WO2017133174A1 - Composition thérapeutique pour le traitement de la leucémie à cellules b et du lymphome à cellules b - Google Patents

Composition thérapeutique pour le traitement de la leucémie à cellules b et du lymphome à cellules b Download PDF

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WO2017133174A1
WO2017133174A1 PCT/CN2016/087488 CN2016087488W WO2017133174A1 WO 2017133174 A1 WO2017133174 A1 WO 2017133174A1 CN 2016087488 W CN2016087488 W CN 2016087488W WO 2017133174 A1 WO2017133174 A1 WO 2017133174A1
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lymphocyte
lymphocytes
cell
transgenic
chimeric antigen
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Chinese (zh)
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严勇朝
朱益林
陈思毅
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北京马力喏生物科技有限公司
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    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
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    • C12N2740/10011—Retroviridae
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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer, and a cancer treatment And a method of increasing lymphocyte activity.
  • TTLs activated cytotoxic T lymphocytes
  • PD1 body 1
  • PD-L1 B7-H1 Programmed Death Ligand 1
  • Cytotoxic T lymphocyte antigen 4 (CTLA4) is another key negative regulator of T cells, which inhibits T cell activation by inhibiting ligands B7.1, B7.2 expressed on antigen-presenting cells. (CD80 and CD86) are implemented in interaction.
  • Adoptive T cell therapy is a therapy in which T cells derived from the body are activated, expanded, and then returned to the body for autoimmune therapy.
  • the present invention aims to solve at least one of the technical problems in the related art to some extent.
  • CD19-targeted chimeric antigen receptor T cells are effective in killing CD19 + malignant B cells.
  • many patients with CD19 + malignant B cell lymphoma still do not respond to CD19-targeted chimeric antigen receptor T cell therapy.
  • the present invention has been made in view of the above problems, and provides a nucleic acid molecule carrying a silent cell surface immunological checkpoint and a nucleic acid molecule encoding a CD19-targeting chimeric antigen receptor, and a transgenic lymphocyte formed by the introduction of the construct.
  • the constructs and transgenic lymphocytes proposed by the present invention are used for immunotherapy of adoptive T cells, which can greatly improve the cytokine production of T lymphocytes and the killing ability of tumor cells, especially for CD19 + malignant B cell lymphoma or leukemia.
  • the killing effect of CD19 + tumor cells was significantly enhanced.
  • the invention proposes a T lymphocyte.
  • the cell surface immunological checkpoint of the T lymphocyte is silenced; and expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: an extracellular region, the extracellular region comprising a heavy chain variable region and a light chain variable region of a single chain antibody, the single chain antibody specifically recognizing an antigen CD19; a transmembrane region, the transmembrane region being linked to the extracellular region, and embedded in the T In the cell membrane of lymphocytes; an intracellular region that is linked to the transmembrane region, and the intracellular region includes an intracellular portion of CD28 and a CD3 ⁇ chain.
  • the cytokine production of the T lymphocytes and the killing ability against tumor cells are significantly enhanced, and the killing effect on CD19 + tumor cells of CD19 + malignant B cell lymphoma or leukemia is greatly improved.
  • 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 which silences the cell surface immunological checkpoint, and the nucleotide sequence of the nucleic acid molecule of the silencing cell surface immunological checkpoint is selected from the group consisting of At least one of SEQ ID NOS: 3 to 68.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the present invention into lymphocytes, the cytokine production of the T lymphocytes and the killing ability to tumor cells are greatly improved, especially for CD19 + malignant B cell lymphoma or leukemia.
  • the killing effect of CD19 + tumor cells is particularly significant.
  • the invention proposes a lentivirus.
  • the lentivirus carries a nucleotide sequence set forth in SEQ ID NO:69.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes, the production of cytokines of the transgenic lymphocytes and the killing ability against tumor cells are greatly improved, especially for CD19 + malignant B cells.
  • the killing effect of CD19 + tumor cells of lymphoma or leukemia is particularly significant.
  • the invention proposes a lentivirus.
  • the lentivirus carries a nucleotide sequence set forth in SEQ ID NO:70.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes, the production of cytokines of the transgenic lymphocytes and the killing ability against tumor cells are greatly improved, especially for CD19 + malignant B cells.
  • the killing effect of CD19 + tumor cells of lymphoma or leukemia is particularly significant.
  • the invention proposes a lentivirus.
  • the lentivirus carries a nucleotide sequence set forth in SEQ ID NO:71.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes, the production of cytokines of the transgenic lymphocytes and the killing ability against tumor cells are greatly improved, especially for CD19 + malignant B cells.
  • the killing effect of CD19 + tumor cells of lymphoma or leukemia is particularly significant.
  • the invention provides a transgenic lymphocyte.
  • the lymphocyte cell surface immunological checkpoint is silenced; and the chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region capable of interacting with the antigen Specific binding; a transmembrane region; and an intracellular region comprising an intracellular segment of an immunostimulatory molecule, the antigen being a tumor antigen, the extracellular region comprising a heavy chain variable region and a light chain of an antibody In the variable region, the antibody binds to the antigen, the antibody is a single chain antibody, and the antigen is CD19.
  • the inventors have surprisingly found that cell surface immunological checkpoints are silenced and express lymphocytes that specifically bind to the chimeric antigen receptor of CD19, the cytokine production of the lymphocytes and the ability to kill CD19 + tumor cells are greatly enhanced.
