Anti-EpCAM nanoantibodies and their applications

Abstract

Anti-EpCAM nanoantibodies and their applications belong to the field of biotechnology and are used to solve the problem of accurate identification of EpCAM. The key points are that the complementary determining region (CDR) of the nanoantibody 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); where X represents a site in the amino acid sequence that can be modified, substituted, or deleted, and can be used to capture and detect EpCAM-positive cells. Through appropriate antibody labeling techniques, it can be applied to immunofluorescence analysis or immunohistochemistry analysis.

Description

Technical Field

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

[0002] Epithelial cell adhesion molecule (EpCAM) is a single-pass transmembrane glycoprotein with a molecular weight of approximately 40 kDa and composed of 314 amino acids. Its molecular structure consists of three parts: an extracellular domain, a single-pass transmembrane domain, and an intracellular domain. It is involved in regulating cell adhesion, proliferation, differentiation, migration, and signal transduction. It interacts with multiple cell adhesion molecules and participates in cell signaling through its intracellular domain (EpICD). Abnormal expression of EpCAM is closely related to the occurrence, development, and prognosis of tumors. In particular, it plays a key role in the epithelial-mesenchymal transition (EMT) process and is one of the important molecular mechanisms of tumor invasion and metastasis. EpCAM is highly expressed in many human malignancies, 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) detach 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] EpCAM-positive cell capture methods can be broadly categorized into two types: antibody-based affinity enrichment and physical enrichment based on cell characteristics. Detection methods can also be divided into two categories: antibody-based and nucleic acid-based. The choice and application of these methods depends on the specific needs of the experiment, including the desired sensitivity, specificity, and throughput, as well as the need for subsequent culture or molecular analysis of the captured cells.

[0005] Antibodies can capture and detect EpCAM-positive cells with high sensitivity and specificity, without damaging the cells and preventing them from being cultured. However, the large size and high production costs of traditional antibodies have limited their practical application in 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 a 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, SEQ ID.37) or

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

[0015] (4) The amino acid sequence of CDR2 as described in (1) of (I) is SRSDRGT, and the amino acid sequence of CDR3 as 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 described in (1) of (II); (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, 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); (SEQ ID.12, SEQ ID.16) or

[0023] (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); (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, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (3) of (II); (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 (3) of (II); (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, 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.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] QLX 41 X 42 SGGGX 43 VQX 44 GX 45 SLRLSCX 46 ASX 47 (4-1);

[0034] X 51 GWFRQX 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] DYWGQGTX 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 Individually selected from Q or V; X 42 Individually selected from E or A; X 43 Individually selected from L or F; X 44 Individually selected from A or P or I; X 45 Individually selected from D or G; X 46 Individually selected from A or V; X 47 selected from G alone or missing;

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

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

[0042] (4) X in the amino acid sequence of FR4 as described in (4) of (IV) 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 as shown in any one of SEQ ID.68 to SEQ ID.76, (2) the amino acid sequence of FR2 is as shown in any one of SEQ ID.77 to SEQ ID.85, (3) the amino acid sequence of FR3 is as shown in any one of SEQ ID.86 to SEQ ID.96, (4) the amino acid sequence of FR4 is as 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 as 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, the 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 compound or conjugate according to some embodiments of the present application comprises the nanobody that 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 conjugate; 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 conjugate; or the conjugate;

[0063] Or the adsorbent, 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 to capture, adsorb and / or detect EpCAM, comprising any one of the nanoantibodies; or the polypeptide; or the nucleic acid molecule;

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

[0066] or the conjugate; or the conjugate;

[0067] or the adsorbent; or the kit.

[0068] In the ninth aspect, the use of the nanobody according to 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 according to 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 nanoantibody according to 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 according to 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 nanobody of the present invention is an anti-EpCAM nanobody with a new amino acid sequence discovered through screening. The nanobody 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. It solves the problems of complex antibody preparation process, high cost, and poor antibody affinity and stability faced in existing EpCAM enrichment, purification, and detection. It can also be applied to immunofluorescence analysis or immunohistochemistry analysis 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 This is the standard curve for sandwich ELISA detection in Example 11 of the present invention.

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

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

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

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

[0083] (1) Unless otherwise indicated, the term "sequence" as used herein (e.g., in similar terms 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 familiar to those skilled in the art. For example, reference is made to the general background art 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 the antigen and the 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 their CDR3s, and have an amino acid sequence identity greater than 75%.

