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WO2017120998A1 - Composition thérapeutique pour traiter un glioblastome - Google Patents

Composition thérapeutique pour traiter un glioblastome Download PDF

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WO2017120998A1
WO2017120998A1 PCT/CN2016/073482 CN2016073482W WO2017120998A1 WO 2017120998 A1 WO2017120998 A1 WO 2017120998A1 CN 2016073482 W CN2016073482 W CN 2016073482W WO 2017120998 A1 WO2017120998 A1 WO 2017120998A1
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lymphocyte
lymphocytes
nucleic acid
acid molecule
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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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

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 and method for treating cancer, and a A method of increasing lymphocyte activity and therapeutic safety.
  • Glioblastoma is the most malignant brain tumor, accounting for ⁇ 81% of central nervous system malignancies.
  • the incidence of malignant glioma is 3-6/100,000.
  • the annual death toll in China is 30,000.
  • Malignant gliomas have invasive growth, and there is no obvious boundary with normal brain tissue, and most of them are not limited to one brain lobe, and the brain tissue is deeply damaged by fingers, and the surgery cannot be completely removed.
  • the most aggressive treatment is used before the mother, the median survival time is still only ⁇ 15 months. Radiotherapy alone, only less than 1% of patients can survive for 5 years, even if new radiotherapy and temozolomide (TMZ) chemotherapy The combined program has only allowed nearly 10% of patients to survive for more than 5 years.
  • TMZ temozolomide
  • EGFR epidermal growth factor receptor mutant EGFRvIII (267 amino acids of the extracellular domain of EGFR were removed, which are the second to seventh exons of the EGFR gene (275 to 1075 nucleotides)
  • the gene encoded by the removal is expressed in about 30% of patients with glioblastoma, but not in normal tissues, and therefore, EGFRvIII may represent a specific mutation of glioblastoma.
  • 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 EGFRvIII. Therefore, the construct of the present invention and the transgenic lymphocytes for the immunotherapy of adoptive T cells of tumors, especially glioblastoma, can greatly enhance the specific killing ability of T lymphocytes to glioblastoma cells. And the safety of treatment is significantly improved.
  • the invention proposes a T lymphocyte.
  • the T a cellular immune checkpoint of lymphocytes is silenced; expressing a non-functional EGFR; and expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: an extracellular region comprising a heavy chain of a single chain antibody a variable region and a light chain variable region, the single chain antibody specifically recognizing an antigen EGFRvIII; a transmembrane region, the transmembrane region being linked to the extracellular region, and intercalating into a cell membrane of the T lymphocyte; An intracellular region, the intracellular region is associated with the transmembrane region, and the intracellular region comprises an intracellular portion of CD28 or 4-1BB and a CD3 ⁇ 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 and intact sequences that bind to anti-EGFR antibodies, and non-functional EGFR can act as lymphocytes. Suicide tag.
  • 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 EGFRvIII mutant glioblastoma with 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 significantly enhanced, especially for EGFRvIII mutant glioblastoma, which has a significant directional killing effect and high safety.
  • the invention proposes a lentivirus.
  • the lentivirus carries The cassette contains the nucleotide sequence set forth in SEQ ID NO: 138, 139, 140 or 141.
  • 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 significantly enhanced, especially for EGFRvIII mutant glioblastoma, which has a significant directional killing effect and is safer.
  • the invention provides a transgenic lymphocyte.
  • the lymphocyte immune checkpoint is silenced; expressing a non-functional EGFR; and expressing a chimeric antigen receptor.
  • the inventors have surprisingly found that cell immunization checkpoints are silenced, express non-functional EGFR and lymphocytes expressing chimeric antigen receptors in vitro, proliferative and viable in tumor patients, and tumor cells in tumor patients.
  • the specific killing ability is greatly improved, especially for the EGFRvIII mutant glioblastoma, which has a significant directional killing effect. High security.
  • the above transgenic lymphocytes may further have at least one of the following additional technical features:
  • the chimeric antigen receptor comprises: an extracellular region capable of specifically binding to an antigen; a transmembrane region; and an intracellular region including immuno-stimulation Intracellular segment of the molecule.