  • the above transgenic lymphocytes may further have at least one of the following additional technical features:
  • the lymphocyte cell surface immunological checkpoint is independently selected from at least one of CTLA4, PD1, TIM3, BTLA, LAG3. These molecules have the effect of negatively regulating and attenuating cellular immune responses.
  • the successful silencing of the cell surface immunological checkpoint further enhances the transgenic lymphocytes, the cytokine production of the transgenic lymphocytes and the further enhancement of the killing ability of the tumor cells.
  • the lymphocyte cell surface immunological checkpoint is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation and zinc finger nuclease.
  • successful silencing of cell surface immunological checkpoints further enhances cytokine production of transgenic lymphocytes and further enhances the killing ability of tumor cells.
  • 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 combination of the expression of the intracellular segment of the immunostimulatory molecule and the silencing of the cell surface immunological checkpoint has a positive regulation and enhances the cellular immune response, so that the effect of the transgenic lymphocytes on the targeted killing effect of the tumor is more significant.
  • the lymphocyte cell surface immunological checkpoint is CTLA4 or PD1.
  • the cell surface surface immunological checkpoint CTLA4 or PD1 is silenced, so that the cytokine production of the transgenic lymphocytes and the killing ability against the tumor cells are more remarkable.
  • silencing of the lymphocyte surface surface immunological checkpoint is achieved by shRNA.
  • the shRNA of the present invention has the function of silencing a cell surface immunological checkpoint with high efficiency and specificity, and the successful silencing of the cell surface immunological checkpoint enables cytokine production of the transgenic lymphocytes and killing of the tumor cells. The ability is further improved, and the effect of directed killing of tumors is more significant.
  • 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 the intracellular segment of CD28 or 4-1BB, which significantly enhances the targeted killing effect of the transgenic lymphocytes.
  • the lymphocytes are CD3 + T lymphocytes or natural killer cells or natural killer T cells.
  • the cell surface immunological checkpoint of the above lymphocytes is silenced and expresses a chimeric antigen receptor, so that the cellular immunity of the lymphocytes is more targeted, the cytokine production of the T lymphocytes and the tumor cells. The killing ability is further improved, and the effect of directed killing on tumors is more significant.
  • the invention proposes a construct.
  • the construct comprises: a first nucleic acid molecule, the first nucleic acid molecule is silenced at a cell surface immunological checkpoint; and a second nucleic acid molecule encoding a chimeric antigen receptor, Wherein the cell surface immunological checkpoint and the chimeric antigen receptor are as described above.
  • the above-mentioned construct successfully introduces the lymphocytes and successfully silences the cell surface immunological checkpoint and expresses the chimeric antigen receptor, thereby causing the cytokine production of the lymphocytes and the killing ability of the tumor cells to be greatly improved, and the tumor cells are substantially improved. The killing effect is more significant.
  • 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 surface checkpoint and to express the chimeric antigen receptor in the lymphocytes described above.
  • lymphocytes that successfully set the above-described first nucleic acid molecule and second nucleic acid molecule have a stronger tumor killing effect.
  • the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; and a second promoter, the second promoter and The second nucleic acid molecule is operably linked.
  • the introduction of the first and second promoters allows the first nucleic acid molecule and the second nucleic acid molecule to be independently expressed, thereby effectively silencing the cell surface immunological checkpoint and the organism ensuring the chimeric antigen receptor The learning effect makes the lymphocyte targeting effect stronger, and the targeted killing effect on the tumor is more significant.
  • the first promoter and the second promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, RSV promoters.
  • the above promoter has the characteristics of high activation efficiency, thereby ensuring efficient silencing of cell surface immunological checkpoints and efficient expression of chimeric antigen receptors, resulting in cytokine production of lymphocytes and killing of tumor cells. The ability is greatly improved, and the targeted killing effect on the tumor is more significant.
  • the construct vector is a non-pathogenic virus.
  • the introduction of a non-pathogenic viral vector greatly enhances the replication and amplification efficiency of the construct in lymphocytes, thereby greatly increasing the silencing of cell surface immunological checkpoints and the efficient expression of chimeric antigen receptors in lymphocytes,
  • the cytokine production of lymphocytes and the ability to kill tumor cells are greatly enhanced, the targeting of lymphocytes is further enhanced, and the killing effect on tumor cells is more significant.
  • the non-pathogenic viral vector is selected from at least one of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-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. Chromosomes, which can be freed from the host chromosome, achieve broad-spectrum and efficient infection efficiency, so that cell surface immune checkpoints are efficiently silenced and chimeric antigen receptors are highly expressed in lymphocytes, resulting in cytokine production by lymphocytes. As well as greatly improving the killing ability of tumor cells, the targeting effect of lymphocytes is further enhanced, and the killing effect on tumor cells is more remarkable.
  • the invention provides a method of preparing a T lymphocyte or a transgenic lymphocyte as described above.
  • 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 cell surface immunoassay of lymphocytes is silenced and the expression of the chimeric antigen receptor specifically binding to the antigen CD19 is obtained, thereby causing the obtained lymphocytes or
  • the cytokine production of T lymphocytes and the ability to kill tumor cells are greatly enhanced, and the targeted killing effect of lymphocytes or T lymphocytes on CD19 + tumor cells is stronger.
  • 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 immunological checkpoints of transgenic lymphocytes or T lymphocytes and efficient binding of chimeric antigen receptors specifically binding antigen CD19 to transgenic lymphocytes or T lymphocytes.
