Cadheprotein-17 single-domain antibody developed based on phage display technology and application of cadheprotein-17 single-domain antibody

Abstract

The invention relates to a cadheprotein-17 single-domain antibody developed based on a phage display technology, which is characterized in that the single-domain antibody has the following determinant complementary regions: CDR1, CDR2 and CDR3; wherein the amino acid sequence of the CDR1 is as shown in any one of SEQ ID NO. 31-60, the amino acid sequence of the CDR2 is as shown in any one of SEQ ID NO. 61-90, and the amino acid sequence of the CDR3 is as shown in any one of SEQ ID NO. 91-120. The cadheprotein-17 single-domain antibody disclosed by the invention is short in research and development period and high in antibody quality, and has outstanding advantages in the aspects of antibody affinity, targeting specificity, small molecular size, stability, modifiability, production cost and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a Cadherin-17 single domain antibody developed based on phage display technology and applications thereof. Background Art

[0002] Cadherin-17 (CDH17) is a calcium-dependent cell adhesion molecule that plays a key role in multiple biological processes. First, CDH17 is important for maintaining the integrity of tissue structure. Intercellular adhesion is the basis for multicellular organisms to maintain normal tissue structure. As a cadherin, CDH17 can interact with CDH17 or other adhesion molecules on adjacent cells through its extracellular domain. For example, in intestinal epithelial cells, CDH17 tightly connects adjacent epithelial cells like "glue". This tight connection helps prevent harmful substances (such as bacteria and toxins in the intestine) from entering the body from the intercellular space and maintains the integrity of the intestinal barrier. It establishes a stable connection between cells, allowing cells to be arranged in an orderly manner, which is essential for the normal morphology and function of organs. In liver tissue, CDH17 is also involved in the adhesion between hepatocytes to ensure the normal lobular structure and metabolic function of the liver. Second, CDH17 helps to establish and maintain cell polarity. CDH17 is involved in the regulation of cell polarity. Cell polarity refers to the asymmetry of cells in morphology, structure and function, which is necessary for the normal physiological functions of many cells, such as absorption and secretion. In polarized epithelial cells, CDH17 is located on the side of the cell, helping to determine the boundaries of the top, bottom and side of the cell. For example, in renal tubular epithelial cells, the distribution of CDH17 helps maintain the polarity of the cell, thereby ensuring the normal reabsorption of water and solutes by the renal tubules. Third, CDH17 also plays a vital role in the development of the embryo. During embryonic development, cells need to migrate and differentiate precisely to form various organs. CDH17 plays an important guiding role in organogenesis. During intestinal development, the expression pattern of CDH17 changes with the morphological development of the intestine. It can promote the aggregation and stratification of intestinal epithelial cells, which is of great significance for the formation of intestinal villi and crypts. During pancreatic development, CDH17 is also involved in the differentiation of pancreatic duct cells and islet cells. Changes in its expression level can affect the fate of pancreatic cells and are indispensable for the formation of normal pancreatic tissue structure. Finally, CDH17 also plays an important role in the regulation of cell migration and differentiation. CDH17 can regulate cell migration. During the migration of neural crest cells, it may affect the migration path and speed of neural crest cells by interacting with the extracellular matrix or other cell surface molecules. In terms of cell differentiation, it acts as a signaling molecule that can activate intracellular signaling pathways, such as the Wnt / β-catenin pathway, to promote cell differentiation into specific cell types. For example, during embryonic liver development, CDH17 is involved in the differentiation of liver progenitor cells into mature hepatocytes.

[0003] CDH17 is associated with many diseases, but there are very few related drugs on the market, and the medical field targeting CDH17 targets is urgently needed to be explored. In tumor biology, CDH17 plays a complex role. On the one hand, its abnormal expression may promote the proliferation of tumor cells. In some digestive system tumors (such as gastric cancer and colorectal cancer), the expression level of CDH17 is increased, which may promote the survival and proliferation of tumor cells by activating related intracellular signaling pathways, such as the PI3K-Akt pathway. On the other hand, CDH17 is closely related to the metastasis of tumor cells. It can change the adhesion properties between tumor cells, making it easier for tumor cells to detach from the primary lesion. Moreover, CDH17 may also interact with the extracellular matrix to promote the migration and invasion of tumor cells, such as playing a role in the intrahepatic metastasis of liver cancer cells.

[0004] Due to the specific expression changes of CDH17 in certain tumors, it may become a potential disease diagnostic marker. By detecting the level of CDH17 in blood or tissue, doctors can be assisted in the early diagnosis of certain cancers. For example, in the diagnosis of gastric cancer, the detection of serum CDH17 content combined with other detection indicators can improve the accuracy of gastric cancer diagnosis. At the same time, it can also be used as a reference indicator for evaluating tumor prognosis. Tumor patients with high expression of CDH17 may have a poor prognosis. High expression of CDH17 is associated with the development of various tumors. CDH17 is often highly expressed in gastric cancer tissue. It can promote the occurrence and development of gastric cancer through various mechanisms. For example, CDH17 can activate intracellular signaling pathways, such as the PI3K-Akt signaling pathway, so that cancer cells gain stronger proliferation ability. At the same time, its abnormal expression will also affect the adhesion between cancer cells, prompting cancer cells to detach from the primary tumor lesion and increase the risk of metastasis. Moreover, the level of CDH17 in the serum of gastric cancer patients will also increase, which provides a potential marker for non-invasive diagnosis of gastric cancer. CDH17 also plays a role in the pathogenesis of colorectal cancer. It is involved in regulating the interaction between tumor cells and surrounding cells and the extracellular matrix. Highly expressed CDH17 may change the migration and invasion characteristics of tumor cells, promote the infiltration of cancer cells in the intestinal wall and metastasis to distant organs. Studies have found that the expression level of CDH17 is closely related to the clinical pathological characteristics of colorectal cancer, such as the stage and lymph node metastasis, and is expected to be an important indicator for evaluating the prognosis of colorectal cancer. In liver cancer tissue, the expression of CDH17 is also changed. It may affect the progression of liver cancer by regulating the adhesion, migration and proliferation of liver cancer cells. For example, CDH17 can promote the adhesion of liver cancer cells to vascular endothelial cells in the liver, which is conducive to the intrahepatic or distant metastasis of liver cancer cells through blood circulation. In addition, its abnormal expression in liver cancer cells is also related to tumor angiogenesis, providing necessary nutritional support for tumor growth. CDH17 is also involved in the occurrence and development of pancreatic cancer. Pancreatic cancer is a highly malignant tumor, and CDH17 may be involved in the abnormal differentiation of pancreatic cancer cells and the regulation of the tumor microenvironment. Its abnormal expression can lead to changes in the biological behavior of pancreatic cancer cells, such as enhancing the invasiveness of cancer cells, making it easier for cancer cells to invade surrounding tissues and organs, such as the duodenum and bile duct, thereby increasing the difficulty of treatment. In pancreatic cystic lesions (such as pancreatic pseudocysts, pancreatic cystic tumors, etc.), the expression pattern of CDH17 may change. This change may be related to factors such as cyst formation, proliferation of cyst wall cells, and the risk of malignant transformation of cysts. Although the specific mechanism of action of CDH17 in pancreatic cystic lesions has not yet been fully clarified, studies have found that its abnormal expression may be a potential factor in the development of pancreatic cystic lesions. In inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease.In the intestinal tissue of IBD patients, the expression of CDH17 may be disordered. Under inflammatory conditions, changes in the expression of CDH17 may affect the barrier function of intestinal epithelial cells, making the intestinal mucosa more vulnerable to invasion by bacteria and toxins, thereby aggravating intestinal inflammation. Moreover, this expression disorder may also be related to abnormal repair of the intestinal mucosa, hindering the normal repair of intestinal tissue after inflammation. Liver fibrosis is an intermediate stage in the development of various chronic liver diseases to cirrhosis. Changes in the expression of CDH17 in hepatic stellate cells and hepatocytes may be involved in the process of liver fibrosis. It may promote the formation of liver fibrosis by regulating cell-to-cell interactions and the synthesis of extracellular matrix. For example, CDH17 can promote the activation of hepatic stellate cells, increase the production of extracellular matrix components such as collagen, and lead to fibrotic changes in liver tissue.

