WO2023044991A1 - Antibody specifically targeting tumor epcam antigen and application thereof - Google Patents

Abstract

Provided is a chimeric antigen receptor specifically targeting a tumor EpCAM antigen, comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen recognition domain comprising a VHH antibody or a fragment thereof, and the VHH antibody or the fragment thereof having a complementarity determining region (CDR) that specifically targets the tumor EpCAM antigen, which can effectively recognize tumor cells with positive EpCAM expression, and does not attack normal epithelial cells with low EpCAM expression.

Description

Antibody and application of a kind of specific targeting tumor EpCAM antigen

priority application

This application claims the priority of the Chinese invention patent application [CN2021111200425] "an antibody specifically targeting tumor EpCAM antigen and its application" submitted on September 24, 2021. The priority invention patent application is incorporated by reference in its entirety.

technical field

The invention belongs to the fields of genetic engineering and biological immunotherapy, and in particular relates to an antibody specifically targeting tumor EpCAM antigen and application thereof.

Background technique

In the process of tumor immunotherapy, inducing patients to produce effective anti-tumor immunity is the key to disease treatment. Tumor cells can develop a series of escape mechanisms to evade immune system surveillance. Including down-regulation of major histocompatibility complex (MHC), down-regulation of self-antigen expression, down-regulation of immune checkpoint molecule ligands, formation of a local inhibitory microenvironment, etc., thereby limiting the recognition and attack of tumor cells by self-effector cells ¹ .

The currently known tumor antigen targets are mainly divided into two categories, one is an antigen that is only expressed in tumor cells but not in normal cells, which is specific to tumor cells and is considered to be tumor-specific antigens; One type is antigens that are highly expressed in tumor cells but low or not expressed in normal cells, and their expression is tumor-associated, called tumor-associated antigens. EpCAM is a kind of tumor-associated antigen. It is highly expressed in tumor cells such as colorectal cancer and ovarian cancer, and is also expressed in human epithelial cells. Although the expression level is relatively low, it also lacks tumor specificity. Therefore, choosing EpCAM as an immunotherapy target needs to consider its immune tolerance.

The treatment targeting EpCAM-positive tumors is currently focused on the prescription and efficacy evaluation of antibody drugs. Such as Edrecolomab, Adecatumumab and Catumaxomab, and antibody-drug conjugates (ADC). The EpCAM monoclonal antibody drug Edrecolomab did not observe its improvement on the overall survival rate and unloaded survival time of patients in the adjuvant treatment of stage III colon cancer ^19-21 . Adecatumumab was found to be dose-dependent and target-antigen-dependent in clinical studies of monotherapy in metastatic breast cancer, but objective tumor regression was not observed ²² . Catumaxomab is a bispecific antibody drug targeting CD3 and EpCAM. It has shown obvious clinical benefits in the treatment of malignant ascites secondary to epithelial cell carcinoma, and its safety is ^{controllable23,24} . It was approved by EMA for marketing in 2009 For the treatment of malignant ascites, it is regrettable that the drug was withdrawn from the market in 2017 due to poor sales. In addition, anti-EpCAM antibody-drug conjugates such as Oportuzumab monatox ²⁵ and Tucotuzumab ²⁶ have shown good tolerance in clinical studies, but more clinical data are needed to confirm the improvement of patients' disease and prolongation of survival. In general, antibody drugs targeting EpCAM have made some progress, but new immunotherapy drugs/methods still need to be developed to supplement the deficiencies of existing medical technologies.

Chimeric antigen receptor-modified T cell (CAR-T) therapy is considered to be an effective means of tumor immunotherapy. The therapy involves expressing in T cells a class of recombinant receptors known as chimeric antigen receptors (CARs), which redirect T cells to tumor cells at a chosen target. The CAR molecule is composed of multiple structural elements, which are the extracellular antigen recognition domain, the hinge region, the transmembrane domain, and the intracellular signal transduction domain. Antigen recognition domains include but are not limited to immunoglobulin-derived single-chain variable fragments (Single-chain variable fragment, scFv), which are connected by variable regions of immunoglobulin-derived heavy and light chains through G4S and other structures composition; and other non-antibody-derived molecules, such as the natural killer cell receptor NKG2D, which can bind to the ligand NKG2DL on the surface of target cells to recognize tumor antigens, etc. ² . The hinge region provides a flexible link between the extracellular domain and the intracellular domain of the CAR molecule, and is usually the source of the _CH2 and _CH3 domains of the immunoglobulin heavy chain. The transmembrane region can anchor the CAR molecule on the cell membrane, and its source is usually the transmembrane region of CD4, CD8, or CD28, 4-1BB and other co-stimulatory molecules. The intracellular signal transduction domain is composed of co-stimulatory molecules such as CD28, 4-1BB, ICOS, etc. and intracellular activating molecules such as CD3ζ or FcRγ, etc., which can transduce and amplify the antigen ligand recognition signal of the extracellular domain, so that T cells or NK Cells etc. are activated.

