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WO2018137295A1 - Lymphocyte transgénique co-exprimant le récepteur antigénique chimérique anti-msln et molécule inhibitrice de point de contrôle immunitaire et leur utilisation - Google Patents

Lymphocyte transgénique co-exprimant le récepteur antigénique chimérique anti-msln et molécule inhibitrice de point de contrôle immunitaire et leur utilisation Download PDF

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WO2018137295A1
WO2018137295A1 PCT/CN2017/081274 CN2017081274W WO2018137295A1 WO 2018137295 A1 WO2018137295 A1 WO 2018137295A1 CN 2017081274 W CN2017081274 W CN 2017081274W WO 2018137295 A1 WO2018137295 A1 WO 2018137295A1
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lymphocyte
lymphocytes
cancer
chimeric antigen
transgenic
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严勇朝
朱益林
陈思毅
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Beijing Marino Biotechnology Pty Ltd
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Beijing Marino Biotechnology Pty Ltd
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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 A method of cell viability.
  • 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 and a nucleic acid molecule encoding a chimeric antigen receptor, and a transgenic lymphocyte formed by the introduction of the construct, encoded by The chimeric antigen receptor 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. It has weaker killing of mesothelial cells with normal MSLN expression levels.
  • the invention proposes a T lymphocyte.
  • the cellular immune checkpoint of the T lymphocyte is silenced; and the chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region including a single a heavy chain variable region and a light chain variable region of a chain antibody, the single chain antibody specifically recognizing an antigen MSLN; a transmembrane region, the transmembrane region being linked to the extracellular region, and embedded in the T lymphocyte In the cell membrane of the cell; an intracellular region that is linked to the transmembrane region, and the intracellular region includes the intracellular portion of CD28 and the CD3 ⁇ chain.
  • the cellular immune checkpoint includes an immune checkpoint on at least one of a cell surface or a cell.
  • 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 tumor cells with high expression of MSLN, which has a significant directional killing effect.
  • the invention proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1, the coding chimera
  • the nucleic acid molecule of the antigen receptor has the nucleotide sequence shown in SEQ ID NO: 2; and the nucleic acid molecule of the silencing cell immunological checkpoint, the nucleotide sequence of the nucleic acid molecule of the silencing cell immunological checkpoint is selected from the group consisting of SEQ ID NO: at least one of 3 to 135.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the present invention into lymphocytes have the characteristics of resisting tumor cell-mediated immunosuppression, proliferative ability in vitro, proliferation and viability in tumor patients.
  • the killing ability of tumor cells is significantly enhanced, especially for tumor cells with high expression of MSLN.
  • the invention proposes a lentivirus.
  • the lentivirus carries a nucleotide sequence as set forth in SEQ ID NO: 136, 137, 138, 139, 140 or 141.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the present invention into lymphocytes have the characteristics of resisting tumor cell-mediated immunosuppression, proliferative ability in vitro, proliferation and viability in tumor patients.
  • the killing ability of tumor cells is significantly enhanced, especially for tumor cells with high expression of MSLN.
  • the invention provides a transgenic lymphocyte.
  • the lymphocyte immune checkpoint is silenced; and a chimeric antigen receptor is expressed, the chimeric antigen receptor comprising: an extracellular region comprising a heavy chain of the antibody a variable region and a light chain variable region, 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 said antibody is a single A chain antibody, the tumor antigen is MSLN.
  • the inventors have surprisingly found that the cell immunological checkpoint is silenced and the in vitro proliferation ability of lymphocytes expressing the chimeric antigen receptor against MSLN, the proliferation and viability in tumor patients, and the specificity of tumor cells in tumor patients. Sexual killing ability is greatly improved, especially for tumor cells with high expression of MSLN.
  • 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, PD1, TIM3, BTLA, LAG-3, IRAK-M, SOCS1, A20, CBL-B.
  • CTLA4, PD1, TIM3, BTLA, LAG-3 are cell surface immune checkpoints
  • IRAK-M, SOCS1, 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.
