Nanobodies that can specifically target human epcam antigen and uses thereof

Abstract

The present application belongs to the field of genetic engineering and biological immunotherapy, and provides a series of chimeric antigen receptors specifically targeting human tumor EpCAM antigens, including an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal domain, wherein the extracellular antigen recognition domain comprises a VHH antibody or a fragment thereof, the VHH antibody or the fragment thereof has a complementarity determining region CDR specifically targeting tumor EpCAM antigens, and can effectively recognize tumor cells positive for EpCAM expression, and does not attack normal epithelial cells with low expression of EpCAM.

Description

[0001] Divisional application This application is a divisional application of Chinese Patent Application No. 2021111200425, filed on September 24, 2021, entitled "An antibody specifically targeting tumor EpCAM antigen and its application". Technical Field

[0002] This invention belongs to the fields of genetic engineering and biological immunotherapy, specifically relating to an antibody that specifically targets the tumor EpCAM antigen and its application. Background Technology

[0003] In tumor immunotherapy, inducing effective anti-tumor immunity in patients is crucial for disease treatment. Tumor cells can develop a series of escape mechanisms to evade the surveillance of the immune system. These include downregulating the major histocompatibility complex (MHC), downregulating the expression of autoantigens, downregulating immune checkpoint ligands, and forming a local inhibitory microenvironment, thereby limiting the recognition and attack of tumor cells by their own effector cells.1

[0004] Currently known tumor antigen targets are mainly divided into two categories. One category consists of antigens expressed only in tumor cells and not in normal cells; these are tumor cell-specific and are considered tumor-specific antigens. The other category consists of antigens highly expressed in tumor cells and poorly expressed or not expressed in normal cells; their expression is tumor-associated and are called tumor-associated antigens (TAAs). EpCAM is one type of TAA. It is highly expressed in colorectal cancer, ovarian cancer, and other tumor cells, and is also expressed in human epithelial cells, although at relatively low levels. However, it also lacks tumor specificity. Therefore, immune tolerance needs to be considered when selecting EpCAM as an immunotherapy target.

[0005] Treatment of EpCAM-positive tumors currently focuses on the administration and efficacy evaluation of antibody drugs, such as Edrecolomab, Adecatumumab, and Catumaxomab, as well as antibody-drug conjugates (ADCs). Edrecolomab, an EpCAM monoclonal antibody, did not show improvement in overall survival or burden-free survival in adjuvant therapy for stage III colon cancer.19-21 Adecatumumab showed dose- and target antigen-dependent effects in clinical studies of monotherapy for metastatic breast cancer, but no objective tumor regression was observed.22 Catumaxomab, a bispecific antibody targeting CD3 and EpCAM, showed significant clinical benefit in the treatment of malignant ascites secondary to epithelial carcinoma, with manageable safety profiles.23,24 It was approved by the EMA in 2009 for the treatment of malignant ascites, but unfortunately, it was withdrawn from the market in 2017 due to poor sales. Furthermore, antibody-drug conjugates targeting EpCAM, such as Oportuzumab monatox25 and Tucotuzumab26, have shown good tolerability in clinical studies; however, more clinical data are needed to confirm their effects on disease improvement and prolonged survival. Overall, antibody drugs targeting EpCAM have made some progress, but the development of new immunotherapies / methods is still needed to supplement the shortcomings of existing medical technologies.

[0006] Chimeric antigen receptor-modified T-cell (CAR-T) therapy is considered an effective tumor immunotherapy. This therapy involves expressing a recombinant receptor called a chimeric antigen receptor (CAR) on T cells, which redirects T cells to tumor cells at selected targets. CAR molecules consist of multiple structural components: an extracellular antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain. The antigen recognition domain includes, but is not limited to, single-chain variable fragments (scFvs) derived from immunoglobulins, which are composed of variable regions of the heavy and light chains of immunoglobulins linked by structures such as G4S; and other non-antibody-derived molecules, such as the natural killer cell receptor NKG2D, which can recognize tumor antigens by binding to the ligand NKG2DL on the surface of target cells.2 The hinge region provides flexible connections between the extracellular and intracellular domains of the CAR molecule and is typically derived from the CH2 and CH3 domains of the immunoglobulin heavy chain. The transmembrane region anchors CAR molecules to the cell membrane and typically originates from the transmembrane regions of co-stimulatory molecules such as CD4, CD8, CD28, and 4-1BB. The intracellular signal transduction domain, composed of co-stimulatory molecules such as CD28, 4-1BB, and ICOS, and intracellular activating molecules such as CD3ζ or FcRγ, transduces and amplifies antigen-ligand recognition signals from the extracellular domain, thereby activating T cells or NK cells.

