CN108342360A - Co-express anti-MSLN Chimeric antigen receptors and the transgenosis lymphocyte of nonfunctional EGFR and application thereof - Google Patents
Co-express anti-MSLN Chimeric antigen receptors and the transgenosis lymphocyte of nonfunctional EGFR and application thereof Download PDFInfo
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- CN108342360A CN108342360A CN201710056385.7A CN201710056385A CN108342360A CN 108342360 A CN108342360 A CN 108342360A CN 201710056385 A CN201710056385 A CN 201710056385A CN 108342360 A CN108342360 A CN 108342360A
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Abstract
本发明提出了一种转基因淋巴细胞、一种构建体和一种治疗癌症的治疗组合物，该转基因淋巴细胞表达无功能EGFR以及表达嵌合抗原受体，其中，所述嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区，所述单链抗体特异性识别抗原MSLN；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述T淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括CD28或4‑1BB的胞内段以及CD3ζ链。该转基因淋巴细胞具有对肿瘤细胞的定向杀伤能力，尤其具有对高表达MSLN的肿瘤细胞的定向杀伤作用。The present invention provides a transgenic lymphocyte, a construct and a therapeutic composition for treating cancer. The transgenic lymphocyte expresses non-functional EGFR and expresses a chimeric antigen receptor, wherein the chimeric antigen receptor includes : the extracellular region, the extracellular region includes the heavy chain variable region and the light chain variable region of the single-chain antibody, and the single-chain antibody specifically recognizes the antigen MSLN; the transmembrane region, the transmembrane region and the an extracellular region connected to and embedded in the cell membrane of the T lymphocyte; an intracellular region connected to the transmembrane region and comprising an intracellular segment of CD28 or 4‑1BB and the CD3ζ chain. The transgenic lymphocyte has the directional killing ability to tumor cells, especially has the directional killing effect to tumor cells with high expression of MSLN.
Description
技术领域technical field
本发明涉及生物医药领域，具体地，本发明涉及一种T淋巴细胞、一种慢病毒、一种转基因淋巴细胞、一种构建体、一种用于治疗癌症的治疗组合物和一种提高淋巴细胞治疗安全性的方法。The present invention relates to the field of biomedicine, in particular, the present invention relates to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer and a lymphatic Approaches to cell therapy safety.
背景技术Background technique
间质素(mesothelin，MSLN)是一种分化抗原，它在人类正常组织的表达仅限于的胸膜、心包膜和腹膜衬里的间皮细胞。然而，间质素却在多种人类癌症组织中高表达，包括几乎所有的间皮瘤和胰腺癌和约70％的卵巢癌和约50％的肺腺癌以及其他癌症，例如胆管癌，胃癌，肠癌，食管癌，乳腺癌。间质素基因编码71KDa的前体蛋白，前体蛋白继而被加工成31KDa的脱落片段和40KDa的蛋白片段，31KDa的脱落片段被称为巨核细胞促进因子(MPF)，而40KDa的蛋白片段即被称为间质素，间质素通过糖基磷脂酰肌醇(glycosyl-phosphatidylinositol，GPI)的锚定作用固定在细胞膜上。Mesothelin (MSLN) is a differentiation antigen whose expression in normal human tissues is limited to mesothelial cells lining the pleura, pericardium and peritoneum. However, interstitin is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancer and about 50% of lung adenocarcinoma and other cancers, such as cholangiocarcinoma, gastric cancer, intestinal cancer , Esophageal cancer, Breast cancer. The mesenchymal gene encodes a 71KDa precursor protein, which is then processed into a 31KDa shedding fragment and a 40KDa protein fragment. The 31KDa shedding fragment is called megakaryocyte-promoting factor (MPF), while the 40KDa protein fragment is Known as interstitium, interstitium is fixed on the cell membrane through the anchoring effect of glycosyl-phosphatidylinositol (GPI).
以间皮瘤为例，间皮瘤有胸膜间皮瘤和腹膜间皮瘤之分，胸膜间皮瘤是胸膜原发肿瘤，有局限型(多为良性)和弥漫型(都是恶性)之分，其中弥漫型恶性间皮瘤是胸部预后最坏的肿瘤之一。腹膜间皮瘤是指原发于腹膜间皮细胞的肿瘤。临床表现不具有特征性，常见的症状和体征有：腹痛、腹水、腹胀及腹部包块等。恶性胸膜间皮瘤的治疗，目前仍然没有有效的根治方法。治疗方法上，有姑息性治疗、外科治疗、化学治疗及放射治疗等，一般认为对于肿瘤相对局限的I期病人，主张做根治的胸膜肺切除术。对于Ⅱ、Ⅲ、Ⅳ期病人，根治性手术已经没有意义了，只有施行姑息性手术。事实上，多数病人到疾病明确诊断时，已处于II期以上。迅速增长的胸水常导致患者严重的呼吸困难，姑息性手术只能暂时提高这些晚期病人的生活质量，而无法根治。Taking mesothelioma as an example, mesothelioma can be divided into pleural mesothelioma and peritoneal mesothelioma. Pleural mesothelioma is a primary tumor of the pleura, which can be divided into localized (mostly benign) and diffuse (all malignant) types. Among them, diffuse malignant mesothelioma is one of the tumors with the worst prognosis in the chest. Peritoneal mesothelioma is a tumor originating from the peritoneal mesothelial cells. The clinical manifestations are not characteristic, and the common symptoms and signs include: abdominal pain, ascites, abdominal distension, and abdominal mass. There is still no effective cure for malignant pleural mesothelioma. In terms of treatment methods, there are palliative treatment, surgical treatment, chemotherapy and radiotherapy, etc. It is generally believed that for stage I patients with relatively limited tumors, radical pleuropneumonectomy is advocated. For stage II, III, and IV patients, radical surgery is meaningless, and only palliative surgery is performed. In fact, most patients are already in stage II or above when the disease is clearly diagnosed. Rapidly growing pleural effusion often leads to severe dyspnea in patients, and palliative surgery can only temporarily improve the quality of life of these advanced patients, but cannot cure it.
由此可见，开发针对间质素高表达肿瘤的治疗方法尤为迫切。It can be seen that it is particularly urgent to develop therapeutic methods for tumors with high expression of interstitial proteins.
发明内容Contents of the invention
本申请是基于发明人对以下事实和问题的发现和认识作出的：This application is based on the inventor's discovery and recognition of the following facts and problems:
间质素却在多种人类癌症组织中高表达，包括几乎所有的间皮瘤和胰腺癌和约70％的卵巢癌和约50％的肺腺癌以及其他癌症，例如胆管癌，胃癌，肠癌，食管癌，乳腺癌。因此，间质素代表着肿瘤免疫治疗领域中一个有着巨大吸引力的靶点。Interstitium is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancer and about 50% of lung adenocarcinoma and other cancers, such as bile duct cancer, gastric cancer, intestinal cancer, esophageal cancer Cancer, breast cancer. Interstitins thus represent an attractive target in the field of tumor immunotherapy.
无功能EGFR(epidermal growth factor receptor，表皮生长因子受体)缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。Non-functional EGFR (epidermal growth factor receptor, epidermal growth factor receptor) lacks the N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane domain of wild-type EGFR and the intact and anti-EGFR antibody The bound sequence, nonfunctional EGFR, acts as a suicide marker for lymphocytes.
基于上述发现，发明人提出了一种编码无功能EGFR的核酸分子和编码嵌合抗原受体的核酸分子的构建体和一种以此构建体导入后形成的转基因淋巴细胞，其编码的嵌合抗原受体特异性结合抗原MSLN。因此，本发明提出的构建体和转基因淋巴细胞可用于肿瘤，尤其是间质素阳性肿瘤的过继T细胞的免疫治疗；本发明所提出转基因淋巴细胞对高表达间质素肿瘤的特异杀伤能力强，对正常MSLN表达水平的间皮细胞具有较弱的杀伤，并且免疫杀伤安全性高。Based on the above findings, the inventors proposed a construct of a nucleic acid molecule encoding a non-functional EGFR and a nucleic acid molecule encoding a chimeric antigen receptor, and a transgenic lymphocyte formed after the introduction of the construct, and the encoded chimeric Antigen receptors specifically bind the antigen MSLN. Therefore, the construct and transgenic lymphocytes proposed by the present invention can be used for immunotherapy of tumors, especially adoptive T cells of mesenchymal positive tumors; the transgenic lymphocytes proposed by the present invention have strong specific killing ability for tumors with high expression of mesenchymal , has weaker killing effect on mesothelial cells with normal MSLN expression level, and has high safety of immune killing.
在本发明的第一方面，本发明提出了一种T淋巴细胞。根据本发明的实施例，所述T淋巴细胞表达无功能EGFR；以及表达嵌合抗原受体，其中，所述嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区，所述单链抗体特异性识别抗原MSLN；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述T淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括CD28或4-1BB的胞内段以及CD3ζ链。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明实施例的T淋巴细胞具有对高表达MSLN的肿瘤细胞的定向杀伤作用，且安全性高。In a first aspect of the present invention, the present invention provides a T lymphocyte. According to an embodiment of the present invention, the T lymphocyte expresses non-functional EGFR; and expresses a chimeric antigen receptor, wherein the chimeric antigen receptor includes: an extracellular region, and the extracellular region includes a single-chain antibody The heavy chain variable region and the light chain variable region, the single-chain antibody specifically recognizes the antigen MSLN; the transmembrane region, the transmembrane region is connected to the extracellular region and embedded in the cell membrane of the T lymphocyte Middle; intracellular region, the intracellular region is connected to the transmembrane region, and the intracellular region includes the intracellular segment of CD28 or 4-1BB and the CD3ζ chain. Non-functional EGFR lacks the N-terminal ligand-binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and the complete sequence that binds to anti-EGFR antibodies. Suicide mark. The T lymphocytes of the embodiments of the present invention have a directional killing effect on tumor cells highly expressing MSLN, and have high safety.
在本发明的第二方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带下列核酸分子：编码嵌合抗原受体的核酸分子，所述嵌合抗原受体具有SEQ ID NO：1所示的氨基酸序列，所述编码嵌合抗原受体的核酸分子具有SEQ ID NO：2所示的核苷酸序列；以及编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：3所示的氨基酸序列，所述编码无功能EGFR的核酸分子具有SEQ ID NO：4所示的核苷酸序列。In the second aspect of the present invention, the present invention provides a lentivirus. According to an embodiment of the present invention, the lentivirus carries the following nucleic acid molecule: a nucleic acid molecule encoding a chimeric antigen receptor, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 1, and the encoding chimeric antigen receptor The nucleic acid molecule of the antigen receptor has the nucleotide sequence shown in SEQ ID NO: 2; and the nucleic acid molecule encoding non-functional EGFR has the amino acid sequence shown in SEQ ID NO: 3, and the coding non-functional EGFR has the amino acid sequence shown in SEQ ID NO: 3 The nucleic acid molecule of functional EGFR has the nucleotide sequence shown in SEQ ID NO:4.
ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ IDNO：2)。ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCG GGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ IDNO：2)。
ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCACGCCCTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGACAGCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCGGCGACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTCTGGACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGCTGATCCAGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGAGATCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCTGAACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGGCACCTCTGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAAGGCAACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAGAGCCACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGTGTAATCTGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGTGTCACCCCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCCCCGACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTGTCCAGCCGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGACACGTGTGCCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGAGGGATGCCCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATGGTGGGGGCACTGCTGCTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAAGGTAA(SEQ ID NO：4)。ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCACGCCCTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGACAGCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCGGCGACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTCTGGACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGCTGATCCAGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGAGATCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCTGAACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGGCACCTCTGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAAGGCAACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAGAGCCACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGTGTAATCTGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGTGTCACCCCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCCCCGACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTGTCCAGCCGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGACACGTGTGCCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGAGGGATGCCCAACCAACGGCCCTAAGATCC CAAGCATCGCCACAGGCATGGTGGGGGCACTGCTGCTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAAGGTAA (SEQ ID NO: 4).
根据本发明的实施例，将本发明实施例的慢病毒导入淋巴细胞所得的转基因淋巴细胞，其具有对肿瘤细胞的特意性杀伤能力尤其具有对高表达MSLN的肿瘤细胞的定向杀伤作用，且安全性高。According to the embodiments of the present invention, the transgenic lymphocytes obtained by introducing the lentivirus of the embodiments of the present invention into lymphocytes have the ability to specifically kill tumor cells, especially have a directional killing effect on tumor cells with high expression of MSLN, and are safe. high sex.
在本发明的第三方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带含有SEQ ID NO：5所示的核苷酸序列。In the third aspect of the present invention, the present invention provides a lentivirus. According to an embodiment of the present invention, the lentivirus carries the nucleotide sequence shown in SEQ ID NO:5.
ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAATCCTACTGCGTCGACACTAGTGAATTCGAATTTAAATCGGATCCGCGGCCGCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGCGTCCGGATCTAGAATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCACGCCCTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGACAGCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCGGCGACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTCTGGACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGCTGATCCAGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGAGATCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCTGAACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGGCACCTCTGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAAGGCAACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAGAGCCACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGTGTAATCTGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGTGTCACCCCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCCCCGACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTGTCCAGCCGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGACACGTGTGCCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGAGGGATGCCCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATGGTGGGGGCACTGCTGCTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAAGGTAA(SEQ ID NO：5)。ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCG TGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAATCCTACTGCGTCGACACTAGTGAATTCGAATTTAAATCGGATCCGCGGCCGCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGA TCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGCGTCCGGATCTAGAATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCACGCCCTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGACAGCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCGGCGACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTCTGGACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGCTGATCCAGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGAGATCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCTGAACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGGCACCTCTGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAAGGCAACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAGAGCCACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGTGTAATCTGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGTGTCACCCCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCCCCGACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTGTCCAGC CGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGACACGTGTGCCACCTGTGCCACCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGAGGGATGCCCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATGGTGGGGCACTGCTGCTGCTGQGTGGTGGCTCTGGTGATTGGAGCTGTTTAT5.(SE)
根据本发明的实施例，将本发明实施例的慢病毒导入淋巴细胞所得的转基因淋巴细胞，具有对肿瘤细胞的定向杀伤能力，尤其具有对高表达MSLN的肿瘤细胞的定向杀伤作用，并且安全性高。According to the embodiments of the present invention, the transgenic lymphocytes obtained by introducing the lentivirus of the embodiments of the present invention into lymphocytes have the ability to kill tumor cells, especially tumor cells with high MSLN expression, and are safe. high.
