WO2018137293A1 - Therapeutic composition for treating mesothelin-positive tumor - Google Patents

Therapeutic composition for treating mesothelin-positive tumor Download PDF

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WO2018137293A1
WO2018137293A1 PCT/CN2017/081272 CN2017081272W WO2018137293A1 WO 2018137293 A1 WO2018137293 A1 WO 2018137293A1 CN 2017081272 W CN2017081272 W CN 2017081272W WO 2018137293 A1 WO2018137293 A1 WO 2018137293A1
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lymphocyte
lymphocytes
nucleic acid
acid molecule
chimeric antigen
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Chinese (zh)
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严勇朝
朱益林
陈思毅
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北京马力喏生物科技有限公司
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Definitions

  • the present invention relates to the field of biomedicine, and in particular to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer, and an augmentation lymph Methods of cell viability and therapeutic safety.
  • MSLN Mesothelin
  • MMSLN Mesothelin
  • interstitial is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancers and about 50% of lung adenocarcinomas and other cancers such as cholangiocarcinoma, gastric cancer, and intestinal cancer. , esophageal cancer, breast cancer.
  • the interstitial gene encodes a precursor protein of 71KDa, which is then processed into a 31KDa exfoliated fragment and a 40KDa protein fragment.
  • the 31KDa exfoliated fragment is called megakaryocyte promoting factor (MPF), and the 40KDa protein fragment is Known as interstitial, interstitial is immobilized on the cell membrane by the anchoring action of glycosyl-phosphatidylinositol (GPI).
  • MPF megakaryocyte promoting factor
  • GPI glycosyl-phosphatidylinositol
  • mesothelioma is divided into pleural mesothelioma and peritoneal mesothelioma.
  • the pleural mesothelioma is the primary tumor of the pleura, which is limited (mostly benign) and diffuse (both malignant). Divided, malignant mesothelioma is one of the worst tumors in the chest.
  • Peritoneal mesothelioma refers to a tumor that originates in the peritoneal mesothelial cells. Clinical manifestations are not characteristic, common symptoms and signs are: abdominal pain, ascites, abdominal distension and abdominal mass.
  • Interstitial is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancers and about 50% of lung adenocarcinomas and other cancers such as cholangiocarcinoma, gastric cancer, intestinal cancer, esophagus Cancer, breast cancer.
  • cholangiocarcinoma cholangiocarcinoma
  • gastric cancer intestinal cancer
  • esophagus Cancer esophagus Cancer
  • the inventors have proposed a nucleic acid molecule carrying a silent cellular immunological checkpoint, a nucleic acid molecule encoding a non-functional EGFR, and a nucleic acid molecule encoding a chimeric antigen receptor, and a construct formed by the introduction of the construct.
  • Transgenic lymphocytes which encode a chimeric antigen receptor that specifically binds to the antigen MSLN. Therefore, the constructs and transgenic lymphocytes proposed by the present invention can be used for immunotherapy of adoptive T cells of tumors, especially mesenchymal positive tumors; the transgenic lymphocytes of the present invention have a strong killing ability for high expression of interstitial tumors.
  • the mesothelial cells with normal MSLN expression levels have weaker killing, and the constructs and transgenic lymphocytes proposed by the present application can significantly increase the safety of treatment by expressing non-functional EGFR.
  • the invention proposes a T lymphocyte.
  • the cellular immune checkpoint of the T lymphocyte is silenced; expressing a non-functional EGFR; and expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: an extracellular region, The extracellular region comprises a heavy chain variable region and a light chain variable region of a single chain antibody that specifically recognizes the antigen MSLN; a transmembrane region, the transmembrane region is linked to the extracellular region, and is embedded Into the cell membrane of the T lymphocyte; an intracellular region, the intracellular region is linked to the transmembrane region, and the intracellular region comprises an intracellular portion of CD28 or 4-1BB and a CD3 ⁇ chain.
  • the cellular immune checkpoint includes an immune checkpoint on at least one of a cell surface or a cell.
  • Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR receptor and intact sequence that binds to anti-EGFR antibody, and non-functional EGFR can act as lymph Cell suicide markers.
  • the T lymphocytes of the embodiments of the present invention have the characteristics of resisting tumor cell-mediated immunosuppression, and the proliferative ability in vitro, the proliferation and viability in tumor patients are significantly improved, and the killing of tumor cells is performed. The ability is significantly enhanced, especially for tumors with high expression of MSLN, which has a significant directional killing effect and high safety.
  • the invention proposes a lentivirus.
  • the lentivirus carries the following nucleic acid molecule: (a) a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1, The nucleic acid molecule encoding the chimeric antigen receptor has the nucleotide sequence shown in SEQ ID NO: 2; (b) the nucleic acid molecule that silences the cellular immunological checkpoint, and the nucleotide sequence of the nucleic acid molecule of the silencing cell immunological checkpoint Is at least one selected from the group consisting of SEQ ID NOS: 3 to 135; and (c) a nucleic acid molecule encoding a non-functional EGFR having the amino acid sequence set forth in SEQ ID NO: 136, encoding a non-functional EGFR
  • the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:
  • the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes have the characteristics of resisting tumor cell-mediated immunosuppression, proliferative ability in vitro, proliferation in tumor patients, and The survivability is significantly enhanced, and the killing ability of tumor cells is remarkably enhanced, especially for tumors with high expression of MSLN, which has a significant directional killing effect and high safety.
  • the invention proposes a lentivirus.
  • the lentivirus carries a nucleotide sequence comprising SEQ ID NO: 138, 139, 140, 141, 142 or 143.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes have the characteristics of resisting tumor cell-mediated immunosuppression, proliferative ability in vitro, proliferation in tumor patients, and The viability is significantly enhanced, and the killing ability of tumor cells is significantly enhanced, especially for tumors with high expression of MSLN, and the safety is higher.
  • the invention provides a transgenic lymphocyte.
  • the lymphocyte immune checkpoint is silenced; expressing a non-functional EGFR; and expressing a chimeric antigen receptor, the chimeric antigen receptor comprising: an extracellular region, the extracellular region comprising a heavy chain variable region and a light chain variable region of an antibody, said antibody being capable of specifically binding to a tumor antigen; a transmembrane region; and an intracellular region comprising an intracellular portion of an immunostimulatory molecule, wherein
  • the antibody is a single chain antibody and the tumor antigen is MSLN.
  • the sexual killing ability is greatly improved, especially for tumors with high expression of MSLN, which has a significant directional killing effect and high safety.
  • the above transgenic lymphocytes may further have at least one of the following additional technical features:
  • the lymphocyte immune checkpoint is independently selected from at least one of CTLA4, PD-1, TIM-3, BTLA, LAG-3IRAK-M, SOCS-1, A20, CBL-B. .
  • CTLA4, PD-1, TIM-3, BTLA, and LAG-3 are cell surface immune checkpoints
  • IRAK-M, SOCS-1, A20, and CBL-B are intracellular immune checkpoints.
  • the immune checkpoint of the embodiment of the invention has the functions of negatively regulating and attenuating the cellular immune response, and the specific binding of the corresponding ligand on the tumor cell leads to down-regulation of the proliferative response of the T lymphocyte and the secretion of the cytokine is reduced.
  • successful silencing of cell surface or intracellular immunological checkpoints according to embodiments of the present invention further enhances the efficacy of transgenic lymphocytes against tumor-mediated immunosuppression, in vitro expansion of transgenic lymphocytes and in tumors
  • the proliferation and viability of the patient's body further enhances the targeted killing effect on tumor cells.
  • the lymphocyte immune checkpoint is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR and zinc finger nuclease.
  • the successful silencing of the cellular immune checkpoint of the embodiment of the present invention can significantly improve the lymphocyte resistance tumor-mediated immunosuppressive property of the embodiment of the present invention, and further improve the transgenic lymphocyte proliferating tumor cell.
  • Directional killing effect is beneficial to be used to the lymphocyte resistance tumor-mediated immunosuppressive property of the embodiment of the present invention.
  • the intracellular segment of the immunocostimulatory 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 immunostimulatory molecule and the silencing of the cellular immune checkpoint in the embodiment of the present invention have the functions of positively regulating and enhancing the cellular immune response, and the expression of the intracellular segment of the immunostimulatory molecule of the present invention is absent.
  • the combination of the expression of functional EGFR and the silencing of cellular immune checkpoints makes the directional killing effect of the transgenic lymphocyte proliferation of the present invention on the tumor more significant and safe.
  • the lymphocyte immune checkpoints are CTLA4, PD-1, CBL-B.
  • CTLA4 and PD-1 are cell surface immune checkpoints
  • CBL-B is an intracellular immune checkpoint.
  • the lymphocyte cell surface immunological checkpoint CTLA4 or PD-1 of the embodiment of the present invention is silenced, or the lymphocyte intracellular immune checkpoint is CBL-B is silenced, preventing the expression of PD1 or CTLA4 molecules and The binding of the corresponding ligands PD-L1 and PD-L2 or CD80 and CD86, thereby effectively inhibiting the inability or apoptosis of T lymphocytes, or silencing through CBL-B, enhancing T cell receptor signaling, allowing transgenic lymphocytes to The proliferation and viability of tumor patients are further improved, and the effect of targeted killing of tumors is more significant.
  • silencing of the lymphocyte immune checkpoint is achieved by shRNA.
  • the shRNA of the embodiment of the present invention carries an siRNA which specifically silences at least one of the cell surface or the intracellular immunological checkpoint, and the shRNA of the embodiment of the present invention has a highly efficient and specific silencing cell surface or The role of at least one of the immune checkpoints in the cell, ie the successful silencing of the cell surface or intracellular immune checkpoint, prevents the specific binding of the immunological checkpoint to the corresponding ligand, thereby effectively inhibiting the immune checkpoint's inability to T lymphocytes.
  • transgenic lymphocytes of the embodiments of the present invention in a tumor patient, and cooperating with the antigen targeting of the chimeric antigen receptor, so that the present invention is implemented
  • a negative regulation mechanism such as apoptosis
  • the intracellular segment of the immunostimulatory molecule is an intracellular segment of 4-1BB or CD28.
  • the intracellular segment of the immunostimulatory molecule of the chimeric antigen receptor of the transgenic lymphocytes of the present invention is the intracellular portion of CD28 or 4-1BB.
  • the intracellular segment of the immunostimulatory molecule is an intracellular segment of CD28 or 4-1BB, which further enhances the targeted killing effect of the transgenic lymphocytes of the embodiments of the present invention.
  • the non-functional EGFR expressed by the transgenic lymphocytes of the present invention lacks an N-terminal ligand binding region and an intracellular receptor tyrosine kinase activity, but includes a transmembrane region of a wild-type EGFR receptor.
  • non-functional EGFR can be used as a suicide marker for transgenic lymphocytes of the present invention.
  • non-functional EGFR combined with the expression of chimeric antigen receptors, and further combined with the silencing of cellular immunological checkpoints, can effectively ensure the targeted killing effect of transgenic lymphocytes, if the patient has serious adverse reactions, transgenic lymphocytes
  • the cells can be cleared by the anti-EGFR antibody, which can further improve the safety of the transgenic lymphocytes of the embodiments of the present invention for treating tumor patients with high expression of MSLN.
  • the lymphocytes are CD3 + T lymphocytes or natural killer cells or natural killer T cells.
  • the cellular immunological checkpoint of the above lymphocytes of the present invention is silenced and expresses non-functional EGFR, while expressing an antigen-specific chimeric antigen receptor, such as the MSLN antigen-specific chimeric antigen receptor of the present invention.
  • an antigen-specific chimeric antigen receptor such as the MSLN antigen-specific chimeric antigen receptor of the present invention.
  • the above-mentioned lymphocyte cell killing effect is more targeted, and the proliferation and viability of the tumor patient are further improved, and the targeted killing effect on the tumor is more significant and safer.
  • the invention proposes a construct.
  • the construct comprises: a first nucleic acid molecule encoding a chimeric antigen receptor; a second nucleic acid molecule, the second nucleic acid molecule silencing a cellular immune checkpoint, and A nucleic acid molecule encoding a non-functional EGFR.
  • the cellular immune checkpoint, the chimeric antigen receptor, and the non-functional EGFR are as described above.
  • the construct of the embodiment of the present invention can effectively silence at least one of the immunological checkpoints on the cell surface or in the cell, and express non-functional EGFR and express antigen-specificity after successfully introducing the lymphocytes of the embodiment of the present invention.
  • the chimeric antigen receptor so that the lymphocytes of the embodiments of the present invention have more targeted killing effect on tumor cells, especially tumor cells with high expression of MSLN, and have high safety.
  • the above-described construct may further include at least one of the following additional technical features:
  • the first nucleic acid molecule and the second nucleic acid molecule and the third nucleic acid molecule are disposed in a lymphocyte as described above to express the chimeric antigen receptor, and silence The cellular immune checkpoint and expression of non-functional EGFR, and the chimeric antigen receptor is in a non-fused form with the non-functional EGFR.
  • the lymphocytes of the first nucleic acid molecule and the second nucleic acid molecule and the third nucleic acid molecule are successfully set, and the immune checkpoint of at least one of the cell surface or the cell of the lymphocyte is successfully silenced and
  • the surface of lymphocytes successfully expressed non-functional EGFR, and the antigen specificity was successfully expressed on the surface of lymphocytes, such as the MSLN-specific chimeric antigen receptor of the present invention, which has a more lethal and specific tumor.
  • the killing effect is more secure.
  • the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; a second promoter, the second promoter and The second nucleic acid molecule is operably linked; and a third promoter operably linked to the third nucleic acid molecule.
  • the introduction of the first promoter and the second and third promoters enables the first nucleic acid molecule and the second nucleic acid molecule and the third nucleic acid molecule to be independently expressed, respectively, thereby effectively ensuring the chimeric antigen receptor
  • the biological role of antigen targeting and the effective silencing of the cell's immune checkpoint and the expression of non-functional EGFR make the lymphocyte targeting effect of the embodiment of the present invention stronger, and the killing effect on the tumor, especially for high expression.
  • the targeted killing of tumor cells of MSLN is more significant and safer.
  • the first promoter, the second promoter and the third promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, LTR, RSV promoters.
  • the above promoter of the embodiment of the invention has the characteristics of high activation efficiency and strong specificity, thereby ensuring efficient silencing and reactive power of the cellular immune checkpoint. High-efficiency expression of EGFR and high-efficiency expression of chimeric antigen receptor, so that the lymphocyte proliferation ability and proliferation and survival ability of the lymphocytes in the embodiment of the invention are greatly improved, and the targeted killing effect on the tumor is more remarkable and safe. More sexual.
  • the construct further comprises: an internal ribosome entry site sequence, the internal ribosome entry site sequence being disposed between the first nucleic acid molecule and the third nucleic acid molecule,
  • the internal ribosome entry site has the nucleotide sequence set forth in SEQ ID NO:144.
  • an internal ribosome entry site sequence allows the first nucleic acid molecule and the third nucleic acid molecule to be expressed independently, respectively.
  • the introduction of an internal ribosome entry site sequence ensures the biological action of the chimeric antigen receptor antigen targeting and the high expression of non-functional EGFR, thereby enabling lymphocytes of the embodiments of the present invention to tumor The targeted killing effect is more pronounced, and lymphocytes are safer for tumor killing.
  • the construct further comprises: a fourth nucleic acid molecule disposed between the first nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encoding a linker peptide,
  • the linker peptide is capable of being cleaved in the lymphocytes.
  • the linker peptide has the amino acid sequence set forth in SEQ ID NO:145.
  • the introduction of the fourth nucleic acid molecule and its correspondingly expressed linker peptide allows the non-functional EGFR and chimeric antigen receptor to be expressed in a non-fusion state on the lymphocyte membrane.
  • the introduction of the linker peptide of the embodiment of the present invention ensures the biological effects of the non-functional EGFR and the chimeric antigen receptor, and has a more specific tumor killing effect and higher safety.
  • the vector of the construct is a non-pathogenic viral vector.
  • the introduction of a non-pathogenic viral vector greatly enhances the replication and amplification efficiency of the construct in lymphocytes, thereby greatly increasing the silencing of cellular immune checkpoints and the expression of non-functional EGFR and chimeric antigen receptors in lymphocytes.
  • the high-efficiency expression of lymphocytes greatly enhances the proliferation of lymphocytes in vitro, the proliferation and viability of tumor patients, and the targeting of lymphocytes is further increased. Strong, the killing effect on tumor cells is more significant, and the safety is further improved.
  • the viral vector comprises at least one selected from the group consisting of a retroviral vector, a lentiviral vector or an adenovirus-associated viral vector.
  • the virus carrier of the embodiment of the invention has a wide range of virus infection during virus packaging and infection, and can infect both terminally differentiated cells and cells in a mitotic phase, and the genome can be integrated into the host chromosome or free.
  • the cellular immune checkpoint is efficiently silenced and the expression of non-functional EGFR is highly expressed in lymphocytes, and the chimeric antigen receptor is highly expressed in lymphocytes, making this
  • the in vitro proliferation ability of the lymphocytes of the invention the proliferation and viability of the tumor patients are greatly improved, the targeting effect of lymphocytes is further enhanced, and the targeted killing effect on tumor cells, especially tumor cells with high expression of MSLN, is more remarkable.
  • the killing effect of lymphocytes is safer.
  • the invention provides a method of preparing the aforementioned T lymphocytes or transgenic lymphocytes.
  • the method comprises introducing the aforementioned construct or the lentivirus described above into lymphocytes or T lymphocytes.
  • the construct or the lentivirus is successfully introduced into the lymphocyte or the T lymphocyte, and the cellular immunological examination of the lymphocyte is silenced and the expression of the non-functional EGFR and the chimeric antigen receptor is expressed, thereby preparing the method of the present invention.
  • the prepared transgenic lymphocytes or T lymphocytes can greatly proliferate in vivo and in vitro of tumor patients and the survival ability of tumor patients, and the targeted killing of tumor cells, especially tumor cells with high expression of MSLN, by transgenic lymphocytes or T lymphocytes More powerful and safer.
  • the invention provides a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: the above construct, lentivirus, T lymphocyte or transgenic lymphocyte.
  • the composition of any of the above therapeutic compositions can achieve silencing of cell surface or intracellular immunological checkpoints of transgenic lymphocytes or T lymphocytes and expression of non-functional EGFR and chimeric antigen receptors in transgenic lymphocytes or T lymphocytes Highly expressed, so that the resulting transgenic lymphocytes or T lymphocytes have significant resistance to tumor cell-mediated immunosuppression, and the proliferation of tumor patients in vitro and in vivo and the survival ability of tumor patients are greatly improved, transgenic lymphocytes or T lymphocytes
  • the targeted killing effect of the cells on tumor cells is stronger, and the targeted killing effect of the therapeutic composition for treating cancer of the present invention on tumor cells is remarkably enhanced, especially the targeted killing effect on tumor cells with high expression of MSLN is significantly enhanced. Safety is further improved.
  • the above therapeutic composition may further comprise at least one of the following additional technical features:
  • the cancer comprises at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.
  • the above tumor cells have high specific expression of MSLN, and the therapeutic composition of the present invention can silence and express non-functional EGFR on lymphocyte cell surface or intracellular immunological checkpoint and efficiently express antigen-specific chimeric antigen receptor,
  • the obtained lymphocyte or T lymphocyte has remarkable characteristics against tumor cell-mediated immunosuppression, and the viability in the microenvironment of mesothelioma is greatly improved.
  • the resulting lymphocytes or T lymphocytes are highly expressed in MSLN The targeted killing effect of tumor cells is stronger and the safety is higher.
  • the invention provides a method of increasing lymphocyte activity and therapeutic safety, said lymphocyte carrying a chimeric antigen receptor, according to an embodiment of the invention, said method comprising:
  • the cellular immune checkpoint of the lymphocytes is silenced and the lymphocytes are expressed as non-functional EGFR.
  • the cellular immune checkpoint, the lymphocyte, the chimeric antigen receptor, and the non-functional EGFR are as defined above, and the lymphocyte activity comprises the ability of the lymphocyte to proliferate in vitro, in a tumor patient The proliferation and viability and at least one of the directional killing ability of the lymphocytes in a tumor patient.
  • the cell surface or intracellular immune checkpoint of lymphocytes according to the embodiment of the present invention is silenced, lymphocytes are activated, proliferative responses are up-regulated, cytokine secretion is increased, and anti-apoptotic ability is enhanced, so that the present invention
  • the lymphocyte expansion of the embodiment in vitro, proliferation in a tumor patient, and survival ability in a tumor patient greatly enhance the silencing of the lymphocyte cell immunological checkpoint and the antigen-specific efficacy of the lymphocyte chimeric antigen receptor, thereby realizing Effectively resisting tumor cell-mediated immunosuppression, the targeted killing effect on tumor cells with high expression of MSLN is significantly enhanced.
  • Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR receptor and intact sequence that binds to anti-EGFR antibody, and non-functional EGFR can act as lymph Cell suicide markers.
  • lymphocytes of the present invention are used for the treatment of tumor cells with high expression of MSLN, if the patient develops a serious adverse reaction, the lymphocytes of the embodiments of the present invention can be cleared by the anti-EGFR antibody, thereby improving the embodiment of the present invention. Lymphocyte treatment for the safety of tumor patients with high expression of MSLN.
  • the invention provides a method of treating cancer.
  • the method comprises: administering to a cancer patient a construct as described above, a lentivirus as described above, a T lymphocyte as described above or a transgenic lymphocyte as described above, wherein The antigen receptor specifically binds to the tumor antigen MSLN.
  • the method for treating cancer according to the embodiment of the invention can effectively achieve targeted killing of tumor cells of cancer patients, in particular, has targeted killing effect on tumor cells with high expression of MSLN, thereby effectively treating cancer, and the therapeutic effect is good and High security.
  • the above method for treating cancer may further comprise at least one of the following additional technical features:
  • the method comprises: isolating lymphocytes from a cancer patient; introducing the aforementioned construct, or the lentivirus described above, into the lymphocytes to obtain transgenic lymphocytes, the transgene The lymphocyte expressing chimeric antigen receptor and the cellular immune checkpoint are silenced; and the transgenic lymphocytes are administered to the cancer patient.
  • the method for treating cancer according to the embodiment of the invention can further effectively achieve targeted killing of tumor cells of cancer patients, especially having targeted killing effect on tumor cells with high expression of MSLN, thereby further effectively treating cancer, and the therapeutic effect Good and safe.
  • the cancer comprises a group selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, stomach At least one of cancer, intestinal cancer, esophageal cancer and breast cancer.
  • the method for treating cancer according to an embodiment of the present invention enables lymphocyte immune checkpoints to be silenced and cells to express chimeric antigen receptors, such as MSLN antigen-specific chimeric antigen receptors of the present invention, resulting lymphocytes or T lymphocytes.
  • the cells have targeted killing of tumor cells of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer or breast cancer which are specifically expressed by MSLN.
  • cell immune checkpoint includes a cell surface immunological checkpoint and an intracellular immunological checkpoint
  • a cell surface immunological checkpoint is a membrane protein on the surface of lymphocytes, which is Ligand interactions expressed on tumor cells can inhibit anti-tumor lymphocyte responses.
  • An "intracellular immune checkpoint” is an intracellular protein that is a negatively regulated cellular signaling machinery that inhibits antitumor lymphocyte responses.
  • FIG. 1 is a schematic diagram showing the structure of a co-expressing MSLN antigen-specific chimeric antigen receptor and a silencing human cell immunological checkpoint and a lentiviral vector expressing a non-functional EGFR according to an embodiment of the present invention
  • FIG. 2 is a diagram showing the results of killing of lymphocytes co-expressing anti-MSLN chimeric antigen receptor, PD1 shRNA, and non-functional EGFR by anti-EGFR antibody according to an embodiment of the present invention
  • Figure 3 is a graph showing the results of the ability of co-expressing MSLN antigen-specific chimeric antigen receptor, PD1 shRNA and non-functional EGFR lymphocytes to kill tumor cells according to an embodiment of the present invention.
  • the invention provides a T lymphocyte or transgenic lymphocyte.
  • a cellular immune checkpoint of a T lymphocyte according to an embodiment of the present invention is silenced; a non-functional EGFR is expressed; and a chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region, a cell
  • the outer region includes the heavy chain variable region and the light chain variable region of the single-chain antibody, the single-chain antibody specifically recognizes the antigen MSLN; the transmembrane region, the transmembrane region is linked to the extracellular region, and is embedded in the cell membrane of the T lymphocyte.
  • the intracellular region is linked to the transmembrane region, and the intracellular region includes the intracellular portion of CD28 or 4-1BB and the CD3 ⁇ chain.
  • the cellular immune checkpoint includes an immune checkpoint on at least one of a cell surface or a cell.
  • Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR receptor and intact sequence that binds to anti-EGFR antibody, and non-functional EGFR can act as lymph Cell suicide markers.
  • the T lymphocyte or transgenic lymphocyte cell of the embodiment of the present invention expresses a non-functional EGFR-expressing MSLN antigen-specific chimeric antigen receptor and the cellular immune checkpoint is silenced, and the T lymphocyte or transgenic lymphocyte of the embodiment of the present invention is Proliferation and viability of tumor patients in vivo and in vitro, as well as in tumor patients
  • the killing ability of the specific tumor cells is remarkably enhanced, especially the specific killing effect on the tumor cells with high expression of MSLN is greatly improved, and the safety is also remarkably improved.