[0007] CAR molecules typically provide specific antigen recognition functionality through extracellular antigen recognition domains, redirecting T cells or NK cells to the location of antigen expression on tumor cells. Recognition of the target antigen activates downstream signal transduction of the CAR molecule, initiating effector functions of T cells or NK cells. Activated T cells or NK cells secrete effector molecules such as IFN-γ, TNF-α, IL-2, granzymes, and perforin, directly acting on target cells or recruiting other immune effector cells to participate in the immune response, ultimately leading to target cell lysis and death. Therefore, current approaches to modifying the structure of CAR molecules primarily focus on enhancing the function of CAR-T cells and their cytotoxicity to target cells.

[0008] In summary, this invention provides a novel nanobody that can be applied to the design of CAR molecular structures, enabling it to specifically recognize EpCAM-positive tumor cells, thereby supplementing the shortcomings of existing medical technologies. Summary of the Invention

[0009] In view of this, the purpose of this invention is to provide a nanobody that can specifically target human EpCAM antigen and its application, and the specific technical solution is as follows.

[0010] A chimeric antigen receptor that specifically targets tumor EpCAM antigen includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen recognition domain includes a VHH antibody, which has a complementarity-determining region (CDR) that specifically targets tumor EpCAM antigen. The amino acid sequence of the VHH antibody is shown in any one of Seq IDs 1-5, 7, and 9 (corresponding sequence names are 1191 / 1201 / 1193 / 1200 / 1194 / 1197 / 1198).

[0011] Furthermore, the amino acid sequence of the CDR region of the VHH antibody sequence is shown in any one of Seq ID NO.10-14, 16, and 18.

[0012] VHH, short for heavy chain single-domain antibody, is a type of antibody derived from alpacas. Unlike traditional IgG, VHH contains only heavy chains, resulting in a smaller size and molecular weight (~15kDa), hence the name nanobody. Compared to traditional monoclonal antibodies, it has unique advantages, such as easier entry into antigenic epitopes, lower immunogenicity, better solubility, higher stability, and affinity comparable to scFv.

[0013] The mechanism by which VHH antibody specifically recognizes the EpCAM target is the process by which the CDR site of the antibody's heavy chain variable region recognizes and binds to the tumor antigen. The CDR structure of VHH determines its specificity in targeting EpCAM.

[0014] Existing research has confirmed that there are cases where multiple targets (usually two) can be targeted. In such cases, recognition regions containing multiple antibodies are designed into the CAR, i.e., CDR regions with multiple antibodies. These CDR regions can be tandemly linked or integrated into a CAR structure, expressed in the cell, and then separated to recognize antigens.

[0015] Further, the transmembrane domain includes one or more of CD3ζ, CD3ε, CD4, CD8α, CD28, CD5, CD16, CD9, CD22, CD33, CD27, CD37, CD45, CD64, CD80, CD86, CD127, CD137, CD134, CD152, CD154, PD-1, or Decin-1; or, the intracellular signaling domain includes one or more of CD3ζ, CD27, CD28, CD30, CD137, CD134, CD154, Decin-1, FcRγ, or ICOS.

[0016] Furthermore, the nucleotide sequence of the VHH antibody is shown in any one of Seq ID NO.19-23, 25, 27 (the corresponding sequence names are 1191 / 1201 / 1193 / 1200 / 1194 / 1197 / 1198).

[0017] A type of CAR-T cell that expresses the aforementioned chimeric antigen receptor.

[0018] Further, the amino acid sequence of the chimeric antigen receptor is shown in any one of Seq ID NO. 28-29, 31-33, 35-36 (the corresponding sequence names are 1191 / 1201 / 1193 / 1200 / 1194 / 1197 / 1198); or, the nucleotide sequence of the chimeric antigen receptor is shown in any one of Seq ID NO. 37-38, 40-42, 44-45 (the corresponding sequence names are 1191 / 1201 / 1193 / 1200 / 1194 / 1197 / 1198).