  • the expression so that the cytokine production of the obtained transgenic lymphocytes or T lymphocytes and the killing ability of the tumor cells are greatly improved, and the targeted killing effect of the transgenic lymphocytes or T lymphocytes on the CD19 + tumor cells is stronger.
  • the above therapeutic composition may further comprise at least one of the following additional technical features:
  • the cancer comprises at least one selected from the group consisting of a B cell lymphoma and a B cell leukemia. Silencing of cell surface immunological checkpoints and efficient expression of chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, resulting in targeted killing of B lymphoma or leukemia CD19 + tumor cells by the resulting lymphocytes or T lymphocytes More powerful.
  • the invention provides a method of increasing lymphocyte activity, according to an embodiment of the invention, the lymphocyte carries a chimeric antigen receptor, characterized in that the method comprises: The cell surface immunological checkpoint of the lymphocyte is silenced, the cell surface immunological checkpoint, the lymphocyte and the chimeric antigen receptor are as defined above, the lymphocyte activity comprising the lymphocyte At least one of the ability to secrete cytokines and the ability of the lymphocytes to kill tumor cells.
  • the cell surface immunological checkpoint of lymphocytes is silenced, the secretion of cytokines is increased, and the targeted killing effect on tumor cells is stronger.
  • 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 CD19.
  • the method proposed in the embodiment of the invention can effectively kill tumor cells having the tumor antigen CD19.
  • the above method for treating cancer may further comprise at least one of the following additional technical features:
  • the method comprises: isolating lymphocytes from a cancer patient; introducing the aforementioned construct, or the lentivirus described above, into the lymphocytes to obtain transgenic lymphocytes, the transgene
  • the lymphocyte cell surface immunological checkpoint is silenced and expresses a chimeric antigen receptor; and the transgenic lymphocytes are administered to the cancer patient.
  • the method of the embodiments of the present invention is capable of further effectively killing tumor cells having the tumor antigen CD19.
  • the cancer comprises at least one selected from the group consisting of a B cell lymphoma and a B cell leukemia.
  • B cell lymphoma and B cell leukemia have specific expression of CD19 tumor cells
  • the method for treating cancer of the present invention can silence lymphocyte cell surface checkpoints and express chimeric antigen receptors.
  • the resulting lymphocytes or T lymphocytes have targeted killing of CD19-specific B cell lymphoma and B cell leukemia tumor cells.
  • cell surface immunological checkpoint is a membrane protein on the surface of lymphocytes, which is a negative regulatory regulatory mechanism that interacts with ligands expressed on tumor cells. It can inhibit the anti-tumor lymphocyte reaction.
  • the "increased secretion of cytokines” mentioned in the present invention is an expression of enhanced physiological functions of cells.
  • FIG. 1 is a schematic diagram showing the structure of a lentiviral vector which co-expresses a chimeric antigen receptor specific for CD19 antigen and silences a human cell surface immunological checkpoint according to an embodiment of the present invention
  • FIG. 2 is a diagram showing the results of co-expression of a CD19 antigen-specific chimeric antigen receptor and PD1-secreting lymphocytes killing tumor cells according to an embodiment of the present invention
  • Figure 3 is a graph showing the results of co-expression of a CD19 antigen-specific chimeric antigen receptor and PD1-secreting lymphocytes secreting interferon- ⁇ according to an embodiment of the present invention.
  • the invention proposes a T lymphocyte or a transgenic lymphocyte.
  • the cell surface immunological checkpoint of the T lymphocyte is silenced; and expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: an extracellular region, the extracellular region comprising a heavy chain variable region and a light chain variable region of a single chain antibody, the single chain antibody specifically recognizing an antigen CD19; a transmembrane region, the transmembrane region being linked to the extracellular region, and embedded in the T In the cell membrane of lymphocytes; an intracellular region that is linked to the transmembrane region, and the intracellular region includes an intracellular portion of CD28 and a CD3 ⁇ chain.
  • the killing ability of the T lymphocytes proposed by the present invention is remarkably enhanced, and the killing effect on CD19 + tumor cells of CD19 + malignant B cell lymphoma or leukemia is greatly enhanced.
  • Tumors can avoid immune surveillance and shut down the immune response by stimulating the expression of immunosuppressive receptors.
  • activated cytotoxic T lymphocytes CTLs
  • CTLs cytotoxic T lymphocytes
  • PD1 B7-H1 Sexual death ligand 1
  • Cytotoxic T lymphocyte antigen 4 (CTLA4) is another key negative regulator of T cells that inhibits T cell activation by interacting with ligands B7.1, B7.2 (CD80) expressed on antigen-presenting cells. Interaction with CD86) inhibits T cell activation. Therefore, the immunological checkpoint of the T lymphocyte or the transgenic lymphocyte proposed in the present invention is silenced, and the proliferation and viability of the T lymphocyte or the transgenic lymphocyte in the tumor patient and the secretion ability of the cytokine are remarkably improved.
  • 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 chimeric antigen receptor of the single-chain antibody expressed by the transgenic lymphocytes of the invention greatly improves the targeted killing effect of the transgenic lymphocytes on the targeted tumor cells.
  • the antigen is CD19. Therefore, the transgenic lymphocytes have a directional killing effect on the cells expressing the antigen CD19, and the specific binding effect of the antigen antibodies is stronger, and the directional killing effect of the transgenic lymphocytes of the present invention on the CD19 antigen-expressing tumor cells is greatly enhanced.