  • Tumors can avoid immune surveillance, shutting down the immune killing response of lymphocytes by stimulating the expression of their immunosuppressive receptors; as a negative immunoregulatory mechanism, activated cytotoxic T lymphocytes (CTLs) also express negative regulatory regulators. , that is, the immune checkpoint molecule on the cell surface or inside the cell.
  • CTLs cytotoxic T lymphocytes
  • the programmed cell death 1 receptor (PD-1) is expressed on activated CTLs, which interact with the programmed death ligand 1 (PD-L1) expressed on tumor cells to inhibit anti-tumor T Cellular response. Many tumors express PD-L1.
  • the binding of PD-L1 to its ligand PD-1 results in down-regulation of proliferative responses to CTLs, decreased secretion of cytokines, and inability or apoptosis of T cells.
  • the cytotoxic T lymphocyte antigen 4 (CTLA-4) of the present invention is a key negative regulator of another T cell, which inhibits T cell activation by binding to a ligand B7 expressed on antigen presenting cells. 1.
  • B7.2 CD80 and CD86
  • CBL-B E3 ubiquitin protein ligase CBL-B in cytotoxic T lymphocytes of the present invention is another key negative regulator in cells by inhibiting T cell receptor (TCR) signaling, To inhibit the activity of T cells. Therefore, the immunological checkpoint of the T lymphocyte or the transgenic lymphocyte of the embodiment of the present invention is silenced, and the proliferation and viability of the T lymphocyte or the transgenic lymphocyte in the tumor patient are remarkably improved.
  • the non-functional EGFR of the present invention lacks an N-terminal ligand binding region and an intracellular receptor tyrosine kinase activity, but includes a transmembrane region of the wild type EGFR receptor and is intact.
  • a sequence that binds to an anti-EGFR antibody, a non-functional EGFR can serve as a suicide marker for lymphocytes.
  • Non-functional EGFR-expressing lymphocytes can be cleared in vivo by anti-EGFR antibodies.
  • the T lymphocytes or transgenic lymphocytes of the embodiments of the present invention express non-functional EGFR.
  • the transgenic lymphocytes can be cleared by the anti-EGFR antibody. Further, the safety of the transgenic lymphocytes or T lymphocytes of the embodiments of the present invention for treating tumor patients with high expression of MSLN can be further improved.
  • the antibody of the chimeric antigen receptor extracellular region is a single chain antibody.
  • the inventors have found that single-chain antibodies can remove non-specifically reactive surface proteins while single-chain antibodies are more permeable to tumor tissue to increase drug treatment concentrations.
  • the transgenic lymphocytes of the embodiments of the present invention express the chimeric antigen receptor of the single-chain antibody, which greatly enhances the targeted killing effect of the transgenic lymphocytes on the targeted tumor cells.
  • the binding antigen of the above antibody is MSLN. Therefore, the transgenic lymphocytes of the embodiments of the present invention have a directional killing effect on cells highly expressing the antigen MSLN, and the specific binding effect of the antigen-antibody is stronger, and the transgenic lymphocytes of the embodiment of the present invention have high expression of tumor cells to the MSLN antigen. Directional killing effect.
  • the cellular immune checkpoint of lymphocytes includes a cell surface and an intracellular immunological checkpoint
  • the lymphocyte cell surface immunological checkpoint of the embodiment of the present invention is independently selected from the group consisting of CTLA4, PD-1, TIM. -3, at least one of BTLA and LAG-3
  • the lymphocyte intracellular immune checkpoint is independently selected from IRAK-M, SOCS-1, A20, At least one of CBL-B.
  • the above molecules can specifically bind to antigens expressed by tumor cells, inhibit lymphocyte activation, promote lymphocyte incompetence or apoptosis, thereby negatively regulating and attenuating cellular immune responses.
  • the successful silencing of the above-mentioned cell surface or intracellular immune checkpoint further improves the proliferation and viability of the transgenic lymphocytes in the tumor patient, and further enhances the directed killing effect on the tumor cells.
  • silencing of a lymphocyte immune checkpoint is achieved by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, zinc finger nuclease, and CRISPR.
  • siRNA small interfering RNA
  • siRNA small interfering RNA
  • siRNA is a small RNA molecule (composed of 21-25 nucleotides), which is composed of Dicer (pair of RNAase III family).
  • Dicer pair of RNAase III family.
  • the RNA of the stranded RNA has a specific cleavage effect; the siRNA plays a central role in the RNA silencing pathway, degrading specific messenger RNA (mRNA) and regulating it at the transcriptional level.
  • mRNA degrading specific messenger RNA
  • Antisense nucleic acids include antisense RNA and antisense DNA.
  • Antisense RNA refers to a small RNA or oligonucleotide fragment that is fully complementary to mRNA.
  • Antisense DNA refers to the sense of being in the double strand of the gene DNA.
  • antisense RNA and antisense DNA mainly function through translation of mRNA and transcription of gene DNA; antisense nucleic acid prevents ribosome by forming steric hindrance effect by binding to target mRNA Binding to mRNA, on the other hand, binding to mRNA activates endogenous RNase or ribozyme, which in turn degrades mRNA; antisense DNA specifically binds to the regulatory region of the double helix of the gene DNA to form a DNA trimer, or with a DNA coding region Binding, termination of the elongation of the mRNA strand being transcribed; antisense nucleic acids also inhibit processing modifications of post-transcriptional mRNA, such as 5' end capping, 3' end tailing, intermediate splicing, and internal base methylation, etc. Mature mRNA is transported from the nucleus to the cytoplasm. Therefore, antisense RNA is an effective technique for silencing the gene of interest.
  • Ribozyme is a catalytically active RNA molecule that is a biocatalyst that degrades specific mRNA sequences.
  • the ribozyme participates in RNA self-cleavage and processing by catalyzing the hydrolysis of transphosphate and phosphodiester bonds, and general antisense RNA.
  • ribozymes have a relatively stable spatial structure and are not susceptible to RNase attack. More importantly, ribozymes can be detached from the hybridization chain and then re-bound and cleave other mRNA molecules.
  • Dominant negative mutations are those in which certain signal transduction proteins are not only self-functional but also inhibit or block the action of wild-type signal transduction proteins in the same cell, mainly by forming dimers with wild-type proteins.
  • the way to achieve this mutation is toxic and can significantly inhibit or block the action of intracellular target signal transduction proteins.
  • the zinc finger nuclease consists of a DNA recognition domain and a non-specific endonuclease.
  • the DNA recognition domain is composed of a series of Cys2-His2 zinc finger proteins in series (generally 3 to 4). Each zinc finger protein recognizes and binds.
  • a specific triplet base, zinc finger protein forms the ⁇ - ⁇ - ⁇ secondary structure, wherein the 16 amino acid residues of the ⁇ helix determine the DNA binding specificity of the zinc finger, the skeleton structure is conserved, and the amino acid determining the DNA binding specificity
  • the introduction of sequence changes can obtain new DNA binding specificity, so that different amino acid introduction sequences can be designed for different genes of interest to achieve specific silencing of different genes of interest.
  • CRISPR Clustered regular interspaced short palindromic repeats
  • the CRISPR cluster is a family of specific DNA repeats that are widely found in the genomes of bacteria and archaea.
  • the sequence consists of a leader, multiple short and highly conserved repeats, and multiple spacers (Spacer). )composition.
  • the leader region is generally located upstream of the CRISPR cluster and is a region rich in AT length of 300-500 bp, which is considered to be a promoter sequence of the CRISPR cluster.
  • the repeat sequence region has a length of 21 to 48 bp and contains a palindromic sequence, which can form a hairpin structure.
  • the repeat sequences are separated by a spacer of length 26 to 72 bp.
  • the Spacer region is composed of captured foreign DNA.
  • CRISPR CRISPR-related genes
  • CRISPR is transcribed into a long RNA precursor (Pre RISPR RNA, pre-crRNA) under the control of the leader region, and then processed into a series of short conserved repeats and spacers.
  • the mature crRNA ultimately recognizes and binds to its complementary foreign DNA sequence to exert a cleavage effect.
  • Processing of pre-crRNA is involved by Cas9 in the Cas family. Cas9 contains two unique active sites, RuvC at the amino terminus and HNH in the middle of the protein, which play a role in crRNA maturation and double-strand DNA cleavage.
  • trans-activating crRNA complementary to its repeat sequence is also transcribed, and Cas9 and double-stranded RNA-specific RNase III nuclease are excited to process pre-crRNA.
  • the crRNA, tracrRNA and Cas9 complexes recognize and bind to the complementary sequence of crRNA, then unwind the DNA double strand to form R-loop, which makes the crRNA hybridize with the complementary strand, and the other strand maintains the free single-stranded state.
  • the complementary DNA strand of the crRNA is then cleaved by the HNH active site in Cas9, and the RuvC active site cleaves the non-complementary strand, eventually introducing a DNA double-strand break (DSB).
  • RNA By artificially designing RNA, it is possible to engineer a sgRNA (short guide RNA) sufficient to guide Cas9 to the targeted gene cleavage of DNA.
  • the shRNA, the antisense nucleic acid, the ribozyme, the dominant negative mutation, and the CRISPR zinc finger nuclease are effective means for specifically silencing the target gene, and the means for silencing the gene is not particularly limited, and those skilled in the art can
  • the experimental purpose and condition selection, such as at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR or zinc finger nuclease used in the embodiments of the present invention, achieve specific silencing of the target gene.
  • the lymphocyte cell surface or intracellular immunological checkpoint is silenced, preferably with shRNA.
  • the siRNA molecule carried by the ShRNA is typically a dual region of base pairs between 10 and 30 in length.
  • the PD1 or CTLA4 or CBL-B siRNA of the embodiments of the present invention is designed to be homologous to the coding region of PD1 or CTLA4 or CBL-B mRNA, through mRNA Degradation to inhibit gene expression.
  • the siRNA is associated with a multiplex protein complex called the Inducible RNA Silencing Complex (RISC), during which the sense strand is cleaved by the enzyme.
  • RISC Inducible RNA Silencing Complex
  • siRNA is introduced into the cell as shRNA (shRNA contains approximately 18-23 nucleotide siRNA sequences followed by a 9-15-length nucleotide loop and a reverse complement of a siRNA sequence), and the shRNA design is better avoided. Matching points in the 3'UTR cell gene; ensuring proper strand selection.
  • RNAi RNA interference
  • the shRNA of the embodiment of the present invention is continuously produced from a cell, and thus the effect thereof is more durable, thereby prolonging the shRNA cycle, and the shRNA used in the embodiment of the present invention has a highly efficient and specific silencing cell surface or intracellular immunity.
  • the role of checkpoints, successful silencing of cell surface or intracellular immune checkpoints makes transgenic lymphocytes significantly resistant to tumor-mediated immunosuppression, and further enhances proliferation and viability in tumor patients. The effect of directional killing is more pronounced.
  • the intracellular segment of the immunocostimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28, and derivatives thereof.
  • the expression of the intracellular segment of the immunostimulatory molecule and the silencing of at least one immunological checkpoint on the cell surface or in the cell have a positive regulation and enhance the cellular immune response, making the transgenic lymphocyte significantly resistant to tumor-mediated immunosuppression.
  • the characteristics of proliferation and viability in tumor patients are further improved, and the targeted killing effect on tumors with high expression of MSLN is more significant.
  • the expression of intracellular segments of immunostimulatory molecules is combined with the expression of non-functional EGFR, making transgenic lymphocytes The immune killing effect is safer and more effective.
  • the lymphocyte cell surface immunological checkpoint is preferably CTLA4 or PD1, and the intralymphocyte immune checkpoint is preferably CBL-B.
  • the lymphocyte cell surface immunological checkpoint CTLA4 or PD1 is silenced or the intracellular immune checkpoint CBL-B is silenced, so that the transgenic lymphocytes have more significant resistance to tumor-mediated immunosuppression. Its proliferation and viability in tumor patients are further improved, and the effect of targeted killing of tumors is more significant.
  • the lymphocytes of the embodiments of the 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
  • natural killer T cells are T cell subsets with T cells and natural killer cell receptors.
  • the immunological checkpoint in the above lymphocytes is silenced and expresses the chimeric antigen receptor, so that the cellular immunity of the lymphocytes is more targeted and killing, and the killing effect on the tumor cells is more significant; the lymphocytes express non-functional EGFR and Expression of the chimeric antigen receptor makes the cellular immune killing effect of the above lymphocytes more safe and effective.
  • the invention proposes a lentivirus or construct.
  • the lentivirus or construct carries the following nucleic acid molecule: (a) a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1, coding chimeric The nucleic acid molecule of the antigen receptor has the nucleotide sequence shown in SEQ ID NO: 2; (b) the nucleic acid molecule which silences the cell surface or the intracellular immunological checkpoint, and the nucleotide sequence of the nucleic acid molecule which silences the cell surface immunological checkpoint a nucleic acid molecule which is selected from at least one of SEQ ID NOS: 3 to 68, wherein the nucleic acid molecule of the intracellular immunological checkpoint is at least one selected from the group consisting of SEQ ID NOS: 69 to 135; and (c) A nucleic acid molecule that functions as EGFR having the amino acid
  • SEQ ID NOs: 3 to 14 are human programmed death receptor 1 (PD1) siRNA nucleotide sequences, and SEQ ID NOs: 15 to 30 are human cytotoxic T lymphocyte-associated antigen 4 (CTLA4) siRNA sequences, SEQ ID NO: 31 to 46 is a human T cell immunoglobulin mucin molecule 3 (TIM3) siRNA sequence, and SEQ ID NOs: 47 to 57 are human T lymphocyte attenuating factor (BTLA) siRNA sequences, SEQ ID NOs: 58-68.
  • PD1 programmed death receptor 1
  • CTLA4 human cytotoxic T lymphocyte-associated antigen 4
  • SEQ ID NO: 31 to 46 is a human T cell immunoglobulin mucin molecule 3 (TIM3) siRNA sequence
  • SEQ ID NOs: 47 to 57 are human T lymphocyte attenuating factor (BTLA) siRNA sequences, SEQ ID NOs: 58-68.
  • SEQ ID NOs: 69-85 are human IRAK-M siRNA (human interleukin-1 receptor-associated kinase 3) nucleotide sequence
  • SEQ ID NO :86-96 is a human SOCS1 siRNA (human cytokine signal transduction inhibitor 1) sequence
  • SEQ ID NOs: 97-116 are human A20 siRNA (human tumor necrosis factor- ⁇ -inducible protein A20) sequences
  • SEQ ID NOs: 117-135 Is a human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence
  • the lentivirus or construct of the embodiment of the present invention is introduced into lymphocytes obtained from lymphocytes according to an embodiment of the present invention, the cell surface thereof Immune checkpoint PD1, CTLA4, TIM3, BTLA, LAG-3 or intracellular Immunological checkpoints IRAK-M, SOCS1, A20, C
  • a retrovirus or construct of an embodiment of the invention carries a nucleotide sequence comprising SEQ ID NO: 138, 139, 140, 141, 142 or 143.
  • SEQ ID NO: 138 represents a nucleic acid molecule (MSLN CAR/iPD1/tEGFR) co-expressing an anti-MSLN chimeric antigen receptor, a non-functional EGFR, a silencing cell immunological checkpoint PD1
  • SEQ ID NO: 139 is a co-expression An anti-MSLN chimeric antigen receptor, a non-functional EGFR, and a nucleic acid molecule (MSLN CAR/iCBL-B/tEGFR) that silences the cellular immunological checkpoint CBL-B
  • SEQ ID NO: 140 is a co-expressing anti-MSLN chimeric antigen receptor, Non-functional EGFR and silencing cellular immunological checkpoint CTLA4 nucleic acid molecule (MSLN C
  • the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes, the cell immunological checkpoints PD1, CTLA4, CBL-B are specifically silenced and express non-functional EGFR and anti-MSLN
  • the expression of the chimeric antigen receptor makes the transgenic lymphocyte have significant anti-tumor-mediated immunosuppressive effect, and its anti-apoptotic ability and proliferative ability are enhanced, the directional killing ability is significantly improved, and the safety of immunological killing is significantly improved, thereby making the transgenic gene
  • the proliferation and viability of lymphocytes in tumor patients in vitro and in vivo and their ability to kill in tumor patients are greatly improved, especially for mesothelioma cells with high expression of MSLN. For mesothelioma with high expression of MSLN.
  • the specific killing safety of cells is significantly improved.
  • the inventors realize that the above-mentioned cell chimeric antigen receptor, surface or intracellular immunological checkpoint shRNA, and non-functional EGFR are independently expressed by at least one of the following methods, wherein , expression herein refers to both protein expression and RNA transcription.
  • the internal ribosome entry site sequence of the present invention is set between a nucleic acid molecule encoding a chimeric antigen receptor and a nucleic acid molecule expressing a non-functional EGFR, and an internal ribosome entry site
  • the dot has the nucleotide sequence shown by SEQ ID NO:144.
  • the internal ribosome entry site is usually located in the 5' untranslated region (UTR) of the RNA viral genome, so that the translation of one viral protein can be independent of the 5' cap structure, and the other protein usually initiates translation by the 5' hat structure.
  • UTR 5' untranslated region
  • the expression of the two genes before and after IRES is usually proportional.
  • an internal ribosome entry site sequence allows expression of a nucleic acid molecule encoding a chimeric antigen receptor independently of a nucleic acid molecule encoding a non-functional EGFR.
  • the internal ribosome entry site sequence effectively ensures the high expression of the chimeric antigen receptor and the non-functional EGFR, and the specific killing effect of lymphocytes on the high expression of MSLN tumor is more significant. The safety of immune killing is further improved.
  • Promoter a first promoter operably linked to a nucleic acid molecule encoding a chimeric antigen receptor; a second promoter operably linked to a nucleic acid molecule that silences the immunological checkpoint; A third promoter, the third promoter is operably linked to a nucleic acid molecule that expresses non-functional EGFR.
  • the first promoter, the second promoter and the third promoter employed are each independently selected from the group consisting of U6, CMV, H1, EF-1, LTR, RSV promoters, first and second
  • the introduction of the promoter and the third promoter enables the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule that silences the immunological checkpoint and the nucleic acid molecule expressing the non-functional EGFR to be independently expressed, thereby effectively silencing the cell surface or intracellular Immunological checkpoints or high-efficiency expression of non-functional EGFR, and ensure the high expression of chimeric antigen receptors, so that the survival rate of lymphocytes in the tumor environment is greatly improved, lymphocyte targeting is stronger, swelling The specific killing effect of the tumor is more significant, and the safety of immune killing is further improved.
  • a fourth nucleic acid molecule is disposed between the first nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encodes a linker peptide capable of being Cutting.
  • the linker peptide has the amino acid sequence set forth in SEQ ID NO:145.
  • the cell surface or intracellular immune checkpoint is efficiently expressed and embedded by highly efficient silencing and non-functional EGFR.
  • the antigen-receptor is efficiently expressed on the transgenic lymphocyte membrane of the present invention, and the non-functional EGFR and the chimeric antigen receptor are expressed in the non-fusion state on the lymphocyte membrane, thereby effectively inhibiting the immunological negative regulation of the immune checkpoint.
  • the biological effect of the chimeric antigen receptor is ensured, and the timely removal of the transgenic lymphocytes is effectively realized, so that the survival rate of lymphocytes in the tumor environment is greatly improved, the targeted killing effect of lymphocytes is more remarkable, and the safety of immune killing is safe.
  • the sex is further improved.
  • the vector of the construct of the embodiment of the present invention is a non-pathogenic viral vector.
  • the non-pathogenic viral vector greatly enhances the replication and amplification efficiency of the construct in lymphocytes, and further, the lymphocyte proliferation and viability of the lymphocytes in the embodiment of the invention are greatly enhanced, and the targeting effect of lymphocytes is further enhanced.
  • the killing effect on tumor cells is more significant, and the safety of immune killing is further improved.
  • the vector of the construct of the embodiment of the invention is a viral vector selected from at least one of a retroviral vector, a lentiviral vector, an adenoviral vector or an adenovirus associated viral vector.
  • the virus carrier of the embodiment of the present invention has a wide range of virus infection during virus packaging and infection, and can infect both terminally differentiated cells and cells in a dividing phase, and can be integrated into the host.
  • the chromosome which can be freed from the host chromosome, achieves a broad-spectrum and efficient infection efficiency, so that cell surface or intracellular immunological checkpoints are efficiently silenced and non-functional EGFR is highly expressed and chimeric antigen receptors are efficiently expressed in lymphocytes.
  • the lymphocyte has a greatly enhanced proliferation and viability in the tumor patient, and the lymphocyte targeting effect is further enhanced, and the killing effect on the tumor cell is more remarkable, and the immune killing safety of the lymphocyte is further improved.
  • the inventors in order to construct a lentiviral vector, the inventors inserted a nucleic acid of interest into a viral genome at a position of a certain viral sequence in order to construct a lentiviral vector, thereby producing a replication-defective virus.
  • the inventors further constructed packaging cell lines (containing the gag, pol and env genes, but excluding LTR and packaging components).
  • the inventors introduced a recombinant plasmid containing the gene of interest, together with the lentiviral LTR and the packaging sequence, into a packaging cell line.
  • the packaging sequence allows the recombinant plasmid RNA transcript to be packaged into viral particles which are then secreted into the culture medium.
  • the inventors collected a matrix containing the recombinant lentivirus, selectively concentrated, and used for gene transfer. Slow vectors can infect a variety of cell types, including cleavable cells and non-dividable cells.
  • the lentivirus of the embodiment of the present invention is a complex lentivirus, and in addition to the common lentiviral genes gag, pol and env, other genes having regulatory and structural functions are also included.
  • 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 produce a biosafety vector by multiple attenuation of HIV-causing genes, such as deletion of the genes env, vif, vpr, vpu and nef.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and are useful for in vivo and in vitro gene transfer and nucleic acid sequence expression.
  • a suitable host cell together with two or more vectors with packaging functions (gag, pol, env, rev and tat), it is possible to infect non-dividing cells.
  • the targeting of recombinant viruses is achieved by binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
  • the targeting of the recombinant virus confers specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, along with another gene encoding a ligand for the receptor on the particular target cell.
  • the lentiviral vector of the present invention can efficiently transport and co-express shRNA (a transport form of siRNA) which can effectively inhibit the expression of PD1 or CTLA4 or CBL-B.
  • shRNA a transport form of siRNA
  • an adeno-associated viral vector (AAV) of an embodiment of the invention may be constructed using one or more DNAs of a well-known serotype adeno-associated viral vector.
  • AAV adeno-associated viral vector
  • One skilled in the art constructs a suitable adeno-associated viral vector to carry and co-express a small hairpin RNA that inhibits the expression of the PDl or CTLA4 or CBL-B genes.
  • the embodiment of the present invention also includes a microgene.
  • Microgenes mean the use of a combination (selected nucleotide sequence and operably necessary related linker sequences) to direct expression of the transform, transcription and/or gene product in a host cell in vivo or in vitro.
  • the "operable ligation" sequence is employed to include expression control sequences for a continuous gene of interest, and expression control sequences for trans- or remote control of the gene of interest.
  • vectors of the embodiments of the invention also include conventional control elements that permit transcription, transformation, and/or expression of small hairpin RNA in cell infection with the plasmid vector or in a cellular infection with the viral vector.
  • a large number of expression control sequences may be used.
  • the shRNA expressing promoter is an RNA polymerase promoter.
  • the promoter is a RAN polymerase promoter selected from the group consisting of U6, H1, pol I, pol II and pol III.
  • the promoter is a tissue-specific promoter.
  • the promoter is an inducible promoter.
  • the promoter is selected from a promoter based on the selected vector.
  • the promoter when a lentiviral vector is selected, the promoter is a U6, H1, CMV IE gene, EF-1 ⁇ , ubiquitin C or phosphoglycerate kinase (PGK) promoter.
  • Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding geneticin, hygromycin, ampicillin or puromycin resistance.
  • Other components of the carrier include an origin of replication.
  • vectors are well known to those skilled in the art and include conventional cloning techniques such as shRNA, polymerase chain reaction and any suitable method for providing the desired nucleotide sequence for use in embodiments of the invention. .
  • the inventors constructed viral vectors that co-express small hairpin RNA (shRNA) (used to suppress immune checkpoints) and non-functional EGFR and chimeric antigen receptor (CAR).
  • shRNA small hairpin RNA
  • CAR chimeric antigen receptor
  • the small hairpin RNA carrying the siRNA silencing PD1 or CTLA4 or CBL-B and the nucleic acid molecule expressing the non-functional EGFR and the viral vector or plasmid expressing the chimeric antigen receptor (CAR) are complexed with the virus of the present invention.
  • the vector or plasmid can be combined with a polymer or other material to increase its stability or assist in its targeted movement.
  • the invention provides a method of preparing a T lymphocyte or a transgenic lymphocyte as described above.
  • the method comprises introducing the construct described above or the lentivirus described above into lymphocytes or T lymphocytes.
  • the mode of introduction can be introduced in a manner selected from the group consisting of electroporation or viral infection of host cells.
  • the construct or lentivirus of the embodiment of the present invention is successfully introduced into the above lymphocyte or T lymphocyte, and the expression of the chimeric antigen receptor against the antigen MSLN and the cell surface or intracellular immune checkpoint of the lymphocyte are silenced and absent.