[0019] A type of CAR-NK cell that expresses the aforementioned chimeric antigen receptor.

[0020] Further, the amino acid sequence of the chimeric antigen receptor is as shown in any one of Seq ID NO. 28-29, 31-33, 35-36 (the corresponding sequence names are 1191 / 1201 / 1193 / 1200 / 1194 / 1197 / 1198); or, the nucleotide sequence of the chimeric antigen receptor is as shown in any one of Seq ID NO. 37-38, 40-42, 44-45 (the corresponding sequence names are 1191 / 1201 / 1193 / 1200 / 1194 / 1197 / 1198).

[0021] An antitumor drug containing the aforementioned CAR-T cells or CAR-NK cells, as well as pharmaceutically permissible excipients and / or adjuvants.

[0022] Preferably, the tumor comprises a solid tumor of epithelial cell origin and is positive for EpCAM expression.

[0023] Preferably, the tumor includes epithelial-derived malignant tumors and circulating tumor cells, as well as tumor stem cells.

[0024] Preferably, the tumor includes colon cancer, lung cancer, ovarian cancer, liver cancer, or stomach cancer.

[0025] EpCAM, short for epithelial cell adhesion molecule, is a 40 kDa single-pass transmembrane glycoprotein encoded by the tumor-associated calcium signaling transduction 1 (TACT) gene. It participates in regulating cell adhesion and migration, controlling proliferation and differentiation, and mediating signal transduction.14 It was identified as a tumor-associated antigen in 1979. 15 It is expressed in most epithelial-derived malignant tumor cells, such as colorectal cancer, lung cancer, prostate cancer, and ovarian cancer. 16 .

[0026] The above-mentioned method for synthesizing chimeric antigen receptors includes the following steps: (1) constructing the complete Anti-EpCAM-VHH-CAR gene; (2) amplifying the Anti-EpCAM-VHH-CAR gene using primer pairs Primer1 and Primer2; (3) digesting the amplified gene sequence using BamHI and EcoRI restriction enzymes and packaging it with a viral vector; the viral vector includes lentiviral vectors, adenovirus vectors or retroviral vectors.

[0027] Furthermore, the sequence of primer Primer1 is shown in Seq ID NO.46, and the sequence of primer Primer2 is shown in Seq ID NO.47.

[0028] The application of a VHH antibody or a fragment thereof that specifically targets the tumor EpCAM antigen in the preparation of bispecific antibodies and antibody-drug conjugates (ADCs), wherein the VHH antibody has a complementarity-determining region (CDR) that specifically targets the tumor EpCAM antigen, and the amino acid sequence of the VHH antibody is shown in any one of Seq ID NOs 1-5, 7, and 9 (the corresponding sequence names are 1191 / 1201 / 1193 / 1200 / 1194 / 1197 / 1198).

[0029] The application of a VHH antibody or fragment thereof that specifically targets the tumor EpCAM antigen in the preparation of a tumor diagnostic kit, wherein the VHH antibody has a complementarity-determining region (CDR) that specifically targets the tumor EpCAM antigen, and the amino acid sequence of the VHH antibody is shown in any one of Seq ID NOs 1-5, 7, and 9 (the corresponding sequence names are 1191 / 1201 / 1193 / 1200 / 1194 / 1197 / 1198).

[0030] Preferably, the tumor is a solid tumor derived from epithelial cells and is positive for EpCAM expression.

[0031] Beneficial technical effects This invention first provides a series of nanobodies that specifically target the tumor antigen EpCAM. As those skilled in the art know, EpCAM is a tumor-associated antigen that is highly expressed in tumor cells, but it is also expressed in normal epithelial cells (at a relatively low level, thus lacking tumor specificity). Based on this, the team of this invention focused on the antibody affinity when selecting antibodies targeting EpCAM. 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 tumor cells expressing EpCAM, leading to treatment failure. Therefore, the anti-EpCAM VHH antibody screened in this invention has moderate affinity, which can ensure effective recognition of tumor cells while ensuring that it will not recognize and attack normal epithelial cells with low EpCAM expression.