  • the lymphocyte cell surface immunological checkpoint is independently selected from at least one of CTLA4, PD1, TIM3, BTLA, LAG3.
  • the above molecules are capable of specifically binding to antigens expressed on the surface of tumor cells, negatively regulating and attenuating cellular immune responses.
  • the successful silencing of the cell surface immunological checkpoint further enhances the targeted killing effect of the transgenic lymphocytes on the tumor cells.
  • the lymphocyte cell surface immunological checkpoint is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation and zinc finger nuclease.
  • 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 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, so 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.
  • 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 are selected, such as at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, or zinc finger nuclease employed in the embodiments of the present invention to achieve specific silencing of the gene of interest.
  • the lymphocyte cell surface 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 siRNA of the examples of the present invention is designed to be homologous to the coding region of PD1 or CTLA4 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
  • RISC is directed to the corresponding mRNA; the same nuclease cleaves to target PD1 or CTLA4 mRNA, resulting in specific gene PD1 or CTLA4 silencing, inhibiting expression of the specific gene PD1 or CTLA4.
  • the siRNA is introduced into the cell as shRNA (shRNA contains approximately 18-23 nucleotide siRNA sequences followed by a 9-15-length nucleotide loop and a reverse complement of a siRNA sequence), and the shRNA design is better avoided. Matching points in the 3'UTR cell gene; ensuring proper strand selection.
  • a single siRNA molecule can be repeatedly applied to the division of multiple targeting mRNA molecules.
  • RNAi can be induced by introducing synthetic siRNA.
  • 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.
  • the shRNA used in the embodiment of the present invention has a highly efficient and specific silencing cell surface immune checkpoint. The successful silencing of cell surface immunological checkpoints makes transgenic lymphocytes significantly resistant to tumor-mediated immunosuppression and targeted killing of tumors. The effect is more significant.
  • 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 combination of the expression of the intracellular segment of the immunostimulatory molecule and the silencing of the cell surface immunological checkpoint has a positive regulation and enhances the cellular immune response, so that the effect of the transgenic lymphocytes on the targeted killing effect of the tumor is more significant.
  • the lymphocyte cell surface immunological checkpoint is CTLA4 or PD1.
  • the cell surface immune checkpoint CTLA4 or PD1 is silenced, so that the proliferation and viability of the transgenic lymphocytes in the tumor patient are further improved, and the effect of the directed killing effect on the tumor is more remarkable.
  • the lymphocytes in the present 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 co-stimulatory molecule in the above lymphocytes is silenced and expresses the chimeric antigen receptor, so that the cellular immune function of the above lymphocytes is more targeted, 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, A nucleic acid molecule encoding a chimeric antigen receptor having the nucleotide sequence set forth in SEQ ID NO: 2; and a nucleic acid molecule that silences a cell surface immunological checkpoint, the nucleotide sequence of the nucleic acid molecule that silences the cell surface immunological checkpoint It is at least one selected from the group consisting of SEQ ID NOS: 3 to 68.
  • SEQ ID NOS: 3 to 14 are nucleotide sequences of human programmed cell death receptor 1 (PD1) siRNA, and SEQ ID NOs: 15 to 30 are nucleus of human cytotoxic T lymphocyte antigen 4 (CTLA4) siRNA.
  • the nucleotide sequence, SEQ ID NOs: 31 to 46 are the nucleotide sequences of human T cell immunoglobulin mucin molecule 3 (TIM3) siRNA, and SEQ ID NOs: 47 to 57 are human T lymphocyte attenuating factor (BTLA) siRNA.
  • the nucleotide sequence of SEQ ID NOS: 58-68 is the nucleotide sequence of human lymphocyte activation gene 3 protein (LAG3) siRNA.
  • the transgenic lymphocytes obtained by introducing the lentivirus or the construct of the present invention into lymphocytes, the cell surface immunological checkpoints PD1, CTLA4, TIM3, BTLA, LAG3 are specifically silenced, and the antigen is specifically bound.
  • the high expression of the chimeric antigen receptor of CD19 makes the anti-apoptotic ability and proliferative ability of transgenic lymphocytes enhanced, and the targeted killing ability is significantly improved, so that the gene lymphocytes can be used for CD19 + malignant B cell lymphoma or leukemia CD19 + tumor cells.
  • the killing effect is particularly significant.
  • the lentivirus or construct proposed by the invention carries a nucleotide sequence as set forth in SEQ ID NO: 69, 70 or 71.
  • SEQ ID NO: 69 is a nucleic acid molecule (CD19-CAR/iPD1) co-expressing an anti-CD19 chimeric antigen receptor and a silencing cell surface immunological checkpoint PD-1
  • SEQ ID NO: 70 is a co-expressing anti-CD19 chimera Antigen receptor and silencing cell surface immunological checkpoint CTLA4 nucleic acid molecule (CD19-CAR/iCTLA4)
  • SEQ ID NO: 71 is a co-expressing anti-CD19 chimeric antigen receptor and silencing cell surface immunological checkpoints PD-1 and CTLA4 Nucleic acid molecule (CD19-CAR/iPD1/iCTLA4).
  • the transgenic lymphocytes obtained by introducing the lentivirus of the present invention into lymphocytes are specifically silenced on the cell surface immunological checkpoints PD1 and CTLA4, and the anti-CD19 chimeric antigen receptor is highly expressed, so that the transgene is transgenic.