  • the presence of the chimeric antigen receptor having the above structure greatly enhances the targeted localization of the transgenic lymphocytes of the embodiments of the present invention, and greatly enhances the targeted killing effect of the transgenic lymphocytes of the embodiments of the present invention on antigen-expressing tumor cells.
  • the antigen is a tumor antigen. Therefore, the targeted killing effect of the transgenic lymphocytes of the embodiments of the present invention on tumors is more remarkable.
  • the extracellular region comprises a heavy chain variable region and a light chain variable region of an antibody, said antibody binding to said antigen.
  • the specific binding effect of the antigen-antibody greatly enhances the targeted localization of the transgenic lymphocytes and the targeted killing effect on the antigen-expressing tumor cells of the present invention.
  • the antibody is a single chain antibody.
  • 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 of the embodiments of the present invention on the targeted tumor cells.
  • the antigen is EGFRvIII. Therefore, the transgenic lymphocytes have a directional killing effect on the cells expressing the antigen EGFRvIII, and the specific binding effect of the antigen antibody is stronger, and the directional killing effect of the transgenic lymphocytes of the embodiment of the present invention on the tumor cells expressing EGFRvIII antigen is greatly improved.
  • the lymphocyte immune checkpoint is independently selected from at least one of CTLA4, PD1, TIM3, BTLA, LAG3IRAK-M, SOCS-1, A20, CBL-B.
  • CTLA4, PD1, TIM3, BTLA, LAG3 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 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 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 the group consisting of 4-1BB, OX-40, CD40L, At least one of 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, 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 Binding of PD-L1 and PD-L2 or CD80 and CD86, thereby effectively inhibiting incompetence or apoptosis of T lymphocytes, or silencing through CBL-B, enhancing T cell receptor signaling, making transgenic lymphocytes in tumor patients
  • the proliferation and viability of the body are further improved, and the effect of directed killing of tumors is more significant.
  • silencing of the lymphocyte intracellular immune checkpoint is achieved by shRNA.
  • the shRNA of the embodiment of the present invention carries a shRNA which specifically silences at least one of the immunological checkpoints on 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 immunological checkpoints in the cell, the successful silencing of the cell's immune site, ie, the cell surface or the intracellular immune checkpoint, prevents the specific binding of the immunological checkpoint to the corresponding ligand, thereby effectively inhibiting the immune checkpoint pair.
  • the negative regulation mechanism of T lymphocyte incompetence or apoptosis, etc. further enhances the proliferation and viability of the transgenic lymphocytes of the embodiments of the present invention in tumor patients, and cooperates with the antigen targeting of chimeric antigen receptors.
  • 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 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 and integrity of wild-type EGFR.
  • the domain that binds to the anti-EGFR antibody, non-functional EGFR can be used as a suicide marker for the transgenic lymphocytes of the examples 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 EGFRvIII mutations.
  • the lymphocyte is a CD3+ T lymphocyte or a natural killer cell or a natural killer T cell.
  • 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 EGFRvIII 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 a more targeted killing effect on tumor cells, especially EGFRvIII mutant tumor cells, 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 EGFRvIII-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 immune checkpoints of cells and the expression of non-functional EGFR make the lymphocytes of the embodiments of the invention more targeted and have a killing effect on tumors, especially EGFRvIII mutations. The targeted killing of tumor cells 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:142.
  • 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:143.
  • 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 vector of the construct is a viral vector comprising 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 proliferative ability of the lymphocytes of the invention the proliferation and the viability of the tumor patients are greatly improved, the targeting effect of the lymphocytes is further enhanced, and the targeted killing effect on the tumor cells, especially the EGFRvIII mutant tumor cells, is more significant.
  • 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 effect of transgenic lymphocytes or T lymphocytes on tumor cells, especially tumor cells with EGFRvIII mutations. Stronger and more secure.
  • 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 the tumor cells is stronger, and the targeted killing effect of the therapeutic composition for treating cancer of the present invention on the tumor cells is remarkably enhanced, especially the targeted killing effect on the EGFRvIII mutant tumor cells is significantly enhanced, Security is further improved.