[0032] Furthermore, this invention utilizes screened nanoscale VHH antibodies or fragments thereof with moderate affinity to construct second- and third-generation chimeric antigen receptor molecule nucleic acid sequences. The chimeric antigen receptor molecules, when expressed in immune effector cells such as T cells and NK cells, can endow these immune cells with the ability to target and lyse tumor cells expressing EpCAM molecules, thereby achieving the purpose of killing tumors.

[0033] Specifically, the Anti-EpCAM-VHH nanobody constructed in this invention has high affinity for EpCAM-expressing positive cells. Transducing T lymphocytes from a patient to be treated with Anti-EpCAM-VHH-CAR and then reinfusing the transduced T lymphocytes into the patient allows successfully transduced T cells to redirect to EpCAM-expressing target cells, generating an immune response. This method enhances the T cell immune response to target cells. The process of transducing T cells to express chimeric antigen receptors using Anti-EpCAM-VHH-CAR can be performed in vitro or in vivo, and the successfully transduced cells are all Anti-EpCAM-VHH-CAR-T cells.

[0034] Finally, the antibodies or fragments screened by this invention can also be used in the preparation of targeted therapy products such as bispecific binding antibodies and antibody-drug conjugates, and can also be applied to antibody detection products. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0036] Figure 1 Molecular weight identification of Anti-EpCAM-VHH hFc protein: VHH-hFc fusion protein expressed in HEK293 cells was reduced with dithiothreitol (DTT), and the relative molecular weight was identified by SDS-PAGE electrophoresis as being between 43-55 kD. Figure 2 ELISA detection results for different VHH hFc combined with EpCAM-His; Figure 3 The Anti-EpCAM-VHH-CAR structure is as follows: The Anti-EpCAM-VHH-CAR gene is loaded into a lentiviral vector constructed in this invention, and CAR gene expression is initiated by the EF1-α promoter. The extracellular domain consists of the IL-2 guide sequence and EpCAM-VHH, and the intracellular domain consists of the CD8a hinge region and transmembrane region, CD28 and 4-1BB, and the intracellular amino acid residues of CD3ζ. Figure 4 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 on the outer side of the cell membrane and are used to recognize target antigens. The CD8a transmembrane region is embedded on the cell membrane and is used to immobilize CAR molecules on the membrane. The signal transduction domain is located inside the membrane and is used to transduce and amplify cell activation signals. Figure 5 Detection of Anti-EpCAM-VHH-CAR expression in T cells: The expression of CAR molecules was detected by flow cytometry using rabbit anti-alpaca antibody labeled with isothiocyanate (FITC). Figure 6 Anti-EpCAM-VHH-CAR-T targets EpCAM-positive cells to release cytokines; Figure 7 For the detection of Anti-EpCAM-VHH-CAR-T cell toxicity. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present 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 the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0039] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0040] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered as having specifically disclosed all possible subranges and independent numerical values ​​within those ranges. For example, range 1... The description of 6 should be considered 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., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0041] Glossary The "specific targeting of tumor EpCAM antigen" mentioned in this invention refers to the VHH antibody or its fragment provided by this invention, which can target EpCAM that is highly expressed on tumor cells, while having a weaker ability to recognize other epithelial cells with low EpCAM expression.

[0042] The "epitope" mentioned in this invention refers to the site on the antigen that is recognized, bound to, and interacts with the antibody.

[0043] In one embodiment of the present invention, the extracellular antigen recognition domain Anti-EpCAM-VHH of the Anti-EpCAM-VHH-CAR molecule is able to recognize and bind to human EpCAM molecules.

[0044] In one embodiment of the present invention, the hinge region and transmembrane region are derived from CD8α, or may be other molecules besides CD8α, such as CD4, CD28, etc. The co-stimulatory signal transduction domain is derived from, but is not limited to, CD28, 4-1BB, or may also be derived from ICOS, OX40, etc., and the number of co-stimulatory molecules can be one or more.

[0045] In one embodiment of the present invention, the CD3ζ signal transduction domain is amino acid residues 61-164 of the intracellular segment of the CD3ζ molecule. The 61-164 amino acid residues of CD3ζ contain three receptor tyrosine activation motifs (ITAMs), namely amino acid residues 61-89, 100-128, and 131-159, each of which can independently mediate CD3ζ activation signal transduction. Therefore, CAR molecules constructed using the intracellular segment of CD3ζ, or using CD3ζ ITAMs alone or in any combination, are all included within the scope of this invention.