在本发明的第四方面，本发明提出了一种转基因淋巴细胞。根据本发明的实施例，所述淋巴细胞细胞表达无功能EGFR；以及表达嵌合抗原受体，所述嵌合抗原受体包括：胞外区，所述胞外区包括抗体的重链可变区和轻链可变区，所述抗体能够与肿瘤抗原特异性结合；跨膜区；以及胞内区，所述胞内区包括免疫共刺激分子胞内段，其中，所述抗体为单链抗体，所述肿瘤抗原为MSLN。发明人惊奇的发现，表达无功能EGFR和表达嵌合抗原受体的淋巴细胞具有对肿瘤细胞的特异性杀伤能力，尤其具有对高表达MSLN的肿瘤细胞的定向杀伤作用，且安全性高。In the fourth aspect of the present invention, the present invention provides a transgenic lymphocyte. According to an embodiment of the present invention, the lymphocyte cell expresses non-functional EGFR; and expresses a chimeric antigen receptor, and the chimeric antigen receptor includes: an extracellular region, and the extracellular region includes an antibody heavy chain variable region and a light chain variable region, the antibody can specifically bind to a tumor antigen; a transmembrane region; and an intracellular region, which includes an intracellular segment of an immune co-stimulatory molecule, wherein the antibody is a single chain Antibody, the tumor antigen is MSLN. The inventors surprisingly found that lymphocytes expressing non-functional EGFR and chimeric antigen receptor have specific killing ability to tumor cells, especially have a directional killing effect on tumor cells with high expression of MSLN, and have high safety.
根据本发明的实施例，上述转基因淋巴细胞还可以具有下列附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned transgenic lymphocytes may also have at least one of the following additional technical features:
根据本发明的实施例，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28以及他们的衍生物的至少一种。本发明实施例的免疫共刺激分子胞内段的表达具有正向调控和增强细胞免疫应答的作用，使得本发明实施例的转基因淋巴细胞对肿瘤的定向杀伤作用效果进一步提高；本发明实施例的免疫共刺激分子胞内段的表达以及无功能EGFR的表达的联合，使得本发明实施例的转基因淋巴细胞增殖对肿瘤的具有更加显著的定向杀伤作用，且更加安全。According to an embodiment of the present invention, the intracellular segment of the immune co-stimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and their derivatives. The expression of the intracellular segment of the immune co-stimulatory molecule in the embodiment of the present invention has the effect of positively regulating and enhancing the cellular immune response, which further improves the directional killing effect of the transgenic lymphocytes in the embodiment of the present invention on tumors; the embodiment of the present invention The combination of the expression of the intracellular segment of the immune co-stimulatory molecule and the expression of non-functional EGFR makes the transgenic lymphocyte proliferation of the embodiment of the present invention have a more significant directional killing effect on tumors and is safer.
根据本发明的实施例，所述免疫共刺激分子胞内段是4-1BB或CD28的胞内段。本发明中的转基因淋巴细胞的嵌合抗原受体的免疫共刺激分子胞内段是CD28或者4-1BB的胞内段。根据本发明的实施例，免疫共刺激分子胞内段是CD28或者4-1BB的胞内段，进一步增强了本发明实施例的转基因淋巴细胞的定向杀伤作用。According to an embodiment of the present invention, the intracellular segment of the immune co-stimulatory molecule is the intracellular segment of 4-1BB or CD28. The intracellular segment of the immune co-stimulatory molecule of the chimeric antigen receptor of the transgenic lymphocyte in the present invention is the intracellular segment of CD28 or 4-1BB. According to the embodiment of the present invention, the intracellular segment of the immune co-stimulatory molecule is the intracellular segment of CD28 or 4-1BB, which further enhances the directional killing effect of the transgenic lymphocytes of the embodiment of the present invention.
根据本发明的实施例，本发明实施例的转基因淋巴细胞表达的无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR结合的结构域，无功能EGFR可作为本发明实施例的转基因淋巴细胞的自杀标记。无功能EGFR的表达，联合嵌合抗原受体的表达可在有效保证转基因淋巴细胞的靶向杀伤作用的前提下，如果病人出现严重不良反应，转基因淋巴细胞可被抗EGFR抗体清除，进而可进一步提高本发明实施例的转基因淋巴细胞治疗高表达MSLN的肿瘤病人的安全性。According to the embodiments of the present invention, the non-functional EGFR expressed by the transgenic lymphocytes of the embodiments of the present invention lacks the N-terminal ligand binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region and intact The domain that binds to anti-EGFR, the non-functional EGFR can be used as the suicide marker of the transgenic lymphocytes of the embodiment of the present invention. The expression of non-functional EGFR combined with the expression of chimeric antigen receptor can effectively ensure the targeted killing effect of transgenic lymphocytes. If the patient has serious adverse reactions, the transgenic lymphocytes can be eliminated by anti-EGFR antibodies, and further Improve the safety of the transgenic lymphocytes of the embodiment of the present invention in treating tumor patients with high expression of MSLN.
根据本发明的实施例，所述淋巴细胞是CD3+T淋巴细胞或自然杀伤细胞或自然杀伤T细胞。本发明实施例的上述淋巴细胞表达无功能EGFR，同时表达抗原特异性的嵌合抗原受体，如本发明的实施例的MSLN抗原特异性的嵌合抗原受体，上述淋巴细胞具有对肿瘤的定向杀伤作用，且安全性更高。According to an embodiment of the present invention, the lymphocytes are CD3 + T lymphocytes or natural killer cells or natural killer T cells. The above-mentioned lymphocytes in the embodiments of the present invention express non-functional EGFR, and at the same time express antigen-specific chimeric antigen receptors, such as the MSLN antigen-specific chimeric antigen receptors in the embodiments of the present invention, and the above-mentioned lymphocytes have anti-tumor Targeted killing effect, and higher security.
在本发明的第五方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体包括：第一核酸分子，所述第一核酸分子编码嵌合抗原受体；以及第二核酸分子，所述第二核酸分子编码无功能EGFR。其中，所述嵌合抗原受体、所述无功能EGFR如前所述。根据本发明的实施例，本发明实施例的构建体成功导入本发明实施例的淋巴细胞后，可高效表达无功能EGFR和表达抗原特异性的嵌合抗原受体，从而本发明实施例的淋巴细胞具有对肿瘤细胞，尤其对高表达MSLN的肿瘤细胞的具有定向杀伤作用，且安全性高。In a fifth aspect of the present invention, the present invention proposes a construct. According to an embodiment of the present invention, the construct comprises: a first nucleic acid molecule encoding a chimeric antigen receptor; and a second nucleic acid molecule encoding a non-functional EGFR. Wherein, the chimeric antigen receptor and the non-functional EGFR are as described above. According to the embodiments of the present invention, after the constructs of the embodiments of the present invention are successfully introduced into the lymphocytes of the embodiments of the present invention, they can efficiently express non-functional EGFR and express antigen-specific chimeric antigen receptors, so that the lymphocytes of the embodiments of the present invention The cells have a directional killing effect on tumor cells, especially tumor cells with high expression of MSLN, and have high safety.
根据本发明的实施例，上述构建体还可以进一步包括下列附加技术特征至少之一：According to an embodiment of the present invention, the above construct may further include at least one of the following additional technical features:
根据本发明的实施例，其特征在于，所述第一核酸分子与所述第二核酸分子被设置在前面所述的淋巴细胞中表达所述嵌合抗原受体和表达无功能EGFR，并且所述嵌合抗原受体与所述无功能EGFR呈非融合形式。根据本发明的实施例，成功设置了上述第一核酸分子以及第二核酸分子的淋巴细胞，其淋巴细胞表面成功表达无功能EGFR，同时在淋巴细胞表面成功表达了抗原特异性嵌合抗原受体，如本发明实施例的MSLN特异性的嵌合抗原受体，且嵌合抗原受体与无功能EGFR在淋巴细胞膜上呈非融合形式，本发明实施例的淋巴细胞具有特异性强的肿瘤杀伤效果，安全性更高。According to an embodiment of the present invention, it is characterized in that, the first nucleic acid molecule and the second nucleic acid molecule are configured to express the chimeric antigen receptor and the non-functional EGFR in the aforementioned lymphocytes, and the The chimeric antigen receptor is non-fused to the non-functional EGFR. According to an embodiment of the present invention, the above-mentioned first nucleic acid molecule and the lymphocyte of the second nucleic acid molecule are successfully set up, and the non-functional EGFR is successfully expressed on the surface of the lymphocyte, and at the same time, the antigen-specific chimeric antigen receptor is successfully expressed on the surface of the lymphocyte , such as the MSLN-specific chimeric antigen receptor of the embodiment of the present invention, and the chimeric antigen receptor and non-functional EGFR are in a non-fused form on the lymphocyte membrane, and the lymphocytes of the embodiment of the present invention have specific and strong tumor killing effect, higher security.
根据本发明的实施例，所述构建体进一步包括：第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；以及第二启动子，所述第二启动子与所述第二核酸分子可操作地连接。根据本发明的实施例，第一启动子以及第二启动子的引入，使得第一核酸分子以及第二核酸分子分别独立的表达，有效保证了嵌合抗原受体抗原靶向性的生物学作用及以有效表达了无功能EGFR，从而有效保证了本发明实施例的淋巴细胞的对肿瘤的靶向杀伤作用，尤其是对高表达MSLN的肿瘤细胞的定向杀伤，且保证了免疫杀伤的安全性。According to an embodiment of the present invention, the construct further includes: a first promoter, which is operably linked to the first nucleic acid molecule; and a second promoter, which is operably linked to the first nucleic acid molecule. The second nucleic acid molecule is operably linked. According to the embodiment of the present invention, the introduction of the first promoter and the second promoter enables the independent expression of the first nucleic acid molecule and the second nucleic acid molecule, effectively ensuring the biological effect of chimeric antigen receptor antigen targeting And the non-functional EGFR is effectively expressed, thereby effectively ensuring the targeted killing effect of the lymphocytes of the embodiment of the present invention on tumors, especially the directional killing of tumor cells with high expression of MSLN, and ensuring the safety of immune killing .
根据本发明的实施例，所述第一启动子、所述第二启动子分别独立地选自CMV，EF-1，LTR，RSV启动子。根据本发明的实施例，本发明实施例的上述启动子具有启动效率高、特异性强的特点，从而保证了无功能EGFR的高效表达和嵌合抗原受体的高效表达，从而高效保证了本发明实施例的淋巴细胞对肿瘤的定向杀伤效果和杀伤安全性。According to an embodiment of the present invention, the first promoter and the second promoter are independently selected from CMV, EF-1, LTR and RSV promoters. According to the embodiments of the present invention, the above-mentioned promoters in the embodiments of the present invention have the characteristics of high efficiency and strong specificity, thereby ensuring the high-efficiency expression of non-functional EGFR and the high-efficiency expression of chimeric antigen receptors, thus ensuring the efficient expression of the present invention. The targeted killing effect and killing safety of the lymphocytes of the embodiment of the invention on tumors.
根据本发明的实施例，所述构建体进一步包括：内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第三核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：6所示的核苷酸序列。According to an embodiment of the present invention, the construct further includes: an internal ribosome entry site sequence, the internal ribosome entry site sequence is arranged between the first nucleic acid molecule and the third nucleic acid molecule, so The internal ribosome entry site has the nucleotide sequence shown in SEQ ID NO:6.
TTTAAATCGGATCCGCGGCCGCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGC(SEQ ID NO：6)。TTTAAATCGGATCCGCGGCCGCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGC(SEQ ID NO：6)。
内部核糖体进入位点序列的引入使得第一核酸分子和第二核酸分子分别独立的表达。根据本发明的实施例，内部核糖体进入位点序列的引入保证了嵌合抗原受体抗原靶向性的生物学作用和无功能EGFR的高效表达，进而使得本发明实施例的淋巴细胞对肿瘤的定向杀伤效果更加显著，淋巴细胞对肿瘤杀伤的安全性更高。The introduction of an internal ribosome entry site sequence allows independent expression of the first nucleic acid molecule and the second nucleic acid molecule. According to the embodiment of the present invention, the introduction of the internal ribosome entry site sequence ensures the biological effect of chimeric antigen receptor antigen targeting and the high-efficiency expression of non-functional EGFR, thereby enabling the lymphocytes of the embodiment of the present invention to be effective against tumors. The directional killing effect is more significant, and the safety of lymphocytes to tumor killing is higher.
根据本发明的实施例，所述构建体进一步包括：第三核酸分子，设置在所述第一核酸分子与所述第二核酸分子之间，并且所述第三核酸分子编码连接肽，所述连接肽能够在所述淋巴细胞中被切割。编码连接肽的第三核酸分子的引入使得无功能EGFR和嵌合抗原受体成非融合状态的形式表达在淋巴细胞膜上，进而进一步保证了无功能EGFR和嵌合抗原受体的生物学作用，本发明实施例的淋巴细胞具有特异性更强的肿瘤杀伤效果，安全性更高。According to an embodiment of the present invention, the construct further includes: a third nucleic acid molecule disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a connecting peptide, the The linker peptide can be cleaved in the lymphocytes. The introduction of the third nucleic acid molecule encoding the connecting peptide makes the non-functional EGFR and the chimeric antigen receptor expressed on the lymphocyte membrane in a non-fusion state, thereby further ensuring the biological effects of the non-functional EGFR and the chimeric antigen receptor, The lymphocytes of the embodiments of the present invention have a more specific tumor killing effect and higher safety.
根据本发明的实施例，所述连接肽具有SEQ ID NO：7所示的氨基酸序列。According to an embodiment of the present invention, the connecting peptide has the amino acid sequence shown in SEQ ID NO:7.
GSGATNFSLLKQAGDVEENPGP(SEQ ID NO：7)。GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 7).
