WO2017120996A1 - 共表达抗EGFRvIII嵌合抗原受体和免疫检查点抑制分子的转基因淋巴细胞及其用途 - Google Patents
共表达抗EGFRvIII嵌合抗原受体和免疫检查点抑制分子的转基因淋巴细胞及其用途 Download PDFInfo
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Definitions
- the present invention relates to the field of biomedicine, and in particular to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition and method for treating cancer, and a A method of increasing lymphocyte activity.
- 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 and a nucleic acid molecule encoding a chimeric antigen receptor, and a transgenic lymphocyte formed by the introduction of the construct, encoded by The chimeric antigen receptor specifically binds to the antigen EGFRvIII. Therefore, the constructs and transgenic lymphocytes proposed by the present invention can be used for immunotherapy of adoptive T cells of tumors, especially glioblastoma, and the transgenic lymphocytes proposed by the present invention have EGFRvIII mutant glioblastoma cells. Strong specific killing ability.
- the invention proposes a T lymphocyte.
- the T a cellular immune checkpoint of lymphocytes is silenced; and a chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region comprising a heavy chain variable region of a single chain antibody and a light a chain variable region, the single-chain antibody specifically recognizing an antigen EGFRvIII; a transmembrane region, the transmembrane region is linked to the extracellular region, and embedded in a cell membrane of the T lymphocyte; an intracellular region, The intracellular region is linked to the transmembrane region, and the intracellular region includes 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.
- 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. Significantly enhanced ability, especially for EGFRvIII mutant glioblastoma with significant directed killing.
- the invention proposes a lentivirus.
- the lentivirus carries a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1, the coding chimera
- the nucleic acid molecule of the antigen receptor has the nucleotide sequence shown in SEQ ID NO: 2; and the nucleic acid molecule of the silencing cell immunological checkpoint, the nucleotide sequence of the nucleic acid molecule of the silencing cell immunological checkpoint is selected from the group consisting of SEQ ID NO: at least one of 3 to 135.
- the transgenic lymphocytes obtained by introducing the lentivirus of the 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 gliding of EGFRvIII mutant glioblastoma.
- the invention proposes a lentivirus.
- the lentivirus carries a nucleotide sequence as set forth in SEQ ID NO: 136, 137, 138 or 139.
- 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 gliding of EGFRvIII mutant glioblastoma.
- the invention provides a transgenic lymphocyte.
- the lymphocyte immune checkpoint is silenced; and the chimeric antigen receptor is expressed.
- the inventors have surprisingly found that the cell immunological checkpoint is silenced and the in vitro proliferation ability of lymphocytes expressing the chimeric antigen receptor, the proliferation and viability in tumor patients, and the specific killing ability to tumor cells in tumor patients. Significantly improved, especially for EGFRvIII mutant glioblastoma with significant directed killing effect.
- 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, LAG3, IRAK-M, SOCS1, A20, CBL-B.
- CTLA4, PD1, TIM3, BTLA, LAG3 are cell surface immune checkpoints
- IRAK-M, SOCS1, A20, and CBL-B are intracellular immune checkpoints.
- the immune checkpoint of the embodiment of the invention has the functions of negatively regulating and attenuating the cellular immune response, and the specific binding of the corresponding ligand on the tumor cell leads to down-regulation of the proliferative response of the T lymphocyte and the secretion of the cytokine is reduced.
- 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 and the targeted killing effect on the tumor cells are further strengthened.
- the lymphocyte cell surface immunological checkpoint is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR and zinc finger nuclease.
- the successful silencing of the cellular immune checkpoint of the embodiment of the present invention can significantly improve the lymphocyte resistance tumor-mediated immunosuppressive property of the embodiment of the present invention, and further improve the transgenic lymphocyte proliferating tumor cell.
- Directional killing effect is beneficial to be used to the lymphocyte resistance tumor-mediated immunosuppressive property of the embodiment of the present invention.
- the intracellular segment of the immunocostimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and their derivatives.
- the expression of the intracellular segment of the immunostimulatory molecule and the silencing of the cellular immune checkpoint in the embodiment of the present invention have the functions of positively regulating and enhancing the cellular immune response, so that the transgenic lymphocyte proliferation of the embodiment of the present invention has a targeted killing effect on the tumor.
- the effect is more remarkable; the combination of the expression of the intracellular segment of the immunostimulatory molecule and the silencing of the cellular immune checkpoint in the embodiment of the present invention makes the proliferative ability of the transgenic lymphocytes of the embodiment of the present invention and the targeted killing effect on the tumor more remarkable.
- the lymphocyte immune checkpoints are CTLA4, PD1, CBL-B.
- CTLA4 and PD1 are cell surface immune checkpoints
- CBL-B is an intracellular immune checkpoint.
- the lymphocyte cell surface immunological checkpoint CTLA4 or PD1 is silenced, or the lymphocyte intracellular immune checkpoint is silenced by CBL-B, preventing the expression of PD1 or CTLA4 molecules from correspondingly
- the combination of PD-L1 and PD-L2 or CD80 and CD86 effectively inhibits the inability or apoptosis of T lymphocytes, or enhances T cell receptor signaling through CBL-B silencing, making transgenic lymphocytes in tumors
- the proliferative and viability of the patient is further improved, and the effect of directed killing of the tumor is more significant.
- shRNA of the embodiment of the present invention carries a shRNA which specifically silences at least one immunological checkpoint on the cell surface or in the cell, and the shRNA of the embodiment of the present invention has a highly efficient and specific silencing cell surface or at least intracellular cells.
- the negative regulation mechanism of T lymphocyte incompetence or apoptosis, etc. further enhances the proliferation and viability of the transgenic lymphocytes of the 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, further The step enhances the targeted killing effect of the transgenic lymphocytes of the embodiments of the present invention.
- the lymphocytes are CD3+ T lymphocytes or natural killer cells or natural killer T cells.
- the cellular immunological checkpoint of the above lymphocytes according to the embodiment of the present invention is silenced while expressing an antigen-specific chimeric antigen receptor, such as the EGFRvIII antigen-specific chimeric antigen receptor of the embodiment of the present invention, the lymphocyte cells.
- an antigen-specific chimeric antigen receptor such as the EGFRvIII antigen-specific chimeric antigen receptor of the embodiment of the present invention.
- the invention proposes a construct.
- the construct comprises: a first nucleic acid molecule encoding a chimeric antigen receptor; and a second nucleic acid molecule, the second nucleic acid molecule silencing a cellular immune checkpoint.
- the cellular immunological checkpoint and the chimeric antigen receptor are as described above.
- the construct of the embodiment of the present invention can effectively silence at least one immunological checkpoint on the cell surface or in the cell and the antigen-specific chimeric antigen receptor after being successfully introduced into the lymphocyte of the embodiment of the present invention.
- the directional killing effect of the lymphocytes of the embodiments of the present invention on tumor cells, particularly EGFRvIII mutant tumor cells is more pronounced.
- the above-described construct may further include at least one of the following additional technical features:
- the first nucleic acid molecule and the second nucleic acid molecule are arranged in a lymphocyte immunological checkpoint and express the chimeric antigen receptor in the lymphocytes described above.
- the lymphocytes of the first nucleic acid molecule and the second nucleic acid molecule are successfully set, and the immunological checkpoint of at least one of the cell surface of the lymphocyte or the cell is successfully silenced, and succeeds on the surface of the lymphocyte.
- the antigen-specific expression such as the EGFRvIII-specific chimeric antigen receptor of the present invention, has a more lethal and specific tumor killing effect and is more safe.
- the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; and a second promoter, the second promoter and The second nucleic acid molecule is operably linked.
- the introduction of the first promoter and the second promoter enables the first nucleic acid molecule and the second nucleic acid molecule to be independently expressed, thereby effectively ensuring the biological effect of the chimeric antigen receptor antigen targeting.
- the lymphocyte targeting effect of the embodiment of the present invention is stronger, and the killing effect on the tumor, especially the directional killing of the EGFRvIII mutant tumor cell is more remarkable.
- the first promoter and the second promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, LTR, RSV promoters.
- the above promoter of the embodiment of the invention has the characteristics of high activation efficiency and high specificity, thereby ensuring efficient silencing of the cellular immune checkpoint and efficient expression of the chimeric antigen receptor, thereby implementing the present invention.
- the in vitro proliferation ability of lymphocytes in the tumor, the proliferation and survival ability in tumor patients are greatly improved, and the targeted killing effect on tumors is more remarkable.
- the vector of the construct is a non-pathogenic viral vector.
- introduction of non-pathogenic viral vectors The efficiency of replication and amplification of the construct in the lymphocyte is greatly improved, thereby greatly improving the silencing of the cellular immune checkpoint and the high-efficiency expression of the chimeric antigen receptor in lymphocytes, so that the lymphocyte proliferation ability in vitro is in the tumor.
- the proliferation and viability of the patient are greatly improved, the targeting effect of lymphocytes is further enhanced, and the killing effect on tumor cells is more significant.
- 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 invention provides a method of preparing the aforementioned T lymphocytes or transgenic lymphocytes.
- the method comprises introducing the aforementioned construct or the lentivirus described above into lymphocytes or T lymphocytes.
- the construct or lentivirus is successfully introduced into the above lymphocytes or T lymphocytes, and the cellular immunological examination of lymphocytes is silenced and the expression of the chimeric antigen receptor is achieved, thereby preparing the transgenic lymphocytes prepared by the preparation method of the present invention.
- the proliferation of T lymphocytes in tumor patients and in vitro and the survival ability of tumor patients are greatly improved, and the transgenic lymphocytes or T lymphocytes have stronger targeted killing effects on tumor cells, especially tumor cells with EGFRvIII mutation.
- the invention provides a therapeutic composition for treating cancer.
- the therapeutic composition comprises: the above construct, lentivirus, T lymphocyte or transgenic lymphocyte.
- the composition of any of the above therapeutic compositions can achieve silencing of cell surface or intracellular immunological checkpoints of transgenic lymphocytes or T lymphocytes and efficient expression of chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, thereby
- the obtained transgenic lymphocytes or T lymphocytes have significant resistance to tumor cell-mediated immunosuppression, and the proliferation of tumor patients in vitro and in vivo and the survival ability of tumor patients are greatly improved, and the target of transgenic lymphocytes or T lymphocytes to tumor cells is improved.
- the killing effect is stronger, and the targeted killing effect of the therapeutic composition for treating cancer of the present invention on tumor cells is remarkably enhanced, especially the targeted killing effect on EGFRvIII mutant tumor cells is remarkably enhanced.
- 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 cells have specific expression of EGFRvIII, and the therapeutic composition of the present invention can silence and efficiently perform lymphocyte cell surface or intracellular immune checkpoints.
- an antigen-specific chimeric antigen receptor such as the EGFRvIII antigen-specific chimeric antigen receptor of the present invention
- obtained lymph Cells or T lymphocytes have significant resistance to tumor cell-mediated immunosuppression, and their ability to survive in the microenvironment of glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer is greatly enhanced.
- T lymphocytes have a stronger targeted killing effect on tumor cells of EGFRvIII mutant glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer.
- 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 in the embodiment of the invention has a significant targeted killing effect on EGFRvIII mutant tumor cells.
- 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 A cellular immune checkpoint of lymphocytes is silenced and expresses 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.
- 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, 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 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 glioblastoma of EGFRvIII mutation, non- Significant targeted killing of tumor cells of small cell lung cancer, breast cancer or ovarian cancer.
- the invention provides a method of increasing lymphocyte activity, the lymphocyte carrying a chimeric antigen receptor, according to an embodiment of the invention, the method comprising: causing the lymphocyte The cellular immune checkpoint was silenced.
- the cellular immune checkpoint, the lymphocyte, and the chimeric antigen receptor are as defined above, and the lymphocyte activity includes the ability of the lymphocyte to proliferate in vitro, proliferation and viability in a tumor patient, and At least one of the directional killing ability of the lymphocytes in a tumor patient.
- the cell surface or intracellular immune checkpoint of lymphocytes according to the embodiment of the present invention is silenced, lymphocytes are activated, proliferative responses are up-regulated, cytokine secretion is increased, and anti-apoptotic ability is enhanced, so that the present invention
- the 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 resistant to tumor cell-mediated immunosuppression, the targeted killing effect on EGFRvIII mutant tumor cells is significantly enhanced.
- the above method for increasing lymphocyte activity may further comprise the following additional technology At least one of the levies:
- 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 protein that inhibits anti-tumor lymphocyte responses.
- FIG. 1 is a schematic diagram showing the structure of a lentiviral vector which co-expresses a chimeric antigen receptor specific for EGFRvIII antigen and silences a human cell immunological checkpoint according to an embodiment of the present invention
- FIG. 2 is a graph showing the results of enhancing the proliferative ability of a chimeric antigen receptor specific for EGFRvIII antigen and silencing PD1 according to an embodiment of the present invention
- FIG. 3 is a graph showing the results of co-expression of a EGFRvIII antigen-specific chimeric antigen receptor and an increase in lymphocyte interferon- ⁇ secretion by silencing PD1 according to an embodiment of the present invention
- Figure 4 is a graph showing the results of enhanced ability of a chimeric antigen receptor that specifically expresses an EGFRvIII antigen and a lymphocyte killing tumor cell that silences PD1 according to an embodiment of the present invention.
- the invention provides a T lymphocyte or transgenic lymphocyte.
- a cellular immune checkpoint of a T lymphocyte according to an embodiment of the present invention is silenced; and a chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region, and the extracellular region includes a single strand The heavy chain variable region and the light chain variable region of the antibody, the single chain antibody specifically recognizes the antigen EGFRvIII; the transmembrane region, the transmembrane region is linked to the extracellular region, and is embedded in the cell membrane of the T lymphocyte; the intracellular region The intracellular region is connected to the transmembrane region, and the intracellular region includes the intracellular portion of CD28 or 4-1BB And a CD3 chain.
- the cellular immune checkpoint includes an immune checkpoint on the cell surface or within the cell.
