, molecular docking was performed to predict the antigen binding epitope of VHH on EpCAM and the key amino acids in the interaction.

[0220] First, the EpCAM structure was obtained from the database, and after combining the EpCAM sequence and the VHH antibody sequence, the complex structure was predicted and searched using Alohafold2 mutimer v3. After the complex structure was generated, Amber was used to perform Relax Process on the complex structure to optimize the side chain structure, ADT was used to check hydrogen atoms and calculate potentials, and Ligplot+ was used to analyze the hydrogen bonds and hydrophobic interaction network between the amino acids in the CDR region of VHH and the VP binding interface, and the atomic contacts between the domains were checked. The reasonable conformation that met the above conditions was selected as the structural model of the VHH antibody and EpCAM protein complex.

[0221] Through molecular docking, the binding epitopes of VHH on EpCAM were calculated: E4 is involved in antigen recognition and binding with SRSXRXX on CDR2 and XXTNYLGANPGRY on CDR3. Moreover, these two sequence regions are strictly conserved within E1 to E11, and have strong van der Waals forces, electrostatic interactions, and hydrogen bond interactions with the side chains of R102, D105, E124, E164, Y227, and Y228 on EpCAM. In addition, the backbones of R103, T104, V167, and Q223 also participate in electrostatic interactions, forming a three-dimensional binding surface together, such as Figure 1 to Figure 4 As shown, the yellow part is EpCAM, the green part is CDR2 of E4, and the blue part is CDR3 of E4.

[0222] The interactions on the binding surface can be divided into five cores, including:

[0223] 1). S50, R51, S52 and N100 on E4 and E124, Q223, Y227 on EpCAM

[0224] 2). R54 on E4 and E164 and V167 on EpCAM

[0225] 3). Y101 on E4 and R102 and Y228 on EpCAM

[0226] 4). N105 on E4 and R103 on EpCAM

[0227] 5). R108 on E4 and D105 on EpCAM

[0228] Therefore, the most important sites of Nanobodies E1 to E11 of the present invention involved in antigen recognition and binding are S50, R51, S52, and R54 on CDR2, and N100, Y101, N105, and R108 on CDR3. That is, CDR2 region SRSXRXX, CDR3 region XXXNYXXXNXXRX.

[0229] Example 5: Preparation of EpCAM Nanobody

[0230] (1) The basic culture medium of the nanobody is TB medium, which is inoculated at a 5% inoculum and cultured at 37°C for 3-5 h, and the inducer IPTG (final concentration 0.25 mM, the same below) is added for overnight induction;

[0231] (2) After induction, the mixture was centrifuged at 4000 rpm for 20 min to obtain wet bacteria containing nanobodies.

[0232] (3) Add lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.40.02M PB) to the obtained wet bacteria at a ratio of 1:10, and use a 700 bar high-pressure homogenizer to disrupt the cells;

[0233] (4) Centrifuge at 4°C and 10,000 rpm for 20 min and collect the supernatant;

[0234] (5) The supernatant was filtered through a 0.45 μm filter and then passed through an affinity chromatography column (GE Healthcare, US) for separation and purification of the EpCAM nanobody, wherein the filler of the affinity chromatography column was Ni Sepharose High Performance;

[0235] (6) The nanoantibodies purified by affinity chromatography were subjected to SDS-PAGE electrophoresis to determine the purity, and the protein solution with higher purity was selected to determine the protein concentration using the BCA method.

[0236] Example 6: Affinity Analysis

[0237] The SPR technique was used to analyze the binding ability of nanobodies to human EpCAM. EpCAM was amino-coupled to a CM5 sensor chip at a density of 500-800 RU, and nanobodies were injected at 7 different concentrations ranging from 1 to 100 nM, with a flow rate of 45 μL / min in all experiments. The chip regeneration condition was glycine-HCl pH 1.5. The binding curves obtained at different nanobody concentrations were used to calculate the kinetic parameter K a , K d and K D . Figure 5 The 11 response curves of nanobodies E1 to E11, each response curve represents the response curve of each antibody at 7 concentrations of 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, and 1.5625nM from top to bottom. The kinetic parameters shown in Table 2 were calculated by equation fitting. The nanobodies have a high affinity for EpCAM, K D Range 10 -8 ~10 -9 (M).