SEQ ID NO：7所示的氨基酸序列是手足口病毒(一种小RNA病毒)的2A肽段。连接肽的引入使得无功能EGFR和嵌合抗原受体成非融合状态表达在淋巴细胞膜上。根据本发明的实施例，连接肽的引入保证了无功能EGFR和嵌合抗原受体的生物学作用，本发明实施例的淋巴细胞具特异性更强的肿瘤杀伤效果，安全性更高。The amino acid sequence shown in SEQ ID NO: 7 is the 2A peptide segment of hand, foot and mouth virus (a small RNA virus). The introduction of the linker peptide allows non-functional EGFR and chimeric antigen receptor to be expressed on the lymphocyte membrane in a non-fused state. According to the embodiment of the present invention, the introduction of the connecting peptide ensures the biological effects of the non-functional EGFR and the chimeric antigen receptor, and the lymphocytes of the embodiment of the present invention have a more specific tumor killing effect and higher safety.
根据本发明的实施例，所述构建体的载体是非致病性病毒载体。非致病性病毒载体的引入大大提高了构建体在淋巴细胞中的复制和扩增效率，从而大大提高了无功能EGFR的表达和嵌合抗原受体在淋巴细胞中的高效表达，使得淋巴细胞的靶向作用进一步增强，安全性进一步提高。According to an embodiment of the present invention, the vector of the construct is a non-pathogenic viral vector. The introduction of non-pathogenic viral vectors greatly improved the replication and amplification efficiency of the construct in lymphocytes, thereby greatly improving the expression of non-functional EGFR and the high-efficiency expression of chimeric antigen receptors in lymphocytes, making lymphocytes The targeting effect is further enhanced, and the safety is further improved.
根据本发明的实施例，所述病毒载体包括选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体的至少之一。本发明实施例的病毒的载体在病毒包装和感染过程中，病毒感染范围广泛，既可感染终末分化细胞，又可感染处于分裂期的细胞，其基因组既可整合到宿主染色体，又可游离在宿主染色体之外，从而可实现广谱而高效的感染效率，无功能EGFR在淋巴细胞中高效表达和嵌合抗原受体在淋巴细胞中的高效表达，使得本发明实施例的淋巴细胞的靶向作用进一步增强，对肿瘤细胞，尤其是高表达MSLN的肿瘤细胞的定向杀伤作用更加显著，淋巴细胞的杀伤作用安全性更高。According to an embodiment of the present invention, the viral vector includes at least one selected from a retroviral vector, a lentiviral vector or an adeno-associated viral vector. During the process of virus packaging and infection, the virus vector of the embodiment of the present invention can infect a wide range of viruses. It can infect terminally differentiated cells and cells in the division stage. Its genome can be integrated into the host chromosome or dissociated. Outside the host chromosome, broad-spectrum and high-efficiency infection efficiency can be achieved, high-efficiency expression of non-functional EGFR in lymphocytes and high-efficiency expression of chimeric antigen receptors in lymphocytes, making the lymphocyte target of the embodiment of the present invention The directional effect is further enhanced, the directional killing effect on tumor cells, especially the tumor cells with high MSLN expression is more significant, and the killing effect on lymphocytes is safer.
在本发明的第六方面，本发明提出了一种制备前面所述的T淋巴细胞或者转基因淋巴细胞的方法。根据本发明的实施例，所述方法包括：将前面所述的构建体或者前面所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。所述构建体或慢病毒成功引入上述淋巴细胞或者T淋巴细胞中，实现了淋巴细胞表达无功能EGFR和嵌合抗原受体的表达，从而本发明实施例的制备方法制备的转基因淋巴细胞或T淋巴细胞具有对肿瘤细胞，尤其具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，且安全性更高。In the sixth aspect of the present invention, the present invention proposes a method for preparing the aforementioned T lymphocytes or transgenic lymphocytes. According to an embodiment of the present invention, the method includes: introducing the aforementioned construct or the aforementioned lentivirus into lymphocytes or T lymphocytes. The construct or lentivirus is successfully introduced into the above-mentioned lymphocytes or T lymphocytes, realizing the expression of non-functional EGFR and chimeric antigen receptors expressed by lymphocytes, so that the transgenic lymphocytes or T lymphocytes prepared by the preparation method of the embodiment of the present invention Lymphocytes have a targeted killing effect on tumor cells, especially tumor cells with high expression of MSLN, and have higher safety.
在本发明的第七方面，本发明提出了一种用于治疗癌症的治疗组合物。根据本发明的实施例，所述治疗组合物包括：上述构建体、慢病毒、T淋巴细胞或者转基因淋巴细胞。上述任意一种治疗组合物的组成均可实现转基因淋巴细胞或T淋巴细胞无功能EGFR的表达和嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达，从而使得所得转基因淋巴细胞或T淋巴细胞具有对肿瘤细胞的靶向杀伤作用，本发明实施例的治疗癌症的治疗组合物具有对肿瘤细胞的靶向杀伤作用，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，且安全性高。In the seventh aspect of the present invention, the present invention proposes a therapeutic composition for treating cancer. According to an embodiment of the present invention, the therapeutic composition includes: the above construct, lentivirus, T lymphocytes or transgenic lymphocytes. The composition of any of the above therapeutic compositions can realize the expression of non-functional EGFR in transgenic lymphocytes or T lymphocytes and the high expression of chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, so that the obtained transgenic lymphocytes or T lymphocytes have a targeted killing effect on tumor cells, and the therapeutic composition for treating cancer according to embodiments of the present invention has a targeted killing effect on tumor cells, especially tumor cells that highly express MSLN. And high security.
根据本发明的实施例，上述治疗组合物还可以进一步包括下列附加技术特征至少之一：According to an embodiment of the present invention, the above therapeutic composition may further include at least one of the following additional technical features:
根据本发明的实施例，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。上述肿瘤细胞具有MSLN的特异性高表达，本发明实施例的治疗组合物可使淋巴细胞细胞表面表达无功能EGFR和高效表达抗原特异性嵌合抗原受体，如本发明实施例的MSLN抗原特异性嵌合抗原受体，所得淋巴细胞或T淋巴细胞具有对高表达MSLN的肿瘤细胞的靶向杀伤，且安全性高。According to an embodiment of the present invention, the cancer includes at least one selected from mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer and breast cancer. The above-mentioned tumor cells have specific and high expression of MSLN. The therapeutic composition of the embodiment of the present invention can make the cell surface of lymphocytes express non-functional EGFR and highly express antigen-specific chimeric antigen receptors, such as the MSLN antigen specificity of the embodiment of the present invention. The resulting lymphocytes or T lymphocytes can target and kill tumor cells with high MSLN expression, and have high safety.
在本发明的第八方面，本发明提出了一种提高淋巴细胞治疗安全性的方法，所述淋巴细胞携带嵌合抗原受体，其特征在于，所述方法包括：使所述淋巴细胞表达无功能EGFR，所述无功能EGFR、所述淋巴细胞、所述嵌合抗原受体如前所述。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明实施例的淋巴细胞在用于治疗治疗高表达MSLN的肿瘤细胞时，如果病人出现严重不良反应，本发明实施例的淋巴细胞可被抗EGFR抗体清除，进而可提高本发明实施例的淋巴细胞治疗高表达MSLN的肿瘤病人的安全性。In the eighth aspect of the present invention, the present invention proposes a method for improving the safety of lymphocyte therapy, said lymphocytes carrying chimeric antigen receptors, characterized in that said method comprises: making said lymphocytes express Functional EGFR, the non-functional EGFR, the lymphocytes, and the chimeric antigen receptor are as described above. Non-functional EGFR lacks the N-terminal ligand-binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and the complete sequence that binds to anti-EGFR antibodies. Suicide mark. When the lymphocytes of the embodiments of the present invention are used to treat tumor cells with high expression of MSLN, if the patient has serious adverse reactions, the lymphocytes of the embodiments of the present invention can be eliminated by anti-EGFR antibodies, thereby improving the lymphocytes of the embodiments of the present invention. Safety of cell therapy in patients with tumors overexpressing MSLN.
附图说明Description of drawings
图1是根据本发明实施例的共表达抗MSLN的嵌合抗原受体和无功能EGFR的慢病毒载体的结构示意图；以及1 is a schematic structural view of a lentiviral vector co-expressing an anti-MSLN chimeric antigen receptor and a non-functional EGFR according to an embodiment of the present invention; and
图2是根据本发明实施例的共表达抗MSLN的嵌合抗原受体和无功能EGFR的淋巴细胞被抗EGFR抗体-介导ADCC杀伤清除的结果图。Fig. 2 is a graph showing the results of anti-EGFR antibody-mediated ADCC killing and clearance of lymphocytes co-expressing anti-MSLN chimeric antigen receptor and non-functional EGFR according to an embodiment of the present invention.
具体实施方式Detailed ways
下面详细描述本发明的实施例，下面描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below, and the embodiments described below are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.
T淋巴细胞或转基因淋巴细胞T lymphocytes or transgenic lymphocytes
在本发明的一方面，本发明提出了一种T淋巴细胞或转基因淋巴细胞。根据本发明的实施例，本发明实施例的T淋巴细胞表达无功能EGFR；以及表达嵌合抗原受体，其中，嵌合抗原受体包括：胞外区，胞外区包括单链抗体的重链可变区和轻链可变区，单链抗体特异性识别抗原MSLN；跨膜区，跨膜区与胞外区相连，并且嵌入到T淋巴细胞的细胞膜中；胞内区，胞内区与跨膜区相连，并且胞内区包括CD28或4-1BB的胞内段以及CD3ζ链。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明实施例的T淋巴细胞或转基因淋巴细胞表达MSLN抗原特异性的嵌合抗原受体，本发明实施例的T淋巴细胞或转基因淋巴细胞具有对特异性肿瘤细胞的杀伤能力，尤其具有对高表达MSLN的肿瘤细胞的特异性杀伤；本发明实施例的T淋巴细胞或转基因淋巴细胞细胞表达无功能EGFR联合表达MSLN抗原特异性的嵌合抗原受体，本发明实施例的T淋巴细胞或转基因淋巴细胞杀伤安全性高。In one aspect of the present invention, the present invention provides a T lymphocyte or a transgenic lymphocyte. According to an embodiment of the present invention, the T lymphocytes of the present invention express non-functional EGFR; and express a chimeric antigen receptor, wherein the chimeric antigen receptor includes: an extracellular region, and the extracellular region includes a heavy single-chain antibody Chain variable region and light chain variable region, the single-chain antibody specifically recognizes the antigen MSLN; the transmembrane region, the transmembrane region is connected to the extracellular region, and embedded in the cell membrane of T lymphocytes; the intracellular region, the intracellular region It is connected to the transmembrane region, and the intracellular region includes the intracellular segment of CD28 or 4-1BB and the CD3ζ chain. Non-functional EGFR lacks the N-terminal ligand-binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and the complete sequence that binds to anti-EGFR antibodies. Suicide mark. The T lymphocytes or transgenic lymphocytes of the embodiments of the present invention express MSLN antigen-specific chimeric antigen receptors, and the T lymphocytes or transgenic lymphocytes of the embodiments of the present invention have the ability to kill specific tumor cells, especially for high Specific killing of tumor cells expressing MSLN; the T lymphocytes or transgenic lymphocytes of the embodiments of the present invention express non-functional EGFR combined with the expression of MSLN antigen-specific chimeric antigen receptors, the T lymphocytes or transgenic lymphocytes of the embodiments of the present invention Lymphocyte killing safety is high.
另外，根据本发明的实施例，本发明实施例的无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。表达无功能EGFR的淋巴细胞可被抗EGFR抗体在体内清除。从而，本发明实施例的T淋巴细胞或转基因淋巴细胞表达无功能EGFR，在保证转基因淋巴细胞的靶向杀伤作用的前提下，如果病人出现严重不良反应，转基因淋巴细胞可被抗EGFR抗体清除，进而可进一步提高本发明实施例的转基因淋巴细胞或T淋巴细胞治疗高表达MSLN的肿瘤病人的安全性。In addition, according to the embodiments of the present invention, the non-functional EGFR of the embodiments of the present invention lacks the N-terminal ligand binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and the complete and anti- The sequence to which EGFR antibodies bind, non-functional EGFR acts as a suicide marker for lymphocytes. Lymphocytes expressing non-functional EGFR can be eliminated in vivo by anti-EGFR antibodies. Therefore, the T lymphocytes or transgenic lymphocytes in the embodiment of the present invention express non-functional EGFR. On the premise of ensuring the targeted killing effect of the transgenic lymphocytes, if the patient has serious adverse reactions, the transgenic lymphocytes can be eliminated by the anti-EGFR antibody. Furthermore, the safety of treating tumor patients with high expression of MSLN by the transgenic lymphocytes or T lymphocytes of the embodiment of the present invention can be further improved.
另外，根据本发明的实施例，上述嵌合抗原受体胞外区的抗体为单链抗体。发明人发现，单链抗体可去除非特异性反应的竞争性表面蛋白，同时单链抗体更易渗透肿瘤组织增加药物治疗浓度。本发明实施例的转基因淋巴细胞表达单链抗体的嵌合抗原受体，进一步提高了转基因淋巴细胞对靶向肿瘤细胞的定向杀伤作用。In addition, according to an embodiment of the present invention, the above-mentioned antibody to the extracellular region of the chimeric antigen receptor is a single-chain antibody. The inventors found that the single-chain antibody can remove non-specifically reacting competitive surface proteins, and at the same time, the single-chain antibody is more likely to penetrate tumor tissue and increase the therapeutic concentration of the drug. The transgenic lymphocytes of the embodiment of the present invention express the chimeric antigen receptor of the single chain antibody, which further improves the directional killing effect of the transgenic lymphocytes on the targeted tumor cells.
根据本发明的另外一些实施例，上述抗体的结合抗原为MSLN。因此本发明实施例的转基因淋巴细胞针对表达抗原MSLN的细胞具有定向性杀伤作用，抗原抗体的特异性结合作用更强，进一步提高了本发明实施例的转基因淋巴细胞对MSLN抗原表达肿瘤细胞的定向杀伤作用。According to some other embodiments of the present invention, the binding antigen of the above antibody is MSLN. Therefore, the transgenic lymphocytes of the embodiment of the present invention have a directional killing effect on the cells expressing the antigen MSLN, and the specific binding effect of the antigen antibody is stronger, which further improves the orientation of the transgenic lymphocytes of the embodiment of the present invention to the tumor cells expressing the MSLN antigen lethal effect.