- the T lymphocyte or transgenic lymphocyte cell immunological checkpoint of the embodiment of the present invention is silenced to jointly express the chimeric antigen receptor specific for the EGFRvIII antigen, and the T lymphocyte or the transgenic lymphocyte of the embodiment of the present invention is in vivo and in vitro of the tumor patient. Proliferation and viability as well as the ability to kill specific tumor cells in tumor patients are significantly enhanced, especially the specific killing effect on EGFRvIII mutant glioblastoma cells is greatly 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 antibody of the chimeric antigen receptor extracellular region is a single chain antibody.
- the inventors have found that single-chain antibodies can remove non-specifically reactive surface proteins while single-chain antibodies are more permeable to tumor tissue to increase drug treatment concentrations.
- the transgenic lymphocytes of the embodiments of the present invention express the chimeric antigen receptor of the single-chain antibody, which greatly enhances the targeted killing effect of the transgenic lymphocytes on the targeted tumor cells.
- the binding antigen of the above antibody is 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 LAG-3 the lymphocyte intracellular immune checkpoint is independently selected from at least one of IRAK-M, SOCS1, A20, and CBL-B.
- the above molecules can specifically bind to antigens expressed by tumor cells, inhibit lymphocyte activation, promote lymphocyte incompetence or apoptosis, thereby negatively regulating and attenuating cellular immune responses.
- the successful silencing of the above cell surface or intracellular immune checkpoint further enhances the transgenic lymphocyte in the tumor
- the proliferation and viability of the patient's body further enhances the targeted killing effect on 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 can be detached from the hybridization chain and then re-bound and cleave other mRNA molecules.
- Dominant negative mutations are those in which certain signal transduction proteins are not only self-functional but also inhibit or block the action of wild-type signal transduction proteins in the same cell, mainly by forming dimers with wild-type proteins.
- the way to achieve this mutation is toxic and can significantly inhibit or block the action of intracellular target signal transduction proteins.
- the zinc finger nuclease consists of a DNA recognition domain and a non-specific endonuclease.
- the DNA recognition domain is composed of a series of Cys2-His2 zinc finger proteins in series (generally 3 to 4). Each zinc finger protein recognizes and binds.
- a specific triplet base, zinc finger protein forms the ⁇ - ⁇ - ⁇ secondary structure, wherein the 16 amino acid residues of the ⁇ helix determine the DNA binding specificity of the zinc finger, the skeleton structure is conserved, and the amino acid determining the DNA binding specificity
- the introduction of sequence changes can obtain new DNA binding specificity, so that different amino acid introduction sequences can be designed for different genes of interest to achieve specific silencing of different genes of interest.
- CRISPR Clustered regular interspaced short palindromic repeats
- the CRISPR cluster is a family of specific DNA repeats that are widely found in the genomes of bacteria and archaea.
- the sequence consists of a leader, multiple short and highly conserved repeats, and multiple spacers (Spacer). )composition.
- the leader region is generally located upstream of the CRISPR cluster and is a region rich in AT length of 300-500 bp, which is considered to be a promoter sequence of the CRISPR cluster.
- the repeat sequence region has a length of 21 to 48 bp and contains a palindromic sequence, which can form a hairpin structure.
- the repeat sequences are separated by a spacer of length 26 to 72 bp.
- the Spacer region is composed of captured foreign DNA.
- CRISPR is transcribed into a long RNA precursor (Pre RISPR RNA, pre-crRNA) under the control of the leader region, and then processed into a series of short conserved repeats and spacers.
- the mature crRNA ultimately recognizes and binds to its complementary foreign DNA sequence to exert a cleavage effect.
- Processing of pre-crRNA is involved by Cas9 in the Cas family. Cas9 contains two unique active sites, RuvC at the amino terminus and HNH in the middle of the protein, which play a role in crRNA maturation and double-strand DNA cleavage.
- trans-activating crRNA complementary to its repeat sequence is also transcribed, and Cas9 and double-stranded RNA-specific RNase III nuclease are excited to process pre-crRNA.
- the crRNA, tracrRNA and Cas9 complexes recognize and bind to the complementary sequence of crRNA, then unwind the DNA double strand to form R-loop, which makes the crRNA hybridize with the complementary strand, and the other strand maintains the free single-stranded state.
- RNA double-strand break
- the shRNA, the antisense nucleic acid, the ribozyme, the dominant negative mutation, the CRISPR, and the zinc finger nuclease are effective means for specifically silencing the target gene, and the means for silencing the gene is not particularly limited, and those skilled in the art can Specific experimental purposes and conditions, such as at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR or zinc finger nuclease employed in the embodiments of the present invention, achieve specific silencing of the gene of interest.
- the lymphocyte cell surface or intracellular immunological checkpoint is silenced, preferably with shRNA.
- the siRNA molecule carried by the ShRNA is typically a dual region of base pairs between 10 and 30 in length.
- the PD1 or CTLA4 or CBL-B siRNA of the present invention is designed to be homologous to the coding region of PD1 or CTLA4 or CBL-B mRNA, and to inhibit gene expression by degradation of mRNA.
- the siRNA is associated with a multiplex protein complex called the Inducible RNA Silencing Complex (RISC), during which the sense strand is cleaved by the enzyme.
- RISC Inducible RNA Silencing Complex
- siRNA 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 directional killing effect on EGFRvIII mutant tumors is more significant.
- the lymphocyte cell surface immunological checkpoint is preferably CTLA4 or PD1, and the intralymphocyte immune checkpoint is preferably CBL-B.
- the lymphocyte cell surface immunological checkpoint CTLA4 or PD1 is silenced or the intracellular immune checkpoint CBL-B is silenced, so that the transgenic lymphocytes have more significant resistance to tumor-mediated immunosuppression. Its proliferation and viability in tumor patients are further improved, and the effect of targeted killing of tumors is more significant.
- the lymphocytes of the embodiments of the invention are CD3+ lymphocytes or natural killer cells or natural killer T cells.
- CD3+ lymphocytes are total T cells
- natural killer cells are a type of immune cells that non-specifically recognize target cells
- natural killer T cells are T cell subsets with T cells and natural killer cell receptors.
- the immune checkpoint is silenced and the chimeric antigen receptor is expressed, so that the cellular immunity of the lymphocytes is more targeted and killing, and the killing effect on the tumor cells is more remarkable.
- the invention proposes a lentivirus or construct.
- the lentivirus or construct carries a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 1 encoding a chimeric antigen receptor
- the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 2; and the nucleic acid molecule which silences the cell surface or the intracellular immunological checkpoint, and the nucleotide sequence of the nucleic acid molecule which silences the cell surface immunological checkpoint is selected from the group consisting of SEQ ID NO: at least one of 3 to 68, silencing the nucleus of the intracellular immune checkpoint
- the nucleotide sequence of the acid molecule is at least one selected from the group consisting of SEQ ID NOS: 69 to 135.
- SEQ ID NOs: 3 to 14 are human programmed death receptor 1 (PD1) siRNA nucleotide sequences, and SEQ ID NOs: 15 to 30 are human cytotoxic T lymphocyte-associated antigen 4 (CTLA4) siRNA sequences, SEQ ID NO: 31 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 (interleukin-1 receptor-associated kinase 3) siRNA nucleotide sequence
- SEQ ID NO: 86 - 96 is a human SOCS1 (cytokine signaling inhibitor 1) siRNA sequence
- SEQ ID NOs: 97-116 are human A20 (tumor necrosis factor- ⁇ -inducible protein A20) siRNA sequences
- SEQ ID NOs: 117-135 are human CBL -B (E3 ubiquitin protein ligase CBL-B) siRNA sequence
- the retrovirus or construct of the present invention is introduced into transgenic lymphocytes obtained from lymphocytes, and cell surface immunity Checkpoint PD1, CTLA4, TIM3, BTLA, LAG3 or intracellular immune checkpoint IRA KM, SOCS1, A20, CBL-B are specifically silenced and inhibited expression, while
- the lentivirus or construct of an embodiment of the invention carries a nucleotide sequence as set forth in SEQ ID NO: 136, 137, 138 or 139.
- SEQ ID NO: 136 is a nucleic acid molecule (EGFRvIII-CAR/iPD1) co-expressing an anti-EGFRvIII chimeric antigen receptor and a silencing cell surface immunological checkpoint PD1
- SEQ ID NO: 137 is a co-expressing anti-EGFRvIII chimeric antigen.
- SEQ ID NO: 138 is a co-expressing anti-EGFRvIII chimeric antigen receptor, silencing cell surface immunological checkpoint PD1 and silencing cells
- Internal immunological checkpoint CBL-B nucleic acid molecule EGFRvIII-CAR/iPD1-CBL-B
- SEQ ID NO: 139 is a co-expressing anti-EGFRvIII chimeric antigen receptor, silencing cell surface immunological checkpoint PD1 and silencing another cell
- the surface immunological checkpoint is the nucleic acid molecule of CTLA4 (EGFRvIII-CAR/iPD1-CTLA4).
- the transgenic lymphocytes obtained by introducing the lentivirus 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, as well as anti-EGFRvIII chimeric antigen receptor expression, make 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, thus enabling transgenic
- the proliferation and viability of lymphocytes in tumor patients in vitro and in vivo and their ability to kill in tumor patients are greatly improved, especially for EGFRvIII mutant glioblastoma cells.
- the inventors realize that the above-mentioned cell chimeric antigen receptor and surface or intracellular immunological checkpoint shRNA are independently expressed, respectively, wherein the expression herein refers to a protein. of Expression also refers to RNA transcription.
- Promoter a first promoter operably linked to a nucleic acid molecule encoding a chimeric antigen receptor; and a second promoter operably linked to a nucleic acid molecule that silences a cellular immunological checkpoint .
- the first promoter and the second promoter employed are each independently selected from the group consisting of U6, CMV, H1, EF-1, LTR, RSV promoter, introduction of the first and second promoters,
- the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule of the silencing cell immunological checkpoint are independently expressed, thereby effectively silencing the cell surface or the intracellular immune checkpoint, and ensuring efficient expression of the chimeric antigen receptor, thereby
- the survival rate of lymphocytes in the tumor environment is greatly improved, the targeting effect of lymphocytes is stronger, and the specific killing effect on tumors is more significant.
- a third promoter may be further introduced, the third promoter being operably linked to the nucleic acid molecule of the immunological checkpoint of the silencing cell, and the silencing cell to which the third promoter and the second promoter are ligated
- the immunologically checked nucleic acid molecules differ in that the third promoter and the second promoter respectively activate shRNAs that silence different immunological checkpoints.
- the introduction of the first or second promoter or the further third promoter described above allows the cell surface or intracellular immunological checkpoint to be efficiently expressed on the transgenic lymphocyte membrane of the present invention by high-efficiency silencing and chimeric antigen receptor.
- the immunological regulation of the immune checkpoint is effectively inhibited and the biological effect of the chimeric antigen receptor is ensured, so that the survival rate of lymphocytes in the tumor environment is greatly improved, and the targeted killing effect of lymphocytes is more remarkable.
- the vector of the construct of the embodiment of the present invention is a non-pathogenic viral vector.
- the non-pathogenic viral vector greatly enhances the replication and amplification efficiency of the construct in lymphocytes, and further, the lymphocyte proliferation and viability of the lymphocytes in the embodiment of the invention are greatly enhanced, and the targeting effect of lymphocytes is further enhanced. The killing effect on tumor cells is more significant.
- the 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 broad-spectrum and high-efficiency infection efficiency, so that the cell surface or intracellular immunological checkpoint is highly efficiently silenced and the chimeric antigen receptor is highly expressed in lymphocytes, and the embodiment of the present invention
- the proliferation and viability of lymphocytes in tumor patients are greatly enhanced, the targeting of lymphocytes is further enhanced, and the killing effect on tumor cells is more significant.
- the inventors in order to construct a lentiviral vector, the inventors inserted a nucleic acid of interest into a viral genome at a position of a certain viral sequence in order to construct a lentiviral vector, thereby producing a replication-defective virus.
- the inventors further constructed packaging cell lines (containing the gag, pol and env genes, but excluding LTR and packaging components).
- the inventors introduced a recombinant plasmid containing the gene of interest, together with the lentiviral LTR and the packaging sequence, into a packaging cell line.
- the packaging sequence allows the recombinant plasmid RNA transcript to be packaged into viral particles and then secreted In the medium.
- the inventors collected a matrix containing the recombinant lentivirus, selectively concentrated, and used for gene transfer. Slow vectors can infect a variety of cell types, including cleavable cells and non-dividable cells.
- the lentivirus of the embodiment of the present invention is a complex lentivirus, and in addition to the common lentiviral genes gag, pol and env, other genes having regulatory and structural functions are also included.
- Lentiviral vectors are well known to those skilled in the art, and lentiviruses include: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors produce a biosafety vector by multiple attenuation of HIV-causing genes, such as deletion of the genes env, vif, vpr, vpu and nef.
- Recombinant lentiviral vectors are capable of infecting non-dividing cells and are useful for in vivo and in vitro gene transfer and nucleic acid sequence expression.
- a suitable host cell together with two or more vectors with packaging functions (gag, pol, env, rev and tat), it is possible to infect non-dividing cells.
- the targeting of recombinant viruses is achieved by binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
- the targeting of the recombinant virus confers specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, along with another gene encoding a ligand for the receptor on the particular target cell.
- the lentiviral vector of the present invention can efficiently transport and co-express shRNA (a transport form of siRNA) which can effectively inhibit the expression of PD1 or CTLA4 or CBL-B.
- shRNA a transport form of siRNA
- an adeno-associated viral vector (AAV) of an embodiment of the invention may be constructed using one or more DNAs of a well-known serotype adeno-associated viral vector.
- AAV adeno-associated viral vector
- One skilled in the art constructs a suitable adeno-associated viral vector to carry and co-express a small hairpin RNA that inhibits the expression of the PDl or CTLA4 or CBL-B genes.
- the embodiment of the present invention also includes a microgene.
