Nanobodies binding to epcam, chimeric antigen receptors and uses thereof

Abstract

The application relates to the technical field of antibodies, in particular to an EpCAM-binding nanobody, a chimeric antigen receptor and application thereof. The EpCAM-binding nanobody provided by the application can efficiently and specifically recognize and bind to EpCAM protein, and can be used for diagnosing and treating diseases taking EpCAM molecules as markers. The application further provides a CAR (chimeric antigen receptor) and a CAR-T cell (T cell) binding to EpCAM protein, the CAR-T cell has a good immune clearance effect of targeting EpCAM, can be used for specifically recognizing and killing cells expressing EpCAM, and can be used for improving and treating diseases taking EpCAM molecules as markers, and has a good application prospect in the diagnosis and treatment of tumors.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more particularly to nanobodies bound to EpCAM, chimeric antigen receptors, and their applications. Background Technology

[0002] Epithelial cell adhesion molecule (EpCAM), also known as CD326 or TACSTD1 (Tumor associated calcium signal transducer 1), is a type I transmembrane glycoprotein that was initially identified as a surface antigen of colorectal cancer[1]. The gene encoding EpCAM, GA733-2, is located on chromosome 2p21 and consists of 9 exons. It belongs to the TACSTD1 gene family, which is known for its important role in cell proliferation, tumorigenesis, and growth. Although EpCAM has similar functions to adhesion molecules, it is not a member of the four major families of cell adhesion molecules and has unique structural features, unlike the classic adhesion molecules in the immunoglobulin superfamily. EpCAM consists of an extracellular domain (EpCAM extracellular domain, EpEX), a single transmembrane domain, and a short intracellular domain (EpCAM intracellular domain, EpICD)[2]. The extracellular domain (EpEX) of EpCAM mediates calcium-independent homophilic cell adhesion and can be cleaved and detached by tumor necrosis factor-α (TNFα)-converting enzyme, while the intracellular domain (EpICD) translocates to the nucleus after being released by a γ-secretase complex containing progerin 2. In the nucleus, EPICD forms a complex with other proteins, including Lef-1, FHL2, and β-catenin. This nuclear complex helps regulate gene expression associated with epithelial-mesenchymal transition (EMT), a key process in cancer metastasis and progression [3, 4].

[0003] In normal human tissues, EpCAM is expressed at varying degrees, but usually at low levels, in most human epithelial cells. EpCAM is expressed on the basolateral membrane of epithelial cells and plays a role in enhancing cell adhesion to the extracellular matrix and other substrates [5, 6]. EpCAM is expressed differentially at different stages of human development. Typically, EpCAM is poorly expressed in human epithelial tissues during the embryonic period; however, it remains persistently expressed at low levels in most epithelial cells after birth. Among various human cancer tissues, the colon shows the most pronounced EpCAM expression, highlighting its significance in colorectal carcinoma (CRC) [5]. In contrast, certain cell types, such as epidermal keratinocytes, hepatocytes, thymic cortical epithelial cells, and squamous stratified epithelial cells, do not express EpCAM.

[0004] EpCAM is widely expressed in human cancer cells, especially adenocarcinoma and squamous cell carcinoma, such as colorectal cancer[7], gastric cancer[8], pancreatic cancer[9, 10], hepatocellular carcinoma[11-13], breast cancer[14, 15], ovarian cancer[16, 17], lung cancer[18-20], renal cancer[21, 22], oral cancer

[23] , etc. EpCAM is involved in various cancer-related processes: cell proliferation, adhesion, drying, metabolism, metastasis, chemotherapy and radiotherapy resistance, angiogenesis, migration, and epithelial-mesenchymal transition (EMT)[24-26]. The versatility of EpCAM makes it an attractive target for novel diagnostic and prognostic approaches in cancer biology and is considered an important prognostic factor[27, 28]. High levels of EpCAM can activate oncogenes such as C-myc and cyclins A and E, accelerate the cell cycle, and promote cell proliferation. Given its consistent expression in cancer cells, EpCAM is often used as a marker for cancer stem cells (CSCs) and is an important marker suitable for the detection of circulating tumor cells, and is therefore also used in human cancer immunotherapy

[29] .

[0005] In gastrointestinal tumors, EpCAM is one of the key targets for immunotherapy.

[0006] In colorectal cancer, analysis of samples from more than 400 patients showed elevated expression of EpCAM and CD166 in 61.5% (246 / 427) and 40.5% (164 / 405) of CRC cases. EpCAM expression was significantly positively correlated with tumor size, tumor stage, tumor differentiation, and invasion of blood vessels, nerves, and lymph nodes. Furthermore, a comprehensive analysis showed a significant mutual correlation between EpCAM and CD166 expression [7].

[0007] In hepatocellular carcinoma (HCC), tumor cells overexpressing EpCAM exhibit high proliferative and aggressive characteristics, and these characteristics can be reduced by downregulating EpCAM through RNA interference

[30] . EpCAM expression in HCC is significantly associated with higher tumor grade and elevated serum AFP levels. Downregulation of EpCAM gene expression significantly reduces the proliferation and invasiveness of HCC cells

[31] . Studies have shown that EpCAM+ cells express liver stem cell markers, effectively form non-adhesive spheroids, and exhibit greater invasiveness and tumorigenicity compared to EpCAM- cells, demonstrating their stem / progenitor cell-like characteristics [32, 33]. Furthermore, proliferative ductal cells (PDCs) expressing EpCAM in inflamed livers have been identified as a potential cellular source for HCC

[34] . There are also studies indicating that EpCAM+ cells are associated with advanced cirrhosis, HCV-related cirrhosis progressing to HCC, and hepatocellular cholangiocarcinoma may also originate from EpCAM+ cells. Studies have found that EpCAM is frequently co-expressed with other markers, and these co-expressing cells may represent a more precise CSC phenotype.

[0008] In pancreatic cancer (mainly pancreatic ductal adenocarcinoma), studies have analyzed cell lines and tissue specimens from pancreatic cancer patients and found that most specimens showed EpCAM overexpression. When patients were divided into LN-positive and LN-negative groups to examine the effect of EpCAM, a strong effect of EpCAM was observed in the LN-negative group. In multivariate analysis, EpCAM expression was an independent prognostic factor, associated with histological and lymph node metastasis. EpCAM expression was associated with inhibition of cancer cell activity in pancreatic cancer and a good prognosis in cancer patients who underwent therapeutic resection

[10] . Subgroup analysis of another study showed that EpCAM overexpression was associated with lower overall survival in patients with ampullary cancer and advanced cancer. In the subgroup analysis, survival gradually worsened with increasing EpCAM score. In addition, EpCAM overexpression was found to be associated with tumor stage in ampullary cancer [9]. Although the conclusions of the two studies differed due to different subgroup analysis methods, it is clear that EpCAM overexpression is significantly associated with the prognosis of pancreatic cancer. One study

[35] evaluated the antitumor efficacy of a bispecific EpCAMxCD3 antibody that connects tumor cells and T lymphocytes. In NOD SCID mice, EpCAMxCD3 had a long serum half-life (t(1 / 2) approximately 7 days). EpCAMxCD3 significantly inhibited the growth of BxPC-3 pancreatic cancer xenografts. Another similar study used the bispecific T cell conjugate (BiTE) MT110 for targeted immunotherapy of EpCAM, through which cytotoxic T cells could be effectively redirected to primary human cancer cells via T cell-conjugated BiTE antibody MT110 (including a highly tumorigenic CSC subset)

[36] . These studies preliminarily demonstrate the feasibility of EpCAM as a target for pancreatic cancer immunotherapy.

[0009] Gastric cancer is also a common tumor that highly expresses EpCAM. Meta-analyses have shown that EpCAM overexpression is significantly associated with gastric cancer tumor size, lymph node metastasis, and poor prognosis

[37] . Histochemical analysis of gastric cancer revealed that more than 90% of the samples showed high expression of EpCAM, suggesting the role of EpCAM as a prognostic indicator. Other studies have shown that strong EpCAM expression was observed in 77% of tumors and 85% of the corresponding lymph nodes. In primary tumors, 58% (n=74) showed homogeneous intratumoral EpCAM expression, while 42% were characterized by heterogeneous expression patterns. Tumors with high EpCAM expression at the invasive front were significantly associated with increased lymph node metastasis and decreased median overall survival. Compared with the tumor center, diffuse tumors showed significantly higher EpCAM expression at the invasive front. Multivariate survival analysis showed that high EpCAM expression at the invasive front was an independent prognostic factor. There was a significant correlation between high EpCAM expression and high tumor cell proliferation

[38] .