  • the above therapeutic composition may further comprise at least one of the following additional technical features:
  • the cancer comprises glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer.
  • Glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer cancer cells have specific expression of EGFRvIII, and the therapeutic composition of the present invention can silence and express lymphocyte cell surface or intracellular immune checkpoint
  • Non-functional EGFR and high expression of antigen-specific chimeric antigen receptors, such as the EGFRvIII antigen-specific chimeric antigen receptor of the present invention the resulting lymphocytes or T lymphocytes have significant resistance to tumor cell-mediated immunosuppression Characteristics, Survival in the microenvironment of glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer is greatly enhanced, and the resulting lymphocytes or T lymphocytes are gliomas of EGFRvIII mutation, non-small cell lung cancer Tumor cells of breast cancer or ovarian cancer have stronger targeted and killing effects and higher safety.
  • 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 EGFRvIII.
  • the method proposed by the embodiment of the invention has significant targeted killing effect on EGFRvIII mutant tumor cells, and has high safety.
  • 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 cellular immune checkpoint of lymphocytes is silenced and co-expressed with non-functional EGFR and the chimeric antigen receptor; and the transgenic lymphocytes are administered to the cancer patient.
  • the method of the present invention further enhances the targeted killing effect on EGFRvIII mutant tumor cells, and the safety is further enhanced.
  • the cancer comprises at least one selected from the group consisting of glioblastoma, non-small cell lung cancer, breast cancer and ovarian cancer.
  • glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer cancer cells have specific expression of EGFRvIII
  • the method for treating cancer of the present invention can make the cellular immune checkpoint of lymphocytes be Efficiently silencing and highly efficient expression of non-functional EGFR and antigen-specific chimeric antigen receptors, such as the EGFRvIII antigen-specific chimeric antigen receptor of the present invention, and the resulting lymphocytes or T lymphocytes have a glial mother of EGFRvIII mutation
  • the tumor cell of cell tumor, non-small cell lung cancer, breast cancer or ovarian cancer has a significant targeted killing effect and is highly safe.
  • 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 resists tumor cell-mediated immunosuppression, The targeted killing effect of EGFRvIII mutant tumor cells was 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 and intact sequences that bind to anti-EGFR antibodies, and non-functional EGFR can act as lymphocytes. Suicide tag.
  • the present invention is a lymphocyte of the embodiment. When the patient is treated with a tumor cell for treating the EGFRvIII mutation, if the patient develops a serious adverse reaction, the lymphocytes of the embodiment of the present invention can be cleared by the anti-EGFR antibody, thereby improving the lymph of the embodiment of the present invention. The safety of cells in patients with cancer treated with EGFRvIII mutations.
  • the tumor comprises at least one selected from the group consisting of an EGFRvIII mutant glioblastoma, an EGFRvIII mutant non-small cell lung cancer, an EGFRvIII mutant breast cancer, or an EGFRvIII mutant ovarian cancer.
  • the method for improving the activity of lymphocytes in the embodiment of the present invention is to make the lymphocytes carry the chimeric antigen receptor specific for the EGFRvIII 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 present invention Specificity further enhances the directed killing ability of EGFRvIII mutant tumor cells, such as the above EGFRvIII mutant tumor cells.
  • 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 view showing the structure of a chimeric antigen receptor which specifically expresses an EGFRvIII antigen and a silent 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 anti-EGFRvIII chimeric antigen receptor, silencing PD1-shRNA, and non-functional EGFR lymphocytes by anti-EGFR antibody according to an embodiment of the present invention
  • Figure 3 is a graph showing the results of co-expression of a EGFRvIII antigen-specific chimeric antigen receptor, silencing of PD1-shRNA, and non-functional EGFR lymphocytes to kill tumor cells, in accordance with 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; a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: an extracellular region, the extracellular region comprising a heavy chain variable region and a light chain variable region of a single chain antibody, and the single chain antibody specifically recognizes the antigen EGFRvIII;
  • the membrane 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 connected to the transmembrane region, and the intracellular region includes the intracellular portion of CD28 or 4-1BB.