CAR molecules usually provide specific antigen recognition function by the extracellular antigen recognition domain, and redirect T cells or NK cells to the tumor cell position where the antigen is expressed. The recognition of the target antigen can activate the signal transduction downstream of the CAR molecule and activate the effector function of T cells or NK cells. Activated T cells or NK cells directly act on target cells or recruit other immune effector cells to participate in the immune response by secreting IFN-γ, TNF-α, IL-2, granzyme, perforin and other effector molecules, eventually leading to target cell lysis die. Therefore, the main idea of modifying the molecular structure of CAR is to enhance the function of CAR-T cells and the cytotoxicity of target cells.

In summary, the present invention will provide a novel nanobody applied to the design of CAR molecular structure, so that it can specifically recognize tumor cells positively expressing EpCAM, so as to supplement the deficiencies of existing medical technologies.

Contents of the invention

In view of this, the present invention aims to provide an antibody specifically targeting tumor EpCAM antigen and its application, and the specific technical scheme is as follows.

A chimeric antigen receptor specifically targeting tumor EpCAM antigens, comprising extracellular antigen recognition domains, transmembrane domains and intracellular signaling domains, the extracellular antigen recognition domains comprising VHH antibodies or fragments thereof, The VHH antibody or its fragment has a complementarity determining region CDR specifically targeting tumor EpCAM antigen.

The full name of VHH is heavy chain single domain antibody, which is a type of antibody derived from alpaca. Unlike traditional IgG, VHH only contains heavy chain, which is small in size and smaller in molecular weight (~15kDa), so it is also called nanobody. Compared with traditional monoclonal antibodies, it has unique advantages, such as easier access to antigenic epitopes, low immunogenicity, good solubility, high stability, and affinity not inferior to scFv, etc.

The mechanism of specific recognition between VHH antibody and EpCAM target is the process of recognizing and binding the CDR of the heavy chain variable region of the antibody to the tumor antigen. The CDR structure of VHH determines its specificity of targeting EpCAM.

Further, the VHH antibody or fragment thereof is optionally selected from polypeptides or epitopes composed of any amino acid sequence in Seq ID NO.1-9.

Further, the complementary determining region CDR is optionally selected from one or more of the amino acid sequences in Seq ID NO.10-18.

Existing studies have confirmed that there are situations where multiple targets (generally two) are targeted. In this case, the recognition region containing multiple antibodies will be designed in the CAR, that is, the CDR region of multiple antibodies. These CDRs The regions can be connected in series, or they can be integrated in a CAR structure and expressed in cells, and then separately recognize antigens.

Further, the transmembrane domain optionally includes CD3ζ, CD3ε, CD4, CD8α, CD28, CD5, CD16, CD9, CD22, CD33, CD27, CD37, CD45, CD64, CD80, CD86, CD127, CD137, CD134, One or more of CD152, CD154, PD-1 or Dectin-1.

Further, the intracellular signaling domain optionally includes one or more of CD3ζ, CD27, CD28, CD30, CD137, CD134, CD154, Dectin-1, FcRγ or ICOS.

Further, the nucleotides of the VHH antibody or fragment thereof are optionally selected from any sequence in Seq ID NO.19-27.

A method of gene transfer comprising the above-mentioned chimeric antigen receptor.

Further, the gene transfer method may optionally include viral vectors, transposon systems, electroporation or CRISPR/Cas9 gene editing tools.

A CAR-T cell expressing the above-mentioned chimeric antigen receptor.