[0010] In recent years, immunotherapy targeting EpCAM has become a promising strategy for cancer treatment, including vaccines targeting EpCAM [39, 40], CAR-T cells (Chimeric Antigen Receptor-T cells) targeting EpCAM [41, 42], and anti-EpCAM antibodies [43, 44]. VB4-845 (Oportuzumab monatox), a conjugated recombinant antibody and immunotoxin targeting EpCAM, has been shown to have antitumor effects

[45] . Studies by Ogawa et al. have shown that VB4-845, when used in combination with 5-FU, can inhibit HCC CSC characteristics and tumor growth in subcutaneous and orthotopic liver xenograft models

[46] . Choi et al. have shown that activation of dendritic cells (DCs) with EpCAM peptides can enhance T cell stimulation, leading to HCC cytotoxicity and tumor growth inhibition

[47] . Anti-EpCAM-BiTE (bispecific T-cell binder) 1H8 / CD3 has been constructed and demonstrated to eradicate HCC cells and HCC CSCs in vitro and in vivo

[48] . These preclinical studies suggest that EpCAM-targeted therapy may provide a promising approach for the treatment of HCC. EpCAM-targeted CAR-T cells have also begun to be explored in cancers such as colorectal cancer[49,50], ovarian cancer

[51] , and lung cancer[52-54], suggesting the potential of EpCAM-targeted CAR-T cell therapy for cancer. Summary of the Invention

[0011] This invention provides nanobodies bound to EpCAM, chimeric antigen receptors, and their applications.

[0012] Specifically, the present invention provides the following technical solutions.

[0013] In a first aspect, the present invention provides a nanobody incorporating EpCAM, the nanobody comprising a heavy chain variable region, wherein the complementarity-determining region (CDR) of the heavy chain variable region is any one of the following (1) to (6):

[0014] (1) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.1, 2 and 3 respectively;

[0015] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.4, 5 and 6 respectively;

[0016] (2) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.7, 8 and 9 respectively;

[0017] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.10, 11 and 12 respectively;

[0018] (3) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.13, 14 and 15 respectively;

[0019] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.16, 17 and 18 respectively;

[0020] (4) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.19, 20 and 21 respectively;

[0021] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.22, 23 and 24 respectively;

[0022] (5) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.25, 26 and 27 respectively;

[0023] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.28, 29 and 30 respectively;

[0024] (6) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.31, 26 and 27 respectively;

[0025] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO.28, 29 and 30 respectively.

[0026] The heavy chain variable region described above also includes a frame region FR, wherein the frame region FR is any one of the following (1) to (9):

[0027] (1) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.32, 33, 34 and 35 respectively;

[0028] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.36, 37, 38 and 39 respectively;

[0029] (2) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.40, 41, 42 and 43 respectively;

[0030] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.44, 45, 46 and 47 respectively;

[0031] (3) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.40, 41, 72 and 43 respectively;

[0032] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.44, 45, 73 and 47 respectively;

[0033] (4) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.48, 49, 50 and 43 respectively;

[0034] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.51, 37, 52 and 47 respectively;

[0035] (5) When defined according to Martin numbering scheme and Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.53, 54, 55 and 56 respectively;

[0036] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.57, 58, 59 and 60 respectively.

[0037] (6) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.61, 54, 55 and 56 respectively;

[0038] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.62, 58, 59 and 60 respectively.

[0039] (7) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.63, 64, 65 and 43 respectively;

[0040] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO. 66, 67, 68 and 47 respectively.

[0041] (8) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.63, 64, 65 and 43 respectively;

[0042] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO. 69, 67, 68 and 47 respectively;

[0043] (9) When defined according to the Martin numbering scheme and the Chothia definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.70, 64, 65 and 43 respectively;

[0044] When defined according to the Martin numbering scheme and the Contact definition scheme, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO.71, 67, 68 and 47 respectively.

[0045] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 74, 75, 76, 77, 78, 79, 80, 81 or 82.

[0046] Preferably, the amino acid sequence of the heavy chain variable region has at least 80% similarity to the sequence shown in SEQ ID NO. 74, 75, 76, 77, 78, 79, 80, 81 or 82.

[0047] The sequence similarity described above is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 98.5%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably at least 99.9%.

[0048] In a second aspect, the present invention provides an antibody that binds to EpCAM, said antibody being a monovalent antibody, bispecific antibody, or multispecific antibody comprising one or more of the EpCAM-binding nanobodies described in the first aspect above.

[0049] When the EpCAM-binding antibody comprises two or more EpCAM-binding nanobodies, the nanobodies can be linked together by a linker peptide. The linker peptide is preferably a flexible peptide rich in glycine and serine. An exemplary linker peptide may be GGGGSGGGGSGGGGS (SEQ ID NO. 89).

[0050] In some embodiments of the present invention, the EpCAM-binding antibody is an antibody formed by two tandemly linked EpCAM-binding nanobodies selected from the first aspect described above. Preferably, the amino acid sequence of the EpCAM-binding antibody is as shown in any one of SEQ ID NO. 83-88.

[0051] Thirdly, the present invention provides a fusion protein, which is obtained by fusing the above-described EpCAM-bound nanobody with Fc.

[0052] The Fc fragment includes the Fc fragment of human IgG, which can be selected from the Fc fragments of IgG1, IgG2, IgG3, and IgG4.

[0053] Fourthly, the present invention provides a chimeric antigen receptor (CAR) targeting EpCAM, wherein the chimeric antigen receptor comprises the EpCAM-binding nanobody or the EpCAM-binding antibody described above.

[0054] In this invention, the chimeric antigen receptor may adopt the structure of currently known chimeric antigen receptors.

[0055] Preferably, the chimeric antigen receptor comprises a signal peptide, the EpCAM-binding nanobody or the EpCAM-binding antibody, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

[0056] Preferably, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a signal peptide, the EpCAM-binding nanobody or the EpCAM-binding antibody, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

[0057] In this invention, the signal peptide, hinge region, transmembrane region, intracellular co-stimulatory domain, and intracellular signal transduction domain can all be selected from known CAR structural sequences.

[0058] In some embodiments of the present invention, the CAR sequentially comprises a CD8α leader membrane receptor signal peptide, a (G4S)3 linker peptide, the EpCAM-binding nanobody or the EpCAM-binding antibody, a CD8α hinge region, a CD8α™ transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ intracellular signal transduction domain.

[0059] Fifthly, the present invention provides a nucleic acid molecule encoding the EpCAM-binding nanobody, the EpCAM-binding antibody, the fusion protein, or the chimeric antigen receptor.

[0060] Based on the amino acid sequences of nanobodies, antibodies, fusion proteins, and chimeric antigen receptors provided by the present invention, those skilled in the art can obtain the nucleotide sequences encoding the aforementioned nanobodies, antibodies, fusion proteins, and chimeric antigen receptors. Due to the degeneracy of codons, the nucleotide sequence encoding a single amino acid sequence is not unique; all nucleic acid molecules capable of encoding the aforementioned nanobodies, antibodies, fusion proteins, and chimeric antigen receptors are within the scope of protection of the present invention.

[0061] In a sixth aspect, the present invention provides a biological material comprising the nucleic acid molecules described above; the biological material is an expression cassette, a vector, or a host cell.

[0062] The aforementioned expression cassette can be obtained by linking a promoter or other transcriptional or translational regulatory element upstream of the nucleic acid molecule and / or a terminator or other transcriptional or translational regulatory element downstream of it.

[0063] The aforementioned vectors include, but are not limited to, plasmid vectors, viral vectors, lipid nanoparticle vectors, transposons, etc. Among them, the viral vectors include lentiviral vectors, retroviral vectors, AAV viral vectors, adenovirus vectors, etc.