另外，根据本发明的实施例，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28以及他们的衍生物的至少一种。免疫共刺激分子胞内段的表达具有正向调控和增强细胞免疫应答的作用，使得转基因淋巴细胞对高表达MSLN的肿瘤的定向杀伤作用效果进一步提高，免疫共刺激分子胞内段的表达联合无功能EGFR的表达，使得转基因淋巴细胞的免疫杀伤作用更加安全有效。In addition, according to an embodiment of the present invention, the intracellular segment of the immune co-stimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and their derivatives. The expression of the intracellular segment of the immune co-stimulatory molecule can positively regulate and enhance the cellular immune response, which further improves the directional killing effect of the transgenic lymphocytes on tumors with high MSLN expression. The expression of the intracellular segment of the immune costimulatory molecule combined with no The expression of functional EGFR makes the immune killing effect of transgenic lymphocytes safer and more effective.
根据本发明的实施例，本发明实施例的淋巴细胞是CD3+淋巴细胞或自然杀伤细胞或自然杀伤T细胞。CD3+淋巴细胞是总T细胞，自然杀伤细胞是免疫细胞的一种，非特异性识别靶细胞，自然杀伤T细胞是具有T细胞和自然杀伤细胞受体的T细胞亚群。上述淋巴细胞表达无功能EGFR和表达嵌合抗原受体，使得上述淋巴细胞的对肿瘤细胞免疫杀伤作用更加安全有效。According to an embodiment of the present invention, the lymphocytes in the embodiment of the present invention are CD3 + lymphocytes or natural killer cells or natural killer T cells. CD3 + lymphocytes are total T cells, natural killer cells are a type of immune cells that non-specifically recognize target cells, and natural killer T cells are a subset of T cells that have T cells and natural killer cell receptors. The above-mentioned lymphocytes express non-functional EGFR and express chimeric antigen receptors, so that the immune killing effect of the above-mentioned lymphocytes on tumor cells is safer and more effective.
慢病毒或构建体lentivirus or construct
在本发明的另一方面，本发明提出了一种慢病毒或构建体。根据本发明的实施例，慢病毒或构建体携带下列核酸分子：编码嵌合抗原受体的核酸分子，嵌合抗原受体具有SEQID NO：1所示的氨基酸序列，编码嵌合抗原受体的核酸分子具有SEQ ID NO：2所示的核苷酸序列；以及编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：3所示的氨基酸序列，所述编码无功能EGFR的核酸分子具有SEQ ID NO：4所示的核苷酸序列。根据本发明的实施例，将本发明实施例的慢病毒或构建体导入淋巴细胞所得的转基因淋巴细胞中，其细胞表面表达无功能EGFR，同时在其细胞表面表达抗MSLN的嵌合抗原受体，从而本发明实施例的转基因淋巴细胞具有了显著的对肿瘤细胞的定向杀伤能力，且免疫杀伤安全性显著提高，本发明实施例的转基因淋巴细胞对高表达MSLN的肿瘤细胞特异性杀伤效果尤为显著。In another aspect of the invention, the invention proposes a lentivirus or construct. According to an embodiment of the present invention, the lentivirus or the construct carries the following nucleic acid molecule: a nucleic acid molecule encoding a chimeric antigen receptor, the chimeric antigen receptor has the amino acid sequence shown in SEQID NO: 1, and the nucleic acid molecule encoding the chimeric antigen receptor The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 2; and the nucleic acid molecule encoding non-functional EGFR has the amino acid sequence shown in SEQ ID NO: 3, and the nucleic acid molecule encoding non-functional EGFR The molecule has the nucleotide sequence shown in SEQ ID NO:4. According to an embodiment of the present invention, the transgenic lymphocytes obtained by introducing the lentivirus or the construct of the embodiment of the present invention into lymphocytes express non-functional EGFR on the cell surface and express an anti-MSLN chimeric antigen receptor on the cell surface , so that the transgenic lymphocytes of the embodiments of the present invention have significant directional killing ability to tumor cells, and the safety of immune killing is significantly improved, and the transgenic lymphocytes of the embodiments of the present invention have a specific killing effect on tumor cells with high MSLN expression. significantly.
根据本发明地实施例，本发明实施例的慢病毒或构建体携带含有SEQ ID NO：5所示的核苷酸序列。SEQ ID NO：5表示的是共表达抗MSLN嵌合抗原受体和无功能EGFR的核苷酸序列(MSLN CAR/tEGFR)。根据本发明的实施例，将本发明实施列的慢病毒导入淋巴细胞所得的转基因淋巴细胞，其表达无功能EGFR以及抗MSLN的嵌合抗原受体表达，使得转基因淋巴细胞对肿瘤的定向杀伤能力，免疫杀伤安全性高，尤其对高表达MSLN的肿瘤细胞具有特异性杀伤效果，对高表达MSLN的肿瘤细胞的特异性杀伤安全性高。According to the embodiment of the present invention, the lentivirus or the construct of the embodiment of the present invention carries the nucleotide sequence shown in SEQ ID NO:5. SEQ ID NO: 5 represents the nucleotide sequence (MSLN CAR/tEGFR) that co-expresses anti-MSLN chimeric antigen receptor and non-functional EGFR. According to an embodiment of the present invention, the transgenic lymphocyte obtained by introducing the lentivirus according to the embodiment of the present invention into lymphocyte expresses non-functional EGFR and anti-MSLN chimeric antigen receptor expression, so that the transgenic lymphocyte can directional kill the tumor , the immune killing safety is high, especially it has a specific killing effect on tumor cells with high MSLN expression, and the specific killing safety on tumor cells with high MSLN expression is high.
根据本发明的实施例，发明人是通过如下方式的至少之一实现上述细胞嵌合抗原受体以及无功能EGFR分别独立地表达的：。According to an embodiment of the present invention, the inventor achieved the expression of the above-mentioned chimeric antigen receptor and non-functional EGFR independently by at least one of the following methods:
内部核糖体进入位点序列(IRES)，本发明实施例的内部核糖体进入位点序列设置在编码嵌合抗原受体的核酸分子与表达无功能EGFR的核酸分子之间，内部核糖体进入位点具有SEQ ID NO：6所示的核苷酸序列。内部核糖体进入位点通常位于RNA病毒基因组的5’非翻译区(UTR)，这样一个病毒蛋白的翻译就可以不依赖于5’帽子结构，另一个蛋白通常靠5’帽子结构起始翻译，IRES前后的两个基因的表达通常是成比例的。内部核糖体进入位点序列的引入使得编码嵌合抗原受体的核酸分子与编码无功能EGFR的核酸分子分别独立的表达。根据本发明的实施例，本发明实施例采用内部核糖体进入位点序列有效保证了嵌合抗原受体和无功能EGFR的高效表达，使得淋巴细胞对肿瘤的特异性杀伤效果进一步提高，免疫杀伤安全性进一步提高。Internal ribosome entry site sequence (IRES), the internal ribosome entry site sequence of the embodiment of the present invention is set between the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule expressing non-functional EGFR, the internal ribosome entry site sequence Dot has the nucleotide sequence shown in SEQ ID NO:6. The internal ribosome entry site is usually located in the 5' untranslated region (UTR) of the RNA virus genome, so that the translation of one viral protein can be independent of the 5' cap structure, and the other protein usually relies on the 5' cap structure to initiate translation, The expression of the two genes before and after the IRES is usually proportional. The introduction of the internal ribosome entry site sequence allows the independent expression of the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the non-functional EGFR. According to the embodiment of the present invention, the embodiment of the present invention adopts the internal ribosome entry site sequence to effectively ensure the high-efficiency expression of chimeric antigen receptor and non-functional EGFR, so that the specific killing effect of lymphocytes on tumors is further improved, and immune killing Safety is further improved.
第三核酸分子，本发明实施例的第三核酸分子设置在第一核酸分子与第二核酸分子之间，并且第三核酸分子编码连接肽，连接肽能够在淋巴细胞中被切割。根据本发明的实施例，连接肽具有SEQ ID NO：7所示的氨基酸序列。SEQ ID NO：7所示的氨基酸序列是手足口病毒(一种小RNA病毒)的2A肽段。第三核酸分子的引入使得无功能性EGFR和嵌合抗原受体成非融合状态表达在淋巴细胞膜上，从而保证了的无功能EGFR和嵌合抗原受体的生物学作用，其具特异性更强的肿瘤杀伤效果，安全性更高。The third nucleic acid molecule, the third nucleic acid molecule in the embodiment of the present invention is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a connecting peptide, which can be cleaved in lymphocytes. According to an embodiment of the present invention, the connecting peptide has the amino acid sequence shown in SEQ ID NO:7. The amino acid sequence shown in SEQ ID NO: 7 is the 2A peptide segment of hand, foot and mouth virus (a small RNA virus). The introduction of the third nucleic acid molecule makes non-functional EGFR and chimeric antigen receptor expressed on the lymphocyte membrane in a non-fusion state, thereby ensuring the biological effect of non-functional EGFR and chimeric antigen receptor, which has more specificity. Strong tumor killing effect, higher safety.
启动子：本发明实施例的第一启动子与编码嵌合抗原受体的核酸分子可操作地连接；以及第二启动子与表达无功能EGFR的核酸分子可操作地连接。根据本发明的实施例，所采用的第一启动子、第二启动子独立地选自CMV，EF-1，LTR，RSV启动子，第一以及第二启动子的引入，使得编码嵌合抗原受体的核酸分子和表达无功能EGFR的核酸分子分别独立的表达，从而保证了嵌合抗原受体的高效表达，淋巴细胞的靶向作用更强，对肿瘤的特异性杀伤作用进一步提高，免疫杀伤的安全性也进一步提高。Promoter: the first promoter of the embodiment of the present invention is operably linked to the nucleic acid molecule encoding chimeric antigen receptor; and the second promoter is operably linked to the nucleic acid molecule expressing non-functional EGFR. According to an embodiment of the present invention, the first promoter and the second promoter used are independently selected from CMV, EF-1, LTR, RSV promoters, and the introduction of the first and second promoters makes it possible to encode a chimeric antigen The nucleic acid molecule of the receptor and the nucleic acid molecule expressing non-functional EGFR are independently expressed, thereby ensuring the high-efficiency expression of the chimeric antigen receptor, the targeting effect of lymphocytes is stronger, and the specific killing effect on tumors is further improved. The safety of killing is also further improved.
通过上述内部核糖体进入位点序列或第一、第二启动子的引入，使得细胞无功能EGFR高效地表达和嵌合抗原受体高效地表达在本发明实施例的转基因淋巴细胞膜上，从而保证了嵌合抗原受体的生物学作用，有效实现了转基因淋巴细胞的及时清除，从而使得淋巴细胞的靶向杀伤作用更加显著，免疫杀伤的安全性进一步提高。Through the introduction of the above-mentioned internal ribosome entry site sequence or the first and second promoters, the non-functional EGFR of the cell is efficiently expressed and the chimeric antigen receptor is efficiently expressed on the transgenic lymphocyte membrane of the embodiment of the present invention, thereby ensuring The biological role of the chimeric antigen receptor is understood, and the timely removal of the transgenic lymphocytes is effectively realized, so that the targeted killing effect of the lymphocytes is more significant, and the safety of immune killing is further improved.
另外，根据本发明的实施例，本发明实施例的构建体的载体是非致病性病毒载体。非致病性病毒载体大大提高了构建体在淋巴细胞中的复制和扩增效率，进而本发明实施例的淋巴细胞靶向作用进一步增强，对肿瘤细胞的杀伤作用进一步提高，免疫杀伤安全性进一步提高。In addition, according to an embodiment of the present invention, the vector of the construct of the embodiment of the present invention is a non-pathogenic viral vector. The non-pathogenic viral vector greatly improves the replication and amplification efficiency of the construct in lymphocytes, and the lymphocyte targeting effect of the embodiments of the present invention is further enhanced, the killing effect on tumor cells is further improved, and the safety of immune killing is further improved. improve.
根据本发明的实施例，本发明实施例的构建体的载体是病毒载体，病毒载体选自反转录病毒载体、慢病毒载体、腺病毒载体或腺病毒关联病毒载体的至少之一。根据本发明的实施例，本发明实施例的病毒的载体在病毒包装和感染过程中，病毒感染范围广泛，既可感染终末分化细胞，又可感染处于分裂期的细胞，既可整合到宿主染色体，又可游离在宿主染色体之外，实现广谱而高效的感染效率，从而无功能EGFR被高效表达和嵌合抗原受体在淋巴细胞中高效表达，本发明实施例的淋巴细胞的靶向作用进一步增强，对肿瘤细胞的杀伤作用更加显著，淋巴细胞的免疫杀伤安全性进一步提高。According to an embodiment of the present invention, the vector of the construct of the embodiment of the present invention is a viral vector, and the viral vector is selected from at least one of a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector. According to the embodiments of the present invention, during the process of virus packaging and infection, the virus vectors of the embodiments of the present invention can infect a wide range of viruses, which can infect terminally differentiated cells and cells in the division stage, and can be integrated into the host Chromosomes can be dissociated outside the host chromosomes to achieve broad-spectrum and high-efficiency infection efficiency, so that non-functional EGFR is highly expressed and chimeric antigen receptors are highly expressed in lymphocytes. The lymphocyte targeting of the embodiment of the present invention The effect is further enhanced, the killing effect on tumor cells is more significant, and the safety of immune killing of lymphocytes is further improved.