[0238] Table 2 Affinity of nanobodies for human EpCAM

[0239] Nanobodies ka(1 / Ms) kd(1 / s) kD(M) E1 1.77E+05 3.63E-04 2.05E-09 E2 3.46E+05 9.15E-03 2.64E-08 E3 2.10E+05 1.97E-03 9.39E-09 E4 9.13E+05 4.01E-03 4.39E-09 E5 4.77E+05 9.98E-04 2.09E-09 E6 5.59E+05 2.05E-03 3.67E-09 E7 1.58E+05 7.72E-03 4.88E-08 E8 3.09E+05 1.46E-02 4.72E-08 E9 3.22E+05 1.31E-02 4.07E-08 E10 6.18E+04 5.75E-03 9.31E-08 E11 7.09E+04 6.56E-03 9.25E-08

[0240] Example 7: Cross-reactivity of Nanobodies

[0241] The cross-species reactivity of an antibody refers to the property that the same antibody can 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, in the process of 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. The docking results in Example 4 show that the nanoantibodies E1~E11 of the present invention have strong van der Waals forces, electrostatic interactions and hydrogen bond interactions with the side chains R102, D105, E124, E164, Y227, and Y228 on EpCAM. These 6 amino acid residues are conserved in EpCAM of humans and commonly used experimental animals (macaques, chimpanzees, pigs, dogs, mice, and rats). Therefore, in theory, VHH E1~11 can bind to EpCAM of the above-mentioned different animals. The present invention was verified by ELISA to be able to bind to EpCAM of humans and commonly used experimental animals (macaques, chimpanzees, pigs, dogs, mice, and rats). The experimental results are shown in Table 3.

[0242] Table 3 Cross-reactivity of Nanobodies

[0243]

[0244] Table 3 shows that the nanoantibodies E1 to E11 of the present invention can bind to EpCAM of humans and commonly used experimental animals (macaques, chimpanzees, pigs, dogs, mice, and rats), have cross-reactivity, strong applicability, and are in line with theoretical expectations.

[0245] Example 8: Semisynthetic library screening

[0246] In the original sequences obtained by screening, there are large differences in CDR1, FR2, and FR3, and the combination has a certain regularity, indicating that the original B cells that produce this family of nanoantibodies have undergone multiple and diverse affinity maturation processes. Mutations in different regions may improve the performance of different aspects of the final nanoantibodies, so they are retained and become dominant mutants. Due to the randomness and irreversibility of affinity maturation (somatic high-frequency mutations), it is difficult for mutants enriched with all advantages to appear in animals. Therefore, based on the hybridization and aggregation mutation method, the CDR1, FR2, CDR2, FR3, and CDR3 characteristics obtained from the original screening are recombined to construct a phage-displayed semi-synthetic nanoantibody library, which is screened under stringent conditions.

[0247] The experimental process is as follows:

[0248] 1) Based on the E1, E4, E7, and E10 DNA sequences, truncated reverse ssDNA chains were designed as templates for hybridization polymerization: rTemp-E1, rTemp-E4, rTemp-E7, and rTemp-E10 (SEQ ID No.39 to SEQ ID No.42), and the total synthesis was commissioned to Shanghai Sangon Biotechnology Co., Ltd.

[0249] SEQ ID No.39: ATACCTCCCCGGGTTAGCACCCAGATAGTTTGTCCATCTGGCGGCACAATAATAAACGGCA GTGTCCTCCAGGTTTCAGGCTGTTCATCTGCAGATACACCGTGTTCTTGGCGTTGTCTCTGGAGATGGTGAATCGGCCCTTCACGGAGTCTGCATAGTATATATCTCTACCACTCCGACTAACAGCTGCTACAAACTCACGCTCCTTCCCTGGAGCCTGGCGGAACCAGCCCATGGCATGGTTACTTTCTCCAGAGGCTGCACAGGAGAGTCTCAGAGA

[0250] SEQ ID No.40:ATACCTCCCCGGGTTAGCACCCAGATAGTTTGTCCATCTGGCCACACAATAATAAACGGCA GTGTCCTCAGGTTTCAGGCTGTTCATCTGCAGATACACCGTGTTCTTGGCGTTGTCTCTGGAGATGGTGAATCGGCCCTTCACGGAGTCTGCATAGTTTATATCTCTACCACTCCGACTAACAGATGCTACAAACTCACGCTGCTTCCCTGGAACCTGGCGGAACCAGCCCATGGCATAGAGACTGAAGGTGCGTCCAGAGGCTGCACAGGAGAGTCTCAGAGA

[0251] SEQ ID No.41:ATACCTCCCCGGGTTAGCACCCAGATAGTTTGTTCCTGAGGCCACACAATAATAAACGGC AGTGTCCTCAGGTTTCAGGGTACTCATCTGCAGATACATCCTGTTCGCGGCGTTGTCTTTGACGATGGTGAATCGGCCCTTCACGGAGTCTCCATACGTTATACCTCTATCACTCCGACTAACAGATGCTACAAACTCACGCTGCTTCCCTGGAACCTGGCGGAACCAGCCCATGCCATAGATATCTACGGTGCGTCCAGAGGCTGCACAGGAGAGTCTCAGAGA

[0252] SEQ ID No.42:ATACCTCCCCGGGTTAGCACCCAGATAGTTTGTTCCTGAGGCGGCACAAAAATAAACGGC AGTGTCCTCAGGTTTCAGGCTGTTCATCTGCAGATACACCGTGTTCTTGGCGTTGTCTCTGGAGATGGTGAATCGGCCCTTCACGGAGCCTGCATACGTTGTACCTCTACCACTCCGACTAACAGCTGCTACAAACTCACGCTGCTTCCCTGGAACCTGGCGGAACCAGCCCATGGCGTTGCTACTACTGGTGCGTCCAGAGGCTGCACAGGAGAGTCTCAGAGA