[0046] In one embodiment of the present invention, the CD3ζ signal transduction domain may also be replaced by other signal transduction structures, such as FcRγ, and the number may be one or more. Chimeric antigen receptors constructed by modifying regions other than the extracellular antigen recognition domain are all within the scope of the present invention.

[0047] In one embodiment of the present invention, the Anti-EpCAM-VHH-CAR molecule can be generated in T cells by expressing the Anti-EpCAM-VHH-CAR nucleic acid sequence in T lymphocytes. Furthermore, this nucleic acid sequence can also be expressed in NK cells, therefore the constructed CAR-NK cells are also within the scope of the present invention.

[0048] In one embodiment of the present invention, the Anti-EpCAM-VHH-CAR nucleic acid molecule is constructed on a lentiviral vector plasmid and the CAR gene expression is initiated by the EF1-α promoter. In addition to lentiviral vectors, other gene transfer methods used to express the CAR of the present invention, such as retroviral vectors, transposon systems, electroporation, CRISPR / Cas9 and other gene editing tools, are also included within the scope of the present invention.

[0049] In one embodiment of the present invention, VHH antibody or a fragment thereof is obtained by periodically immunizing alpacas with E. coli-expressed protein.

[0050] In one embodiment of the present invention, the VHH antibody or a fragment thereof is transcribed and amplified to form a single-domain antibody gene fragment.

[0051] Example 1 Preparation of VHH antibodies specifically targeting human tumor EpCAM antigen 1) Alpaca Immunization and cDNA Library Construction. Human EpCAM antigen 1.5 mg was mixed with adjuvant at a 1:1 ratio and injected at multiple sites near the cervical lymph nodes of alpacas for immunization. Immunization was performed twice a week for a total of 7 weeks. Starting from the 4th immunization, peripheral blood was collected each time for immune assessment and peripheral blood lymphocytes were isolated. Lymphocytes were lysed with Trizol solution, and total RNA was extracted. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit, and then an E. coli cDNA library was constructed using a cDNA library construction kit.

[0052] (2) Preparation of phage library. *E. coli* cDNA library cells were inoculated into 2XYT medium containing tetracycline and ampicillin, and cultured at 37°C with shaking until DO600 = 0.5. Helper phage M13KO7 was added to the culture system to a final concentration of 1E12 cfu / ml, and the culture was incubated at 37°C for one hour. Kanamycin was then added, followed by overnight incubation at 30°C. The culture solution was then centrifuged at 5000g for 10 minutes, and the supernatant was collected. PEG / NaCl solution was added, mixed, and allowed to stand at 4°C for 20 minutes. The solution was then centrifuged at 4000g for 20 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS. The phage library was then obtained by centrifugation at 16000g for 10 minutes.

[0053] (3) Phage screening for VHH. Immunotherapy tubes containing the library phage and pre-coated with EpCAM-camFc (human EpCAM and alpaca Fc recombinant protein) were blocked with 3% BSA at room temperature for 2 hours. The blocked tubes were washed multiple times with PBS, and then the blocked phage was added to the tubes and incubated at room temperature for 1 hour by rotation. After washing the tubes multiple times with PBS-T, 100 mM TEA was added for elution, and the tubes were incubated at room temperature for 10 minutes. Then, 1 M Tris-HCl was added, and the resulting eluent was the selected phage. This screening process needs to be repeated twice to remove non-specific phages.

[0054] (4) Phage ELISA identification of VHH. The phage obtained in step (3) was diluted and inoculated into SS320 bacterial culture with OD600=0.5. After incubation at 37°C for 30 minutes, the culture was spread onto 2X YT plates. After incubation at 37°C overnight, single clones were picked and inoculated into 48-well plates containing 2X YT. After incubation at 37°C for 3-4 hours, kanamycin and helper phage were added to the wells. After incubation at 30°C overnight, the culture was centrifuged to obtain the supernatant. 96-well plates were pre-coated with EpCAM-camFc and the wells were blocked with 3% BSA. The supernatant was added and incubated at room temperature for 1 hour. After washing with PBS, anti-M13 HRP was used for binding, followed by TMB color development. The OD450 value was measured, and the affinity of different VHHs for EpCAM was determined based on the OD450 value. By comparing the OD450 values ​​of the sample wells and the negative control, the protein samples corresponding to wells with significantly elevated OD450 were sequenced, yielding different VHH amino acid sequences. After removing sequences with identical CDR regions and redundant sequences through sequence alignment, nine single amino acid sequences (ID NO. 1-9) were obtained. The CDR regions of each sequence were determined using the IGMT database (International Immunogenetic Database). The segments highlighted in bold in the sequence listing below are, from left to right, CDR1, CDR2, and CDR3. Different combinations of CDR region sequences exhibit different VHH-EpCAM affinities.