[0064] The host cells mentioned above include prokaryotic or eukaryotic cells. Prokaryotic cells include, but are not limited to, *Escherichia coli*. Eukaryotic cells include, but are not limited to, yeast, insect cells, or other mammalian cells. Yeast includes, but is not limited to, *Pichia pastoris* and *Saccharomyces cerevisiae*; mammalian cells include, but are not limited to, immune cells (e.g., T cells, NK cells), stem cells or progenitor cells capable of differentiating into immune cells, young hamster embryonic kidney cells (BHK), Chinese hamster ovary cells (CHO), African green monkey kidney cells (Vero), mouse myeloma cells (SP0 / 2), and human embryonic kidney 293 cells (HEK293), etc.

[0065] In a seventh aspect, the present invention provides a recombinant cell expressing the chimeric antigen receptor described above.

[0066] Preferably, the recombinant cells are immune cells expressing the chimeric antigen receptors described above.

[0067] Preferably, the immune cells are T cells or NK cells.

[0068] Eighthly, the present invention provides antibody conjugates obtained by conjugating the EpCAM-bound nanobody, the EpCAM-bound antibody, the fusion protein, or the chimeric antigen receptor with a detectable marker, drug, toxin, or cytokine.

[0069] The markers mentioned above are selected from one or more of enzyme labeling, biotin labeling, chemiluminescent dye labeling, fluorescent dye labeling, radioactive labeling, and colloidal gold labeling.

[0070] The drugs described above are cytotoxic drugs. These cytotoxic drugs include, but are not limited to, topoisomerase inhibitors, DNA replication inhibitors, antibiotics, anti-microtubule drugs, alkylating agents, and folic acid antagonists.

[0071] The toxins mentioned above include, but are not limited to, paclitaxel, cisplatin, ricin B, vincristine, colchicine, diphtheria toxin, absinthecin, and actinomycin.

[0072] In a ninth aspect, the present invention provides a method for generating the EpCAM-binding nanobody, the EpCAM-binding antibody, the fusion protein, or the chimeric antigen receptor, the method comprising: culturing a host cell containing the nucleic acid molecule, and collecting the EpCAM-binding nanobody, the EpCAM-binding antibody, the fusion protein, or the chimeric antigen receptor from the culture.

[0073] The process of collecting from the culture includes steps such as separation and purification.

[0074] In a tenth aspect, the present invention provides any one of the following applications of the EpCAM-bound nanobody, the EpCAM-bound antibody, the fusion protein, the chimeric antigen receptor, the nucleic acid molecule, the biomaterial, the recombinant cell, or the antibody conjugate:

[0075] (1) Use in the preparation of medicaments for the prevention or treatment of diseases associated with EpCAM expression;

[0076] (2) Application in the preparation of CAR-T cells;

[0077] (3) Application in the preparation of reagents for detecting the presence or level of EpCAM in a sample;

[0078] (4) Application in the preparation of reagents for detecting the presence or level of EpCAM+ cells in a sample;

[0079] (5) Application in the preparation of tumor detection reagents.

[0080] In (1) above, the disease associated with EpCAM expression is preferably a disease that uses EpCAM molecules as a marker, preferably a tumor, more preferably a malignant tumor; preferably a tumor that highly expresses EpCAM, especially an epithelial malignant tumor that highly expresses EpCAM; more preferably adenocarcinoma or squamous cell carcinoma. The malignant tumor includes at least one selected from colorectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, lung cancer, kidney cancer, oral cancer, etc.

[0081] In (5) above, the tumor is a tumor expressing EpCAM, preferably an epithelial malignant tumor that highly expresses EpCAM. More preferably, it is adenocarcinoma or squamous cell carcinoma. The malignant tumor includes at least one selected from colorectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, lung cancer, kidney cancer, oral cancer, etc.

[0082] Eleventhly, the present invention provides a pharmaceutical composition comprising the EpCAM-binding nanobody or the EpCAM-binding antibody or the fusion protein or the chimeric antigen receptor or the recombinant cell or the antibody conjugate.

[0083] Optionally, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients.

[0084] Preferably, the pharmaceutical composition is used for the prevention or treatment of diseases associated with EpCAM expression.

[0085] Among them, the diseases associated with EpCAM expression are preferably diseases that use EpCAM molecules as markers, preferably tumors, more preferably malignant tumors; preferably tumors that highly express EpCAM, especially epithelial malignant tumors that highly express EpCAM; more preferably adenocarcinoma or squamous cell carcinoma. The malignant tumors include at least one selected from colorectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, lung cancer, kidney cancer, oral cancer, etc.

[0086] In a twelfth aspect, the present invention provides a detection reagent comprising the EpCAM-bound nanobody, the EpCAM-bound antibody, the fusion protein, or the antibody conjugate.

[0087] The detection reagents mentioned above include, but are not limited to, ELISA detection reagents or kits, chemiluminescence detection reagents or kits, radioimmunoassay detection reagents or kits, and fluorescence immunoassay detection reagents or kits.

[0088] In a thirteenth aspect, the present invention provides a method for treating a disease associated with EpCAM expression, the method comprising: administering a subject a therapeutically effective amount of the EpCAM-binding nanobody or the EpCAM-binding antibody or the fusion protein or the chimeric antigen receptor or the recombinant cell or the antibody conjugate.

[0089] Among them, the diseases associated with EpCAM expression are preferably diseases that use EpCAM molecules as markers, preferably tumors, more preferably malignant tumors; preferably tumors that highly express EpCAM, especially epithelial malignant tumors that highly express EpCAM; more preferably adenocarcinoma or squamous cell carcinoma. The malignant tumors include at least one selected from colorectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, lung cancer, kidney cancer, oral cancer, etc.

[0090] In a fourteenth aspect, the present invention provides a method for detecting EpCAM or EpCAM+ cells, the method comprising: contacting the EpCAM-bound nanobody or the EpCAM-bound antibody or the fusion protein or the antibody conjugate with a sample to be tested to determine the presence or level of EpCAM or EpCAM+ cells in the sample to be tested.

[0091] The beneficial effects of this invention include at least the following: This invention provides nanobodies bound to EpCAM. Compared to traditional monoclonal antibodies and single-chain variable fragments (scFv), nanobodies have advantages such as small molecular weight, less susceptibility to aggregation and precipitation, and high stability. While maintaining high affinity for antigens, the long and flexible complementarity-determining region (CDR3) of the nanobody allows it to bind to the cracks and cavities of the target antigen, thereby recognizing some hidden epitopes. The anti-EpCAM nanobody provided by this invention can efficiently and specifically recognize and bind to the EpCAM protein. Based on this nanobody, this invention also provides drugs prepared from it, including but not limited to bispecific antibodies, antibody-drug conjugates, chimeric antigen receptors, and oncolytic viruses, which can be used to specifically recognize or kill cells expressing EpCAM, such as EpCAM-positive cells in various malignant tumors such as colorectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, lung cancer, kidney cancer, and oral cancer, for the diagnosis and treatment of diseases using EpCAM molecules as markers.

[0092] This invention also provides a CAR that binds to EpCAM, which can efficiently target EpCAM. Moreover, it uses nanobodies that bind to EpCAM as the antigen-specific recognition structural component. Nanobodies have the advantage of small molecular weight, which is conducive to recombinant design to generate multispecific nanobodies and multispecific CARs, improves the recognition specificity of the target antigen to avoid the on-target, off-tumor effect, improves the safety of CAR in tumor immunotherapy, and obtains a wider range of therapeutic applications.

[0093] Based on the aforementioned CAR, this invention also provides CAR-T cells that bind to EpCAM. Cell-level experiments have verified that these CAR-T cells have a good immune clearance effect targeting EpCAM and can be used to specifically recognize and kill EpCAM-expressing cells, such as EpCAM-positive cells in various malignant tumors including colorectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, lung cancer, kidney cancer, and oral cancer, for the purpose of improving and treating diseases that use EpCAM molecules as markers. Attached Figure Description

[0094] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0095] Figure 1This invention, in Example 1, uses phage ELISA to screen nine anti-human EpCAM VHH antibodies from an alpaca antibody library. 450 The reading results are as follows: EpCAM represents the detection results of positive clones binding to the EpCAM protein antigen, and BSA represents the detection results using BSA as a control antigen.