[0005] It can be seen that CDH17 is a very promising drug target, especially in the field of cancer treatment. However, there are serious restrictions on the research of drugs related to CDH17 immunodiagnosis in my country, and traditional antibodies can no longer meet the needs of patients. First, there are problems in targeting specificity. CDH17 is expressed in both normal tissues and tumor tissues, but abnormal high expression or abnormal cell localization may occur in tumor tissues. This makes it difficult for drugs targeting CDH17 to act only on tumor cells without affecting normal cells at all. For example, in intestinal tissue, normal intestinal epithelial cells also express CDH17, and drugs may interfere with these normal cells to a certain extent, causing adverse reactions such as intestinal dysfunction. There is a certain homology with other members of the cadherin family. CDH17 has structural similarities with other cadherins (such as CDH1, CDH2, etc.). It is difficult to accurately identify only CDH17 when designing drugs, and off-target effects may occur, affecting the normal physiological functions of other cadherins. This may lead to unexpected interference in processes such as cell-to-cell adhesion and signal transduction, and then trigger a series of complex biological consequences. Second, there are challenges in tumor heterogeneity. The expression and functional status of CDH17 may differ between tumor cells of different patients and between different cells within the same tumor. Some tumor cells may be highly sensitive to CDH17 targeted drugs, while others may be resistant to drugs due to factors such as mutations, modification states, or the microenvironment in which they are located in CDH17. This tumor heterogeneity makes it difficult for drugs to produce consistent therapeutic effects on all tumor cells, affecting the overall success rate of treatment. Third, traditional antibody therapy has limited effects. In many cases, CDH17 targeted drugs are difficult to completely inhibit tumor growth and metastasis when used as monotherapy. For example, in the treatment of gastric cancer, relying solely on traditional monoclonal antibody drugs targeting CDH17 may only partially slow the proliferation of tumor cells, but not completely regress the tumor. This is because the occurrence and development of tumors is a complex multi-factor, multi-step process involving multiple signaling pathways and cell biological behaviors. For tumors that have already undergone distant metastasis, the effect of traditional monotherapy with antibodies is even less than ideal. The cells in the tumor metastasis may have adapted to the new microenvironment, and their biological characteristics may change further. CDH17 targeted drugs may not be able to effectively respond to these changes, resulting in poor treatment of metastases. Finally, some drugs have immune-related adverse reactions. Some CDH17 targeted drugs are based on the principles of immunotherapy, such as antibody-drug conjugates (ADCs) or immune checkpoint inhibitors combined with CDH17 targeted therapy. These treatments may induce immune-related adverse reactions. For example, activating the body's immune system to attack normal tissue cells expressing CDH17, leading to autoimmune disease-like reactions, including skin inflammation, endocrine disorders, etc.Overactivation of the immune system may also cause cytokine release syndrome, which can cause patients to experience severe symptoms such as high fever and low blood pressure, and even endanger their lives. These problems indicate that traditional CDH17 monoclonal antibodies can no longer meet the medication needs of patients.

[0006] Since Hamers et al. discovered heavy chain antibodies with naturally missing light chains in camel blood in 1993, single domain antibodies (sdAb) have gradually replaced other small antibodies and become a hot spot in the research and development of new antibody drugs. Single domain antibodies, also known as nanobodies, are usually only about 15KDa, about one-tenth the size of traditional antibodies. They have disulfide bonds inside and a large number of hydrophilic residues on the surface, and have strong resistance to heat and pH. The lack of Fc segments and light chains in sdAb enables it to recognize hidden epitopes or small epitopes that traditional antibodies cannot recognize, and avoid complement reactions. In addition, single domain antibodies also have many advantages such as high stability, low toxicity, strong solubility, easy target screening, and easy direct expression in prokaryotic microorganisms, and good economy. Sequence homology analysis showed that the VHH germline gene sequence of camel sdAb was highly homologous to human VH3, but CDR1 and CDR3 were slightly longer than those of humans, and CDR3 protruded outward in the tertiary structure, so it was speculated that it had higher antigen binding specificity and affinity.

[0007] There are many advantages to developing single-domain antibodies targeting CDH17 for cancer treatment. The drug forms are diverse and each has its own advantages. Single-domain antibody-drug conjugates (ADCs) can accurately target the CDH17 antigen on the surface of tumor cells, deliver cytotoxins to cancer cells to achieve specific killing, enhance efficacy while reducing damage to normal cells and toxic side effects. Moreover, the small size of single-domain antibodies allows them to spread more easily in these complex tumor microenvironments, penetrate deep into tumor tissues, and more effectively contact and bind to cancer cells expressing CDH17. Bispecific antibodies can act on multiple targets, such as binding to CDH17 and other related targets at the same time, exerting synergistic anti-tumor effects, and can also improve the specificity of identifying tumor cells and reduce off-target effects. Single-domain antibody-modified immune cell therapy can enhance the targeting of immune cells to tumor cells expressing CDH17, effectively activate the killing mechanism, and enhance the killing activity. As an imaging probe, single-domain antibodies can image with high specificity, which is conducive to early detection of tumors and determination of their range. They can also monitor tumor progression and changes in CDH17 expression in real time to guide the adjustment of treatment plans. Summary of the invention

[0008] In view of this, a CDH17 single-domain antibody developed based on phage display technology and its application are provided, which has a short research and development cycle, high antibody quality, and outstanding advantages in antibody affinity and targeting specificity, small molecule size, stability and modifiability, and production cost.

[0009] A single-domain antibody for E-cadherin-17 developed based on phage display technology, characterized in that the single-domain antibody has the following complementary regions of determinants: CDR1, CDR2 and CDR3; Among them, the amino acid sequence of CDR1 is shown in any one of SEQ ID NOs.31-60, the amino acid sequence of CDR2 is shown in any one of SEQ ID NOs.61-90, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs.91-120.