  • lymphocytes or T lymphocytes have significant anti-tumor-mediated immunosuppressive effects, and the proliferation of tumor patients in vitro and in vivo and the survival ability of tumor patients are greatly improved, lymphocytes or T lymphocytes
  • the targeted killing effect on tumor cells, especially tumor cells with high expression of MSLN, is stronger, and the safety of immune killing is high.
  • the invention provides a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: the above construct, the above lentivirus, the above T lymphocyte or the above transgenic lymphocyte.
  • the composition of any of the above therapeutic compositions can achieve high expression of the antigen MSLN chimeric antigen receptor in transgenic lymphocytes or T lymphocytes and silencing of transgenic lymphocytes or T lymphocyte cells or intracellular immune checkpoints.
  • the expression of non-functional EGFR on the surface of transgenic lymphocytes or T lymphocytes so that the obtained transgenic lymphocytes or T lymphocytes can be expanded in vitro, proliferate in tumor patients and survive in tumor patients, and transgenic lymphocytes are greatly improved.
  • T lymphocytes have stronger targeted killing effect on tumor cells with high expression of MSLN, and the safety of immune killing is higher.
  • the therapeutic composition of the embodiments of the invention provided to a patient is preferably applied to a biocompatible solution or an acceptable pharmaceutical carrier.
  • the various therapeutic compositions prepared are suspended or dissolved in a pharmaceutically or physiologically acceptable carrier, such as physiological saline; an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
  • a pharmaceutically or physiologically acceptable carrier such as physiological saline; an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
  • physiological saline such as physiological saline
  • an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
  • the appropriate carrier will depend to a large extent on the route of administration.
  • Other isotonic sterile injections with water and anhydrous, and sterile suspensions with water and anhydrous are pharmaceutically acceptable carriers.
  • a sufficient number of viral vectors are transduced into targeted T cells and provide sufficient strength Transgenic, silencing PD1 or CTLA4 or CBL-B and expressing non-functional EGFR as well as expressing a unique MSLN chimeric antigen receptor.
  • the dosage of the therapeutic agent depends primarily on the condition of treatment, age, weight, and the health of the patient, which may result in patient variability.
  • These methods of silencing PD1 or CTLA4 or CBL-B and expressing non-functional EGFR as well as expressing a specific receptor for the antigenic MSLN chimeric antigen are part of a combination therapy.
  • These viral vectors and anti-tumor T cells for adoptive immunotherapy can be performed alone or in combination with other methods of treating cancer. Under appropriate conditions, one treatment involves the use of one or more drug therapies.
  • the cancer comprises mesothelioma.
  • the survival ability is greatly improved, and the lymphocyte or T lymphocyte has stronger targeted killing effect on tumor cells with high expression of MSLN, especially for tumor cells with high expression of MSLN, and for tumor cells with high expression of MSLN. Immune killing is safer and more effective.
  • the invention provides a method of increasing lymphocyte activity and therapeutic safety, wherein the lymphocytes of the embodiments of the invention carry a chimeric antigen receptor, according to an embodiment of the invention, the method comprising: Causing at least one of the cell surface or intracellular immune checkpoint of the lymphocyte; and causing the lymphocyte to express non-functional EGFR, cell surface or intracellular immunological checkpoint, lymphocyte, chimeric antigen receptor, none Functional EGFR is as previously defined.
  • lymphocyte activity according to an embodiment of the present invention includes at least one of lymphocyte proliferation ability in vitro, proliferation and viability in a tumor patient, and killing ability of lymphocytes in a tumor patient.
  • the cell surface or intracellular immune checkpoint of the lymphocytes of the embodiment of the present invention is silenced, lymphocytes are activated, the proliferative response is up-regulated, the cytokine secretion is increased, and the anti-apoptotic ability is enhanced.
  • the lymphocytes of the embodiments of the present invention are expanded and propagated in vitro, and the targeted killing effect on tumor cells is remarkably enhanced.
  • Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR receptor and intact sequence that binds to anti-EGFR antibody, and non-functional EGFR can act as lymph Cell suicide markers.
  • lymphocytes of the present invention are used for treating tumor cells having high expression of MSLN, if the patient develops a serious adverse reaction, the lymphocytes of the embodiments of the present invention can be cleared by the anti-EGFR antibody, thereby further improving the lymph of the embodiment of the present invention. Cellular therapy for the safety of tumor patients with high expression of MSLN.
  • the invention provides a method of treating cancer.
  • the method comprises: administering to a cancer patient a construct as described above, a lentivirus as described above, a T lymphocyte as described above or a transgenic lymphocyte as described above, wherein the chimeric The antigen receptor specifically binds to the tumor antigen MSLN.
  • the method for treating cancer according to the embodiment of the invention can effectively achieve targeted killing of tumor cells of cancer patients, especially having The targeted killing effect on tumor cells with high expression of MSLN can further effectively treat cancer, and the therapeutic effect is good and the safety is high.
  • the method comprises: isolating lymphocytes from a cancer patient; introducing the aforementioned construct, or the lentivirus described above, into the lymphocytes to obtain transgenic lymphocytes,
  • the transgenic lymphocytes express the chimeric antigen receptor and the cellular immune checkpoint is silenced; and the transgenic lymphocytes are administered to the cancer patient.
  • the method for treating cancer according to the embodiment of the invention can further effectively achieve targeted killing of tumor cells of cancer patients, especially having targeted killing effect on tumor cells with high expression of MSLN, thereby further effectively treating cancer, and the therapeutic effect Good and safe.
  • the cancer includes at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.
  • the method for treating cancer according to an embodiment of the present invention enables lymphocyte immune checkpoints to be silenced and cells to express chimeric antigen receptors, such as MSLN antigen-specific chimeric antigen receptors of the present invention, resulting lymphocytes or T lymphocytes.
  • the cells have targeted killing of tumor cells of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer or breast cancer which are specifically expressed by MSLN.
  • a lentiviral vector having a replication defect is produced, and the lentiviral vector is collected by centrifugation for transduction of human T lymphocytes.
  • the following is a brief introduction to the experimental procedure for the generation and collection of lentiviral vectors: 293T cells are plated in cell culture dishes with a bottom area of 150-cm 2 and using Express-In according to the instructions (purchased from Open Biosystems/Thermo Scientific, Waltham) , MA) Virus transduction of 293T cells.
  • lentiviral transgenic plasmid 15 ⁇ g of lentiviral transgenic plasmid, 5 ⁇ g of pVSV-G (VSV glycoprotein expression plasmid), 10 ⁇ g of pCMVR8.74 plasmid (Gag/Pol/Tat/Rev expression plasmid) and 174 ⁇ l of Express to each plate.
  • -In concentration is 1 ⁇ g/ ⁇ l.
  • the supernatants were collected at 24 and 48 hours, respectively, and using an ultracentrifuge at 28,000 rpm (the centrifuge rotor was Beckman SW 32Ti, available from Beckman Coulter, Brea, Centrifuge for 2 hours under conditions of CA). Finally, the viral plasmid pellet was resuspended in 0.75 ml of RPMI-1640 medium.
  • Human primary T lymphocytes were isolated from healthy volunteer donors. Human T lymphocytes were cultured in RPMI-1640 medium and challenged with monoclonal antibody coated beads of anti-CD3 and CD28 (purchased from Invitrogen, Carlsbad, CA). T-lymphocytes were transduced by spin-inoculation 18 to 24 hours after activation of human T lymphocytes. The transduction process was as follows: in a 24-well plate, 0.5 x 106 T lymphocytes per well were plated. To each well of the cells, 0.75 ml of the above-mentioned resuspended virus supernatant and Polybrene (concentration: 8 ⁇ g/ml) were added.
  • IL-2 Human recombinant interleukin-2
  • T lymphocyte culture medium every 2 to 3 days.
  • the final concentration of IL-2 was 100-IU/ml in T lymphocytes.
  • the density of the cells is maintained at 0.5 x 106 to 1 x 106 / ml.
  • T lymphocytes are dormant, for example, the cell growth rate is slowed down and the cells become smaller, wherein the cell growth rate and size are assessed by Coulter Counter (purchased from Beckman Coulter), or transduced T lymphocytes.
  • Coulter Counter purchased from Beckman Coulter
  • T lymphocytes can be used for functional analysis.
  • the flow cytometer used in the examples of the present application was BD FACSCanto II (purchased from BD Biosciences), and flow cytometric data was analyzed using FlowJo version 7.2.5 software (purchased from Tree Star, Ashland, OR).
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • the ability of anti-EGFR antibodies to induce cell-dependent lysis of lymphocytes expressing non-functional EGFR was assessed using the 4 hour- 51 Cr-release method.
  • Human T lymphocytes transduced with a lentiviral vector were used as target cells.
  • 100 ⁇ Ci Na 2 51 CrO 4 available from GE Healthcare Life Sciences, Marlborough, MA
  • the cells were washed three times with PBS and resuspended in medium (cell density was 1 x 10 5 /ml).
  • the calibrated cells were then plated in 96-well plates (5 x 10 3 cells per well, plus 50 ⁇ l of medium) and 50 ⁇ l of anti-EGFR antibody (purchased from Erbitux, Genentech) ( The final concentration was 20 ⁇ g/ml, and preculture was carried out for 30 minutes under normal temperature conditions. Then, the medium containing the antibody was changed to a normal medium, thereby detecting the spontaneous release of 51 Cr. Triton X-100 was added to a final concentration of 1% to ensure maximum release of 51 Cr.
  • human PBMCs effector cells
  • % specific lysis (experimental release cpm data - spontaneous release of cpm data) / (maximum release cpm data - spontaneous release of cpm data) * 100, wherein the maximum release cpm data was added through the target cells
  • the spontaneous release of cpm data by Triton X-100 was measured in the absence of anti-EGFR antibodies and effector cells.
  • anti-MSLN CAR T lymphocytes The cytotoxic activity of anti-MSLN chimeric antigen receptor T cells (anti-MSLN CAR T lymphocytes) was evaluated in the Examples using a 4 - hour 51 chromium release assay. The specific steps are as follows: Target test cells were labeled with 51 Cr at 37 degrees Celsius for 1 hour. After labeling, the cells were rinsed with RPMI medium containing 10% fetal bovine serum (FCS). After rinsing, the cells were resuspended in the same medium, and the concentration of the resuspended cells was 1 ⁇ 10 5 /ml.
  • FCS fetal bovine serum
  • T cells were added to the target test cell suspension at different target cell ratios (E:T), and the cells were seeded in 96-wells at a volume of 200 microliters per well.
  • the cells were cultured for 4 hours in a 37 degree incubator. After 4 hours, 30 microliters of the supernatant was taken from each well and placed in a counter 96-well plate for counting analysis.
  • the analytical instrument was a top-level counting NXT micro-scintillator counter (purchased from Packard Bioscience). The number of effector cells in all counting wells was calculated based on the total number of T cells.
  • the target test cell to be labeled is MSLN + MSTO-211H (human pleural mesothelioma cells (ATCC)).
  • Example 2 Construction of a vector for co-expression of shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor
  • the inventors cloned the sequence encoding the single-chain antibody against human MSLN, the 4-1BB intracellular domain and the T cell receptor combined ⁇ -strand sequence into a lentiviral vector containing the EF-1 promoter ( On the lentiviral vector), during the cloning process, the restriction enzyme digestion is the double digestion of XbaI and NotI, and the double digestion of NotI and XhoI, and the expression of anti-MSLN is generated by restriction enzyme digestion, ligation, screening and amplification of the plasmid of interest.
  • Anti-receptor lentiviral plasmid (LV-MSLN CAR).
  • the sequence containing the U6 promoter and human PD1 shRNA (iPD1) or CBL-B shRNA (iCBL-B) or CTLA4 shRNA (iCTLA4) was cloned into the LV-MSLN CAR vector plasmid and constructed into LV-MSLN CAR/iPD1 or LV- MSLN CAR/iCBL-B or LV-MSLN CAR/iCTLA4, including synthetic IRES and sequences expressing non-functional EGFR cloned into LV-MSLN CAR/iPD1 or LV-MSLN CAR/iCBL-B or LV-MSLN CAR/iCTLA4 vector plasmid , constructed as LV-MSLN CAR/iPD1/tEGFR (M) or LV-MSLN CAR/iCBL-B/tEGFR (M) or LV-MSLN CAR/iCTLA4/tEGFR (M); comprising synthetic IRES and expressing non-functional
  • Figure 1 is a schematic representation of a lentiviral vector comprising a sequence encoding an anti-MSLN chimeric antigen receptor, an IRES, U6 and H1 promoter sequence, a PD1 shRNA or CBL-B shRNA or a CTLA4 shRNA sequence, and a coding non-functional EGFR sequence.
  • the sequence of the anti-MSLN chimeric antigen receptor is regulated by the promoter EF-1, and the CTLA4, PD1 or CBL-B shRNA sequence is expressed under the promoter of promoter U6 or H1, and the sequence of non-functional EGFR is expressed as a single
  • the mRNA transcription unit begins translation after the IRES sequence.
  • Anti-EGFR antibody effectively kills T lymphocytes that secrete PD1 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptors
  • peripheral blood lymphocytes are taken from an unnamed blood donor. Peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. Activated T lymphocytes were transduced with lentiviral vector and expanded in vitro in the presence of T lymphocyte activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) as described in Example 1. After the culture was activated for 72 hours, the cells were washed with a washing solution, and the magnetic beads were washed away.
  • T lymphocyte activator magnetic beads CD3/CD28 purchased from Invitrogen, Carlsbad, CA
  • T cells were seeded on a recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were LV-MSLN CAR/iPD1/tEGFR, LV-MSLN CAR, respectively.
  • the /iPD1 or no-load (LV-GFP) transduction process is as described in Example 1.
  • T cells expressing non-functional EGFR after transfection were stained with anti-EGFR antibody and then isolated by FACS. After isolation, T cells were cultured in RPMI-1640 medium and recombinant human IL-2 factor (100 ng/ml; purchased from R&D Systems).
  • Induction amplification was carried out for 7-10 days and then used as a target cell for the experiment.
  • the inventors measured the killing effect of anti-EGFR antibody-differentiated ADCC on T cells (target cells) transduced with different lentiviruses by ADCC assay using standard 4–hour 51 chromium release method, 4–hour 51 chromium release. The method is as described in Example 1. The result is shown in Figure 2.
  • anti-EGFR antibodies are effective in dissociating T lymphocytes that co-express anti-MSLN chimeric antigen receptors, PD1 shRNA (iPD1) and non-functional EGFR (LV-MSLN-CAR/iPD1/M), but Anti-EGFR antibodies do not mediate killing of T lymphocytes expressing the anti-MSLN chimeric antigen receptor and PD1 shRNA (LV-MSLN-CAR/iPD1). Anti-EGFR antibodies also do not mediate T lymphocyte killing of anti-MSLN chimeric antigen receptor (LV-MSLN-CAR) alone.
  • Example 4 T lymphocyte tumor cell lysis ability co-expressing PD1 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor.
  • peripheral blood lymphocytes are taken from an unnamed blood donor. Peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. T lymphocytes were incubated with T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) for 72 hours at 5% CO 2 at 37 ° C. The medium was supplemented with 2 mmol/L glutamine, 10%. High temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U/ml penicillin/streptomycin double antibody in RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012).
  • FCS High temperature inactivated fetal calf serum
  • T cells were seeded on a recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were LV-MSLN CAR/iPD1/tEGFR, LV-MSLN CAR, respectively.
  • FN ch-296; Retronectin fibronectin fragment
  • lentiviruses were LV-MSLN CAR/iPD1/tEGFR, LV-MSLN CAR, respectively.
  • the /iPD1, LV-tEGFR, or no-load (LV-GFP) transduction process is as described in Example 1.
  • the transduced T cells were cultured in RPMI-1640 medium and induced for amplification for 7-10 days with recombinant human IL-2 factor (100 ng/ml; purchased from R&D Systems), followed by a functional test.
  • the inventors measured the killing effect of T cells (effector cells) transduced with different lentiviruses on mesothelioma target cells (MSLN + MSTO-211H) with high expression of MSLN.
  • the ratio of target cells was 50:1, 25:1 or 10:1, the measurement method uses a standard 4 - hour 51 chromium release method, and the 4 - hour 51 chromium release method is as described in Example 1. The result is shown in Figure 3.
  • T lymphocytes transduced with anti-MSLN chimeric antigen receptor, PD1 shRNA (iPD1) and non-functional EGFR lentivirus (LV-MSLN-CAR/iPD1/M) were co-expressed to express anti-MSLN inlays.
  • T lymphocytes combined with antigen receptor and PD1 shRNA (iPD1) T lymphocytes (LV-MSLN-CAR/iPD1) are also effective in killing mesothelioma target cells with high expression of MSLN.
  • Non-functional EGFR lentiviral transduced T lymphocytes (LV-M T lymphocytes) have no significant killing effect on mesothelial cells with high expression of MSLN.
  • T lymphocyte killing of T lymphocytes (LV-MSLN-CAR) expressing only the anti-MSLN chimeric antigen receptor has the ability to kill mesenchymal target cells with high expression of MSLN, but weaker than co-expression of anti-MSLN chimeric antigen T lymphocytes of T1 lymphocytes (LV-MSLN-CAR/iPD1) of PD1 shRNA (iPD1).
  • iPD1 shRNA PD1 shRNA
  • Example 5 Co-expressing PD1 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor T cells, co-expressing CTLA4 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor T cells, co-expressing CBL-B shRNA, Non-functional EGFR and anti-MSLN chimeric antigen receptor T cells, co-expressing PD1 shRNA, CTLA4 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor T cells, co-expressing PD1 shRNA, CBL-B shRNA, Anti-MSLN chimeric antigen receptor and non-functional EGFR T cells, with enhanced solvency and more cytokine secretion and stronger cell proliferation
  • the inventors also examined T cells co-expressing one shRNA (CBL-B shRNA or PD1 shRNA or CTLA4 shRNA), non-functional EGFR and anti-MSLN chimeric antigen receptor, and co-expressed two shRNAs ( Tumor lytic capacity, cytokine secretion capacity of lymphocytes of two PD1 shRNA sequences, PD1 shRNA and CBL-B shRNA or PD1 shRNA and CTLA4 shRNA), non-functional EGFR and anti-MSLN chimeric antigen receptors in different PD1 regions Cell proliferation ability.
  • the above T cells have enhanced cytolysis ability, more cytokine secretion and stronger cell proliferation than T cells expressing the anti-MSLN chimeric antigen receptor alone.
  • T cells with no functional EGFR and anti-MSLN chimeric antigen receptor were co-expressed.
  • T cells sh1 shRNA or CBL-B shRNA or CTLA4 shRNA
  • non-functional EGFR and anti-MSLN chimeric antigen receptors have stronger cytolysis ability, more cytokine secretion and stronger cell proliferation.
  • Example 6 Effect of expression of non-functional EGFR on cytolysis, cytokine secretion and cell proliferation of T cells
  • the inventors examined the effects of expressing non-functional EGFR on the cytolytic ability, cytokine secretion ability, and cell proliferation ability of T lymphocytes.
  • the inventors found that cytosolic ability, cells co-expressing two shRNAs (two PD1 shRNAs, PD1 shRNA and CBL-B shRNA or PD1 shRNA and CTLA4 shRNA), anti-MSLN chimeric antigen receptor and non-functional EGFR T cells Factor secretion capacity and cell proliferation ability and co-expression of 2 shRNAs (2 PD1 shRNAs, PD1 shRNA and CBL-B shRNA or PD1 shRNA and CTLA4 shRNA) are equivalent to T cells against MSLN chimeric antigen receptor; co-expression of 1 shRNA (PD1 shRNA or CBL-B shRNA or CTLA4 shRNA), none The cytosolic ability, cytokine secretion ability and cell proliferative ability of functional EGFR and anti-MSLN chimeric antigen receptor T cells
  • non-functional EGFR does not affect the cytolytic ability, cytokine secretion ability and cell proliferation ability of T lymphocytes, and the inventors introduced non-functional EGFR in T cells, so that the lymphocytes of the examples of the present invention are used.
  • the lymphocytes of the present invention can be cleared by the anti-EGFR antibody, thereby improving the safety of the lymphocyte treatment of the tumor patient with high expression of MSLN in the embodiment of the present invention. .

Abstract

Provided are a transgenic lymphocyte, a construct, and a therapeutic composition for treating a cancer. A cell checkpoint of the transgenic lymphocyte is silenced, and the transgenic lymphocyte expresses a non-functional EGFR and a chimeric antigen receptor. The chimeric antigen receptor comprises an extracellular domain, and the extracellular domain comprises a heavy chain variable region and a light chain variable region of a single-chain antibody. The single-chain antibody specifically recognizes an antigen, mesothelin. The chimeric antigen receptor further comprises a transmembrane domain. The transmembrane domain is connected with the extracellular domain and is embedded in the membrane of the T lymphocyte. The chimeric antigen receptor further comprises an intracellular domain. The intracellular domain is connected with the transmembrane domain and comprises an intracellular segment of CD28 or 4-1BB and a CD3ζ chain.

Description

治疗间质素阳性肿瘤的治疗组合物Therapeutic composition for treating interstitial positive tumors
优先权信息Priority information
本申请请求2017年01月25日向中国国家知识产权局提交的、专利申请号为201710056392.7的专利申请的优先权和权益，并且通过参照将其全文并入此处。The present application claims priority to and the benefit of the patent application No. PCT Application No.
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活性和治疗安全性的方法。The present invention relates to the field of biomedicine, and in particular to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer, and an augmentation lymph Methods of cell viability and therapeutic 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 human normal tissues is limited to the pleural, pericardial and peritoneal lining mesothelial cells. However, interstitial is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancers and about 50% of lung adenocarcinomas and other cancers such as cholangiocarcinoma, gastric cancer, and intestinal cancer. , esophageal cancer, breast cancer. The interstitial gene encodes a precursor protein of 71KDa, which is then processed into a 31KDa exfoliated fragment and a 40KDa protein fragment. The 31KDa exfoliated fragment is called megakaryocyte promoting factor (MPF), and the 40KDa protein fragment is Known as interstitial, interstitial is immobilized on the cell membrane by the anchoring action 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。In the case of mesothelioma, mesothelioma is divided into pleural mesothelioma and peritoneal mesothelioma. The pleural mesothelioma is the primary tumor of the pleura, which is limited (mostly benign) and diffuse (both malignant). Divided, malignant mesothelioma is one of the worst tumors in the chest. Peritoneal mesothelioma refers to a tumor that originates in the peritoneal mesothelial cells. Clinical manifestations are not characteristic, common symptoms and signs are: 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 radiation therapy. It is generally considered that for stage I patients with relatively limited tumors, radical pleural pneumonectomy is advocated. For patients with stage II, III, and IV, radical surgery is no longer meaningful, and only palliative surgery is performed. In fact, most patients are already in stage II or above when the disease is clearly diagnosed. The rapidly growing pleural effusion often causes severe dyspnea in patients, and palliative surgery can only temporarily improve the quality of life of these advanced patients, and cannot be cured.
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。Thus, it is particularly urgent to develop a treatment method for interstitial high expression tumors.
发明内容Summary 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 discovery and recognition of the following facts and issues by the inventors:
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。Interstitial is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancers and about 50% of lung adenocarcinomas and other cancers such as cholangiocarcinoma, gastric cancer, intestinal cancer, esophagus Cancer, breast cancer. Cause Therefore, interstitial represents a highly attractive target in the field of tumor immunotherapy.
基于上述发现，发明人提出了一种携带沉默细胞免疫检查点的核酸分子和编码无功能EGFR的核酸分子以及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，且本申请所提出的构建体和转基因淋巴细胞通过表达无功能EGFR，可显著增加治疗安全性。Based on the above findings, the inventors have proposed a nucleic acid molecule carrying a silent cellular immunological checkpoint, a nucleic acid molecule encoding a non-functional EGFR, and a nucleic acid molecule encoding a chimeric antigen receptor, and a construct formed by the introduction of the construct. Transgenic lymphocytes, which encode a chimeric antigen receptor that specifically binds to the antigen MSLN. Therefore, the constructs and transgenic lymphocytes proposed by the present invention can be used for immunotherapy of adoptive T cells of tumors, especially mesenchymal positive tumors; the transgenic lymphocytes of the present invention have a strong killing ability for high expression of interstitial tumors. The mesothelial cells with normal MSLN expression levels have weaker killing, and the constructs and transgenic lymphocytes proposed by the present application can significantly increase the safety of treatment by expressing non-functional EGFR.