[0087] (5) Amino acid substitutions can generally be described as substitutions where an amino acid residue can be substituted with an amino acid of similar chemical structure or with an amino acid of 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 of 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 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 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; and (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] The framework regions are more conserved than the complementarity determining regions. Those skilled in the art will perform reasonable screening of the sequence structure of the framework regions based on the actual use and function of the Nanobody. As amino acid sequences in the framework regions, amino acid sequences with a homology of 50% or more are preferred, further preferably amino acid sequences with a homology of 70% or more, and further preferably amino acid sequences with a homology of 95% or more are preferred.

[0092] The contribution of the framework region to affinity is relatively small, so 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 acids in the framework region are also suitable for the above-mentioned amino acid substitution methods. Among them, humanization is a typical example of amino acid substitution 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 range of 110 to 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 the framework regions FR and antigen-binding regions CDR have the same length and high sequence identity, similar structures, and have substantially equivalent antigen-binding abilities.

[0095] The structure of the nanoantibody of the present invention can be determined by any appropriate known method, 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 simulations), Relax Process (in molecular dynamics simulations, Relax Process usually refers to the process of energy minimization of protein structure to optimize the geometric configuration of side chains and reduce conflicts and poor geometric configurations within the molecule), 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] The method for preparing a "Nanobody" in its broadest sense is not limited to a specific biological source or a specific method of preparation. For example, the Nanobodies 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 protein encoding the humanized V HH (4) preparing a protein, polypeptide or other amino acid sequence using synthetic or semi-synthetic techniques; (5) preparing a nucleic acid encoding a Nanobody by applying nucleic acid synthesis techniques and then expressing the nucleic acid thus obtained; and / or (6) by any combination of the foregoing.

[0097] In addition, based on a variant 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 present 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 essentially consists of a Nanobody. "Essentially consists 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, is added to the amino terminus (N-terminus) and / or carboxyl terminus (C-terminus) of said 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 affinity techniques directed against said sequence or residue. 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 an N-terminal amino acid residue, for example, Met, Ala, Gln or MetAlaGln, AlaGln, thereby enabling expression 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, an IgG hinge, an IgA hinge, or other artificially synthesized hinges;

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

[0106] The polypeptides of the present invention may also comprise two or more of said Nanobodies, also known as multivalent polypeptides.

[0107] A bivalent polypeptide comprises two Nanobodies, optionally linked by a hinge sequence, a trivalent polypeptide comprises three Nanobodies, optionally linked by two hinge sequences, and a tetravalent polypeptide comprises four Nanobodies, optionally linked by three hinge sequences. Multivalent polypeptides can bind to the same antigenic epitope or to different antigen-binding epitopes; the latter are also called 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] Hinges for multivalent and multispecific polypeptides are well known to those skilled in the art and may include, for example, Gly-Ser, such as (Gly4Ser)3 or (Gly3Ser2)3 described in WO 99 / 42077, or naturally occurring heavy chain antibody hinge regions or portions thereof. For other suitable hinges, reference is also made to the general background art 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, etc.

[0111] In addition, the dissociation equilibrium constant (K D ) is 10 -8 ~10 -9 Mole / L (M). The dissociation equilibrium constant of the present invention is measured using the plasma resonance technique.

[0112] The specific binding between the antigen and the antigen-binding molecule can be determined using any suitable known method, including Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), as well as other novel methods known in the art, such as plasma resonance imaging (SPR) and / or biomembrane interferometry (BLI). It will be appreciated by those skilled in the art that affinity parameters measured using different methods can vary significantly, sometimes by 2 to 3 orders of magnitude.

[0113] The Nanobodies, polypeptides, and nucleic acids encoding the same of the invention can be prepared in a known manner, as will be apparent to those skilled in the art from the further description herein. A particularly useful method for preparing such Nanobodies, polypeptides, and nucleic acids generally comprises the following steps:

[0114] (1) expressing a nucleic acid encoding said 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, another method may include the following steps:

[0117] (3) culturing and / or maintaining the host of the present invention under 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;

[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 utilization that is particularly suitable for expressing in the host cell that will use or host organism, 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. For example, for naturally occurring V 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 such form, is present in and / or is the part of genetic construct, and this is well known to those skilled in the art.Such genetic construct usually comprises at least a nucleic acid of the present invention, can be vector form, as plasmid, YAC, viral vector or transposon.Especially, said carrier can be an expression vector, that is, can provide the carrier (as in suitable host cell, host organism and / or expression system) of external and internal expression.