  • the anti-apoptotic ability and proliferative ability of lymphocytes are enhanced, and the directed killing ability is significantly improved, so that the transgenic lymphocytes have a particularly significant killing effect on CD19 + malignant B cell lymphoma or leukemia CD19 + tumor cells.
  • the inventors independently realize the above-mentioned cell chimeric antigen receptor and cell surface immunological checkpoint shRNA, respectively, wherein, in addition, expression herein refers to protein 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, the second promoter and a nucleic acid molecule that silences the immunological checkpoint Operatively connected.
  • the first promoter and the second promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, RSV promoters, introduction of first and second promoters, such that Nucleic acid molecules encoding chimeric antigen receptors and nucleic acid molecules that silence cell surface immunological checkpoints are independently expressed, effectively silencing cell surface immunological checkpoints and ensuring efficient expression of chimeric antigen receptors, enabling lymphocyte targeting The effect is stronger, and the targeted killing effect on the tumor 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 on the surface of the silenced cell, and the silencing cell to which the third promoter and the second promoter are ligated
  • the nucleic acid molecules of the surface are immunologically examined, and the third promoter and the second promoter respectively activate shRNAs that silence different immune checkpoints.
  • the immunological checkpoint is efficiently silenced and the chimeric antigen receptor is efficiently expressed on the transgenic lymphocyte membrane, thereby effectively inhibiting the immunological checkpoint. Negative immunoregulation and the biological role of the chimeric antigen receptor are ensured, so that the lymphocyte targeted killing effect is more significant.
  • the vector of the construct is a non-pathogenic viral vector.
  • the introduction of non-pathogenic viral vectors greatly enhances the replication and amplification efficiency of the constructs in lymphocytes, and further enhances the targeting of lymphocytes, and the killing effect on tumor cells is more significant.
  • the non-pathogenic viral vector is selected from at least one of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adenovirus associated viral vector.
  • the above-mentioned construct virus has a wide range of infection, which can infect both terminally differentiated cells and cells in a mitotic phase, and can be integrated into the host chromosome or freed from the host chromosome to achieve a wide range.
  • the efficient and efficient infection efficiency so that the cell surface immune checkpoint is efficiently silenced and the anti-CD19 chimeric antigen receptor is highly expressed in lymphocytes, which further enhances the targeting of lymphocytes and kills CD19 + tumor cells. The effect 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 which are then secreted into the culture medium.
  • the inventors collected a matrix containing the recombinant lentivirus, selectively concentrated, and used for gene transfer. Slow vectors can infect a variety of cell types, including cleavable cells and non-dividable cells.
  • the lentivirus of the embodiment of the present invention is a complex lentivirus, and in addition to the common lentiviral genes gag, pol and env, other genes having regulatory and structural functions are also included.
  • Lentiviral vectors are well known to those skilled in the art, and lentiviruses include: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors produce a biosafety vector by multiple attenuation of HIV-causing genes, such as deletion of the genes env, vif, vpr, vpu and nef.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and are useful for in vivo and in vitro gene transfer and nucleic acid sequence expression.
  • a suitable host cell together with two or more vectors with packaging functions (gag, pol, env, rev and tat), it is possible to infect non-dividing cells.
  • the targeting of recombinant viruses is achieved by binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
  • the targeting of the recombinant virus confers specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, along with another gene encoding a ligand for the receptor on the particular target cell.
  • the lentiviral vector of the present invention can efficiently transport and co-express shRNA (a transport form of siRNA) which can effectively inhibit the expression of PD1 or CTLA4.
  • 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 to carry and co-express a small hairpin RNA that inhibits the expression of the PDl or CTLA4 gene.
  • 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 gene.
  • the embodiment of the present invention also includes a microgene.
  • Microgenes mean the use of a combination (selected nucleotide sequence and operably necessary related linker sequences) to direct expression of the transform, transcription and/or gene product in a host cell in vivo or in vitro.
  • the "operable ligation" sequence is employed to include expression control sequences for a continuous gene of interest, and expression control sequences for trans- or remote control of the gene of interest.
  • vectors of the embodiments of the invention also include conventional control elements that permit transcription, transformation, and/or expression of small hairpin RNA in cell infection with the plasmid vector or in a cellular infection with the viral vector.
  • a large number of expression control sequences may be used.
  • the promoter that expresses shRNA is the RNA polymerase promoter.
  • the promoter is a RAN polymerase promoter selected from the group consisting of U6, H1, polI, polII and polIII.
  • the promoter is a tissue-specific promoter.
  • the promoter is an inducible promoter.
  • the promoter is selected from a promoter based on the selected vector.
  • the promoter when a lentiviral vector is selected, the promoter is a U6, H1, CMV IE gene, EF-1 ⁇ , ubiquitin C, or phosphoglycerate kinase (PGK) promoter.
  • Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding geneticin, hygromycin, ampicillin or puromycin resistance.
  • Other components of the carrier include an origin of replication.
  • vectors are well known to those skilled in the art and include conventional cloning techniques such as shRNA, polymerase chain reaction and any suitable method for providing the desired nucleotide sequence for use in embodiments of the invention. .
  • the inventors constructed viral vectors that co-express small hairpin RNA (shRNA) (used to suppress immune checkpoints) as well as chimeric antigen receptors (CAR).
  • shRNA small hairpin RNA
  • CAR chimeric antigen receptors
  • the small hairpin RNA carrying the siRNA for silencing PD1 or CTLA4 and the viral vector or plasmid expressing the chimeric antigen receptor (CAR) are complexed in the embodiments of the present invention, and the viral vector or plasmid may be combined with a polymer or other material to increase Its stability, or assist its targeted movement.