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淋巴细胞的细胞免疫检查点被沉默；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。其中，细胞免疫检查点包括细胞表面或细胞内至少之一的免疫检查点。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受体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。根据本发明的实施例，本发明实施例的T淋巴细胞具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著提高，对肿瘤细胞的杀伤能力显著增强，尤其对高表达MSLN的肿瘤具有显著的定向杀伤作用，安全性高。In a first aspect of the invention, the invention proposes a T lymphocyte. According to an embodiment of the present invention, the cellular immune checkpoint of the T lymphocyte is silenced; expressing a non-functional EGFR; and expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: an extracellular region, The extracellular region comprises a heavy chain variable region and a light chain variable region of a single chain antibody that specifically recognizes the antigen MSLN; a transmembrane region, the transmembrane region is linked to the extracellular region, and is embedded Into the cell membrane of the T lymphocyte; an intracellular region, the intracellular region is linked to the transmembrane region, and the intracellular region comprises an intracellular portion of CD28 or 4-1BB and a CD3 ζ chain. Wherein, the cellular immune checkpoint includes an immune checkpoint on at least one of a cell surface or a cell. Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR receptor and intact sequence that binds to anti-EGFR antibody, and non-functional EGFR can act as lymph Cell suicide markers. According to an embodiment of the present invention, the T lymphocytes of the embodiments of the present invention have the characteristics of resisting tumor cell-mediated immunosuppression, and the proliferative ability in vitro, the proliferation and viability in tumor patients are significantly improved, and the killing of tumor cells is performed. The ability is significantly enhanced, especially for tumors with high expression of MSLN, which has a significant directional killing effect and 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：(a)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；(b)沉默细胞免疫检查点的核酸分子，所述沉默细胞免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：3～135的至少之一；以及(c)编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：136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：137所示的核苷酸序列。In a second aspect of the invention, the invention proposes a lentivirus. According to an embodiment of the invention, the lentivirus carries the following nucleic acid molecule: (a) a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1, The nucleic acid molecule encoding the chimeric antigen receptor has the nucleotide sequence shown in SEQ ID NO: 2; (b) the nucleic acid molecule that silences the cellular immunological checkpoint, and the nucleotide sequence of the nucleic acid molecule of the silencing cell immunological checkpoint Is at least one selected from the group consisting of SEQ ID NOS: 3 to 135; and (c) a nucleic acid molecule encoding a non-functional EGFR having the amino acid sequence set forth in SEQ ID NO: 136, encoding a non-functional EGFR The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:137.
Figure PCTCN2017081272-appb-000001
Figure PCTCN2017081272-appb-000001
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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，其具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著增强，对肿瘤细胞的杀伤能力显著增强，尤其对高表达MSLN的肿瘤具有显著的定向杀伤作用，安全性高。According to an embodiment of the present invention, the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes have the characteristics of resisting tumor cell-mediated immunosuppression, proliferative ability in vitro, proliferation in tumor patients, and The survivability is significantly enhanced, and the killing ability of tumor cells is remarkably enhanced, especially for tumors with high expression of MSLN, which has a significant directional killing effect and high safety.
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：138、139、140、141、142或143所示的核苷酸序列。In a third aspect of the invention, the invention proposes a lentivirus. According to an embodiment of the invention, the lentivirus carries a nucleotide sequence comprising SEQ ID NO: 138, 139, 140, 141, 142 or 143.
Figure PCTCN2017081272-appb-000009
Figure PCTCN2017081272-appb-000009
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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，其具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著增强，对肿瘤细胞的杀伤能力显著增强，尤其对高表达MSLN的肿瘤具有显著的定向杀伤作用，且安全性更高。According to an embodiment of the present invention, the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes have the characteristics of resisting tumor cell-mediated immunosuppression, proliferative ability in vitro, proliferation in tumor patients, and The viability is significantly enhanced, and the killing ability of tumor cells is significantly enhanced, especially for tumors with high expression of MSLN, and the safety is higher.
在本发明的第八方面，本发明提出了一种转基因淋巴细胞。根据本发明的实施例，所述淋巴细胞细胞免疫检查点被沉默；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。发明人发现，细胞免疫检查点被沉默、表达无功能EGFR和表达嵌合抗原受体的淋巴细胞的体外增殖能力、在肿瘤病人体内的增殖和生存能力以及在肿瘤病人体内的对肿瘤细胞的特异性杀伤能力大大提高，尤其对高表达MSLN的肿瘤具有显著的定向杀伤作用，安全性高。In an eighth aspect of the invention, the invention provides a transgenic lymphocyte. According to an embodiment of the present invention, the lymphocyte immune checkpoint is silenced; expressing a non-functional EGFR; and expressing a chimeric antigen receptor, the chimeric antigen receptor comprising: an extracellular region, the extracellular region comprising a heavy chain variable region and a light chain variable region of an antibody, said antibody being capable of specifically binding to a tumor antigen; a transmembrane region; and an intracellular region comprising an intracellular portion of an immunostimulatory molecule, wherein The antibody is a single chain antibody and the tumor antigen is MSLN. The inventors found that the cell immune checkpoint is silenced, expresses the ability of non-functional EGFR and lymphocytes expressing chimeric antigen receptors to proliferate in vitro, proliferation and viability in tumor patients, and tumor cell specificity in tumor patients. The sexual killing ability is greatly improved, especially for tumors with high expression of MSLN, which has a significant directional killing effect and 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 transgenic lymphocytes may further have at least one of the following additional technical features:
根据本发明的实施例，所述淋巴细胞细胞免疫检查点独立地选自CTLA4、PD-1、TIM-3、BTLA、LAG-3IRAK-M、SOCS-1、A20、CBL-B的至少之一。其中，CTLA4、PD-1、TIM-3、BTLA、LAG-3为细胞表面免疫检查点，IRAK-M、SOCS-1、A20、CBL-B为细胞内免疫检查点。本发明实施例的免疫检查点具有负向调控和减弱细胞免疫应答的作用，其通过与肿瘤细胞上相应的配体的特异结合，导致T淋巴细胞增生性反应的下调,细胞因子的分泌减少,和T细胞的无能或凋亡。根据本发明的实施例，本发明实施例的细胞表面或细胞内免疫检查点的成功沉默，进一步提高了转基因淋巴细胞抵抗肿瘤介导的免疫抑制的功效，转基因淋巴细胞在体外扩增及在肿瘤病人体内的增殖和生存能力，对肿瘤细胞的定向杀伤作用进一步加强。According to an embodiment of the present invention, the lymphocyte immune checkpoint is independently selected from at least one of CTLA4, PD-1, TIM-3, BTLA, LAG-3IRAK-M, SOCS-1, A20, CBL-B. . Among them, CTLA4, PD-1, TIM-3, BTLA, and LAG-3 are cell surface immune checkpoints, and IRAK-M, SOCS-1, A20, and CBL-B are intracellular immune checkpoints. The immune checkpoint of the embodiment of the invention has the functions of negatively regulating and attenuating the cellular immune response, and the specific binding of the corresponding ligand on the tumor cell leads to down-regulation of the proliferative response of the T lymphocyte and the secretion of the cytokine is reduced. And the inability or apoptosis of T cells. According to an embodiment of the present invention, successful silencing of cell surface or intracellular immunological checkpoints according to embodiments of the present invention further enhances the efficacy of transgenic lymphocytes against tumor-mediated immunosuppression, in vitro expansion of transgenic lymphocytes and in tumors The proliferation and viability of the patient's body further enhances the targeted killing effect on tumor cells.
根据本发明的实施例，所述淋巴细胞细胞免疫检查点被沉默是通过shRNA、反义核酸、核酶、显性负突变、CRISPR和锌指核酸酶至少之一实现的。根据本发明的实施例，本发明实施例的细胞免疫检查点的成功沉默，可显著提高本发明实施例的淋巴细胞抵抗肿瘤介导的免疫抑制的特性，进一步提高了转基因淋巴细胞增殖肿瘤细胞的定向杀伤作用。 According to an embodiment of the invention, the lymphocyte immune checkpoint is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR and zinc finger nuclease. According to an embodiment of the present invention, the successful silencing of the cellular immune checkpoint of the embodiment of the present invention can significantly improve the lymphocyte resistance tumor-mediated immunosuppressive property of the embodiment of the present invention, and further improve the transgenic lymphocyte proliferating tumor cell. Directional killing effect.
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以及细胞免疫检查点的沉默联合具有正向调控和增强细胞免疫应答的作用，本发明实施例的免疫共刺激分子胞内段的表达、无功能EGFR的表达以及细胞免疫检查点的沉默的联合，使得本发明实施例的转基因淋巴细胞增殖对肿瘤的定向杀伤作用更加显著和安全。According to an embodiment of the invention, the intracellular segment of the immunocostimulatory 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 immunostimulatory molecule and the silencing of the cellular immune checkpoint in the embodiment of the present invention have the functions of positively regulating and enhancing the cellular immune response, and the expression of the intracellular segment of the immunostimulatory molecule of the present invention is absent. The combination of the expression of functional EGFR and the silencing of cellular immune checkpoints makes the directional killing effect of the transgenic lymphocyte proliferation of the present invention on the tumor more significant and safe.
根据本发明的实施例，所述淋巴细胞细胞免疫检查点是CTLA4、PD-1、CBL-B。其中，CTLA4、PD-1是细胞表面免疫检查点，CBL-B是细胞内免疫检查点。根据本发明的实施例，本发明实施例的淋巴细胞细胞表面免疫检查点CTLA4或PD-1被沉默，或淋巴细胞细胞内免疫检查点是CBL-B被沉默，阻止了PD1或CTLA4分子表达与其相应配体PD-L1及PD-L2或CD80及CD86的结合，从而有效抑制了对T淋巴细胞无能或凋亡，或通过CBL-B沉默,增强T细胞受体信号传导，使得转基因淋巴细胞在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著。According to an embodiment of the invention, the lymphocyte immune checkpoints are CTLA4, PD-1, CBL-B. Among them, CTLA4 and PD-1 are cell surface immune checkpoints, and CBL-B is an intracellular immune checkpoint. According to an embodiment of the present invention, the lymphocyte cell surface immunological checkpoint CTLA4 or PD-1 of the embodiment of the present invention is silenced, or the lymphocyte intracellular immune checkpoint is CBL-B is silenced, preventing the expression of PD1 or CTLA4 molecules and The binding of the corresponding ligands PD-L1 and PD-L2 or CD80 and CD86, thereby effectively inhibiting the inability or apoptosis of T lymphocytes, or silencing through CBL-B, enhancing T cell receptor signaling, allowing transgenic lymphocytes to The proliferation and viability of tumor patients are further improved, and the effect of targeted killing of tumors is more significant.
根据本发明的实施例，所述淋巴细胞细胞免疫检查点被沉默是通过shRNA实现的。根据本发明的实施例，本发明实施例的shRNA携带特异性沉默细胞表面或细胞内免疫检查点至少之一免疫检查点的siRNA，本发明实施例的shRNA具有高效、特异性的沉默细胞表面或细胞内至少之一免疫检查点的作用，即细胞表面或细胞内免疫检查点的成功沉默，阻止了免疫检查点与相应配体的特异性结合，从而有效抑制了免疫检查点对T淋巴细胞无能或凋亡等的负调控机制，进而使得本发明实施例的转基因淋巴细胞在在肿瘤病人体内的增殖殖和生存能力得到进一步提高，配合嵌合抗原受体的抗原靶向性，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更加显著。According to an embodiment of the invention, silencing of the lymphocyte immune checkpoint is achieved by shRNA. According to an embodiment of the present invention, the shRNA of the embodiment of the present invention carries an siRNA which specifically silences at least one of the cell surface or the intracellular immunological checkpoint, and the shRNA of the embodiment of the present invention has a highly efficient and specific silencing cell surface or The role of at least one of the immune checkpoints in the cell, ie the successful silencing of the cell surface or intracellular immune checkpoint, prevents the specific binding of the immunological checkpoint to the corresponding ligand, thereby effectively inhibiting the immune checkpoint's inability to T lymphocytes. Or a negative regulation mechanism such as apoptosis, thereby further enhancing the proliferation and viability of the transgenic lymphocytes of the embodiments of the present invention in a tumor patient, and cooperating with the antigen targeting of the chimeric antigen receptor, so that the present invention is implemented The effect of transgenic lymphocytes on the targeted killing effect of tumors is more pronounced.
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 invention, the intracellular segment of the immunostimulatory molecule is an intracellular segment of 4-1BB or CD28. The intracellular segment of the immunostimulatory molecule of the chimeric antigen receptor of the transgenic lymphocytes of the present invention is the intracellular portion of CD28 or 4-1BB. According to an embodiment of the present invention, the intracellular segment of the immunostimulatory molecule is an intracellular segment of CD28 or 4-1BB, which further enhances the targeted killing effect of the transgenic lymphocytes of the embodiments 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受体的跨膜区和完整的与抗EGFR抗体结合的结构域，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，并进一步联合细胞免疫检查点的沉默，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 an embodiment of the present invention, the non-functional EGFR expressed by the transgenic lymphocytes of the present invention lacks an N-terminal ligand binding region and an intracellular receptor tyrosine kinase activity, but includes a transmembrane region of a wild-type EGFR receptor. As a complete domain that binds to an anti-EGFR antibody, non-functional EGFR can be used as a suicide marker for transgenic lymphocytes of the present invention. The expression of non-functional EGFR, combined with the expression of chimeric antigen receptors, and further combined with the silencing of cellular immunological checkpoints, can effectively ensure the targeted killing effect of transgenic lymphocytes, if the patient has serious adverse reactions, transgenic lymphocytes The cells can be cleared by the anti-EGFR antibody, which can further improve the safety of the transgenic lymphocytes of the embodiments of the present invention for 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的细胞免疫检查点被沉默和表达无功能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，上述淋巴细胞的细胞免疫杀伤作用的靶向性更强，在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著，安全性更高。According to an embodiment of the invention, the lymphocytes are CD3 + T lymphocytes or natural killer cells or natural killer T cells. The cellular immunological checkpoint of the above lymphocytes of the present invention is silenced and expresses non-functional EGFR, while expressing an antigen-specific chimeric antigen receptor, such as the MSLN antigen-specific chimeric antigen receptor of the present invention. The above-mentioned lymphocyte cell killing effect is more targeted, and the proliferation and viability of the tumor patient are further improved, and the targeted killing effect on the tumor is more significant and safer.
在本发明的第九方面，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；第二核酸分子，所述第二核酸分子沉默细胞免疫检查点，以及第三核酸分子，所述第三核酸分子编码无功能EGFR。其中，所述细胞免疫检查点、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，可有效沉默细胞表面或细胞内至少之一免疫检查点和表达无功能EGFR以及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，安全性高。In a ninth aspect of the invention, the invention proposes a construct. According to an embodiment of the invention, the construct comprises: a first nucleic acid molecule encoding a chimeric antigen receptor; a second nucleic acid molecule, the second nucleic acid molecule silencing a cellular immune checkpoint, and A nucleic acid molecule encoding a non-functional EGFR. Wherein, the cellular immune checkpoint, the chimeric antigen receptor, and the non-functional EGFR are as described above. According to an embodiment of the present invention, the construct of the embodiment of the present invention can effectively silence at least one of the immunological checkpoints on the cell surface or in the cell, and express non-functional EGFR and express antigen-specificity after successfully introducing the lymphocytes of the embodiment of the present invention. The chimeric antigen receptor, so that the lymphocytes of the embodiments of the present invention have more targeted 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-described 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和所述第三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、沉默细胞免疫检查点和表达无功能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和第三核酸分子的淋巴细胞，其淋巴细胞的细胞表面或细胞内至少之一的免疫检查点被成功沉默并在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，如本发明实施例的MSLN特异性的嵌合抗原受体，其具有杀伤力更强和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 first nucleic acid molecule and the second nucleic acid molecule and the third nucleic acid molecule are disposed in a lymphocyte as described above to express the chimeric antigen receptor, and silence The cellular immune checkpoint and expression of non-functional EGFR, and the chimeric antigen receptor is in a non-fused form with the non-functional EGFR. According to an embodiment of the present invention, the lymphocytes of the first nucleic acid molecule and the second nucleic acid molecule and the third nucleic acid molecule are successfully set, and the immune checkpoint of at least one of the cell surface or the cell of the lymphocyte is successfully silenced and The surface of lymphocytes successfully expressed non-functional EGFR, and the antigen specificity was successfully expressed on the surface of lymphocytes, such as the MSLN-specific chimeric antigen receptor of the present invention, which has a more lethal and specific tumor. The killing effect is more secure.
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；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；以及第三启动子，所述第三启动子与所述第三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和第三启动子的引入，使得第一核酸分子以及第二核酸分子和第三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沉默了细胞的免疫检查点和表达了无功能EGFR，使得本发明实施例的淋巴细胞的靶向作用更强，对肿瘤的杀伤作用，尤其对高表达MSLN的肿瘤细胞的定向杀伤更加显著，安全性更高。According to an embodiment of the invention, the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; a second promoter, the second promoter and The second nucleic acid molecule is operably linked; and a third promoter operably linked to the third nucleic acid molecule. According to an embodiment of the present invention, the introduction of the first promoter and the second and third promoters enables the first nucleic acid molecule and the second nucleic acid molecule and the third nucleic acid molecule to be independently expressed, respectively, thereby effectively ensuring the chimeric antigen receptor The biological role of antigen targeting and the effective silencing of the cell's immune checkpoint and the expression of non-functional EGFR make the lymphocyte targeting effect of the embodiment of the present invention stronger, and the killing effect on the tumor, especially for high expression. The targeted killing of tumor cells of MSLN is more significant and safer.
根据本发明的实施例，所述第一启动子、所述第二启动子和所述第三启动子分别独立地选自U6，H1,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，从而保证了细胞免疫检查点的高效沉默和无功 能EGFR的高效表达以及嵌合抗原受体的高效表达，从而本发明实施例的淋巴细胞的体外增殖能力、在肿瘤病人体内的增殖和生存能力大大提高，对肿瘤的定向杀伤效果更加显著，安全性更高。According to an embodiment of the invention, the first promoter, the second promoter and the third promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, LTR, RSV promoters. According to an embodiment of the present invention, the above promoter of the embodiment of the invention has the characteristics of high activation efficiency and strong specificity, thereby ensuring efficient silencing and reactive power of the cellular immune checkpoint. High-efficiency expression of EGFR and high-efficiency expression of chimeric antigen receptor, so that the lymphocyte proliferation ability and proliferation and survival ability of the lymphocytes in the embodiment of the invention are greatly improved, and the targeted killing effect on the tumor is more remarkable and safe. More sexual.
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：144所示的核苷酸序列。According to an embodiment of the present invention, the construct further comprises: an internal ribosome entry site sequence, the internal ribosome entry site sequence being disposed between the first nucleic acid molecule and the third nucleic acid molecule, The internal ribosome entry site has the nucleotide sequence set forth in SEQ ID NO:144.
Figure PCTCN2017081272-appb-000023
Figure PCTCN2017081272-appb-000023
内部核糖体进入位点序列的引入使得第一核酸分子和第三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 the first nucleic acid molecule and the third nucleic acid molecule to be expressed independently, respectively. According to an embodiment of the present invention, the introduction of an internal ribosome entry site sequence ensures the biological action of the chimeric antigen receptor antigen targeting and the high expression of non-functional EGFR, thereby enabling lymphocytes of the embodiments of the present invention to tumor The targeted killing effect is more pronounced, and lymphocytes are safer for tumor killing.
根据本发明的实施例，所述构建体进一步包括：第四核酸分子，设置在所述第一核酸分子与所述第三核酸分子之间，并且所述第四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。所述连接肽具有SEQ ID NO：145所示的氨基酸序列。According to an embodiment of the present invention, the construct further comprises: a fourth nucleic acid molecule disposed between the first nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encoding a linker peptide, The linker peptide is capable of being cleaved in the lymphocytes. The linker peptide has the amino acid sequence set forth in SEQ ID NO:145.
Figure PCTCN2017081272-appb-000024
Figure PCTCN2017081272-appb-000024
第四核酸分子及其相应表达的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，本发明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，其具特异性更强的肿瘤杀伤效果，安全性更高。The introduction of the fourth nucleic acid molecule and its correspondingly expressed linker peptide allows the non-functional EGFR and chimeric antigen receptor to be expressed in a non-fusion state on the lymphocyte membrane. According to an embodiment of the present invention, the introduction of the linker peptide of the embodiment of the present invention ensures the biological effects of the non-functional EGFR and the chimeric antigen receptor, and has 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，从而大大提高了细胞免疫检查点的沉默和无功能EGFR的表达以及嵌合抗原受体在淋巴细胞中的高效表达，使得淋巴细胞体外增殖能力、在肿瘤病人体内的增殖和生存能力大大提高，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，安全性进一步提高。According to an embodiment of the invention, the vector of the construct is a non-pathogenic viral vector. The introduction of a non-pathogenic viral vector greatly enhances the replication and amplification efficiency of the construct in lymphocytes, thereby greatly increasing the silencing of cellular immune checkpoints and the expression of non-functional EGFR and chimeric antigen receptors in lymphocytes. The high-efficiency expression of lymphocytes greatly enhances the proliferation of lymphocytes in vitro, the proliferation and viability of tumor patients, and the targeting of lymphocytes is further increased. Strong, the killing effect on tumor cells is more significant, 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，细胞免疫检查点被高效沉默和无功能EGFR在淋巴细胞中高效表达以及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体外增殖能力、在肿瘤病人体内的增殖和生存能力大大提高，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 invention, the viral vector comprises at least one selected from the group consisting of a retroviral vector, a lentiviral vector or an adenovirus-associated viral vector. The virus carrier of the embodiment of the invention has a wide range of virus infection during virus packaging and infection, and can infect both terminally differentiated cells and cells in a mitotic phase, and the genome can be integrated into the host chromosome or free. Beyond the host chromosome, a broad-spectrum and efficient infection efficiency can be achieved, the cellular immune checkpoint is efficiently silenced and the expression of non-functional EGFR is highly expressed in lymphocytes, and the chimeric antigen receptor is highly expressed in lymphocytes, making this The in vitro proliferation ability of the lymphocytes of the invention, the proliferation and viability of the tumor patients are greatly improved, the targeting effect of lymphocytes is further enhanced, and the targeted killing effect on tumor cells, especially tumor cells with high expression of MSLN, is more remarkable. The killing effect of lymphocytes is safer.
在本发明的第十方面，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，实现了淋巴细胞的细胞免疫检查被沉默和表达无功能EGFR以及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淋巴细胞在肿瘤病人体内和体外的增殖及肿瘤病人体内存活能力大大提高，转基因淋巴细胞或T淋巴细胞对肿瘤细胞，尤其是对高表达MSLN的肿瘤细胞的靶向杀伤作用更强，安全性更高。In a tenth aspect of the invention, the invention provides a method of preparing the aforementioned T lymphocytes or transgenic lymphocytes. According to an embodiment of the invention, the method comprises introducing the aforementioned construct or the lentivirus described above into lymphocytes or T lymphocytes. The construct or the lentivirus is successfully introduced into the lymphocyte or the T lymphocyte, and the cellular immunological examination of the lymphocyte is silenced and the expression of the non-functional EGFR and the chimeric antigen receptor is expressed, thereby preparing the method of the present invention. The prepared transgenic lymphocytes or T lymphocytes can greatly proliferate in vivo and in vitro of tumor patients and the survival ability of tumor patients, and the targeted killing of tumor cells, especially tumor cells with high expression of MSLN, by transgenic lymphocytes or T lymphocytes More powerful and safer.
在本发明的第十一方面，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淋巴细胞的细胞表面或细胞内免疫检查点的沉默和无功能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淋巴细胞具有显著的抵抗肿瘤细胞介导的免疫抑制，在肿瘤病人体外和体内的增殖及肿瘤病人体内存活能力大大提高，转基因淋巴细胞或T淋巴细胞对肿瘤细胞的靶向杀伤作用更强，本发明实施例的治疗癌症的治疗组合物对肿瘤细胞的靶向杀伤作用显著增强，尤其是对高表达MSLN的肿瘤细胞的靶向杀伤作用显著增强、安全性进一步提高。In an eleventh aspect of the invention, the invention provides a therapeutic composition for treating cancer. According to an embodiment of the invention, the therapeutic composition comprises: the above construct, lentivirus, T lymphocyte or transgenic lymphocyte. The composition of any of the above therapeutic compositions can achieve silencing of cell surface or intracellular immunological checkpoints of transgenic lymphocytes or T lymphocytes and expression of non-functional EGFR and chimeric antigen receptors in transgenic lymphocytes or T lymphocytes Highly expressed, so that the resulting transgenic lymphocytes or T lymphocytes have significant resistance to tumor cell-mediated immunosuppression, and the proliferation of tumor patients in vitro and in vivo and the survival ability of tumor patients are greatly improved, transgenic lymphocytes or T lymphocytes The targeted killing effect of the cells on tumor cells is stronger, and the targeted killing effect of the therapeutic composition for treating cancer of the present invention on tumor cells is remarkably enhanced, especially the targeted killing effect on tumor cells with high expression of MSLN is significantly enhanced. Safety is further improved.
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 invention, the above therapeutic composition may further comprise 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或细胞内免疫检查点的沉默和表达无功能EGFR以及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淋巴细胞具有显著的抵抗肿瘤细胞介导的免疫抑制的特性，在间皮瘤的微环境中的存活能力大大提高，所得淋巴细胞或T淋巴细胞对高表达MSLN的 肿瘤细胞的靶向杀伤作用更强、安全性更高。According to an embodiment of the present invention, the cancer comprises at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer. The above tumor cells have high specific expression of MSLN, and the therapeutic composition of the present invention can silence and express non-functional EGFR on lymphocyte cell surface or intracellular immunological checkpoint and efficiently express antigen-specific chimeric antigen receptor, According to the MSLN antigen-specific chimeric antigen receptor of the embodiment of the present invention, the obtained lymphocyte or T lymphocyte has remarkable characteristics against tumor cell-mediated immunosuppression, and the viability in the microenvironment of mesothelioma is greatly improved. , the resulting lymphocytes or T lymphocytes are highly expressed in MSLN The targeted killing effect of tumor cells is stronger and the safety is higher.