[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 produced in prokaryotic systems, and suitable prokaryotic expression systems, vectors, host cells, etc. are well known to those skilled in the art, for example, as cited in the references 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 pharmaceutical 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 can be produced intracellularly (e.g. in the cytoplasm or periplasmic space), then isolated from the host cell and optionally further purified; or can 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 (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 the conversion, it is possible to detect and select those hosts that have successfully converted nucleotide sequence of the present invention / genetic construct. The transformed host cell (which can be the form of a stable cell line) or the host organism (which can be the form of a 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 said 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 nanobody, polypeptide or adsorbent 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 complementarity determining region CDR3 includes SEQ ID No: 58 or SEQ ID No: 59, and the amino acid sequence of the complementarity 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 as shown in any one of SEQ ID.68 to SEQ ID.76, (2) the amino acid sequence of FR2 is as shown in any one of SEQ ID.77 to SEQ ID.85, (3) the amino acid sequence of FR3 is as shown in any one of SEQ ID.86 to SEQ ID.96, (4) the amino acid sequence of FR4 is as 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 comprises: 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 N-terminal and / or C-terminal amino acids of the Nanobody comprises:

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

[0172] Method 2: After the N-terminal and / or C-terminal amino acids of the Nanobody are tagged, the tags are further linked to protect the amino acids via a hinge;

[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 nanoantibodies.

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

[0176] In a fifth aspect, the present invention provides an expression vector comprising an expression cassette of the nucleic acid according to 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 to capture and detect 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. Any selection and change can be made within the scope that does not affect the technical effects 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 the carrier, and the establishment steps are as follows:

[0185] (1) Alpacas (numbered 2309-1 and 2309-2) were immunized with EpCAM. After four immunizations, jugular vein blood was collected from two alpacas, 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, with UP primer 1 and DOWN primer 1 as upstream and downstream primers, respectively. After amplification, a band of 650 to 750 bp was recovered. This band was used as a template for the second round of PCR, with UP primer 2 and DOWN primer 2 as upstream and downstream primers, respectively. A PCR product of 450 to 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 EcoRI and HindIII, 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 the original phage 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, the antigen EpCAM was diluted to 10 μg / mL with TBS, 100 μL was added to a 96-well plate, and incubated at 4°C for 12 hours. The antigen dilution in the well was aspirated, the plate was washed three times with TBS, patted dry, and 1% protein-free blocking solution (purchased from Sangon Biotech Co., Ltd.) was added at 300 μL / well and incubated at room temperature for 2 hours (1% protein-free blocking solution and 1% BSA were used alternately during screening). The blocking agent in the well was aspirated, the plate was washed six times with TBST, patted dry, and the amplified phage was added at 100 μL / well and incubated at room temperature for 30 minutes. The plate was washed 10 times with TBST, the phage was eluted with T7 elution buffer (1% SDS), incubated at room temperature for 30 minutes, and the eluate was amplified 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 was 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 nanobody naming and sequence number information are shown in Table 1.

[0203] (3) Another portion of the PCR product was double-digested with NdeI and XhoI, and the digestion product was recovered. Simultaneously, the digestion product and vector were recovered using the same method. The digestion product and vector were ligated with T4 ligase, and the ligation product was transformed into Escherichia coli to obtain a genetically engineered bacterium expressing the EpCAM-specific nanobody.

[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 RTVDIYG SRSDRGI SGTNYLGANPGRY E8 SEQ ID No.8 RTVDIYG SRSDRGL SGTNYLGANPGRY E9 SEQ ID No.9 RTVDIYG 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, used to distinguish FR from CDR; there are no vacant sites. 2. The "--" in "ESNH--A" in the CDR1 region represents a placeholder, 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 a database. After combining the EpCAM sequence with the VHH antibody sequence, the complex structure was predicted and searched using Alohafold2 mutimer v3. After generating the complex structure, the side chain structure was optimized using Amber's Relax Process. ADT was used to analyze hydrogen atoms and calculate potentials. Ligplot+ was used to analyze the hydrogen bonding and hydrophobic interaction networks between the VHH CDR amino acids and the VP binding interface, and to examine atomic contacts between the domains. A plausible conformation that met these criteria was selected as the structural model for the VHH antibody-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. Furthermore, these two sequence regions are strictly conserved within E1-E11, and have strong van der Waals, electrostatic, and hydrogen bonding interactions with the side chains of R102, D105, E124, E164, Y227, and Y228 on EpCAM. Furthermore, the backbone of R103, T104, V167, and Q223 also participates in electrostatic interactions, forming a three-dimensional binding surface. Figures 1 to 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 involved in antigen recognition and binding of Nanobodies E1 to E11 of the present invention 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 Nanobodies