[0010] Preferably, the complementary determining region of the E-cadherin-17 single domain antibody is as shown in any one of the following (1) to (30): (1) CDR1 is shown in SEQ ID NO.31, CDR2 is shown in SEQ ID NO.61, and CDR3 is shown in SEQ ID NO.91; (2) CDR1 is shown in SEQ ID NO.32, CDR2 is shown in SEQ ID NO.62, and CDR3 is shown in SEQ ID NO.92; (3) CDR1 is shown in SEQ ID NO.33, CDR2 is shown in SEQ ID NO.63, and CDR3 is shown in SEQ ID NO.93; (4) CDR1 is shown in SEQ ID NO.34, CDR2 is shown in SEQ ID NO.64, and CDR3 is shown in SEQ ID NO.94; (5) CDR1 is shown in SEQ ID NO.35, CDR2 is shown in SEQ ID NO.65, and CDR3 is shown in SEQ ID NO.95; (6) CDR1 is shown in SEQ ID NO.36, CDR2 is shown in SEQ ID NO.66, and CDR3 is shown in SEQ ID NO.96; (7) CDR1 is shown in SEQ ID NO.37, CDR2 is shown in SEQ ID NO.67, and CDR3 is shown in SEQ ID NO.97; (8) CDR1 is shown in SEQ ID NO.38, CDR2 is shown in SEQ ID NO.68, and CDR3 is shown in SEQ ID NO.98; (9) CDR1 is shown in SEQ ID NO.39, CDR2 is shown in SEQ ID NO.69, and CDR3 is shown in SEQ ID NO.99; (10) CDR1 is shown in SEQ ID NO.40, CDR2 is shown in SEQ ID NO.70, and CDR3 is shown in SEQ ID NO.100; (11) CDR1 is shown in SEQ ID NO.41, CDR2 is shown in SEQ ID NO.71, and CDR3 is shown in SEQ ID NO.101; (12) CDR1 is shown in SEQ ID NO.42, CDR2 is shown in SEQ ID NO.72, and CDR3 is shown in SEQ ID NO.102; (13) CDR1 is shown in SEQ ID NO.43, CDR2 is shown in SEQ ID NO.73, and CDR3 is shown in SEQ ID NO.103; (14) CDR1 is shown in SEQ ID NO.44, CDR2 is shown in SEQ ID NO.74, and CDR3 is shown in SEQ ID NO.104; (15) CDR1 is shown in SEQ ID NO.45, CDR2 is shown in SEQ ID NO.75, and CDR3 is shown in SEQ ID NO.105; (16) CDR1 is shown in SEQ ID NO.46, CDR2 is shown in SEQ ID NO.76, and CDR3 is shown in SEQ ID NO.106; (17) CDR1 is shown in SEQ ID NO.47, CDR2 is shown in SEQ ID NO.77, and CDR3 is shown in SEQ ID NO.107; (18) CDR1 is shown in SEQ ID NO.48, CDR2 is shown in SEQ ID NO.78, and CDR3 is shown in SEQ ID NO.108; (19) CDR1 is shown in SEQ ID NO.49, CDR2 is shown in SEQ ID NO.79, and CDR3 is shown in SEQ ID NO.109; (20) CDR1 is shown in SEQ ID NO.50, CDR2 is shown in SEQ ID NO.80, and CDR3 is shown in SEQ ID NO.110; (21) CDR1 is shown in SEQ ID NO.51, CDR2 is shown in SEQ ID NO.81, and CDR3 is shown in SEQ ID NO.111; (22) CDR1 is shown in SEQ ID NO.52, CDR2 is shown in SEQ ID NO.82, and CDR3 is shown in SEQ ID NO.112; (23) CDR1 is shown in SEQ ID NO.53, CDR2 is shown in SEQ ID NO.83, and CDR3 is shown in SEQ ID NO.113; (24) CDR1 is shown in SEQ ID NO.54, CDR2 is shown in SEQ ID NO.84, and CDR3 is shown in SEQ ID NO.114; (25) CDR1 is shown in SEQ ID NO.55, CDR2 is shown in SEQ ID NO.85, and CDR3 is shown in SEQ ID NO.115; (26) CDR1 is shown in SEQ ID NO.56, CDR2 is shown in SEQ ID NO.86, and CDR3 is shown in SEQ ID NO.116; (27) CDR1 is shown in SEQ ID NO.57, CDR2 is shown in SEQ ID NO.87, and CDR3 is shown in SEQ ID NO.117; (28) CDR1 is shown in SEQ ID NO.58, CDR2 is shown in SEQ ID NO.88, and CDR3 is shown in SEQ ID NO.118; (29) CDR1 is shown in SEQ ID NO.59, CDR2 is shown in SEQ ID NO.89, and CDR3 is shown in SEQ ID NO.119; (30) CDR1 is shown in SEQ ID NO.60, CDR2 is shown in SEQ ID NO.90, and CDR3 is shown in SEQ ID NO.120.

[0011] Preferably, the E-cadherin-17 single-domain antibody has the following framework regions: FR1, FR2, FR3, and FR4; wherein the amino acid sequence of FR1 is shown in any one of SEQ ID NOs.121-150; the amino acid sequence of FR2 is shown in any one of SEQ ID NOs.151-180; the amino acid sequence of FR3 is shown in any one of SEQ ID NOs.181-210; and the amino acid sequence of FR4 is shown in any one of SEQ ID NOs.211-240.

[0012] Preferably, the framework region of the single domain antibody is as shown in any one of the following (31)-(60): (31) FR1 is shown in SEQ ID NO.121, FR2 is shown in SEQ ID NO.151, FR3 is shown in SEQ ID NO.181; FR4 is shown in SEQ ID NO.211; (32) FR1 is shown in SEQ ID NO.122, FR2 is shown in SEQ ID NO.152, FR3 is shown in SEQ ID NO.182; FR4 is shown in SEQ ID NO.212; (33) FR1 is shown in SEQ ID NO.123, FR2 is shown in SEQ ID NO.153, FR3 is shown in SEQ ID NO.183; FR4 is shown in SEQ ID NO.213; (34) FR1 is shown in SEQ ID NO.124, FR2 is shown in SEQ ID NO.154, FR3 is shown in SEQ ID NO.184; FR4 is shown in SEQ ID NO.214; (35) FR1 is shown in SEQ ID NO.125, FR2 is shown in SEQ ID NO.155, FR3 is shown in SEQ ID NO.185; FR4 is shown in SEQ ID NO.215; (36) FR1 is shown in SEQ ID NO.126, FR2 is shown in SEQ ID NO.156, FR3 is shown in SEQ ID NO.186; FR4 is shown in SEQ ID NO.216; (37) FR1 is shown in SEQ ID NO.127, FR2 is shown in SEQ ID NO.157, FR3 is shown in SEQ ID NO.187; FR4 is shown in SEQ ID NO.217; (38) FR1 is shown in SEQ ID NO.128, FR2 is shown in SEQ ID NO.158, FR3 is shown in SEQ ID NO.188; FR4 is shown in SEQ ID NO.218; (39) FR1 is shown in SEQ ID NO.129, FR2 is shown in SEQ ID NO.159, FR3 is shown in SEQ ID NO.189; FR4 is shown in SEQ ID NO.219; (40) FR1 is shown in SEQ ID NO.130, FR2 is shown in SEQ ID NO.160, FR3 is shown in SEQ ID NO.190; FR4 is shown in SEQ ID NO.220; (41) FR1 is shown in SEQ ID NO.131, FR2 is shown in SEQ ID NO.161, FR3 is shown in SEQ ID NO.191; FR4 is shown in SEQ ID NO.221; (42) FR1 is shown in SEQ ID NO.132, FR2 is shown in SEQ ID NO.162, FR3 is shown in SEQ ID NO.192; FR4 is shown in SEQ ID NO.222; (43) FR1 is shown in SEQ ID NO.133, FR2 is shown in SEQ ID NO.163, FR3 is shown in SEQ ID NO.193; FR4 is shown in SEQ ID NO.223; (44) FR1 is shown in SEQ ID NO.134, FR2 is shown in SEQ ID NO.164, FR3 is shown in SEQ ID NO.194; FR4 is shown in SEQ ID NO.224; (45) FR1 is shown in SEQ ID NO.135, FR2 is shown in SEQ ID NO.165, FR3 is shown in SEQ ID NO.195; FR4 is shown in SEQ ID NO.225; (46) FR1 is shown in SEQ ID NO.136, FR2 is shown in SEQ ID NO.166, FR3 is shown in SEQ ID NO.196; FR4 is shown in SEQ ID NO.226; (47) FR1 is shown in SEQ ID NO.137, FR2 is shown in SEQ ID NO.167, FR3 is shown in SEQ ID NO.197; FR4 is shown in SEQ ID NO.227; (48) FR1 is shown in SEQ ID NO.138, FR2 is shown in SEQ ID NO.168, FR3 is shown in SEQ ID NO.198; FR4 is shown in SEQ ID NO.228; (49) FR1 is shown in SEQ ID NO.139, FR2 is shown in SEQ ID NO.169, FR3 is shown in SEQ ID NO.199; FR4 is shown in SEQ ID NO.229; (50) FR1 is shown in SEQ ID NO.140, FR2 is shown in SEQ ID NO.170, FR3 is shown in SEQ ID NO.200; FR4 is shown in SEQ ID NO.230; (51) FR1 is shown in SEQ ID NO.141, FR2 is shown in SEQ ID NO.171, FR3 is shown in SEQ ID NO.201; FR4 is shown in SEQ ID NO.231; (52) FR1 is shown in SEQ ID NO.142, FR2 is shown in SEQ ID NO.172, FR3 is shown in SEQ ID NO.202; FR4 is shown in SEQ ID NO.232; (53) FR1 is shown in SEQ ID NO.143, FR2 is shown in SEQ ID NO.173, FR3 is shown in SEQ ID NO.203; FR4 is shown in SEQ ID NO.233; (54) FR1 is shown in SEQ ID NO.144, FR2 is shown in SEQ ID NO.174, FR3 is shown in SEQ ID NO.204; FR4 is shown in SEQ ID NO.234; (55) FR1 is shown in SEQ ID NO.145, FR2 is shown in SEQ ID NO.175, FR3 is shown in SEQ ID NO.205; FR4 is shown in SEQ ID NO.235; (56) FR1 is shown in SEQ ID NO.146, FR2 is shown in SEQ ID NO.176, FR3 is shown in SEQ ID NO.206; FR4 is shown in SEQ ID NO.236; (57) FR1 is shown in SEQ ID NO.147, FR2 is shown in SEQ ID NO.177, FR3 is shown in SEQ ID NO.207; FR4 is shown in SEQ ID NO.237; (58) FR1 is shown in SEQ ID NO.148, FR2 is shown in SEQ ID NO.178, FR3 is shown in SEQ ID NO.208; FR4 is shown in SEQ ID NO.238; (59) FR1 is shown in SEQ ID NO.149, FR2 is shown in SEQ ID NO.179, FR3 is shown in SEQ ID NO.209; FR4 is shown in SEQ ID NO.239; (60) FR1 is shown in SEQ ID NO.150, FR2 is shown in SEQ ID NO.180, FR3 is shown in SEQ ID NO.210; FR4 is shown in SEQ ID NO.240.

