WO2018137293A1 - Composition thérapeutique pour le traitement d'une tumeur à mésothéline positive - Google Patents

Composition thérapeutique pour le traitement d'une tumeur à mésothéline positive 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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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. .

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Abstract

La présente invention décrit un lymphocyte transgénique, un produit de recombinaison, et une composition thérapeutique pour le traitement d'un cancer. Un point de contrôle cellulaire du lymphocyte transgénique est inactivé, et le lymphocyte transgénique exprime un EGFR non fonctionnel et un récepteur antigénique chimérique. Le récepteur antigénique chimérique comprend un domaine extracellulaire, et le domaine extracellulaire comprend une région variable à chaîne lourde et une région variable à chaîne légère d'un anticorps monocaténaire. L'anticorps monocaténaire reconnaît spécifiquement un antigène, la mésothéline. Le récepteur antigénique chimérique comprend en outre un domaine transmembranaire. Le domaine transmembranaire est relié au domaine extracellulaire et est intégré dans la membrane du lymphocyte T. Le récepteur antigénique chimérique comprend en outre un domaine intracellulaire. Le domaine intracellulaire est relié au domaine transmembranaire et comprend un segment intracellulaire de CD28 ou 4-1 BB et une chaîne CD3ζ.
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US11332713B2 (en) 2016-11-16 2022-05-17 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11261428B2 (en) 2018-03-15 2022-03-01 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11421228B2 (en) 2018-03-15 2022-08-23 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11459544B2 (en) 2018-03-15 2022-10-04 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11608500B2 (en) 2018-03-15 2023-03-21 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US12084682B2 (en) 2018-03-15 2024-09-10 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
WO2020083282A1 (fr) * 2018-10-24 2020-04-30 艾生命序公司 Anticorps pd-l1 sécrétant des lymphocytes car-t anti-mésothéline pour l'immunothérapie antitumorale
US12054531B2 (en) 2020-03-20 2024-08-06 Lyell Immunopharma, Inc. Recombinant cell surface markers
US12123021B2 (en) 2022-03-29 2024-10-22 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy

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