[0230] (1) The basal culture medium for the nanobody was TB medium, which was inoculated at a 5% inoculum and cultured at 37°C for 3–5 h. The inducer IPTG (final concentration 0.25 mM, the same below) was 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) Lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.4, 0.02 M PB) was added to the wet bacteria at a ratio of 1:10, and the cells were disrupted using a 700 bar high-pressure homogenizer;

[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 EpCAM nanoantibodies, wherein the affinity chromatography column was filled with 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 binding ability of the nanobody to human EpCAM was analyzed using SPR technology. EpCAM was amino-coupled to a CM5 sensor chip at a density of 500 to 800 RU. The nanobody was injected at seven different concentrations ranging from 1 to 100 nM, with a flow rate of 45 μL / min in all experiments. The chip was regenerated using 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 These are the 11 response curves of nanobodies E1 to E11. From top to bottom, each response curve represents the response curve of each antibody at 7 concentrations, namely 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, and 1.5625nM. The kinetic parameters shown in Table 2 were calculated by equation fitting. Nanobodies all have high affinity for EpCAM, with K D Range 10 -8 ~10 -9 (M).

[0238] Table 2 Affinity of Nanobodies to 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-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, 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. The docking results in Example 4 show that the nanoantibodies E1 to E11 of the present invention 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. These six 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 to 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] Among the original sequences obtained through screening, there are significant differences in CDR1, FR2, and FR3, and the combinations have a certain regularity, indicating that the original B cells that produced this family of nanoantibodies have undergone multiple and diverse affinity maturation processes. Mutations in different regions may have improved the performance of different aspects of the final nanoantibody, and thus have been 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 polymerization mutagenesis method, the CDR1, FR2, CDR2, FR3, and CDR3 characteristics obtained through the original screening were recombined to construct a phage-displayed semi-synthetic nanoantibody library, which was then 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). The synthesis was commissioned to Sangon Biotechnology (Shanghai) 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 (see Table 4), some of which contain degenerate bases. Dissolve them in ddH2O and mix in a 1.5ml EP microcentrifuge tube according to the following ratios. Add 2.0 μl of 10× T4 Polynucleotide Kinase Buffer (Takara 2021S, included with T4 Polynucleotide Kinase) and add water to a total volume of 18 μl. Add 20 U of T4 Polynucleotide Kinase and mix thoroughly. Incubate at 37°C for 1 hour.

[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: CAGCTTGCAGGAGTCTGGGGGAGGATTGGTGCAGSCTGGGGRCTCTCTGAGACTCTCCTGT 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) Use 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. The titer of the original library was tested to be 2.75×10 7 pfu / mL, and the library was sequenced for high-throughput monitoring data of 1G, with an efficiency of >90%, and more than 11,000 different sequences were obtained.

[0282] After screening, the obtained single clones were sequenced and it was found that most of the clone sequences were identical to 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] Sequences E20-E30 (SEQ ID No: 12-SEQ ID No: 22) obtained from the screening were outsourced for synthesis. Vector preparation, genetically engineered bacterial preparation, periplasmic expression, large-scale production and expression purification, and affinity determination were performed as described in Examples 5 and 6. The affinity results for Nanobodies E20-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 nanoantibody 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 highly functional 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 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 were 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 has not changed significantly, and the affinity for related antigens remains at the same order of magnitude. For example, the affinity of the nanobody E9 to EpCAM is 4.07×10 -8 The affinity of its four humanized sequences, E9-ah, E9-FLAG, E9-com, and E9-hs, to EpCAM is still ranked as 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 in this field include enzyme labeling, biotin labeling, fluorescein labeling, colloidal gold labeling, and radioisotope labeling. Each labeling technology has its own specific application scenarios and advantages. Choosing the appropriate labeling technology 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: Dissolve 1 mg / mL of E1 in PBS, add 10-fold molar amount of NHS-biotin, mix well, incubate overnight at 4°C in the dark, and replace the solution by ultrafiltration.