Further, the amino acid sequence of the expressed chimeric antigen receptor is shown in any one of Seq ID NO.28-36.

Further, the nucleotide sequence of the expressed chimeric antigen receptor is shown in any one of Seq ID NO.37-45.

A CAR-NK cell expressing the above-mentioned chimeric antigen receptor.

Further, the amino acid sequence of the expressed chimeric antigen receptor is shown in any one of Seq ID NO.28-36.

Further, the nucleotide sequence of the expressed chimeric antigen receptor is shown in any one of Seq ID NO.37-45.

An anti-tumor drug, containing the CAR-T cells described in the present invention and pharmaceutically allowed adjuvants and/or adjuvants.

Further, the tumors include solid tumors derived from epithelial cells, and the expression of EpCAM is positive.

Further, the tumors include epithelial-derived malignant tumors and circulating tumor cells, as well as tumor stem cells.

Further, the tumor includes intestinal cancer, lung cancer, ovarian cancer, liver cancer or gastric cancer.

EpCAM, the full name of epithelial cell adhesion molecule (Epithelial cell adhesion molecule), is a 40KD single transmembrane glycoprotein encoded by the tumor-associated calcium signal transduction 1 gene, which is involved in regulating cell adhesion and migration, regulating proliferation and differentiation, and mediating Signal Transduction ¹⁴ . It was identified as a tumor-associated antigen in 197915 and expressed in most malignant tumor cells of epithelial origin such ^as intestinal cancer, lung cancer, prostate cancer and ovarian ^cancer16 .

The synthesis method of the chimeric antigen receptor of the present invention comprises the following steps: (1) constructing the complete gene of Anti-EpCAM-VHH-CAR; (2) using primers to Primer1 and Primer2 to amplify the Anti-EpCAM-VHH -CAR gene; (3) using BamHI and EcoRI restriction enzymes to digest the amplified gene sequence and package it with a viral vector.

Further, the viral vectors include lentiviral vectors, adenoviral vectors or retroviral vectors.

Further, the sequence of the primer Primer1 is shown in Seq ID NO.46, and the sequence of the primer Primer2 is shown in Seq ID NO.47.

Application of a VHH antibody or its fragment specifically targeting tumor EpCAM antigen in the preparation of bispecific antibody and antibody-drug conjugated drug ADC, the VHH antibody or its fragment has a complementary antibody specifically targeting tumor EpCAM antigen Determining region CDR, the complementary determining region CDR is optionally selected from one or more of the amino acid sequences in Seq ID NO.10-18.

Application of a VHH antibody or fragment thereof specifically targeting tumor EpCAM antigen in the preparation of a tumor diagnostic kit, wherein the VHH antibody or fragment thereof has a complementarity-determining region CDR specifically targeting tumor EpCAM antigen, and the complementarity-determining region CDR is optionally selected from one or more of the amino acid sequences in Seq ID NO.10-18.

Further, the tumor is a solid tumor derived from epithelial cells, and the expression of EpCAM is positive.

Beneficial effect

The present invention firstly provides an antibody specifically targeting tumor EpCAM antigen. As known to those skilled in the art, EpCAM is a tumor-associated antigen, which is highly expressed in tumor cells, but also expressed in normal epithelial cells. Although the expression level is relatively low, this target lacks tumor specificity. When the team of the present invention selected the antibody targeting EpCAM, they focused on the affinity of the antibody. When the antibody affinity is too high, although it can recognize tumor cells with high EpCAM expression, it will also cause CAR-T cells to recognize and attack normal epithelial cells with low EpCAM expression; while when the antibody affinity is too low, it cannot effectively recognize EpCAM-expressing tumor cells, leading to treatment failure. Therefore, the anti-EpCAM VHH antibody screened by the present invention has a moderate affinity, which can ensure that tumor cells can be effectively recognized, and at the same time, it cannot recognize and attack normal epithelial cells with low EpCAM expression.

The present invention utilizes the nano-scale VHH antibody or fragment thereof with moderate affinity screened to construct the nucleic acid sequence of the second-generation and third-generation chimeric antigen receptor molecules. Expression in effector cells can endow these immune cells with the ability to target, recognize and lyse tumor cells expressing EpCAM molecules, so as to achieve the purpose of treating tumor diseases.