[0055] NO.1: DVQLVESGGGSVQSGGSLRLSCAAS GYTYRRYYMG WFRQAPGEQRE GVAVINNDGRTNYADSVKG RFRISRDNAENTLHLEMNSLKPEDTAMYYC AATGNILPPMTAVPPLGRQWYPY WGRGTLVTVSS NO.2: HVQLVESGGGSVQSGGSLRLSCAAS GYAVKNCMG WFRQAPGKERE GVAVINRNGITTYADSVKG RFTISQDKDKNTLDLQMNSLKPEDTAMYYC AATPTLLTIPARFLCDVRNPSGFTD WGQGTLVTVSS NO.3: QVQLVESGGGSVQAGGSLRLSCVVS AYSAYTYKTMCMG WFRQAPGKERE GVAAIYRGGLNTY YADSVKGRFIISRDNAESTMYLQMNSLKPEDTAMYYC AADWLRGDDCNIGANFDY WGQGTQVTVSS NO.4: QVQLVESGGGSVQAGGSLRLSCVAT GFTISRKCMG WFREAPGKKREVIA TINTGSSSPYYADGVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYC AATKGVVVGTGYCGGPYVERPNSAY WGQGTQVTVSS NO. 5: DVQLVESGGGSVQAGRSLRLSCELS DYTWSTVCMG WFRQAPGKEREGVAV IYTRSGGTTYADSAKG RFTISRDNAKDTLYLQMDSLKPEDTAMYYC AAGPLYDGRCTYRSPAFHY WGQGTQVTVSS NO. 6: DVQLVESGGGSAQAGGSLRLSCAAS GPTSSLRTMG WFRQASGKERERVA VIWDGRTTDYDDSVQD RFTISQDNAKSTVYLQMNTLKPEDTAMYYC AASPRIVPFASTYFQH WGQGTQVTVSS NO. 7: HVQLVESGGGSVQAGGSLKLSCAAS GSIFSGSIFSRCGMR WYRQAPGKERELVS STSKDGFTSYTDSVKG RFTISQDNANNTLYLQMSSLKTEDTAVYSC AAICAVGGYSLSTYTY WGQGTQVTVSS NO. 8: EVQLVESGGDSVQAGGSLRLSCAAS GYSPGSYCMG WFRQAPGKERERVA IIESRGTVTYVDSVKG RFTISKDNAKNTLYLQMNSLKPEDTAMYYC AASRPWSGVRCLHDKYDY WGQGTQVTVSS NO. 9: HVQLVESGGGSVQSGGSLRLSCAVS GYAYSSLA WFRQAPGKEREGVA ALLTAIGGPTRTTYADSVKG RLAISQDHAKNTLYLQMSSLKPEDTAMYYC AAGRPAGTPRWLLLAPRDYNY WGQGTQVTVSS Example 2 Anti-EpCAM-VHH antibody identification. To identify Anti-EpCAM-VHH at the molecular and cellular levels, the selected Anti-EpCAM-VHH was fused to the N-terminus of a human signal peptide and human Fc (hFc, approximately 29 kDa), and transduced into the mammalian transient expression vector pQDFc for transient expression in HEK293 cells. After affinity purification, the VHH-hFc fusion protein was obtained. The VHH-hFc fusion protein expressed in HEK293 cells was reduced with dithiothreitol (DTT), and the relative molecular weight was identified by SDS-PAGE electrophoresis as being between 43-55 kDa. Figure 1 ).