[0096] Figure 2 This is a flow cytometry analysis of the expression of the target antigen EpCAM in human pancreatic-related cell lines, colorectal cancer cell lines, and gastric cancer cell lines in Example 2 of the present invention; wherein, Anti-EpCAM is the detection result of the target antigen EpCAM, and Blank is the detection result of the blank control.

[0097] Figure 3 This is a schematic diagram of the molecular structure of the CAR composed of a single VHH (a) and two VHHs (b) against EpCAM in Example 3 of the present invention.

[0098] Figure 4 The positive rate of the anti-EpCAM CAR (CAR54) transduced dual reporter cell line J-NN was detected by flow cytometry in Example 4 of this invention, where NC represents the negative control.

[0099] Figure 5 , Figure 6 , Figure 7 and Figure 8 This invention, in Example 4, describes the detection of downstream signal activation levels of nine anti-EpCAM CAR J-NN receptors under different target cell stimuli, and a comparison of peak activation levels of GFP and / or mCherry signals. Figure 5 The peak intensity of green fluorescence signal of 9 different anti-EpCAM CAR J-NNs when co-cultured with EpCAM-positive target cells AsPC-1, BxPC-3, CFPAC-1, Capan-2, and EpCAM-negative target cells hTERT-HPNE is shown. Figure 6 and Figure 7 The changes in green and red fluorescence signal intensity of four different anti-EpCAM CAR J-NN antibodies when co-cultured with EpCAM-positive target cells AsPC-1, BxPC-3, CFPAC-1, Capan-2, and EpCAM-negative target cells hTERT-HPNE were shown. Figure 8 This study compared the green fluorescence intensity at 12 hours and the red fluorescence intensity at 18 hours of co-culture with different anti-EpCAM CAR J-NNs activated on different target cells. "au" represents an arbitrary unit. "NONE" represents a negative control.

[0100] Figure 9This illustrates the downstream signal activation of the dual VHH tandem anti-EpCAM CAR J-NNs (such as 5355, 5553, 5455, 5554, 5356, and 5653) composed of anti-EpCAM VHHs tandemly linked with 53, 54, 55, and 56 (as in Example 4 of this invention) under stimulation by EpCAM-positive target cells AsPC-1, BxPC-3, CFPAC-1, Capan-2, and EpCAM-negative target cells hTERT-HPNE. The upper and lower figures show the green fluorescence intensity of the untransduced CAR J-NN and the red fluorescence intensity of the six anti-EpCAM CAR J-NNs after 12 hours and 18 hours of co-culture with the aforementioned target cells, respectively.

[0101] Figure 10 In Example 5 of this invention, flow cytometry was used to detect the positive rate of human primary T cells transduced with anti-EpCAM CAR (taking CAR 54, 55, and 58 as examples), where NC represents the negative control.

[0102] Figure 11 and Figure 12 This presents the in vitro killing kinetics and killing efficiency results of anti-EpCAM CAR-T cells against different target cells in Example 6 of this invention. Figure 11 Effector cells at different effector-to-target ratios: control T cells without CAR transfection, 9 anti-EpCAM CAR-T cells (51-59), and EpCAM-positive human pancreatic cancer cells BxPC-3-GFP were co-cultured with the target cells to the analysis endpoint (48h). Figure 12 The killing efficiency of effector cells at different effector-to-target ratios at the end of the co-culture period (72h or 96h) of untransfected CAR control T cells, anti-EpCAM CAR-T 53, 54, 55, and 56 cells with EpCAM-positive target cells (human pancreatic cancer cells BxPC-3-GFP, Capan-2-GFP, human colorectal cancer cells HT-29-GFP, human gastric cancer cells MKN-45-GFP) and EpCAM-negative target cells (hTERT-HPNE-GFP).

[0103] Figure 13 and Figure 14 This presents the statistical results of the killing kinetics and killing efficiency of anti-EpCAM CAR-T cells against different target cells in Example 6 of this invention. Figure 13The changes in green fluorescence signal intensity (normalized) of untransfected CAR control T cells, anti-EpCAM CAR-T 5355, 5553, 5455, 5554, 5356, and 5653 cells co-cultured with EpCAM-positive target cells (human pancreatic cancer cells BxPC-3-GFP, Capan-2-GFP, human colorectal cancer cells HT-29-GFP, human gastric cancer cells MKN-45-GFP) and EpCAM-negative target cells (hTERT-HPNE-GFP), respectively. Figure 14 Effector cells at the end of the co-culture period (72h or 96h) of untransfected CAR control T cells (T), anti-EpCAMCAR-T 5355, 5553, 5455, 5554, 5356, and 5653 with EpCAM-positive target cells (human pancreatic cancer cells BxPC-3-GFP, Capan-2-GFP, human colorectal cancer cells HT-29-GFP, human gastric cancer cells MKN-45-GFP) and EpCAM-negative target cells (hTERT-HPNE-GFP), respectively: killing efficiency of target cells at different effector-to-target ratios.

[0104] Figure 15 This presents the kinetics of the killing effect of anti-EpCAM CAR-T on different target cell tumor spheres in Example 7 of the present invention. a and b show the in vitro killing effects of anti-EpCAM CAR-T 53, 54, 55, 56 and anti-EpCAM CAR-T 5355, 5553, 5455, 5554, 5356, 5653 cells on BxPC-3-GFP 3D tumor spheres, respectively. c and d show the killing effects of anti-EpCAM CAR-T 53, 54, 55, 56 and anti-EpCAM CAR-T 5355, 5553, 5455, 5554, 5356, 5653 cells on Capan-2-GFP 3D tumor spheres, respectively.

[0105] Figure 16 This invention demonstrates the in vivo killing effect of anti-EpCAM CAR-T 5455 and 5554 cells on MKN-45-GFP tumors in the cNKG mouse CDX model in Example 8 of this invention. Detailed Implementation

[0106] In a specific embodiment of the present invention, an antibody binding to EpCAM protein is provided. This antibody is a low-molecular-weight antibody derived from alpacas, containing only a single variable domain of heavy chain antibody (VHH). It was obtained by screening from an alpaca immune library using phage display technology. First, alpacas are immunized with purified EpCAM protein. After successful immunization, peripheral blood containing mature B cells is collected from alpacas, and lymphocytes are extracted. RNA is extracted and reverse transcribed into a cDNA library. The heavy chain variable domain of the alpaca antibody is amplified by PCR to obtain a specific gene fragment, which is then ligated into a phage vector and transformed into *E. coli* to obtain a nanobody gene library, i.e., an immune library. After four rounds of screening using phage display technology, positive clones are identified using indirect ELISA, and sequencing confirms the antibody sequence binding to the EpCAM antigen.

[0107] The antibodies that bind to the EpCAM protein obtained above contain the VHH of the heavy chain antibody. The amino acid sequences of the VHH of each antibody (SEQ ID NO. 74-82) are shown in Table 1. Tandem nanobodies were constructed by linking the two nanobodies mentioned above via a linker peptide, and their sequences are shown in SEQ ID NO. 83-88 (Table 1). The amino acid sequence of each antibody was numbered using the Martin antibody numbering scheme, and the CDR regions were defined using both the Chothia and Contact schemes to obtain the CDR1, CDR2, CDR3 regions and the frame regions FR1, FR2, FR3, and FR4. The amino acid sequences of the CDR regions (SEQ ID NO. 1-31) are shown in Table 2, and the amino acid sequences of FR1, FR2, FR3, and FR4 (SEQ ID NO. 32-73) are shown in Table 3.

[0108] Table 1

[0109]

[0110] Table 2

[0111]

[0112] Table 3

[0113]

[0114] The antibodies that bind to the EpCAM protein described above can be used to specifically recognize or kill cells expressing EpCAM, such as EpCAM-positive cells in various malignant tumors including colorectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, lung cancer, kidney cancer, and oral cancer, for the diagnosis and treatment of diseases that use EpCAM molecules as markers.