[0253] 2) Design 15 forward primers (as shown in Table 4), some of which contain degenerate bases, dissolve them in ddH2O, mix them in a 1.5ml EP centrifuge tube according to the following ratio, add 2.0μl of 10×T4 Polynucleotide Kinase Buffer (Takara2021S, included with T4 Polynucleotide Kinase), and add water to a total volume of 18μl. Add 20U of T4 Polynucleotide Kinase and mix well. Incubate at 37℃ for 1h.

[0254] Table 4 Primer sequences and addition amounts

[0255] Primers sequence Addition amount pmol >FR1 SEQ ID No.43 10 >CDR1(1) SEQ ID No.44 2.5 >CDR1(2) SEQ ID No.45 2.5 >CDR1(3) SEQ ID No.46 2.5 >CDR1(4) SEQ ID No.47 2.5 >CDR2(1) SEQ ID No.48 2 >CDR2(2) SEQ ID No.49 2 >CDR2(3) SEQ ID No.50 2 >CDR2(4) SEQ ID No.51 2 >CDR2(5) SEQ ID No.52 2 >CDR2(6) SEQ ID No.53 2 >FR3(1) SEQ ID No.54 5 >FR3(2) SEQ ID No.55 5 >CDR3(1) SEQ ID No.56 5 >CDR3(2) SEQ ID No.57 5

[0256] SEQ ID No.43: CAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGSCTGGGGRCTCTCTGAGACTCTCCTGT GCA

[0257] SEQ ID No.44: GCCTCTGGAGAAAGTAACCATGCCATGGGCTGGTTCCGCCAGGYTCCAGGGAAGSAGCG TGAG

[0258] SEQ ID No.45: GCCTCTGGACGCACCTTCAGTCTCTATGCCATGGGCTGGTTCCGCCAGGYTCCAGGGAAG SAGCGTGAG

[0259] SEQ ID No.46: GCCTCTGGACGCACCGTAGATATCTATGGCATGGGCTGGTTCCGCCAGGYTCCAGGGAAG SAGCGTGAG

[0260] SEQ ID No.47: GCCTCTGGACGCACCAGTAGTAGCAACGCCATGGGCTGGTTCCGCCAGGYTCCAGGGAA GSAGCGTGAG

[0261] SEQ ID No.48:TTTGTAGCAKCTGTTAGTCGGAGTGGTAGAGATATAWACTATGCAGACTCCGTGAAG

[0262] SEQ ID No.49:TTTGTAGCAKCTGTTAGTCGGAGTGGTAGAGATATAACGTATGSAGRCTCCGTGAAG

[0263] SEQ ID No.50: TTTGTAGCAKCTGTTAGTCGGAGTGATAGAGGTMTAWACTATGCAGACTCCGTGAAG

[0264] SEQ ID No.51: TTTGTAGCAKCTGTTAGTCGGAGTGATAGAGGTMTAACGTATGSAGRCTCCGTGAAG

[0265] SEQ ID No.52: TTTGTAGCAKCTGTTAGTCGGAGTGGTAGAGGTACAWACTATGCAGACTCCGTGAAG

[0266] SEQ ID No.53: TTTGTAGCAKCTGTTAGTCGGAGTGGTAGAGGTACAACGTATGSAGRCTCCGTGAAG

[0267] SEQ ID No.54: GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGYGTATCTGCAGATGAACAG CCTGAAACCTGAGGACACT

[0268] SEQ ID No.55: GGCCGATTCACCATCGTCAAAGACAACGCCGCGAACAGGATGTATCTGCAGATGAGTACC CTGARACCTGAGGACACT

[0269] SEQ ID No.56: GCCGTTTATTATTGTGTGGCCAGATGGACAAACTATCTGGGTGCTAACCCGGGGAGGTATG ACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0270] SEQ ID No.57: GCCGTTTATTWTTGTGTGGCCTCAGGAACAAACTATCTGGGTGCTAACCCGGGGAGGTAT GACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0271] 3) Add 2.5 pmol of each of the 4 ssDNA templates to a 20 μl phosphorylation reaction mixture and add water to a total volume of 180 μl.

[0272] 4) Incubate at 90°C for 3 min, 50°C for 3 min, and 20°C for 5 min.

[0273] 5) Add 20 μl of 10×T4 DNA Ligase Buffer (Takara 2011A, included in the T4 DNA ligase kit) and 30 Weiss Units of T4 DNA ligase to the annealed oligonucleotide and template mixture.

[0274] 6) Incubate at 20°C overnight.

[0275] 7) The DNA was purified and recovered using a DNA purification kit (Takara 9761).