Specifically, the Anti-EpCAM-VHH nanobody constructed in the present invention has high affinity to EpCAM positive cells, and Anti-EpCAM-VHH-CAR is used to transduce the T lymphocytes of the patient to be treated, and the transduced T lymphocytes Reinfusion to patients can redirect the successfully transduced T cells to EpCAM-positive target cells to generate an immune response. This method can enhance the immune response of T cells to target cells. The process of using Anti-EpCAM-VHH-CAR to transduce T cells to express chimeric antigen receptors can be completed in vitro or in vivo, and the cells that are finally successfully transduced are Anti-EpCAM-VHH-CAR-T cells.

In addition, the antibodies or fragments thereof screened by the present invention can also be used in the research of targeted therapy products such as bispecific binding antibodies and antibody-drug conjugated drugs, and can also be applied to antibody detection products.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the description of the embodiments or the prior art. Apparently, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative effort.

Figure 1 shows the identification of the molecular weight of the Anti-EpCAM-VHH hFc protein: the VHH-hFc fusion protein expressed by HEK293 cells was reduced by dithiothreitol (DTT), and the relative molecular weight was identified by SDS-PAGE electrophoresis between 43-55kD;

Figure 2 is the ELISA detection results of different VHH hFc binding to EpCAM-His;

Figure 3 shows the structure of Anti-EpCAM-VHH-CAR: the Anti-EpCAM-VHH-CAR gene is loaded on the self-constructed lentiviral vector of the present invention, the EF1-α promoter is used to promote the expression of the CAR gene, and the extracellular domain is guided by IL-2 Sequence and EpCAM-VHH, the intracellular domain is the hinge region and transmembrane region of CD8a, CD28 and 4-1BB, and the amino acid residues of the intracellular segment of CD3ζ;

Figure 4 shows the expression pattern of Anti-EpCAM-VHH-CAR on the cell membrane: the EpCAM-VHH and CD8a hinge regions of Anti-EpCAM-VHH-CAR are located outside the cell membrane for recognizing target antigens, and the CD8a transmembrane region is embedded on the cell membrane , for immobilizing CAR molecules on the membrane, the signal transduction domain is located in the membrane, and is used to transmit and amplify cell activation signals;

Figure 5 is the expression detection of Anti-EpCAM-VHH-CAR in T cells: using flow cytometry, the expression of CAR molecules can be detected using isothiocyanate (FITC)-labeled rabbit anti-alpaca antibody;

Figure 6 shows that Anti-EpCAM-VHH-CAR-T targets EpCAM positive cells to release cytokines;

Figure 7 is the cytotoxicity detection of Anti-EpCAM-VHH-CAR-T.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

It should be noted that, in this document, the term "comprising", "comprising" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, It also includes other elements not expressly listed, or elements inherent in the process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising that element.

As used in this specification, the term "about" typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 4% of the stated value /-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.

的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。 In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description "within a certain range" is merely for convenience and brevity, and should not be construed as an inflexible limitation on the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, range

The description should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and within this range Individual numbers such as 1, 2, 3, 4, 5 and 6. The above rules apply regardless of the breadth of the scope.

Glossary

The "specifically targeting tumor EpCAM antigen" in the present invention means that the VHH antibody or its fragments provided by the present invention can target EpCAM with high abundance expression on tumor cells, and have the ability to recognize epithelial cells with low expression of other EpCAM weaker.

The "epitope" in the present invention refers to the site on the antigen that is recognized, bound by and interacts with the antibody.

In one embodiment of the present invention, the extracellular antigen recognition domain Anti-EpCAM-VHH of the Anti-EpCAM-VHH-CAR molecule can recognize and bind to the human EpCAM molecule.

In one embodiment of the present invention, the hinge region and the transmembrane region are derived from CD8α, or other molecules other than CD8α, such as CD4, CD28, etc. Sources of co-stimulatory signal transduction domains include but are not limited to CD28, 4-1BB, ICOS, OX40, etc., and the number of co-stimulatory molecules can be one or more.