- Microgenes mean the use of a combination (selected nucleotide sequence and operably necessary related linker sequences) to direct expression of the transform, transcription and/or gene product in a host cell in vivo or in vitro.
- the "operable ligation" sequence is employed to include expression control sequences for a continuous gene of interest, and expression control sequences for trans- or remote control of the gene of interest.
- vectors of the embodiments of the invention also include conventional control elements that permit transcription, transformation, and/or expression of small hairpin RNA in cell infection with the plasmid vector or in a cellular infection with the viral vector.
- a large number of expression control sequences may be used.
- the shRNA expressing promoter is an RNA polymerase promoter.
- the promoter is a RAN polymerase promoter selected from the group consisting of U6, H1, pol I, pol II and pol III.
- the promoter is a tissue-specific promoter.
- the promoter is an inducible promoter.
- the promoter is selected from a promoter based on the selected vector.
- the promoter when a lentiviral vector is selected, the promoter is a U6, H1, CMV IE gene, EF-1 ⁇ , ubiquitin C, or phosphoglycerate kinase (PGK) promoter.
- Other conventional expression control sequences include selectable markers or reporter genes, including the coding for geneticin, hygromycin, ampicillin or guanidine A nucleotide sequence such as puromycin resistance.
- Other components of the carrier include an origin of replication.
- vectors are well known to those skilled in the art and include conventional cloning techniques such as shRNA, polymerase chain reaction and any suitable method for providing the desired nucleotide sequence for use in embodiments of the invention. .
- the inventors constructed viral vectors that co-express small hairpin RNA (shRNA) (used to suppress immune checkpoints) as well as chimeric antigen receptors (CAR).
- shRNA small hairpin RNA
- CAR chimeric antigen receptors
- the small hairpin RNA carrying the siRNA silencing PD1 or CTLA4 or CBL-B and the viral vector or plasmid expressing the chimeric antigen receptor (CAR) are complexed in the embodiment of the present invention, and the viral vector or plasmid can bind to the polymer or Other materials to increase its stability or assist in its targeted movement.
- the invention provides a method of preparing a T lymphocyte or a transgenic lymphocyte as described above.
- the method comprises introducing the construct described above or the lentivirus described above into lymphocytes or T lymphocytes.
- the mode of introduction can be introduced in a manner selected from the group consisting of electroporation or viral infection of host cells.
- the construct or lentivirus of the embodiment of the present invention is successfully introduced into the above lymphocyte or T lymphocyte, and the expression of the chimeric antigen receptor against the antigen EGFRvIII and the cell surface or intracellular immune checkpoint of the lymphocyte are silenced, thereby
- the obtained lymphocytes or T lymphocytes have remarkable anti-tumor-mediated immunosuppressive effects, and the proliferation of tumor patients in vitro and in vivo and the survival ability of tumor patients are greatly improved, and lymphocytes or T lymphocytes are suitable for tumor cells, especially The targeted killing effect of EGFRvIII mutant glioblastoma cells is stronger.
- 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. Therefore, the obtained transgenic lymphocytes or T lymphocytes are expanded in vitro, the proliferation in tumor patients and the survival ability of tumor patients are greatly improved, and the targeted killing effect of transgenic lymphocytes or T lymphocytes on EGFRvIII tumor cells is stronger.
- the therapeutic composition of the embodiments of the invention provided to a patient is preferably applied to a biocompatible solution or an acceptable pharmaceutical carrier.
- the various therapeutic compositions prepared are suspended or dissolved in a pharmaceutically or physiologically acceptable carrier, such as physiological saline; an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
- a pharmaceutically or physiologically acceptable carrier such as physiological saline; an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
- physiological saline such as physiological saline
- an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
- the appropriate carrier will depend to a large extent on the route of administration.
- Other isotonic sterile injections with water and anhydrous, and sterile suspensions with water and anhydrous are pharmaceutically acceptable carriers.
- a sufficient number of viral vectors are transduced into targeted T cells and provide sufficient transgenes to silence PD1 or CTLA4 or CBL-B and express a unique EGFRvIII chimeric antigen receptor.
- the dosage of the therapeutic agent depends primarily on the condition of treatment, age, weight, and the health of the patient, which may result in patient variability.
- Silencing PD1 or CTLA4 or CBL-B and expressing specific methods for the antigen 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.
- At least one of the following biological effects: silencing of cell surface or intracellular immune checkpoints, combined with high expression of chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, resulting in the resulting lymphocytes or T lymphocytes in the glial Survival in the environment of blastoma, non-small cell lung cancer, breast cancer or ovarian cancer is greatly enhanced, lymphocytes or T lymphocytes are tumor cells of glioblastoma, non-small cell lung cancer, breast cancer or ovarian cancer
- the targeted killing effect is stronger, especially the killing effect on the above tumor cells of EGFRvIII mutation is more significant.
- 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 in the embodiment of the invention has a significant targeted killing effect on EGFRvIII mutant tumor cells.
- 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 A cellular immune checkpoint of lymphocytes is silenced and expresses 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.
- 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, 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 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 glioblastoma of EGFRvIII mutation, non- Significant targeted killing of tumor cells of small cell lung cancer, breast cancer or ovarian cancer.
- 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 may utilize specific delivery of the active ingredient to target cells. The device is administered.
- 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 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 the activity of lymphocytes, the lymphocytes of the embodiments of the invention carrying a chimeric antigen receptor, according to an embodiment of the invention, the method comprising: causing the lymph The cell surface or intracellular immune checkpoint of the cell is silenced, and the cell surface or intracellular immune checkpoint, lymphocyte, and chimeric antigen receptor are as previously defined.
- lymphocyte activity according to an embodiment of the present invention includes lymphocyte proliferation ability in vitro, proliferation and viability in a tumor patient body, and killing ability of lymphocytes in a tumor patient to One less.
- 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.
- 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).
- T cells (number of cells 1 ⁇ 10 6 /well) that were non-transduced or transduced with chimeric antigen receptor plasmid were co-cultured with EGFRvIII-U87 brain tumor cells. The ratio of different target cells is changed during the process.
- the production of cytokines in the cell supernatant was determined using a specific enzyme-linked immunosorbent assay (cytokine enzyme-linked immunosorbent assay kit, purchased from R&D Systems, Inc., Minneapolis, MN, USA). The above cell supernatant was taken from the supernatant of cells after 24 hours, 48 hours, and 72 hours of culture, and the results were used to measure the yield of a representative cytokine (interferon- ⁇ ) (IFN ⁇ ).
- IFN ⁇ interferon- ⁇
- the measurement procedure was as follows: 100 ⁇ l/well of a cytokine dilution solution (such as IFN ⁇ ) or a supernatant solution of the test cell to be tested was added to the plate, and the plate was placed at room temperature for 2 hours. After 2 hours, the solution in the plate was discarded and the plate was rinsed with 400 ⁇ l of the washing solution and rinsed four times. After rinsing, 200 microliters of enzyme-linked anti-cytokine antibody was added to each well of the plate. Continue to stand at room temperature for 2 hours, then add 200 microliters of substrate solution to each well. After the addition of the substrate solution, the plate was allowed to stand at room temperature for 30 minutes, after which 50 ⁇ l of the termination reaction solution was added to each well. The optical density of each well of the microplate was measured within 30 minutes. The microplate reader was set at 450 nm.
- anti-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 a silencing cell immunological checkpoint shRNA and an 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).
- FIG. 1 is a schematic representation of a lentiviral vector comprising a sequence encoding an anti-EGFRvIII chimeric antigen receptor, a U6 and H1 promoter sequence, a PD1-shRNA or a CBL-B-shRNA sequence.
- the sequence of the anti-EGFRvIII chimeric antigen receptor is under the initiation of the promoter EF-1, and the CTLA4, PD1 or CBL-B shRNA sequence is under the initiation of promoter U6 or H1.
- T lymphocytes co-expressing PD1-shRNA and anti-EGFRvIII chimeric antigen receptor have higher cell proliferation ability
- peripheral blood lymphocytes are taken from an unnamed blood donor. Peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. Activated T lymphocytes were transduced with lentiviral vector and expanded in vitro in the presence of T lymphocyte activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) as described in Example 1. 2-7 days after lentiviral vector transduction, transduced T cells (number of cells were 1 ⁇ 10 6 /well) were co-cultured with EGFRvIII-U87 brain tumor cells, and after 4 days, the number of cells was preceded by flowmeter Detection. The experimental results are shown in Figure 2.
- T lymphocytes co-expressing PD1-shRNA and anti-EGFRvIII chimeric antigen receptor have more cytokine secretion characteristics
- T lymphocyte activator magnetic beads CD3/CD28 the activated T lymphocytes were transduced by lentiviral vector and expanded in vitro, as described in Example 1. 2-7 days after lentiviral vector transduction, transduced T cells (number of cells were 1 ⁇ 10 6 /well) were co-cultured with EGFRvIII-U87 brain tumor cells, and after 4 days, cytokine secretion was detected by ELISA. .
- the experimental results are shown in Figure 3.
- Figure 3 shows that T lymphocytes transduced with LV-EGFRvIII-CAR/iPD1 secrete more IFNy than T lymphocytes transduced with LV-EGFRvIII-CAR or empty LV-GFP.
- Example 5 Enhanced lysis of T lymphocyte tumor cells with PD1-shRNA 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. T lymphocytes were incubated with T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) for 72 hours at 5% CO 2 at 37 ° C. The medium was supplemented with 2 mmol/L glutamine, 10%. High temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U/ml penicillin/streptomycin double antibody in RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012).
- FCS High temperature inactivated fetal calf serum
- the T cells were seeded on a recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were LV-EGFRvIII CAR/iPD1, LV-EGFRvIII CAR, respectively.
- the no-load (LV-GFP) transduction process is as described in Example 1.
- the transduced T cells were cultured in RPMI-1640 medium and induced for amplification for 7-10 days with recombinant human IL-2 factor (100 ng/ml; purchased from R&D Systems), followed by a functional test.
- the inventors measured the killing effect of T cells (effector cells) transduced with different lentiviruses on glial glioma target cells with EGFRvIII mutation, and the ratio of target cells was 10:1 or 1:1, and the standard method was 4–hour. 51 chromium release method, 4 - hour 51 chromium release method as described in Example 1. The result is shown in Figure 4.
- T lymphocytes co-expressing the anti-EGFRvIII chimeric antigen receptor and PD1-shRNA (iPD1) are more effective at killing the EGFRvIII mutation than T lymphocytes expressing the anti-EGFRvIII chimeric antigen receptor alone.
- the vacant lentivirus-transduced T lymphocytes (control LV-GFP T lymphocytes) had no significant killing effect on EGFRvIII mutant brain tumor cells.
- the statistical data represents the mean ⁇ SEM of the three wells.
- Example 6 Co-expressing CBL-B-shRNA and T cells against the EGFRvIII chimeric antigen receptor, co-expressing PD1-shRNA, CBL-B-shRNA and T cells against the EGFRvIII chimeric antigen receptor, co-expressing PD1-shRNA
- the cells of CTLA4-shRNA and T cells resistant to the EGFRvIII chimeric antigen receptor have enhanced solvency and are characterized by more cytokine secretion and stronger cell proliferation.
- the inventors also examined T cells co-expressing CBL-B-shRNA and anti-EGFRvIII chimeric antigen receptors, co-expressing two shRNAs (PD1-shRNA and CBL-B-shRNA or PD1-shRNA and Tumor lytic ability, cytokine secretion ability, and cell proliferation ability of CTLA4-shRNA) and T cells resistant to the EGFRvIII chimeric antigen receptor.
- the experimental procedure was the same as in Examples 3, 4 and 5.
- 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.
Abstract
提出了一种转基因淋巴细胞、一种构建体和一种治疗癌症的治疗组合物及方法。该转基因淋巴细胞的细胞免疫检查点被沉默；以及表达嵌合抗原受体，其中，所述嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区，所述单链抗体特异性识别抗原EGFRvIII；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述T淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括CD28或4-1BB的胞内段以及CD3ζ链。
Description
优先权信息
本申请请求2016年01月13日向中国国家知识产权局提交的、专利申请号为201610022379.5的专利申请的优先权和权益，并且通过参照将其全文并入此处。
本发明涉及生物医药领域，具体地，本发明涉及一种T淋巴细胞、一种慢病毒、一种转基因淋巴细胞、一种构建体、一种用于治疗癌症的治疗组合物及方法和一种提高淋巴细胞活性的方法。
脑胶质母细胞瘤是恶性度最高的脑瘤，占中枢神经系统恶性肿瘤的～81％，恶性胶质瘤的发病率为3-6/10万,每年中国因此病死亡人数为3万人。恶性胶质瘤呈浸润性生长，与正常脑组织无明显界限，并且多数不限于一个脑叶，呈指状深入破坏脑组织，手术不可能完全切除。虽然母前采用了最为积极的治疗手段，但中位生存期仍只有<15个月，单独使用放疗，仅有不足1％的患者可以存活5年，即使新出现的放疗与替莫唑胺(TMZ)化疗的联用方案，也只使近10％患者存活至5年以上。
由此可见，开发有效治疗脑胶质母细胞瘤的方法尤为迫切.