[0276] 8) Take the purified DNA (0.2 pmol) as a template, add the upstream and downstream primers UPprimer2 and DOWNprimer2 respectively, and recover the PCR product of 450-500 bp after PCR;

[0277] UP primer2:

[0278] TATCTAGTC GAATTC CGCCCAGGTGCAGCTC

[0279] DOWNprimer2:

[0280] AGCGACT AAGCTT TGAGGAGACGGTGAC

[0281] 9) The library construction method is the same as in Example 1. Therefore, the theoretical diversity of the semi-synthetic library does not exceed 15,000, and the library capacity is greater than 1×10 5 pfu is sufficient to meet the experimental requirements. After testing, the titer of the original library is 2.75×10 7 pfu / mL, and the library was sequenced by high-throughput monitoring data of 1G, with an efficiency of >90%, and more than 11,000 different sequences were obtained by analysis.

[0282] After screening, the obtained single clones were sequenced and it was found that most of the clone sequences were the same as E1 to E11, and only a few new combinations were obtained, numbered E20 to E30 (SEQ ID No: 12 to SEQ ID No: 22).

[0283] SEQ ID No.12:QLQESGGGLVQAGGSLRLSCAASG-RTVDIYG-MGWFRQAPGKEREFVAAV-SRSGRDI-YYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0284] SEQ ID No.13:QLQESGGGLVQAGGSLRLSCAASG-RTFSLYA-MGWFRQAPGKEREFVAAV-SRSGRGT-TYAGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0285] SEQ ID No.14:QLQESGGGLVQAGDSLRLSCAASG-RTSSSNA-MGWFRQVPGKEREFVAAV-SRSDRGI-TYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0286] SEQ ID No.15:QLQESGGGLVQAGDSLRLSCVASG-ESNH--A-VGWFRQAPGKEREFVAAV-SRSGRDI-NYADSVKGRFTISRDNAANTVYLQMNSLKPEDTAVYYCVA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0287] SEQ ID No.16:QLQESGGGLVQAGGSLRLSCAASG-RTVDIYG-MGWFRQAPGKEREFVAAV-SRSGRDI-NYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0288] SEQ ID No.17:QLQESGGGLVQPGGSLRLSCAASG-RTFSLYA-MGWFRQAPGKEREFVAAV-SRSGRDI-NYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0289] SEQ ID No.18: QLQESGGGLVQAGGSLRLSCVAS--ESNH--A-VGWFRQAPGKEREFVAAV-SRSGRGT-TYGDSVKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0290] SEQ ID No.19: QLQESGGGLVQAGGSLRLSCAASG-RTFSLYA-MGWFRQAPGKEREFVAAV-SRSGRGT-TYGDSVKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0291] SEQ ID No.20: QLQESGGGLVQAGDSLRLSCAASG-RTSSSNA-MGWFRQAPGKEREFVAAV-SRSGRGT-TYGDSVKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0292] SEQ ID No.21: QLQESGGGLVQPGGSLRLSCAASG-RTFSLYA-MGWFRQAPGKEREFVAAV-SRSDRGL-TYGD SVKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0293] SEQ ID No.22: QLQESGGGLVQAGGSLRLSCAASG-RTSSSNA-MGWFRQAPGKEREFVAAV-SRSGRGT-TYA GSVKGRFTIVKDNAANRMYLQMSTLKPEDTAVYYCVA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0294] The sequences E20 to E30 (SEQ ID No: 12 to SEQ ID No: 22) obtained by screening were outsourced for synthesis. Vector preparation, genetic engineering bacteria preparation, periplasmic expression, large-scale preparation and expression purification, and affinity determination were carried out in the manner described in Examples 5 and 6. The affinity results of nanobodies E20 to E30 are shown in Table 5.

[0295] Table 5 Affinity of Nanobodies Screened by Semisynthetic Library Method for Human EpCAM

[0296] Nanobodies ka(1 / Ms) kd(1 / s) kD(M) E20 1.74E+05 3.22E-04 1.85E-09 E21 1.32E+05 4.41E-03 3.34E-08 E22 1.39E+05 7.97E-03 5.73E-08 E23 9.48E+04 4.50E-03 4.75E-08 E24 1.45E+05 7.95E-03 5.49E-08 E25 6.28E+04 3.72E-03 5.92E-08 E26 3.00E+05 1.08E-02 3.59E-08 E27 1.22E+05 9.01E-03 7.39E-08 E28 1.35E+05 6.37E-03 4.71E-08 E29 5.96E+04 1.46E-02 2.45E-07 E30 5.49E+04 1.39E-02 2.52E-07

[0297] Example 9: Stability and humanization of nanobodies and affinity determination.

[0298] In order to reduce the immunogenicity of nanobodies while maintaining or improving the stability and biological activity of nanobodies, the present invention performs CDR region transplantation based on commonly used humanized or highly stable nanobody frameworks to complete the humanization and stability modification of nanobodies.