[0115] In a specific embodiment of the present invention, a chimeric antigen receptor (CAR) binding to the EpCAM protein, an expression vector comprising the chimeric antigen receptor, and a host cell are also provided. The EpCAM-binding CAR uses the aforementioned antibody binding to the EpCAM protein as the antigen-specific recognition structural component. The molecular structure of the anti-EpCAM CAR is SP-anti-EpCAM VHH-HINGE-TM-CD-SD, wherein SP is a signal peptide, HINGE is a hinge region, TM is a transmembrane region, CD is a co-stimulatory domain, SD is a signal transduction domain, and "-" represents a linker peptide or peptide bond.

[0116] In this invention, SP, HINGE, TM, CD, and SD can all be selected from known sequences in the CAR technology field. SP can be derived from signal peptides such as CD8α and GM-CSFR; HINGE can be derived from the hinge region of CD8α and IgG; TM can be derived from the transmembrane region of CD8α, CD28, 4-1BB, etc.; CD can be derived from the co-stimulatory domains of CD28, 4-1BB, OX40, 2B4, DAP10, DAP12, etc., or combinations thereof; and SD can be derived from the activation domains of CD3ζ, CD3γ, CD3δ, CD3ε, MyD88, etc. In a specific embodiment, the CAR molecule is composed of, in series, CD8α leader membrane receptor signal peptide, (G4S)3 linker peptide, anti-EpCAM VHH, CD8α Hinge region, CD8α TM transmembrane region, CD28 co-stimulatory domain, and CD3ζ intracellular signal transduction domain.

[0117] The chimeric antigen receptor binding to the EpCAM protein described above uses an EpCAM-binding nanobody as the antigen-specific recognition structural component. This chimeric antigen receptor is transduced into immune-responsive host cells using vectors such as lentiviruses. It can target and recognize cells expressing EpCAM. Upon antigen stimulation, it releases various pro-apoptotic cytokines such as granzymes, perforin, and interferon-γ, inducing target cell death and exerting an immune clearance function. Therefore, the chimeric antigen receptor binding to the EpCAM protein of this invention can be used to specifically recognize and kill EpCAM-expressing cells, such as EpCAM-positive cells in various malignant tumors including colorectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, lung cancer, kidney cancer, and oral cancer, for the improvement and treatment of diseases using EpCAM molecules as markers.

[0118] In a specific embodiment of the present invention, an expression vector is also provided, which contains a nucleic acid molecule encoding the chimeric antigen receptor or antibody, and may be a plasmid vector, a lipid nanoparticle vector, or a viral vector (such as a lentiviral vector, a retroviral vector, etc.).

[0119] In specific embodiments of the present invention, a host cell is also provided, wherein the host cell is an immune cell or a stem cell or progenitor cell capable of differentiating into an immune cell. Immune cells include, but are not limited to, T cells, NK cells, NKT cells, macrophages, and dendritic cells, possessing cytotoxic or phagocytic functions, immunomodulatory functions, and cytokine release functions. Stem cells may be hematopoietic stem cells or induced pluripotent stem cells, wherein the stem cells are capable of differentiating into one or more of the aforementioned immune cells.

[0120] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0121] Example 1: Screening of nanobodies bound to EpCAM protein

[0122] Healthy adult alpacas were immunized with human EpCAM / TROP1 protein (Fc Tag, PEP-H5223, catalog number EPM-H5223) as an antigen. Four immunizations were administered, 20 days apart. Peripheral blood was collected from the alpacas before each immunization to measure serum antibody titers. After immunization, peripheral blood was collected again, and peripheral blood mononuclear cells (PBMCs) were extracted. RNA was extracted, and cDNA was obtained through RT-PCR reverse transcription. The VHH gene fragment was then amplified by PCR to obtain the heavy chain variable region. The fragment was then cloned and ligated into the phage vector pComb3X, transformed into *E. coli*, and a nanobody gene library was obtained.

[0123] Phage display technology was used for bioscreening of anti-EpCAM nanobodies. The specific steps were as follows: 1) Antigen coating: EpCAM antigen was coated onto immunotubes (30µg / tube), and incubated overnight at 4°C with slow rotation. Simultaneously, 30µg of 5% non-fat milk powder was coated as a control. 2) Washing away unbound antigen: The coating solution was discarded, and the immunotubes were washed three times with 2mL PBS buffer at room temperature, rotating for 5min each time. 3) Blocking: 5mL blocking solution was added, and the tubes were blocked by rotation at room temperature for 2h. 4) Washing away blocking solution: The liquid in the blocked immunotubes was discarded, and the tubes were washed three times with 5mL PBS buffer at room temperature, rotating for 5min each time. 5) Adding phage display library for binding: The washing solution in the immunotubes was discarded, 2mL PBS buffer was added, and the prepared phage library was added as the first-round screening input phage library. The tubes were incubated by rotation at room temperature for 1h. 6) Washing away unbound phage: The liquid in the immunotubes was discarded, and 5mL PBST (1×PBS plus 0.1%) was added. 7) Wash the immunotubes 20 times with Tween 20 buffer at room temperature, rotating for 5 minutes each time. Discard the liquid in the immunotubes and remove as much residual liquid as possible. 8) Elute the bound phages: Add 1 mL of pH 2.2 Glycine-HCl solution, rotate and elute for 30 minutes at room temperature, then add 90 μL of pH 8.8 Tris-HCl to stop elution. Transfer the solution from the immunotubes to a new 1.5 mL centrifuge tube; this is the phage elution buffer for the first round of screening. 9) The first-round phages are amplified and used for subsequent second-round screening and detection. The above screening process is performed in 3 rounds. The phages from each round are plated and the enrichment efficiency is calculated. Single clones are selected for antigen-specific detection.

[0124] Positive monoclonal antibodies were screened and identified using phage ELISA. The specific steps were as follows: After each round of phage elution enrichment, the ELISA plate was coated with EpCAM protein antigen, with BSA as the control antigen. Antibodies displayed by selected cultured monoclonal phages were used as the test antibody binding antigen. Unbound phages were eluted, and HRP-labeled anti-phage M13 antibody was used as the secondary antibody to bind the phages. Substrate was added for color development, and the ELISA reader readings were performed. The OD values ​​of the EpCAM test group and the BSA control group were compared. 450 The difference value initially indicates a positive clone.

[0125] Ninety-six positive clones from the phage ELISA results were selected and sent to Qingke Biotechnology Co., Ltd. for sequencing. Eighty-two valid sequencing results were obtained. The diversity of the positive clone sequences was assessed. The successfully sequenced sequences were converted into amino acid sequences, and identical amino acid sequences were merged to obtain nine positive antibody clones selected for EpCAM phage screening. The ELISA results of these nine positive clones are shown below. Figure 1As shown. For the nine EpCAM phage-selected positive antibody clones, their corresponding amino acid sequences were synthesized, and each clone was further tagged with an amino acid tag / linker (GGGGS) × 3, facilitating subsequent detection. The nine tagged EpCAM VHH genes were synthesized, and the correspondence between their CAR names (51-59) and clone numbers is shown below. Figure 1 As shown.

[0126] Example 2: Flow cytometry detection of EpCAM-positive and negative target cells

[0127] The expression levels of EpCAM protein on the surface of five representative human pancreatic cancer, gastric cancer, and colorectal cancer cell lines were detected using a flow cytometry method with EpCAM antibody (CD326 (EpCAM) Monoclonal Antibody (1B7), PE, eBioscience Thermo Fisher Scientific, catalog number 12-9326-42). These included two human pancreatic cancer cell lines, BxPC-3 and Capan-2; human colorectal cancer cell line HT-29; human gastric cancer cell line MKN-45; and an immortalized human normal pancreatic ductal epithelial cell line, hTERT-HPNE. The procedure involved digesting the cells with trypsin, neutralizing them, transferring them to centrifuge tubes, centrifuging, resuspending the cells in culture medium, and counting. 1×10⁶ cells were collected from both the stained group and the blank control group. 6 Cells were centrifuged, the supernatant was removed, and the cells were washed once with pre-prepared FACS buffer (1×PBS + 2% FBS or 1×PBS + 0.5% BSA). EpCAM flow cytometry antibody diluted in FACS buffer was added to the stained cells, and the cells were incubated at 4°C for 30 minutes. After centrifugation and removal of the supernatant, the cells were washed three times with FACS buffer and resuspended in 200 μL for flow cytometry analysis.

