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WO2023044991A1 - Anticorps ciblant de manière spécifique un antigène epcam tumoral et son utilisation - Google Patents

Anticorps ciblant de manière spécifique un antigène epcam tumoral et son utilisation Download PDF

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WO2023044991A1
WO2023044991A1 PCT/CN2021/123768 CN2021123768W WO2023044991A1 WO 2023044991 A1 WO2023044991 A1 WO 2023044991A1 CN 2021123768 W CN2021123768 W CN 2021123768W WO 2023044991 A1 WO2023044991 A1 WO 2023044991A1
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epcam
tumor
vhh
cells
antigen receptor
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王玮
魏于全
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Sichuan University
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Definitions

  • 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.
  • 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 .
  • MHC major histocompatibility complex
  • 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.
  • EpCAM-positive tumors are currently focused on the prescription and efficacy evaluation of antibody drugs.
  • Edrecolomab Adecatumumab and Catumaxomab
  • ADC antibody-drug conjugates
  • 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.
  • 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.
  • CARs chimeric antigen receptors
  • 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 CD3 ⁇ or FcR ⁇ , 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- ⁇ , TNF- ⁇ , 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.
  • 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.
  • 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.
  • VHH 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.
  • VHH antibody and EpCAM target 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.
  • 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 complementary determining region CDR is optionally selected from one or more of the amino acid sequences in Seq ID NO.10-18.
  • the recognition region containing multiple antibodies will be designed in the CAR, that is, the CDR region of multiple antibodies.
  • 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.
  • the transmembrane domain optionally includes CD3 ⁇ , CD3 ⁇ , CD4, CD8 ⁇ , 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.
  • the intracellular signaling domain optionally includes one or more of CD3 ⁇ , CD27, CD28, CD30, CD137, CD134, CD154, Dectin-1, FcR ⁇ or ICOS.
  • 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.
  • 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 A CAR-T cell expressing the above-mentioned chimeric antigen receptor.
  • amino acid sequence of the expressed chimeric antigen receptor is shown in any one of Seq ID NO.28-36.
  • 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 is provided.
  • amino acid sequence of the expressed chimeric antigen receptor is shown in any one of Seq ID NO.28-36.
  • 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.
  • the tumors include solid tumors derived from epithelial cells, and the expression of EpCAM is positive.
  • the tumors include epithelial-derived malignant tumors and circulating tumor cells, as well as tumor stem cells.
  • 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.
  • viral vectors include lentiviral vectors, adenoviral vectors or retroviral vectors.
  • sequence of the primer Primer1 is shown in Seq ID NO.46
  • sequence of the primer Primer2 is shown in Seq ID NO.47.
  • 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.
  • 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.
  • the tumor is a solid tumor derived from epithelial cells, and the expression of EpCAM is positive.
  • the present invention firstly provides an antibody specifically targeting tumor EpCAM antigen.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • DTT dithiothreitol
  • 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- ⁇ 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 CD3 ⁇ ;
  • 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;
  • FITC isothiocyanate
  • 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.
  • 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.
  • 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.
  • epitopope in the present invention refers to the site on the antigen that is recognized, bound by and interacts with the antibody.
  • the extracellular antigen recognition domain Anti-EpCAM-VHH of the Anti-EpCAM-VHH-CAR molecule can recognize and bind to the human EpCAM molecule.
  • the hinge region and the transmembrane region are derived from CD8 ⁇ , or other molecules other than CD8 ⁇ , 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.
  • the CD3 ⁇ signal transduction domain is the 61-164 amino acid residues of the intracellular segment of the CD3 ⁇ molecule.
  • the 61-164 amino acid residues of CD3 ⁇ 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 CD3 ⁇ Signal Transduction 28 . Therefore, CAR molecules constructed by using CD3 ⁇ intracellular segment in the CAR molecular structure, or using CD3 ⁇ ITAM alone or in any combination are included in the scope of the present invention.
  • ITAM receptor tyrosine activation motifs
  • the CD3 ⁇ signal transduction domain can also be replaced by other signal transduction structures, such as FcR ⁇ , 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.
  • Anti-EpCAM-VHH-CAR molecules can be produced in T cells.
  • 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.
  • the Anti-EpCAM-VHH-CAR nucleic acid molecule is constructed on a lentiviral vector plasmid, and the EF1- ⁇ promoter is used to promote CAR gene expression.
  • the EF1- ⁇ promoter is used to promote CAR gene expression.
  • 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.
  • the VHH antibody or its fragments are obtained by immunizing alpacas regularly by immunizing proteins expressed in Escherichia coli.
  • the VHH antibody or its fragment is transcribed and amplified into a single domain antibody gene fragment.
  • VHH antibody specifically targeting human tumor EpCAM antigen
  • 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.
  • EpCAM-camFc human EpCAM and alpaca Fc recombinant protein
  • the sequences were analyzed using the IGMT database (International Immunogenetics Database) to determine the CDR regions of each sequence.
  • 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.
  • 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).
  • DTT dithiothreitol
  • 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.
  • Anti-EpCAM-VHH-CAR Construction of chimeric antigen receptor targeting EpCAM (Anti-EpCAM-VHH-CAR) and lentiviral vector construction.
  • Anti-EpCAM-VHH-CAR the extracellular segment of Anti-EpCAM-VHH, CD8 ⁇ transmembrane region and intracellular signal transduction domain (8 ⁇ 28BB ⁇ ) 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.
  • 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.
  • 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- ⁇ , TNF- ⁇ , 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.
  • 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.