[0013] Preferably, the amino acid sequence of the single-domain antibody is as shown in any one of SEQ ID NOs. 1-30.

[0014] Preferably, the framework region is a heavy chain framework region, and at least a portion of the heavy chain framework region is independently derived from an alpaca-derived antibody.

[0015] Preferably, the Cadherin-17 single domain antibody is prepared by immunizing camels with Cadherin-17 recombinant protein antigen, collecting peripheral blood cells of the immunized camels, isolating Cadherin-17 affinity lymphocytes therefrom, extracting total RNA and reverse transcribing it into cDNA, cloning the V region of the camel heavy chain antibody using Nest-PCR technology, inserting it into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the CDH17 antigen by phage display technology, and verifying the binding ability of the obtained single domain antibody by enzyme-linked immunosorbent assay, thereby screening out Cadherin-17 single domain antibodies with high affinity activity.

[0016] Preferably, the DNA sequence of the E-cadherin-17 single domain antibody is as shown in any one of SEQ ID NOs. 241-270.

[0017] Another aspect of the present application provides a fusion protein comprising the E-cadherin-17 single domain antibody as described above.

[0018] Furthermore, the present application provides a product, which contains the Cadherin-17 single-domain antibody as described above or the fusion protein as described above, wherein the product is an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs to sites expressing Cadherin-17.

[0019] This application uses the recombinant protein antigen of Cadherin-17 (CDH17) to immunize camels and successfully screens out CDH17 single-domain antibodies with high affinity activity. The heavy chain variable region of the single-domain antibody has three CDR regions. Among them, CDR1 and CDR3 are slightly longer than those of humans, and CDR3 protrudes outward in the tertiary structure. Therefore, single-domain antibodies have higher antigen binding specificity and affinity than traditional antibodies. In addition, the CDH17 single-domain antibodies and their applications developed by the phage display technology of this application have a short research and development cycle, high antibody quality, and outstanding advantages in antibody affinity and targeting specificity, small molecule size, stability and modifiability, and production cost.

[0020] In addition, the present invention provides a scheme for preparing the above-mentioned CDH17 single-domain antibody supernatant, expresses the CDH17 single-domain antibody in small quantities, and effectively reduces the development and production costs of the CDH17 antibody. The single-domain antibody is verified by the ELISA system to have high specificity and high affinity for targeting CDH17, indicating that the CDH17 single-domain antibody obtained by the present invention has further development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the titer test results of camel immune serum of E-cadherin-17 single domain antibody.

[0022] Figure 2 This is a schematic diagram of the results of detecting PCR products using agarose gel electrophoresis.

[0023] Figure 3 This is an ELISA test result diagram for evaluating the enrichment degree of specific antibodies.

[0024] Figure 4 This is a diagram showing the screening results of positive clones of single-domain antibodies specific to CDH17.

[0025] Figure 5 Unique sequence analysis results of positive clones are shown.

[0026] Figure 6 The results of analyzing the binding ability of CDH17 single domain antibodies by ELISA are shown. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0028] The embodiment of the present invention provides a single-domain antibody of E-cadherin-17 developed based on phage display technology, characterized in that the single-domain antibody has the following complementary regions of determinants: CDR1, CDR2 and CDR3; wherein the amino acid sequence of CDR1 is shown in any one of SEQ ID NO.31-60, the amino acid sequence of CDR2 is shown in any one of SEQ ID NO.61-90, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NO.91-120. Preferably, the complementarity determining regions of the E-cadherin-17 single-domain antibody, CDR1, CDR2, CDR3, each group matches as shown in the sequence table. Preferably, the Cadherin-17 single domain antibody has the following heavy chain framework regions, namely FR1, FR2, and FR3; wherein the amino acid sequence of FR1 is shown in any one of SEQ ID NO.121-150; the amino acid sequence of FR2 is shown in any one of SEQ ID NO.151-180; the amino acid sequence of FR3 is shown in any one of SEQ ID NO.181-210; the amino acid sequence of FR4 is shown in any one of SEQ ID NO.211-240. Preferably, the amino acid sequence of the single domain antibody is shown in any one of SEQ ID NO.1-30. Preferably, the framework region is a heavy chain framework region, and at least a part of the heavy chain framework region is independently derived from an alpaca-derived antibody. Preferably, the DNA sequence of the Cadherin-17 single domain antibody is shown in any one of SEQ ID NO.241-270.

[0029] Preferably, the Cadherin-17 single domain antibody is prepared by immunizing camels with Cadherin-17 recombinant protein antigen, collecting peripheral blood cells of the immunized camels, isolating Cadherin-17 affinity lymphocytes therefrom, extracting total RNA and reverse transcribing it into cDNA, cloning the V region of the camel heavy chain antibody using Nest-PCR technology, inserting it into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the CDH17 antigen by phage display technology, and verifying the binding ability of the obtained single domain antibody by enzyme-linked immunosorbent assay, thereby screening out Cadherin-17 single domain antibodies with high affinity activity.