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
EGFR(epidermal growth factor receptor，表皮生长因子受体)突变体EGFRvIII(EGFR胞外段的267个氨基酸被去除，其是由EGFR基因的第2～7个外显子(275～1075位核苷酸)去除后的基因编码的)在约30％的胶质母细胞瘤患者中表达，但在正常组织中未检测到，因此，EGFRvIII可代表胶质母细胞瘤的特异性突变。
基于上述发现，发明人提出了一种携带沉默细胞免疫检查点的核酸分子和编码嵌合抗原受体的核酸分子的构建体和一种以此构建体导入后形成的转基因淋巴细胞，其编码的嵌合抗原受体特异性结合抗原EGFRvIII。因此，本发明提出的构建体和转基因淋巴细胞可用于肿瘤，尤其是胶质母细胞瘤的过继T细胞的免疫治疗，本发明提出的转基因淋巴细胞对EGFRvIII突变的脑胶质母细胞瘤细胞具有强的特异杀伤能力。
在本发明的第一方面，本发明提出了一种T淋巴细胞。根据本发明的实施例，所述T
淋巴细胞的细胞免疫检查点被沉默；以及表达嵌合抗原受体，其中，所述嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区，所述单链抗体特异性识别抗原EGFRvIII；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述T淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括CD28或4-1BB的胞内段以及CD3ζ链。其中，细胞免疫检查点包括细胞表面或细胞内至少之一的免疫检查点。根据本发明的实施例，本发明实施例的T淋巴细胞具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著提高，对肿瘤细胞的杀伤能力显著增强，尤其对EGFRvIII突变的脑胶质母细胞瘤具有显著的定向杀伤作用。
在本发明的第二方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带下列核酸分子：编码嵌合抗原受体的核酸分子，所述嵌合抗原受体具有SEQ ID NO：1所示的氨基酸序列，所述编码嵌合抗原受体的核酸分子具有SEQ ID NO：2所示的核苷酸序列；以及沉默细胞免疫检查点的核酸分子，所述沉默细胞免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：3～135的至少之一。
根据本发明的实施例，将本发明实施例的慢病毒导入淋巴细胞所得的转基因淋巴细胞，其具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著增强，对肿瘤细胞的杀伤能力显著增强，尤其对EGFRvIII突变的脑胶质母细胞瘤具有显著的定向杀伤作用。
在本发明的第三方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带含有SEQ ID NO：136、137、138或139所示的核苷酸序列。
根据本发明的实施例，将本发明实施例的慢病毒导入淋巴细胞所得的转基因淋巴细胞，其具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著增强，对肿瘤细胞的杀伤能力显著增强，尤其对EGFRvIII突变的脑胶质母细胞瘤具有显著的定向杀伤作用。
在本发明的第四方面，本发明提出了一种转基因淋巴细胞。根据本发明的实施例，所述淋巴细胞细胞免疫检查点被沉默；以及表达嵌合抗原受体。发明人惊奇的发现，细胞免疫检查点被沉默和表达嵌合抗原受体的淋巴细胞的体外增殖能力、在肿瘤病人体内的增殖和生存能力以及在肿瘤病人体内的对肿瘤细胞的特异性杀伤能力大大提高，尤其对EGFRvIII突变的脑胶质母细胞瘤具有显著的定向杀伤作用。
根据本发明的实施例，上述转基因淋巴细胞还可以具有下列附加技术特征至少之一：
根据本发明的实施例，所述嵌合抗原受体包括：胞外区，所述胞外区能够与抗原特异性结合；跨膜区；以及胞内区，所述胞内区包括免疫共刺激分子胞内段。具有上述结构的嵌合抗原受体的存在，大大提高了本发明实施例的转基因淋巴细胞的靶向定位，大大提高了本发明实施例的转基因淋巴细胞对抗原表达肿瘤细胞的定向杀伤作用。
根据本发明的实施例，所述抗原是肿瘤抗原。因此，本发明实施例的转基因淋巴细胞对肿瘤的定向杀伤作用更加显著。
根据本发明的实施例，所述胞外区包括抗体的重链可变区和轻链可变区，所述抗体结合所述抗原。抗原抗体的特异性结合作用，大大提高了本发明实施例的转基因淋巴细胞的靶向定位和对表达抗原的肿瘤细胞的定向杀伤作用。
根据本发明的实施例，所述抗体为单链抗体。单链抗体可去除非特异性反应的竞争性表面蛋白，同时单链抗体更易渗透肿瘤组织增加药物治疗浓度。本发明实施例的转基因淋巴细胞表达单链抗体的嵌合抗原受体，大大提高了本发明实施例的转基因淋巴细胞对靶向肿瘤细胞的定向杀伤作用。
根据本发明的实施例，所述抗原为EGFRvIII。因此所述转基因淋巴细胞针对表达抗原EGFRvIII的细胞具有定向性杀伤作用，抗原抗体的特异性结合作用更强，大大提高了本发明实施例的转基因淋巴细胞对EGFRvIII抗原表达肿瘤细胞的定向杀伤作用。
根据本发明的实施例，所述淋巴细胞细胞免疫检查点独立地选自CTLA4、PD1、TIM3、BTLA、LAG3、IRAK-M、SOCS1、A20、CBL-B的至少之一。其中，CTLA4、PD1、TIM3、BTLA、LAG3为细胞表面免疫检查点，IRAK-M、SOCS1、A20、CBL-B为细胞内免疫检查点。本发明实施例的免疫检查点具有负向调控和减弱细胞免疫应答的作用，其通过与肿瘤细胞上相应的配体的特异结合，导致T淋巴细胞增生性反应的下调,细胞因子的分泌减少,
和T细胞的无能或凋亡。根据本发明的实施例，本发明实施例的细胞表面或细胞内免疫检查点的成功沉默，进一步提高了转基因淋巴细胞抵抗肿瘤介导的免疫抑制的功效，转基因淋巴细胞在体外扩增及在肿瘤病人体内的增殖和生存能力、对肿瘤细胞的定向杀伤作用进一步加强。
根据本发明的实施例，所述淋巴细胞细胞表面免疫检查点被沉默是通过shRNA、反义核酸、核酶、显性负突变、CRISPR和锌指核酸酶至少之一实现的。根据本发明的实施例，本发明实施例的细胞免疫检查点的成功沉默，可显著提高本发明实施例的淋巴细胞抵抗肿瘤介导的免疫抑制的特性，进一步提高了转基因淋巴细胞增殖肿瘤细胞的定向杀伤作用。
根据本发明的实施例，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28以及他们的衍生物的至少一种。本发明实施例的免疫共刺激分子胞内段的表达以及细胞免疫检查点的沉默联合具有正向调控和增强细胞免疫应答的作用，使得本发明实施例的转基因淋巴细胞增殖对肿瘤的定向杀伤作用效果更加显著；本发明实施例的免疫共刺激分子胞内段的表达以及细胞免疫检查点的沉默的联合，使得本发明实施例的转基因淋巴细胞的增殖能力以及对肿瘤的定向杀伤作用更加显著。
根据本发明的实施例，所述淋巴细胞细胞免疫检查点是CTLA4、PD1、CBL-B。其中，CTLA4、PD1是细胞表面免疫检查点，CBL-B是细胞内免疫检查点。根据本发明的实施例，本发明实施例的淋巴细胞细胞表面免疫检查点CTLA4或PD1被沉默，或淋巴细胞细胞内免疫检查点是CBL-B被沉默，阻止了PD1或CTLA4分子表达与其相应配体PD-L1及PD-L2或CD80及CD86的结合，从而有效抑制了对T淋巴细胞的无能或凋亡，或通过CBL-B沉默,增强T细胞受体信号传导，使得转基因淋巴细胞在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著。
根据本发明的实施例，所述淋巴细胞细胞内免疫检查点被沉默是通过shRNA实现的。根据本发明的实施例，本发明实施例的shRNA携带特异性沉默细胞表面或细胞内至少之一免疫检查点的shRNA，本发明实施例的shRNA具有高效、特异性的沉默细胞表面或细胞内至少之一免疫检查点的作用，细胞免疫检查点的成功沉默，即细胞表面或细胞内免疫检查点的成功沉默，阻止了免疫检查点与相应配体的特异性结合，从而有效抑制了免疫检查点对T淋巴细胞无能或凋亡等的负调控机制，进而使得本发明实施例的转基因淋巴细胞在在肿瘤病人体内的增殖和生存能力得到进一步提高，配合嵌合抗原受体的抗原靶向性，使得本发明实施例的转基因淋巴细胞对肿瘤的定向杀伤作用效果更加显著。
根据本发明的实施例，所述免疫共刺激分子胞内段是4-1BB或CD28的胞内段。本发明中的转基因淋巴细胞的嵌合抗原受体的免疫共刺激分子胞内段是CD28或者4-1BB的胞内段。根据本发明的实施例，免疫共刺激分子胞内段是CD28或者4-1BB的胞内段，进一
步增强了本发明实施例的转基因淋巴细胞的定向杀伤作用。
根据本发明的实施例，所述淋巴细胞是CD3+T淋巴细胞或自然杀伤细胞或自然杀伤T细胞。本发明实施例的上述淋巴细胞的细胞免疫检查点被沉默同时表达抗原特异性的嵌合抗原受体，如本发明的实施例的EGFRvIII抗原特异性的嵌合抗原受体，上述淋巴细胞的细胞免疫杀伤作用的靶向性更强，在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著。
在本发明的第九方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体包括：第一核酸分子，所述第一核酸分子编码嵌合抗原受体；以及第二核酸分子，所述第二核酸分子沉默细胞免疫检查点。其中，所述细胞免疫检查点、所述嵌合抗原受体如前所述。根据本发明的实施例，本发明实施例的构建体成功导入本发明实施例的淋巴细胞后，可有效沉默细胞表面或细胞内至少之一免疫检查点和表达抗原特异性的嵌合抗原受体，从而本发明实施例的淋巴细胞对肿瘤细胞，尤其是EGFRvIII突变的肿瘤细胞的定向杀伤作用更加显著。
根据本发明的实施例，上述构建体还可以进一步包括下列附加技术特征至少之一：
根据本发明的实施例，其特征在于所述第一核酸分子与所述第二核酸分子被设置在前面所述的淋巴细胞中沉默细胞免疫检查点和表达所述嵌合抗原受体。根据本发明的实施例，成功设置了上述第一核酸分子以及第二核酸分子的淋巴细胞，其淋巴细胞的细胞表面或细胞内至少之一的免疫检查点被成功沉默，同时在淋巴细胞表面成功表达了抗原特异性，如本发明实施例的EGFRvIII特异性的嵌合抗原受体，其具有杀伤力更强和特异性更强的肿瘤杀伤效果，安全性更高。
根据本发明的实施例，所述构建体进一步包括：第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；以及第二启动子,所述第二启动子与所述第二核酸分子可操作地连接。根据本发明的实施例，第一启动子以及第二启动子的引入，使得第一核酸分子以及第二核酸分子分别独立的表达，有效保证了嵌合抗原受体抗原靶向性的生物学作用及以有效沉默了细胞免疫检查点，使得本发明实施例的淋巴细胞的靶向作用更强，对肿瘤的杀伤作用，尤其对EGFRvIII突变的肿瘤细胞的定向杀伤更加显著。
根据本发明的实施例，所述第一启动子、所述第二启动子分别独立地选自U6，H1,CMV，EF-1,LTR,RSV启动子。根据本发明的实施例，本发明实施例的上述启动子具有启动效率高、特异性强的特点，从而保证了细胞免疫检查点的高效沉默和嵌合抗原受体的高效表达，从而本发明实施例的淋巴细胞的体外增殖能力、在肿瘤病人体内的增殖和生存能力大大提高，对肿瘤的定向杀伤效果更加显著。
根据本发明的实施例，所述构建体的载体是非致病性病毒载体。非致病性病毒载体的引
入大大提高了构建体在淋巴细胞中的复制和扩增效率，从而大大提高了细胞免疫检查点的沉默和嵌合抗原受体在淋巴细胞中的高效表达，使得淋巴细胞体外增殖能力、在肿瘤病人体内的增殖和生存能力大大提高，淋巴细胞的靶向作用进一步增强，对肿瘤细胞的杀伤作用更加显著。
根据本发明的实施例，所述构建体的载体是病毒载体，所述病毒载体包括选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体的至少之一。本发明实施例的病毒的载体在病毒包装和感染过程中，病毒感染范围广泛，既可感染终末分化细胞，又可感染处于分裂期的细胞，其基因组既可整合到宿主染色体，又可游离在宿主染色体之外，从而可实现广谱而高效的感染效率，细胞免疫检查点被高效沉默和嵌合抗原受体在淋巴细胞中的高效表达，使得本发明实施例的淋巴细胞的体外增殖能力、在肿瘤病人体内的增殖和生存能力大大提高，淋巴细胞的靶向作用进一步增强，对肿瘤细胞，尤其是EGFRvIII突变的肿瘤细胞的定向杀伤作用更加显著。
在本发明的第十方面，本发明提出了一种制备前面所述的T淋巴细胞或者转基因淋巴细胞的方法。根据本发明的实施例，所述方法包括：将前面所述的构建体或者前面所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。所述构建体或慢病毒成功引入上述淋巴细胞或者T淋巴细胞中，实现了淋巴细胞的细胞免疫检查被沉默和嵌合抗原受体的表达，从而本发明实施例的制备方法制备的转基因淋巴细胞或T淋巴细胞在肿瘤病人体内和体外的增殖及肿瘤病人体内存活能力大大提高，转基因淋巴细胞或T淋巴细胞对肿瘤细胞，尤其是对EGFRvIII突变的肿瘤细胞的靶向杀伤作用更强。
在本发明的第十一方面，本发明提出了一种用于治疗癌症的治疗组合物。根据本发明的实施例，所述治疗组合物包括：上述构建体、慢病毒、T淋巴细胞或者转基因淋巴细胞。上述任意一种治疗组合物的组成均可实现转基因淋巴细胞或T淋巴细胞的细胞表面或细胞内免疫检查点的沉默和嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达，从而使得所得转基因淋巴细胞或T淋巴细胞具有显著的抵抗肿瘤细胞介导的免疫抑制，在肿瘤病人体外和体内的增殖及肿瘤病人体内存活能力大大提高，转基因淋巴细胞或T淋巴细胞对肿瘤细胞的靶向杀伤作用更强，本发明实施例的治疗癌症的治疗组合物对肿瘤细胞的靶向杀伤作用显著增强，尤其是对EGFRvIII突变的肿瘤细胞的靶向杀伤作用显著增强。
根据本发明的实施例，上述治疗组合物还可以进一步包括下列附加技术特征至少之一：