[0299] (1) Sequence design

[0300] The present invention selects four nano antibody skeletons for modification, namely ah, FGLA, hs2dAb (hereinafter referred to as hs) and com skeletons.

[0301] The ah skeleton is a universal fully humanized skeleton 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).).

[0302] FGLA is a universal 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).).

[0303] hs is a highly stable, highly expressed, partially humanized skeleton from the literature (Moutel, S. et al. NaLi-H1: Auniversal synthetic library of humanized nanobodies providing highlyfunctional antibodies and intrabodies. Elife 5, doi: 10.7554 / eLife.16228 (2016).).

[0304] com is a universal skeleton with high stability and high expression, which comes from the literature (Ferrari, D., Garrapa, V., Locatelli, M. & Bolchi, AA 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).).

[0305] The sequence names and sequence numbers of the modified humanized sequences are shown in Table 6.

[0306] Table 6 Humanized sequence names and sequence numbers

[0307] Antibody Name Serial Number Antibody Name Serial Number E1-ah SEQ ID No.23 E1-com SEQ ID No.31 E4-ah SEQ ID No.24 E4-com SEQ ID No.32 E7-ah SEQ ID No.25 E7-com SEQ ID No.33 E9-ah SEQ ID No.26 E9-com SEQ ID No.34 E1-FGLA SEQ ID No.27 E1-hs SEQ ID No.35 E4-FGLA SEQ ID No.28 E4-hs SEQ ID No.36 E7-FGLA SEQ ID No.29 E7-hs SEQ ID No.37 E9-FGLA SEQ ID No.30 E9-hs SEQ ID No.38

[0308] SEQ ID No.23: QLVESGGGLVQPGGSLRLSCAASG-ESNH--A-MGWFRQAPGKGLEAVAAI-SRSGRDI-YYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTLVTVSS

[0309] SEQ ID No.24: QLVESGGGLVQPGGSLRLSCAASG-RTFSLYA-MGWFRQAPGKGLEAVAAI-SRSGRDI-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTLVTVSS

[0310] SEQ ID No.25:QLVESGGGLVQPGGSLRLSCAASG-RTVDIYG-MGWFRQAPGKGLEAVAAI-SRSDRGI-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA-SGTNYLGANPGRY-DYWGQGTLVTVSS

[0311] SEQ ID No.26:QLVESGGGLVQPGGSLRLSCAASG-RTSSSNA-MGWFRQAPGKGLEAVAAI-SRSGRGT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA-SGTNYLGANPGRY-DYWGQGTLVTVSS

[0312] SEQ ID No.27:QLVESGGGLVQPGGSLRLSCAASG-ESNH--A-LGWFRQAPGQGLEAVAAV-SRSGRDI-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTLVTVSS

[0313] SEQ ID No.28:QLVESGGGLVQPGGSLRLSCAASG-RTFSLYA-LGWFRQAPGQGLEAVAAV-SRSGRDI-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTLVTVSS

[0314] SEQ ID No.29:QLVESGGGLVQPGGSLRLSCAASG-RTVDIYG-LGWFRQAPGQGLEAVAAV-SRSDRGI-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA-SGTNYLGANPGRY-DYWGQGTLVTVSS

[0315] SEQ ID No.30:QLVESGGGLVQPGGSLRLSCAASG-RTSSSNA-LGWFRQAPGQGLEAVAAV-SRSGRGT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA-SGTNYLGANPGRY-DYWGQGTLVTVSS

[0316] (2) Nanobody preparation and affinity determination

[0317] The vector construction process is as described in Example 3, the nanobody preparation process is as described in Example 5, and the affinity characterization of the nanobody is as described in Example 6. The binding curves obtained at different nanobody concentrations are used to calculate the kinetic parameter K a , K d and K D Table 7 is obtained.

[0318] Table 7 Affinity results of nanobodies before and after humanization

[0319]

[0320] SEQ ID No.31: QLQESGGGLVQAGGSLRLSCAASG-ESNH--A-MGWFRQAPGKEREFVAAI-SRSGRDI-YYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0321] SEQ ID No.32: QLQESGGGLVQAGGSLRLSCAASG-RTFSLYA-MGWFRQAPGKEREFVAAI-SRSGRDI-YYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0322] SEQ ID No.33: QLQESGGGLVQAGGSLRLSCAASG-RTVDIYG-MGWFRQAPGKEREFVAAI-SRSDRGI-YYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0323] SEQ ID No.34: QLQESGGGLVQAGGSLRLSCAASG-RTSSSNA-MGWFRQAPGKEREFVAAI-SRSGRGT-YYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0324] SEQ ID No.35: QLQASGGGFVQPGGSLRLSCAASG-ESNH--A-MGWFRQAPGKEREFVSAV-SRSGRDI-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0325] SEQ ID No.36: QLQASGGGFVQPGGSLRLSCAASG-RTFSLYA-MGWFRQAPGKEREFVSAV-SRSGRDI-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAA-RWTNYLGANPGRY-DYWGQGTQVTVSS