[0128] Flow cytometry analysis results of EpCAM expression in five cell types are as follows: Figure 2 As shown, human pancreatic cancer cells BxPC-3 and Capan-2, human colorectal cancer cells HT-29, and human gastric cancer cells MKN-45 highly expressed EpCAM and were used as EpCAM-positive target cells in subsequent experiments; human pancreatic ductal epithelial cells hTERT-HPNE hardly expressed EpCAM and were used as EpCAM-negative target cells in subsequent experiments.

[0129] Example 3: Construction of plasmids against EpCAM CAR and packaging of lentiviruses

[0130] The anti-EpCAM VHH nanobody obtained in Example 1 was synthesized into a VHH fragment with restriction enzyme sites after codon optimization according to the amino acid sequence, or the original PCR product with restriction enzyme sites obtained by phage screening was amplified by PCR and spliced ​​with the CAR expression plasmid backbone after restriction enzyme digestion to construct a lentiviral CAR expression vector (or master plasmid vector, which was modified with the EF1α promoter). After sequencing confirmed the accuracy of the plasmid sequence, the lentiviral master plasmid expressing anti-EpCAM CAR was obtained. The complete molecular composition of anti-EpCAM CAR is as follows: Figure 3 As shown in Figure a, the CAR molecules include, in tandem, CD8α leader signal peptide (SP), (G4S)3 linker peptide (L), anti-EpCAM VHH, CD8α Hinge region, CD8α TM transmembrane region (Tm), CD28 co-stimulatory domain (28), and CD3ζ intracellular signal transduction domain (ζ). These CAR molecules, or plasmids, viral vectors, CAR-T cells, and CAR-Jurkat cells containing these molecules, are designated as 51, 52, 53, 54, 55, 56, 57, 58, and 59 in this and subsequent examples. For some tandem dual VHH CARs, their structures are as follows... Figure 3 As shown in b, the CAR molecules include, in sequence, the CD8α leader signal peptide (SP), the pre-anti-EpCAM VHH (anti-EpCAM VHH1), the (G4S)3 linker peptide (L), the post-anti-EpCAM VHH (anti-EpCAM VHH2), the CD8α Hinge region, the CD8α TM transmembrane region (Tm), the CD28 co-stimulatory domain (28), and the CD3ζ intracellular signal transduction domain (ζ). These CAR molecules, or plasmids, viral vectors, CAR-T cells, and CAR-Jurkat cells containing these molecules, are designated as 5355, 5553, 5455, 5554, 5356, and 5653 in this embodiment and subsequent embodiments.

[0131] Lentiviral cells were prepared using a three-plasmid co-transfection method on 293T cells. The transfection reagent was PEI MAX, or linearized polyethyleneimine PEI 40000 (MW40000, Polysciences, catalog number 24765-1). The three plasmids included a lentiviral main plasmid expressing anti-EpCAM CAR and two packaging plasmids, psPAX2 and pMD2.G. Healthy 293T cells were plated one day in advance, and transfection was performed when the cell confluence was approximately 80%. The plasmids and transfection reagent were diluted with serum-free medium at a mass ratio of lentiviral main plasmid: psPAX2: pMD2.G = 3:2:1, and the ratio of transfection reagent to plasmid was 2.5 μL: 1 μg. After mixing the diluted transfection reagent and plasmid, the mixture was incubated at room temperature for 20 min. The transfection suspension was then added to the 293T cells and gently shaken to mix. The culture medium was changed 6-8 hours after transfection, and the culture supernatant was collected at 48 hours and 72 hours, respectively. Cell debris was removed by centrifugation. The collected supernatant was added to lentiviral concentrate (PEG8000 and NaCl), mixed well, and incubated overnight at 4°C to concentrate the virus. The virus was then centrifuged at 4°C and 4000g for 20 minutes. After centrifugation, the supernatant was discarded, and the virus pellet was resuspended in an appropriate amount of PBS or culture medium to obtain the recombinant lentiviral vector containing anti-EpCAM CAR. The lentiviral titer was verified to meet the requirements of subsequent experiments using a rapid lentiviral titer test card (Biodragon, catalog number BF06202-100).

[0132] Example 4: Expression and downstream signal activation detection of the anti-EpCAM CAR transduced dual reporter cell line J-NN

[0133] The Jurkat-N / N dual reporter cell line is a Jurkat dual reporter signaling cell line (J-NN) carrying pNFAT-EGFP and pNFκB-mCherry. It can rapidly and in high throughput identify activation signal characteristics for the design of novel CAR molecules, and is an experimental method for rapid screening of novel CARs.

[0134] Using the packaging and concentration method described in Example 3, a series of lentiviral vectors were obtained and used to transduce anti-EpCAM CAR into J-NN cells, constructing a series of anti-EpCAM CAR J-NN cells. Cells were collected 48 hours after transduction, and the positive rate of anti-EpCAM CAR J-NN cells was detected using flow cytometry staining as described in Example 2, with G4S antibody (Anti-(G4S)n-(BO2H1)m-Ab(PE), Heyousheng Biotechnology, catalog number GS-ARPE100). Taking CAR 54 as an example... Figure 4 As shown, the positive rate of anti-EpCAM CAR expression was over 20%.

[0135] The aforementioned anti-EpCAM CAR J-NN cells were co-cultured with EpCAM-positive target cells AsPC-1, BxPC-3, CFPAC-1, Capan-2, and EpCAM-negative target cells hTERT-HPNE, respectively. Simultaneously, an anti-EpCAM CAR J-NN cell group without target cells and a control group of J-NN cells without CAR transduction were established. One day in advance, target cells were seeded in 96-well plates at a density of 5000-10000 cells per well, with three parallel wells per group. On the second day, anti-EpCAM CAR J-NN cells and J-NN cells were added at an effector-to-target ratio of 5:1. The changes in green and red fluorescence intensity of J-NN cells were dynamically monitored using the Incucyte S3 live cell analysis system (Sartorius). The imaging channel was BF+GFP+RFP, with an imaging interval of 2 hours and a recording duration of 3 days. Results are as follows: Figure 5 As shown, anti-EpCAM CAR J-NN cells 51-59 (i.e., HCAR051-059 in the figure) were co-cultured with EpCAM-positive target cells AsPC-1, BxPC-3, CFPAC-1, and Capan-2, respectively. Most of these cells were activated, suggesting that anti-EpCAM CAR 52-59 can recognize EpCAM and activate CAR J-NN cells. The strength of the activation signal is related to the VHH clone of the anti-EpCAM CAR and the abundance of EpCAM expression in EpCAM-positive target cells. In one experiment of this embodiment (e.g....), Figure 6 and Figure 7 As shown in the figure, EpCAM CARs 53, 54, 55, and 56 activated various EpCAM-positive target cells, including BxPC-3 and Capan-2, and all significantly activated anti-EpCAM CAR J-NN cells to express green and red fluorescent reporter genes. No green or red fluorescent signals were produced in the EpCAM-negative target cell group, the untreated target cell group, and the J-NN cell control group, indicating that the activation of the two downstream signals in anti-EpCAM CAR J-NN cells depends on the specific binding of EpCAM antigen and anti-EpCAM CAR. Similarly, the activation of downstream signals was detected after co-culturing nine types or four types of anti-EpCAM CAR J-NN cells with BxPC-3, etc. The results are as follows: Figure 8 As shown, the anti-EpCAM CARs prepared from the four clones of anti-EpCAM nanobodies (53-56) were all able to bind to the EpCAM antigen and activate J-NN.

[0136] In some subgroups of this embodiment, the same method described above was used to transfect J-NN reporter cells with a tandem dual VHH CAR lentiviral vector. Pancreatic cancer, gastric cancer, and colorectal cancer cell lines were used as target cells to activate the J-NN reporter cells. The changes in the green and red fluorescence intensity of the J-NN cells were dynamically monitored using the Incucyte S3 live cell analysis system (Sartorius). The results are as follows: Figure 9 As shown, the dual VHH tandem CARs with codes 5355, 5553, 5455, 5554, 5356, and 5653 can all bind to EpCAM-positive cells and activate J-NN.