In one embodiment of the present invention, the CD3ζ signal transduction domain is the 61-164 amino acid residues of the intracellular segment of the CD3ζ molecule. The 61-164 amino acid residues of CD3ζ contain three receptor tyrosine activation motifs (ITAM), which are amino acid residues 61-89, 100-128, and 131-159, respectively, and the three ITAMs can independently mediate the activation of CD3ζ Signal Transduction ²⁸ . Therefore, CAR molecules constructed by using CD3ζ intracellular segment in the CAR molecular structure, or using CD3ζITAM alone or in any combination are included in the scope of the present invention.

In one embodiment of the present invention, the CD3ζ signal transduction domain can also be replaced by other signal transduction structures, such as FcRγ, and the number can be one or more. Chimeric antigen receptors constructed by altering regions other than the extracellular antigen recognition domain are within the scope of the present invention.

In one embodiment of the present invention, by expressing the Anti-EpCAM-VHH-CAR nucleic acid sequence in T lymphocytes, Anti-EpCAM-VHH-CAR molecules can be produced in T cells. In addition, the nucleic acid sequence can also be expressed in NK cells, so the constructed CAR-NK cells are also within the scope of the present invention.

In one of the embodiments of the present invention, the Anti-EpCAM-VHH-CAR nucleic acid molecule is constructed on a lentiviral vector plasmid, and the EF1-α promoter is used to promote CAR gene expression. In addition to the lentiviral vector, other gene transfer methods are used The CAR of the present invention can be expressed by any means, such as retroviral vectors, transposon systems, electroporation, CRISPR/Cas9 and other gene editing tools, which are also included in the scope of the present invention.

In one embodiment of the present invention, the VHH antibody or its fragments are obtained by immunizing alpacas regularly by immunizing proteins expressed in Escherichia coli.

In one embodiment of the present invention, the VHH antibody or its fragment is transcribed and amplified into a single domain antibody gene fragment.

Illustrate below by specific embodiment:

Example 1

Preparation of VHH antibody specifically targeting human tumor EpCAM antigen

(1) Alpaca immunization and cDNA library construction. Mix 1.5mg of human EpCAM antigen with adjuvant 1:1 and inject it into the vicinity of the alpaca cervical lymph nodes for immunization, twice a week for a total of 7 immunizations. Peripheral blood was collected every time from the fourth immunization for immune evaluation and peripheral blood lymphocytes were isolated. Lymphocytes were lysed with Trizol solution, and the total RNA of lymphocytes was extracted. The extracted RNA was reverse-transcribed into cDNA using a reverse transcription kit, and then a cDNA library of E. coli was constructed using a cDNA library construction kit.

(2) Phage library preparation. Escherichia coli cDNA library bacteria were inoculated into 2X YT medium containing tetracycline and ampicillin antibiotics, and cultured with shaking at 37°C until DO ₆₀₀ =0.5. Add helper phage M13KO7 to the culture system to a final concentration of 1E12cfu/ml, add kanamycin after culturing at 37°C for one hour, and then culture at 30°C overnight. Afterwards, the culture solution was centrifuged at 5000 g for 10 minutes to take the supernatant, added with PEG/NaCl solution and mixed evenly, and then allowed to stand at 4° C. for 20 minutes. Then the solution was centrifuged at 4000g for 20 minutes, the supernatant was discarded, PBS was added to the precipitate to suspend the precipitate, and then centrifuged at 16000g for 10 minutes to obtain the phage library.

(3) Phage selection of VHH. Block the library phage and the immunotube coated with EpCAM-camFc (human EpCAM and alpaca Fc recombinant protein) with 3% BSA solution at room temperature for 2 hours. Wash the blocked immunotube several times with PBS, and then add the blocked phage to the immune tube, and incubate at room temperature for 1 h. After washing the immunotube several times with PBS-T solution, add 100mM TEA to elute, incubate at room temperature for 10 minutes, then add 1M Tris-HCl, and the obtained eluate is the screened phage. This screening process needs to be repeated twice to remove non-specific phages.