[0333] Denoted as E1-biotin.

[0334] b) Fluorescent labeling: Dissolve 1 mg / mL of E1 in PBS, add a 10-fold molar amount of fluorescein isothiocyanate (FITC), mix thoroughly, incubate overnight at 4°C in the dark, and replace the solution by ultrafiltration. This is referred to as E1-FITC.

[0335] c) HRP labeling: Nanobody E1 was exchanged into PBS (pH 7.4) at a concentration of about 10 mg / mL and labeled using an HRP conjugation kit (abcam, Lightning- It was labeled with ab102890 and dialyzed thoroughly with exchange buffer. This was designated 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) Coat a highly hydrophobic 96-well plate with 10 μg / mL E1-HRP overnight and wash the plate.

[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, or 512 ng / mL. 50 μL of the sample 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) Develop the color using TBM, measure OD450, draw a standard curve or calculate 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. R 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] Streptavidin-coated magnetic beads were mixed with E1-biotin (0.1 mg / mL) and incubated for 30 minutes, then thoroughly washed with PBST buffer for later use. MCF-7 cells (EpCAM, HER2-positive cells) were used as model cells. The experimental group consisted of 5 mL of fresh healthy human whole blood with 100 MCF-7 cells added, and the control group consisted of 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 and commercial fluorescently labeled HER2 antibodies according to conventional experimental methods and observed under a fluorescence microscope. Figure 7 As shown, the control group ( Figure 7 (a)) cells without HER2 fluorescence signals on the magnetic beads; while the experimental group ( Figure 7 (b) All cells on the magnetic beads have HER2 fluorescence signals, that is, all captured cells are MCF-7 cells. This proves that the Nanobody of the present invention can be used for capturing EpCAM-positive cells.

[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, DAPI signals can be seen in the cell nucleus and FITC signals can be seen in the cell membrane, indicating that E1-FITC labels EpCAM on the cell membrane. This demonstrates that the nanobody of the present invention can be used for 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 then excised and paraffin sections were prepared. Paraffin sections were labeled with E1-HRP and developed using conventional experimental methods. Figure 9 As shown, the staining intensity of EpCAM protein in tumor tissue sections 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 labeling and immunohistochemical analysis of EpCAM-positive cells.

[0351] Industrial applicability

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

[0353] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. An anti-EpCAM nanobody, characterized in that The complementary determining regions (CDRs) of the Nanobody include 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); Wherein, X represents a site in the amino acid sequence that can be modified, substituted or deleted; in: (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; 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; in: (III): (1) the amino acid sequence of the CDR1 is ESNHA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the same as 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 same as the amino acid sequence 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 (2) of (II); 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 same as the amino acid sequence 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 (3) of (II); or (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 (3) of (II); or (8) the amino acid sequence of the CDR1 is ESNHA, and the amino acid sequence of the CDR2 and the amino acid sequence of the CDR3 are the amino acid sequences described in (4) of (II); 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 (4) of (II); 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 (4) of (II); 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).

2. The Nanobody according to claim 1, 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.

3. The Nanobody according to claim 2, 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 alone from D or G; X 46 Individually selected from A or V; X 47 G alone or missing; (2) X in the amino acid sequence of FR2 as described in (2) of (IV) 51 Selected alone from M or L; X 52 Individually selected from A or V; X 53 Selected alone from K or Q; X 54 Individually selected from E or Q or G; X 55 Selected alone from R or L; X 56 Individually selected from F or A; 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) X in the amino acid sequence of FR3 as described in (3) of (IV) 61 Individually selected from Y or N or T; X 62 Alone selected from A or G; X 63 Selected alone from D or G; 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 Individually selected 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 alone from S or T; X 612 Selected alone from K or R; X 613 Selected alone from A or P; X 614 Individually selected from V or T; X 615 Individually selected from Y or F; X 616 Individually selected from A or V; (4) X in the amino acid sequence of FR4 as described in (4) of (IV) 71 Selected from Q or L alone.