[0056] Example 3 VHH-hFc binding capacity was determined using an ELISA method. EpCAM-His protein (1 ng / µl) was coated onto a 96-well plate and incubated overnight at 4°C. Then, serially diluted VHH-hFc protein (initial concentration 20 ng / µl) was used to bind the EpCAM-His protein in the wells for 1 hour. Finally, anti-hFc-HRP secondary antibody was used to bind VHH-hFc. After color development, the OD value at 450 nm was measured. The binding capacity of VHH-hFc to EpCAM-His protein was determined based on OD450.

[0057] Experimental conclusion: The nine screened Anti-EpCAM-VHH sequences showed varying degrees of affinity for EpCAM, with 1194 exhibiting the strongest affinity, 1201 the weakest, and VHH 1191 showing a moderate affinity for EpCAM. Figure 2 ).

[0058] Example 4 Construction of the chimeric antigen receptor targeting EpCAM (Anti-EpCAM-VHH-CAR) and its lentiviral vector. During the construction of Anti-EpCAM-VHH-CAR, the complete gene containing the extracellular segment, CD8α transmembrane region, and intracellular signal transduction domain (8α28BBζ) of Anti-EpCAM-VHH was synthesized. The Anti-EpCAM-VHH-CAR gene was amplified using Primer 1: 5'-CGGGATCCATGTACCGGATGCAG-3' (SEQ ID NO.46) and Primer 2: 5'-CGGAATTCTTAGCGAGGGGGC-3' (SEQ ID NO.47). BamHI and EcoRI restriction endonuclease sites were added to primer sequences ID NO.46 and ID NO.47, respectively. The amplified Anti-EpCAM-VHH-CAR nucleotides had a BamHI site at the 5' end and an EcoRI site at the 3' end. The Anti-EpCAM-VHH-CAR product and the self-constructed vector PCLK plasmid were digested with BamHI and EcoRI restriction enzymes, which enabled the same BamHI and EcoRI sticky ends to be obtained in both products. This facilitated the ligation of the two gene fragments into a complete DNA plasmid loop using T4 ligase at the same sticky ends.

[0059] Example 5 Human lymphocyte culture and lentiviral transduction. Peripheral blood mononuclear cells were extracted from healthy individuals and cultured in human lymphocyte culture medium X-vivo-15, supplemented with 5% FBS and 100 IU / ml human IL-2. Cells were stimulated with CD3 / CD28 antibody-coated magnetic beads (beads:cells = 3:1) for 24 hours to activate T cells. Lentiviral particles and activated T cells were then added to wells coated with fibronectin (50 μg / ml), with a multiplicity of infection (MOI) of 2-4. Centrifugation at 1000 x g for 2 hours promoted lentiviral infection of T cells. Successfully lentivirally infected T cells expressed the Anti-EpCAM-VHH-CAR gene, thus becoming Anti-EpCAM-VHH-CAR-T cells.

[0060] Example 6 The expression of chimeric antigen receptor in human lymphocytes was detected 48-72 hours after retrofection. Since the extracellular domain of Anti-EpCAM-VHH-CAR is derived from alpaca, it can be detected using rabbit anti-alpaca VHH antibody (Genscript, Nanjing) by flow cytometry.

[0061] Example 7 Targeted Specific Cytokine Release Detection. Since Anti-EpCAM-VHH-CAR-T has specific cytotoxic effects on EpCAM-positive cells, this invention selected three types of tumor cells as target cells: HT29 human colon cancer cells that highly express EpCAM, SKOV3 human ovarian cancer cells that lowly express EpCAM, and HeLa human cervical cancer cells that do not express EpCAM. Before cytokine detection, 1*10... 5 One (E:T=10:1) or 5*10 4 (E:T=5:1) Anti-EpCAM-VHH-CAR-T cells were co-cultured with three types of tumor cells in 96-well plates, with a tumor cell count of 1*103. 4 Each well was used to co-culture for 24 hours. The co-culture supernatant was then used to detect IFN-γ, TNF-α, and IL-2 using enzyme-linked immunosorbent assay (ELISA).

[0062] Experimental conclusion: Anti-EpCAM-VHH-CAR-T cells recognize and bind to EpCAM on the surface of target cells in the co-culture system, promoting the activation of CAR-T cell effector function. This results in the release of large amounts of cytokines, such as IFN-γ and TNF-α. The degree of target-specific activation of CAR-T cells can be assessed by detecting the amount of cytokine secretion in the co-culture supernatant. Figure 6 CAR-T cells can release cytokines when stimulated by EpCAM-positive HT29 and SKOV3 cells, but cannot be activated by EpCAM-negative HeLa cells. Furthermore, the cytokine release level of CAR-T cells increases with the enhancement of target cell antigen expression, exhibiting antigen dependence.