根据本发明的实施例，所述癌症包括脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌。脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌癌细胞具有EGFRvIII的特异性表达，本发明实施例的治疗组合物可使淋巴细胞细胞表面或细胞内免疫检查点的沉默和高效表达抗原特异性嵌合抗原受体，如本发明实施例的EGFRvIII抗原特异性嵌合抗原受体，所得淋巴
细胞或T淋巴细胞具有显著的抵抗肿瘤细胞介导的免疫抑制的特性，在脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌的微环境中的存活能力大大提高，所得淋巴细胞或T淋巴细胞对EGFRvIII突变的脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌癌的肿瘤细胞的靶向杀伤作用更强。
在本发明的第十二方面，本发明提出了一种治疗癌症的方法。根据本发明的实施例，所述方法包括：为癌症患者给药前面所述的构建体、前面所述的慢病毒、前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞，其中，嵌合抗原受体特异性结合肿瘤抗原EGFRvIII。本发明实施例提出的方法对EGFRvIII突变的肿瘤细胞具有显著的靶向杀伤作用。
根据本发明的实施例，上述治疗癌症的方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述方法包括：从癌症患者体内分离淋巴细胞；将前面所述的构建体，或前面所述的慢病毒导入所述淋巴细胞，以便获得转基因淋巴细胞，所述转基因淋巴细胞的细胞免疫检查点被沉默和表达所述嵌合抗原受体；以及为所述癌症患者给药所述转基因淋巴细胞。本发明实施例的方法对EGFRvIII突变的肿瘤细胞的靶向杀伤作用进一步增强。
根据本发明的实施例，所述癌症包括选自脑胶质母细胞瘤、非小细胞肺癌、乳腺癌和卵巢癌的至少之一。如前所述，脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌癌细胞具有EGFRvIII的特异性表达，本发明实施例的治疗癌症的方法可使淋巴细胞的细胞免疫检查点被高效沉默和高效表达抗原特异性嵌合抗原受体，如本发明实施例的EGFRvIII抗原特异性嵌合抗原受体，所得淋巴细胞或T淋巴细胞具有对EGFRvIII突变的脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌癌的肿瘤细胞的显著的靶向杀伤作用。
在本发明的第十三方面，本发明提出了一种提高淋巴细胞活性的方法，所述淋巴细胞携带嵌合抗原受体，根据本发明的实施例，所述方法包括：使所述淋巴细胞的细胞免疫检查点被沉默。所述细胞免疫检查点、所述淋巴细胞和所述嵌合抗原受体是如前所定义的，所述淋巴细胞活性包括所述淋巴细胞体外增殖能力、在肿瘤病人体内的增殖和生存能力以及所述淋巴细胞在肿瘤病人体内的定向杀伤能力的至少一种。根据本发明的实施例，本发明实施例的淋巴细胞的细胞表面或细胞内免疫检查点被沉默，淋巴细胞被活化、增生性反应上调、细胞因子分泌增多、抗调亡能力增强，使得本发明实施例的淋巴细胞在体外扩增、在肿瘤病人体内的增殖及肿瘤病人体内存活能力大大提高上述淋巴细胞细胞免疫检查点的沉默配合淋巴细胞嵌合抗原受体的抗原特异性功效，从而实现了有效抵抗肿瘤细胞介导的免疫抑制，对EGFRvIII突变的肿瘤细胞的靶向杀伤作用显著增强。
根据本发明的实施例，上述提高淋巴细胞活性的方法还可以进一步包括如下附加技术特
征至少之一：
根据本发明的实施例，所述肿瘤包括选自EGFRvIII突变的脑胶质母细胞瘤、EGFRvIII突变的非小细胞肺癌、EGFRvIII突变的乳腺癌或EGFRvIII突变的卵巢癌的至少之一。本发明实施例的提高淋巴细胞活性的方法，是使淋巴细胞携带EGFRvIII抗原特异性的嵌合抗原受体，同时使淋巴细胞的免疫检查点被沉默，本发明实施例的提高淋巴细胞活性的方法特异性进一步提高了对EGFRvIII突变的肿瘤细胞的定向杀伤能力，如上述EGFRvIII突变的肿瘤细胞。
需要说明的是，本发明中所使用的术语“细胞免疫检查点”包括细胞表面免疫检查点和细胞内免疫检查点，“细胞表面免疫检查点”是一种淋巴细胞表面的膜蛋白，其与肿瘤细胞上表达的配体相互作用，可以抑制抗肿瘤淋巴细胞反应。“细胞内免疫检查点”是一种细胞内蛋白，此种细胞免疫检查点蛋白一种负调控的细胞信号传导机构蛋白，可以抑制抗肿瘤淋巴细胞反应。
图1是根据本发明实施例的共表达EGFRvIII抗原特异性的嵌合抗原受体和沉默人类细胞免疫检查点的慢病毒载体的结构示意图；
图2是根据本发明实施例的共表达EGFRvIII抗原特异性的嵌合抗原受体和沉默PD1的淋巴细胞增殖能力增强的结果图；
图3是根据本发明实施例的共表达EGFRvIII抗原特异性的嵌合抗原受体和沉默PD1的淋巴细胞干扰素-γ分泌增多的结果图；以及
图4是根据本发明实施例的共表达EGFRvIII抗原特异性的嵌合抗原受体和沉默PD1的淋巴细胞杀伤肿瘤细胞能力增强的结果图。
下面详细描述本发明的实施例，下面描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
T淋巴细胞或转基因淋巴细胞
在本发明的一方面，本发明提出了一种T淋巴细胞或转基因淋巴细胞。根据本发明的实施例，本发明实施例的T淋巴细胞的细胞免疫检查点被沉默；以及表达嵌合抗原受体，其中，嵌合抗原受体包括：胞外区，胞外区包括单链抗体的重链可变区和轻链可变区，单链抗体特异性识别抗原EGFRvIII；跨膜区，跨膜区与胞外区相连，并且嵌入到所T淋巴细胞的细胞膜中；胞内区，胞内区与跨膜区相连，并且胞内区包括CD28或4-1BB的胞内段
以及CD3ζ链。其中，细胞免疫检查点包括细胞表面或细胞内的免疫检查点。本发明实施例的T淋巴细胞或转基因淋巴细胞细胞免疫检查点被沉默联合表达EGFRvIII抗原特异性的嵌合抗原受体，本发明实施例的T淋巴细胞或转基因淋巴细胞在肿瘤病人体内和体外的增殖和生存能力以及在肿瘤病人体内的对特意性肿瘤细胞的杀伤能力显著增强，尤其对EGFRvIII突变的脑胶质母细胞瘤细胞的特异性杀伤效果大大提高。
肿瘤可以避免免疫监视，通过刺激其免疫抑制性受体的表达而关闭淋巴细胞对其的免疫杀伤反应；作为免疫负调节机制,激活的细胞毒性T淋巴细胞(CTLs)也表达负调控的监管机构,即细胞表面或细胞内的免疫检查点分子。如本发明实施例的程序性细胞死亡1受体(PD1)表达在活化CTLs上，其与肿瘤细胞上表达的程序性死亡配体1(PD-L1)相互作用,可以抑制抗肿瘤T细胞反应。许多肿瘤,包括淋巴瘤、肺癌、卵巢癌、黑色素瘤、胰腺肿瘤,表达PD-L1。PD-L1与其配体PD1的结合，导致CTLs增生性反应的下调,细胞因子的分泌减少,和T细胞的无能或凋亡。本发明实施例的细胞毒性T淋巴细胞抗原4(CTLA4)是另一个T细胞的关键负面调节因子，其可抑制T细胞活化，其通过与表达在抗原递呈细胞上的配体B7.1、B7.2(CD80和CD86)的相互作用而抑制T细胞的活化。本发明实施例的细胞毒性T淋巴细胞内的CBL-B(E3泛素蛋白连接酶CBL-B)是细胞内的另一个关键负面调节因子，其通过抑制T细胞受体(TCR)信号传导，来抑制T细胞的活性。因此，本发明实施例的T淋巴细胞或转基因淋巴细胞的免疫检查点被沉默，T淋巴细胞或转基因淋巴细胞的在肿瘤病人体内的增殖和生存能力显著提高。
另外，根据本发明的实施例，上述嵌合抗原受体胞外区的抗体为单链抗体。发明人发现，单链抗体可去除非特异性反应的竞争性表面蛋白，同时单链抗体更易渗透肿瘤组织增加药物治疗浓度。本发明实施例的转基因淋巴细胞表达单链抗体的嵌合抗原受体，大大提高了转基因淋巴细胞对靶向肿瘤细胞的定向杀伤作用。
根据本发明的另外一些实施例，上述抗体的结合抗原为EGFRvIII。因此本发明实施例的转基因淋巴细胞针对表达抗原EGFRvIII的细胞具有定向性杀伤作用，抗原抗体的特异性结合作用更强，大大提高了本发明实施例的转基因淋巴细胞对EGFRvIII抗原表达肿瘤细胞的定向杀伤作用。
根据本发明的另外一些实施例，淋巴细胞的细胞免疫检查点包括细胞表面和细胞内的免疫检查点，本发明实施例的淋巴细胞细胞表面免疫检查点独立地选自CTLA4、PD1、TIM3、BTLA、LAG-3至少之一，淋巴细胞细胞内免疫检查点独立地选自IRAK-M、SOCS1、A20、CBL-B的至少之一。上述分子能够与肿瘤细胞表达的抗原特异性结合，抑制淋巴细胞的活化，促进淋巴细胞的无能或凋亡，从而负向调控和减弱细胞免疫应答。根据本发明的实施例，上述细胞表面或细胞内免疫检查点的成功沉默，进一步提高了转基因淋巴细胞在肿瘤
病人体内的增殖和生存能力，对肿瘤细胞的定向杀伤作用进一步加强。
根据本发明的另外一些实施例，本发明实施例的淋巴细胞细胞表面免疫检查点被沉默是通过shRNA、反义核酸、核酶、显性负突变、锌指核酸酶和CRISPR至少之一实现的。
小发夹RNA或短发夹RNA(shRNA)是siRNA(小干扰RNA)的导入形式，siRNA是一种小RNA分子(由21～25个核苷酸组成)，由Dicer(RNAaseⅢ家族中对双链RNA具有特异性剪切作用的酶)加工而成；siRNA在RNA沉默通路中起中心作用，对特定信使RNA(mRNA)进行降解，为转录水平后调控。
反义核酸包括反义RNA和反义DNA，反义RNA是指能和mRNA完全互补的一段小分子RNA或寡聚核苷酸片段，反义DNA是指能与基因DNA双链中的有义链互补结合的短小DNA分子，反义RNA和反义DNA主要是通过mRNA的翻译和基因DNA的转录而发挥作用的；反义核酸一方面通过与靶mRNA结合形成空间位阻效应，阻止核糖体与mRNA结合，另一方面其与mRNA结合后激活内源性RNA酶或核酶，进而降解mRNA；反义DNA与基因DNA双螺旋的调控区特异结合形成DNA三聚体，或与DNA编码区结合，终止正在转录的mRNA链的延长；反义核酸还可抑制转录后mRNA的加工修饰，如5'端加帽、3'端加尾、中间剪接和内部碱基甲基化等，并阻止成熟mRNA由细胞核向细胞浆内运输，因此，反义RNA是一种有效的沉默目的基因的技术。
核酶是具有催化功能的RNA分子，是生物催化剂，可降解特异的mRNA序列，核酶通过催化转磷酸酯和磷酸二酯键水解反应参与RNA自身剪切、加工过程，与一般的反义RNA相比，核酶具有较稳定的空间结构，不易受到RNA酶的攻击，更重要的是，核酶在切断mRNA后，又可从杂交链上解脱下来，重新结合和切割其它的mRNA分子。
显性负性突变是指某些信号转导蛋白突变后不仅自身无功能，还能抑制或阻断同一细胞内的野生型信号转导蛋白的作用，其主要通过和野生型蛋白形成二聚物的方式实现，这种突变毒性作用大，能显著抑制或阻断细胞内目标信号转导蛋白的作用。
锌指核酸酶由一个DNA识别域和一个非特异性核酸内切酶构成，DNA识别域是由一系列Cys2-His2锌指蛋白串联组成(一般3～4个)，每个锌指蛋白识别并结合一个特异的三联体碱基，锌指蛋白形成α-β-β二级结构，其中α螺旋的16氨基酸残基决定锌指的DNA结合特异性，骨架结构保守，对决定DNA结合特异性的氨基酸引入序列的改变可以获得新的DNA结合特异性，从而可以针对不同的目的基因设计不同的氨基酸引入序列，实现不同目的基因的特异性沉默。
CRISPR(Clustered regularly interspaced short palindromic repeats,规律成簇间隔短回文重复)，是一种基因编辑器，是细菌用以保护自身对抗病毒的一个系统。它可以用来删除、添加、激活或抑制其他生物体的目标基因，这些目标基因包括人细胞内的目标基因。
CRISPR簇是一个广泛存在于细菌和古生菌基因组中的特殊DNA重复序列家族，其序列由一个前导区(Leader)、多个短而高度保守的重复序列(Repeat)和多个间隔区(Spacer)组成。前导区一般位于CRISPR簇上游，是富含AT长度为300～500bp的区域，被认为可能是CRISPR簇的启动子序列。重复序列区长度为21～48bp，含有回文序列，可形成发卡结构。重复序列之间被长度为26～72bp的间隔区隔开。Spacer区域由俘获的外源DNA组成，当含有同样序列的外源DNA入侵时，可被细菌机体识别，并进行剪切使之表达沉默，达到保护自身安全的目的。通过对CRISPR簇的侧翼序列分析发现，在其附近存在一个多态性家族基因。该家族编码的蛋白质均含有可与核酸发生作用的功能域(具有核酸酶、解旋酶、整合酶和聚合酶等活性)，并且与CRISPR区域共同发挥作用，因此被命名为CRISPR关联基(CRISPR associated)，缩写为Cas。目前发现的Cas包括Cas1～Cas10等多种类型。Cas基因与CRISPR共同进化，共同构成一个高度保守的系统。当细菌抵御噬菌体等外源DNA入侵时，在前导区的调控下，CRISPR被转录为长的RNA前体(Pre RISPR RNA，pre-crRNA)，然后加工成一系列短的含有保守重复序列和间隔区的成熟crRNA，最终识别并结合到与其互补的外源DNA序列上发挥剪切作用。pre-crRNA的加工由Cas家族中的Cas9参与。Cas9含有在氨基末端的RuvC和蛋白质中部的HNH2个独特的活性位点，在crRNA成熟和双链DNA剪切中发挥作用。pre-crRNA转录的同时，与其重复序列互补的反式激活crRNA(Trans-activating crRNA，tracrRNA)也转录出来，并且激发Cas9和双链RNA特异性RNase III核酸酶对pre-crRNA进行加工。加工成熟后，crRNA、tracrRNA和Cas9组成复合体，识别并结合于crRNA互补的序列，然后解开DNA双链，形成R-loop，使crRNA与互补链杂交，另一条链保持游离的单链状态，然后由Cas9中的HNH活性位点剪切crRNA的互补DNA链，RuvC活性位点剪切非互补链，最终引入DNA双链断裂(DSB)。通过人工设计RNA，可以改造形成具有引导作用的sgRNA(short guide RNA)，以引导Cas9对DNA的定点目标基因切割。
综上所述，shRNA、反义核酸、核酶、显性负突变、CRISPR、锌指核酸酶为特异性沉默目标基因的有效手段，沉默基因的手段不受特别限制，本领域技术人员可根据具体的实验目的和条件选择，如本发明实施例所采用的shRNA、反义核酸、核酶、显性负突变，CRISPR或锌指核酸酶的至少之一，实现目的基因的特异性沉默。