在本发明的第十二方面，本发明提出了一种提高淋巴细胞活性和治疗安全性的方法，所述淋巴细胞携带嵌合抗原受体，根据本发明的实施例，所述方法包括：使所述淋巴细胞的细胞免疫检查点被沉默，以及使所述淋巴细胞表达无功能EGFR。所述细胞免疫检查点、所述淋巴细胞、所述嵌合抗原受体和所述无功能EGFR是如前所定义的，所述淋巴细胞活性包括所述淋巴细胞体外增殖能力、在肿瘤病人体内的增殖和生存能力以及所述淋巴细胞在肿瘤病人体内的定向杀伤能力的至少一种。根据本发明的实施例，本发明实施例的淋巴细胞的细胞表面或细胞内免疫检查点被沉默，淋巴细胞被活化、增生性反应上调、细胞因子分泌增多、抗调亡能力增强，使得本发明实施例的淋巴细胞在体外扩增、在肿瘤病人体内的增殖及肿瘤病人体内存活能力大大提高上述淋巴细胞细胞免疫检查点的沉默配合淋巴细胞嵌合抗原受体的抗原特异性功效，从而实现了有效抵抗肿瘤细胞介导的免疫抑制，对高表达MSLN的肿瘤细胞的靶向杀伤作用显著增强。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受体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。本发明是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 a twelfth aspect of the invention, the invention provides a method of increasing lymphocyte activity and therapeutic safety, said lymphocyte carrying a chimeric antigen receptor, according to an embodiment of the invention, said method comprising: The cellular immune checkpoint of the lymphocytes is silenced and the lymphocytes are expressed as non-functional EGFR. The cellular immune checkpoint, the lymphocyte, the chimeric antigen receptor, and the non-functional EGFR are as defined above, and the lymphocyte activity comprises the ability of the lymphocyte to proliferate in vitro, in a tumor patient The proliferation and viability and at least one of the directional killing ability of the lymphocytes in a tumor patient. According to an embodiment of the present invention, the cell surface or intracellular immune checkpoint of lymphocytes according to the embodiment of the present invention is silenced, lymphocytes are activated, proliferative responses are up-regulated, cytokine secretion is increased, and anti-apoptotic ability is enhanced, so that the present invention The lymphocyte expansion of the embodiment in vitro, proliferation in a tumor patient, and survival ability in a tumor patient greatly enhance the silencing of the lymphocyte cell immunological checkpoint and the antigen-specific efficacy of the lymphocyte chimeric antigen receptor, thereby realizing Effectively resisting tumor cell-mediated immunosuppression, the targeted killing effect on tumor cells with high expression of MSLN is significantly enhanced. Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR receptor and intact sequence that binds to anti-EGFR antibody, and non-functional EGFR can act as lymph Cell suicide markers. When the lymphocytes of the present invention are used for the treatment of tumor cells with high expression of MSLN, if the patient develops a serious adverse reaction, the lymphocytes of the embodiments of the present invention can be cleared by the anti-EGFR antibody, thereby improving the embodiment of the present invention. Lymphocyte treatment for the safety of tumor patients with high expression of MSLN.
在本发明的第十三方面，本发明提出了一种治疗癌症的方法。根据本发明的实施例，所述方法包括：为癌症患者给药前面所述的构建体、前面所述的慢病毒、前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞，其中，嵌合抗原受体特异性结合肿瘤抗原MSLN。利用本发明实施例的治疗癌症的方法，可有效实现对癌症患者肿瘤细胞的靶向杀伤，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，进而能够有效治疗癌症，治疗效果好且安全性高。In a thirteenth aspect of the invention, the invention provides a method of treating cancer. According to an embodiment of the invention, the method comprises: administering to a cancer patient a construct as described above, a lentivirus as described above, a T lymphocyte as described above or a transgenic lymphocyte as described above, wherein The antigen receptor specifically binds to the tumor antigen MSLN. The method for treating cancer according to the embodiment of the invention can effectively achieve targeted killing of tumor cells of cancer patients, in particular, has targeted killing effect on tumor cells with high expression of MSLN, thereby effectively treating cancer, and the therapeutic effect is good and High security.
根据本发明的实施例，上述治疗癌症的方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above method for treating cancer may further comprise at least one of the following additional technical features:
根据本发明的实施例，所述方法包括：从癌症患者体内分离淋巴细胞；将前面所述的构建体，或前面所述的慢病毒导入所述淋巴细胞，以便获得转基因淋巴细胞，所述转基因淋巴细胞表达嵌合抗原受体和细胞免疫检查点被沉默；以及为所述癌症患者给药所述转基因淋巴细胞。利用本发明实施例的治疗癌症的方法，可进一步有效实现对癌症患者肿瘤细胞的靶向杀伤，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，进而能够进一步有效治疗癌症，治疗效果好且安全性高。According to an embodiment of the present invention, the method comprises: isolating lymphocytes from a cancer patient; introducing the aforementioned construct, or the lentivirus described above, into the lymphocytes to obtain transgenic lymphocytes, the transgene The lymphocyte expressing chimeric antigen receptor and the cellular immune checkpoint are silenced; and the transgenic lymphocytes are administered to the cancer patient. The method for treating cancer according to the embodiment of the invention can further effectively achieve targeted killing of tumor cells of cancer patients, especially having targeted killing effect on tumor cells with high expression of MSLN, thereby further effectively treating cancer, and the therapeutic effect Good and safe.
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。本发明实施例的治疗癌症的方法可使淋巴细胞细胞免疫检查点被沉默和细胞表达嵌合抗原受体，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MSLN特异性表达的间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌或乳腺癌的肿瘤细胞的靶向杀伤。According to an embodiment of the present invention, the cancer comprises a group selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, stomach At least one of cancer, intestinal cancer, esophageal cancer and breast cancer. The method for treating cancer according to an embodiment of the present invention enables lymphocyte immune checkpoints to be silenced and cells to express chimeric antigen receptors, such as MSLN antigen-specific chimeric antigen receptors of the present invention, resulting lymphocytes or T lymphocytes. The cells have targeted killing of tumor cells of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer or breast cancer which are specifically expressed by MSLN.
需要说明的是，本发明中所使用的术语“细胞免疫检查点”包括细胞表面免疫检查点和细胞内免疫检查点，“细胞表面免疫检查点”是一种淋巴细胞表面的膜蛋白，其与肿瘤细胞上表达的配体相互作用，可以抑制抗肿瘤淋巴细胞反应。“细胞内免疫检查点”是一种细胞内蛋白，此种细胞免疫检查点蛋白一种负调控的细胞信号传导机构，可以抑制抗肿瘤淋巴细胞反应。It should be noted that the term "cell immune checkpoint" as used in the present invention includes a cell surface immunological checkpoint and an intracellular immunological checkpoint, and a "cell surface immunological checkpoint" is a membrane protein on the surface of lymphocytes, which is Ligand interactions expressed on tumor cells can inhibit anti-tumor lymphocyte responses. An "intracellular immune checkpoint" is an intracellular protein that is a negatively regulated cellular signaling machinery that inhibits antitumor lymphocyte responses.
附图说明DRAWINGS
图1是根据本发明实施例的共表达MSLN抗原特异性的嵌合抗原受体和沉默人类细胞免疫检查点以及表达无功能EGFR的慢病毒载体的结构示意图；1 is a schematic diagram showing the structure of a co-expressing MSLN antigen-specific chimeric antigen receptor and a silencing human cell immunological checkpoint and a lentiviral vector expressing a non-functional EGFR according to an embodiment of the present invention;
图2是根据本发明实施例的共表达抗MSLN的嵌合抗原受体、PD1 shRNA和无功能EGFR的淋巴细胞被抗EGFR抗体杀伤清除的结果图；以及2 is a diagram showing the results of killing of lymphocytes co-expressing anti-MSLN chimeric antigen receptor, PD1 shRNA, and non-functional EGFR by anti-EGFR antibody according to an embodiment of the present invention;
图3是根据本发明实施例的共表达MSLN抗原特异性的嵌合抗原受体、PD1 shRNA和无功能EGFR的淋巴细胞杀伤肿瘤细胞能力的结果图。Figure 3 is a graph showing the results of the ability of co-expressing MSLN antigen-specific chimeric antigen receptor, PD1 shRNA and non-functional EGFR lymphocytes to kill tumor cells according to an embodiment of the present invention.
具体实施方式detailed description
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。The embodiments of the present invention are described in detail below, and the embodiments described below are intended to be illustrative of the invention and are not to be construed as limiting.
T淋巴细胞或转基因淋巴细胞T lymphocyte or transgenic lymphocyte
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淋巴细胞的细胞免疫检查点被沉默；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，并且嵌入到所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。其中，细胞免疫检查点包括细胞表面或细胞内至少之一的免疫检查点。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受体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和细胞免疫检查点被沉默，本发明实施例的T淋巴细胞或转基因淋巴细胞在肿瘤病人体内和体外的增殖和生存能力以及在肿瘤病人体内的 对特意性肿瘤细胞的杀伤能力显著增强，尤其对高表达MSLN的肿瘤细胞的特异性杀伤效果大大提高，并且安全性也显著提高。In one aspect of the invention, the invention provides a T lymphocyte or transgenic lymphocyte. According to an embodiment of the present invention, a cellular immune checkpoint of a T lymphocyte according to an embodiment of the present invention is silenced; a non-functional EGFR is expressed; and a chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region, a cell The outer region includes the heavy chain variable region and the light chain variable region of the single-chain antibody, the single-chain antibody specifically recognizes the antigen MSLN; the transmembrane region, the transmembrane region is linked to the extracellular region, and is embedded in the cell membrane of the T lymphocyte. The intracellular region, the intracellular region is linked to the transmembrane region, and the intracellular region includes the intracellular portion of CD28 or 4-1BB and the CD3 ζ chain. Wherein, the cellular immune checkpoint includes an immune checkpoint on at least one of a cell surface or a cell. Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR receptor and intact sequence that binds to anti-EGFR antibody, and non-functional EGFR can act as lymph Cell suicide markers. The T lymphocyte or transgenic lymphocyte cell of the embodiment of the present invention expresses a non-functional EGFR-expressing MSLN antigen-specific chimeric antigen receptor and the cellular immune checkpoint is silenced, and the T lymphocyte or transgenic lymphocyte of the embodiment of the present invention is Proliferation and viability of tumor patients in vivo and in vitro, as well as in tumor patients The killing ability of the specific tumor cells is remarkably enhanced, especially the specific killing effect on the tumor cells with high expression of MSLN is greatly improved, and the safety is also remarkably improved.
肿瘤可以避免免疫监视，通过刺激其免疫抑制性受体的表达而关闭淋巴细胞对其的免疫杀伤反应；作为免疫负调节机制，激活的细胞毒性T淋巴细胞(CTLs)也表达负调控的监管机构,即细胞表面或细胞内的免疫检查点分子。如本发明实施例的程序性细胞死亡1受体(PD-1)表达在活化CTLs上，其与肿瘤细胞上表达的程序性死亡配体1(PD-L1)相互作用,可以抑制抗肿瘤T细胞反应。许多肿瘤表达PD-L1。PD-L1与其配体PD-1的结合，导致CTLs增生性反应的下调,细胞因子的分泌减少，和T细胞的无能或凋亡。本发明实施例的细胞毒性T淋巴细胞抗原4(CTLA-4)是另一个T细胞的关键负面调节因子，其可抑制T细胞活化，其通过与表达在抗原递呈细胞上的配体B7.1、B7.2(CD80和CD86)的相互作用而抑制T细胞的活化。本发明实施例的细胞毒性T淋巴细胞内的CBL-B(E3泛素蛋白连接酶CBL-B)是细胞内的另一个关键负面调节因子，其通过抑制T细胞受体(TCR)信号传导，来抑制T细胞的活性。因此，本发明实施例的T淋巴细胞或转基因淋巴细胞的免疫检查点被沉默，T淋巴细胞或转基因淋巴细胞的在肿瘤病人体内的增殖和生存能力显著提高。Tumors can avoid immune surveillance, shutting down the immune killing response of lymphocytes by stimulating the expression of their immunosuppressive receptors; as a negative immunoregulatory mechanism, activated cytotoxic T lymphocytes (CTLs) also express negative regulatory regulators. , that is, the immune checkpoint molecule on the cell surface or inside the cell. The programmed cell death 1 receptor (PD-1), as in the embodiment of the present invention, is expressed on activated CTLs, which interact with the programmed death ligand 1 (PD-L1) expressed on tumor cells to inhibit anti-tumor T Cellular response. Many tumors express PD-L1. The binding of PD-L1 to its ligand PD-1 results in down-regulation of proliferative responses to CTLs, decreased secretion of cytokines, and inability or apoptosis of T cells. The cytotoxic T lymphocyte antigen 4 (CTLA-4) of the present invention is a key negative regulator of another T cell, which inhibits T cell activation by binding to a ligand B7 expressed on antigen presenting cells. 1. The interaction of B7.2 (CD80 and CD86) inhibits the activation of T cells. CBL-B (E3 ubiquitin protein ligase CBL-B) in cytotoxic T lymphocytes of the present invention is another key negative regulator in cells by inhibiting T cell receptor (TCR) signaling, To inhibit the activity of T cells. Therefore, the immunological checkpoint of the T lymphocyte or the transgenic lymphocyte of the embodiment of the present invention is silenced, and the proliferation and viability of the T lymphocyte or the transgenic lymphocyte in the tumor patient are remarkably improved.
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受体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。无功能EGFR表达淋巴细胞可被抗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。Further, according to an embodiment of the present invention, the non-functional EGFR of the present invention lacks an N-terminal ligand binding region and an intracellular receptor tyrosine kinase activity, but includes a transmembrane region of the wild type EGFR receptor and is intact. A sequence that binds to an anti-EGFR antibody, a non-functional EGFR can serve as a suicide marker for lymphocytes. Non-functional EGFR-expressing lymphocytes can be cleared in vivo by anti-EGFR antibodies. Thus, the T lymphocytes or transgenic lymphocytes of the embodiments of the present invention express non-functional EGFR. Under the premise of ensuring the targeted killing effect of the transgenic lymphocytes, if the patient has serious adverse reactions, the transgenic lymphocytes can be cleared by the anti-EGFR antibody. Further, the safety of the transgenic lymphocytes or T lymphocytes of the embodiments of the present invention for treating tumor patients with high expression of MSLN 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，大大提高了转基因淋巴细胞对靶向肿瘤细胞的定向杀伤作用。Further, according to an embodiment of the present invention, the antibody of the chimeric antigen receptor extracellular region is a single chain antibody. The inventors have found that single-chain antibodies can remove non-specifically reactive surface proteins while single-chain antibodies are more permeable to tumor tissue to increase drug treatment concentrations. The transgenic lymphocytes of the embodiments of the present invention express the chimeric antigen receptor of the single-chain antibody, which greatly enhances the targeted 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高表达抗原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，大大提高了本发明实施例的转基因淋巴细胞对MSLN抗原高表达肿瘤细胞的定向杀伤作用。According to still further embodiments of the present invention, the binding antigen of the above antibody is MSLN. Therefore, the transgenic lymphocytes of the embodiments of the present invention have a directional killing effect on cells highly expressing the antigen MSLN, and the specific binding effect of the antigen-antibody is stronger, and the transgenic lymphocytes of the embodiment of the present invention have high expression of tumor cells to the MSLN antigen. Directional killing effect.
根据本发明的另外一些实施例，淋巴细胞的细胞免疫检查点包括细胞表面和细胞内的免疫检查点，本发明实施例的淋巴细胞细胞表面免疫检查点独立地选自CTLA4、PD-1、TIM-3、BTLA、LAG-3至少之一，淋巴细胞细胞内免疫检查点独立地选自IRAK-M、SOCS-1、A20、 CBL-B的至少之一。上述分子能够与肿瘤细胞表达的抗原特异性结合，抑制淋巴细胞的活化，促进淋巴细胞的无能或凋亡，从而负向调控和减弱细胞免疫应答。根据本发明的实施例，上述细胞表面或细胞内免疫检查点的成功沉默，进一步提高了转基因淋巴细胞在肿瘤病人体内的增殖和生存能力，对肿瘤细胞的定向杀伤作用进一步加强。According to still other embodiments of the present invention, the cellular immune checkpoint of lymphocytes includes a cell surface and an intracellular immunological checkpoint, and the lymphocyte cell surface immunological checkpoint of the embodiment of the present invention is independently selected from the group consisting of CTLA4, PD-1, TIM. -3, at least one of BTLA and LAG-3, the lymphocyte intracellular immune checkpoint is independently selected from IRAK-M, SOCS-1, A20, At least one of CBL-B. The above molecules can specifically bind to antigens expressed by tumor cells, inhibit lymphocyte activation, promote lymphocyte incompetence or apoptosis, thereby negatively regulating and attenuating cellular immune responses. According to an embodiment of the present invention, the successful silencing of the above-mentioned cell surface or intracellular immune checkpoint further improves the proliferation and viability of the transgenic lymphocytes in the tumor patient, and further enhances the directed killing effect on the tumor cells.
根据本发明的另外一些实施例，本发明实施例的淋巴细胞细胞免疫检查点被沉默是通过shRNA、反义核酸、核酶、显性负突变、锌指核酸酶和CRISPR至少之一实现的。According to further embodiments of the present invention, silencing of a lymphocyte immune checkpoint according to an embodiment of the present invention is achieved by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, zinc finger nuclease, and CRISPR.
小发夹RNA或短发夹RNA(shRNA)是siRNA(小干扰RNA)的导入形式，siRNA是一种小RNA分子(由21～25个核苷酸组成)，由Dicer(RNAaseⅢ家族中对双链RNA具有特异性剪切作用的酶)加工而成；siRNA在RNA沉默通路中起中心作用，对特定信使RNA(mRNA)进行降解，为转录水平后调控。Small hairpin RNA or short hairpin RNA (shRNA) is a introduced form of siRNA (small interfering RNA). siRNA is a small RNA molecule (composed of 21-25 nucleotides), which is composed of Dicer (pair of RNAase III family). The RNA of the stranded RNA has a specific cleavage effect; the siRNA plays a central role in the RNA silencing pathway, degrading specific messenger RNA (mRNA) and regulating it at the transcriptional level.
反义核酸包括反义RNA和反义DNA，反义RNA是指能和mRNA完全互补的一段小分子RNA或寡聚核苷酸片段，反义DNA是指能与基因DNA双链中的有义链互补结合的短小DNA分子，反义RNA和反义DNA主要是通过mRNA的翻译和基因DNA的转录而发挥作用的；反义核酸一方面通过与靶mRNA结合形成空间位阻效应，阻止核糖体与mRNA结合，另一方面其与mRNA结合后激活内源性RNA酶或核酶，进而降解mRNA；反义DNA与基因DNA双螺旋的调控区特异结合形成DNA三聚体，或与DNA编码区结合，终止正在转录的mRNA链的延长；反义核酸还可抑制转录后mRNA的加工修饰，如5'端加帽、3'端加尾、中间剪接和内部碱基甲基化等，并阻止成熟mRNA由细胞核向细胞浆内运输，因此，反义RNA是一种有效的沉默目的基因的技术。Antisense nucleic acids include antisense RNA and antisense DNA. Antisense RNA refers to a small RNA or oligonucleotide fragment that is fully complementary to mRNA. Antisense DNA refers to the sense of being in the double strand of the gene DNA. Short DNA molecules with complementary strands, antisense RNA and antisense DNA mainly function through translation of mRNA and transcription of gene DNA; antisense nucleic acid prevents ribosome by forming steric hindrance effect by binding to target mRNA Binding to mRNA, on the other hand, binding to mRNA activates endogenous RNase or ribozyme, which in turn degrades mRNA; antisense DNA specifically binds to the regulatory region of the double helix of the gene DNA to form a DNA trimer, or with a DNA coding region Binding, termination of the elongation of the mRNA strand being transcribed; antisense nucleic acids also inhibit processing modifications of post-transcriptional mRNA, such as 5' end capping, 3' end tailing, intermediate splicing, and internal base methylation, etc. Mature mRNA is transported from the nucleus to the cytoplasm. Therefore, antisense RNA is an effective technique for silencing the gene of interest.
核酶是具有催化功能的RNA分子，是生物催化剂，可降解特异的mRNA序列，核酶通过催化转磷酸酯和磷酸二酯键水解反应参与RNA自身剪切、加工过程，与一般的反义RNA相比，核酶具有较稳定的空间结构，不易受到RNA酶的攻击，更重要的是，核酶在切断mRNA后，又可从杂交链上解脱下来，重新结合和切割其它的mRNA分子。Ribozyme is a catalytically active RNA molecule that is a biocatalyst that degrades specific mRNA sequences. The ribozyme participates in RNA self-cleavage and processing by catalyzing the hydrolysis of transphosphate and phosphodiester bonds, and general antisense RNA. In contrast, ribozymes have a relatively stable spatial structure and are not susceptible to RNase attack. More importantly, ribozymes can be detached from the hybridization chain and then re-bound and cleave other mRNA molecules.
显性负性突变是指某些信号转导蛋白突变后不仅自身无功能，还能抑制或阻断同一细胞内的野生型信号转导蛋白的作用，其主要通过和野生型蛋白形成二聚物的方式实现，这种突变毒性作用大，能显著抑制或阻断细胞内目标信号转导蛋白的作用。Dominant negative mutations are those in which certain signal transduction proteins are not only self-functional but also inhibit or block the action of wild-type signal transduction proteins in the same cell, mainly by forming dimers with wild-type proteins. The way to achieve this mutation is toxic and can significantly inhibit or block the action of intracellular target signal transduction proteins.
锌指核酸酶由一个DNA识别域和一个非特异性核酸内切酶构成，DNA识别域是由一系列Cys2-His2锌指蛋白串联组成(一般3～4个)，每个锌指蛋白识别并结合一个特异的三联体碱基，锌指蛋白形成α-β-β二级结构，其中α螺旋的16氨基酸残基决定锌指的DNA结合特异性，骨架结构保守，对决定DNA结合特异性的氨基酸引入序列的改变可以获得新的DNA结合特异性，从而可以针对不同的目的基因设计不同的氨基酸引入序列，实现不同目的基因的特异性沉默。 The zinc finger nuclease consists of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain is composed of a series of Cys2-His2 zinc finger proteins in series (generally 3 to 4). Each zinc finger protein recognizes and binds. A specific triplet base, zinc finger protein forms the α-β-β secondary structure, wherein the 16 amino acid residues of the α helix determine the DNA binding specificity of the zinc finger, the skeleton structure is conserved, and the amino acid determining the DNA binding specificity The introduction of sequence changes can obtain new DNA binding specificity, so that different amino acid introduction sequences can be designed for different genes of interest to achieve specific silencing of different genes of interest.
CRISPR(Clustered regularly interspaced short palindromic repeats规律成簇间隔短回文重复)，是一种基因编辑器，是细菌用以保护自身对抗病毒的一个系统。它可以用来删除、添加、激活或抑制其他生物体的目标基因，这些目标基因包括人细胞内的目标基因。CRISPR (Clustered regular interspaced short palindromic repeats) is a gene editor, a system used by bacteria to protect itself against viruses. It can be used to delete, add, activate or inhibit target genes of other organisms, including target genes in human cells.