4. The Nanobody according to any one of claims 1 to 3, characterized in that in: (VI): (1) the amino acid sequence of FR1 is as shown in any one of SEQ ID.68 to SEQ ID.76, (2) the amino acid sequence of FR2 is as shown in any one of SEQ ID.77 to SEQ ID.85, (3) the amino acid sequence of FR3 is as shown in any one of SEQ ID.86 to SEQ ID.96, (4) the amino acid sequence of FR4 is as 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).

5. A nanobody, characterized in that in: (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 as 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.

6. A polypeptide, characterized in that The nanobody according to any one of claims 1 to 5 is obtained by adding a tag to the N-terminal and / or C-terminal amino acid.

7. The polypeptide according to claim 6, characterized in that The tag includes at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag, and Flag-tag.

8. The polypeptide according to claim 6, characterized in that The tag is connected to protected amino acids via a hinge.

9. The polypeptide according to claim 8, characterized in that The hinge includes at least one of a GS hinge, an IgG hinge, an 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.

10. A polypeptide, characterized in that The nanobody according to any one of claims 1 to 5 is obtained by multivalent synthesis.

11. A nucleic acid molecule encoding the Nanobody according to any one of claims 1 to 5.

12. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 11.

13. A host cell transformed or transfected with the expression vector according to claim 12.

14. A conjugate or a coupling substance, characterized in that: Composed of the Nanobody according to any one of claims 1 to 5 that is chemically labeled or biologically labeled.

15. An adsorbent, characterized in that Comprising the Nanobody of any one of claims 1 to 5; or the polypeptide of any one of claims 6 to 10; or the nucleic acid molecule of claim 11; or the expression vector of claim 12; or the host cell of claim 13; or the conjugate of claim 14; or the conjugate of claim 14, and a carrier.

16. A kit, characterized in that Comprising the Nanobody of any one of claims 1 to 5; or the polypeptide of any one of claims 6 to 10; or the nucleic acid molecule of claim 11; or the expression vector of claim 12; or the host cell of claim 13; or the conjugate of claim 14; or the conjugate of claim 14; or the adsorbent of claim 15, and an auxiliary agent acceptable in detection.

17. A device, characterized in that For capturing, adsorbing and / or detecting EpCAM, comprising the Nanobody of any one of claims 1 to 5; or the polypeptide of any one of claims 6 to 10; or the nucleic acid molecule of claim 11; or the expression vector of claim 12; or the host cell of claim 13; or the conjugate of claim 14; or the conjugate of claim 14; or the adsorbent of claim 15; or the kit of claim 16.

18. Use of the Nanobody according to any one of claims 1 to 5; or the polypeptide according to any one of claims 6 to 10; or the nucleic acid molecule according to claim 11; or the expression vector according to claim 12; or the host cell according to claim 13; or the conjugate according to claim 14; or the conjugate according to claim 14; or the adsorbent according to claim 15; or the kit according to claim 16 in the preparation of a preparation for specific capture, adsorption and / or detection of EpCAM.

19. Use of the Nanobody according to any one of claims 1 to 5; or the polypeptide according to any one of claims 6 to 10; or the nucleic acid molecule according to claim 11; or the expression vector according to claim 12; or the host cell according to claim 13; or the conjugate according to claim 14; or the conjugate according to claim 14; or the adsorbent according to claim 15; or the kit according to claim 16 in the preparation of a tumor detection preparation that specifically captures, adsorbs and / or detects EpCAM.

20. Use of the Nanobody according to any one of claims 1 to 5; or the polypeptide according to any one of claims 6 to 10; or the nucleic acid molecule according to claim 11; or the expression vector according to claim 12; or the host cell according to claim 13; or the conjugate according to claim 14; or the conjugate according to claim 14; or the adsorbent according to claim 15; or the kit according to claim 16 in the preparation of an enriched and / or purified cell preparation for specific capture, adsorption and / or detection of EpCAM.

21. Use of the Nanobody according to any one of claims 1 to 5; or the polypeptide according to any one of claims 6 to 10; or the nucleic acid molecule according to claim 11; or the expression vector according to claim 12; or the host cell according to claim 13; or the conjugate according to claim 14; or the conjugate according to claim 14; or the adsorbent according to claim 15; or the kit according to claim 16 in the preparation of immunofluorescence analysis or immunohistochemistry analysis reagents for heterosexual capture, adsorption and / or detection of EpCAM.

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