根据本发明的实施例，淋巴细胞细胞表面或细胞内免疫检查点被沉默优选采用shRNA实现。ShRNA所携带的siRNA分子通常是一个长度在10和30之间的碱基对的双重区域。本发明实施例的PD1或CTLA4或CBL-B siRNA被设计为同源于PD1或CTLA4或CBL-B mRNA的编码区域，通过mRNA的降解来抑制基因表达。siRNA关联于被称为诱导RNA沉默复合物(RISC)的多重蛋白复合物，在此期间正义链被酶裂解。被激活的RISC中基于序列同源性，指引RISC到对应的mRNA；相同的核酸酶切割靶向PD1或CTLA4或CBL-B
mRNA，产生特定基因PD1或CTLA4或Cb1沉默，抑制特定基因PD1或CTLA4或CBL-B的表达。siRNA以shRNA的形式导入细胞(shRNA包含大约18-23的核苷酸siRNA序列，后跟一个9-15长度的核苷酸环和一个siRNA序列的反向补充)，shRNA的设计较好的避免了在3’UTR细胞基因中的匹配点；确保了适当的链选择。一个单一siRNA分子可被重复应用于多靶向mRNA分子的分裂。RNAi(RNA干扰)可通过引入合成siRNA的方式被诱导。根据本发明的实施例，本发明实施例的shRNA不断产自细胞内，因此其效果更加持久，从而延长shRNA周期，本发明实施例采用的shRNA具有高效、特异性的沉默细胞表面或细胞内免疫检查点的作用，细胞表面或细胞内免疫检查点的成功沉默，使得转基因淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的特性，在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著。
另外，根据本发明的实施例，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28以及他们的衍生物的至少一种。免疫共刺激分子胞内段的表达和细胞表面或细胞内至少之一免疫检查点的沉默联合具有正向调控和增强细胞免疫应答的作用，使得转基因淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的特性，在肿瘤病人体内的增殖和生存能力得到进一步提高，对EGFRvIII突变的肿瘤的定向杀伤作用效果更加显著。
根据本发明的另外一些实施例，淋巴细胞细胞表面免疫检查点优选CTLA4或PD1，淋巴细胞内免疫检查点优选CBL-B。根据本发明的时施例，淋巴细胞细胞表面免疫检查点CTLA4或PD1被沉默或细胞内免疫检查点CBL-B被沉默，使得转基因淋巴细胞具有更加显著的抵抗肿瘤介导的免疫抑制的特性，其在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著。
根据本发明的实施例，本发明实施例的淋巴细胞是CD3+淋巴细胞或自然杀伤细胞或自然杀伤T细胞。CD3+淋巴细胞是总T细胞，自然杀伤细胞是免疫细胞的一种，非特异性性识别靶细胞，自然杀伤T细胞是具有T细胞和自然杀伤细胞受体的T细胞亚群。上述淋巴细胞中免疫检查点被沉默和表达嵌合抗原受体，使得上述淋巴细胞的细胞免疫的靶向杀伤性更强，对肿瘤细胞的杀伤作用效果更加显著。
慢病毒或构建体
在本发明的另一方面，本发明提出了一种慢病毒或构建体。根据本发明的实施例，慢病毒或构建体携带下列核酸分子：编码嵌合抗原受体的核酸分子，嵌合抗原受体具有SEQ ID NO：1所示的氨基酸序列，编码嵌合抗原受体的核酸分子具有SEQ ID NO：2所示的核苷酸序列；以及沉默细胞表面或细胞内免疫检查点的核酸分子，沉默细胞表面免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：3～68的至少之一，沉默细胞内免疫检查点的核
酸分子的核苷酸序列为选自SEQ ID NO：69～135的至少之一。其中，SEQ ID NO：3～14是人类程序性死亡受体1(PD1)siRNA核苷酸序列，SEQ ID NO：15～30是人类细胞毒T淋巴细胞相关抗原4(CTLA4)siRNA序列，SEQ ID NO：31～46是人类T细胞免疫球蛋白粘蛋白分子3(TIM3)siRNA序列，SEQ ID NO：47～57是人类T淋巴细胞衰减因子(BTLA)siRNA序列，SEQ ID NO：58～68是人类淋巴细胞活化基因3蛋白(LAG3)siRNA序列，SEQ ID NO：69～85是人类IRAK-M(白细胞介素-1受体相关激酶3)siRNA核苷酸序列，SEQ ID NO：86～96是人类SOCS1(细胞因子信号转导抑制因子1)siRNA序列，SEQ ID NO：97～116是人类A20(肿瘤坏死因子-α诱导蛋白A20)siRNA序列，SEQ ID NO：117～135是人类CBL-B(E3泛素蛋白连接酶CBL-B)siRNA序列，根据本发明的实施例，将本发明实施例的逆转录病毒或构建体导入淋巴细胞所得的转基因淋巴细胞中，其细胞表面的免疫检查点PD1、CTLA4、TIM3、BTLA、LAG3或细胞内免疫检查点IRAK-M、SOCS1、A20、CBL-B被特异性沉默和抑制表达，同时在其细胞表面表达抗EGFRvIII的嵌合抗原受体，从而本发明实施例的转基因淋巴细胞具有了显著的抵抗肿瘤介导的免疫抑制的功效，抗调亡能力和增殖能力增强、定向杀伤能力显著提高，本发明实施例的转基因淋巴细胞在肿瘤病人体内和体外的增殖和生存能力以及在肿瘤病人体内的杀伤能力大大提高，尤其对EGFRvIII突变的脑胶质母细胞瘤细胞特异性杀伤效果尤为显著。
根据本发明地实施例，本发明实施例的慢病毒或构建体携带含有SEQ ID NO：136、137、138或139所示的核苷酸序列。其中，SEQ ID NO：136是共表达抗EGFRvIII嵌合抗原受体和沉默细胞表面免疫检查点PD1的核酸分子(EGFRvIII-CAR/iPD1)，SEQ ID NO：137是共表达抗EGFRvIII嵌合抗原受体和沉默细胞内免疫检查点CBL-B的核酸分子(EGFRvIII-CAR/iCBL-B)，SEQ ID NO：138是共表达抗EGFRvIII嵌合抗原受体、沉默细胞表面免疫检查点PD1和沉默细胞内免疫检查点CBL-B的核酸分子(EGFRvIII-CAR/iPD1-CBL-B)，SEQ ID NO：139是共表达抗EGFRvIII嵌合抗原受体、沉默细胞表面免疫检查点PD1和沉默另一个细胞表面免疫检查点CTLA4的核酸分子(EGFRvIII-CAR/iPD1-CTLA4)。根据本发明的实施例，将本发明实施列的慢病毒导入淋巴细胞所得的转基因淋巴细胞，其细胞表面的免疫检查点PD1或CTLA4被特异性沉默，或细胞内的免疫检查CBL-B被特异性沉默，以及抗EGFRvIII的嵌合抗原受体表达，使得转基因淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的功效，其抗调亡能力和增殖能力增强、定向杀伤能力显著提高，从而使得转基因淋巴细胞在肿瘤病人体外和体内的增殖和生存能力以及在肿瘤病人体内的杀伤能力大大提高，尤其对EGFRvIII突变的脑胶质母细胞瘤细胞的特异性杀伤效果尤为显著。
根据本发明的实施例，发明人是通过如下方式实现上述细胞嵌合抗原受体以及表面或细胞内免疫检查点shRNA分别独立地表达的，其中，需要说明的是，此处的表达既指蛋白的
表达又指RNA转录。
启动子：第一启动子，第一启动子与编码嵌合抗原受体的核酸分子可操作地连接；以及第二启动子，第二启动子与沉默细胞免疫检查点的核酸分子可操作地连接。根据本发明的实施例，所采用的第一启动子、第二启动子分别独立地选自U6，CMV,H1，EF-1,LTR,RSV启动子，第一以及第二启动子的引入，使得编码嵌合抗原受体的核酸分子和沉默细胞免疫检查点的核酸分子分别独立的表达，从而有效沉默了细胞表面或细胞内免疫检查点，并且保证了嵌合抗原受体的高效表达，使得淋巴细胞在肿瘤环境中的成活率大大提高，淋巴细胞的靶向作用更强，对肿瘤的特异性杀伤作用更加显著。
根据本发明的实施例，也可进一步引入第三启动子，第三启动子与沉默细胞的免疫检查点的核酸分子可操作的连接，第三启动子和第二启动子所连接的沉默细胞的免疫检查的核酸分子不同，第三启动子和第二启动子分别启动沉默不同免疫检查点的shRNA。
通过上述第一、第二启动子或进一步第三启动子的引入，使得细胞表面或细胞内免疫检查点被高效沉默和嵌合抗原受体高效地表达在本发明实施例的转基因淋巴细胞膜上，从而高效抑制了免疫检查点的免疫负调控和保证了嵌合抗原受体的生物学作用，从而使得淋巴细胞在肿瘤环境中的成活率大大提高，淋巴细胞的靶向杀伤作用更加显著。
另外，根据本发明的实施例，本发明实施例的构建体的载体是非致病性病毒载体。非致病性病毒载体大大提高了构建体在淋巴细胞中的复制和扩增效率，进而本发明实施例的淋巴细胞在肿瘤病人体内的增殖和生存能力大大提高，淋巴细胞的靶向作用进一步增强，对肿瘤细胞的杀伤作用更加显著。
根据本发明的实施例，本发明实施例的构建体的载体是病毒载体，病毒载体选自反转录病毒载体、慢病毒载体、腺病毒载体或腺病毒关联病毒载体的至少之一。根据本发明的实施例，本发明实施例的病毒的载体在病毒包装和感染过程中，病毒感染范围广泛，既可感染终末分化细胞，又可感染处于分裂期的细胞，既可整合到宿主染色体，又可游离在宿主染色体之外，实现广谱而高效的感染效率，从而细胞表面或细胞内免疫检查点被高效沉默和嵌合抗原受体在淋巴细胞中高效表达，本发明实施例的淋巴细胞的在肿瘤病人体内的增殖和生存能力大大提高，淋巴细胞的靶向作用进一步增强，对肿瘤细胞的杀伤作用更加显著。
根据本发明的具体实施例，以构建一个慢病毒载体为例，发明人为了构建一个慢病毒载体，在某些病毒序列的位置，将目的核酸插入到病毒基因组中，从而产生复制缺陷的病毒。为了产生病毒体，发明人进而构建包装细胞系(包含gag，pol和env基因，但不包括LTR和包装成分)。发明人将含有目的基因的重组质粒，连同慢病毒LTR和包装序列，一起引入包装细胞系中。包装序列允许重组质粒RNA转录产物被包装到病毒颗粒中，然后被分泌到
培养基中。进而发明人收集包含重组慢病毒的基质，有选择性地浓缩，并用于基因转移。慢载体可以感染多种细胞类型，包括可分裂细胞和不可分裂细胞。
另外，根据本发明的实施例，本发明实施例的慢病毒是复合慢病毒，除了常见的慢病毒基因gag，pol和env，还包含有调控和结构功能的其他基因。慢病毒载体是本领域技术人员所熟知的，慢病毒包括：人类免疫缺陷病毒HIV–1，HIV–2和猿猴免疫缺陷病毒SIV。慢病毒载体通过多重衰减艾滋病毒致病基因产生，例如全部删除基因env，vif，vpr，vpu和nef，使慢病毒载体形成生物安全型载体。重组慢病毒载体能够感染非分裂细胞，同时可用于体内和体外基因转移和核酸序列表达。例如：在合适的宿主细胞中，和带有包装功能(gag，pol，env，rev和tat)的两个或更多的载体一起，能够感染非分裂细胞。重组病毒的靶向性，是通过抗体或特定配体(靶向特定细胞类型受体)与膜蛋白的结合来实现的。同时，重组病毒的靶向性通过插入一个有效序列(包括调控区域)到病毒载体中，连同另一个编码了特定靶细胞上的受体的配体的基因，使载体具有了特定的靶向。各种有用的慢病毒载体，以及各种方法和操作等产生的载体，用于改变细胞的表达。根据本发明的实施例，本发明实施例的慢病毒载体可有效运送和共表达shRNA(siRNA的转运形式)，该小shRNA可以有效抑制PD1或CTLA4或CBL-B的表达。
根据本发明的实施例，本发明实施例的腺关联病毒载体(AAV)可使用一种或多种为人熟知的血清类型腺关联病毒载体的DNA构建。本领域技术人员构建一个合适的腺关联病毒载体，以此携带和共表达小发夹RNA，该小发夹RNA可以抑制PD1或CTLA4或CBL-B基因的表达。
另外，根据本发明的实施例，本发明实施例的也包含微基因。微基因意味着用组合(选定的核苷酸序列和可操作的必要的相关连接序列)来指导转化、转录和/或基因产物在体内或体外的宿主细胞中的表达。应用“可操作的连接”序列包含连续目的基因的表达控制序列，和作用于反式或远距离控制目的基因的表达控制序列。
另外，本发明实施例的载体还包括常规控制元素，在和质粒载体一起的细胞转染或/和病毒载体一起的细胞感染中，这些元素允许转录、转化和/或小发夹RNA的表达。大量的表达控制序列(包括天然的，可诱导和/或特定组织的启动子)可能被使用。根据本发明的实施例，表达shRNA的启动子为RNA聚合酶启动子。同时，根据本发明的实施例，启动子为选自U6,H1,pol I,pol II and pol III的RAN聚合酶启动子。根据本发明的实施例，启动子为组织特异型启动子。根据本发明的实施例，启动子为诱导型启动子。根据本发明的实施例，启动子为选自基于所选载体的启动子。根据本发明的实施例，当选择慢病毒载体时，启动子为U6,H1,CMV IE基因,EF-1α,泛素C,或磷酸甘油激酶(PGK)启动子。其他常规表达控制序列包括可选标记或报告基因，包括编码遗传霉素，潮霉素，氨苄青霉素或嘌
呤霉素耐药性等的核苷酸序列。载体的其他组件包括复制起点。
构建载体的技术为本领域技术人员所熟知的，这些技术包括常规克隆技术，例如在本发明实施例中所使用的shRNA、聚合酶链反应和任何适当的提供所需的核苷酸序列的方法。
根据本发明的实施例，发明人构建了共表达小发夹RNA(shRNA)(用来抑制免疫检查点)以及嵌合抗原受体(CAR)的病毒载体。本发明实施例的运送沉默PD1或CTLA4或CBL-B的siRNA的小发夹RNA以及表达嵌合抗原受体(CAR)的病毒载体或质粒是复合的，此病毒载体或质粒可结合聚合物或其他材料来增加其稳定性，或协助其靶向运动。
制备转基因淋巴细胞的方法
在本发明的另一方面，本发明提出了一种制备前面所述的T淋巴细胞或者转基因淋巴细胞的方法。根据本发明的实施例，该方法包括：将前面所述的构建体或者前面所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。引入方式可以选自电转或病毒感染宿主细胞的方式引入。本发明实施例的构建体或慢病毒成功引入上述淋巴细胞或者T淋巴细胞中，实现了针对抗原EGFRvIII的嵌合抗原受体的表达和淋巴细胞的细胞表面或细胞内免疫检查点被沉默，从而使得所得淋巴细胞或T淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的功效，在肿瘤病人体内和体外的增殖及肿瘤病人体内存活能力大大提高，淋巴细胞或T淋巴细胞对肿瘤细胞，尤其是EGFRvIII突变的脑胶质母细胞瘤细胞的靶向杀伤作用更强。
治疗癌症的治疗组合物