CRISPR簇是一个广泛存在于细菌和古生菌基因组中的特殊DNA重复序列家族，其序列由一个前导区(Leader)、多个短而高度保守的重复序列(Repeat)和多个间隔区(Spacer)组成。前导区一般位于CRISPR簇上游，是富含AT长度为300～500bp的区域，被认为可能是CRISPR簇的启动子序列。重复序列区长度为21～48bp，含有回文序列，可形成发卡结构。重复序列之间被长度为26～72bp的间隔区隔开。Spacer区域由俘获的外源DNA组成，当含有同样序列的外源DNA入侵时，可被细菌机体识别，并进行剪切使之表达沉默，达到保护自身安全的目的。通过对CRISPR簇的侧翼序列分析发现，在其附近存在一个多态性家族基因。该家族编码的蛋白质均含有可与核酸发生作用的功能域(具有核酸酶、解旋酶、整合酶和聚合酶等活性)，并且与CRISPR区域共同发挥作用，因此被命名为CRISPR关联基因(CRISPR associated)，缩写为Cas。目前发现的Cas包括Cas1～Cas10等多种类型。Cas基因与CRISPR共同进化，共同构成一个高度保守的系统。当细菌抵御噬菌体等外源DNA入侵时，在前导区的调控下，CRISPR被转录为长的RNA前体(Pre RISPR RNA，pre-crRNA)，然后加工成一系列短的含有保守重复序列和间隔区的成熟crRNA，最终识别并结合到与其互补的外源DNA序列上发挥剪切作用。pre-crRNA的加工由Cas家族中的Cas9参与。Cas9含有在氨基末端的RuvC和蛋白质中部的HNH2个独特的活性位点，在crRNA成熟和双链DNA剪切中发挥作用。pre-crRNA转录的同时，与其重复序列互补的反式激活crRNA(Trans-activating crRNA，tracrRNA)也转录出来，并且激发Cas9和双链RNA特异性RNase III核酸酶对pre-crRNA进行加工。加工成熟后，crRNA、tracrRNA和Cas9组成复合体，识别并结合于crRNA互补的序列，然后解开DNA双链，形成R-loop，使crRNA与互补链杂交，另一条链保持游离的单链状态，然后由Cas9中的HNH活性位点剪切crRNA的互补DNA链，RuvC活性位点剪切非互补链，最终引入DNA双链断裂(DSB)。通过人工设计RNA，可以改造形成具有引导作用的sgRNA(short guide RNA)，足以引导Cas9对DNA的定点目标基因切割。The CRISPR cluster is a family of specific DNA repeats that are widely found in the genomes of bacteria and archaea. The sequence consists of a leader, multiple short and highly conserved repeats, and multiple spacers (Spacer). )composition. The leader region is generally located upstream of the CRISPR cluster and is a region rich in AT length of 300-500 bp, which is considered to be a promoter sequence of the CRISPR cluster. The repeat sequence region has a length of 21 to 48 bp and contains a palindromic sequence, which can form a hairpin structure. The repeat sequences are separated by a spacer of length 26 to 72 bp. The Spacer region is composed of captured foreign DNA. When the foreign DNA containing the same sequence is invaded, it can be recognized by the bacteria organism and cut to silence the expression to protect itself. By analyzing the flanking sequence of the CRISPR cluster, it was found that there is a polymorphic family gene in its vicinity. The proteins encoded by this family contain functional domains (having activities such as nuclease, helicase, integrase, and polymerase) that interact with nucleic acids, and interact with the CRISPR region, and are therefore named CRISPR-related genes (CRISPR). Associated), abbreviated as Cas. Currently discovered Cas includes many types such as Cas1 to Cas10. The Cas gene and CRISPR have evolved together to form a highly conservative system. When the bacteria resist invasion by foreign DNA such as phage, CRISPR is transcribed into a long RNA precursor (Pre RISPR RNA, pre-crRNA) under the control of the leader region, and then processed into a series of short conserved repeats and spacers. The mature crRNA ultimately recognizes and binds to its complementary foreign DNA sequence to exert a cleavage effect. Processing of pre-crRNA is involved by Cas9 in the Cas family. Cas9 contains two unique active sites, RuvC at the amino terminus and HNH in the middle of the protein, which play a role in crRNA maturation and double-strand DNA cleavage. At the same time as the pre-crRNA is transcribed, trans-activating crRNA (tracrRNA) complementary to its repeat sequence is also transcribed, and Cas9 and double-stranded RNA-specific RNase III nuclease are excited to process pre-crRNA. After processing, the crRNA, tracrRNA and Cas9 complexes, recognize and bind to the complementary sequence of crRNA, then unwind the DNA double strand to form R-loop, which makes the crRNA hybridize with the complementary strand, and the other strand maintains the free single-stranded state. The complementary DNA strand of the crRNA is then cleaved by the HNH active site in Cas9, and the RuvC active site cleaves the non-complementary strand, eventually introducing a DNA double-strand break (DSB). By artificially designing RNA, it is possible to engineer a sgRNA (short guide RNA) sufficient to guide Cas9 to the targeted gene cleavage of DNA.
综上所述，shRNA、反义核酸、核酶、显性负突变、CRISPR锌指核酸酶为特异性沉默目标基因的有效手段，沉默基因的手段不受特别限制，本领域技术人员可根据具体的实验目的和条件选择，如本发明实施例所采用的shRNA、反义核酸、核酶、显性负突变，CRISPR或锌指核酸酶的至少之一，实现目的基因的特异性沉默。根据本发明的实施例，淋巴细胞细胞表面或细胞内免疫检查点被沉默优选采用shRNA实现。ShRNA所携带的siRNA分子通常是一个长度在10和30之间的碱基对的双重区域。本发明实施例的PD1或CTLA4或CBL-B siRNA被设计为同源于PD1或CTLA4或CBL-B mRNA的编码区域，通过mRNA 的降解来抑制基因表达。siRNA关联于被称为诱导RNA沉默复合物(RISC)的多重蛋白复合物，在此期间正义链被酶裂解。被激活的RISC中基于序列同源性，指引RISC到对应的mRNA；相同的核酸酶切割靶向PD1或CTLA4或CBL-B mRNA，产生特定基因PD1或CTLA4或Cb1沉默，抑制特定基因PD1或CTLA4或CBL-B的表达。siRNA以shRNA的形式导入细胞(shRNA包含大约18-23的核苷酸siRNA序列，后跟一个9-15长度的核苷酸环和一个siRNA序列的反向补充)，shRNA的设计较好的避免了在3’UTR细胞基因中的匹配点；确保了适当的链选择。一个单一siRNA分子可被重复应用于多靶向mRNA分子的分裂。RNAi(RNA干扰)可通过引入合成siRNA的方式被诱导。根据本发明的实施例，本发明实施例的shRNA不断产自细胞内，因此其效果更加持久，从而延长shRNA周期，本发明实施例采用的shRNA具有高效、特异性的沉默细胞表面或细胞内免疫检查点的作用，细胞表面或细胞内免疫检查点的成功沉默，使得转基因淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的特性，在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著。In summary, the shRNA, the antisense nucleic acid, the ribozyme, the dominant negative mutation, and the CRISPR zinc finger nuclease are effective means for specifically silencing the target gene, and the means for silencing the gene is not particularly limited, and those skilled in the art can The experimental purpose and condition selection, such as at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR or zinc finger nuclease used in the embodiments of the present invention, achieve specific silencing of the target gene. According to an embodiment of the invention, the lymphocyte cell surface or intracellular immunological checkpoint is silenced, preferably with shRNA. The siRNA molecule carried by the ShRNA is typically a dual region of base pairs between 10 and 30 in length. The PD1 or CTLA4 or CBL-B siRNA of the embodiments of the present invention is designed to be homologous to the coding region of PD1 or CTLA4 or CBL-B mRNA, through mRNA Degradation to inhibit gene expression. The siRNA is associated with a multiplex protein complex called the Inducible RNA Silencing Complex (RISC), during which the sense strand is cleaved by the enzyme. Based on sequence homology in activated RISC, direct RISC to the corresponding mRNA; the same nuclease cleaves to target PD1 or CTLA4 or CBL-B mRNA, resulting in specific gene PD1 or CTLA4 or Cb1 silencing, inhibiting specific gene PD1 or CTLA4 Or the expression of CBL-B. The siRNA is introduced into the cell as shRNA (shRNA contains approximately 18-23 nucleotide siRNA sequences followed by a 9-15-length nucleotide loop and a reverse complement of a siRNA sequence), and the shRNA design is better avoided. Matching points in the 3'UTR cell gene; ensuring proper strand selection. A single siRNA molecule can be repeatedly applied to the division of multiple targeting mRNA molecules. RNAi (RNA interference) can be induced by introducing synthetic siRNA. According to an embodiment of the present invention, the shRNA of the embodiment of the present invention is continuously produced from a cell, and thus the effect thereof is more durable, thereby prolonging the shRNA cycle, and the shRNA used in the embodiment of the present invention has a highly efficient and specific silencing cell surface or intracellular immunity. The role of checkpoints, successful silencing of cell surface or intracellular immune checkpoints, makes transgenic lymphocytes significantly resistant to tumor-mediated immunosuppression, and further enhances proliferation and viability in tumor patients. The effect of directional killing is more pronounced.
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和细胞表面或细胞内至少之一免疫检查点的沉默联合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具有显著的抵抗肿瘤介导的免疫抑制的特性，在肿瘤病人体内的增殖和生存能力得到进一步提高，对高表达MSLN的肿瘤的定向杀伤作用效果更加显著，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。Further, according to an embodiment of the present invention, the intracellular segment of the immunocostimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28, and derivatives thereof. The expression of the intracellular segment of the immunostimulatory molecule and the silencing of at least one immunological checkpoint on the cell surface or in the cell have a positive regulation and enhance the cellular immune response, making the transgenic lymphocyte significantly resistant to tumor-mediated immunosuppression. The characteristics of proliferation and viability in tumor patients are further improved, and the targeted killing effect on tumors with high expression of MSLN is more significant. The expression of intracellular segments of immunostimulatory molecules is combined with the expression of non-functional EGFR, making transgenic lymphocytes The immune killing effect is safer and more effective.
根据本发明的另外一些实施例，淋巴细胞细胞表面免疫检查点优选CTLA4或PD1，淋巴细胞内免疫检查点优选CBL-B。根据本发明的时施例，淋巴细胞细胞表面免疫检查点CTLA4或PD1被沉默或细胞内免疫检查点CBL-B被沉默，使得转基因淋巴细胞具有更加显著的抵抗肿瘤介导的免疫抑制的特性，其在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著。According to further embodiments of the invention, the lymphocyte cell surface immunological checkpoint is preferably CTLA4 or PD1, and the intralymphocyte immune checkpoint is preferably CBL-B. According to the embodiment of the present invention, the lymphocyte cell surface immunological checkpoint CTLA4 or PD1 is silenced or the intracellular immune checkpoint CBL-B is silenced, so that the transgenic lymphocytes have more significant resistance to tumor-mediated immunosuppression. Its proliferation and viability in tumor patients are further improved, and the effect of targeted killing of tumors is more significant.
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。上述淋巴细胞中免疫检查点被沉默和表达嵌合抗原受体，使得上述淋巴细胞的细胞免疫的靶向杀伤性更强，对肿瘤细胞的杀伤作用效果更加显著；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细胞免疫杀伤作用更加安全有效。According to an embodiment of the invention, the lymphocytes of the embodiments of the 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 T cell subsets with T cells and natural killer cell receptors. The immunological checkpoint in the above lymphocytes is silenced and expresses the chimeric antigen receptor, so that the cellular immunity of the lymphocytes is more targeted and killing, and the killing effect on the tumor cells is more significant; the lymphocytes express non-functional EGFR and Expression of the chimeric antigen receptor makes the cellular immune killing effect of the above lymphocytes more safe and 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：(a)编码嵌合抗原受体的核酸分子，嵌合抗原受体具有SEQ ID 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；(b)沉默细胞表面或细胞内免疫检查点的核酸分子，沉默细胞表面免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：3～68的至少之一，沉默细胞内免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：69～135的至少之一；以及(c)编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：136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：137所示的核苷酸序列。其中，SEQ ID NO：3～14是人类程序性死亡受体1(PD1)siRNA核苷酸序列，SEQ ID NO：15～30是人类细胞毒T淋巴细胞相关抗原4(CTLA4)siRNA序列，SEQ ID NO：31～46是人类T细胞免疫球蛋白粘蛋白分子3(TIM3)siRNA序列，SEQ ID NO：47～57是人类T淋巴细胞衰减因子(BTLA)siRNA序列，SEQ ID NO：58～68是人类淋巴细胞活化基因3蛋白(LAG-3)siRNA序列，SEQ ID NO：69～85是人类IRAK-M siRNA(人类白细胞介素-1受体相关激酶3)核苷酸序列，SEQ ID NO：86～96是人类SOCS1siRNA(人类细胞因子信号转导抑制因子1)序列，SEQ ID NO：97～116是人类A20siRNA(人类肿瘤坏死因子-α诱导蛋白A20)序列，SEQ ID NO：117～135是人类CBL-B siRNA(E3泛素蛋白连接酶CBL-B)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，其细胞表面的免疫检查点PD1、CTLA4、TIM3、BTLA、LAG-3或细胞内免疫检查点IRAK-M、SOCS1、A20、CBL-B、被特异性沉默和抑制表达和表达无功能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抵抗肿瘤介导的免疫抑制的功效，抗调亡能力和增殖能力增强、定向杀伤能力显著提高，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在肿瘤病人体内和体外的增殖和生存能力以及在肿瘤病人体内的杀伤能力大大提高，尤其对高表达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 invention, the lentivirus or construct carries the following nucleic acid molecule: (a) a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1, coding chimeric The nucleic acid molecule of the antigen receptor has the nucleotide sequence shown in SEQ ID NO: 2; (b) the nucleic acid molecule which silences the cell surface or the intracellular immunological checkpoint, and the nucleotide sequence of the nucleic acid molecule which silences the cell surface immunological checkpoint a nucleic acid molecule which is selected from at least one of SEQ ID NOS: 3 to 68, wherein the nucleic acid molecule of the intracellular immunological checkpoint is at least one selected from the group consisting of SEQ ID NOS: 69 to 135; and (c) A nucleic acid molecule that functions as EGFR having the amino acid sequence set forth in SEQ ID NO: 136, the nucleic acid molecule encoding a non-functional EGFR having the nucleotide sequence set forth in SEQ ID NO:137. Wherein SEQ ID NOs: 3 to 14 are human programmed death receptor 1 (PD1) siRNA nucleotide sequences, and SEQ ID NOs: 15 to 30 are human cytotoxic T lymphocyte-associated antigen 4 (CTLA4) siRNA sequences, SEQ ID NO: 31 to 46 is a human T cell immunoglobulin mucin molecule 3 (TIM3) siRNA sequence, and SEQ ID NOs: 47 to 57 are human T lymphocyte attenuating factor (BTLA) siRNA sequences, SEQ ID NOs: 58-68. Is a human lymphocyte activation gene 3 protein (LAG-3) siRNA sequence, and SEQ ID NOs: 69-85 are human IRAK-M siRNA (human interleukin-1 receptor-associated kinase 3) nucleotide sequence, SEQ ID NO :86-96 is a human SOCS1 siRNA (human cytokine signal transduction inhibitor 1) sequence, and SEQ ID NOs: 97-116 are human A20 siRNA (human tumor necrosis factor-α-inducible protein A20) sequences, SEQ ID NOs: 117-135 Is a human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence, and the lentivirus or construct of the embodiment of the present invention is introduced into lymphocytes obtained from lymphocytes according to an embodiment of the present invention, the cell surface thereof Immune checkpoint PD1, CTLA4, TIM3, BTLA, LAG-3 or intracellular Immunological checkpoints IRAK-M, SOCS1, A20, CBL-B, specifically silenced and inhibited expression and expression of non-functional EGFR, while expressing a chimeric antigen receptor against MSLN on the cell surface thereof, thereby transgenes of the present invention Lymphocytes have remarkable anti-tumor-mediated immunosuppressive effects, anti-apoptotic ability and proliferative ability are enhanced, directional killing ability is significantly improved, and immune killing safety is remarkably improved. The transgenic lymphocytes of the embodiments of the present invention are in tumor patients. The proliferation and viability in vitro and the killing ability in tumor patients are greatly improved, especially for tumor cells with high expression of MSLN.
根据本发明地实施例，本发明实施例的逆转录病毒或构建体携带含有SEQ ID NO：138、139、140、141、142或143所示的核苷酸序列。其中，SEQ ID NO：138表示的是共表达抗MSLN嵌合抗原受体、无功能EGFR、沉默细胞免疫检查点PD1的核酸分子(MSLN CAR/iPD1/tEGFR),SEQ ID NO：139是共表达抗MSLN嵌合抗原受体、无功能EGFR和沉默细胞免疫检查点CBL-B的核酸分子(MSLN CAR/iCBL-B/tEGFR)，SEQ ID NO：140是共表达抗MSLN嵌合抗原受体、无功能EGFR和沉默细胞免疫检查点CTLA4的核酸分子(MSLN CAR/CTLA4/tEGFR)，SEQ ID NO：141表示的是共表达抗MSLN嵌合抗原受体、无功能EGFR、沉默细胞免疫检查点PD1和沉默细胞另一个免疫检查点CBL-B的核酸分子 (MSLN CAR/iPD1-CBL-B/tEGFR)，SEQ ID NO：142是共表达抗MSLN嵌合抗原受体、无功能EGFR、沉默细胞免疫检查点PD1和沉默细胞另一个免疫检查点CTLA4的核酸分子(MSLN CAR/i PD1-CTLA4/tEGFR)，SEQ ID NO：143是共表达抗MSLN嵌合抗原受体、无功能EGFR、第一沉默细胞表面免疫检查点PD1的核酸分子和第二沉默细胞免疫检查点PD1的核酸分子(MSLN CAR/i PD1-PD1/tEGFR)。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，其细胞的免疫检查点PD1、CTLA4、CBL-B被特异性沉默以及表达无功能EGFR和抗MSLN的嵌合抗原受体表达，使得转基因淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的功效，其抗调亡能力和增殖能力增强、定向杀伤能力显著提高，免疫杀伤安全性显著提高，从而使得转基因淋巴细胞在肿瘤病人体外和体内的增殖和生存能力以及在肿瘤病人体内的杀伤能力大大提高，尤其对高表达MSLN的间皮瘤细胞的特异性杀伤效果尤为显著，对高表达MSLN的间皮瘤细胞的特异性杀伤安全性显著提高。According to an embodiment of the invention, a retrovirus or construct of an embodiment of the invention carries a nucleotide sequence comprising SEQ ID NO: 138, 139, 140, 141, 142 or 143. Wherein SEQ ID NO: 138 represents a nucleic acid molecule (MSLN CAR/iPD1/tEGFR) co-expressing an anti-MSLN chimeric antigen receptor, a non-functional EGFR, a silencing cell immunological checkpoint PD1, and SEQ ID NO: 139 is a co-expression An anti-MSLN chimeric antigen receptor, a non-functional EGFR, and a nucleic acid molecule (MSLN CAR/iCBL-B/tEGFR) that silences the cellular immunological checkpoint CBL-B, SEQ ID NO: 140 is a co-expressing anti-MSLN chimeric antigen receptor, Non-functional EGFR and silencing cellular immunological checkpoint CTLA4 nucleic acid molecule (MSLN CAR/CTLA4/tEGFR), SEQ ID NO: 141 indicates co-expression of anti-MSLN chimeric antigen receptor, non-functional EGFR, silencing cellular immune checkpoint PD1 And silent cell another immunological checkpoint CBL-B nucleic acid molecule (MSLN CAR/iPD1-CBL-B/tEGFR), SEQ ID NO: 142 is a nucleic acid that co-expresses anti-MSLN chimeric antigen receptor, non-functional EGFR, silencing cellular immunological checkpoint PD1, and another immunological checkpoint CTLA4 Molecule (MSLN CAR/i PD1-CTLA4/tEGFR), SEQ ID NO: 143 is a nucleic acid molecule co-expressing anti-MSLN chimeric antigen receptor, non-functional EGFR, first silencing cell surface immunological checkpoint PD1 and second silencing cell The nucleic acid molecule of the immunological checkpoint PD1 (MSLN CAR/i PD1-PD1/tEGFR). According to an embodiment of the present invention, the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes, the cell immunological checkpoints PD1, CTLA4, CBL-B are specifically silenced and express non-functional EGFR and anti-MSLN The expression of the chimeric antigen receptor makes the transgenic lymphocyte have significant anti-tumor-mediated immunosuppressive effect, and its anti-apoptotic ability and proliferative ability are enhanced, the directional killing ability is significantly improved, and the safety of immunological killing is significantly improved, thereby making the transgenic gene The proliferation and viability of lymphocytes in tumor patients in vitro and in vivo and their ability to kill in tumor patients are greatly improved, especially for mesothelioma cells with high expression of MSLN. For mesothelioma with high expression of MSLN. The specific killing safety of cells is significantly improved.
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、表面或细胞内免疫检查点shRNA以及无功能EGFR分别独立地表达的，其中，需要说明的是，此处的表达既指蛋白的表达又指RNA转录。According to an embodiment of the present invention, the inventors realize that the above-mentioned cell chimeric antigen receptor, surface or intracellular immunological checkpoint shRNA, and non-functional EGFR are independently expressed by at least one of the following methods, wherein , expression herein refers to both protein expression and RNA transcription.
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：144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，使得淋巴细胞对高表达MSLN肿瘤的特异性杀伤效果更加显著，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 present invention is set between a nucleic acid molecule encoding a chimeric antigen receptor and a nucleic acid molecule expressing a non-functional EGFR, and an internal ribosome entry site The dot has the nucleotide sequence shown by SEQ ID NO:144. The internal ribosome entry site is usually located in the 5' untranslated region (UTR) of the RNA viral genome, so that the translation of one viral protein can be independent of the 5' cap structure, and the other protein usually initiates translation by the 5' hat structure. The expression of the two genes before and after IRES is usually proportional. The introduction of an internal ribosome entry site sequence allows expression of a nucleic acid molecule encoding a chimeric antigen receptor independently of a nucleic acid molecule encoding a non-functional EGFR. According to an embodiment of the present invention, the internal ribosome entry site sequence effectively ensures the high expression of the chimeric antigen receptor and the non-functional EGFR, and the specific killing effect of lymphocytes on the high expression of MSLN tumor is more significant. The safety of immune killing is further improved.
启动子：第一启动子，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；第二启动子，第二启动子与沉默免疫检查点的核酸分子可操作地连接；以及第三启动子，第三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、第二启动子和第三启动子分别独立地选自U6，CMV,H1，EF-1,LTR,RSV启动子，第一以及第二启动子和第三启动子的引入，使得编码嵌合抗原受体的核酸分子和沉默免疫检查点的核酸分子以及表达无功能EGFR的核酸分子分别独立的表达，从而有效沉默了细胞表面或细胞内免疫检查点或高效表达了无功能EGFR，并且保证了嵌合抗原受体的高效表达，使得淋巴细胞在肿瘤环境中的成活率大大提高，淋巴细胞的靶向作用更强，对肿 瘤的特异性杀伤作用更加显著，免疫杀伤的安全性进一步提高。Promoter: a first promoter operably linked to a nucleic acid molecule encoding a chimeric antigen receptor; a second promoter operably linked to a nucleic acid molecule that silences the immunological checkpoint; A third promoter, the third promoter is operably linked to a nucleic acid molecule that expresses non-functional EGFR. According to an embodiment of the invention, the first promoter, the second promoter and the third promoter employed are each independently selected from the group consisting of U6, CMV, H1, EF-1, LTR, RSV promoters, first and second The introduction of the promoter and the third promoter enables the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule that silences the immunological checkpoint and the nucleic acid molecule expressing the non-functional EGFR to be independently expressed, thereby effectively silencing the cell surface or intracellular Immunological checkpoints or high-efficiency expression of non-functional EGFR, and ensure the high expression of chimeric antigen receptors, so that the survival rate of lymphocytes in the tumor environment is greatly improved, lymphocyte targeting is stronger, swelling The specific killing effect of the tumor is more significant, and the safety of immune killing is further improved.
第四核酸分子:第四核酸分子设置在所述第一核酸分子与所述第三核酸分子之间，并且所述第四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。连接肽具有SEQ ID NO：145所示的氨基酸序列。第四核酸分子及其相应表达的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，本发明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，其具特异性更强的肿瘤杀伤效果，安全性更高。a fourth nucleic acid molecule: a fourth nucleic acid molecule is disposed between the first nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encodes a linker peptide capable of being Cutting. The linker peptide has the amino acid sequence set forth in SEQ ID NO:145. The introduction of the fourth nucleic acid molecule and its correspondingly expressed linker peptide allows the non-functional EGFR and chimeric antigen receptor to be expressed in a non-fusion state on the lymphocyte membrane. According to an embodiment of the present invention, the introduction of the linker peptide of the embodiment of the present invention ensures the biological effects of the non-functional EGFR and the chimeric antigen receptor, and has a more specific tumor killing effect and higher safety.
通过上述内部核糖体进入位点序列、或第一、第二、第三启动子或第三核酸分子的引入，使得细胞表面或细胞内免疫检查点被高效沉默和无功能EGFR高效地表达以及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，并且无功能EGFR和嵌合抗原受体呈非融合状态表达在淋巴细胞膜上，从而高效抑制了免疫检查点的免疫负调控和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，从而使得淋巴细胞在肿瘤环境中的成活率大大提高，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 internal ribosome entry site sequence, or the introduction of the first, second, third promoter or third nucleic acid molecule, the cell surface or intracellular immune checkpoint is efficiently expressed and embedded by highly efficient silencing and non-functional EGFR. The antigen-receptor is efficiently expressed on the transgenic lymphocyte membrane of the present invention, and the non-functional EGFR and the chimeric antigen receptor are expressed in the non-fusion state on the lymphocyte membrane, thereby effectively inhibiting the immunological negative regulation of the immune checkpoint. The biological effect of the chimeric antigen receptor is ensured, and the timely removal of the transgenic lymphocytes is effectively realized, so that the survival rate of lymphocytes in the tumor environment is greatly improved, the targeted killing effect of lymphocytes is more remarkable, and the safety of immune killing is safe. The sex 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在肿瘤病人体内的增殖和生存能力大大提高，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，免疫杀伤安全性进一步提高。Further, 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 enhances the replication and amplification efficiency of the construct in lymphocytes, and further, the lymphocyte proliferation and viability of the lymphocytes in the embodiment of the invention are greatly enhanced, and the targeting effect of lymphocytes is further enhanced. The killing effect on tumor cells is more significant, and the safety of immune killing is further improved.