  • the present invention proposes 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 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 is successfully introduced into the above lymphocytes or T lymphocytes, and the cell surface immunoassay of lymphocytes is silenced and the expression of the anti-CD19 chimeric antigen receptor is obtained, thereby obtaining the obtained lymphocytes or T lymphocytes.
  • the targeted killing effect on tumor cells is stronger.
  • the present 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 immunological checkpoints of transgenic lymphocytes or T lymphocytes and efficient expression of anti-CD19 chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, thereby The resulting transgenic lymphocytes or T lymphocytes have a stronger targeted killing effect on tumor cells.
  • compositions are provided to a patient and are 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 and express a unique anti-CD19 chimeric antigen receptor.
  • the dosage of the therapeutic agent depends primarily on the condition of treatment, age, weight, and the health of the patient, which may result in patient variability.
  • These methods of silencing PD1 or CTLA4 or silencing PD1 and CTLA4 and expressing a specific CD19 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.
  • the invention of a method of treatment involves the use of one or more drug therapies.
  • the cancer comprises at least one selected from the group consisting of a B cell lymphoma or a B cell leukemia.
  • Silencing of cell surface immunological checkpoints and efficient expression of anti-CD19 chimeric antigen receptors in transgenic lymphocytes or T lymphocytes, resulting in cytokine production of the resulting lymphocytes or T lymphocytes and against B-cell lymphoma or leukemia The targeted killing effect of CD19 + tumor cells is stronger.
  • the present invention provides a method of increasing lymphocyte activity, according to an embodiment of the present invention, the lymphocyte carries a chimeric antigen receptor, characterized in that the method comprises: subjecting the lymphocyte cell surface immunological examination The point is silenced, the cell surface immunological checkpoint, the lymphocyte and the chimeric antigen receptor are as defined above, the lymphocyte activity, the lymphocyte activity comprising the cytokine of the lymphocyte The ability to secrete and at least one of the ability of the lymphocytes to kill tumor cells.
  • the cell surface immunological checkpoint of lymphocytes is silenced, the secretion of cytokines is increased, and the targeted killing effect on tumor cells is stronger.
  • 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 CD19.
  • the method proposed in the embodiment of the invention can effectively kill tumor cells having the tumor antigen CD19.
  • the method comprises: isolating lymphocytes from a cancer patient; introducing the aforementioned construct, or the lentivirus described above, into the lymphocytes to obtain transgenic lymphocytes, the transgene
  • the lymphocyte cell surface immunological checkpoint is silenced and expresses a chimeric antigen receptor; and the transgenic lymphocytes are administered to the cancer patient.
  • the method of the embodiments of the present invention is capable of further effectively killing tumor cells having the tumor antigen CD19.
  • the cancer comprises at least one selected from the group consisting of a B cell lymphoma and a B cell leukemia.
  • B cell lymphoma and B cell leukemia have specific expression of CD19 tumor cells
  • the method for treating cancer of the present invention can silence lymphocyte cell surface checkpoints and express chimeric antigen receptors.
  • the resulting lymphocytes or T lymphocytes have targeted killing of CD19-specific B cell lymphoma and B cell leukemia tumor cells.
  • administering refers to introducing a predetermined amount of a substance into a patient in some suitable manner.
  • the therapeutic composition of the invention can be administered by any conventional route as long as it can reach the intended tissue.
  • Various modes of administration are contemplated, including peritoneal, venous, muscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the invention is not limited to these exemplary modes of administration.
  • the active ingredient of the orally administered composition should be coated or formulated to prevent its degradation in the stomach.
  • the compositions of the invention may be administered as an injectable preparation.
  • the therapeutic compositions of the present invention can be administered using a particular device that delivers the active ingredient to the target cells.
  • the frequency and dosage of the therapeutic composition of the present invention can be determined by a number of relevant factors including the type of disease to be treated, the route of administration, the age, sex, weight and severity of the disease as well as the active ingredient. Type of drug. According to some embodiments of the invention, the daily dose may be divided into 1 dose, 2 doses or multiple doses in a suitable form for administration once, twice or more times throughout the time period, as long as a therapeutically effective amount is achieved. .
  • terapéuticaally effective amount refers to an amount of a compound that is sufficient to significantly ameliorate certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • a therapeutic composition that reduces, prevents, delays, inhibits, or blocks any symptoms of a disease or condition is therapeutically effective.
  • 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 the treatment of CD19 + B-cell lymphoma and B-cell leukemia), including: (a) in an individual who is susceptible to the disease but has not yet been diagnosed with the disease.
  • Prevent disease eg, treatment of CD19 + B cell lymphoma and B cell leukemia
  • condition e.g., treatment of CD19 + B cell lymphoma and B cell leukemia
  • inhibit disease such as arrest disease progression
  • relieve disease such as alleviate disease-related symptoms
  • 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.
  • OCI-LY3 cells CD19 + diffuse large B cell lymphoma cell line (DLBCL)
  • Jurkat cells CD19 - T lymphoma cell line
  • K562 natural killer cells
  • All of the above cells were obtained from ATCC (American Cell Bank) and DSMZ (German Cell Bank), and cultured in RPMI-1640 medium supplemented with 10% or 20% fetal bovine serum and 2 ml of L-glutamine. From Gibco-BRL, San Francisco, CA, USA).
  • 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 subjected to stimulation activation using magnetic beads coated with monoclonal antibodies against 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.