  • 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 and intact sequences that bind to anti-EGFR antibodies, and non-functional EGFR can act as lymphocytes. Suicide tag.
  • the T lymphocyte or transgenic lymphocyte cell of the embodiment of the present invention expresses a non-functional EGFR, and the chimeric antigen receptor specific for the EGFRvIII antigen and the cellular immune checkpoint are silenced, and the T lymphocyte or the transgenic lymphocyte of the embodiment of the present invention is The proliferation and viability of tumor patients in vivo and in vitro, as well as the ability to kill specific tumor cells in tumor patients, significantly enhance the specific killing effect of EGFRvIII mutant glioblastoma cells, and safety. Also significantly 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.
  • the programmed cell death 1 receptor (PD1) as in the embodiment of the present invention, is expressed on activated CTLs, which interact with the programmed death ligand 1 (PD-L1) expressed on tumor cells to inhibit anti-tumor T cell responses. .
  • Many tumors including lymphoma, lung cancer, ovarian cancer, melanoma, and pancreatic tumors, express PD-L1.
  • the binding of PD-L1 to its ligand PD1 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 (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 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 wild-type EGFR and an intact anti-antibody.
  • the sequence of EGFR antibody binding, non-functional EGFR can be used 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 EGFRvIII mutations can be further improved.
  • the antibody of the chimeric antigen receptor extracellular region is a single chain antibody.
  • 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 EGFRvIII. Therefore, the transgenic lymphocytes of the embodiments of the present invention have a directional killing effect on the cells expressing the antigen EGFRvIII, and the specific binding effect of the antigen antibodies is stronger, and the orientation of the transgenic lymphocytes of the present invention to the tumor cells expressing EGFRvIII antigen 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 LAG3, the lymphocyte intracellular immune checkpoint is independently selected from at least one of IRAK-M, SOCS-1, 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 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 are cut off. After the mRNA, it can be released from the hybridization chain to recombine 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 form a complex, recognize and bind to the complementary sequence of the crRNA, then unwind the DNA double strand to form the R-loop, so that the crRNA hybridizes with the complementary strand, and the other chain protects Holding 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 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 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 EGFRvIII mutant tumors is more significant.
  • the expression of intracellular segments of immunostimulatory molecules is combined with the expression of non-functional EGFR, making transgenic lymphocytes Immune killing is safer and more effective.
  • the lymphocyte cell surface immunological checkpoint is preferably CTLA4 or PD1
  • the intralymphocyte immune checkpoint is preferably CBL-B.
  • Lymphocyte cell surface immunological checkpoint according to the time embodiment of the present invention CTLA4 or PD1 is silenced or the intracellular immune checkpoint CBL-B is silenced, making transgenic lymphocytes more resistant to tumor-mediated immunosuppression, and their proliferation and viability in tumor patients are further improved. The effect of targeted killing of tumors is more pronounced.
  • 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 are human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence
  • a lentivirus or a construct of the present invention is introduced into a transgenic lymphocyte obtained from a lymphocyte, and the cell surface is immunized Checkpoint PD1, CTLA4, TIM3, BTLA, LAG3 or intracellular Epileptic checkpoints IRAK-M, SOCS1, A
  • a retrovirus or construct of an embodiment of the invention carries a nucleotide sequence as set forth in SEQ ID NO: 138, 139, 140 or 141.
  • SEQ ID NO: 138 represents a nucleic acid molecule (EvIII-CAR/iPD1) which co-expresses an anti-EGFRvIII chimeric antigen receptor, a non-functional EGFR, a silencing cell surface immunological checkpoint PD1, and a silencing intracellular immunological checkpoint CBL-B.