  • the successful silencing of the epidemic checkpoint further enhances the efficacy of transgenic lymphocytes against tumor-mediated immunosuppression.
  • the transgenic lymphocytes are expanded in vitro and proliferated and viable in tumor patients, and the targeted killing effect on tumor cells is further strengthened. .
  • the lymphocyte cell surface immunological 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 to the tumor cell.
  • Directional killing effect is beneficial to be used.
  • 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, so that the transgenic lymphocyte proliferation of the embodiment of the present invention has a targeted killing effect on the tumor.
  • the effect is more remarkable; the combination of 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 makes the proliferative ability of the transgenic lymphocytes of the embodiment of the present invention and the targeted killing effect on the tumor more remarkable.
  • the lymphocyte immune checkpoints are CTLA4, PD1, CBL-B.
  • CTLA4 and PD1 are cell surface immune checkpoints
  • CBL-B is an intracellular immune checkpoint.
  • the lymphocyte cell surface immunological checkpoint CTLA4 or PD1 is silenced, or the lymphocyte intracellular immune checkpoint is silenced by CBL-B, preventing the expression of PD1 or CTLA4 molecules from correspondingly
  • the combination of PD-L1 and PD-L2 or CD80 and CD86 effectively inhibits the inability or apoptosis of T lymphocytes, or enhances T cell receptor signaling through CBL-B silencing, making transgenic lymphocytes in tumors
  • the proliferative and viability of the patient is further improved, and the effect of directed killing of the tumor is more significant.
  • shRNA of the embodiment of the present invention carries a shRNA which specifically silences at least one immunological checkpoint on the cell surface or in the cell, and the shRNA of the embodiment of the present invention has a highly efficient and specific silencing cell surface or at least intracellular cells.
  • the negative regulation mechanism of T lymphocyte incompetence or apoptosis, etc. further enhances the proliferation and viability of the transgenic lymphocytes of the present invention in tumor patients, and cooperates with the antigen targeting of the chimeric antigen receptor, thereby making The effect of the transgenic lymphocytes of the embodiments of the present invention on the targeted killing effect of tumors is more remarkable.
  • 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 lymphocytes are CD3 + T lymphocytes or natural killer cells or natural killer T cells.
  • the cellular immunological checkpoint of the lymphocytes of the present invention is silenced while expressing an antigen-specific chimeric antigen receptor, such as the MSLN antigen-specific chimeric antigen receptor of the embodiment of the present invention, the lymphocyte.
  • an antigen-specific chimeric antigen receptor such as the MSLN antigen-specific chimeric antigen receptor of the embodiment of the present invention.
  • the invention proposes a construct.
  • the construct comprises: a first nucleic acid molecule encoding a chimeric antigen receptor; and a second nucleic acid molecule, the second nucleic acid molecule silencing a cellular immune checkpoint.
  • the cellular immunological checkpoint and the chimeric antigen receptor are as described above.
  • the construct of the embodiment of the present invention can effectively silence at least one immunological checkpoint on the cell surface or in the cell and the antigen-specific chimeric antigen receptor after being successfully introduced into the lymphocyte of the embodiment of the present invention.
  • the directional killing effect of the lymphocytes of the embodiments of the present invention on tumor cells, especially tumor cells highly expressing MSLN is more remarkable.
  • 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 are arranged in a lymphocyte immunological checkpoint and express the chimeric antigen receptor in the lymphocytes described above.
  • the lymphocytes of the first nucleic acid molecule and the second nucleic acid molecule are successfully set, and the immunological checkpoint of at least one of the cell surface of the lymphocyte or the cell is successfully silenced, and succeeds on the surface of the lymphocyte.
  • An antigen-specific chimeric antigen receptor such as the MSLN-specific chimeric antigen receptor of the present invention, is expressed, which has a more lethal and specific tumor killing effect.
  • the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; and a second promoter, the second promoter and The second nucleic acid molecule is operably linked.
  • the introduction of the first promoter and the second promoter enables the first nucleic acid molecule and the second nucleic acid molecule to be independently expressed, thereby effectively ensuring the biological effect of the chimeric antigen receptor antigen targeting.