在本发明的另一方面，本发明的提出了一种用于治疗癌症的治疗组合物。根据本发明的实施例，该治疗组合物包括：上述构建体、上述慢病毒、上述T淋巴细胞或者上述转基因淋巴细胞。上述任意一种治疗组合物的组成均可实现针对抗原EGFRvIII嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达和转基因淋巴细胞或T淋巴细胞细胞表面或细胞内免疫检查点的沉默，从而使得所得转基因淋巴细胞或T淋巴细胞在体外扩增、在肿瘤病人体内的增殖及肿瘤病人体内存活能力大大提高，转基因淋巴细胞或T淋巴细胞对EGFRvIII肿瘤细胞的靶向杀伤作用更强。
根据本发明的实施例，提供给患者的本发明实施例的治疗组合物,较好的应用于生物兼容溶液或可接受的药学运载载体。作为准备的各种治疗组合物被悬浮或溶解在医药上或生理上可接受的载体，如生理盐水；等渗的盐溶液或其他精于此道的人的比较明显的配方中。适当的载体在很大程度上取决于给药途径。其他有水和无水的等渗无菌注射液和有水和无水的无菌悬浮液，是医药上可接受的载体。
根据本发明的实施例，足够数量的病毒载体被转导入靶向T细胞中，并提供足够强度的转基因，沉默PD1或CTLA4或CBL-B和表达特有的EGFRvIII嵌合抗原受体。治疗试剂的剂量主要取决于治疗状况,年龄，体重，病人的健康程度，从而可能造成病人的变异性。
沉默PD1或CTLA4或CBL-B以及表达特有的针对抗原EGFRvIII嵌合抗原受体的这些方法是联合治疗的一部分。这些病毒载体和用于过继免疫治疗的抗肿瘤T细胞，可以被单独或结合其他治疗癌症的方法一起执行。在合适的条件下，一个治疗方法的包括使用一个或多个药物疗法。
根据本发明的实施例，所述癌症包括脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌。下列生物效应的至少之一：细胞表面或细胞内免疫检查点的沉默，联合嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达，使得所得淋巴细胞或T淋巴细胞在脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌的环境中的存活能力大大提高，淋巴细胞或T淋巴细胞对脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌的肿瘤细胞的靶向杀伤作用更强，尤其对EGFRvIII突变的上述肿瘤细胞的杀伤作用更加显著。
治疗癌症的方法
在本发明的另一方面，本发明提出了一种治疗癌症的方法。根据本发明的实施例，该方法包括：为癌症患者给药前面所述的构建体、前面所述的慢病毒、前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞，其中，嵌合抗原受体特异性结合肿瘤抗原EGFRvIII。本发明实施例提出的方法对EGFRvIII突变的肿瘤细胞具有显著的靶向杀伤作用。
根据本发明的实施例，所述方法包括：从癌症患者体内分离淋巴细胞；将前面所述的构建体，或前面所述的慢病毒导入所述淋巴细胞，以便获得转基因淋巴细胞，所述转基因淋巴细胞的细胞免疫检查点被沉默和表达所述嵌合抗原受体；以及为所述癌症患者给药所述转基因淋巴细胞。本发明实施例的方法对EGFRvIII突变的肿瘤细胞的靶向杀伤作用进一步增强。
根据本发明的实施例，所述癌症包括选自脑胶质母细胞瘤、非小细胞肺癌、乳腺癌和卵巢癌的至少之一。如前所述，脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌癌细胞具有EGFRvIII的特异性表达，本发明实施例的治疗癌症的方法可使淋巴细胞的细胞免疫检查点被高效沉默和高效表达抗原特异性嵌合抗原受体，如本发明实施例的EGFRvIII抗原特异性嵌合抗原受体，所得淋巴细胞或T淋巴细胞具有对EGFRvIII突变的脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌癌的肿瘤细胞的显著的靶向杀伤作用。
在本文中所使用的术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的治疗组合物可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但是本发明不限于这些已举例的给药方式。然而，由于口服给药时，口服给药的组合物的活性成分应该被包被或被配制以防止其在胃部被降解。优选地，本发明的组合物可以注射制剂被给药。此外，本发明的治疗组合物可以使用将活性成分传送到靶细胞的特定
器械来给药。
本发明的治疗组合物的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些实施例，日剂量可分为适宜形式的1剂、2剂或多剂，以在整个时间段内以1次、2次或多次给药，只要达到治疗有效量即可。
术语“治疗有效量”是指化合物足以显著改善某些与疾病或病症相关的症状的量，也即为给定病症和给药方案提供治疗效果的量。例如，在EGFRvIII+的脑胶质母细胞瘤、非小细胞肺癌、乳腺癌和卵巢癌的治疗中，减少、预防、延缓、抑制或阻滞疾病或病症的任何症状的治疗组合物是治疗有效的。治疗有效量的治疗组合物不需要治愈疾病或病症，但将为疾病或病症提供治疗，使得个体的疾病或病症的发作被延缓、阻止或预防，或者疾病或病症的症状得以缓解，或者疾病或病症的期限被改变，或者例如疾病或病症变得不严重，或者加速康复。
术语“治疗”用于指获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病(主要指EGFRvIII+的脑胶质母细胞瘤、非小细胞肺癌、乳腺癌和卵巢癌的治疗)的治疗，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病(例如预防EGFRvIII+的脑胶质母细胞瘤、非小细胞肺癌、乳腺癌和卵巢癌的治疗)或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将治疗组合物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述治疗组合物给予有需要的个体。
根据本发明的实施例，本发明实施例的治疗组合物可与常规治疗方法和/或疗法相结合使用，或者可与常规治疗方法和/或疗法分开使用。当本发明的治疗组合物在采用与其它药物的联合疗法中给药时，它们可序贯地或同时地给予个体。或者，本发明的治疗方法可包含本发明的治疗组合物、药学上可接受的载体或药学上可接受的赋形剂以及本领域已知的其它治疗药或预防药的组合。
提高淋巴细胞活性的方法
在本发明的另一方面，本发明提出了一种提高淋巴细胞活性的方法，本发明实施例的淋巴细胞携带嵌合抗原受体，根据本发明的实施例，该方法包括:使所述淋巴细胞的细胞表面或细胞内免疫检查点被沉默，细胞表面或细胞内免疫检查点、淋巴细胞、嵌合抗原受体是如前所定义的。根据本发明的实施例，本发明实施例的淋巴细胞活性包括淋巴细胞体外增殖能力，在肿瘤病人体内的增殖和生存能力以及淋巴细胞在肿瘤病人体内的杀伤能力的至
少一种。根据本发明的实施例，本发明实施例的淋巴细胞的细胞表面或细胞内免疫检查点被沉默，淋巴细胞被活化、增生性反应上调、细胞因子分泌增多、抗调亡能力增强。本发明实施例的淋巴细胞在体外扩增和增殖、对肿瘤细胞的靶向杀伤作用显著增强。
下面将结合实施例对本发明的方案进行解释。
本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
本发明实施例中所用到的细胞系和基本实验技术如下所述：
慢病毒的产生和人T淋巴细胞的转导
产生复制缺陷的慢病毒载体，并将慢病毒载体离心收集用于人T淋巴细胞的转导。下面简要介绍慢病毒载体的产生、收集的实验过程：将293T细胞铺在底面积为150-平方厘米的细胞培养皿中，并根据说明书，使用Express-In(购自Open Biosystems/Thermo Scientific,Waltham,MA)对293T细胞进行病毒转导。每盘细胞加入15μg的慢病毒转基因质粒、5μg的pVSV-G(VSV糖蛋白表达质粒)、10μg的pCMVR8.74质粒(Gag/Pol/Tat/Rev表达质粒)和174μl的Express-In(浓度为1μg/μl)。分别于24小时和48小时收集上清，并使用超速离心机在28,000rpm(离心机转子为Beckman SW 32Ti，购自Beckman Coulter,Brea,CA)的条件下离心2小时。最后用0.75ml的RPMI-1640培养基对病毒质粒沉淀进行重悬。
从健康志愿者供体上分离人原代T淋巴细胞。人T淋巴细胞培养在RPMI-1640培养基中并使用抗CD3和CD28的单克隆抗体包被的珠(购自Invitrogen,Carlsbad,CA)进行刺激激活。人T淋巴细胞激活后的18～24小时，采用自旋-接种的方法对T淋巴细胞进行转导，转导过程如下所述：在24-孔板中，每孔铺有0.5×106T淋巴细胞，向每孔细胞中加入0.75ml的上述重悬的病毒上清和Polybrene(浓度为8μg/ml)。细胞和病毒质粒的混合液在台式离心机(购自Sorvall ST 40；Thermo Scientific)中离心，离心条件是室温，2500rpm，时间为90分钟。人重组白细胞介素-2(IL-2；购自Novartis,Basel,Switzerland)每隔2～3天加入T淋巴细胞培养液中，IL-2的终浓度为100-IU/ml,在T淋巴细胞培养过程中，保持细胞的密度为0.5×106～1×106/ml。一旦被转导的T淋巴细胞出现休眠，例如细胞生长速度变慢和细胞变小，其中，细胞
生长速度和大小是通过Coulter Counter(购自Beckman Coulter)评估的，或被转导的T淋巴细胞在某个计划的时间点上，T淋巴细胞即可用来做功能分析。
本申请的实施例中所用的流式细胞仪为BD FACSCanto II(购自BD Biosciences)，并且流式细胞分析数据使用FlowJo version 7.2.5软件(购自Tree Star,Ashland,OR)进行分析。
测定细胞因子的分泌
在质粒转导后的2-7天，非转导或转导了嵌合抗原受体质粒的T细胞(细胞个数是1×106/孔)与EGFRvIII-U87脑瘤细胞共培养，实验过程中改变不同的效靶细胞比例。应用特定的酶联免疫吸附测定法(细胞因子酶联免疫吸附测试剂盒，购自R&D Systems,Inc.,Minneapolis,MN,USA)检测细胞上清液中细胞因子的产量。上述细胞上清液取自培养了24小时、48小时和72小时之后的细胞的上清，测定结果用来衡量有代表性的细胞因子(干扰素-γ)(IFNγ)的产量。
简要测定过程如下：酶标板中加入100微升/孔的作为一系列标准对照的细胞因子稀释溶液(如IFNγ)或待测细胞上清溶液，并将酶标板在室温下放置2小时。2小时后吸弃酶标板中溶液并用400微升的洗液润洗酶标板，润洗四次。润洗后，向酶标板每个孔中加入200微升的酶联抗细胞因子抗体。继续在室温下放置2个小时，之后向每孔中加入200微升的底物溶液。加入底物溶液后，将酶标板在室温下放置30分钟，之后，向每孔中加入50微升的终止反应液。在30分钟内测定酶标板每孔的光密度。酶标仪设置在450nm。
铬释放实验
实施例中应用4–小时51铬释放法分析评估抗EGFRvIII嵌合抗原受体T细胞(抗EGFRvIII CAR T淋巴细胞)的细胞毒活性。具体步骤如下：目标测试细胞用51Cr在37摄氏度下标记1小时。标记后，用含有10％胎牛血清(FCS)的RPMI培养基润洗细胞。润洗后，将细胞重悬在相同的培养基中，重悬细胞的浓度是1×105/ml。转导后T细胞以不同的效靶细胞比值(E：T)加入目标测试细胞悬浮液中，并将细胞种在96-孔中，每孔体积是200微升。将细胞在37度培养箱中培养4小时。4小时后，从每孔中取出30微升的上清放于计数器的96-微孔板进行计数分析。分析仪器是顶级计数NXT微闪烁计数器(购自Packard Bioscience)。所有计数孔中效应细胞的数目是基于T细胞总数来计算的。被标记的目标测试细胞是EGFRvIII-U87脑瘤细胞。
实施例2构建共表达沉默细胞免疫检查点shRNA和抗EGFRvIII嵌合抗原受体的载体
本实施例中，发明人将编码有抗人EGFRvIII的单链抗体的序列、4-1BB胞内段和T细胞受体组合的ζ-链序列克隆到含有EF-1启动子的慢病毒载体(lentiviral vector)上，克隆过程中，选择的限制性酶切是XbaI和NotI双酶切,以及NotI和XhoI双酶切,通过酶切、连接、筛选和目的质粒的扩增，生成表达抗EGFRvIII嵌合抗原受体的慢病毒质粒(LV-EGFRvIII CAR)。包含U6启动子和人PD1-shRNA(iPD1)或CBL-B-shRNA(iCBL-B)的序列被克隆进LV-EGFRvIII CAR载体质粒,构建成LV-EGFRvIII CAR/iPD1或LV-EGFRvIII CAR/iCBL-B。.图1是慢病毒载体的示意图，包含编码抗EGFRvIII嵌合抗原受体的序列,U6and H1启动子序列,PD1-shRNA或CBL-B-shRNA序列。抗EGFRvIII嵌合抗原受体的序列在启动子EF-1的启动调控下，CTLA4,PD1或者CBL-B shRNA序列在启动子U6或H1的启动调控下。
实施例3共表达PD1-shRNA和抗EGFRvIII嵌合抗原受体的T淋巴细胞具有细胞增殖能力更高的特点
在本实施例中，外周血淋巴细胞取自不记名供血者。外周血淋巴细胞通过梯度离心进行分离，梯度离心机为Ficoll-Hypaque。在T淋巴细胞激活因子磁珠CD3/CD28(购自Invitrogen,Carlsbad,CA)存在下，被激活的T淋巴细胞经过慢病毒载体转导、体外扩增培养，方法如实施例1所述。在慢病毒载体转导后的2-7天，转导的T细胞(细胞个数是1×106/孔)与EGFRvIII-U87脑瘤细胞共培养，4天后，细胞数目前由流式仪检测。实验结果如图2所示。图2结果表明，转导了LV-EGFRvIII-CAR/iPD1的T淋巴细胞比转导了LV-EGFRvIII-CAR或空载LV-GFP的T淋巴细胞的细胞数显著增加。标注代表每3个孔的平均值±SD。(P<0.05；LV-EGFRvIII-CAR/iPD1vs.LV-EGFRvIII-CAR).