[0063] Example 8 Cytotoxicity assay. The cytotoxicity of Anti-EpCAM-VHH-CAR-T cells against tumor cells was determined using a real-time cytotoxicity assay. The specific protocol involved seeding HT29 and HeLa cells into labeled assay plates at a cell count of 1*10⁴ cells / well. Cell adhesion was recorded on a TRCA assay instrument. After 24 hours of stable cell adhesion, Anti-EpCAM-CAR-T cells were added to the plates at the set effector cell:target cell (E:T) ratio, and cell adhesion was recorded again on a TRCA assay instrument. Target cells lose their adhesion after being killed by CAR-T cells.

[0064] Experimental conclusion: The cytotoxicity of CAR-T cells can be assessed by real-time detection of the adherence of target cells. Figure 7The results showed that after adding T cells to target cells HT29 and HeLa, the number of HT29 cells in the ControlT cell group increased slightly and then slowly decreased, while the number of HT29 cells in the CAR-T cell group decreased rapidly. When Control T cells and CAR-T cells were added to HeLa cells, the number of cells decreased to the same extent, with no significant change. This confirms that CAR-T cells have EpCAM+ cytotoxicity.

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Nat Med25, 82-88, doi:10.1038 / s41591-018-0290-5 (2019) The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor that specifically targets tumor EpCAM antigen, comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, characterized in that, The extracellular antigen recognition domain is a VHH antibody, which has a complementarity-determining region (CDR) that specifically targets the tumor EpCAM antigen; the amino acid sequence of the VHH antibody is shown in Seq ID NO.

5.

2. The chimeric antigen receptor as described in claim 1, characterized in that, The transmembrane domain includes one or more of CD3ζ, CD3ε, CD4, CD8α, CD28, CD5, CD16, CD9, CD22, CD33, CD27, CD37, CD45, CD64, CD80, CD86, CD127, CD137, CD134, CD152, CD154, PD-1, or Decin-1; or, the intracellular signaling domain includes one or more of CD3ζ, CD27, CD28, CD30, CD137, CD134, CD154, Decin-1, FcRγ, or ICOS.

3. A CAR-T cell, characterized in that, The chimeric antigen receptor as described in claim 1 or 2 is expressed.

4. The CAR-T cells as described in claim 3, characterized in that, The amino acid sequence of the chimeric antigen receptor is shown in Seq ID NO. 33; or, the nucleotide sequence of the chimeric antigen receptor is shown in Seq ID NO.

42.

5. A CAR-NK cell, characterized in that, The chimeric antigen receptor as described in claim 1 or 2 is expressed.

6. The CAR-NK cells as described in claim 5, characterized in that, The amino acid sequence of the chimeric antigen receptor is shown in Seq ID NO. 33; or, the nucleotide sequence of the chimeric antigen receptor is shown in Seq ID NO.

42.

7. An antitumor drug, characterized in that, It contains the CAR-T cells of claim 3 or the CAR-NK cells of claim 5, as well as pharmaceutically permissible excipients and / or adjuvants.

8. The method for synthesizing the chimeric antigen receptor according to claim 1 or 2, characterized in that, Includes the following steps: (1) Construct the complete Anti-EpCAM-VHH-CAR gene; (2) Amplify the complete Anti-EpCAM-VHH-CAR gene using primer pairs Primer1 and Primer2; (3) Digest the amplified gene sequence using BamHI and EcoRI restriction enzymes and package it with a viral vector; the viral vector is a retroviral vector.

9. The synthesis method as described in claim 8, characterized in that, The sequence of primer Primer1 is shown in Seq ID NO.46, and the sequence of primer Primer2 is shown in Seq ID NO.

47.

10. The application of a VHH antibody specifically targeting the tumor EpCAM antigen in the preparation of a diagnostic kit for EpCAM-positive tumors, characterized in that, The VHH antibody has a complementarity-determining region (CDR) that specifically targets the tumor EpCAM antigen, and the amino acid sequence of the VHH antibody is shown in Seq ID NO. 5.

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