[0326] SEQ ID No.37: QLQASGGGFVQPGGSLRLSCAASG-RTVDIYG-MGWFRQAPGKEREFVSAV-SRSDRGI-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0327] SEQ ID No.38: QLQASGGGFVQPGGSLRLSCAASG-RTSSSNA-MGWFRQAPGKEREFVSAV-SRSGRGT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAA-SGTNYLGANPGRY-DYWGQGTQVTVSS

[0328] Compared with the original sequence, the affinity of the humanized nanobody to the antigen EpCAM did not change significantly, and the affinity to the related antigen remained at the same order of magnitude. For example, the affinity of the nanobody E9 to EpCAM was 4.07×10 -8 M, the affinity ranking of its four humanized sequences E9-ah, E9-FLAG, E9-com and E9-hs for EpCAM is still 10 -8 M. This indicates that the humanized nanobody of the present invention has been successfully transformed.

[0329] Example 10: Antibody labeling

[0330] Antibody labeling techniques well known to those skilled 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 an appropriate labeling technique can improve the sensitivity and specificity of the experiment.

[0331] The present invention adopts three commonly used antibody labeling methods, as follows:

[0332] a) Biotinylation: 1 mg / mL E1 was dissolved in PBS, and 10-fold molar amount of NHS-biotin was added. After mixing, the mixture was incubated at 4°C in the dark overnight, and the solution was replaced by ultrafiltration.

[0333] Denoted as E1-biotin.

[0334] b) Fluorescence labeling: 1 mg / mL E1 was dissolved in PBS, and 10 times the molar amount of fluorescein isothiocyanate FITC was added. After mixing, the mixture was incubated at 4°C in the dark overnight, and the solution was replaced by ultrafiltration. This was recorded as E1-FITC.

[0335] c) HRP labeling: Nanobody E1 was replaced with PBS (pH 7.4) to a concentration of about 10 mg / mL and conjugated using an HRP conjugation kit (abcam, Lightning- ab102890) and dialyzed thoroughly for replacement of the solution. It is denoted as E1-HRP.

[0336] Example 11: EpCAM detection (competitive ELISA)

[0337] 1) HRP-labeled EPCAM was used as a competitor and diluted to 10 ng / mL with PBS.

[0338] 2) A highly hydrophobic 96-well plate was coated with 10 μg / mL E1-HRP overnight and the plate was washed.

[0339] 3) Block with 3% BSA at room temperature for 2 hours and wash the plate.

[0340] 4) Sample preparation: The test sample or EPCAM standard was diluted with PBS to 0, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512 ng / mL, 50 μL was taken, and an equal volume of competitor (10 ng / mL HRP-labeled EPCAM) was added.

[0341] 5) Add the prepared samples to the 96-well plate, incubate at room temperature for 2 hours, and wash the plate.

[0342] 6) TBM color development, determination of OD450, drawing of a standard curve or calculation of the concentration of the sample to be tested.

[0343] The standard curve of E1-HRP for measuring EpCAM of the present invention is as follows Figure 6As shown, the fitting curve used is a four-parameter Logistic curve fitting, and the fitting result is: y = (AD) / [1 + (x / C) ^ B] + D, where A = 3.46172, B = 5.87825, C = 4.08176, D = 0.04565, R 2 =0.99989. 2 It can reach above 0.99, indicating that the ELISA detection method established based on the nanobody of the present invention has strong reliability and high sensitivity.

[0344] Example 12: EpCAM-positive cell capture

[0345] The streptavidin-coated magnetic beads were mixed with E1-biotin (0.1 mg / mL) and incubated for 30 minutes, and then washed thoroughly with PBST buffer for later use; MCF-7 cells (EpCAM, HER2 positive cells) were used as model cells, the experimental group was 5 mL of fresh healthy human whole blood with 100 MCF-7 cells added, and the control group was 5 mL of fresh healthy human whole blood without MCF-7 cells added.

[0346] 20 μL of the above magnetic beads were added to the experimental group and the control group, respectively, and mixed and incubated at 37°C for 30 min. The magnetic beads were fully washed with PBS buffer, and then labeled with DAPI using conventional experimental methods and commercial fluorescently labeled HER2 antibodies, and observed under a fluorescence microscope. Figure 7 As shown, the control group ( Figure 7 (a)) cells without HER2 fluorescence signal on the magnetic beads; while the experimental group ( Figure 7 (b)) All cells on the magnetic beads have HER2 fluorescent signals, that is, all captured cells are MCF-7 cells. This proves that the nanobody of the present invention can be used for EpCAM positive cell capture.