根据本发明的具体实施例，以构建一个慢病毒载体为例，发明人为了构建一个慢病毒载体，在某些病毒序列的位置，将目的核酸插入到病毒基因组中，从而产生复制缺陷的病毒。为了产生病毒体，发明人进而构建包装细胞系(包含gag，pol和env基因，但不包括LTR和包装成分)。发明人将含有目的基因的重组质粒，连同慢病毒LTR和包装序列，一起引入包装细胞系中。包装序列允许重组质粒RNA转录产物被包装到病毒颗粒中，然后被分泌到培养基中。进而发明人收集包含重组慢病毒的基质，有选择性地浓缩，并用于基因转移。慢载体可以感染多种细胞类型，包括可分裂细胞和不可分裂细胞。According to a specific embodiment of the present invention, taking the construction of a lentiviral vector as an example, in order to construct a lentiviral vector, the inventor inserted the target nucleic acid into the viral genome at the position of certain viral sequences, thereby producing a replication-deficient virus. To produce virions, the inventors proceeded to construct a packaging cell line (comprising gag, pol and env genes, but excluding LTR and packaging components). The inventors introduced the recombinant plasmid containing the gene of interest, together with the lentiviral LTR and packaging sequence, into the packaging cell line. The packaging sequence allows the recombinant plasmid RNA transcript to be packaged into viral particles and then secreted into the culture medium. The inventors further collected the matrix containing the recombinant lentivirus, selectively concentrated it, and used it for gene transfer. Lentivectors can infect a variety of cell types, including both dividing and non-dividing cells.
另外，根据本发明的实施例，本发明实施例的慢病毒是复合慢病毒，除了常见的慢病毒基因gag，pol和env，还包含有调控和结构功能的其他基因。慢病毒载体是本领域技术人员所熟知的，慢病毒包括：人类免疫缺陷病毒HIV–1，HIV–2和猿猴免疫缺陷病毒SIV。慢病毒载体通过多重衰减艾滋病毒致病基因产生，例如全部删除基因env，vif，vpr，vpu和nef，使慢病毒载体形成生物安全型载体。重组慢病毒载体能够感染非分裂细胞，同时可用于体内和体外基因转移和核酸序列表达。例如：在合适的宿主细胞中，和带有包装功能(gag，pol，env，rev和tat)的两个或更多的载体一起，能够感染非分裂细胞。重组病毒的靶向性，是通过抗体或特定配体(靶向特定细胞类型受体)与膜蛋白的结合来实现的。同时，重组病毒的靶向性通过插入一个有效序列(包括调控区域)到病毒载体中，连同另一个编码了特定靶细胞上的受体的配体的基因，使载体具有了特定的靶向。各种有用的慢病毒载体，以及各种方法和操作等产生的载体，用于改变细胞的表达。In addition, according to the embodiment of the present invention, the lentivirus of the embodiment of the present invention is a composite lentivirus, which includes other genes with regulatory and structural functions in addition to the common lentiviral genes gag, pol and env. Lentiviral vectors are well known to those skilled in the art, and lentiviruses include human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors are produced by multiple attenuation of HIV-causing genes, such as the complete deletion of genes env, vif, vpr, vpu and nef, so that lentiviral vectors form biosafety vectors. Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression in vivo and in vitro. For example, in suitable host cells, two or more vectors with packaging functions (gag, pol, env, rev and tat) can infect non-dividing cells. Targeting of recombinant viruses is achieved through the binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins. At the same time, the targeting of the recombinant virus enables the vector to have specific targeting by inserting an effective sequence (including the regulatory region) into the viral vector together with another gene encoding the ligand of the receptor on the specific target cell. A variety of lentiviral vectors are useful, as well as vectors produced by various methods and manipulations, etc., for altering expression in cells.
根据本发明的实施例，本发明实施例的腺关联病毒载体(AAV)可使用一种或多种为人熟知的血清类型腺关联病毒载体的DNA构建。本领域技术人员构建一个合适的腺关联病毒载体，以此携带共表达嵌合抗原受体和无功能EGFR的核苷酸分子。According to the embodiment of the present invention, the adeno-associated virus vector (AAV) of the embodiment of the present invention can be constructed using DNA of one or more well-known serotype AAV vectors. Those skilled in the art construct a suitable adeno-associated virus vector to carry the nucleotide molecule co-expressing chimeric antigen receptor and non-functional EGFR.
另外，根据本发明的实施例，本发明实施例的也包含微基因。微基因意味着用组合(选定的核苷酸序列和可操作的必要的相关连接序列)来指导转化、转录和/或基因产物在体内或体外的宿主细胞中的表达。应用“可操作的连接”序列包含连续目的基因的表达控制序列，和作用于反式或远距离控制目的基因的表达控制序列。In addition, according to the embodiments of the present invention, the embodiments of the present invention also include minigenes. Minigene means the use of combinations (selected nucleotide sequences and operatively necessary associated linking sequences) to direct transformation, transcription and/or expression of gene products in host cells in vivo or in vitro. Use of "operably linked" sequences comprises expression control sequences for the gene of interest contiguously, and expression control sequences that act to control the gene of interest in trans or remotely.
另外，本发明实施例的载体还包括常规控制元素，在和质粒载体一起的细胞转染或/和病毒载体一起的细胞感染中，这些元素允许转录、转化mRNA的表达。大量的表达控制序列(包括天然的，可诱导和/或特定组织的启动子)可能被使用。根据本发明的实施例，启动子为选pol I，pol II and pol III的RAN聚合酶启动子。根据本发明的实施例，启动子为组织特异型启动子。根据本发明的实施例，启动子为诱导型启动子。根据本发明的实施例，启动子为选自基于所选载体的启动子。根据本发明的实施例，当选择慢病毒载体时，启动子为CMV IE基因，EF-1α，泛素C，或磷酸甘油激酶(PGK)启动子。其他常规表达控制序列包括可选标记或报告基因，包括编码遗传霉素，潮霉素，氨苄青霉素或嘌呤霉素耐药性等的核苷酸序列。载体的其他组件包括复制起点。In addition, the vectors of the embodiments of the present invention also include conventional control elements that allow transcription and expression of transformed mRNA during cell transfection with plasmid vectors or/with viral vectors. A variety of expression control sequences (including native, inducible and/or tissue-specific promoters) may be used. According to an embodiment of the present invention, the promoter is a RAN polymerase promoter selected from pol I, pol II and pol III. According to an embodiment of the present invention, the promoter is a tissue-specific promoter. According to an embodiment of the present invention, the promoter is an inducible promoter. According to an embodiment of the present invention, the promoter is a promoter selected from a selected vector. According to an embodiment of the present invention, when the lentiviral vector is selected, the promoter is the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerol kinase (PGK) promoter. Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding resistance to geneticin, hygromycin, ampicillin or puromycin, among others. Other components of vectors include origins of replication.
构建载体的技术为本领域技术人员所熟知的，这些技术包括常规克隆技术，例如在本发明实施例中所使用聚合酶链反应和任何适当的提供所需的核苷酸序列的方法。Techniques for constructing vectors are well known to those skilled in the art, and these techniques include conventional cloning techniques, such as the polymerase chain reaction used in the embodiments of the present invention and any appropriate method for providing the desired nucleotide sequence.
根据本发明的实施例，发明人构建了共表达无功能EGFR以及嵌合抗原受体(CAR)的病毒载体。本发明实施例的表达无功能EGFR的核酸分子以及表达嵌合抗原受体(CAR)的病毒载体或质粒是复合的，此病毒载体或质粒可结合聚合物或其他材料来增加其稳定性，或协助其靶向运动。According to an embodiment of the present invention, the inventors constructed a viral vector that co-expresses non-functional EGFR and a chimeric antigen receptor (CAR). The nucleic acid molecule expressing non-functional EGFR and the viral vector or plasmid expressing chimeric antigen receptor (CAR) in the embodiment of the present invention are composite, and the viral vector or plasmid can be combined with polymer or other materials to increase its stability, or assist in its targeting.
制备转基因淋巴细胞的方法Method for preparing transgenic lymphocytes
在本发明的另一方面，本发明提出了一种制备前面所述的T淋巴细胞或者转基因淋巴细胞的方法。根据本发明的实施例，该方法包括：将前面所述的构建体或者前面所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。引入方式可以选自电转或病毒感染宿主细胞的方式引入。本发明实施例的构建体或慢病毒成功引入上述淋巴细胞或者T淋巴细胞中，实现了针对抗原MSLN的嵌合抗原受体的表达和无功能EGFR的表达，从而使得所得淋巴细胞或T淋巴细胞具有对肿瘤细胞，尤其是高表达MSLN的肿瘤细胞的靶向杀伤作用，免疫杀伤的安全性高。In another aspect of the present invention, the present invention proposes a method for preparing the aforementioned T lymphocytes or transgenic lymphocytes. According to an embodiment of the present invention, the method includes: introducing the aforementioned construct or the aforementioned lentivirus into lymphocytes or T lymphocytes. The introduction method can be selected from electroporation or virus infection of host cells. The construct or lentivirus of the embodiment of the present invention is successfully introduced into the above-mentioned lymphocytes or T lymphocytes, realizing the expression of the chimeric antigen receptor for the antigen MSLN and the expression of non-functional EGFR, so that the resulting lymphocytes or T lymphocytes It has a targeted killing effect on tumor cells, especially tumor cells with high expression of MSLN, and the safety of immune killing is high.
治疗癌症的治疗组合物Therapeutic compositions for treating cancer
在本发明的另一方面，本发明的提出了一种用于治疗癌症的治疗组合物。根据本发明的实施例，该治疗组合物包括：上述构建体、上述慢病毒、上述T淋巴细胞或者上述转基因淋巴细胞。上述任意一种治疗组合物的组成均可实现针对抗原MSLN嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达和无功能EGFR在转基因淋巴细胞或T淋巴细胞表面的表达，从而使得所得转基因淋巴细胞或T淋巴细胞具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，免疫杀伤的安全性高。In another aspect of the present invention, the present invention provides a therapeutic composition for treating cancer. According to an embodiment of the present invention, the therapeutic composition includes: the above-mentioned construct, the above-mentioned lentivirus, the above-mentioned T lymphocyte or the above-mentioned transgenic lymphocyte. The composition of any of the above-mentioned therapeutic compositions can realize the high expression of the antigen MSLN chimeric antigen receptor in transgenic lymphocytes or T lymphocytes and the expression of non-functional EGFR on the surface of transgenic lymphocytes or T lymphocytes, so that The obtained transgenic lymphocytes or T lymphocytes have a targeted killing effect on tumor cells highly expressing MSLN, and the safety of immune killing is high.
根据本发明的实施例，提供给患者的本发明实施例的治疗组合物，较好的应用于生物兼容溶液或可接受的药学运载载体。作为准备的各种治疗组合物被悬浮或溶解在医药上或生理上可接受的载体，如生理盐水；等渗的盐溶液或其他精于此道的人的比较明显的配方中。适当的载体在很大程度上取决于给药途径。其他有水和无水的等渗无菌注射液和有水和无水的无菌悬浮液，是医药上可接受的载体。According to the embodiments of the present invention, the therapeutic composition of the embodiments of the present invention provided to patients is preferably applied to a biocompatible solution or an acceptable pharmaceutical carrier. Various therapeutic compositions are prepared by being suspended or dissolved in pharmaceutically or physiologically acceptable carriers such as physiological saline; isotonic saline solutions or other formulations apparent to those skilled in the art. The appropriate carrier depends largely on the route of administration. Other aqueous and anhydrous isotonic sterile injections and aqueous and anhydrous sterile suspensions are pharmaceutically acceptable carriers.
根据本发明的实施例，足够数量的病毒载体被转导入靶向T细胞中，并提供足够强度的转基因，表达无功能EGFR和表达特有的MSLN嵌合抗原受体。治疗试剂的剂量主要取决于治疗状况，年龄，体重，病人的健康程度，从而可能造成病人的变异性。According to an embodiment of the present invention, a sufficient number of viral vectors are transduced into targeted T cells and provide sufficient strength of the transgene to express non-functional EGFR and express a specific MSLN chimeric antigen receptor. The dosage of the therapeutic agent depends mainly on the treatment condition, age, weight, and health of the patient, which may cause patient variability.
表达无功能EGFR以及表达特有的针对抗原MSLN嵌合抗原受体的这些方法是联合治疗的一部分。这些病毒载体和用于过继免疫治疗的抗肿瘤T细胞，可以被单独或结合其他治疗癌症的方法一起执行。在合适的条件下，一个治疗方法的包括使用一个或多个药物疗法。These approaches expressing non-functional EGFR as well as expressing a chimeric antigen receptor specific for the antigen MSLN are part of a combination therapy. These viral vectors and antitumor T cells for adoptive immunotherapy can be administered alone or in combination with other approaches to treating cancer. Under appropriate conditions, a method of treatment includes the use of one or more drug therapies.
根据本发明的实施例，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。无功能EGFR的表达联合嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达，使得所得淋巴细胞或T淋巴细胞具有对肿瘤细胞的靶向杀伤作用，尤其具有对高表达MSLN的上述肿瘤细胞的杀伤作用更加显著，对高表达MSLN的上述肿瘤细胞的免疫杀伤作用更加安全有效。According to an embodiment of the present invention, the cancer includes at least one selected from mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer and breast cancer. The expression of non-functional EGFR combined with the high expression of chimeric antigen receptor in transgenic lymphocytes or T lymphocytes makes the resulting lymphocytes or T lymphocytes have a targeted killing effect on tumor cells, especially the above-mentioned effects on highly expressed MSLN The killing effect on tumor cells is more significant, and the immune killing effect on the above-mentioned tumor cells with high expression of MSLN is safer and more effective.
提高淋巴细胞治疗安全性的方法Ways to improve the safety of lymphocyte therapy
在本发明的另一方面，本发明提出了一种提高淋巴细胞治疗安全性的方法，所述淋巴细胞携带嵌合抗原受体，其特征在于，所述方法包括：使所述淋巴细胞表达无功能EGFR，所述无功能EGFR、所述淋巴细胞、所述嵌合抗原受体如前所述。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明实施例的淋巴细胞在用于治疗高表达MSLN的肿瘤细胞时，如果病人出现严重不良反应，本发明实施例的淋巴细胞可被抗EGFR抗体清除，进而可提高本发明实施例的淋巴细胞治疗高表达MSLN的肿瘤病人的安全性。In another aspect of the present invention, the present invention proposes a method for improving the safety of lymphocyte therapy, said lymphocyte carrying chimeric antigen receptor, characterized in that said method comprises: making said lymphocyte express Functional EGFR, the non-functional EGFR, the lymphocytes, and the chimeric antigen receptor are as described above. Non-functional EGFR lacks the N-terminal ligand-binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and the complete sequence that binds to anti-EGFR antibodies. Suicide mark. When the lymphocytes of the embodiments of the present invention are used to treat tumor cells with high expression of MSLN, if the patient has serious adverse reactions, the lymphocytes of the embodiments of the present invention can be eliminated by the anti-EGFR antibody, and the lymphocytes of the embodiments of the present invention can be improved. Safety of treatment in patients with tumors overexpressing MSLN.