(4) Identification of VHH by phage ELISA. Dilute the phage obtained in step (3) and inoculate into SS320 bacterial solution with OD ₆₀₀ =0.5, incubate at 37° C. for 30 minutes, and then take the culture solution and spread it on a 2X YT plate. After culturing overnight at 37°C, pick a single clone and inoculate it in a 48-well plate containing 2X YT, culture at 37°C for 3-4 hours, then add kanamycin and helper phage to the well plate, culture overnight at 30°C, and centrifuge The cultures were obtained as supernatants. Coat the 96-well plate with EpCAM-camFc in advance, seal the well plate with 3% BSA, add the supernatant and incubate at room temperature for 1 hour, wash with PBS, bind with anti-M13 HRP, develop color with TMB, and measure the OD ₄₅₀ value , Judging the affinity of different VHHs and EpCAM according to the OD ₄₅₀ value. Comparing the OD ₄₅₀ values of the sample wells and the negative control, the protein samples corresponding to the wells with significantly increased OD ₄₅₀ were sequenced to obtain different VHH amino acid sequences. After removing sequences with the same CDR region and redundant sequences by sequence comparison, 9 single amino acid sequences (ID NO.1-9) were obtained. The sequences were analyzed using the IGMT database (International Immunogenetics Database) to determine the CDR regions of each sequence. In the sequence listed below, the fragments marked in black and bold are CDR1, CDR2, and CDR3 from left to right. Combinations of different CDR region sequences have different VHH-EpCAM affinities.

NO.1:

NO.2:

NO.3:

NO.4:

NO.5:

NO.6:

NO.7:

NO.8:

NO.9:

Example 2

Anti-EpCAM-VHH antibody identification. In order to identify Anti-EpCAM-VHH at the molecular and cellular levels, the screened Anti-EpCAM-VHH was fused to the human signal peptide and the N-terminus of human Fc (hFc, about 29kD), and transformed into a mammalian transient expression vector HEK293 cells were transiently expressed on pQDFc. After affinity purification, the VHH-hFc fusion protein was obtained. The VHH-hFc fusion protein expressed by HEK293 cells was reduced by dithiothreitol (DTT), and the relative molecular weight was identified by SDS-PAGE electrophoresis between 43-55kD (Figure 1).

Example 3

Identification of VHH-hFc binding ability. The binding ability of VHH-hFc was identified by ELISA method to identify the binding ability of VHH-hFc and EpCAM antigen. Coat EpCAM-His protein 1ng/ul in a 96-well plate, place it overnight at 4 degrees, and then use 5-fold serially diluted VHH-hFc protein (initial solubility is 20ng/ul) to mix with EpCAM-His in the well plate His protein was bound for 1 hour, and then anti-hFc-HRP secondary antibody was used to bind VHH-hFc. After color development, the OD value at a wavelength of 450nm was detected, and the binding ability of VHH-hFc and EpCAM-His protein was judged according to OD450.

Experimental conclusion: The nine Anti-EpCAM-VHH sequences screened have different degrees of affinity to EpCAM, among which 1194 has the strongest affinity, 1201 the weakest affinity, and VHH 1191 has a moderate affinity to EpCAM (Figure 2).

Example 4

Construction of chimeric antigen receptor targeting EpCAM (Anti-EpCAM-VHH-CAR) and lentiviral vector construction. During the construction of Anti-EpCAM-VHH-CAR, the extracellular segment of Anti-EpCAM-VHH, CD8α transmembrane region and intracellular signal transduction domain (8α28BBζ) were synthesized as a complete gene. Anti-EpCAM-VHH-CAR gene was amplified using Primer1: 5'-CGGGATCCATGTACCGGATGCAG-3' (SEQ ID NO.46) and Primer2: 5'-CGGAATTCTTAGCGAGGGGGC-3' (SEQ ID NO.47). BamHI and EcoRI restriction endonuclease sites were added to the primer sequences ID NO.46 and ID NO.47 respectively, and the amplified Anti-EpCAM-VHH-CAR nucleotide was introduced into the BamHI site at the 5' segment , an EcoRI site was introduced at the 3' end, and the Anti-EpCAM-VHH-CAR product and the self-constructed vector PCLK plasmid were digested with BamHI and EcoRI restriction enzymes, and the same BamHI and EcoRI cohesive ends could be obtained in the two products, which is convenient for both The two gene fragments were ligated into a complete DNA plasmid circle using T4 ligase at the same cohesive ends.