  • transduced T lymphocytes appear dormant, such as slower cell growth and smaller cells, the cell growth rate and size are assessed by Multisizer 3 Coulter Counter (purchased from Beckman Coulter), or transduced T Lymphocytes can be used for functional analysis at a planned time point.
  • the flow cytometer used in the examples of the present application is BD FACSCanto II (purchased from BD Biosciences), and Flow cytometric data was analyzed using FlowJo version 7.2.5 software (available from Tree Star, Ashland, OR).
  • the lentiviral vector transduced T cells (the number of cells is 1 ⁇ 10 6 /well) were co-cultured with OCI-LY3 lymphoma cells (OCI-LY3 cells expressing CD19 and PD-L1), and different target targets were changed during the experiment. Cell ratio.
  • 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.
  • the cytotoxic activity of anti-CD19 chimeric antigen receptor T cells was assessed using the 4 - hour 51 chromium release assay in the Examples.
  • Target test cells were labeled with 51 Cr at 37 degrees Celsius for 1 hour. After labeling, the cells were rinsed with RPMI medium containing 10% fetal bovine serum (FCS). After rinsing, the cells were resuspended in the same medium, and the concentration of the resuspended cells was 1 ⁇ 10 5 /ml. After transduction, T cells were added to the target test cell suspension at different target cell ratios (E:T), and the cells were seeded in 96-wells at a volume of 200 microliters per well. The cells were cultured for 4 hours in a 37 degree incubator.
  • E:T target cell ratios
  • 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 cells to be labeled include OCI-LY3 (CD19 + , PD-L1 + ), and Jurkat (CD19 – ) cells.
  • the inventors cloned the sequence encoding the single-chain antibody against human CD19, the CD28 intracellular domain and the T cell receptor combination ⁇ -strand sequence into the LV lentiviral vector, and during the cloning process, the selection restriction
  • the restriction enzyme digestion is a double digestion of XbaI and NotI, and double digestion with NotI and XhoI.
  • the restriction enzyme digestion, ligation, screening and amplification of the plasmid of interest generate a slow DNA sequence encoding the anti-CD19 chimeric antigen receptor.
  • the viral plasmid (LV-CD19 CAR); the sequence of the U6 promoter and PD1-shRNA (shPD1) was cloned into the lentiviral plasmid of the above LV-CD19 CAR, and the shPD1 was generated. Sequence and lentiviral plasmid (LV-CD19 CAR-shPD1) encoding the nucleotide sequence of the anti-CD19 chimeric antigen receptor.
  • the schematic of the carrier is shown in Figure 1.
  • Figure 1 shows a schematic representation of the construction of a related LV lentiviral vector (containing the U6 promoter, the sequence encoding human shPD1 and the anti-CD19 chimeric antigen receptor sequence).
  • Example 3 Enhanced killing ability of T cells co-expressing PD1-shRNA and anti-CD19 chimeric antigen receptor
  • peripheral blood lymphocytes are taken from an unnamed blood donor. Peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. T lymphocytes were incubated with T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) for 72 hours at 5% CO 2 at 37 ° C. The medium was supplemented with 2 mmol/L glutamine, 10 % high temperature inactivated fetal bovine 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 bovine serum
  • T cells were seeded on a cell culture dish containing recombinant fibronectin fragments (FN ch-296; Retronectin) and transduced with a lentiviral vector.
  • the transduction vectors were LV-CD19 CAR-shPD1, LV-CD19 CAR, LV. -shPD1 or no load (LV-GFP). The 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 transfected by different lentiviral vectors on (CD19 - and CD19 + ) lymphoma cells, the ratio of target cells was 10:1, and the standard method was 4-hour 51 chromium release method.
  • the 4 - hour 51 chromium release method is as described in Example 1. The result is shown in Figure 2.
  • human T cells co-expressing shPD1 and anti-CD19 chimeric antigen receptors were more effective at killing OCI-LY3 lymphoma than T cells expressing anti-CD19 chimeric antigen receptor or shPD1 alone.
  • T cells expressing shPD1 alone have no significant cytotoxicity against CD19 + lymphoma cells.
  • T cells expressing an anti-CD19 chimeric antigen receptor or T cells co-expressing shPD1 and anti-CD19 chimeric antigen receptors have no significant killing effect on Jurkat (CD19 - ) lymphoma cells.
  • the empty plasmid transduced T lymphocytes (control T lymphocytes) showed significant killing effect on CD19 + or CD19 - lymphoma cells.
  • T lymphocytes co-expressing shPD1 and anti-CD19 chimeric antigen receptors have more cytokine secretion characteristics
  • T lymphocyte activator magnetic beads CD3/CD28 activated T lymphocytes are transduced by lentiviral vector and expanded in vitro, as described above.
  • Transduction of T cells in lentiviral vectors (the number of cells is 1 ⁇ 10 6 /well) co-cultured with CD19 + /PDL1 + OCI-LY3 lymphoma cells, and after 3 days, cytokine secretion and cell activity were evaluated by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Figure 3 shows that T lymphocytes transduced with LV-CD19 CAR-shPD1 secrete more IFN ⁇ than T lymphocytes transduced with LV-CD19 CAR or LV-shPD1 vector (P ⁇ 0.05; LV-CD19 CAR- shPD1 vs.LV-CD19 CAR).