  • the lymphocyte targeting effect of the embodiment of the present invention is stronger, and the killing effect on the tumor, especially the targeted killing of the tumor cell with high expression of MSLN, is more remarkable.
  • the first promoter and the second 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 high specificity, thereby ensuring efficient silencing of the cellular immune checkpoint and efficient expression of the chimeric antigen receptor, thereby implementing the present invention.
  • the in vitro proliferation ability of lymphocytes in the tumor, the proliferation and survival ability in tumor patients are greatly improved, and the targeted killing effect on tumors is more remarkable.
  • 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 improving the cellular immune checkpoint.
  • the high expression of silencing and chimeric antigen receptors in lymphocytes makes lymphocyte proliferation in vitro, proliferation and viability in tumor patients greatly improved, lymphocyte targeting is further enhanced, and the killing effect on tumor cells is further enhanced.
  • 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 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 lentivirus is successfully introduced into the above lymphocytes or T lymphocytes, and the cellular immunological examination of lymphocytes is silenced and the expression of the chimeric antigen receptor is achieved, thereby preparing the transgenic lymphocytes prepared by the preparation method of the present invention.
  • the proliferation of T lymphocytes in tumor patients and in vitro and the survival ability of tumor patients are greatly improved, and transgenic lymphocytes or T lymphocytes have stronger targeted killing effects on tumor cells, especially tumor cells with high expression of MSLN.
  • 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 efficient expression of chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, thereby
  • the obtained 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, and the target of transgenic lymphocytes or T lymphocytes to tumor cells is improved.
  • the killing effect 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 remarkably enhanced.
  • 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 efficiently express antigen-specific chimeric antigen receptors on the surface of lymphocyte cells or intracellular immunological checkpoints, as in the embodiment of the present invention.
  • the MSLN antigen-specific chimeric antigen receptor, the resulting lymphocytes or T lymphocytes have significant resistance to tumor cell-mediated immunosuppression, and the viability in the microenvironment of the tumor is greatly enhanced, and the resulting lymphocytes or T lymphocytes are obtained.
  • the invention provides a method of increasing lymphocyte activity, the lymphocyte carrying a chimeric antigen receptor, according to an embodiment of the invention, the method comprising: causing the lymphocyte The cellular immune checkpoint was silenced.
  • the cellular immune checkpoint, the lymphocyte, and the chimeric antigen receptor are as defined above, and the lymphocyte activity includes the ability of the lymphocyte to proliferate in vitro, proliferation and viability in a tumor patient, 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 lymphocytes of the examples are expanded in vitro, the proliferation in tumor patients and the survival ability of tumor patients are greatly improved, and the silencing of the lymphocyte cell immunological checkpoints cooperates with the antigen-specific efficacy of the lymphocyte chimeric antigen receptor, thereby realizing It is effective against tumor cell-mediated immunosuppression and significantly enhances the targeted killing effect on tumor cells with high expression of MSLN.
  • the above method for increasing lymphocyte activity may further comprise at least one of the following additional technical features:
  • the tumor 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 specifically express MSLN.
  • the method for increasing the activity of lymphocytes in the embodiment of the invention enables the lymphocytes to express the chimeric antigen receptor specific to the MSLN antigen, and at the same time, the immune checkpoint of the lymphocytes is silenced, and the method for increasing lymphocyte activity in the embodiment of the invention is further
  • the targeted killing ability of tumor cells with high expression of MSLN such as mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer or breast cancer cells, is improved.
  • 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.
  • 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 it is good.