[0030] Therefore, the screening of the E-cadherin-17 single domain antibody of the present application is divided into the following stages: ① camel immunization and serum titer determination; ② camel single domain antibody phage display library construction; ③ camel single domain antibody phage display library amplification and rescue; ④ selection of single domain antibodies specifically binding to CDH17 using phage display technology; ⑤ identification of CDH17-specific single domain antibody positive clones; ⑥ positive clone sequence analysis; ⑦ verification of unique clones that specifically bind to CDH17.

[0031] The above-mentioned stages are illustrated below by means of specific embodiments.

[0032] ① Camel immunization and serum titer determination (1) CDH17 immunization of camels 1 mg of CDH17 was mixed with an equal volume of Freund's adjuvant and injected at 3-5 points under the skin of the camel's neck. Blood was collected from the ear vein of the camel before immunization. Immunization was performed once every two weeks, for a total of 5 times; 5 mL of peripheral blood was collected from the camel each time. When collecting blood, the camel's head was fixed to one side, the skin of the animal's blood collection site was shaved first, and 75% alcohol was disinfected. After drying, blood was collected. The jugular vein groove was pressed with a finger. After the blood vessel was dilated, the needle was disinfected at the blood collection site to collect blood. 5 mL of blood was collected and left to stand for the preparation of serum for titer evaluation. 7 days after the last immunization, 50 mL of blood was collected in an EDTA anticoagulant tube, and it was immediately shaken continuously and slowly, mixed thoroughly, placed on ice, and transported back to the laboratory.

[0033] (2) Serum titer detection 100 ng CDH17 antigen was coated on a 96-well high-adsorption ELISA plate at 4 °C overnight. The coated antigen was washed 3 times with PBST, and then blocked with 200 uL 2% BSA at room temperature for 2 hours. The serum collected before and after each immunization was diluted to different concentration gradients, and the blocking solution was discarded. The ELISA plate was washed 3-5 times with PBST, and 100 uL serum samples with different dilution gradients were added to each well, incubated at room temperature for 2 hours, and then the serum was discarded, and washed 5 times with PBST. Camelid IgG-HRP was diluted with 1% BSA, and 100 uL was added to each well. Incubated at room temperature for 1.5 hours in the dark, and then the plate was washed 5 times. 100 uL TMB colorimetric solution was added to each well, incubated at room temperature for 10-30 min, and OD450 readings were taken after adding the stop solution. The results of serum titer detection are attached. Figure 1 .

[0034] (3) Separation of blood lymphocyte samples Lymphocytes were isolated from blood samples collected after the last immunization using the following method: i. Add 7 mL of Ficoll, a lymphocyte separation medium, to each 15 mL centrifuge tube; ii. Add an equal volume of PBS (1×) or saline to the fresh whole blood to which the anticoagulant (EDTA) has been added and mix thoroughly; iii. Take the 15 mL centrifuge tube with lymphocyte separation solution and slowly and carefully transfer it to another 15 mL centrifuge tube with lymphocyte separation solution, and make the mixed solution above the surface of lymphocyte separation solution (i.e., the two liquids should not be mixed and a clear interface should be retained), and centrifuge at 3,000 g for 20 min; iv. Use a 1 mL pipette to carefully transfer the supernatant to a 1.5 mL cell cryopreservation tube, write the animal number and plasma, put it in a small cloth bag with a rope, and store it in a liquid nitrogen tank. Use a 1 mL pipette to carefully separate the white blood cell layer into a 15 mL centrifuge tube; fill it up to 15 mL with PBS (1×); wash the white blood cells with PBS (1×), centrifuge (3,000 g, centrifuge for 20 min), carefully pour off the supernatant, do not stir the cell clumps at the bottom of the tube, and recover the white blood cells in the remaining 0.1 - 0.2 mL PBS.

[0035] vi. Add 5 times the volume of RNA later, gently dissolve the cell clumps, divide into 2 portions into 1.5 mL cell cryopreservation tubes, and store in liquid nitrogen tank.

[0036] ② Construction of camel single domain antibody phage display library (1) Total RNA extraction Take a portion of frozen lymphocytes, add 1 mL Trizol, let it stand at room temperature for 10 min, then add 0.2 mL chloroform, shake vigorously, let it stand at room temperature, wait for the solution to separate (about 10 min), centrifuge at 12,000 rpm, collect the upper aqueous phase, add an equal volume of isopropanol, mix, let it stand at room temperature for 15 min, wait for the nucleic acid to precipitate, centrifuge at high speed to remove the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) to wash the RNA precipitate, centrifuge at high speed to remove the supernatant, control the water, dissolve the RNA in nuclease-free water, and take 1 uL for concentration and purity determination.

[0037] (2) cDNA synthesis 1 ug RNA was taken and cDNA was synthesized using the cDNA first-strand synthesis kit (Super Script TMIII First-StrandSynthesis SuperMix (Invitrogen)). Oligo dT was used as the reverse transcription primer and the synthesized cDNA was frozen at -20°C; (3) Phage display library construction and PCR amplification The above synthesized cDNA was used as a template to amplify the V region (VHH) of the camel heavy chain antibody using Nest-PCR. The following table lists the names and sequences of the Nest-PCR primers:

[0038] a. First round PCR reaction system: cDNA 1 uL; Mix 12.5 uL; CALL001 0.5 uL; CALL002 0.5 uL; water to 25 uL. First round PCR reaction conditions: 95 ℃ 5 min; 94 ℃ 1 min, 57 ℃ 1 min, 72 ℃ 1 min, 35 cycles; 72 ℃ 5 min. b. Second round PCR reaction system: 40 ng of the first round PCR product; 25 uL of Mix; 1 uL of VHH-Back; 1 uL of VHH-For; water to 50 uL. Second round PCR reaction conditions: 95 ℃ 5min; 94 ℃ 45 s, 60 ℃ 45 s, 72 ℃ 45 s, 15 cycles; 72 ℃ 5 min. c. After the PCR reaction, the PCR product was detected by 1.5% agarose gel electrophoresis. The target gene fragment of the first round of PCR was at 700 bp. The gel was cut and the target band was recovered by the QIAEX II Gel Extraction kit. The second round of PCR was performed. The target gene fragment was at 500 bp. The target band was recovered by cutting the gel, i.e., the VHH fragment. The electrophoresis results are attached. Figure 2 .

[0039] (4) Phagemid vector digestion and target fragment ligation The VHH fragment and pMES4 vector were double-digested with restriction endonucleases Eco91I and PstI, respectively. The reaction system was as follows: a. Vector enzyme digestion system: pMES4 vector 20 ug; PstI 10 uL; Eco91I 20 uL; Cutsmartbuffer 50 uL; add H2O to 500 uL. b. Fragment digestion system: VHH fragment 5 ug; PstI 7 uL; Eco91I 14 uL; Cutsmart buffer 50 uL; add H2O to 500 uL. Digest overnight at 37 ℃, and after agarose gel electrophoresis, cut the gel for recovery; mix the digestion products of the vector and VHH fragment, and connect them with T4 DNA Ligase at 16 ℃ overnight.

[0040] ③ Amplification and rescue of camel single domain antibody phage display library (1) Construction of phage display library After the ligation product was purified by PCR Purification Kit, 1 uL was taken to transform E. coli TG1 competent cells, revived at 37 ℃ for 2 h, gradiently diluted to 101, 102, and 103, and 300 uL was taken to coat the plate, cultured at 37 ℃ overnight, and the number of clones was calculated, about 105 clones / plate. The same transformation method was used for large-scale transformation until the number of clones in the library reached more than 108. All clones were eluted with LB, centrifuged at 5,000g for 5 min, and the precipitate was suspended with 2 mL LB, an equal volume of 30% glycerol was added, and frozen at -80 ℃.