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Abstract

L'invention concerne un récepteur antigénique chimérique ciblant de manière spécifique un antigène EpCAM tumoral, comprenant un domaine de reconnaissance d'antigène extracellulaire, un domaine transmembranaire et un domaine de signalisation intracellulaire, le domaine de reconnaissance d'antigène extracellulaire comprenant un anticorps VHH ou un fragment de celui-ci, et l'anticorps VHH ou un fragment de celui-ci ayant une région déterminant la complémentarité (CDR) qui cible de manière spécifique l'antigène EpCAM tumoral, qui peut reconnaître de manière efficace des cellules tumorales avec une expression de l'EpCAM positif, et n'attaque pas les cellules épithéliales normales ayant une faible expression de l'EpCAM.
PCT/CN2021/123768 2021-09-24 2021-10-14 Anticorps ciblant de manière spécifique un antigène epcam tumoral et son utilisation Ceased WO2023044991A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150329640A1 (en) * 2012-12-20 2015-11-19 Bluebird Bio, Inc. Chimeric antigen receptors and immune cells targeting b cell malignancies
CN105384825A (zh) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 一种基于单域抗体的双特异性嵌合抗原受体及其应用
CN107602703A (zh) * 2016-08-12 2018-01-19 四川大学 靶向人EpCAM的基因工程化淋巴细胞及其制备方法和用途
CN108484771A (zh) * 2018-04-24 2018-09-04 南京市妇幼保健院 EpCAM单域抗体G7
CN109694413A (zh) * 2019-01-17 2019-04-30 深圳市前海精准生物科技有限公司 一种基于bmca纳米抗体序列的嵌合抗原受体及其应用
CN111333730A (zh) * 2020-03-11 2020-06-26 南京融捷康生物科技有限公司 可特异性结合EpCAM的单域抗体及其应用
CN113166274A (zh) * 2020-02-13 2021-07-23 四川大学 一种嵌合抗原受体及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108586614B (zh) * 2018-04-24 2021-07-23 南京市妇幼保健院 EpCAM单域抗体D7
CN108530538B (zh) * 2018-04-24 2021-07-23 南京市妇幼保健院 EpCAM单域抗体E6

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150329640A1 (en) * 2012-12-20 2015-11-19 Bluebird Bio, Inc. Chimeric antigen receptors and immune cells targeting b cell malignancies
CN105384825A (zh) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 一种基于单域抗体的双特异性嵌合抗原受体及其应用
CN107602703A (zh) * 2016-08-12 2018-01-19 四川大学 靶向人EpCAM的基因工程化淋巴细胞及其制备方法和用途
CN108484771A (zh) * 2018-04-24 2018-09-04 南京市妇幼保健院 EpCAM单域抗体G7
CN109694413A (zh) * 2019-01-17 2019-04-30 深圳市前海精准生物科技有限公司 一种基于bmca纳米抗体序列的嵌合抗原受体及其应用
CN113166274A (zh) * 2020-02-13 2021-07-23 四川大学 一种嵌合抗原受体及应用
CN111333730A (zh) * 2020-03-11 2020-06-26 南京融捷康生物科技有限公司 可特异性结合EpCAM的单域抗体及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FU JUAN, SHANG YUHONG, QIAN ZHANG, HOU JINPING, YAN FENG, LIU GUODI, DEHUA LI, TIAN XIAOLI: "Chimeric Antigen receptor-T (CAR-T) cells targeting Epithelial cell adhesion molecule (EpCAM) can inhibit tumor growth in ovarian cancer mouse model", JOURNAL OF VETERINARY MEDICAL SCIENCE, JAPANESE SOCIETY OF VETERINARY SCIENCE, TOKYO., JP, vol. 83, no. 2, 1 January 2021 (2021-01-01), JP , pages 241 - 247, XP093054852, ISSN: 0916-7250, DOI: 10.1292/jvms.20-0455 *

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