  • 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 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 lentiviral vector which co-expresses a chimeric antigen receptor specific for MSLN antigen and silences a human cell immunological checkpoint according to an embodiment of the present invention
  • FIG. 2 is a graph showing the results of enhancing the proliferative ability of a chimeric antigen receptor specific for MSLN antigen and a lymphocyte silencing PD1 according to an embodiment of the present invention
  • Figure 3 is a graph showing the results of co-expression of a chimeric antigen receptor specific for MSLN antigen and lymphocytes silencing PD1 with increased secretion of interferon- ⁇ ;
  • Figure 4 is a graph showing the results of co-expression of a MSLN antigen-specific chimeric antigen receptor and PD1-silent lymphocytes, which enhances the ability 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; and a chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region, and the extracellular region includes a single strand The heavy chain variable region and the light chain variable region of the 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 ⁇ chain.
  • the cellular immune checkpoint includes an immune checkpoint on the cell surface or within the cell.
  • the T lymphocyte or transgenic lymphocyte cell immunological checkpoint of the embodiment of the present invention is silenced to jointly express the chimeric antigen receptor specific to the MSLN antigen, and the T lymphocyte or the transgenic lymphocyte of the embodiment of the present invention is in vivo and in vitro of the tumor patient. Proliferation and Survivability and the ability to kill specific tumor cells in tumor patients are significantly enhanced, especially for tumor cells that express MSLN efficiently.
  • 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 (PD1) is expressed on activated CTLs, which interact with the programmed death ligand 1 (PD-L1) expressed on tumor cells to inhibit anti-tumor T cell responses. .
  • cytotoxic T lymphocyte antigen 4 (CTLA4) of the present invention is a key negative regulator of another T cell, which inhibits T cell activation by binding to a ligand B7.1 expressed on antigen presenting cells, 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.
  • 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 the cells expressing the antigen MSLN, and the specific binding effect of the antigen antibodies is stronger, and the orientation of the transgenic lymphocytes to the tumor cells expressing the MSLN antigen in the embodiment of the present invention is greatly improved. 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, PD1, TIM3, BTLA.
  • At least one of LAG-3 the lymphocyte intracellular immune checkpoint is independently selected from at least one of IRAK-M, SOCS1, A20, and 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.
  • the lymphocyte cell surface immunological checkpoint of the embodiment of the present invention is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, zinc finger nuclease, and CRISPR. .
  • siRNA small interfering RNA
  • siRNA is A small RNA molecule (composed of 21-25 nucleotides), which is processed by Dicer (an enzyme that specifically cleaves double-stranded RNA in the RNAaseIII family); siRNA plays a central role in the RNA silencing pathway, Specific messenger RNA (mRNA) is degraded and regulated at the transcriptional level.
  • Dicer an enzyme that specifically cleaves double-stranded RNA in the RNAaseIII family
  • 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 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.
  • RNA double-strand break
  • the shRNA, the antisense nucleic acid, the ribozyme, the dominant negative mutation, the CRISPR, and the 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 Specific experimental purposes and conditions, such as at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR or zinc finger nuclease employed in the embodiments of the present invention, achieve specific silencing of the gene of interest.
  • 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 present invention is designed to be homologous to the coding region of PD1 or CTLA4 or CBL-B mRNA, and to inhibit gene expression by degradation of mRNA.
  • 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 is well designed. The matching points in the 3'UTR cell gene are avoided; proper chain selection is ensured.
  • 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 directional killing effect on tumors with high expression of MSLN is more significant.
  • 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, The proliferation and viability of tumor patients are further improved, and the effect of directed 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 immune checkpoint is silenced and the chimeric antigen receptor is expressed, so that the cellular immunity of the lymphocytes is more targeted and killing, and the killing effect on the tumor cells is more remarkable.
  • the invention proposes a lentivirus or construct.
  • the lentivirus or construct carries a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1 encoding a chimeric antigen receptor
  • the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 2; and 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 is selected from the group consisting of SEQ ID NO: at least one of 3 to 68, wherein the nucleotide sequence of the nucleic acid molecule which silences the intracellular immunological checkpoint is at least one selected from the group consisting of SEQ ID NOS: 69 to 135.