[0041] (2) Library diversity detection 60 clones of (1) were randomly selected for sequencing to detect the recombination rate and evaluate the quality of the library. The recombination rate of the constructed CDH17 single domain antibody library was 95.7%. The diversity of the CDH17 single domain antibody library was analyzed. The sequencing results showed that 45 monoclones had 45 kinds of amino acid sequences, indicating that the constructed library had good diversity.

[0042] (3) Phage amplification and rescue The phage library of CDH17 single domain antibody was amplified and rescued using helper phage. The preserved monoclonal library was inoculated into 100 mL culture medium and cultured to the logarithmic growth phase, and helper phage with an MOI of 20 was added. The precipitate was suspended with culture medium at room temperature for 30 minutes, centrifuged at low speed, and inoculated into 300 mL culture medium for overnight culture. The next day, centrifuged at 3,000g for 30 minutes, the supernatant was collected, PEG was added to precipitate phage, the phage was inoculated on ice for 30 minutes, and centrifuged at 3,000g for 30 minutes to precipitate the CDH17 single domain antibody phage library. After suspending the precipitate with PBS, the titer was determined to be 2.05 x1013 pfu / mL.

[0043] ④ Use phage display technology to select single-domain antibodies that specifically bind to CDH17 (1) Affinity CDH17 single domain antibody phage library panning A total of 4 rounds of panning were performed, and the CDH17 antigen was coated on the ELISA plate and incubated overnight at 4°C. The next day, the rescued CDH17 single-domain antibody phage was added and incubated at room temperature for 2 h; the wells were washed 10 times with PBST, 100 uL of triethylamine was added, and incubated at room temperature for 30 min. The collected phages were the CDH17 single-domain antibody phage library obtained by affinity panning; 10 uL of infected TG1 cells was coated on the plate for determining the number of clones after screening, and the remaining screened phages were used for amplification.

[0044] (2) Amplification and rescue of phage after screening The amplification and rescue methods are the same as above. The obtained PBS suspension, i.e. the phage after the first round of screening, is stored at 4°C and used for the next round of screening. According to the same screening steps as above, the amount of antigen is gradually reduced for 3-4 rounds of screening.

[0045] (3) ELISA to evaluate the enrichment of specific antibodies ELISA plates were coated with 100 ng of CDH17 antigen at 4°C overnight; the next day, 2% BSA was added for blocking at room temperature for 1 h; the experimental group added the amplified phages after each round of panning, and the control group added an equal amount of wild-type phages, incubated at room temperature for 2 h; washed 10 times with PBST to remove unbound phages; HRP-labeled anti-M13 antibody was added and incubated at room temperature for 1 h; color development solution was added, reacted in the dark for 60 min, and the absorbance was measured. The absorbance gradually increased with the number of panning times, indicating that specific antibodies were enriched. ELISA results are attached. Figure 3 .

[0046] ⑤ Identification of CDH17-specific single domain antibody positive clones The ELISA plate of the experimental group was coated with 100 ng of CDH17 antigen, and the ELISA plate of the control group was coated with 100 uL PBS and incubated at 4 ℃ overnight; the plates coated with phages obtained in the third and last rounds of screening were taken, 77 monoclones were randomly selected in 1 mL of culture medium, cultured at 37 ℃ to the logarithmic phase, and 1 mM IPTG was added for induction overnight; the next day, the bacterial pellet was collected by centrifugation, and after breaking, it was centrifuged at 5,000 g for 15 min, and the supernatant was collected; at the same time, the ELISA plate was taken, and 2% BSA was added to block at room temperature for 2 h; the monoclonal supernatant was added to each well of the experimental group and the control group, and incubated at room temperature for 2 h; PBST was washed 5 times, and VHH antibody was added, and the reaction was carried out at room temperature for 1 h; PBST was washed 3-5 times; TMB substrate was added, reacted for 10 min, and the absorbance value was read on the microplate reader; when the absorbance value was greater than 2 compared with the control well, it was determined to be a positive clone; the ELISA verification results showed that 36 positive clones were obtained. The screening results of positive clones are attached. Figure 4 .

[0047] ⑥ Sequence analysis of positive clones The 36 positive clones were sequenced, and the sequencing results showed that 30 nucleotide sequences were obtained. The amino acid sequences were analyzed, and all 30 sequences had a typical single-domain antibody structure, which consisted of a framework region (FR1, FR2, FR3 and FR4) and a complementary determining region (CDR1, CDR2 and CDR3). The unique sequence analysis of the positive clones is shown in the attached figure. Figure 5 .

[0048] ⑦ Verify the unique clone specific to CDH17 The experimental group ELISA plate was coated with 100 ng CDH17 antigen, and the control group ELISA plate was coated with 100 uL PBS and incubated at 4 ℃ overnight; the phage-coated plates obtained in the third and last rounds of screening were taken, 30 monoclones were randomly selected in 1 mL culture medium, cultured at 37 ℃ to the logarithmic phase, and M13 was added for infection at room temperature for 30 min, and then centrifuged to replace fresh culture medium; the next day, the supernatant was collected by centrifugation; at the same time, the ELISA plate was taken, and 2% BSA was added for blocking at room temperature for 2 h; the supernatant was added to each well of the experimental group and the control group, incubated at room temperature for 2 h; washed 5 times with PBST, and M13 antibody was added for 1 h at room temperature; washed 3-5 times with PBST; TMB substrate was added, reacted for 10 min, and the absorbance value was read on the microplate reader; when the absorbance value was greater than 2 compared with the control well, it was determined to be a positive clone; the ELISA verification results showed that the secondary verification results of the 30 unique clones were all positive. This shows that all 30 positive sequences can specifically bind to the CDH17 antigen. The verification results of Unique clone are attached. Figure 6 .

[0049] The present invention successfully screened a single-domain antibody sequence that specifically binds to CDH17 through phage display technology, and analyzed the binding force of the CDH17 single-domain antibody through the ELISA method.

[0050] Example 1: Detection of CDH17-specific antibody enrichment by polyclonal phage ELISA The enrichment of CDH17 antibodies in the original library and the library rescued after the 1st, 2nd, 3rd, and 4th rounds of enrichment was detected by polyclonal phage ELISA. The experimental group was coated with 100 ng of CDH17 on the ELISA plate, and the blank group was coated with PBS on the ELISA plate without antigen, and incubated at 4°C overnight; the next day, 2% BSA was added to block at room temperature for 1 h; the phage supernatant collected from the library rescued in each round of CDH17 selection and the original library was taken as the primary antibody for incubation, and the antibody was added to the control group and the experimental group respectively, and incubated at room temperature for 2 h; PBST was washed 10 times, and the secondary antibody was added at room temperature for 1 h; TMB substrate was added, and the reaction was 10-20 min, and the OD 450 absorbance was read on the microplate reader. The results showed that after 4 rounds of selection, the antibody that specifically binds to CDH17 was effectively enriched (see Appendix Figure 1 ).