  • SEQ ID NO: 139 is a nucleic acid molecule (EvIII-CAR/iCBL-B/tEGFR) that co-expresses the anti-EGFRvIII chimeric antigen receptor, non-functional EGFR, and silences the intracellular immunological checkpoint CBL-B
  • SEQ ID NO: 140 is a nucleic acid molecule (EvIII-CAR/iPD1/tEGFR) co-expressing an anti-EGFRvIII chimeric antigen receptor, a non-functional EGFR, and a silencing cell surface immunological checkpoint PD1, SEQ ID NO: 141
  • the anti-EGFRvIII chimeric antigen receptor, non-functional EGFR, the silencing cell surface immunological checkpoint PD1, and the nucleic acid molecule (EvIII-CAR/iPD1-CTLA4/tEGFR) of another immunological checkpoint CTLA4 on the cell surface were co-expressed.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes are specifically silenced on the cell surface immunological checkpoint PD1 or CTLA4, or the intracellular immunological test CBL-B is specifically Silencing and expression of chimeric antigen receptors expressing non-functional EGFR and anti-EGFRvIII, the transgenic lymphocytes have significant anti-tumor-mediated immunosuppressive effects, and their anti-apoptotic and proliferative ability is enhanced, and the directional killing ability is significantly improved.
  • the safety of immune killing is significantly improved, so that the proliferation and viability of transgenic lymphocytes in vitro and in vivo of tumor patients and the killing ability in tumor patients are greatly improved, especially for EGFRvIII mutant glioblastoma cells.
  • the killing effect is particularly significant, and the specific killing safety of EGFRvIII mutant glioblastoma 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: 142.
  • the internal ribosome entry site is usually located in the 5' untranslated region (UTR) of the RNA viral genome, so that the translation of one viral protein can be independent of the 5' cap structure, and the other protein usually initiates translation by the 5' hat structure.
  • the expression of the two genes before and after IRES is usually proportional.
  • 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, so that the specific killing effect of lymphocytes on the tumor is more significant, and the immune killing effect is more obvious. Security is further improved.
  • Promoter a first promoter operably linked to a nucleic acid molecule encoding a chimeric antigen receptor; a promoter, the second promoter being operably linked to a nucleic acid molecule that silences the immunological checkpoint; and a third promoter operably linked to the nucleic acid molecule expressing the 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
  • the immune checkpoint or high-efficiency expression of non-functional EGFR and ensure the high expression of chimeric antigen receptor, so that the survival rate of lymphocytes in the tumor environment is greatly improved, the lymphocyte targeting effect is stronger, and the specificity to the tumor
  • the killing effect 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:143.
  • 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 of the embodiment of the invention has greatly improved proliferation and viability in tumor patients, and the targeting effect of lymphocytes is further enhanced. The killing effect on tumor cells is more significant, and the immune killing safety of lymphocytes 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 promoter that expresses shRNA is the 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 to 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 lymphocytes or T lymphocytes, and the expression of the chimeric antigen receptor against the antigen EGFRvIII and the cell surface or intracellular immune checkpoint of lymphocytes 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 EGFRvIII mutant glioblastoma cells, 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 EGFRvIII chimeric antigen receptor in transgenic lymphocytes or T lymphocytes and silencing of transgenic lymphocytes or T lymphocyte cells or intracellular immune checkpoints. And the expression of non-functional EGFR on the surface of transgenic lymphocytes or T lymphocytes, so that the obtained transgenic lymphocytes or T lymphocytes can be expanded in vitro, proliferate in tumor patients and survive in tumor patients, and transgenic lymphocytes are greatly improved. Or the targeted killing effect of T lymphocytes on EGFRvIII tumor cells Stronger, immune killer is safer.
  • 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 non-functional EGFR and express a unique EGFRvIII chimeric antigen. body.
  • 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 non-functional EGFR and expressing specific antibodies against the antigenic EGFRvIII chimeric antigen receptor 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 glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer.
  • Silencing of cell surface or intracellular immune checkpoints and expression of non-functional EGFR, combined with high expression of chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, resulting in lymphocytes or T lymphocytes in glioblastoma Survival in the environment of neoplasms, non-small cell lung cancer, breast cancer or ovarian cancer is greatly enhanced, and lymphocytes or T lymphocytes are targets for tumor cells of glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer. It has a stronger killing effect, especially for the above-mentioned tumor cells with EGFRvIII mutation, and it is safer and more effective for the immune killing effect of the above-mentioned tumor cells with EGFRvIII mutation.