  • 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 ⁇ 46 is human T cell immunoglobulin adhesion Protein molecule 3 (TIM3) siRNA sequence, SEQ ID NOs: 47-57 are human T lymphocyte attenuation factor (BTLA) siRNA sequences, and SEQ ID NOs: 58-68 are human lymphocyte activation gene 3 protein (LAG-3) siRNA Sequence, SEQ ID NOs: 69-85 are human IRAK-M (interleukin-1 receptor-associated kinase 3) siRNA nucleotide sequences, and SEQ ID NOs: 86-96 are human SOCS1 (cytokine signaling inhibitors) 1) siRNA sequences, SEQ ID NOs: 97-116 are human A20 (tumor necrosis factor- ⁇ -inducible protein A
  • the lentivirus or construct of an embodiment of the invention carries a nucleotide sequence as set forth in SEQ ID NO: 136, 137, 138, 139, 140 or 141.
  • SEQ ID NO: 136 is a nucleic acid molecule (MSLN-CAR/iPD1) co-expressing an anti-MSLN chimeric antigen receptor and a silencing cellular immunological checkpoint PD-1
  • SEQ ID NO: 137 is a co-expressing anti-MSLN chimeric antigen Receptor and silencing cellular immunological checkpoint CBL-B nucleic acid molecule (MSLN-CAR/iCBL-B)
  • SEQ ID NO: 138 is a nucleic acid molecule that co-expresses the anti-MSLN chimeric antigen receptor and silences the cellular immunological checkpoint CTLA4 ( MSLN-CAR/iCTLA4)
  • SEQ ID NO: 139 is a nucleic acid molecule
  • the transgenic lymphocytes obtained by introducing the lentivirus of the present invention into lymphocytes are specifically silenced by the cell immunological checkpoint PD1 or CTLA4 or CBL-B, and the chimeric antigen against MSLN is subjected to
  • the expression of the transgenic lymphocytes has significant anti-tumor-mediated immunosuppressive effects, and its anti-apoptotic ability and proliferative ability are enhanced, and the directional killing ability is significantly improved, so that the transgenic lymphocytes can proliferate in vitro and in vivo in tumor patients.
  • the viability and the ability to kill in tumor patients are greatly improved, especially for tumor cells with high expression of MSLN.
  • the inventors realize that the above-mentioned cell chimeric antigen receptor and surface or intracellular immunological checkpoint shRNA are independently expressed, respectively, wherein the expression herein refers to a protein. of Expression also refers to RNA transcription.
  • Promoter a first promoter operably linked to a nucleic acid molecule encoding a chimeric antigen receptor; and a second promoter operably linked to a nucleic acid molecule that silences a cellular immunological checkpoint .
  • the first promoter and the second promoter employed are each independently selected from the group consisting of U6, CMV, H1, EF-1, LTR, RSV promoter, introduction of the first and second promoters,
  • the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule of the silencing cell immunological checkpoint are independently expressed, thereby effectively silencing the cell surface or the intracellular immune checkpoint, and ensuring efficient expression of the chimeric antigen receptor, thereby
  • the survival rate of lymphocytes in the tumor environment is greatly improved, the targeting effect of lymphocytes is stronger, and the specific killing effect on tumors is more significant.
  • a third promoter may be further introduced, the third promoter being independently selected from at least one of U6, CMV, H1, EF-1, LTR, RSV promoter, third promoter and silence
  • the nucleic acid molecule of the immunological checkpoint of the cell is operably linked, and the nucleic acid molecule of the immunoassay of the silencing cell to which the third promoter and the second promoter are linked is different, and the third promoter and the second promoter respectively initiate silencing of different immunological tests.
  • Point shRNA Point shRNA.
  • the introduction of the first or second promoter or the further third promoter described above allows the cell surface or intracellular immunological checkpoint to be efficiently expressed on the transgenic lymphocyte membrane of the present invention by high-efficiency silencing and chimeric antigen receptor.
  • the immunological regulation of the immune checkpoint is effectively inhibited and the biological effect of the chimeric antigen receptor is ensured, so that the survival rate of lymphocytes in the tumor environment is greatly improved, and the targeted killing effect of lymphocytes is more remarkable.
  • 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.