[0347] Example 13: Immunofluorescence analysis

[0348] MCF-7 cells (EpCAM positive cells) were cultured in 24-well plates, and were labeled with DAPI and E1-FITC using conventional experimental methods and observed under laser confocal microscopy. Figure 8 As shown, it can be seen that the cell nucleus has a DAPI signal and the cell membrane has a FITC signal, that is, E1-FITC marks EpCAM on the cell membrane. This proves that the nanobody of the present invention can be used for the labeling and fluorescence analysis of EpCAM positive cells.

[0349] Example 14: Immunohistochemical analysis

[0350] MCF-7 cells (EpCAM positive cells) were inoculated into the tail vein of mice and fed normally until tumor tissue could be observed. Tumor tissue was cut and paraffin sections were prepared. Paraffin sections were labeled with E1-HRP and developed using conventional experimental methods. Fig. 9 As shown, in tumor tissue sections, the staining intensity of EpCAM protein is strongly positive, and the positively stained cells are 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 EpCAM positive cells.

[0351] Industrial Applicability

[0352] The nanoantibody of the present invention is an anti-EpCAM nanoantibody with a new amino acid sequence discovered by screening a phage library. The nanoantibody and its polypeptide have high affinity and activity, and can specifically recognize and bind to EpCAM. The nanoantibody prepared by the present invention can be used for the capture and detection of EpCAM-positive cells, and can be applied to immunofluorescence analysis or immunohistochemistry analysis, etc. through appropriate antibody labeling technology.

[0353] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An anti-EpCAM nanobody, characterized in that: The complementary determining region CDR of the Nanobody includes CDR1, CDR2 and CDR3 sequences: (I): (1) the amino acid sequence of the CDR2 is as shown in formula (1-1), (2) the amino acid sequence of the CDR3 is as shown in formula (1-2); SRSX 21 RX 22 X 23 (1-1); X 31 X 32 X 33 NYX 34 X 35 X 36 NX 37 X 38 RX 39 (1-2); In the formula, X represents a site in the amino acid sequence that can be modified, substituted or deleted.

2. The Nanobody according to claim 1, characterized in that in: (II): (1) the amino acid sequence of CDR2 described in (1) of (I) is SRSGRDI, and the amino acid sequence of CDR3 described in (2) of (I) is RWTNYLGANPGRY; or (2) the amino acid sequence of CDR2 described in (1) of (I) is SRSDRGI, and the amino acid sequence of CDR3 described in (2) of (I) is SGTNYLGANPGRY; or (3) the amino acid sequence of CDR2 described in (1) of (I) is SRSDRGL, and the amino acid sequence of CDR3 described in (2) of (I) is SGTNYLGANPGRY; or (4) the amino acid sequence of CDR2 described in (1) of (I) is SRSDRGT, and the amino acid sequence of CDR3 described in (2) of (I) is SGTNYLGANPGRY; or (5) The amino acid sequence of CDR2 described in (1) of (I) is SRSDRGT, and the amino acid sequence of CDR3 described in (2) of (I) is RWTNYLGANPGRY. (6) An amino acid sequence having 75% or more homology with any one of the amino acid sequences described in (1) to (5) of (II).

3. The Nanobody according to claim 2, characterized in that in: (III): (1) the amino acid sequence of the CDR1 is ESNH--A, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (1) of (II); or (2) the amino acid sequence of the CDR1 is RTFSLYA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (1) of (II); or (3) the amino acid sequence of the CDR1 is RTVDIYG, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (1) of (II); or (4) the amino acid sequence of the CDR1 is RTVDIYG, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (2); or (5) the amino acid sequence of the CDR1 is RTSSSNA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (2) of (II); (6) the amino acid sequence of the CDR1 is RTFSLYA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (3); or (7) the amino acid sequence of the CDR1 is RTVDIYG, the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (3); or (8) the amino acid sequence of the CDR1 is ESNH--A, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (4); or (9) the amino acid sequence of the CDR1 is RTFSLYA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (4); or (10) the amino acid sequence of the CDR1 is RTSSSNA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (II) (4); or (11) The amino acid sequence of the CDR1 is RTSSSNA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (5) of (II).

4. The Nanobody according to any one of claims 1 to 3, characterized in that The framework region FR of the Nanobody comprises FR1, FR2, FR3 and FR4 sequences, wherein: (IV) (1) the amino acid sequence of FR1 is as shown in formula (4-1), (2) the amino acid sequence of FR2 is as shown in formula (4-2), (3) the amino acid sequence of FR3 is as shown in formula (4-3), (4) the amino acid sequence of FR4 is as shown in formula (4-4); QLX 41 X 42 SGGGX 43 VQX 44 GX 45 SLRLSCX 46 ASX 47 (4-1); X 51 GWFRQX 52 PGX 53 X 54 X 55 EX 56 VX 57 X 58 X 59 (4-2); X 61 YX 62 X 63 SVKGRFTIX 64 X 65 DNX 66 X 67 NX 68 X 69 YLQMX 610 X 611 LX 612 X 613 EDTAX 614 YX 615 CX 616 A(4-3); DYWGQGTX 71 VTVSS(4-4); In the formula, X represents a site in the amino acid sequence that can be modified, substituted or deleted.