  • 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 EGFRvIII.
  • the method proposed by the embodiment of the invention has significant targeted killing effect on EGFRvIII mutant tumor cells, and has high safety.
  • 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 cellular immune checkpoint of lymphocytes is silenced and co-expressed with non-functional EGFR and the chimeric antigen receptor; and the transgenic lymphocytes are administered to the cancer patient.
  • the method of the embodiment of the present invention further enhances the targeted killing effect on EGFRvIII mutant tumor cells, and the safety is further improved.
  • the cancer comprises a group selected from the group consisting of glioblastoma, non-small cell lung cancer, breast cancer and eggs At least one of the nest cancers.
  • glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer cancer cells have specific expression of EGFRvIII, and the method for treating cancer of the present invention can make the cellular immune checkpoint of lymphocytes be Efficiently silencing and highly efficient expression of non-functional EGFR and antigen-specific chimeric antigen receptors, such as the EGFRvIII antigen-specific chimeric antigen receptor of the present invention, and the resulting lymphocytes or T lymphocytes have a glial mother of EGFRvIII mutation
  • the tumor cell of cell tumor, non-small cell lung cancer, breast cancer or ovarian cancer has a significant targeted killing effect and is highly safe.
  • administering refers to introducing a predetermined amount of a substance into a patient in some suitable manner.
  • the therapeutic composition of the invention can be administered by any conventional route as long as it can reach the intended tissue.
  • Various modes of administration are contemplated, including peritoneal, venous, muscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the invention is not limited to these exemplary modes of administration.
  • the active ingredient of the orally administered composition should be coated or formulated to prevent its degradation in the stomach.
  • the compositions of the invention may be administered as an injectable preparation.
  • the therapeutic compositions of the present invention can be administered using a particular device that delivers the active ingredient to the target cells.
  • the frequency and dosage of the therapeutic composition of the present invention can be determined by a number of relevant factors including the type of disease to be treated, the route of administration, the age, sex, weight and severity of the disease as well as the active ingredient. Type of drug. According to some embodiments of the invention, the daily dose may be divided into 1 dose, 2 doses or multiple doses in a suitable form for administration once, twice or more times throughout the time period, as long as a therapeutically effective amount is achieved. .
  • terapéuticaally effective amount refers to an amount of a compound that is sufficient to significantly ameliorate certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • a therapeutic composition that reduces, prevents, delays, inhibits, or arrests any symptoms of a disease or condition is therapeutically effective. of.
  • a therapeutically effective amount of the therapeutic composition does not require a cure for the disease or condition, but will provide a treatment for the disease or condition such that the onset of the disease or condition of the individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the disease or The duration of the condition is altered, or for example the disease or condition becomes less severe, or the recovery is accelerated.
  • treatment is used to mean obtaining the desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing the disease and/or the adverse effects caused by the disease.
  • treatment encompasses the treatment of a mammalian, particularly human, disease (mainly referred to as EGFRvIII + glioblastoma, non-small cell lung cancer, breast cancer, and ovarian cancer), including: (a) Prevention of disease (eg, prevention of EGFRvIII + glioblastoma, non-small cell lung cancer, breast cancer, and ovarian cancer) or disease in individuals who are susceptible to disease but have not yet been diagnosed; (b) inhibition of disease, such as resistance Stagnation of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease.
  • a mammalian, particularly human, disease mainly referred to as EGFRvIII + glioblastoma, non-small cell lung cancer, breast cancer, and ovarian cancer
  • Prevention of disease eg, prevention of EGFRvIII + glioblastoma, non-small cell lung cancer, breast cancer, and ovarian cancer
  • disease mainly referred to as EGFRvIII
  • "treatment&quot encompasses any administration of a therapeutic composition to an individual to treat, cure, ameliorate, ameliorate, ameliorate, or inhibit a disease in an individual, including, but not limited to, administering a therapeutic composition comprising a subject described herein to an individual in need thereof.