  • the viral vector of the construct of the embodiment of the invention is 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 broad-spectrum and high-efficiency infection efficiency, so that the cell surface or intracellular immunological checkpoint is highly efficiently silenced and the chimeric antigen receptor is highly expressed in lymphocytes, and the embodiment of the present invention
  • the proliferation and viability of lymphocytes in tumor patients are greatly enhanced, the targeting of lymphocytes is further enhanced, and the killing effect on tumor cells is more significant.
  • 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 and then secreted In the 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 the coding for geneticin, hygromycin, and ampicillin Nucleotide sequence such as hormone 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) as well as chimeric antigen receptors (CAR).
  • shRNA small hairpin RNA
  • CAR chimeric antigen receptors
  • the small hairpin RNA carrying the siRNA silencing PD1 or CTLA4 or CBL-B and the viral vector or plasmid expressing the chimeric antigen receptor (CAR) are complexed in the embodiment of the present invention, and the viral vector or plasmid can bind to the polymer or Other materials 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, thereby
  • the obtained lymphocytes or T lymphocytes have remarkable 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, and lymphocytes or T lymphocytes are especially suitable for tumor cells. Tumor cells with high expression of MSLN have a stronger targeted killing effect.
  • 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. Therefore, the obtained transgenic lymphocytes or T lymphocytes are expanded in vitro, the proliferation in tumor patients and the survival ability of tumor patients are greatly improved, and the targeted killing effect of transgenic lymphocytes or T lymphocytes on tumor cells with high expression of MSLN is obtained. Stronger.
  • 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 transgenes to silence PD1 or CTLA4 or CBL-B and express a unique anti-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.
  • Silencing PD1 or CTLA4 or CBL-B and expressing specific methods for antigenic MSLN chimeric antigen receptors 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 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 following biological effects silencing of cellular immune checkpoints, combined with high expression of chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, greatly increasing the viability of the resulting lymphocytes or T lymphocytes in the microenvironment of the above tumors
  • the lymphocyte or T lymphocyte has a stronger targeted killing effect on tumor cells, especially the above-mentioned tumor cells with high expression of MSLN.
  • the invention provides a method of increasing the activity of lymphocytes, the lymphocytes of the embodiments of the invention carrying a chimeric antigen receptor, according to an embodiment of the invention, the method comprising: causing the lymph The cellular immune checkpoint of the cell is silenced, and the cellular immune checkpoint, lymphocyte, and chimeric antigen receptor are 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 cellular immune checkpoint of the lymphocytes of the embodiment of the present invention is silenced, the 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.
  • 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, in particular, has targeted killing effect on tumor cells with high expression of MSLN, thereby effectively treating cancer, and the therapeutic effect is good.
  • 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 it is good.
  • 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 the embodiment of the present invention can make lymphocyte cell immune checkpoint
  • the chimeric antigen receptor is silenced and expressed by a cell, such as the MSLN antigen-specific chimeric antigen receptor of the present invention, and the obtained lymphocyte or T lymphocyte has mesothelioma, pancreatic cancer, ovarian cancer which specifically expresses 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 supernatant was collected at 24 hours and 48 hours, respectively, and centrifuged for 2 hours using an ultracentrifuge at 28,000 rpm (the centrifuge rotor was Beckman SW 32Ti, available from Beckman Coulter, Brea, 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, each well was plated with 0.5 ⁇ 10 6 T For lymphocytes, 0.75 ml of the above-mentioned resuspended virus supernatant and Polybrene (concentration of 8 ⁇ g/ml) were added to each well of cells.
  • 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 10 6 to 1 x 10 6 /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).
  • T cells (number of cells are 1 ⁇ 10 6 /well) that are non-transduced or transduced with chimeric antigen receptor plasmid and human pleural mesothelioma cells, human pleural mesothelioma Cells (from ATCC) were co-cultured, and the proportion of different target cells was changed during the experiment.