[0137] Example 5: Preparation of anti-EpCAM CAR-T cells

[0138] T cells were sorted from freshly isolated or resuscitated human peripheral blood mononuclear cells (PBMCs) using a magnetic bead negative selection method (Pan T Cell Isolation Kit, Miltenyi, catalog number 130-096-535). CD3 / CD28 magnetic beads (Dynabeads Human T-activator CD3 / CD28, Thermo Fisher Scientific, catalog number 11132D) were added to purified CD3 cells at a bead:cell ratio of 1:1. + T cells were activated and cultured in serum-free medium (X-VIVO 15, Lonza, catalog number 04-418Q) supplemented with IL-2 (Tetracycline Biotech, catalog number S10970015) at a final concentration of 200 IU / mL. After 24 h of magnetic bead activation culture, anti-EpCAM CAR lentivirus from Example 3 and polybrene (Merck, catalog number TR-1003-G) staining agent at a final concentration of 6 μg / mL were added. After 16 h of lentivirus infection, the medium was changed and cultured further.

[0139] After 4 days of activation and culture, the magnetic beads were removed. 72 hours after lentiviral infection, the positive rate of anti-EpCAM CAR-T cells was detected using flow cytometry staining as described in Example 2, with G4S antibody (Anti-(G4S)n-(BO2H1)m-Ab(PE), Heyousheng Biotechnology, catalog number GS-ARPE100). Taking CAR 54, 55, and 58 as examples... Figure 10 As shown, the positive rate of anti-EpCAM CAR-T expression was above 20%. T cells were further cultured in serum-free medium containing 200 IU / mL IL-2 for subsequent CAR-T function assays.

[0140] Example 6: In vitro killing of different target cells by anti-EpCAM CAR-T cells

[0141] Reporter target cells carrying green fluorescent protein were constructed to obtain EpCAM-positive target cells (human pancreatic cancer cells BxPC-3-GFP, Capan-2-GFP, human colorectal cancer cells HT-29-GFP, human gastric cancer cells MKN-45-GFP) and EpCAM-negative target cells (hTERT-HPNE-GFP). The anti-EpCAM CAR-T cells obtained in Example 5 were co-cultured with BxPC-3-GFP, Capan-2-GFP, HT-29-GFP, MKN-45-GFP, and hTERT-HPNE-GFP, respectively. A control group of T cells without CAR transduction and a target cell group without effector cells were also set up. One day in advance, the target cells were seeded in 96-well plates at a density of 2000-5000 cells per well, with three parallel wells per group. On the second day, anti-EpCAM CAR-T cells and T cells were added at effector-to-target ratios of 1:1, 2:1, and 4:1, respectively. The changes in green fluorescence intensity of the target cells were dynamically monitored using the Incucyte S3 live cell analysis system (Sartorius), with the imaging channel selected as BF+GFP. The imaging interval was 4 hours, and the imaging duration was 3-4 days. The in vitro killing efficiency at 72 hours or 96 hours was calculated using the following formula: In vitro killing efficiency (Cytotoxicity) = (Normalized single-target fluorescence intensity - Normalized experimental group fluorescence intensity) / Normalized single-target fluorescence intensity × 100%.

[0142] In one experiment of this embodiment, the killing effect of several CAR-T cells (52, 53, 54, 55, 56, 57, 58, and 59) on target cells BxPC-3 was verified, and their killing efficiency on target cells over 48 hours was summarized. Figure 11 As shown, based on the kill efficiency range, these CAR-Ts can be roughly divided into four groups: 52 and 53 in one group, 54 in another, 55 and 56 in another, and 57, 58, and 59 in yet another. Combined with the CDRs sequence information in Table 2, it can be seen that the CDRs within each group are relatively consistent. CAR-T 51 did not show significant effects in activation and early kill experiments, and was not further investigated.

[0143] In another experiment of this embodiment, compared with the T cell control group, anti-EpCAM CAR-T cells showed higher specific killing effect on EpCAM-positive target cells BxPC-3-GFP, Capan-2-GFP, HT-29-GFP, and MKN-45-GFP. The in vitro killing efficiency increased with the increase of effector-to-target ratio and time, while having no killing effect on EpCAM-negative target cells hTERT-HPNE-GFP. The killing efficiency results are as follows: Figure 12As shown, at the endpoint of the killing experiment, compared with the T cell control group, anti-EpCAM CAR-T cells showed high specific killing of four EpCAM-positive target cells: BxPC-3-GFP, Capan-2-GFP, HT-29-GFP, and MKN-45-GFP, and exhibited a significant dose-response effect. The in vitro killing efficiency increased with the increase of the effector-to-target ratio, while it had no killing effect on EpCAM-negative target cells hTERT-HPNE-GFP.

[0144] Similarly, the in vitro killing efficacy of six dual-VHH tandem anti-EpCAM CAR-T cells (5355, 5455, 5356, etc.) against BxPC-3-GFP, Capan-2-GFP, HT-29-GFP, and MKN-45-GFP was tested. The results showed that anti-EpCAM CAR-T cells had good specific killing effect on EpCAM-positive target cells at the in vitro cellular level. Figure 13 The results of the kill kinetics changes are shown at an effective-to-target ratio of 4:1. Figure 14 The study demonstrates the killing efficiency of different anti-EpCAM CAR-T agents under different effective-to-target ratios at the endpoint.

[0145] Example 7: In vitro killing of tumor spheres by anti-EpCAM CAR-T cells

[0146] EpCAM-positive target cells carrying GFP fluorescence were seeded at a density of 3000 cells per well in 96-well transparent round-bottom ultra-low adsorption microplates (Corning, catalog number 7007). Tumor spheres with a diameter of 200-500 μm were observed 1-3 days after seeding. Anti-EpCAM CAR-T cells and T cells were added at effector-to-target ratios of 1:1, 2:1, and 4:1, respectively. The changes in the green fluorescence intensity of the tumor spheres were dynamically monitored using the Incucyte S3 live cell analysis system (Sartorius). The imaging mode was Spheroid, the imaging channel was BF+GFP, the imaging interval was 4 hours, and the imaging duration was 4 days.

[0147] In one experiment of this embodiment, the CAR-T cells used were 53, 54, 55, and 56 CAR-T cells, or dual VHH tandem CAR-T cells 5355, 5553, 5455, 5554, 5356, and 5653. The EpCAM-positive target cells used were BxPC-3-GFP and Capan-2-GFP. The in vitro killing results of anti-EpCAM CAR-T cells on tumor spheres are as follows: Figure 15As shown, compared with the T cell control group, anti-EpCAM CAR-T cells can effectively eliminate GFP fluorescence in tumor spheres, achieving a three-dimensional in vitro killing effect on tumor cells. When comparing the killing effect of CAR-T 53, 54, 55, and 56, or CAR-T 5355, 5553, 5455, 5554, 5356, and 5653 (with dual VHH tandem), on tumor spheres of BxPC-3-GFP and Capan-2-GFP cells, it can be seen that CAR-T tandem with dual VHH is superior to CAR-T with a single VHH. Specifically, this is reflected in the speed of tumor killing and elimination. For example, the half-maximum killing time (KT50) of CAR-T 54 alone on BxPC-3-GFP tumor spheres is about 24 hours, the KT50 of CAR-T 53, 55, and 56 is about 40 hours, while the KT50 of CAR-T 5355, 5455, 5554, and 5356 tandem with dual VHH on BxPC-3-GFP tumor spheres is about 20 hours. The KT50 of both types of CAR-T cells against Capan-2-GFP tumor spheres increased from 28 h to 24 h. These results suggest that the tandem VHH increases the binding affinity to EpCAM, thereby further enhancing the CAR-T cell-killing ability.