Example 5

Human lymphocyte culture and lentiviral transduction. Peripheral blood mononuclear cells were extracted from healthy people and cultured in human lymphocyte medium X-vivo-15, supplemented with 5% FBS and 100IU/ml human IL-2. The cells were stimulated with CD3/CD28 antibody-coated magnetic beads (magnetic beads: cells = 3:1) for 24 hours to activate T cells. Add lentiviral particles and activated T cells to the well plate coated with fibronectin (50ug/ml), the multiplicity of infection is 2-4, and centrifuge at 1000xg for 2 hours to promote lentiviral infection of T cells. T cells successfully infected by lentivirus express the Anti-EpCAM-VHH-CAR gene, namely Anti-EpCAM-VHH-CAR-T cells.

Example 6

Detection of chimeric antigen receptor expression on human lymphocytes was performed 48-72 hours after retroinfection. Since the Anti-EpCAM-VHH of the ectodomain of Anti-EpCAM-VHH-CAR is derived from alpaca, it can be detected using a rabbit anti-alpaca VHH antibody (Genscript, Nanjing), and the detection method is flow cytometry.

Example 7

Target-specific cytokine release assay. Since Anti-EpCAM-VHH-CAR-T has specific cytotoxic effect on cells positively expressing EpCAM, the present invention selects three kinds of tumor cells as target cells, which are human colon cancer tumor cell HT29 with high expression of EpCAM and human colon cancer tumor cell with low expression of EpCAM. EpCAM human ovarian cancer cell SKOV3 and human cervical cancer cell Hela not expressing EpCAM. Before cytokine detection, 1*10 ⁵ (E:T=10:1) or 5*10 ⁴ (E:T=5:1) Anti-EpCAM-VHH-CAR-T cells were mixed with three Tumor cells were co-cultured in a 96-well plate, and the number of tumor cells was 1* ¹⁰⁴ /well. After 24 hours of co-culture, the supernatant of the co-culture was taken to detect IFN-γ, TNF-α and IL-2, and the detection method was ELISA Adsorption test.

Experimental conclusion: Anti-EpCAM-VHH-CAR-T cells recognize and bind to EpCAM on the surface of target cells in a co-culture system, which promotes the activation of CAR-T cell effector functions. A large number of cytokines are released, such as IFN-γ, TNF-α, etc., and the degree of target-specific activation of CAR-T cells can be evaluated by detecting the secretion of cytokines in the co-culture supernatant. The CAR-T cells in Figure 6 can be stimulated by EpCAM-positive HT29 and SKOV3 cells to release cytokines, but cannot be activated by EpCAM-negative Hela cells, and the level of factor release of CAR-T cells increases with the expression of target cell antigens And increased, with antigen dependence.

Example 8

Cytotoxicity assay. Anti-EpCAM-VHH-CAR-T cytotoxicity to tumor cells was determined using a real-time cytotoxicity assay. The specific plan is to take HT29 and Hela cells and place them on the labeling detection plate. The number of cells is 1*10 ⁴ cells/well. As for the cell adhesion status recorded on the TRCA detector, after 24 hours of plating, the cell adhesion is stable according to the set effector cells. : Anti-EpCAM-CAR-T cells were added to the target cell (E:T) ratio, and then placed on a TRCA detector to record the cell adhesion. Target cells will lose their adherence after being killed by CAR-T cells.

Experimental conclusion: The cytotoxicity of CAR-T cells was evaluated by detecting the adherence of target cells in real time. Figure 7 shows that after adding T cells to target cells HT29 and Hela, the number of HT29 cells in the Control T cell group increased and then decreased slowly, while the number of HT29 cells in the CAR-T cell group decreased rapidly; adding Control T cells and CAR to Hela cells After -T cells, the cells all decreased in the same degree without significant change. It was confirmed that CAR-T cells have targeting EpCAM+ cytotoxicity.

The sequences involved in the present invention are shown in the following table:

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Embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementations, and the above-mentioned specific implementations are only illustrative, rather than restrictive, and those of ordinary skill in the art will Under the enlightenment of the present invention, many forms can also be made without departing from the gist of the present invention and the protection scope of the claims, and these all belong to the protection of the present invention.