  • the therapeutic compositions of the embodiments of the invention may be used in conjunction with conventional methods of treatment and/or therapy, or may be used separately from conventional methods of treatment and/or therapy.
  • the therapeutic compositions of the invention are administered in combination therapy with other drugs, they can be administered to the subject sequentially or simultaneously.
  • the methods of treatment of the invention may comprise a therapeutic composition of the invention, a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient, and combinations of other therapeutic or prophylactic agents known in the art.
  • 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 and intact sequences that bind to anti-EGFR antibodies, and non-functional EGFR can act as lymphocytes. Suicide tag.
  • the present invention is a lymphocyte of the embodiment. When the patient is treated with a tumor cell for treating the EGFRvIII mutation, if the patient develops a serious adverse reaction, the lymphocytes of the embodiment of the present invention can be cleared by the anti-EGFR antibody, thereby improving the lymph of the embodiment of the present invention. The safety of cells in patients with cancer treated with EGFRvIII mutations.
  • 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.
  • 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).
  • 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) (final concentration) It was 20 ⁇ g/ml), and precultured for 30 minutes under normal temperature conditions. Then, the medium containing the antibody was replaced with 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-EGFRvIII CAR T lymphocytes The cytotoxic activity of anti-EGFRvIII chimeric antigen receptor T cells (anti-EGFRvIII 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 labeled is EGFRvIII-U87 brain tumor cells.
  • Example 2 Construction of a vector for co-expression of shRNA, non-functional EGFR and anti-EGFRvIII chimeric antigen receptor
  • the inventors cloned the sequence encoding the single-chain antibody against human EGFRvIII, the 4-1BB intracellular domain and the T cell receptor combination into a lentiviral vector containing the EF-1 promoter ( On the lentiviral vector), during the cloning process, the restriction enzyme digestion is double digestion with XbaI and NotI, and double digestion with NotI and XhoI, and the expression of anti-EGFRvIII is expressed by restriction enzyme digestion, ligation, screening and amplification of the plasmid of interest.
  • Anti-receptor lentiviral plasmid (LV-EGFRvIII CAR).
  • the sequence containing the U6 promoter and human PD1-shRNA (iPD1) or CBL-B-shRNA (iCBL-B) was cloned into the LV-EGFRvIII CAR vector plasmid and constructed into LV-EGFRvIII CAR/iPD1 or LV-EGFRvIII CAR/iCBL -B, or a sequence comprising a synthetic IRES and a sequence expressing a non-functional EGFR, a U6 promoter and a human PD1-shRNA sequence, and an H1 promoter and a CBL-B-shRNA were cloned into the LV-EGFRvIII CAR vector plasmid to construct LV- EGFRvIII CAR/iPD1-Cbl/tEGFR.
  • Figure 1 is a schematic representation of a lentiviral vector comprising a sequence encoding an anti-EGFRvIII chimeric antigen receptor, an IRES, U6 and H1 promoter sequence, a PD1-shRNA or a CBL-B-shRNA sequence, and A non-functional EGFR sequence is encoded.
  • the sequence of the anti-EGFRvIII 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 is effective in killing T lymphocytes that secrete PD1-shRNA, non-functional EGFR and anti-EGFRvIII 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-EGFRvIII-CAR/iPD1/tEGFR, LV-EGFRvIII, respectively.
  • the -CAR/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 can effectively conjugate to anti-EGFRvIII chimeric antigen receptors, PD1-shRNA (iPD1), and T lymphocytes without function EGFR, but anti-EGFR antibodies cannot mediate anti-expression.
  • Example 4 T lymphocyte tumor cell lysis ability co-expressing PD1-shRNA, non-functional EGFR and anti-EGFRvIII 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-EGFRvIII CAR/iPD1/tEGFR, LV-EGFRvIII 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 different lentivirus-derived T cells (effector cells) on EGFRvIII mutant glioma target cells, and the ratio of target cells was 50: 1, 25: 1, or 10: 1, measurement method.