下面将结合实施例对本发明的方案进行解释。The solutions of the present invention will be explained below in conjunction with examples.
本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific techniques or conditions are indicated in the examples, according to the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang, third edition, Science Press) or follow the product instructions. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
实施例1Example 1
本发明实施例中所用到的细胞系和基本实验技术如下所述：Cell lines and basic experimental techniques used in the embodiments of the present invention are as follows:
慢病毒的产生和人T淋巴细胞的转导Generation of lentivirus and transduction of human T lymphocytes
产生复制缺陷的慢病毒载体，并将慢病毒载体离心收集用于人T淋巴细胞的转导。下面简要介绍慢病毒载体的产生、收集的实验过程：将293T细胞铺在底面积为150-平方厘米的细胞培养皿中，并根据说明书，使用Express-In(购自Open Biosystems/ThermoScientific，Waltham，MA)对293T细胞进行病毒转导。每盘细胞加入15微克的慢病毒转基因质粒、5μg的pVSV-G(VSV糖蛋白表达质粒)、10微克的pCMVR8.74质粒(Gag/Pol/Tat/Rev表达质粒)和174微升的Express-In(浓度为1微克/微升)。分别于24小时和48小时收集上清，并使用超速离心机在28，000rpm(离心机转子为Beckman SW 32Ti，购自Beckman Coulter，Brea，CA)的条件下离心2小时。最后用0.75ml的RPMI-1640培养基对病毒质粒沉淀进行重悬。Replication-defective lentiviral vectors were generated and collected by centrifugation for transduction of human T lymphocytes. The following is a brief introduction to the production and collection of lentiviral vectors: 293T cells were placed on a cell culture dish with a bottom area of 150-cm2, and according to the instructions, Express-In (purchased from Open Biosystems/ThermoScientific, Waltham, MA) Viral transduction of 293T cells. Add 15 μg of lentiviral transgenic plasmid, 5 μg of pVSV-G (VSV glycoprotein expression plasmid), 10 μg of pCMVR8.74 plasmid (Gag/Pol/Tat/Rev expression plasmid) and 174 μl of Express- In (concentration is 1 μg/μl). The supernatants were collected at 24 hours and 48 hours, and centrifuged for 2 hours using an ultracentrifuge at 28,000 rpm (Beckman SW 32Ti rotor, purchased from Beckman Coulter, Brea, CA). Finally, resuspend the viral plasmid pellet with 0.75ml of RPMI-1640 medium.
从健康志愿者供体上分离人原代T淋巴细胞。人T淋巴细胞培养在RPMI-1640培养基中并使用抗CD3和CD28的单克隆抗体包被的珠(购自Invitrogen，Carlsbad，CA)进行刺激激活。人T淋巴细胞激活后的18～24小时，采用自旋-接种的方法对T淋巴细胞进行转导，转导过程如下所述：在24-孔板中，每孔铺有0.5×106T淋巴细胞，向每孔细胞中加入0.75ml的上述重悬的病毒上清和Polybrene(浓度为8微克/ml)。细胞和病毒质粒的混合液在台式离心机(购自Sorvall ST 40；Thermo Scientific)中离心，离心条件是室温，2500rpm，时间为90分钟。人重组白细胞介素-2(IL-2；购自Novartis，Basel，Switzerland)每隔2～3天加入T淋巴细胞培养液中，IL-2的终浓度为100-IU/ml，在T淋巴细胞培养过程中，保持细胞的密度为0.5×106～1×106/ml。一旦被转导的T淋巴细胞出现休眠，例如细胞生长速度变慢和细胞变小，其中，细胞生长速度和大小是通过Coulter Counter(购自Beckman Coulter)评估的，或被转导的T淋巴细胞在某个计划的时间点上的T淋巴细胞即可用来做功能分析。Human primary T lymphocytes were isolated from healthy volunteer donors. Human T lymphocytes were cultured in RPMI-1640 medium and stimulated with beads coated with anti-CD3 and CD28 monoclonal antibodies (purchased from Invitrogen, Carlsbad, CA). 18 to 24 hours after the activation of human T lymphocytes, the T lymphocytes were transduced by the spin-seeding method, and the transduction process was as follows: In a 24-well plate, each well was plated with 0.5×10 6 T For lymphocytes, add 0.75 ml of the above-mentioned resuspended virus supernatant and Polybrene (concentration: 8 μg/ml) to each well of cells. The mixture of cells and viral plasmids was centrifuged in a desktop centrifuge (purchased from Sorvall ST 40; Thermo Scientific) at room temperature at 2500 rpm for 90 minutes. Human recombinant interleukin-2 (IL-2; purchased from Novartis, Basel, Switzerland) was added to T lymphocyte culture medium every 2 to 3 days, and the final concentration of IL-2 was 100-IU/ml. During cell culture, keep the cell density at 0.5×10 6 -1×10 6 /ml. Once the transduced T lymphocytes appear dormant, such as slowed cell growth rate and cell size, wherein the cell growth rate and size were assessed by a Coulter Counter (purchased from Beckman Coulter), or the transduced T lymphocytes T lymphocytes at a planned time point are ready for functional analysis.
本申请的实施例中所用的流式细胞仪为BD FACSCanto II(购自BDBiosciences)，并且流式细胞分析数据使用FlowJo version 7.2.5软件(购自Tree Star，Ashland，OR)进行分析。The flow cytometer used in the examples of the present application was BD FACSCanto II (purchased from BD Biosciences), and the flow cytometric analysis data was analyzed using FlowJo version 7.2.5 software (purchased from Tree Star, Ashland, OR).
抗体依赖性细胞介导的细胞毒作用(ADCC)Antibody-dependent cell-mediated cytotoxicity (ADCC)
在有关ADCC实施例中，采用4小时-51Cr-释放法评估抗-EGFR抗体诱导表达无功能EGFR的淋巴细胞的细胞依赖性裂解的能力。被转导了慢病毒载体的人类T淋巴细胞被用作靶细胞。100μCi Na2 51CrO4(购自GE Healthcare Life Sciences，Marlborough，MA)标定2～5x106靶细胞，标定条件是37℃下震荡孵育1小时。细胞采用PBS润洗三次，并且用培养基重悬(细胞密度是1x105/ml)。继而，被标定的细胞铺在96-孔板中(每孔铺有5×103个细胞，加有50微升培养基)，并加入50微升的抗-EGFR抗体(购自Erbitux，Genentech)(终浓度为20微克/ml)，在常温条件下预培养30分钟.继而将含有抗体的培养基换成普通培养基，由此来检测51Cr的自发释放。加入终浓度为1％的Triton X-100以保证51Cr的最大释放量。在以下有关ADCC实施中，人PBMC(效应细胞)加入孔板中(每孔5×105个细胞)并将细胞在37℃培养过夜。第二天，收集细胞上清，并利用γ计数器计算cpm以此来确定51Cr的释放。细胞毒性比例用以下公式计算：％特异性裂解＝(实验释放cpm数据-自发释放cpm数据)/(最大释放cpm数据-自发释放cpm数据)*100，其中，最大释放cpm数据通过靶细胞中加入Triton X-100实现的，自发释放cpm数据是在没有抗EGFR抗体和效应细胞的条件下测量的。In the ADCC example, the ability of anti-EGFR antibodies to induce cell-dependent lysis of lymphocytes expressing non-functional EGFR was assessed using the 4 hr- 51Cr -release assay. Human T lymphocytes transduced with lentiviral vectors were used as target cells. 100 μCi Na 2 51 CrO 4 (purchased from GE Healthcare Life Sciences, Marlborough, MA) was used to calibrate 2-5×10 6 target cells, and the calibration condition was to incubate with shaking at 37° C. for 1 hour. Cells were rinsed three times with PBS and resuspended in culture medium (cell density was 1x10 5 /ml). Then, the labeled cells were plated in a 96-well plate (5×10 cells per well , plus 50 microliters of culture medium), and 50 microliters of anti-EGFR antibody (purchased from Erbitux, Genentech ) (final concentration of 20 μg/ml), pre-incubated at room temperature for 30 minutes. Then, the culture medium containing the antibody was replaced with normal culture medium to detect the spontaneous release of 51 Cr. Triton X-100 was added at a final concentration of 1% to ensure the maximum release of 51 Cr. In the ADCC implementation below, human PBMCs (effector cells) were added to well plates (5×10 5 cells per well) and the cells were incubated overnight at 37°C. The next day, cell supernatants were collected and cpm was calculated using a gamma counter to determine 51 Cr release. The ratio of cytotoxicity is calculated with the following formula: %specific lysis=(experimental release cpm data-spontaneous release cpm data)/(maximum release cpm data-spontaneous release cpm data)*100, wherein, the maximum release cpm data is added to target cells Triton X-100 achieves spontaneous release cpm data measured in the absence of anti-EGFR antibody and effector cells.
铬释放实验Chromium release test
实施例中应用4–小时51铬释放法分析评估抗MSLN嵌合抗原受体T细胞(抗MSLN CART淋巴细胞)的细胞毒活性。具体步骤如下：目标测试细胞用51Cr在37摄氏度下标记1小时。标记后，用含有10％胎牛血清(FCS)的RPMI培养基润洗细胞。润洗后，将细胞重悬在相同的培养基中，重悬细胞的浓度是1×105/ml。转导后T细胞以不同的效靶细胞比值(E：T)加入目标测试细胞悬浮液中，并将细胞种在96-孔中，每孔体积是200微升。将细胞在37度培养箱中培养4小时。4小时后，从每孔中取出30微升的上清放于计数器的96-微孔板进行计数分析。分析仪器是顶级计数NXT微闪烁计数器(购自Packard Bioscience)。所有计数孔中效应细胞的数目是基于T细胞总数来计算的。被标记的目标测试细胞是MSLN+MSTO-211H(人胸膜间皮瘤细胞，human pleural mesothelioma cells(来自ATCC))。In the embodiment, the cytotoxic activity of anti-MSLN chimeric antigen receptor T cells (anti-MSLN CART lymphocytes) was analyzed and evaluated by the 4-hour 51 chromium release method. The specific steps are as follows: target test cells were labeled with 51 Cr at 37°C for 1 hour. After labeling, cells were rinsed with RPMI medium containing 10% fetal calf serum (FCS). After rinsing, the cells were resuspended in the same medium at a concentration of 1×10 5 /ml. After transduction, T cells were added to the target test cell suspension with different effector-to-target cell ratios (E:T), and the cells were seeded in 96-wells with a volume of 200 microliters per well. Cells were incubated for 4 hours in a 37°C incubator. After 4 hours, 30 microliters of the supernatant was taken from each well and placed in a 96-microwell plate of a counter for counting analysis. The analytical instrument was a top count NXT microscintillation counter (purchased from Packard Bioscience). The number of effector cells in all counted wells was calculated based on the total number of T cells. The labeled target test cells are MSLN + MSTO-211H (human pleural mesothelioma cells, human pleural mesothelioma cells (from ATCC)).
实施例2构建共表达无功能EGFR和抗MSLN嵌合抗原受体的载体Example 2 Construction of vectors co-expressing non-functional EGFR and anti-MSLN chimeric antigen receptors
本实施例中，发明人将编码有抗人MSLN的单链抗体的序列、4-1BB胞内段和T细胞受体组合的ζ-链序列克隆到含有EF-1启动子的慢病毒载体(lentiviral vector)上，克隆过程中，选择的限制性酶切是XbaI和NotI双酶切，以及NotI和XhoI双酶切，通过酶切、连接、筛选和目的质粒的扩增，生成表达抗MSLN嵌合抗原受体的慢病毒质粒(LV-MSLN CAR)。包含合成IRES和表达无功能EGFR的序列被克隆进LV-MSLN CAR载体质粒，构建成LV-MSLN CAR/tEGFR。图1是慢病毒载体的示意图，包含编码抗MSLN嵌合抗原受体的序列，IRES、及编码无功能EGFR序列。抗MSLN嵌合抗原受体的序列在启动子EF-1的启动调控下，表达无功能EGFR的序列作为一个单独的mRNA转录单元从IRES序列后开始翻译。In this example, the inventors cloned the sequence encoding the single-chain antibody against human MSLN, the ζ-chain sequence of the 4-1BB intracellular segment and the T cell receptor combination into a lentiviral vector containing the EF-1 promoter ( lentiviral vector), during the cloning process, the selected restriction enzymes were XbaI and NotI double enzyme digestion, and NotI and XhoI double enzyme digestion. Lentiviral plasmid with antigen receptor complex (LV-MSLN CAR). The sequence containing synthetic IRES and expressing non-functional EGFR was cloned into LV-MSLN CAR vector plasmid to construct LV-MSLN CAR/tEGFR. Figure 1 is a schematic diagram of a lentiviral vector, comprising a sequence encoding an anti-MSLN chimeric antigen receptor, an IRES, and a sequence encoding a non-functional EGFR. The sequence of the anti-MSLN chimeric antigen receptor is under the control of the promoter EF-1, and the sequence of the non-functional EGFR is expressed as a single mRNA transcription unit to start translation after the IRES sequence.