实施例4共表达PD1-shRNA和抗EGFRvIII嵌合抗原受体的T淋巴细胞具有细胞因子分泌更多的特点
在T淋巴细胞激活因子磁珠CD3/CD28存在下，被激活的T淋巴细胞经过慢病毒载体转导、体外扩增培养，方法如实施例1所述。在慢病毒载体转导后的2-7天，转导的T细胞(细胞个数是1×106/孔)与EGFRvIII-U87脑瘤细胞共培养，4天后，具有细胞因子分泌由ELISA检测。实验结果如图3所示。图3结果表明，转导了LV-EGFRvIII-CAR/iPD1的T淋巴细胞比转导了LV-EGFRvIII-CAR或空载LV-GFP的T淋巴细胞分泌更多的IFNγ。(P<0.05；LV-EGFRvIII-CAR/iPD1vs.LV-EGFRvIII-CAR).这说明转导了LV-EGFRvIII-CAR/iPD1的T淋
巴细胞比转导LV-EGFRvIII-CAR的T淋巴细胞的产生细胞因子能力显著提高。
实施例5共表达PD1-shRNA和抗EGFRvIII嵌合抗原受体的T淋巴细胞肿瘤细胞溶解能力增强。
在本实施例中，外周血淋巴细胞取自不记名供血者。外周血淋巴细胞通过梯度离心进行分离，梯度离心机为Ficoll-Hypaque。T淋巴细胞与T细胞激活因子磁珠CD3/CD28(购自Invitrogen,Carlsbad,CA)在5％CO2、37摄氏度下孵育培养72小时，培养基是加有2mmol/L谷氨酰胺,10％高温灭活的胎牛血清(FCS)(购自Sigma-Aldrich Co.)和100U/ml的青霉素/链霉素双抗的RPMI培养基1640(购自Invitrogen Gibco Cat.no.12633-012)。激活培养72小时后，用洗液润洗细胞，将磁珠洗去。将T细胞种在铺有重组纤连蛋白片段(FN ch-296；Retronectin)细胞培养皿上，并用慢病毒转导,转导慢病毒分别为LV-EGFRvIII CAR/iPD1,LV-EGFRvIII CAR,或空载(LV-GFP)转导过程如实施例1所述。转导后的T细胞培养在RPMI-1640培养基中并用重组人类IL-2因子(100ng/ml；购自R&D Systems)进行诱导扩增7-10天，然后进行功能测试实验。发明人测量转导了不同慢病毒的T细胞(效应细胞)对EGFRvIII突变的脑胶质瘤靶细胞的杀伤作用，效靶细胞比例是10：1或1:1，测量方法采用标准4–小时51铬释放法，4–小时51铬释放法如实施例1所述。结果如图4所示。如图4所示,共表达抗EGFRvIII嵌合抗原受体和PD1-shRNA(iPD1)的T淋巴细胞比单独表达抗EGFRvIII嵌合抗原受体的T淋巴细胞,可更为有效的杀死EGFRvIII突变的脑瘤靶细胞。空载慢病毒转导的T淋巴细胞(对照LV-GFP T淋巴细胞)对EGFRvIII突变的脑瘤细胞无明显杀伤作用。统计数据代表三个孔的平均值±SEM。
实施例6共表达CBL-B-shRNA和抗EGFRvIII嵌合抗原受体的T细胞，共表达PD1-shRNA，CBL-B-shRNA和抗EGFRvIII嵌合抗原受体的T细胞,共表达PD1-shRNA，CTLA4-shRNA和抗EGFRvIII嵌合抗原受体的T细胞的细胞，溶解能力增强并且具有细胞因子分泌更多和细胞增殖更强的特点。
在本实施例中，发明人还考察了共表达CBL-B-shRNA和抗EGFRvIII嵌合抗原受体的T细胞、共表达2个shRNA(PD1-shRNA和CBL-B-shRNA或PD1-shRNA和CTLA4-shRNA)和抗EGFRvIII嵌合抗原受体的T细胞的肿瘤溶解能力、细胞因子分泌能力和细胞增殖能力。实验过程与实施例3，4和5相同。上述T细胞比单独表达抗EGFRvIII嵌合抗原受体的T细胞的细胞溶解能力增强，细胞因子分泌更多和细胞增殖更强。共表达2个shRNA(PD1-shRNA和CTLA4-shRNA或PD1-shRNA和CBL-B-shRNA)和抗EGFRvIII嵌合抗原受体的T细胞比共表
达1个shRNA(PD1-shRNA或CBL-B-shRNA)和抗EGFRvIII嵌合抗原受体的T细胞的细胞溶解能力更强，细胞因子分泌更多和细胞增殖更强。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (32)
- 一种T淋巴细胞，其特征在于，所述T淋巴细胞的细胞免疫检查点被沉默；以及表达嵌合抗原受体，其中，所述嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区，所述单链抗体特异性识别抗原EGFRvIII；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述T淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括CD28或4-1BB的胞内段以及CD3ζ链。
- 一种慢病毒，其特征在于，所述慢病毒携带下列核酸分子：编码嵌合抗原受体的核酸分子，所述嵌合抗原受体具有SEQ ID NO：1所示的氨基酸序列，所述编码嵌合抗原受体的核酸分子具有SEQ ID NO：2所示的核苷酸序列；以及沉默细胞免疫检查点的核酸分子，所述沉默细胞免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：3～135的至少之一。
- 一种慢病毒，其特征在于，所述慢病毒携带含有SEQ ID NO：136、137、138或139所示的核苷酸序列。
- 一种转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点被沉默；以及表达嵌合抗原受体。
- 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述嵌合抗原受体包括：胞外区，所述胞外区能够与抗原特异性结合；跨膜区；以及胞内区，所述胞内区包括免疫共刺激分子胞内段。
- 根据权利要求5所述的转基因淋巴细胞，其特征在于，所述抗原是肿瘤抗原。
- 根据权利要求6所述的转基因淋巴细胞，其特征在于，所述胞外区包括抗体的重链可变区和轻链可变区，所述抗体结合所述抗原。
- 根据权利要求7所述的转基因淋巴细胞，其特征在于，所述抗体为单链抗体。
- 根据权利要求8所述的转基因淋巴细胞，其特征在于，所述抗原为EGFRvIII。
- 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点独立地选自CTLA4、PD1、TIM3、BTLA、LAG-3、IRAK-M、SOCS1、A20、CBL-B的至少之一。
- 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查 点被沉默是通过shRNA、反义核酸、核酶、显性负突变、CRISPR和锌指核酸酶至少之一实现的。
- 根据权利要求5所述的转基因淋巴细胞，其特征在于，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28以及他们的衍生物的至少一种。
- 根据权利要求10所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点是CTLA4、PD1、CBL-B。
- 根据权利要求11所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点被沉默是通过shRNA实现的。
- 根据权利要求12所述的转基因淋巴细胞，其特征在于，所述免疫共刺激分子胞内段是4-1BB或CD28的胞内段。
- 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞是CD3+T淋巴细胞。
- 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞是自然杀伤细胞。
- 根据权利要求4所述的转基因淋巴细胞，其特征在于，所述淋巴细胞是自然杀伤T细胞。
- 一种构建体，其特征在于，所述构建体包括：第一核酸分子，所述第一核酸分子编码嵌合抗原受体；以及第二核酸分子，所述第二核酸分子沉默细胞免疫检查点，其中，所述细胞免疫检查点、所述嵌合抗原受体是如权利要求4～18任一项中所定义的。
- 根据权利要求19所述的构建体，其特征在于，所述第一核酸分子以及所述第二核酸分子被设置在权利要求4～18任一项所述的淋巴细胞中表达所述嵌合抗原受体和沉默细胞免疫检查点。
- 根据权利要求19所述的构建体，其特征在于，进一步包括：第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；以及第二启动子，所述第二启动子与所述第二核酸分子可操作地连接。
- 根据权利要求21所述的构建体，其特征在于，所述第一启动子、所述第二启动子分别独立地选自U6，H1,CMV,EF-1,LTR,或RSV启动子。
- 根据权利要求19所述的构建体，其特征在于，所述构建体的载体是非致病性病毒载体。
- 根据权利要求23所述的构建体，其特征在于，所述构建体的载体是病毒载体，所述病毒载体包括选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体的至少之一。
- 一种制备权利要求1所述的T淋巴细胞或者权利要求4～18任一项所述的转基因淋巴细胞的方法，其特征在于，包括：将权利要求19～24任一项所述的构建体或者权利要求2～3任一项所述的慢病毒引入到淋巴细胞或者T淋巴细胞中。
- 一种用于治疗癌症的治疗组合物，其特征在于，包括：权利要求19～24任一项所述的构建体、权利要求2～3任一项所述的慢病毒、权利要求1所述的T淋巴细胞或者权利要求4～18任一项所述的转基因淋巴细胞。
- 根据权利要求26所述的治疗组合物，其特征在于，所述癌症包括脑胶质母细胞瘤、非小细胞肺癌、乳腺癌或卵巢癌。
- 一种治疗癌症的方法，其特征在于，包括：为癌症患者给药权利要求19～24任一项所述的构建体、权利要求2～3任一项所述的慢病毒、权利要求1所述的T淋巴细胞或者权利要求4～18任一项所述的转基因淋巴细胞，其中，嵌合抗原受体特异性结合肿瘤抗原EGFRvIII。
- 根据权利要求28所述的方法，其特征在于，包括：从癌症患者体内分离淋巴细胞；将权利要求19～24任一项所述的构建体，或权利要求2～3任一项所述的慢病毒导入所述淋巴细胞，以便获得转基因淋巴细胞，所述转基因淋巴细胞的细胞免疫检查点被沉默和表达所述嵌合抗原受体；以及为所述癌症患者给药所述转基因淋巴细胞。
- 根据权利要求29所述的方法，其特征在于，所述癌症包括选自脑胶质母细胞瘤、非小细胞肺癌、乳腺癌和卵巢癌的至少之一。
- 一种提高淋巴细胞活性的方法，所述淋巴细胞携带嵌合抗原受体，其特征在于，所述方法包括：使所述淋巴细胞的细胞免疫检查点被沉默,所述细胞免疫检查点、所述淋巴细胞、所述嵌合抗原受体如权利要求4～18任一项中所定义的，所述淋巴细胞活性包括所述淋巴细胞在体外的增殖能力、在肿瘤病人体内的增殖和生存能力以及所述淋巴细胞在肿瘤病人体内的定向杀伤能力的至少一种。
- 根据权利要求31所述的方法，其特征在于，所述肿瘤包括选自EGFRvIII突变的脑胶质母细胞瘤、EGFRvIII突变的非小细胞肺癌、EGFRvIII突变的乳腺癌或EGFRvIII突变的卵巢癌的至少之一。
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