[0148] Example 8: In vivo tumor killing by anti-EpCAM CAR-T cells

[0149] EpCAM-positive Luc-GFP dual reporter gene target cells, including pancreatic cancer BxPC-3-GFP and Capan-2-GFP, colorectal cancer HT-29-GFP, and gastric cancer MKN-45-GFP, were selected at a ratio of 1×10⁻⁶. 6 cNKG mice (Cyagen Biosciences) were inoculated with a dose of CAR-T cells / mouse and, approximately 5 days later, were divided into CAR-T treatment group, T treatment group, and non-treatment group. The CAR-T treatment group and the T treatment group were respectively injected into the tail vein with 5 × 10⁶ CAR-T cells and 5 × 10⁶ T cells. 6 Each mouse was given one dose of PBS, while the non-treatment group received the same volume. Mice underwent bioluminescence imaging or tumor size measurement before and every week after administration. The major axis *a* and the minor axis *b* perpendicular to the major axis were calculated using the formula 1 / 2ab. 2 Tumor volume was calculated. In one experiment of this embodiment, the EpCAM-positive Luc-GFP dual reporter gene target cells used were gastric cancer MKN-45-GFP cells, and the CAR-T cells used were dual VHH tandem CAR-T cells 5455 and 5554. It can be seen that tandem VHH has a significant killing and inhibitory effect on tumors. The results are as follows: Figure 16 As shown, CAR-T 5455 and 5554 completely eliminated tumors in mice.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0151] The references involved in this invention are as follows:

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Claims

1. A Nanobody binding to EpCAM, characterized in that, The nanobody comprises a heavy chain variable region, the complementarity determining regions CDR of which are as follows: The amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO. 19, 20, 21 in turn when numbered according to the Martin numbering scheme, defined according to the Chothia definition scheme; The amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO. 22, 23, 24 in turn when numbered according to the Martin numbering scheme, defined according to the Contact definition scheme.

2. The EpCAM-binding Nanobody according to claim 1, characterized in that, The heavy chain variable region further comprises a framework region FR, which is as follows: The amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO. 53, 54, 55, 56 in turn when numbered according to the Martin numbering scheme, defined according to the Chothia definition scheme; The amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NO. 57, 58, 59, 60 in turn when numbered according to the Martin numbering scheme, defined according to the Contact definition scheme.

3. The EpCAM-binding Nanobody according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.

78.

4. An antibody that binds EpCAM, characterized in that, The antibody is an antibody formed by the tandem of the EpCAM-binding nanobody according to any one of claims 1-3 and any one of the following (1) and (2): (1) the nanobody comprises a heavy chain variable region, the complementarity determining regions CDR of which are as follows: the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO. 13, 14, 15 in turn when numbered according to the Martin numbering scheme, defined according to the Chothia definition scheme; the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO. 16, 17, 18 in turn when numbered according to the Martin numbering scheme, defined according to the Contact definition scheme; (2) the nanobody comprises a heavy chain variable region, the complementarity determining regions CDR of which are as follows: the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO. 7, 8, 9 in turn when numbered according to the Martin numbering scheme, defined according to the Chothia definition scheme; The amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO. 10, 11, 12 in turn when numbered according to the Martin numbering scheme, defined according to the Contact definition scheme.

5. The antibody binding EpCAM according to claim 4, characterized in that, The heavy chain variable region of the nanobody in (1) further comprises framework regions FR, which are: in the order of FR1, FR2, FR3 and FR4, the amino acid sequences of which are shown in SEQ ID NO. 48, 49, 50, 43, respectively, when numbered according to the Martin numbering scheme, defined according to the Chothia definition scheme; in the order of FR1, FR2, FR3 and FR4, the amino acid sequences of which are shown in SEQ ID NO. 51, 37, 52, 47, respectively, when numbered according to the Martin numbering scheme, defined according to the Contact definition scheme. The heavy chain variable region of the nanobody in (2) further comprises framework regions FR, which are: in the order of FR1, FR2, FR3 and FR4, the amino acid sequences of which are shown in SEQ ID NO. 40, 41, 72, 43, respectively, when numbered according to the Martin numbering scheme, defined according to the Chothia definition scheme; in the order of FR1, FR2, FR3 and FR4, the amino acid sequences of which are shown in SEQ ID NO. 44, 45, 73, 47, respectively, when numbered according to the Martin numbering scheme, defined according to the Contact definition scheme. The amino acid sequence of the heavy chain variable region of the nanobody in (1) is shown in SEQ ID NO. 77; the amino acid sequence of the heavy chain variable region of the nanobody in (2) is shown in SEQ ID NO.

76.

6. The antibody binding EpCAM according to claim 4 or 5, characterized in that, The chimeric antigen receptor comprises the EpCAM-binding nanobody of any one of claims 1-3 or the EpCAM-binding antibody of any one of claims 4-6.

7. A chimeric antigen receptor targeting EpCAM, characterized in that, The chimeric antigen receptor comprises a signal peptide, the EpCAM-binding nanobody of any one of claims 1-3 or the EpCAM-binding antibody of any one of claims 4-6, a hinge region, a transmembrane region, an intracellular costimulatory domain and an intracellular signaling domain.

8. The chimeric antigen receptor of claim 7, wherein, The nucleic acid molecule of claim 9; the biological material is an expression cassette, a vector or a host cell.

9. A nucleic acid molecule, characterized in that, The recombinant cell expresses the chimeric antigen receptor of claim 7 or 8.

10. Biomaterials characterized in that, The method comprises: culturing the host cell comprising the nucleic acid molecule of claim 9, and collecting the EpCAM-binding nanobody or the EpCAM-binding antibody or the chimeric antigen receptor from the culture.

11. A recombinant cell, wherein, The disease associated with EpCAM expression is colorectal cancer, gastric cancer or pancreatic cancer.

12. A method of producing the EpCAM-binding Nanobody according to any one of claims 1 to 3 or the EpCAM-binding antibody according to any one of claims 4 to 6 or the chimeric antigen receptor according to claim 7 or 8, characterized in that, The disease associated with EpCAM expression is colorectal cancer, gastric cancer or pancreatic cancer.

13. Use of the EpCAM-binding Nanobody according to any one of claims 1 to 3 or the EpCAM-binding antibody according to any one of claims 4 to 6 or the chimeric antigen receptor according to claim 7 or 8 or the nucleic acid molecule according to claim 9 or the biological material according to claim 10 or the recombinant cell according to claim 11 for the manufacture of a medicament for the prevention or treatment of a disease associated with EpCAM expression; wherein, ​ 14. Use of the EpCAM-binding Nanobody according to any one of claims 1 to 3 or the EpCAM-binding antibody according to any one of claims 4 to 6 or the chimeric antigen receptor according to claim 7 or 8 or the nucleic acid molecule according to claim 9 or the biological material according to claim 10 or the recombinant cell according to claim 11 for the manufacture of CAR-T cells for the treatment of a disease associated with EpCAM expression; wherein, ​ 15. Use of the EpCAM-binding Nanobody according to any one of claims 1 to 3 or the EpCAM-binding antibody according to any one of claims 4 to 6 or the chimeric antigen receptor according to claim 7 or 8 or the nucleic acid molecule according to claim 9 or the biological material according to claim 10 or the recombinant cell according to claim 11 for the manufacture of a reagent for detecting the presence or level of EpCAM in a sample.

16. Use of the EpCAM-binding Nanobody according to any one of claims 1 to 3 or the EpCAM-binding antibody according to any one of claims 4 to 6 or the chimeric antigen receptor according to claim 7 or 8 or the nucleic acid molecule according to claim 9 or the biological material according to claim 10 or the recombinant cell according to claim 11 for the manufacture of a reagent for detecting the presence or level of EpCAM+ cells in a sample.

17. Use of the EpCAM-binding Nanobody according to any one of claims 1 to 3 or the EpCAM-binding antibody according to any one of claims 4 to 6 or the chimeric antigen receptor according to claim 7 or 8 or the nucleic acid molecule according to claim 9 or the biological material according to claim 10 or the recombinant cell according to claim 11 for the manufacture of a tumour detection reagent; wherein, The tumor is a colorectal cancer, a gastric cancer or a pancreatic cancer.

18. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the EpCAM-binding Nanobody according to any one of claims 1 to 3 or the EpCAM-binding antibody according to any one of claims 4 to 6 or the chimeric antigen receptor according to claim 7 or 8 or the recombinant cell according to claim 11.

19. An assay reagent, characterized in that, The detection reagent comprises the EpCAM-binding Nanobody according to any one of claims 1 to 3 or the EpCAM-binding antibody according to any one of claims 4 to 6.

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