[0051] Example 2: ELISA analysis of the binding capacity of CDH17 single domain antibody The experimental group was coated with 100 ng of CDH17 protein, and the blank group was coated with PBS, and incubated at 4 ℃ overnight; the next day, 2% BSA was added for blocking at room temperature for 1 h; the M13 monoclonal supernatant of CDH17 was taken as the primary antibody for incubation, and the antibody was added to the control group and the experimental group respectively, and incubated at room temperature for 2 h; PBST was washed 10 times, and the secondary antibody was added for 1 h at room temperature; the substrate was added, and the reaction was 10-20 minutes, and the absorbance value was read on the microplate reader. The ELISA test results showed that the CDH17 single domain antibody had a good specific binding to the CDH17 antigen, and the signal value of the experimental group was much higher than that of the blank group (see Appendix). Figure 3 ).

[0052] Another aspect of the present application provides a fusion protein comprising the E-cadherin-17 single domain antibody as described above.

[0053] Furthermore, the present application provides a product, which contains the Cadherin-17 single-domain antibody as described above or the fusion protein as described above, wherein the product is an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs to sites expressing Cadherin-17.

[0054] This application uses the recombinant protein antigen of Cadherin-17 (CDH17) to immunize camels and successfully screens out CDH17 single-domain antibodies with high affinity activity. The heavy chain variable region of the single-domain antibody has three CDR regions. Among them, CDR1 and CDR3 are slightly longer than those of humans, and CDR3 protrudes outward in the tertiary structure. Therefore, single-domain antibodies have higher antigen binding specificity and affinity than traditional antibodies. In addition, this application provides a CDH17 single-domain antibody developed based on phage display technology and its application, which has a short R&D cycle, high antibody quality, and outstanding advantages in antibody affinity and targeting specificity, small molecule size, stability and modifiability, and production cost.

[0055] It should be noted that the present invention is not limited to the above-mentioned embodiments. Based on the creative spirit of the present invention, those skilled in the art may also make other changes. These changes made based on the creative spirit of the present invention should be included in the scope of protection required by the present invention.

Claims

1. A single-domain antibody of E-cadherin-17 developed based on phage display technology, characterized in that: The single domain antibody has the following complementary region determining clusters: CDR1, CDR2 and CDR3; Among them, the amino acid sequence of CDR1 is shown in any one of SEQ ID NOs.31-60, the amino acid sequence of CDR2 is shown in any one of SEQ ID NOs.61-90, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs.91-120.

2. The E-cadherin-17 single domain antibody according to claim 1, characterized in that The complementary determining region of the Cadherin-17 single domain antibody is as shown in any one of the following (1) to (30): (1) CDR1 is shown in SEQ ID NO.31, CDR2 is shown in SEQ ID NO.61, and CDR3 is shown in SEQ ID NO.91; (2) CDR1 is shown in SEQ ID NO.32, CDR2 is shown in SEQ ID NO.62, and CDR3 is shown in SEQ ID NO.92; (3) CDR1 is shown in SEQ ID NO.33, CDR2 is shown in SEQ ID NO.63, and CDR3 is shown in SEQ ID NO.93; (4) CDR1 is shown in SEQ ID NO.34, CDR2 is shown in SEQ ID NO.64, and CDR3 is shown in SEQ ID NO.94; (5) CDR1 is shown in SEQ ID NO.35, CDR2 is shown in SEQ ID NO.65, and CDR3 is shown in SEQ ID NO.95; (6) CDR1 is shown in SEQ ID NO.36, CDR2 is shown in SEQ ID NO.66, and CDR3 is shown in SEQ ID NO.96; (7) CDR1 is shown in SEQ ID NO.37, CDR2 is shown in SEQ ID NO.67, and CDR3 is shown in SEQ ID NO.97; (8) CDR1 is shown in SEQ ID NO.38, CDR2 is shown in SEQ ID NO.68, and CDR3 is shown in SEQ ID NO.98; (9) CDR1 is shown in SEQ ID NO.39, CDR2 is shown in SEQ ID NO.69, and CDR3 is shown in SEQ ID NO.99; (10) CDR1 is shown in SEQ ID NO.40, CDR2 is shown in SEQ ID NO.70, and CDR3 is shown in SEQ ID NO.100; (11) CDR1 is shown in SEQ ID NO.41, CDR2 is shown in SEQ ID NO.71, and CDR3 is shown in SEQ ID NO.101; (12) CDR1 is shown in SEQ ID NO.42, CDR2 is shown in SEQ ID NO.72, and CDR3 is shown in SEQ ID NO.102; (13) CDR1 is shown in SEQ ID NO.43, CDR2 is shown in SEQ ID NO.73, and CDR3 is shown in SEQ ID NO.103; (14) CDR1 is shown in SEQ ID NO.44, CDR2 is shown in SEQ ID NO.74, and CDR3 is shown in SEQ ID NO.104; (15) CDR1 is shown in SEQ ID NO.45, CDR2 is shown in SEQ ID NO.75, and CDR3 is shown in SEQ ID NO.105; (16) CDR1 is shown in SEQ ID NO.46, CDR2 is shown in SEQ ID NO.76, and CDR3 is shown in SEQ ID NO.106; (17) CDR1 is shown in SEQ ID NO.47, CDR2 is shown in SEQ ID NO.77, and CDR3 is shown in SEQ ID NO.107; (18) CDR1 is shown in SEQ ID NO.48, CDR2 is shown in SEQ ID NO.78, and CDR3 is shown in SEQ ID NO.108; (19) CDR1 is shown in SEQ ID NO.49, CDR2 is shown in SEQ ID NO.79, and CDR3 is shown in SEQ ID NO.109; (20) CDR1 is shown in SEQ ID NO.50, CDR2 is shown in SEQ ID NO.80, and CDR3 is shown in SEQ ID NO.110; (21) CDR1 is shown in SEQ ID NO.51, CDR2 is shown in SEQ ID NO.81, and CDR3 is shown in SEQ ID NO.111; (22) CDR1 is shown in SEQ ID NO.52, CDR2 is shown in SEQ ID NO.82, and CDR3 is shown in SEQ ID NO.112; (23) CDR1 is shown in SEQ ID NO.53, CDR2 is shown in SEQ ID NO.83, and CDR3 is shown in SEQ ID NO.113; (24) CDR1 is shown in SEQ ID NO.54, CDR2 is shown in SEQ ID NO.84, and CDR3 is shown in SEQ ID NO.114; (25) CDR1 is shown in SEQ ID NO.55, CDR2 is shown in SEQ ID NO.85, and CDR3 is shown in SEQ ID NO.115; (26) CDR1 is shown in SEQ ID NO.56, CDR2 is shown in SEQ ID NO.86, and CDR3 is shown in SEQ ID NO.116; (27) CDR1 is shown in SEQ ID NO.57, CDR2 is shown in SEQ ID NO.87, and CDR3 is shown in SEQ ID NO.117; (28) CDR1 is shown in SEQ ID NO.58, CDR2 is shown in SEQ ID NO.88, and CDR3 is shown in SEQ ID NO.118; (29) CDR1 is shown in SEQ ID NO.59, CDR2 is shown in SEQ ID NO.89, and CDR3 is shown in SEQ ID NO.119; (30) CDR1 is shown in SEQ ID NO.60, CDR2 is shown in SEQ ID NO.90, and CDR3 is shown in SEQ ID NO.

120.

3. The E-cadherin-17 single domain antibody according to claim 1, characterized in that: The E-cadherin-17 single domain antibody has the following framework regions: FR1, FR2, FR3, FR4; in, The amino acid sequence of FR1 is shown in any one of SEQ ID NOs. 121-150; The amino acid sequence of FR2 is shown in any one of SEQ ID NOs. 151-180; The amino acid sequence of FR3 is shown in any one of SEQ ID NOs. 181-210; The amino acid sequence of FR4 is shown in any one of SEQ ID NOs. 211-240.