实施例3抗EGFR抗体可有效杀伤清除共表达无功能EGFR和抗MSLN嵌合抗原受体的T淋巴细胞Example 3 Anti-EGFR antibody can effectively kill and eliminate T lymphocytes co-expressing non-functional EGFR and anti-MSLN chimeric antigen receptor
在本实施例中，外周血淋巴细胞取自不记名供血者。外周血淋巴细胞通过梯度离心进行分离，梯度离心机为Ficoll-Hypaque。T淋巴细胞与T细胞激活因子磁珠CD3/CD28(购自Invitrogen，Carlsbad，CA)在5％CO2、37摄氏度下孵育培养72小时，培养基是加有2mmol/L谷氨酰胺，10％高温灭活的胎牛血清(FCS)(购自Sigma-Aldrich Co.)和100U/ml的青霉素/链霉素双抗的RPMI培养基1640(购自Invitrogen Gibco Cat.no.12633-012)。激活培养72小时后，用洗液润洗细胞，将磁珠洗去。将T细胞种在铺有重组纤连蛋白片段(FN ch-296；Retronectin)细胞培养皿上，并用慢病毒转导，转导慢病毒分别为LV-MSLN CAR/tEGFR，LV-MSLN CAR或空载(LV-GFP)转导过程如实施例1所述。转导后表达无功能EGFR的T细胞用抗EGFR抗体染色后，然后流式细胞细胞(FACS)分离，分离后T细胞培养在RPMI-1640培养基中并用重组人类IL-2因子(100ng/ml；购自R&D Systems)进行诱导扩增7-10天，然后作为实验的靶细胞。发明人用ADCC检测法测量抗EGFR抗体介异的对转导了不同慢病毒的T细胞的杀伤作用，测量方法采用标准4–小时51铬释放法，4–小时51铬释放法如实施例1所述。结果如图2所示。如图2所示，抗EGFR抗体可有效介异杀伤共表达抗MSLN嵌合抗原受体和无功能EGFR的T淋巴细胞，但抗EGFR抗体不能介异杀伤只表达抗MSLN嵌合抗原受体的T淋巴细胞，抗EGFR抗体不能介异杀伤空载慢病毒转导的T淋巴细胞，统计数据代表三个孔的平均值±SEM。In this example, peripheral blood lymphocytes were obtained from anonymous blood donors. Peripheral blood lymphocytes were separated by gradient centrifugation using Ficoll-Hypaque. T lymphocytes and T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) were incubated at 5% CO 2 at 37 degrees Celsius for 72 hours, and the medium was supplemented with 2mmol/L glutamine, 10% High temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U/ml penicillin/streptomycin double antibody RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012). After activating and culturing for 72 hours, the cells were rinsed with washing solution to wash away the magnetic beads. The T cells were planted on cell culture dishes covered with recombinant fibronectin fragments (FN ch-296; Retronectin), and transduced with lentiviruses. The transduced lentiviruses were LV-MSLN CAR/tEGFR, LV-MSLN CAR or empty The vector (LV-GFP) transduction process was as described in Example 1. T cells expressing non-functional EGFR after transduction were stained with anti-EGFR antibody, and then separated by flow cytometry (FACS). After separation, T cells were cultured in RPMI-1640 medium and treated with recombinant human IL-2 factor (100ng/ml ; purchased from R&D Systems) for induction and amplification for 7-10 days, and then used as the target cells of the experiment. The inventors used the ADCC detection method to measure the anti-EGFR antibody-mediated killing effect on T cells transduced with different lentiviruses. The measurement method used the standard 4-hour 51 chromium release method, and the 4-hour 51 chromium release method was as in Example 1 mentioned. The result is shown in Figure 2. As shown in Figure 2, anti-EGFR antibodies can effectively mediate killing of T lymphocytes co-expressing anti-MSLN chimeric antigen receptor and non-functional EGFR, but anti-EGFR antibodies cannot mediate killing of T lymphocytes expressing only anti-MSLN chimeric antigen receptor For T lymphocytes, anti-EGFR antibodies cannot mediate killing of T lymphocytes transduced with empty lentivirus, statistical data represent the mean ± SEM of three wells.
实施例4共表达无功能EGFR和抗MSLN嵌合抗原受体的T淋巴细胞肿瘤细胞溶解能力。Example 4 The ability of tumor cell lysis of T lymphocytes co-expressing non-functional EGFR and anti-MSLN chimeric antigen receptor.
在本实施例中，外周血淋巴细胞取自不记名供血者。外周血淋巴细胞通过梯度离心进行分离，梯度离心机为Ficoll-Hypaque。T淋巴细胞与T细胞激活因子磁珠CD3/CD28(购自Invitrogen，Carlsbad，CA)在5％CO2、37摄氏度下孵育培养72小时，培养基是加有2mmol/L谷氨酰胺，10％高温灭活的胎牛血清(FCS)(购自Sigma-Aldrich Co.)和100U/ml的青霉素/链霉素双抗的RPMI培养基1640(购自Invitrogen Gibco Cat.no.12633-012)。激活培养72小时后，用洗液润洗细胞，将磁珠洗去。将T细胞种在铺有重组纤连蛋白片段(FN ch-296；Retronectin)细胞培养皿上，并用慢病毒转导，转导慢病毒分别为LV-MSLN CAR，LV-tEGFR，或空载(LV-GFP)转导过程如实施例1所述。转导后的T细胞培养在RPMI-1640培养基中并用重组人类IL-2因子(100ng/ml；购自R&D Systems)进行诱导扩增7-10天，然后进行功能测试实验。发明人测量转导了不同慢病毒的T细胞对MSLN阳性MSTO-211H肿瘤细胞的杀伤作用，效靶细胞比例是50：1或25:1或1:1，测量方法采用标准4–小时51铬释放法，4–小时51铬释放法如实施例1所述。结果显示，共表达抗MSLN嵌合抗原受体和无功能EGFR慢病毒转导的T淋巴细胞(效应细胞)可杀死MSLN阳性MSTO-211H肿瘤细胞(靶细胞)。无功能EGFR慢病毒转导的T淋巴细胞(LV-tEGFR T淋巴细胞)或空载慢病毒转导的T淋巴细胞(对照LV-GFP T淋巴细胞)对MSLN阳性肿瘤细胞无明显杀伤作用。In this example, peripheral blood lymphocytes were obtained from anonymous blood donors. Peripheral blood lymphocytes were separated by gradient centrifugation using Ficoll-Hypaque. T lymphocytes and T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) were incubated at 5% CO 2 at 37 degrees Celsius for 72 hours, and the medium was supplemented with 2mmol/L glutamine, 10% High temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U/ml penicillin/streptomycin double antibody RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012). After activating and culturing for 72 hours, the cells were rinsed with washing solution to wash away the magnetic beads. The T cells were planted on cell culture dishes covered with recombinant fibronectin fragments (FN ch-296; Retronectin), and transduced with lentiviruses. The transduced lentiviruses were LV-MSLN CAR, LV-tEGFR, or empty ( LV-GFP) transduction process as described in Example 1. The transduced T cells were cultured in RPMI-1640 medium and induced and expanded with recombinant human IL-2 factor (100 ng/ml; purchased from R&D Systems) for 7-10 days, and then functional test experiments were performed. The inventors measured the killing effect of T cells transduced with different lentiviruses on MSLN-positive MSTO-211H tumor cells. The effective target cell ratio was 50:1 or 25:1 or 1:1. The measurement method used standard 4-hour 51Cr Release method, 4-hour 51 Chromium release method is as described in Example 1. The results showed that lentivirally transduced T lymphocytes (effector cells) co-expressing anti-MSLN chimeric antigen receptor and nonfunctional EGFR killed MSLN-positive MSTO-211H tumor cells (target cells). Non-functional EGFR lentivirus-transduced T lymphocytes (LV-tEGFR T lymphocytes) or empty lentivirus-transduced T lymphocytes (control LV-GFP T lymphocytes) had no obvious killing effect on MSLN-positive tumor cells.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 北京马力喏生物科技有限公司<110> Beijing Marino Biotechnology Co., Ltd.
<120> 共表达抗MSLN嵌合抗原受体和无功能EGFR的转基因淋巴细胞及其用途<120> Transgenic lymphocytes co-expressing anti-MSLN chimeric antigen receptor and non-functional EGFR and use thereof
<130> PIDC1160952<130> PIDC1160952
<160> 7<160> 7
<170> PatentIn version 3.3<170> PatentIn version 3.3
<210> 1<210> 1
<211> 507<211> 507
<212> PRT<212> PRT
<213> Artificial<213> Artificial
<220><220>
<223> 嵌合抗原受体的氨基酸序列<223> amino acid sequence of chimeric antigen receptor
<400> 1<400> 1
Met Val Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His ProMet Val Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 151 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Ala Gln Val Gln Leu Val GlnAla Phe Leu Leu Ile Pro Asp Ile Gln Ala Gln Val Gln Leu Val Gln
20 25 30 20 25 30
Ser Gly Ala Glu Val Lys Arg Pro Gly Ala Ser Val Gln Val Ser CysSer Gly Ala Glu Val Lys Arg Pro Gly Ala Ser Val Gln Val Ser Cys
35 40 45 35 40 45
Arg Ala Ser Gly Tyr Ser Ile Asn Thr Tyr Tyr Met Gln Trp Val ArgArg Ala Ser Gly Tyr Ser Ile Asn Thr Tyr Tyr Met Gln Trp Val Arg
50 55 60 50 55 60
Gln Ala Pro Gly Ala Gly Leu Glu Trp Met Gly Val Ile Asn Pro SerGln Ala Pro Gly Ala Gly Leu Glu Trp Met Gly Val Ile Asn Pro Ser
65 70 75 8065 70 75 80
Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Leu ThrGly Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Leu Thr
85 90 95 85 90 95
Asn Asp Thr Ser Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu ThrAsn Asp Thr Ser Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu Thr
100 105 110 100 105 110
Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ala Leu Trp GlySer Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ala Leu Trp Gly
115 120 125 115 120 125
Asp Phe Gly Met Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val SerAsp Phe Gly Met Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser
130 135 140 130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly SerSer Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160145 150 155 160
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile GlyAsp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly
165 170 175 165 170 175
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His TrpAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His Trp
180 185 190 180 185 190
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleLeu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205 195 200 205
Tyr Lys Ala Ser Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser GlyTyr Lys Ala Ser Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly
210 215 220 210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
225 230 235 240225 230 235 240
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro LeuAsp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu
245 250 255 245 250 255
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ser Phe ValThr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ser Phe Val
260 265 270 260 265 270
Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg ProPro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro
275 280 285 275 280 285
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg ProPro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
290 295 300 290 295 300
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly LeuGlu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
305 310 315 320305 310 315 320
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr CysAsp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
325 330 335 325 330 335
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His ArgGly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg
340 345 350 340 345 350
Asn Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro PheAsn Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
355 360 365 355 360 365
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys ArgMet Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
370 375 380 370 375 380
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe SerPhe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
385 390 395 400385 390 395 400
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu TyrArg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
405 410 415 405 410 415
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp LysAsn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
420 425 430 420 425 430
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys AsnArg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
435 440 445 435 440 445
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala GluPro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
450 455 460 450 455 460
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys GlyAla Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
465 470 475 480465 470 475 480
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr TyrHis Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
485 490 495 485 490 495
Asp Ala Leu His Met Gln Ala Leu Pro Pro ArgAsp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505 500 505
<210> 2<210> 2
<211> 1524<211> 1524
<212> DNA<212>DNA
<213> Artificial<213> Artificial
<220><220>
<223> 编码嵌合抗原受体的核酸的核苷酸序列<223> Nucleotide sequence of nucleic acid encoding chimeric antigen receptor
<400> 2<400> 2
atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60
attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120
ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180
cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240
ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300
acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360
tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420
gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480
gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540
atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600
ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660
cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720
gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780
gggaccaagc tggagatcaa acgtgctagc ttcgtgccgg tcttcctgcc agcgaagccc 840gggaccaagc tggagatcaa acgtgctagc ttcgtgccgg tcttcctgcc agcgaagccc 840
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 900accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 900
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 960tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 960
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 1020gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 1020
ctgtcactgg ttatcaccct ttactgcaac cacaggaaca aacggggcag aaagaaactc 1080ctgtcactgg ttatcaccct ttactgcaac cacaggaaca aacggggcag aaagaaactc 1080
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1140ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1140
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1200tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1200
aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1260aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1260
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1320ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1320
gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1380gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1380
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1440aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1440
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1500cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1500
atgcaggccc tgccccctcg ctaa 1524atgcaggccc tgccccctcg ctaa 1524
<210> 3<210> 3
<211> 360<211> 360
<212> PRT<212> PRT
<213> Artificial<213> Artificial
<220><220>
<223> 无功能EGFR的氨基酸序列<223> Amino acid sequence of non-functional EGFR
<400> 3<400> 3
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu LeuMet Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 151 5 10 15
His Ala Ala Arg Pro Gly Ser Arg Lys Val Cys Asn Gly Ile Gly IleHis Ala Ala Arg Pro Gly Ser Arg Lys Val Cys Asn Gly Ile Gly Ile
20 25 30 20 25 30
Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys HisGly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His
35 40 45 35 40 45
Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro ValPhe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val
50 55 60 50 55 60
Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro GlnAla Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln
65 70 75 8065 70 75 80
Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu LeuGlu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu
85 90 95 85 90 95
Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu AsnIle Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn
100 105 110 100 105 110
Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser LeuLeu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu
115 120 125 115 120 125
Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu LysAla Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys
130 135 140 130 135 140
Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu CysGlu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys
145 150 155 160145 150 155 160
Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly GlnTyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln
165 170 175 165 170 175
Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala ThrLys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr
180 185 190 180 185 190
Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly ProGly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro
195 200 205 195 200 205
Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg GluGlu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu
210 215 220 210 215 220
Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe ValCys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val
225 230 235 240225 230 235 240
Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln AlaGlu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala
245 250 255 245 250 255
Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln CysMet Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys
260 265 270 260 265 270
Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala GlyAla His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly
275 280 285 275 280 285
Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala GlyVal Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly
290 295 300 290 295 300
His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr GlyHis Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly
305 310 315 320305 310 315 320
Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser IlePro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile
325 330 335 325 330 335
Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala LeuAla Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu
340 345 350 340 345 350
Gly Ile Gly Leu Phe Met Arg ArgGly Ile Gly Leu Phe Met Arg Arg
355 360 355 360
<210> 4<210> 4
<211> 1083<211> 1083
<212> DNA<212>DNA
<213> Artificial<213> Artificial
<220><220>
<223> 编码无功能EGFR的核酸分子的核苷酸序列<223> Nucleotide sequence of nucleic acid molecule encoding non-functional EGFR
<400> 4<400> 4
atggctctgc ccgtcaccgc tctgctgctg cctctggctc tgctgctgca cgccgcacgc 60atggctctgc ccgtcaccgc tctgctgctg cctctggctc tgctgctgca cgccgcacgc 60
cctgggagtc gcaaagtctg taatgggatc ggcatcggcg agttcaagga cagcctgtcc 120cctgggagtc gcaaagtctg taatgggatc ggcatcggcg agttcaagga cagcctgtcc 120
atcaacgcca ccaatatcaa gcactttaag aattgcacat ctatcagcgg cgacctgcac 180atcaacgcca ccaatatcaa gcactttaag aattgcacat ctatcagcgg cgacctgcac 180
atcctgccag tggccttccg gggcgattct tttacccaca caccccctct ggaccctcag 240atcctgccag tggccttccg gggcgattct tttacccaca caccccctct ggaccctcag 240
gagctggata tcctgaagac cgtgaaggag atcacaggct tcctgctgat ccaggcctgg 300gagctggata tcctgaagac cgtgaaggag atcacaggct tcctgctgat ccaggcctgg 300
cctgagaaca gaaccgatct gcacgccttt gagaatctgg agatcatccg gggcagaaca 360cctgagaaca gaaccgatct gcacgccttt gagaatctgg agatcatccg gggcagaaca 360
aagcagcacg gccagttctc cctggccgtg gtgtctctga acatcaccag cctgggcctg 420aagcagcacg gccagttctc cctggccgtg gtgtctctga acatcaccag cctgggcctg 420
aggtccctga aggagatctc tgacggcgat gtgatcatct ccggcaacaa gaacctgtgc 480aggtccctga aggagatctc tgacggcgat gtgatcatct ccggcaacaa gaacctgtgc 480
tacgccaaca caatcaattg gaagaagctg tttggcacct ctggccagaa gacaaagatc 540tacgccaaca caatcaattg gaagaagctg tttggcacct ctggccagaa gacaaagatc 540
atctctaacc ggggcgagaa tagctgcaag gcaaccggac aggtgtgcca cgcactgtgc 600atctctaacc ggggcgagaa tagctgcaag gcaaccggac aggtgtgcca cgcactgtgc 600
agcccagagg gatgttgggg cccagagcca cgggactgcg tgagctgtag aaacgtgtcc 660agcccagagg gatgttgggg cccagagcca cgggactgcg tgagctgtag aaacgtgtcc 660
aggggccgcg agtgcgtgga taagtgtaat ctgctggagg gcgagccaag ggagttcgtg 720aggggccgcg agtgcgtgga taagtgtaat ctgctggagg gcgagccaag ggagttcgtg 720
gagaactccg agtgcatcca gtgtcacccc gagtgcctgc ctcaggccat gaacatcacc 780gagaactccg agtgcatcca gtgtcacccc gagtgcctgc ctcaggccat gaacatcacc 780
tgtacaggcc gcggccccga caattgcatc cagtgtgccc actatatcga tggccctcac 840tgtacaggcc gcggccccga caattgcatc cagtgtgccc actatatcga tggccctcac 840
tgcgtgaaga cctgtccagc cggcgtgatg ggcgagaaca atacactggt gtggaagtac 900tgcgtgaaga cctgtccagc cggcgtgatg ggcgagaaca atacactggt gtggaagtac 900
gcagacgcag gacacgtgtg ccacctgtgc caccccaatt gcacctatgg ctgtacagga 960gcagacgcag gacacgtgtg ccacctgtgc caccccaatt gcacctatgg ctgtacagga 960
ccaggcctgg agggatgccc aaccaacggc cctaagatcc caagcatcgc cacaggcatg 1020ccaggcctgg agggatgccc aaccaacggc cctaagatcc caagcatcgc cacaggcatg 1020
gtgggggcac tgctgctgct gctggtggtg gctctgggga ttgggctgtt tatgagaagg 1080gtgggggcac tgctgctgct gctggtggtg gctctgggga ttgggctgtt tatgagaagg 1080
taa 1083taa 1083
<210> 5<210> 5
<211> 3223<211> 3223
<212> DNA<212>DNA
<213> Artificial<213> Artificial
<220><220>
<223> MSLN CAR/tEGFR的核苷酸序列<223> Nucleotide sequence of MSLN CAR/tEGFR
<400> 5<400> 5
atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60
attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120
ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180
cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240
ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300
acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360
tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420
gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480
gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540
atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600
ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660
cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720
gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780
gggaccaagc tggagatcaa acgtgctagc accactaccc cagcaccgag gccacccacc 840gggaccaagc tggagatcaa acgtgctagc accactaccc cagcaccgag gccaccccacc 840
ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900
gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960
cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020
cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080
actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140
ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200
ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260
ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320
aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380aacgagctcc aaaaggtaa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380
cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440
acctatgacg ctcttcacat gcaggccctg ccgcctcggt aatcctactg cgtcgacact 1500acctatgacg ctcttcacat gcaggccctg ccgcctcggt aatcctactg cgtcgacact 1500
agtgaattcg aatttaaatc ggatccgcgg ccgcgcccct ctccctcccc cccccctaac 1560agtgaattcg aatttaaatc ggatccgcgg ccgcgcccct ctccctcccc cccccctaac 1560
gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 1620gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 1620
accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 1680accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 1680
agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 1740agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 1740
aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 1800aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 1800
aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 1860aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 1860
gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 1920gatacacctg caaaggcggc acaacccccag tgccacgttg tgagttggat agttgtggaa 1920
agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 1980agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 1980
ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg 2040ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg 2040
aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 2100aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 2100
cgatgataat atggccacaa ccatggcgtc cggatctaga atggctctgc ccgtcaccgc 2160cgatgataat atggccacaa ccatggcgtc cggatctaga atggctctgc ccgtcaccgc 2160
tctgctgctg cctctggctc tgctgctgca cgccgcacgc cctgggagtc gcaaagtctg 2220tctgctgctg cctctggctc tgctgctgca cgccgcacgc cctgggagtc gcaaagtctg 2220
taatgggatc ggcatcggcg agttcaagga cagcctgtcc atcaacgcca ccaatatcaa 2280taatgggatc ggcatcggcg agttcaagga cagcctgtcc atcaacgcca ccaatatcaa 2280
gcactttaag aattgcacat ctatcagcgg cgacctgcac atcctgccag tggccttccg 2340gcactttaag aattgcacat ctatcagcgg cgacctgcac atcctgccag tggccttccg 2340
gggcgattct tttacccaca caccccctct ggaccctcag gagctggata tcctgaagac 2400gggcgattct tttacccaca caccccctct ggaccctcag gagctggata tcctgaagac 2400
cgtgaaggag atcacaggct tcctgctgat ccaggcctgg cctgagaaca gaaccgatct 2460cgtgaaggag atcacaggct tcctgctgat ccaggcctgg cctgagaaca gaaccgatct 2460
gcacgccttt gagaatctgg agatcatccg gggcagaaca aagcagcacg gccagttctc 2520gcacgccttt gagaatctgg agatcatccg gggcagaaca aagcagcacg gccagttctc 2520
cctggccgtg gtgtctctga acatcaccag cctgggcctg aggtccctga aggagatctc 2580cctggccgtg gtgtctctga acatcaccag cctgggcctg aggtccctga aggagatctc 2580
tgacggcgat gtgatcatct ccggcaacaa gaacctgtgc tacgccaaca caatcaattg 2640tgacggcgat gtgatcatct ccggcaacaa gaacctgtgc tacgccaaca caatcaattg 2640
gaagaagctg tttggcacct ctggccagaa gacaaagatc atctctaacc ggggcgagaa 2700gaagaagctg tttggcacct ctggccagaa gacaaagatc atctctaacc ggggcgagaa 2700
tagctgcaag gcaaccggac aggtgtgcca cgcactgtgc agcccagagg gatgttgggg 2760tagctgcaag gcaaccggac aggtgtgcca cgcactgtgc agcccagagg gatgttgggg 2760
cccagagcca cgggactgcg tgagctgtag aaacgtgtcc aggggccgcg agtgcgtgga 2820cccagagcca cgggactgcg tgagctgtag aaacgtgtcc aggggccgcg agtgcgtgga 2820
taagtgtaat ctgctggagg gcgagccaag ggagttcgtg gagaactccg agtgcatcca 2880taagtgtaat ctgctggagg gcgagccaag ggagttcgtg gagaactccg agtgcatcca 2880
gtgtcacccc gagtgcctgc ctcaggccat gaacatcacc tgtacaggcc gcggccccga 2940gtgtcacccc gagtgcctgc ctcaggccat gaacatcacc tgtacaggcc gcggccccga 2940
caattgcatc cagtgtgccc actatatcga tggccctcac tgcgtgaaga cctgtccagc 3000caattgcatc cagtgtgccc actatatcga tggccctcac tgcgtgaaga cctgtccagc 3000
cggcgtgatg ggcgagaaca atacactggt gtggaagtac gcagacgcag gacacgtgtg 3060cggcgtgatg ggcgagaaca atacactggt gtggaagtac gcagacgcag gacacgtgtg 3060
ccacctgtgc caccccaatt gcacctatgg ctgtacagga ccaggcctgg agggatgccc 3120ccacctgtgc caccccaatt gcacctatgg ctgtacagga ccaggcctgg agggatgccc 3120
aaccaacggc cctaagatcc caagcatcgc cacaggcatg gtgggggcac tgctgctgct 3180aaccaacggc cctaagatcc caagcatcgc cacaggcatg gtgggggcac tgctgctgct 3180
gctggtggtg gctctgggga ttgggctgtt tatgagaagg taa 3223gctggtggtg gctctgggga ttgggctgtt tatgagaagg taa 3223
<210> 6<210> 6
<211> 615<211>615
<212> DNA<212>DNA
<213> Artificial<213> Artificial
<220><220>
<223> 内部核糖体进入位点的核苷酸序列<223> Nucleotide sequence of internal ribosome entry site
<400> 6<400> 6
tttaaatcgg atccgcggcc gcgcccctct ccctcccccc cccctaacgt tactggccga 60tttaaatcgg atccgcggcc gcgcccctct ccctcccccc cccctaacgt tactggccga 60
agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg 120agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg 120
tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg 180tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg 180
ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt 240ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt 240
cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac 300cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac 300
cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca 360cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca 360
aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg 420aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg 420
ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg 480ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg 480
ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa 540ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa 540
cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat 600cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat 600
ggccacaacc atggc 615ggccacaacc atggc 615
<210> 7<210> 7
<211> 22<211> 22
<212> PRT<212> PRT
<213> Artificial<213> Artificial
<220><220>
<223> 连接肽的氨基酸序列<223> Amino acid sequence of linker peptide
<400> 7<400> 7
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp ValGly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 10 151 5 10 15
Glu Glu Asn Pro Gly ProGlu Glu Asn Pro Gly Pro
20 20
Claims (22)
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710056385.7A CN108342360A (en) | 2017-01-25 | 2017-01-25 | Co-express anti-MSLN Chimeric antigen receptors and the transgenosis lymphocyte of nonfunctional EGFR and application thereof |
| PCT/CN2017/081273 WO2018137294A1 (en) | 2017-01-25 | 2017-04-20 | Transgenic lymphocyte co-expressing anti-msln chimeric antigen receptor and non-functional egfr and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710056385.7A CN108342360A (en) | 2017-01-25 | 2017-01-25 | Co-express anti-MSLN Chimeric antigen receptors and the transgenosis lymphocyte of nonfunctional EGFR and application thereof |
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| WO (1) | WO2018137294A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057158A (en) * | 2020-03-16 | 2020-04-24 | 南京蓝盾生物科技有限公司 | Chimeric antigen receptor targeting human GPC3 and application thereof |
| CN111088231A (en) * | 2018-10-24 | 2020-05-01 | 艾生命序公司 | Anti-mesothelin CAR-T cell tumor immunotherapy secreted by PD-L1 antibody |
| CN111518771A (en) * | 2020-03-19 | 2020-08-11 | 浙江帝格生物科技有限责任公司 | Transgenic lymphocyte co-expressing anti-CD 19 chimeric antigen receptor and Nur77 inhibitory molecule and application thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342360A (en) * | 2017-01-25 | 2018-07-31 | 北京马力喏生物科技有限公司 | Co-express anti-MSLN Chimeric antigen receptors and the transgenosis lymphocyte of nonfunctional EGFR and application thereof |
| WO2021189008A1 (en) | 2020-03-20 | 2021-09-23 | Lyell Immunopharma, Inc. | Novel recombinant cell surface markers |
| WO2022182891A1 (en) | 2021-02-25 | 2022-09-01 | Lyell Immunopharma, Inc. | Ror1 targeting chimeric antigen receptor |
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| WO2018137294A1 (en) * | 2017-01-25 | 2018-08-02 | 北京马力喏生物科技有限公司 | Transgenic lymphocyte co-expressing anti-msln chimeric antigen receptor and non-functional egfr and use thereof |
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| CN106535925A (en) * | 2014-05-23 | 2017-03-22 | 佛罗里达大学研究基金会有限公司 | CAR based immunotherapy |
| CN105331586B (en) * | 2015-11-20 | 2020-09-15 | 上海细胞治疗研究院 | A tumor precision T cell containing a high-efficiency killing initiation mechanism and its use |
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2017
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| WO2018137294A1 (en) * | 2017-01-25 | 2018-08-02 | 北京马力喏生物科技有限公司 | Transgenic lymphocyte co-expressing anti-msln chimeric antigen receptor and non-functional egfr and use thereof |
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| LEONID CHERKASSKY等: "Human car-t cells with intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
| XIULI WANG等: "A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells", 《BLOOD》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111088231A (en) * | 2018-10-24 | 2020-05-01 | 艾生命序公司 | Anti-mesothelin CAR-T cell tumor immunotherapy secreted by PD-L1 antibody |
| CN111057158A (en) * | 2020-03-16 | 2020-04-24 | 南京蓝盾生物科技有限公司 | Chimeric antigen receptor targeting human GPC3 and application thereof |
| CN111518771A (en) * | 2020-03-19 | 2020-08-11 | 浙江帝格生物科技有限责任公司 | Transgenic lymphocyte co-expressing anti-CD 19 chimeric antigen receptor and Nur77 inhibitory molecule and application thereof |
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| WO2018137294A1 (en) | 2018-08-02 |
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