Claims (20)

A chimeric antigen receptor specifically targeting tumor EpCAM antigens, comprising extracellular antigen recognition domains, transmembrane domains and intracellular signaling domains, characterized in that the extracellular antigen recognition domains include VHH antibodies or a fragment thereof, the VHH antibody or fragment thereof has a complementarity determining region CDR specifically targeting tumor EpCAM antigen. The chimeric antigen receptor according to claim 1, wherein the VHH antibody or fragment thereof is optionally selected from polypeptides or epitopes composed of any amino acid sequence in Seq ID NO.1-9. The chimeric antigen receptor according to claim 1, wherein the complementarity determining region CDR is optionally selected from one or more of the amino acid sequences in Seq ID NO.10-18. The chimeric antigen receptor according to claim 1, wherein the transmembrane domain optionally includes CD3ζ, CD3ε, CD4, CD8α, CD28, CD5, CD16, CD9, CD22, CD33, CD27, CD37 One or more of CD45, CD64, CD80, CD86, CD127, CD137, CD134, CD152, CD154, PD-1 or Dectin-1. The chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain optionally includes CD3ζ, CD27, CD28, CD30, CD137, CD134, CD154, Dectin-1, FcRγ or ICOS one or more of . The chimeric antigen receptor according to claim 1, wherein the nucleotides of the VHH antibody or fragment thereof are optionally selected from any sequence in Seq ID NO.19-27. The gene transfer method comprising the chimeric antigen receptor according to any one of claims 1-6, wherein the gene transfer method optionally comprises a viral vector, a transposon system, electroporation or CRISPR/Cas9 Gene editing tools. A CAR-T cell, characterized in that it expresses the chimeric antigen receptor according to any one of claims 1-6. The CAR-T cell according to claim 8, wherein the expressed chimeric antigen receptor has an amino acid sequence as shown in any one of Seq ID NO.28-36; or, the expressed chimeric antigen receptor The nucleotide sequence of the antigen receptor is shown in any one of Seq ID NO.37-45. A CAR-NK cell, characterized in that it expresses the chimeric antigen receptor according to any one of claims 1-6. The CAR-NK cell according to claim 10, wherein the amino acid sequence of the expressed chimeric antigen receptor is as shown in any one of Seq ID NO.28-36; or, the expressed chimeric antigen receptor The nucleotide sequence of the antigen receptor is shown in any one of Seq ID NO.37-45. An anti-tumor drug, characterized in that it contains the CAR-T cell according to claim 8 and pharmaceutically allowed adjuvants and/or adjuvants. The antitumor drug according to claim 12, wherein the tumor comprises a solid tumor derived from epithelial cells, and the expression of EpCAM is positive. The antitumor drug according to claim 12, wherein the tumor comprises epithelial-derived malignant tumors and circulating tumor cells, and tumor stem cells; or, the tumor comprises intestinal cancer, lung cancer, ovarian cancer, liver cancer or gastric cancer . The synthetic method of the chimeric antigen receptor described in any one of claims 1-6, is characterized in that, comprises the following steps: (1) construct Anti-EpCAM-VHH-CAR complete gene; (2) use primer pair Primer1 and Primer2 amplifies the Anti-EpCAM-VHH-CAR gene; (3) digests the amplified gene sequence with BamHI and EcoRI restriction enzymes and packages it with a viral vector. The synthesis method according to claim 15, wherein the viral vector comprises a lentiviral vector, an adenoviral vector or a retroviral vector. The synthetic method according to claim 15, wherein the sequence of the primer Primer1 is as shown in Seq ID NO.46, and the sequence of the primer Primer2 is as shown in Seq ID NO.47. Application of a VHH antibody or its fragment specifically targeting tumor EpCAM antigen in the preparation of bispecific antibody and antibody-drug conjugated drug ADC, characterized in that the VHH antibody or its fragment has specific tumor targeting The complementarity determining region CDR of the EpCAM antigen, the complementarity determining region CDR is optionally selected from one or more of the amino acid sequences in Seq ID NO.10-18. Application of a VHH antibody or fragment thereof specifically targeting tumor EpCAM antigen in the preparation of a tumor diagnostic kit, characterized in that the VHH antibody or fragment thereof has a complementarity determining region CDR specifically targeting tumor EpCAM antigen, so The complementary determining region CDR is optionally selected from one or more of the amino acid sequences in Seq ID NO.10-18. The use according to claim 19, wherein the tumor is a solid tumor derived from epithelial cells, and the expression of EpCAM is positive.

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