5. The Nanobody according to claim 4, characterized in that in: (V): (1) X in the amino acid sequence of FR1 as described in (1) of (IV) 41 Selected from Q or V alone; X 42 Selected from E or A alone; X 43 Selected from L or F alone; X 44 Individually selected from A or P or I; X 45 Selected from D or G alone; X 46 Selected from A or V alone; X 47 selected from G alone or missing; (2) In the amino acid sequence of FR2 as described in (IV) (2), X 51 Selected from M or L alone; X 52 Selected from A or V alone; X 53 Select K or Q alone; X 54 Individually selected from E or Q or G; X 55 Selected from R or L alone; X 56 Selected from F or A alone; X 57 Selected from A or S alone; X 58 Selected from A or S alone; X 59 Individually selected from V or I; (3) In the amino acid sequence of FR3 described in (IV) (3), X 61 Individually selected from Y or N or T; X 62 Selected from A or G alone; X 63 Selected from D or G alone; X 64 Selected singly from S or V; X 65 Selected alone from R or K; X 66 Selected from A or S alone; X 67 Selected from K or A alone; X 68 Selected from T or R alone; X 69 Individually selected from V or M or A; X 610 Selected alone from N or S; X 611 Selected from S or T alone; X 612 Selected alone from K or R; X 613 Selected from A or P alone; X 614 Selected from V or T alone; X 615 Selected from Y or F alone; X 616 Selected from A or V alone; (4) In the amino acid sequence of FR4 as described in (IV) (4), X 61 Selected from Q or L alone.

6. The Nanobody according to any one of claims 1 to 5, characterized in that in: (VI): (1) the amino acid sequence of FR1 is shown in any one of SEQ ID.68 to SEQ ID.76, (2) the amino acid sequence of FR2 is shown in any one of SEQ ID.77 to SEQ ID.85, (1) the amino acid sequence of FR3 is shown in any one of SEQ ID.86 to SEQ ID.96, (4) the amino acid sequence of FR4 is shown in any one of SEQ ID.97 to SEQ ID.98; (2) or an amino acid sequence having 50% or more homology with the amino acid sequence described in (1), (2), (3) or (4).

7. The Nanobody according to any one of claims 1 to 6, characterized in that in: (VII): (1) the amino acid sequence of the Nanobody is as shown in any one of SEQ ID.1, SEQ ID.2, SEQ ID.3, SEQ ID.4, SEQ ID.5, SEQ ID.6, SEQ ID.12, SEQ ID.15, SEQ ID.16, SEQ ID.17, SEQ ID.23, SEQ ID.24, SEQ ID.27, SEQ ID.28, SEQ ID.31, SEQ ID.32, SEQ ID.35, and SEQ ID.36; or (2) the amino acid sequence of the Nanobody is shown in any one of SEQ ID.7, SEQ ID.14, SEQ ID.25, SEQ ID.29, SEQ ID.33, and SEQ ID.37; or (3) the amino acid sequence of the Nanobody is as shown in any one of SEQ ID.8, SEQ ID.9, and SEQ ID.21; or (4) the amino acid sequence of the Nanobody is as shown in any one of SEQ ID.10, SEQ ID.11, SEQ ID.13, SEQ ID.18, SEQ ID.19, SEQ ID.20, SEQ ID.26, SEQ ID.30, SEQ ID.34, and SEQ ID.38; or (5) The amino acid sequence of the Nanobody is shown in SEQ ID.

22.

8. A polypeptide, characterized in that Comprising the Nanobody according to 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 vector, characterized in that: Comprising the nucleic acid molecule of claim 9.

11. A host cell transformed or transfected with the expression vector according to claim 10.

12. A conjugate or a coupling substance, characterized in that: The nanobody according to any one of claims 1 to 7 is chemically or biologically labeled.

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

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

15. A device, characterized in that For capturing, adsorbing and / or detecting EpCAM, comprising the Nanobody according to any one of claims 1 to 7; or the polypeptide according to claim 8; or the nucleic acid molecule according to claim 9; or the expression vector according to claim 10; or the host cell according to claim 11; or the conjugate as claimed in claim 12; or the conjugate as claimed in claim 12; Or the adsorbent according to claim 13; or the kit according to 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 according to claim 9; or the expression vector according to claim 10; or the host cell according to claim 11; or the conjugate as claimed in claim 12; or the conjugate as claimed in claim 12; or the adsorbent according to claim 13; or the kit according to claim 14 Application in preparing specific capture, adsorption and / or detection of EpCAM preparations; or Application in the preparation of tumor detection preparations for specific capture, adsorption and / or detection of EpCAM; or Application in the preparation of enriched and / or purified cell preparations for specific capture, adsorption and / or detection of EpCAM; or Application in the preparation of immunofluorescence analysis or immunohistochemistry analysis reagents for heterogeneous capture, adsorption and / or detection of EpCAM.

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