4. The Cadherin-17 single domain antibody according to claim 3, characterized in that The framework region of the single domain antibody is as shown in any one of the following (31)-(60): (31) FR1 is shown in SEQ ID NO.121, FR2 is shown in SEQ ID NO.151, FR3 is shown in SEQ ID NO.181; FR4 is shown in SEQ ID NO.211; (32) FR1 is shown in SEQ ID NO.122, FR2 is shown in SEQ ID NO.152, FR3 is shown in SEQ ID NO.182; FR4 is shown in SEQ ID NO.212; (33) FR1 is shown in SEQ ID NO.123, FR2 is shown in SEQ ID NO.153, FR3 is shown in SEQ ID NO.183; FR4 is shown in SEQ ID NO.213; (34) FR1 is shown in SEQ ID NO.124, FR2 is shown in SEQ ID NO.154, FR3 is shown in SEQ ID NO.184; FR4 is shown in SEQ ID NO.214; (35) FR1 is shown in SEQ ID NO.125, FR2 is shown in SEQ ID NO.155, FR3 is shown in SEQ ID NO.185; FR4 is shown in SEQ ID NO.215; (36) FR1 is shown in SEQ ID NO.126, FR2 is shown in SEQ ID NO.156, FR3 is shown in SEQ ID NO.186; FR4 is shown in SEQ ID NO.216; (37) FR1 is shown in SEQ ID NO.127, FR2 is shown in SEQ ID NO.157, FR3 is shown in SEQ ID NO.187; FR4 is shown in SEQ ID NO.217; (38) FR1 is shown in SEQ ID NO.128, FR2 is shown in SEQ ID NO.158, FR3 is shown in SEQ ID NO.188; FR4 is shown in SEQ ID NO.218; (39) FR1 is shown in SEQ ID NO.129, FR2 is shown in SEQ ID NO.159, FR3 is shown in SEQ ID NO.189; FR4 is shown in SEQ ID NO.219; (40) FR1 is shown in SEQ ID NO.130, FR2 is shown in SEQ ID NO.160, FR3 is shown in SEQ ID NO.190; FR4 is shown in SEQ ID NO.220; (41) FR1 is shown in SEQ ID NO.131, FR2 is shown in SEQ ID NO.161, FR3 is shown in SEQ ID NO.191; FR4 is shown in SEQ ID NO.221; (42) FR1 is shown in SEQ ID NO.132, FR2 is shown in SEQ ID NO.162, FR3 is shown in SEQ ID NO.192; FR4 is shown in SEQ ID NO.222; (43) FR1 is shown in SEQ ID NO.133, FR2 is shown in SEQ ID NO.163, FR3 is shown in SEQ ID NO.193; FR4 is shown in SEQ ID NO.223; (44) FR1 is shown in SEQ ID NO.134, FR2 is shown in SEQ ID NO.164, FR3 is shown in SEQ ID NO.194; FR4 is shown in SEQ ID NO.224; (45) FR1 is shown in SEQ ID NO.135, FR2 is shown in SEQ ID NO.165, FR3 is shown in SEQ ID NO.195; FR4 is shown in SEQ ID NO.225; (46) FR1 is shown in SEQ ID NO.136, FR2 is shown in SEQ ID NO.166, FR3 is shown in SEQ ID NO.196; FR4 is shown in SEQ ID NO.226; (47) FR1 is shown in SEQ ID NO.137, FR2 is shown in SEQ ID NO.167, FR3 is shown in SEQ ID NO.197; FR4 is shown in SEQ ID NO.227; (48) FR1 is shown in SEQ ID NO.138, FR2 is shown in SEQ ID NO.168, FR3 is shown in SEQ ID NO.198; FR4 is shown in SEQ ID NO.228; (49) FR1 is shown in SEQ ID NO.139, FR2 is shown in SEQ ID NO.169, FR3 is shown in SEQ ID NO.199; FR4 is shown in SEQ ID NO.229; (50) FR1 is shown in SEQ ID NO.140, FR2 is shown in SEQ ID NO.170, FR3 is shown in SEQ ID NO.200; FR4 is shown in SEQ ID NO.230; (51) FR1 is shown in SEQ ID NO.141, FR2 is shown in SEQ ID NO.171, FR3 is shown in SEQ ID NO.201; FR4 is shown in SEQ ID NO.231; (52) FR1 is shown in SEQ ID NO.142, FR2 is shown in SEQ ID NO.172, FR3 is shown in SEQ ID NO.202; FR4 is shown in SEQ ID NO.232; (53) FR1 is shown in SEQ ID NO.143, FR2 is shown in SEQ ID NO.173, FR3 is shown in SEQ ID NO.203; FR4 is shown in SEQ ID NO.233; (54) FR1 is shown in SEQ ID NO.144, FR2 is shown in SEQ ID NO.174, FR3 is shown in SEQ ID NO.204; FR4 is shown in SEQ ID NO.234; (55) FR1 is shown in SEQ ID NO.145, FR2 is shown in SEQ ID NO.175, FR3 is shown in SEQ ID NO.205; FR4 is shown in SEQ ID NO.235; (56) FR1 is shown in SEQ ID NO.146, FR2 is shown in SEQ ID NO.176, FR3 is shown in SEQ ID NO.206; FR4 is shown in SEQ ID NO.236; (57) FR1 is shown in SEQ ID NO.147, FR2 is shown in SEQ ID NO.177, FR3 is shown in SEQ ID NO.207; FR4 is shown in SEQ ID NO.237; (58) FR1 is shown in SEQ ID NO.148, FR2 is shown in SEQ ID NO.178, FR3 is shown in SEQ ID NO.208; FR4 is shown in SEQ ID NO.238; (59) FR1 is shown in SEQ ID NO.149, FR2 is shown in SEQ ID NO.179, FR3 is shown in SEQ ID NO.209; FR4 is shown in SEQ ID NO.239; (60) FR1 is shown in SEQ ID NO.150, FR2 is shown in SEQ ID NO.180, FR3 is shown in SEQ ID NO.210; FR4 is shown in SEQ ID NO.

240.

5. The E-cadherin-17 single domain antibody according to claim 1, characterized in that The amino acid sequence of the single domain antibody is shown in any one of SEQ ID NOs. 1-30.

6. The Cadherin-17 single domain antibody according to claim 4, characterized in that The framework region is a heavy chain framework region, and at least a portion of the heavy chain framework region is independently derived from an alpaca-derived antibody.

7. The E-cadherin-17 single domain antibody according to claim 1, characterized in that: The Cadherin-17 single domain antibody is prepared by immunizing camels with Cadherin-17 recombinant protein antigen, collecting peripheral blood cells of the immunized camels, isolating Cadherin-17 affinity lymphocytes therefrom, extracting total RNA and reversely transcribing it into cDNA, cloning the V region of the camel heavy chain antibody using the Nest-PCR technology, inserting it into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the CDH17 antigen through phage display technology, and verifying the binding force of the obtained single domain antibody through enzyme-linked immunosorbent assay, thereby screening out the Cadherin-17 single domain antibody with high affinity activity.

8. The E-cadherin-17 single domain antibody according to claim 1, characterized in that The DNA sequence of the E-cadherin-17 single domain antibody is shown in any one of SEQ ID NOs. 241-270.

9. A fusion protein, characterized in that It contains the E-cadherin-17 single domain antibody as described in any one of claims 1 to 8.

10. A product, characterized in that It contains the Cadherin-17 single domain antibody as described in any one of claims 1 to 8 or the fusion protein as described in claim 9, and the product is an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs to a site expressing Cadherin-17.

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