  • the production of cytokines in the cell supernatant was determined using a specific enzyme-linked immunosorbent assay (cytokine enzyme-linked immunosorbent assay kit, purchased from R&D Systems, Inc., Minneapolis, MN, USA). The above cell supernatant was taken from the supernatant of cells after 24 hours, 48 hours, and 72 hours of culture, and the results were used to measure the yield of a representative cytokine (interferon- ⁇ ) (IFN ⁇ ).
  • IFN ⁇ interferon- ⁇
  • the measurement procedure was as follows: 100 ⁇ l/well of a cytokine dilution solution (such as IFN ⁇ ) or a supernatant solution of the test cell to be tested was added to the plate, and the plate was placed at room temperature for 2 hours. After 2 hours, the solution in the plate was discarded and the plate was rinsed with 400 ⁇ l of the washing solution and rinsed four times. After rinsing, 200 microliters of enzyme-linked anti-cytokine antibody was added to each well of the plate. Continue to stand at room temperature for 2 hours, then add 200 microliters of substrate solution to each well. After the addition of the substrate solution, the plate was allowed to stand at room temperature for 30 minutes, after which 50 ⁇ l of the termination reaction solution was added to each well. The optical density of each well of the microplate was measured within 30 minutes. The microplate reader was set at 450 nm.
  • 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 51Cr 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 (T:E) 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 (from ATCC)).
  • Example 2 Construction of a vector for co-expression of a silencing cell immunological checkpoint shRNA and an 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. Lentiviral plasmid with antigen receptor (LV-MSLN) CAR).
  • Lentiviral plasmid with antigen receptor (LV-MSLN) CAR Lentiviral plasmid with antigen receptor
  • FIG. 1 is a schematic representation of a lentiviral vector comprising a sequence encoding an anti-MSLN chimeric antigen receptor, a U6 or H1 promoter sequence, a PD1 shRNA or a CBL-B shRNA or a CTLA4 shRNA sequence.
  • the sequence of the anti-MSLN chimeric antigen receptor is under the initiation of the promoter EF-1, and the CTLA4, PD1 or CBL-B shRNA sequence is under the initiation of promoter U6 or H1.
  • T lymphocytes co-expressing PD1 shRNA and anti-MSLN chimeric antigen receptor have higher cell proliferation ability
  • 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. Two to seven days after lentiviral vector transduction, transduced T cells (number of cells were 1 ⁇ 10 6 /well) were co-cultured with MSLN + MSTO-211H, and after 4 days, the number of cells was detected by flow cytometry. . The experimental results are shown in Figure 2.
  • Figure 2 shows that the number of T lymphocytes transduced with LV-MSLN CAR/iPD1 was significantly increased compared to the number of T lymphocytes transduced with LV-MSLN CAR or LV-GFP.
  • the labels represent the mean ⁇ SD of every 3 wells. (P ⁇ 0.05; LV-MSLN CAR/iPD1vs.LV-MSLN CAR).
  • T lymphocytes co-expressing PD1 shRNA and anti-MSLN chimeric antigen receptor have more cytokine secretion characteristics
  • T lymphocyte activator magnetic beads CD3/CD28 the activated T lymphocytes were transduced by lentiviral vector and expanded in vitro, as described in Example 1. 2-7 days after lentiviral vector transduction, transduced T cells (number of cells were 1 ⁇ 10 6 /well) were co-cultured with MSLN + MSTO-211H cells, and after 4 days, cytokine secretion was detected by ELISA. .
  • the experimental results are shown in Figure 3.
  • FIG. 3 shows that T lymphocytes transduced with LV-MSLN CAR/iPD1 secrete more IFN ⁇ than T lymphocytes transduced with LV-MSLN CAR or empty LV-GFP (P ⁇ 0.05; LV-MSLN CAR /iPD1vs.LV-MSLN CAR). This indicates that T lymphocytes transduced with LV-MSLN CAR/iPD1 have significantly increased cytokine production capacity compared to T lymphocytes transduced with LV-MSLN CAR.
  • Example 5 Enhanced lysis of T lymphocyte tumor cells with PD1 shRNA 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
  • the T cells were seeded on a culture dish containing recombinant fibronectin fragments (FN ch-296; Retronectin) and transduced with lentivirus, and the lentiviruses were LV-MSLN CAR/iPD1, LV-MSLN CAR or empty.