  • a standard 4 - hour 51 chromium release method was used, and a 4 - hour 51 chromium release method was as described in Example 1. The result is shown in Figure 3.
  • anti-EGFRvIII chimeric antigen receptor, PD1-shRNA (iPD1) and non-functional EGFR lentiviral transduced T lymphocytes were co-expressed, and anti-EGFRvIII chimeric antigen receptor and PD1-shRNA were co-expressed ( iPD1)
  • Receptor lentiviral transduced T lymphocytes are effective in killing EGFRvIII mutant brain tumor target cells.
  • Non-functional EGFR lentivirus-transduced T lymphocytes (LV-tEGFR T lymphocytes) or empty lentiviral-transduced T lymphocytes (control LV-GFP T lymphocytes) showed no significant killing of EGFRvIII mutant brain tumor target cells effect.
  • the statistical data represents the mean ⁇ SEM of the three wells.
  • Example 5 T cells co-expressing PD1-shRNA, non-functional EGFR and anti-EGFRvIII chimeric antigen receptor, co-expressing CBL-B-shRNA, non-functional EGFR and anti-EGFRvIII chimeric antigen receptor T cells, co-expressing PD1 -shRNA, CTLA4-shRNA, non-functional EGFR and anti-EGFRvIII chimeric antigen receptor T cells, co-expressing PD1-shRNA, CBL-B-shRNA, anti-EGFRvIII chimeric antigen receptor, non-functional EGFR T cells , 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), non-functional EGFR and anti-EGFRvIII chimeric antigen receptor, and co-expressing two shRNAs (PD1) -shRNA and CBL-B-shRNA or PD1-shRNA and CTLA4-shRNA), lymphocytes without functional EGFR and anti-EGFRvIII chimeric antigen receptor, tumor lysis ability, cytokine secretion ability and cell proliferation ability.
  • the above T cells have enhanced cytolysis ability, more cytokine secretion and stronger cell proliferation than T cells expressing the anti-EGFRvIII chimeric antigen receptor alone.
  • T cells co-expressing two shRNAs PD1-shRNA and CTLA4-shRNA or PD1-shRNA and CBL-B-shRNA
  • non-functional EGFR and anti-EGFRvIII chimeric antigen receptors co-expressed 1 shRNA PD1-shRNA or T cells with CBL-B-shRNA
  • non-functional EGFR and anti-EGFRvIII 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, cytokines, of T cells co-expressing two shRNAs (PD1-shRNA and CBL-B-shRNA or PD1-shRNA and CTLA4-shRNA), anti-EGFRvIII chimeric antigen receptor and non-functional EGFR Secretion capacity and cell proliferation ability are equivalent to T cells co-expressing two shRNAs (PD1-shRNA and CBL-B-shRNA or PD1-shRNA and CTLA4-shRNA) and anti-EGFRvIII chimeric antigen receptor; co-expression of 1 shRNA (PD1) -shRNA or CBL-B-shRNA), non-functional EGFR and anti-EGFRvIII chimeric antigen receptor T cells have cytolysis ability, cytokine secretion ability and cell proliferation ability
  • 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 embodiment 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 the EGFRvIII mutation of the embodiment of the present invention.

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Abstract

L'invention concerne un lymphocyte transgénique, une construction et une composition thérapeutique ainsi qu'un procédé pour traiter un cancer. Le lymphocyte transgénique voit son point de contrôle cellulaire inactivé, il exprime un EGFR non fonctionnel et il exprime un récepteur d'antigène chimérique, le récepteur d'antigène chimérique comprenant : une région extracellulaire, la région extracellulaire comprenant une région variable de chaîne lourde et une région variable de chaîne légère d'un anticorps monocaténaire, et l'anticorps monocaténaire identifiant précisément l'antigène EGFRvIII; une région transmembranaire, la région transmembranaire étant reliée à la région extracellulaire et étant incorporée dans la membrane cellulaire des lymphocytes T; et une région intracellulaire, la région intracellulaire étant reliée à la région transmembranaire et la région intracellulaire comprenant un segment intracellulaire de CD28 ou 4-1BB et une chaîne CD3ζ.
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