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，从而细胞表面或细胞内免疫检查点被高效沉默和无功能EGFR被高效表达以及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在肿瘤病人体内的增殖和生存能力大大提高，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 invention, the vector of the construct of the embodiment of the invention is a viral vector selected from at least one of a retroviral vector, a lentiviral vector, an adenoviral vector or an adenovirus associated viral vector. According to an embodiment of the present invention, the virus carrier of the embodiment of the present invention has a wide range of virus infection during virus packaging and infection, and can infect both terminally differentiated cells and cells in a dividing phase, and can be integrated into the host. The chromosome, which can be freed from the host chromosome, achieves a broad-spectrum and efficient infection efficiency, so that cell surface or intracellular immunological checkpoints are efficiently silenced and non-functional EGFR is highly expressed and chimeric antigen receptors are efficiently expressed in lymphocytes. The lymphocyte has a greatly enhanced proliferation and viability in the tumor patient, and the lymphocyte targeting effect is further enhanced, and the killing effect on the tumor cell is more remarkable, and the immune killing safety of the lymphocyte 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。In accordance with a specific embodiment of the present invention, in order to construct a lentiviral vector, the inventors inserted a nucleic acid of interest into a viral genome at a position of a certain viral sequence in order to construct a lentiviral vector, thereby producing a replication-defective virus. To generate virions, the inventors further constructed packaging cell lines (containing the gag, pol and env genes, but excluding LTR and packaging components). The inventors introduced a recombinant plasmid containing the gene of interest, together with the lentiviral LTR and the packaging sequence, into a packaging cell line. The packaging sequence allows the recombinant plasmid RNA transcript to be packaged into viral particles which are then secreted into the culture medium. Further, the inventors collected a matrix containing the recombinant lentivirus, selectively concentrated, and used for gene transfer. Slow vectors can infect a variety of cell types, including cleavable cells and non-dividable 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。根据本发明的实施例，本发明实施例的慢病毒载体可有效运送和共表达shRNA(siRNA的转运形式)，该小shRNA可以有效抑制PD1或CTLA4或CBL-B的表达。Further, according to an embodiment of the present invention, the lentivirus of the embodiment of the present invention is a complex lentivirus, and in addition to the common lentiviral genes gag, pol and env, other genes having regulatory and structural functions are also included. 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 produce a biosafety vector by multiple attenuation of HIV-causing genes, such as deletion of the genes env, vif, vpr, vpu and nef. Recombinant lentiviral vectors are capable of infecting non-dividing cells and are useful for in vivo and in vitro gene transfer and nucleic acid sequence expression. For example, in a suitable host cell, together with two or more vectors with packaging functions (gag, pol, env, rev and tat), it is possible to infect non-dividing cells. The targeting of recombinant viruses is achieved by binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins. At the same time, the targeting of the recombinant virus confers specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, along with another gene encoding a ligand for the receptor on the particular target cell. A variety of useful lentiviral vectors, as well as vectors produced by various methods and procedures, are used to alter the expression of cells. According to an embodiment of the present invention, the lentiviral vector of the present invention can efficiently transport and co-express shRNA (a transport form of siRNA) which can effectively inhibit the expression of PD1 or CTLA4 or CBL-B.
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，以此携带和共表达小发夹RNA，该小发夹RNA可以抑制PD1或CTLA4或CBL-B基因的表达。According to an embodiment of the invention, an adeno-associated viral vector (AAV) of an embodiment of the invention may be constructed using one or more DNAs of a well-known serotype adeno-associated viral vector. One skilled in the art constructs a suitable adeno-associated viral vector to carry and co-express a small hairpin RNA that inhibits the expression of the PDl or CTLA4 or CBL-B genes.
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。Further, according to an embodiment of the present invention, the embodiment of the present invention also includes a microgene. Microgenes mean the use of a combination (selected nucleotide sequence and operably necessary related linker sequences) to direct expression of the transform, transcription and/or gene product in a host cell in vivo or in vitro. The "operable ligation" sequence is employed to include expression control sequences for a continuous gene of interest, and expression control sequences for trans- or remote control of the gene of interest.
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、转化和/或小发夹RNA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，表达shRNA的启动子为RNA聚合酶启动子。同时，根据本发明的实施例，启动子为选自U6,H1,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U6,H1,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, vectors of the embodiments of the invention also include conventional control elements that permit transcription, transformation, and/or expression of small hairpin RNA in cell infection with the plasmid vector or in a cellular infection with the viral vector. A large number of expression control sequences (including native, inducible and/or specific tissue promoters) may be used. According to an embodiment of the invention, the shRNA expressing promoter is an RNA polymerase promoter. Meanwhile, according to an embodiment of the present invention, the promoter is a RAN polymerase promoter selected from the group consisting of U6, H1, pol I, pol II and pol III. According to an embodiment of the invention, the promoter is a tissue-specific promoter. According to an embodiment of the invention, the promoter is an inducible promoter. According to an embodiment of the invention, the promoter is selected from a promoter based on the selected vector. According to an embodiment of the present invention, when a lentiviral vector is selected, the promoter is a U6, H1, CMV IE gene, EF-1α, ubiquitin C or phosphoglycerate kinase (PGK) promoter. Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding geneticin, hygromycin, ampicillin or puromycin resistance. Other components of the carrier include an origin 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的shRNA、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 include conventional cloning techniques such as shRNA, polymerase chain reaction and any suitable method for providing the desired nucleotide sequence for use in embodiments of the invention. .
根据本发明的实施例，发明人构建了共表达小发夹RNA(shRNA)(用来抑制免疫检查点)和无功能EGFR以及嵌合抗原受体(CAR)的病毒载体。本发明实施例的运送沉默PD1或CTLA4或CBL-B的siRNA的小发夹RNA和表达无功能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。In accordance with an embodiment of the invention, the inventors constructed viral vectors that co-express small hairpin RNA (shRNA) (used to suppress immune checkpoints) and non-functional EGFR and chimeric antigen receptor (CAR). The small hairpin RNA carrying the siRNA silencing PD1 or CTLA4 or CBL-B and the nucleic acid molecule expressing the non-functional EGFR and the viral vector or plasmid expressing the chimeric antigen receptor (CAR) are complexed with the virus of the present invention. The vector or plasmid can be combined with a polymer or other material to increase its stability or assist in its targeted movement.
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淋巴细胞的细胞表面或细胞内免疫检查点被沉默以及无功能EGFR的表达，从而使得所得淋巴细胞或T淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的功效，在肿瘤病人体内和体外的增殖及肿瘤病人体内存活能力大大提高，淋巴细胞或T淋巴细胞对肿瘤细胞，尤其是高表达MSLN的肿瘤细胞的靶向杀伤作用更强，免疫杀伤的安全性高。In another aspect of the invention, the invention provides a method of preparing a T lymphocyte or a transgenic lymphocyte as described above. According to an embodiment of the invention, the method comprises introducing the construct described above or the lentivirus described above into lymphocytes or T lymphocytes. The mode of introduction can be introduced in a manner selected from the group consisting of electroporation or viral infection of host cells. The construct or lentivirus of the embodiment of the present invention is successfully introduced into the above lymphocyte or T lymphocyte, and the expression of the chimeric antigen receptor against the antigen MSLN and the cell surface or intracellular immune checkpoint of the lymphocyte are silenced and absent. Functional EGFR expression, so that the resulting lymphocytes or T lymphocytes have significant anti-tumor-mediated immunosuppressive effects, and the proliferation of tumor patients in vitro and in vivo and the survival ability of tumor patients are greatly improved, lymphocytes or T lymphocytes The targeted killing effect on tumor cells, especially tumor cells with high expression of MSLN, is stronger, and the safety of immune killing is high.
治疗癌症的治疗组合物Therapeutic composition 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转基因淋巴细胞或T淋巴细胞细胞表面或细胞内免疫检查点的沉默；以及无功能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淋巴细胞在体外扩增、在肿瘤病人体内的增殖及肿瘤病人体内存活能力大大提高，转基因淋巴细胞或T淋巴细胞对高表达MSLN的肿瘤细胞的靶向杀伤作用更强，免疫杀伤的安全性更高。In another aspect of the invention, the invention provides a therapeutic composition for treating cancer. According to an embodiment of the present invention, the therapeutic composition comprises: the above construct, the above lentivirus, the above T lymphocyte or the above transgenic lymphocyte. The composition of any of the above therapeutic compositions can achieve high expression of the antigen MSLN chimeric antigen receptor in transgenic lymphocytes or T lymphocytes and silencing of transgenic lymphocytes or T lymphocyte cells or intracellular immune checkpoints. 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 can be expanded in vitro, proliferate in tumor patients and survive in tumor patients, and transgenic lymphocytes are greatly improved. Or T lymphocytes have stronger targeted killing effect on tumor cells with high expression of MSLN, and the safety of immune killing is higher.
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 an embodiment of the invention, the therapeutic composition of the embodiments of the invention provided to a patient is preferably applied to a biocompatible solution or an acceptable pharmaceutical carrier. The various therapeutic compositions prepared are suspended or dissolved in a pharmaceutically or physiologically acceptable carrier, such as physiological saline; an isotonic saline solution or other relatively obvious formulation of a person skilled in the art. The appropriate carrier will depend to a large extent on the route of administration. Other isotonic sterile injections with water and anhydrous, and sterile suspensions with water and anhydrous 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，沉默PD1或CTLA4或CBL-B和表达无功能EGFR以及表达特有的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 invention, a sufficient number of viral vectors are transduced into targeted T cells and provide sufficient strength Transgenic, silencing PD1 or CTLA4 or CBL-B and expressing non-functional EGFR as well as expressing a unique MSLN chimeric antigen receptor. The dosage of the therapeutic agent depends primarily on the condition of treatment, age, weight, and the health of the patient, which may result in patient variability.
沉默PD1或CTLA4或CBL-B和表达无功能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 methods of silencing PD1 or CTLA4 or CBL-B and expressing non-functional EGFR as well as expressing a specific receptor for the antigenic MSLN chimeric antigen are part of a combination therapy. These viral vectors and anti-tumor T cells for adoptive immunotherapy can be performed alone or in combination with other methods of treating cancer. Under appropriate conditions, one treatment involves the use of one or more drug therapies.
根据本发明的实施例，所述癌症包括间皮瘤。细胞表面或细胞内免疫检查点的沉默和无功能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淋巴细胞在间皮瘤的环境中的存活能力大大提高，淋巴细胞或T淋巴细胞对高表达MSLN的肿瘤细胞的靶向杀伤作用更强，尤其对高表达MSLN的肿瘤细胞的杀伤作用更加显著，对高表达MSLN的肿瘤细胞的免疫杀伤作用更加安全有效。According to an embodiment of the invention, the cancer comprises mesothelioma. Silencing of cell surface or intracellular immunological checkpoints and expression of non-functional EGFR, combined with high expression of chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, resulting in the appearance of lymphocytes or T lymphocytes in mesothelioma The survival ability is greatly improved, and the lymphocyte or T lymphocyte has stronger targeted killing effect on tumor cells with high expression of MSLN, especially for tumor cells with high expression of MSLN, and for tumor cells with high expression of MSLN. Immune killing is safer and more effective.
提高淋巴细胞活性和治疗安全性的方法Method for increasing lymphocyte activity and therapeutic safety
在本发明的另一方面，本发明提出了一种提高淋巴细胞活性和治疗安全性的方法，本发明实施例的淋巴细胞携带嵌合抗原受体，根据本发明的实施例，该方法包括：使所述淋巴细胞的细胞表面或细胞内至少之一免疫检查点被沉默；以及使所述淋巴细胞表达无功能EGFR，细胞表面或细胞内免疫检查点、淋巴细胞、嵌合抗原受体、无功能EGFR是如前所定义的。根据本发明的实施例，本发明实施例的淋巴细胞活性包括淋巴细胞体外增殖能力，在肿瘤病人体内的增殖和生存能力以及淋巴细胞在肿瘤病人体内的杀伤能力的至少一种。根据本发明的实施例，本发明实施例的淋巴细胞的细胞表面或细胞内免疫检查点被沉默，淋巴细胞被活化、增生性反应上调、细胞因子分泌增多、抗调亡能力增强。本发明实施例的淋巴细胞在体外扩增和增殖、对肿瘤细胞的靶向杀伤作用显著增强。In another aspect of the invention, the invention provides a method of increasing lymphocyte activity and therapeutic safety, wherein the lymphocytes of the embodiments of the invention carry a chimeric antigen receptor, according to an embodiment of the invention, the method comprising: Causing at least one of the cell surface or intracellular immune checkpoint of the lymphocyte; and causing the lymphocyte to express non-functional EGFR, cell surface or intracellular immunological checkpoint, lymphocyte, chimeric antigen receptor, none Functional EGFR is as previously defined. According to an embodiment of the present invention, lymphocyte activity according to an embodiment of the present invention includes at least one of lymphocyte proliferation ability in vitro, proliferation and viability in a tumor patient, and killing ability of lymphocytes in a tumor patient. According to an embodiment of the present invention, the cell surface or intracellular immune checkpoint of the lymphocytes of the embodiment of the present invention is silenced, lymphocytes are activated, the proliferative response is up-regulated, the cytokine secretion is increased, and the anti-apoptotic ability is enhanced. The lymphocytes of the embodiments of the present invention are expanded and propagated in vitro, and the targeted killing effect on tumor cells is remarkably enhanced.
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受体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。本发明是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。Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR receptor and intact sequence that binds to anti-EGFR antibody, and non-functional EGFR can act as lymph Cell suicide markers. When the lymphocytes of the present invention are used for treating tumor cells having high expression of MSLN, if the patient develops a serious adverse reaction, the lymphocytes of the embodiments of the present invention can be cleared by the anti-EGFR antibody, thereby further improving the lymph of the embodiment of the present invention. Cellular therapy for the safety of tumor patients with high expression of MSLN.
治疗癌症的方法Method of treating cancer
在本发明的另一方面，本发明提出了一种治疗癌症的方法。根据本发明的实施例，该方法包括：为癌症患者给药前面所述的构建体、前面所述的慢病毒、前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞，其中，嵌合抗原受体特异性结合肿瘤抗原MSLN。利用本发明实施例的治疗癌症的方法，可有效实现对癌症患者肿瘤细胞的靶向杀伤，尤其是具有 对高表达MSLN的肿瘤细胞的靶向杀伤作用，进而能够有效治疗癌症，治疗效果好且安全性高。In another aspect of the invention, the invention provides a method of treating cancer. According to an embodiment of the invention, the method comprises: administering to a cancer patient a construct as described above, a lentivirus as described above, a T lymphocyte as described above or a transgenic lymphocyte as described above, wherein the chimeric The antigen receptor specifically binds to the tumor antigen MSLN. The method for treating cancer according to the embodiment of the invention can effectively achieve targeted killing of tumor cells of cancer patients, especially having The targeted killing effect on tumor cells with high expression of MSLN can further effectively treat cancer, and the therapeutic effect is good and the safety is high.
根据本发明的具体实施例，所述方法包括：从癌症患者体内分离淋巴细胞；将前面所述的构建体，或前面所述的慢病毒导入所述淋巴细胞，以便获得转基因淋巴细胞，所述转基因淋巴细胞表达嵌合抗原受体和细胞免疫检查点被沉默；以及为所述癌症患者给药所述转基因淋巴细胞。利用本发明实施例的治疗癌症的方法，可进一步有效实现对癌症患者肿瘤细胞的靶向杀伤，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，进而能够进一步有效治疗癌症，治疗效果好且安全性高。According to a particular embodiment of the invention, the method comprises: isolating lymphocytes from a cancer patient; introducing the aforementioned construct, or the lentivirus described above, into the lymphocytes to obtain transgenic lymphocytes, The transgenic lymphocytes express the chimeric antigen receptor and the cellular immune checkpoint is silenced; and the transgenic lymphocytes are administered to the cancer patient. The method for treating cancer according to the embodiment of the invention can further effectively achieve targeted killing of tumor cells of cancer patients, especially having targeted killing effect on tumor cells with high expression of MSLN, thereby further effectively treating cancer, and the therapeutic effect Good and safe.
具体地，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。本发明实施例的治疗癌症的方法可使淋巴细胞细胞免疫检查点被沉默和细胞表达嵌合抗原受体，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MSLN特异性表达的间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌或乳腺癌的肿瘤细胞的靶向杀伤。Specifically, the cancer includes at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer. The method for treating cancer according to an embodiment of the present invention enables lymphocyte immune checkpoints to be silenced and cells to express chimeric antigen receptors, such as MSLN antigen-specific chimeric antigen receptors of the present invention, resulting lymphocytes or T lymphocytes. The cells have targeted killing of tumor cells of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer or breast cancer which are specifically expressed by MSLN.
下面将结合实施例对本发明的方案进行解释。The solution of the present invention will be explained below in conjunction with the embodiments.
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 appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not mentioned, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
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：The cell lines and basic experimental techniques used in the examples of the invention are as follows:
慢病毒的产生和人T淋巴细胞的转导Lentivirus production 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/Thermo Scientific,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、5微克的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。A lentiviral vector having a replication defect is produced, and the lentiviral vector is collected by centrifugation for transduction of human T lymphocytes. The following is a brief introduction to the experimental procedure for the generation and collection of lentiviral vectors: 293T cells are plated in cell culture dishes with a bottom area of 150-cm 2 and using Express-In according to the instructions (purchased from Open Biosystems/Thermo Scientific, Waltham) , MA) Virus 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 to each plate. -In (concentration is 1 μg/μl). The supernatants were collected at 24 and 48 hours, respectively, and using an ultracentrifuge at 28,000 rpm (the centrifuge rotor was Beckman SW 32Ti, available from Beckman Coulter, Brea, Centrifuge for 2 hours under conditions of CA). Finally, the viral plasmid pellet was resuspended in 0.75 ml 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8μg/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，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 challenged with monoclonal antibody coated beads of anti-CD3 and CD28 (purchased from Invitrogen, Carlsbad, CA). T-lymphocytes were transduced by spin-inoculation 18 to 24 hours after activation of human T lymphocytes. The transduction process was as follows: in a 24-well plate, 0.5 x 106 T lymphocytes per well were plated. To each well of the cells, 0.75 ml of the above-mentioned resuspended virus supernatant and Polybrene (concentration: 8 μg/ml) were added. A mixture of cells and viral plasmids was centrifuged in a bench top centrifuge (purchased from Sorvall ST 40; Thermo Scientific) at room temperature, 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. The final concentration of IL-2 was 100-IU/ml in T lymphocytes. During cell culture, the density of the cells is maintained at 0.5 x 106 to 1 x 106 / ml. Once the transduced T lymphocytes are dormant, for example, the cell growth rate is slowed down and the cells become smaller, wherein the cell growth rate and size are assessed by Coulter Counter (purchased from Beckman Coulter), or transduced T lymphocytes. At a planned time point, T lymphocytes can be used 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BD Biosciences)，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 flow cytometric 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的实施例中，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2～5*106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50μl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实施例中，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 an example relating to ADCC, the ability of anti-EGFR antibodies to induce cell-dependent lysis of lymphocytes expressing non-functional EGFR was assessed using the 4 hour- 51 Cr-release method. Human T lymphocytes transduced with a lentiviral vector were used as target cells. 100 μCi Na 2 51 CrO 4 (available from GE Healthcare Life Sciences, Marlborough, MA) was used to calibrate 2 to 5*10 6 target cells under the conditions of shaking incubation at 37 ° C for 1 hour. The cells were washed three times with PBS and resuspended in medium (cell density was 1 x 10 5 /ml). The calibrated cells were then plated in 96-well plates (5 x 10 3 cells per well, plus 50 μl of medium) and 50 μl of anti-EGFR antibody (purchased from Erbitux, Genentech) ( The final concentration was 20 μg/ml, and preculture was carried out for 30 minutes under normal temperature conditions. Then, the medium containing the antibody was changed to a normal medium, thereby detecting the spontaneous release of 51 Cr. Triton X-100 was added to a final concentration of 1% to ensure maximum release of 51 Cr. In the following ADCC examples, human PBMCs (effector cells) were added to the wells (5 x 105 cells per well) and the cells were incubated overnight at 37 °C. On the next day, the cell supernatant was collected and cpm was calculated using a gamma counter to determine the release of 51 Cr. The cytotoxicity ratio was calculated using the following formula: % specific lysis = (experimental release cpm data - spontaneous release of cpm data) / (maximum release cpm data - spontaneous release of cpm data) * 100, wherein the maximum release cpm data was added through the target cells The spontaneous release of cpm data by Triton X-100 was measured in the absence of anti-EGFR antibodies and effector cells.
铬释放实验Chromium release experiment
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 CAR T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(ATCC))。The cytotoxic activity of anti-MSLN chimeric antigen receptor T cells (anti-MSLN CAR T lymphocytes) was evaluated in the Examples using a 4 - hour 51 chromium release assay. The specific steps are as follows: Target test cells were labeled with 51 Cr at 37 degrees Celsius for 1 hour. After labeling, the cells were rinsed with RPMI medium containing 10% fetal bovine serum (FCS). After rinsing, the cells were resuspended in the same medium, and the concentration of the resuspended cells was 1 × 10 5 /ml. After transduction, T cells were added to the target test cell suspension at different target cell ratios (E:T), and the cells were seeded in 96-wells at a volume of 200 microliters per well. The cells were cultured for 4 hours in a 37 degree incubator. After 4 hours, 30 microliters of the supernatant was taken from each well and placed in a counter 96-well plate for counting analysis. The analytical instrument was a top-level counting NXT micro-scintillator counter (purchased from Packard Bioscience). The number of effector cells in all counting wells was calculated based on the total number of T cells. The target test cell to be labeled is MSLN + MSTO-211H (human pleural mesothelioma cells (ATCC)).
实施例2构建共表达shRNA、无功能EGFR和抗MSLN嵌合抗原受体的载体Example 2 Construction of a vector for co-expression of shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor
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U6启动子和人PD1 shRNA(iPD1)或CBL-B shRNA(iCBL-B)或CTLA4 shRNA(iCTLA4)的序列被克隆进LV-MSLN CAR载体质粒,构建成LV-MSLN CAR/iPD1或LV-MSLN CAR/iCBL-B或LV-MSLN CAR/iCTLA4，包含合成IRES和表达无功能EGFR的序列克隆进LV-MSLN CAR/iPD1或LV-MSLN CAR/iCBL-B或LV-MSLN CAR/iCTLA4载体质粒，构建成LV-MSLN CAR/iPD1/tEGFR(M)或LV-MSLN CAR/iCBL-B/tEGFR(M)或LV-MSLN CAR/iCTLA4/tEGFR(M)；包含合成IRES和表达无功能EGFR的序列、H1启动子和PD1 shRNA序列、H1启动子和CBL-B shRNA序列或H1启动子和CTLA4 shRNA序列被克隆进LV-MSLN CAR载体质粒，构建成LV-MSLN CAR/iPD1-CTLA4/tEGFR(M)和LV-MSLN CAR/iPD1-CBL-B/tEGFR(M)。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、U6和H1启动子序列、PD1 shRNA或CBL-B shRNA或CTLA4 shRNA序列,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，CTLA4,PD1或者CBL-B shRNA序列在启动子U6或H1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 the present example, the inventors cloned the sequence encoding the single-chain antibody against human MSLN, the 4-1BB intracellular domain and the T cell receptor combined ζ-strand sequence into a lentiviral vector containing the EF-1 promoter ( On the lentiviral vector), during the cloning process, the restriction enzyme digestion is the double digestion of XbaI and NotI, and the double digestion of NotI and XhoI, and the expression of anti-MSLN is generated by restriction enzyme digestion, ligation, screening and amplification of the plasmid of interest. Anti-receptor lentiviral plasmid (LV-MSLN CAR). The sequence containing the U6 promoter and human PD1 shRNA (iPD1) or CBL-B shRNA (iCBL-B) or CTLA4 shRNA (iCTLA4) was cloned into the LV-MSLN CAR vector plasmid and constructed into LV-MSLN CAR/iPD1 or LV- MSLN CAR/iCBL-B or LV-MSLN CAR/iCTLA4, including synthetic IRES and sequences expressing non-functional EGFR cloned into LV-MSLN CAR/iPD1 or LV-MSLN CAR/iCBL-B or LV-MSLN CAR/iCTLA4 vector plasmid , constructed as LV-MSLN CAR/iPD1/tEGFR (M) or LV-MSLN CAR/iCBL-B/tEGFR (M) or LV-MSLN CAR/iCTLA4/tEGFR (M); comprising synthetic IRES and expressing non-functional EGFR The sequence, H1 promoter and PD1 shRNA sequence, H1 promoter and CBL-B shRNA sequence or H1 promoter and CTLA4 shRNA sequence were cloned into LV-MSLN CAR vector plasmid to construct LV-MSLN CAR/iPD1-CTLA4/tEGFR ( M) and LV-MSLN CAR/iPD1-CBL-B/tEGFR (M). Figure 1 is a schematic representation of a lentiviral vector comprising a sequence encoding an anti-MSLN chimeric antigen receptor, an IRES, U6 and H1 promoter sequence, a PD1 shRNA or CBL-B shRNA or a CTLA4 shRNA sequence, and a coding non-functional EGFR sequence. The sequence of the anti-MSLN chimeric antigen receptor is regulated by the promoter EF-1, and the CTLA4, PD1 or CBL-B shRNA sequence is expressed under the promoter of promoter U6 or H1, and the sequence of non-functional EGFR is expressed as a single The mRNA transcription unit begins translation after the IRES sequence.