  • the loading (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 MSLN + MSTO-211H target cells.
  • the target cell ratio was 1:25 or 1:5, and the standard method was 4–hour 51 chrome. Release method, 4 - hour 51 chromium release method as described in Example 1.
  • the result is shown in Figure 4.
  • T lymphocytes co-expressing the anti-MSLN chimeric antigen receptor and PD1 shRNA (iPD1) can kill MSLN + MSTO- more effectively than T lymphocytes expressing the anti-MSLN chimeric antigen receptor alone.
  • iPD1 shRNA iPD1 shRNA
  • Example 6 Co-expressing CBL-B shRNA and T-cell against anti-MSLN chimeric antigen receptor, co-expressing CTLA4 shRNA and T-cell against anti-MSLN chimeric antigen receptor, co-expressing PD1 shRNA, CBL-B shRNA and anti-MSLN chimeric antigen Receptor T cells, which co-express PD1 shRNA, CTLA4 shRNA and T cells against the MSLN chimeric antigen receptor, have enhanced solvency and are characterized by more cytokine secretion and stronger cell proliferation.
  • the inventors also examined T cells co-expressing CBL-B shRNA and anti-MSLN chimeric antigen receptor, T cells co-expressing CTLA4 shRNA and anti-MSLN chimeric antigen receptor, and co-expressing two shRNAs ( Tumor lytic capacity, cytokine secretion ability, and cell proliferation ability of PD1 shRNA and CBL-B shRNA or PD1 shRNA and CTLA4 shRNA or 2 PD1 shRNAs against different PD1 regions) and T cells against the MSLN chimeric antigen receptor.
  • the experimental procedure was the same as in Examples 3, 4 and 5.
  • T cells have enhanced cytolysis ability, more cytokine secretion and stronger cell proliferation than T cells expressing the anti-MSLN chimeric antigen receptor alone.
  • Co-expressing 2 shRNAs (PD1 shRNA and CTLA4 shRNA or PD1 shRNA and CBL-B shRNA or 2 PD1 shRNAs against different PD1 regions) and anti-MSLN chimeric antigen receptor T cells co-express 1 shRNA (PD1 shRNA or CBL-B shRNA) T cells with or with CTLA4 shRNA) and anti-MSLN chimeric antigen receptors are more cytosolic, with more cytokine secretion and stronger cell proliferation.

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Abstract

L'invention concerne un lymphocyte transgénique, une construction et une composition thérapeutique pour le traitement du cancer. Un point de contrôle immunitaire cellulaire du lymphocyte transgénique est inhibé, et le lymphocyte transgénique exprime un récepteur antigénique chimérique. Le récepteur antigénique chimérique comprend des domaines extracellulaire, trans-membranaire et intracellulaire. Le domaine extracellulaire comprend une région variable de chaîne lourde et une région variable de chaîne légère d'un anticorps à chaîne unique. L'anticorps à chaîne unique reconnaît spécifiquement l'antigène MSLN. Le domaine trans-membranaire est relié au domaine extracellulaire et est intégré à la membrane du lymphocyte T. Le domaine intracellulaire est relié au domaine trans-membranaire et comprend un segment intracellulaire du CD28 et une chaîne CD3ζ.
PCT/CN2017/081274 2017-01-25 2017-04-20 Lymphocyte transgénique co-exprimant le récepteur antigénique chimérique anti-msln et molécule inhibitrice de point de contrôle immunitaire et leur utilisation Ceased WO2018137295A1 (fr)

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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
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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
US12123021B2 (en) 2018-03-15 2024-10-22 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US12280111B2 (en) 2018-03-15 2025-04-22 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
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WO2020191336A1 (fr) * 2019-03-21 2020-09-24 Yamaguchi, Yukiko Composition et procédé pour réduire l'expression d'inhibiteurs de points de contrôle dans des lymphocytes t exprimant un car ou un ctl

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