实施例3抗EGFR抗体可有效杀伤清除共表达PD1 shRNA、无功能EGFR和抗MSLN嵌合抗原受体的T淋巴细胞 Example 3 Anti-EGFR antibody effectively kills T lymphocytes that secrete PD1 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptors
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。在T淋巴细胞激活因子磁珠CD3/CD28(购自Invitrogen,Carlsbad,CA)存在下，被激活的T淋巴细胞经过慢病毒载体转导、体外扩增培养，方法如实施例1所述。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/iPD1/tEGFR,LV-MSLN CAR/iPD1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ADCC的对转导了不同慢病毒的T细胞(靶细胞)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,PD1 shRNA(iPD1)和无功能EGFR(LV-MSLN-CAR/iPD1/M)的T淋巴细胞,但抗EGFR抗体不能介异对表达抗MSLN嵌合抗原受体和PD1 shRNA(LV-MSLN-CAR/iPD1)的T淋巴细胞杀伤。抗EGFR抗体也不能介异对仅表达抗MSLN嵌合抗原受体(LV-MSLN-CAR)的T淋巴细胞杀伤.In this embodiment, peripheral blood lymphocytes are taken from an unnamed blood donor. Peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. Activated T lymphocytes were transduced with lentiviral vector and expanded in vitro in the presence of T lymphocyte activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) as described in Example 1. After the culture was activated for 72 hours, the cells were washed with a washing solution, and the magnetic beads were washed away. T cells were seeded on a recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were LV-MSLN CAR/iPD1/tEGFR, LV-MSLN CAR, respectively. The /iPD1 or no-load (LV-GFP) transduction process is as described in Example 1. T cells expressing non-functional EGFR after transfection were stained with anti-EGFR antibody and then isolated by FACS. After isolation, T cells were cultured in RPMI-1640 medium and recombinant human IL-2 factor (100 ng/ml; purchased from R&D Systems). Induction amplification was carried out for 7-10 days and then used as a target cell for the experiment. The inventors measured the killing effect of anti-EGFR antibody-differentiated ADCC on T cells (target cells) transduced with different lentiviruses by ADCC assay using standard 4–hour 51 chromium release method, 4–hour 51 chromium release. The method is as described in Example 1. The result is shown in Figure 2. As shown in Figure 2, anti-EGFR antibodies are effective in dissociating T lymphocytes that co-express anti-MSLN chimeric antigen receptors, PD1 shRNA (iPD1) and non-functional EGFR (LV-MSLN-CAR/iPD1/M), but Anti-EGFR antibodies do not mediate killing of T lymphocytes expressing the anti-MSLN chimeric antigen receptor and PD1 shRNA (LV-MSLN-CAR/iPD1). Anti-EGFR antibodies also do not mediate T lymphocyte killing of anti-MSLN chimeric antigen receptor (LV-MSLN-CAR) alone.
实施例4共表达PD1 shRNA、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 T lymphocyte tumor cell lysis ability co-expressing PD1 shRNA, 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/iPD1/tEGFR,LV-MSLN CAR/iPD1,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(效应细胞)对高表达MSLN的间皮瘤靶细胞(MSLN+MSTO-211H)的杀伤作用，效靶细胞比例是50：1，25:1或10:1，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。结果如图3所示。如图3所示,共表达抗MSLN嵌合抗原受体、PD1 shRNA(iPD1)和无功能EGFR慢病毒(LV-MSLN-CAR/iPD1/M)转导的T淋巴细胞，共表达抗MSLN嵌合抗原受体和 PD1 shRNA(iPD1)的T淋巴细胞(LV-MSLN-CAR/iPD1)的T淋巴细胞也可有效的杀死高表达MSLN的间皮瘤靶细胞。无功能EGFR慢病毒转导的T淋巴细胞(LV-M T淋巴细胞)对高表达MSLN的间皮靶细胞无明显杀伤作用。仅表达抗MSLN嵌合抗原受体的T淋巴细胞(LV-MSLN-CAR)的T淋巴细胞杀死具有杀死高表达MSLN的间皮靶细胞能力，但弱于共表达抗MSLN嵌合抗原受体和PD1 shRNA(iPD1)的T淋巴细胞(LV-MSLN-CAR/iPD1)的T淋巴细胞。这些结果表明共表达PD1 shRNA、无功能EGFR和抗MSLN嵌合抗原受体的T淋巴细胞能够高效杀伤肿瘤细胞，共表达无功能EGFR标记不影响抗MSLN CAR淋巴细胞对间皮瘤细胞的杀伤作用.In this embodiment, peripheral blood lymphocytes are taken from an unnamed blood donor. Peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. T lymphocytes were incubated with T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) for 72 hours at 5% CO 2 at 37 ° C. The medium was supplemented with 2 mmol/L glutamine, 10%. High temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U/ml penicillin/streptomycin double antibody in RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012). After the culture was activated for 72 hours, the cells were washed with a washing solution, and the magnetic beads were washed away. T cells were seeded on a recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were LV-MSLN CAR/iPD1/tEGFR, LV-MSLN CAR, respectively. The /iPD1, LV-tEGFR, or no-load (LV-GFP) transduction process is as described in Example 1. The transduced T cells were cultured in RPMI-1640 medium and induced for amplification for 7-10 days with recombinant human IL-2 factor (100 ng/ml; purchased from R&D Systems), followed by a functional test. The inventors measured the killing effect of T cells (effector cells) transduced with different lentiviruses on mesothelioma target cells (MSLN + MSTO-211H) with high expression of MSLN. The ratio of target cells was 50:1, 25:1 or 10:1, the measurement method uses a standard 4 - hour 51 chromium release method, and the 4 - hour 51 chromium release method is as described in Example 1. The result is shown in Figure 3. As shown in Figure 3, T lymphocytes transduced with anti-MSLN chimeric antigen receptor, PD1 shRNA (iPD1) and non-functional EGFR lentivirus (LV-MSLN-CAR/iPD1/M) were co-expressed to express anti-MSLN inlays. T lymphocytes combined with antigen receptor and PD1 shRNA (iPD1) T lymphocytes (LV-MSLN-CAR/iPD1) are also effective in killing mesothelioma target cells with high expression of MSLN. Non-functional EGFR lentiviral transduced T lymphocytes (LV-M T lymphocytes) have no significant killing effect on mesothelial cells with high expression of MSLN. T lymphocyte killing of T lymphocytes (LV-MSLN-CAR) expressing only the anti-MSLN chimeric antigen receptor has the ability to kill mesenchymal target cells with high expression of MSLN, but weaker than co-expression of anti-MSLN chimeric antigen T lymphocytes of T1 lymphocytes (LV-MSLN-CAR/iPD1) of PD1 shRNA (iPD1). These results indicate that T lymphocytes co-expressing PD1 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor can effectively kill tumor cells, and co-expression of non-functional EGFR markers does not affect the killing effect of anti-MSLN CAR lymphocytes on mesothelioma cells. .
实施例5共表达PD1 shRNA、无功能EGFR和抗MSLN嵌合抗原受体的T细胞，共表达CTLA4 shRNA、无功能EGFR和抗MSLN嵌合抗原受体的T细胞，共表达CBL-B shRNA、无功能EGFR和抗MSLN嵌合抗原受体的T细胞，共表达PD1 shRNA、CTLA4 shRNA、无功能EGFR和抗MSLN嵌合抗原受体的T细胞的细胞，共表达PD1 shRNA、CBL-B shRNA、抗MSLN嵌合抗原受体和无功能EGFR的T细胞，溶解能力增强并且具有细胞因子分泌更多和细胞增殖更强的特点Example 5 Co-expressing PD1 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor T cells, co-expressing CTLA4 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor T cells, co-expressing CBL-B shRNA, Non-functional EGFR and anti-MSLN chimeric antigen receptor T cells, co-expressing PD1 shRNA, CTLA4 shRNA, non-functional EGFR and anti-MSLN chimeric antigen receptor T cells, co-expressing PD1 shRNA, CBL-B shRNA, Anti-MSLN chimeric antigen receptor and non-functional EGFR T cells, with enhanced solvency and more cytokine secretion and stronger cell proliferation
在本实施例中，发明人还考察了共表达1个shRNA(CBL-B shRNA或PD1 shRNA或CTLA4 shRNA)、无功能EGFR和抗MSLN嵌合抗原受体的T细胞，共表达2个shRNA(针对不同PD1区域的2个PD1 shRNA序列,PD1 shRNA和CBL-B shRNA或PD1 shRNA和CTLA4 shRNA)、无功能EGFR和抗MSLN嵌合抗原受体的淋巴细胞的肿瘤溶解能力、细胞因子分泌能力和细胞增殖能力。上述T细胞，比单独表达抗MSLN嵌合抗原受体的T细胞的细胞溶解能力增强，细胞因子分泌更多和细胞增殖更强。共表达2个shRNA(针对不同PD1区域的2个PD1 shRNAs,PD1 shRNA和CTLA4 shRNA或PD1 shRNA和CBL-B shRNA)、无功能EGFR和抗MSLN嵌合抗原受体的T细胞比共表达1个shRNA(PD1 shRNA或CBL-B shRNA或CTLA4 shRNA)、无功能EGFR和抗MSLN嵌合抗原受体的T细胞的细胞溶解能力更强，细胞因子分泌更多和细胞增殖更强。In this example, the inventors also examined T cells co-expressing one shRNA (CBL-B shRNA or PD1 shRNA or CTLA4 shRNA), non-functional EGFR and anti-MSLN chimeric antigen receptor, and co-expressed two shRNAs ( Tumor lytic capacity, cytokine secretion capacity of lymphocytes of two PD1 shRNA sequences, PD1 shRNA and CBL-B shRNA or PD1 shRNA and CTLA4 shRNA), non-functional EGFR and anti-MSLN chimeric antigen receptors in different PD1 regions Cell proliferation ability. The above T cells have enhanced cytolysis ability, more cytokine secretion and stronger cell proliferation than T cells expressing the anti-MSLN chimeric antigen receptor alone. Two shRNAs (two PD1 shRNAs, PD1 shRNA and CTLA4 shRNA or PD1 shRNA and CBL-B shRNA for different PD1 regions), T cells with no functional EGFR and anti-MSLN chimeric antigen receptor were co-expressed. T cells (sh1 shRNA or CBL-B shRNA or CTLA4 shRNA), non-functional EGFR and anti-MSLN chimeric antigen receptors have stronger cytolysis ability, more cytokine secretion and stronger cell proliferation.
实施例6无功能EGFR的表达对T细胞的细胞溶解能力、细胞因子分泌能力和细胞增殖能力的影响Example 6 Effect of expression of non-functional EGFR on cytolysis, cytokine secretion and cell proliferation of T cells
在本实施例中，发明人考察了表达无功能EGFR对T淋巴细胞的细胞溶解能力、细胞因子分泌能力和细胞增殖能力的影响。发明人发现，共表达2个shRNA(2个PD1 shRNAs,PD1 shRNA和CBL-B shRNA或PD1 shRNA和CTLA4 shRNA)、抗MSLN嵌合抗原受体和无功能EGFR的T细胞的细胞溶解能力、细胞因子分泌能力和细胞增殖能力与共表达2个shRNA(2 个PD1 shRNAs,PD1 shRNA和CBL-B shRNA或PD1 shRNA和CTLA4 shRNA)和抗MSLN嵌合抗原受体的T细胞相当；共表达1个shRNA(PD1 shRNA或CBL-B shRNA或CTLA4 shRNA)、无功能EGFR和抗MSLN嵌合抗原受体的T细胞的细胞溶解能力、细胞因子分泌能力和细胞增殖能力与共表达1个shRNA(PD1 shRNA或CBL-B shRNA或CTLA4 shRNA)和抗MSLN嵌合抗原受体的T细胞相当；且共表达抗MSLN嵌合抗原受体和无功能EGFR的T细胞与单独表达抗MSLN嵌合抗原受体的T细胞的细胞溶解能力、细胞因子分泌能力和细胞增殖能力相当。由此可见无功能EGFR的表达不影响T淋巴细胞的细胞溶解能力、细胞因子分泌能力和细胞的增殖能力，发明人在T细胞中引入无功能EGFR，从而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 the present example, the inventors examined the effects of expressing non-functional EGFR on the cytolytic ability, cytokine secretion ability, and cell proliferation ability of T lymphocytes. The inventors found that cytosolic ability, cells co-expressing two shRNAs (two PD1 shRNAs, PD1 shRNA and CBL-B shRNA or PD1 shRNA and CTLA4 shRNA), anti-MSLN chimeric antigen receptor and non-functional EGFR T cells Factor secretion capacity and cell proliferation ability and co-expression of 2 shRNAs (2 PD1 shRNAs, PD1 shRNA and CBL-B shRNA or PD1 shRNA and CTLA4 shRNA) are equivalent to T cells against MSLN chimeric antigen receptor; co-expression of 1 shRNA (PD1 shRNA or CBL-B shRNA or CTLA4 shRNA), none The cytosolic ability, cytokine secretion ability and cell proliferative ability of functional EGFR and anti-MSLN chimeric antigen receptor T cells co-expressed with one shRNA (PD1 shRNA or CBL-B shRNA or CTLA4 shRNA) and anti-MSLN chimeric antigen T cells are equivalent; and T cells co-expressing the anti-MSLN chimeric antigen receptor and non-functional EGFR are comparable to the cytostatic ability, cytokine secretion ability and cell proliferation ability of T cells expressing the anti-MSLN chimeric antigen receptor alone. . Thus, it can be seen that the expression of non-functional EGFR does not affect the cytolytic ability, cytokine secretion ability and cell proliferation ability of T lymphocytes, and the inventors introduced non-functional EGFR in T cells, so that the lymphocytes of the examples of the present invention are used. When treating a tumor cell with high expression of MSLN, if the patient develops a serious adverse reaction, the lymphocytes of the present invention can be cleared by the anti-EGFR antibody, thereby improving the safety of the lymphocyte treatment of the tumor patient with high expression of MSLN in the embodiment of the present invention. .
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 the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms is not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, various embodiments or examples described in the specification, as well as features of various embodiments or examples, may be combined and combined.
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, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the scope of the invention. The embodiments are subject to variations, modifications, substitutions and variations.

Claims (29)

  1. 一种T淋巴细胞，其特征在于，所述T淋巴细胞的免疫检查点被沉默；表达无功能EGFR；以及表达嵌合抗原受体，其中，A T lymphocyte characterized in that the immune checkpoint of the T lymphocyte is silenced; expressing a non-functional EGFR; and expressing a chimeric antigen receptor, wherein
    所述嵌合抗原受体包括：The chimeric antigen receptor comprises:
    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间质素；An extracellular region comprising a heavy chain variable region and a light chain variable region of a single chain antibody, the single chain antibody specifically recognizing an antigen interferon;
    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；a transmembrane region, the transmembrane region is linked to the extracellular region, and embedded in a cell membrane of the T lymphocyte;
    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。An intracellular region, the intracellular region is associated with the transmembrane region, and the intracellular region comprises an intracellular portion of CD28 or 4-1BB and a CD3 ζ chain.
  2. 一种慢病毒，其特征在于，所述慢病毒携带下列核酸分子：A lentivirus characterized in that the lentivirus carries the following nucleic acid molecules:
    (a)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；(a) a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1, the nucleic acid molecule encoding the chimeric antigen receptor having SEQ ID NO: Nucleotide sequence;
    (b)沉默细胞免疫检查点的核酸分子，所述沉默细胞免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：3～135的至少之一；以及(b) a nucleic acid molecule that silences a cell immunological checkpoint, the nucleotide sequence of the nucleic acid molecule of the silencing cell immunological checkpoint being at least one selected from the group consisting of SEQ ID NOS: 3 to 135;
    (c)编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：136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：137所示的核苷酸序列。(c) a nucleic acid molecule encoding a non-functional EGFR having the amino acid sequence set forth in SEQ ID NO: 136, the nucleic acid molecule encoding the non-functional EGFR having the nucleotide sequence set forth in SEQ ID NO:137 .
  3. 一种慢病毒，其特征在于，所述慢病毒携带含有SEQ ID NO：138～143所示的核苷酸序列。A lentivirus characterized in that the lentivirus carries a nucleotide sequence represented by SEQ ID NOS: 138 to 143.
  4. 一种转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点被沉默；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：A transgenic lymphocyte characterized in that said lymphocyte immune checkpoint is silenced; expressing a non-functional EGFR; and expressing a chimeric antigen receptor, said chimeric antigen receptor comprising:
    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，An extracellular region comprising a heavy chain variable region and a light chain variable region of an antibody, said antibody being capable of specifically binding to a tumor antigen; a transmembrane region; and an intracellular region, said intracellular region comprising Immune co-stimulatory molecule intracellular segment,
    其中，所述抗体为单链抗体，所述肿瘤抗原为MSLN。Wherein the antibody is a single chain antibody and the tumor antigen is MSLN.
  5. 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点独立地选自CTLA4、PD1、TIM3、BTLA、LAG-3、IRAK-M、SOCS1、A20、CBL-B的至少之一。The transgenic lymphocyte according to claim 4, wherein the lymphocyte immune checkpoint is independently selected from the group consisting of CTLA4, PD1, TIM3, BTLA, LAG-3, IRAK-M, SOCS1, A20, CBL-B. At least one of them.
  6. 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点被沉默是通过shRNA、反义核酸、核酶、显性负突变、CRISPR和锌指核酸酶至少之一实现的。The transgenic lymphocyte according to claim 4, wherein the lymphocyte immune checkpoint is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR and zinc finger nuclease Realized.
  7. 根据权利要求4所述的转基因淋巴细胞，其特征在于，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。The transgenic lymphocyte according to claim 4, wherein the intracellular segment of the immunostimulatory molecule is independently selected from at least 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and at least one of their derivatives. One.
  8. 根据权利要求5所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点独立地选自CTLA4、PD1、CBL-B的至少之一。The transgenic lymphocyte according to claim 5, wherein the lymphocyte immune checkpoint is independently selected from at least one of CTLA4, PD1, CBL-B.
  9. 根据权利要求6所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查 点被沉默是通过shRNA实现的。The transgenic lymphocyte according to claim 6, wherein said lymphocyte immune test The point is silenced by shRNA.
  10. 根据权利要求7所述的转基因淋巴细胞，其特征在于，所述免疫共刺激分子胞内段是4-1BB或CD28的胞内段。The transgenic lymphocyte according to claim 7, wherein the intracellular segment of the immunostimulatory molecule is an intracellular segment of 4-1BB or CD28.
  11. 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞是CD3+T淋巴细胞。The transgenic lymphocyte according to claim 4, wherein the lymphocyte is a CD3 + T lymphocyte.
  12. 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞是自然杀伤细胞。The transgenic lymphocyte according to claim 4, wherein the lymphocyte is a natural killer cell.
  13. 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞是自然杀伤T细胞。The transgenic lymphocyte according to claim 4, wherein the lymphocyte is a natural killer T cell.
  14. 一种构建体，其特征在于，所述构建体包括：A construct characterized in that the construct comprises:
    第一核酸分子，所述第一核酸分子编码嵌合抗原受体；a first nucleic acid molecule encoding a chimeric antigen receptor;
    第二核酸分子，所述第二核酸分子沉默细胞免疫检查点；以及a second nucleic acid molecule that silences a cellular immune checkpoint;
    第三核酸分子，所述第三核酸分子编码无功能EGFR，a third nucleic acid molecule encoding a non-functional EGFR,
    其中，所述细胞免疫检查点、所述嵌合抗原受体、所述无功能EGFR是如权利要求2、4～13任一项中所定义的。Wherein the cellular immunological checkpoint, the chimeric antigen receptor, and the non-functional EGFR are as defined in any one of claims 2, 4 to 13.
  15. 根据权利要求14所述的构建体，其特征在于，所述第一核酸分子、所述第二核酸分子和所述第三核酸分子被设置为在权利要求4～13任一项所述的淋巴细胞中表达所述嵌合抗原受体、沉默细胞免疫检查点和表达无功能EGFR，并且所述嵌合抗原受体与所述无功能EGFR呈非融合形式。The construct according to claim 14, wherein the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are provided in the lymph of any one of claims 4 to 13 The chimeric antigen receptor, the silencing cellular immune checkpoint, and the expression non-functional EGFR are expressed in the cell, and the chimeric antigen receptor is in a non-fused form with the non-functional EGFR.
  16. 根据权利要求14所述的构建体，其特征在于，进一步包括：The construct of claim 14 further comprising:
    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；a first promoter operably linked to the first nucleic acid molecule;
    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；以及a second promoter operably linked to the second nucleic acid molecule;
    第三启动子，所述第三启动子与所述第三核酸分子可操作地连接。A third promoter operably linked to the third nucleic acid molecule.
  17. 根据权利要求16所述的构建体，其特征在于，所述第一启动子、所述第二启动子、所述第三启动子分别独立地选自U6，H1,CMV,EF-1,LTR或RSV启动子。The construct according to claim 16, wherein the first promoter, the second promoter, and the third promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, and LTR. Or the RSV promoter.
  18. 根据权利要求14所述的构建体，其特征在于，进一步包括：The construct of claim 14 further comprising:
    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：144所示的核苷酸序列。An internal ribosome entry site sequence, the internal ribosome entry site sequence disposed between the first nucleic acid molecule and the third nucleic acid molecule, the internal ribosome entry site having SEQ ID NO:144 The nucleotide sequence shown.
  19. 根据权利要求14所述的构建体，其特征在于，进一步包括:The construct of claim 14 further comprising:
    第四核酸分子，设置在所述第一核酸分子与所述第三核酸分子之间，并且所述第四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。A fourth nucleic acid molecule disposed between the first nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encoding a linker peptide capable of being cleaved in the lymphocyte.
  20. 根据权利要求19所述的构建体，其特征在于，所述连接肽具有SEQ ID NO：145所示的氨基酸序列。The construct according to claim 19, wherein the linker peptide has the amino acid sequence set forth in SEQ ID NO:145.
  21. 根据权利要求14所述的构建体，其特征在于，所述构建体的载体是非致病性病毒载体。The construct according to claim 14, wherein the vector of the construct is a non-pathogenic viral vector.
  22. 根据权利要求21所述的构建体，其特征在于，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。The construct according to claim 21, wherein said viral vector comprises a retrovirus selected from the group consisting of At least one of a vector, a lentiviral vector or an adenovirus-associated viral vector.
  23. 一种制备权利要求1所述的T淋巴细胞或者权利要求4～13任一项所述的转基因淋巴细胞的方法，其特征在于，包括：A method of preparing the T lymphocyte of claim 1 or the transgenic lymphocyte of any one of claims 4 to 13, comprising:
    将权利要求14～22任一项所述的构建体或者权利要求2～3任一项所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。The construct according to any one of claims 14 to 22 or the lentivirus according to any one of claims 2 to 3 is introduced into lymphocytes or T lymphocytes.
  24. 一种用于治疗癌症的治疗组合物，其特征在于，包括：A therapeutic composition for treating cancer, comprising:
    权利要求14～22任一项所述的构建体、权利要求2～3任一项所述的慢病毒、权利要求1所述的T淋巴细胞或者权利要求4～13任一项所述的转基因淋巴细胞。The construct according to any one of claims 14 to 22, the lentivirus according to any one of claims 2 to 3, the T lymphocyte according to claim 1, or the transgene according to any one of claims 4 to 13. Lymphocytes.
  25. 根据权利要求24所述的治疗组合物，其特征在于，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。The therapeutic composition according to claim 24, wherein the cancer comprises at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer. .
  26. 一种提高淋巴细胞活性和治疗安全性的方法，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：A method of increasing lymphocyte activity and therapeutic safety, the lymphocyte carrying a chimeric antigen receptor, characterized in that the method comprises:
    使所述淋巴细胞的细胞免疫检查点被沉默,以及Causing the cellular immune checkpoint of the lymphocyte to be silenced, and
    使所述淋巴细胞表达无功能EGFR，Causing the lymphocytes to express non-functional EGFR,
    所述细胞内免疫检查点、所述淋巴细胞、所述嵌合抗原受体、所述无功能EGFR如权利要求2、4～13任一项中所定义的，The intracellular immune checkpoint, the lymphocyte, the chimeric antigen receptor, the non-functional EGFR, as defined in any one of claims 2, 4 to 13,
    所述淋巴细胞活性包括所述淋巴细胞在体外的增殖能力、在肿瘤病人体内的增殖和生存能力以及所述淋巴细胞在肿瘤病人体内的定向杀伤能力的至少一种。The lymphocyte activity includes at least one of proliferative ability of the lymphocytes in vitro, proliferation and viability in a tumor patient, and directional killing ability of the lymphocytes in a tumor patient.
  27. 一种治疗癌症的方法，其特征在于，包括：A method of treating cancer, comprising:
    为癌症患者给药权利要求14～22任一项所述的构建体、权利要求2～3任一项所述的慢病毒、权利要求1所述的T淋巴细胞或者权利要求4～13任一项所述的转基因淋巴细胞，The polypeptide according to any one of claims 14 to 22, the lentivirus according to any one of claims 2 to 3, the T lymphocyte according to claim 1, or any one of claims 4 to 13 is administered to a cancer patient. Transgenic lymphocytes as described in
    其中，嵌合抗原受体特异性结合肿瘤抗原MSLN。Among them, the chimeric antigen receptor specifically binds to the tumor antigen MSLN.
  28. 根据权利要求27所述的方法，其特征在于，包括：The method of claim 27, comprising:
    从癌症患者体内分离淋巴细胞；Isolating lymphocytes from cancer patients;
    将权利要求14～22任一项所述的构建体，或权利要求2～3任一项所述的慢病毒导入所述淋巴细胞，以便获得转基因淋巴细胞，所述转基因淋巴细胞表达嵌合抗原受体和细胞免疫检查点被沉默；以及The construct according to any one of claims 14 to 22, or the lentivirus according to any one of claims 2 to 3, is introduced into the lymphocytes to obtain transgenic lymphocytes, and the transgenic lymphocytes express chimeric antigen Receptors and cellular immune checkpoints are silenced;
    为所述癌症患者给药所述转基因淋巴细胞。The transgenic lymphocytes are administered to the cancer patient.
  29. 根据权利要求28所述的方法，其特征在于，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。 The method according to claim 28, wherein the cancer comprises at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.
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