  • T cells co-expressing CTLA4-shRNA and anti-CD19 chimeric antigen receptors have enhanced killing ability and cells Factor secretion more features
  • the inventors also examined the targeted killing ability and cytokine secretion ability of T cells co-expressing CTLA4-shRNA and anti-CD19 chimeric antigen receptor, and the experimental procedure was the same as that of Example 3 and Example 4, and the experiment was carried out.
  • T cells co-expressing PD1-shRNA and anti-CD19 chimeric antigen receptors T cells co-expressing CTLA4-shRNA and anti-CD19 chimeric antigen receptors express anti-CD19 chimeric antigen receptor or CTLA4-shRNA alone.
  • the targeted killing ability of T cells is enhanced, and the secretion of cytokines is increased.
  • the inventors also investigated the targeted killing ability and cytokine secretion ability of T cells co-expressing CTLA4-shRNA, PD1-shRNA and anti-CD19 chimeric antigen receptors, and the experimental procedure was the same as in Example 3 and Example 4, and co-expression was found.
  • the targeted killing ability and cytokine secretion ability of CTLA4-shRNA, PD1-shRNA and anti-CD19 chimeric antigen receptor T cells are stronger than T cells co-expressing CTLA4-shRNA and anti-CD19 chimeric antigen receptor or co-expressing PD1 -shRNA and T cells against the anti-CD19 chimeric antigen receptor.

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Abstract

L'invention concerne un lymphocyte transgénique, une construction et une composition thérapeutique pour traiter les cancers, le point de contrôle immunitaire de la surface cellulaire du lymphocyte transgénique décrit dans la présente invention étant désactivé et exprimant un récepteur d'antigène chimérique.
PCT/CN2016/087488 2016-02-03 2016-06-28 Composition thérapeutique pour le traitement de la leucémie à cellules b et du lymphome à cellules b WO2017133174A1 (fr)

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CN201610079061.0A CN107034193B (zh) 2016-02-03 2016-02-03 治疗b细胞白血病及b细胞淋巴瘤的治疗组合物
CN201610079061.0 2016-02-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114957475A (zh) * 2018-09-26 2022-08-30 福州拓新天成生物科技有限公司 抗b7-h3的单克隆抗体及其在细胞治疗中的应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019033249A1 (fr) * 2017-08-14 2019-02-21 深圳市博奥康生物科技有限公司 Sharn du gène btla humain et utilisation correspondante
WO2019037055A1 (fr) * 2017-08-24 2019-02-28 深圳市博奥康生物科技有限公司 Arnsh du gène grd4 humain et application d'arnsh
CN108424927A (zh) * 2018-02-10 2018-08-21 武汉波睿达生物科技有限公司 一种能同时表达cd19 car和敲低t细胞表面pd-1表达的质粒结构及其构建方法
CN110526983B (zh) * 2018-05-24 2022-02-11 北京马力喏生物科技有限公司 改良型抗cd19 car-t细胞
EP4025597A2 (fr) * 2019-09-06 2022-07-13 Avectas Limited Ingénierie de cellules immunitaires pour des applications de thérapie cellulaire ex vivo

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014130657A1 (fr) * 2013-02-20 2014-08-28 The Trustees Of The University Of Pennsylvania Traitement du cancer au moyen d'un récepteur d'antigènes chimériques anti-egfrviii humanisés
WO2014153270A1 (fr) * 2013-03-16 2014-09-25 Novartis Ag Traitement du cancer à l'aide d'un récepteur d'antigène chimérique anti-cd19 humanisé
WO2014201021A2 (fr) * 2013-06-10 2014-12-18 Dana-Farber Cancer Institute, Inc. Procédés et compositions pour réduire l'immunodépression par des cellules tumorales
WO2015164759A2 (fr) * 2014-04-25 2015-10-29 Bluebird Bio, Inc. Récepteurs d'antigènes chimériques de promoteur mnd

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10934550B2 (en) * 2013-12-02 2021-03-02 Phio Pharmaceuticals Corp. Immunotherapy of cancer
CN105177031B (zh) * 2015-06-12 2018-04-24 北京艺妙神州医疗科技有限公司 嵌合抗原受体修饰的t细胞及其用途
CN106554414B (zh) * 2015-09-18 2019-04-23 上海科济制药有限公司 抗cd19全人抗体以及靶向cd19的免疫效应细胞

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014130657A1 (fr) * 2013-02-20 2014-08-28 The Trustees Of The University Of Pennsylvania Traitement du cancer au moyen d'un récepteur d'antigènes chimériques anti-egfrviii humanisés
WO2014153270A1 (fr) * 2013-03-16 2014-09-25 Novartis Ag Traitement du cancer à l'aide d'un récepteur d'antigène chimérique anti-cd19 humanisé
WO2014201021A2 (fr) * 2013-06-10 2014-12-18 Dana-Farber Cancer Institute, Inc. Procédés et compositions pour réduire l'immunodépression par des cellules tumorales
WO2015164759A2 (fr) * 2014-04-25 2015-10-29 Bluebird Bio, Inc. Récepteurs d'antigènes chimériques de promoteur mnd

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAUD, C. ET AL.: "Tumor-Targeted Human T Cells Expressing CD 28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition", PLOS ONE, vol. 10, no. 6, 25 June 2015 (2015-06-25), pages 1 - 15, XP055403799, ISSN: 1932-6203 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114957475A (zh) * 2018-09-26 2022-08-30 福州拓新天成生物科技有限公司 抗b7-h3的单克隆抗体及其在细胞治疗中的应用
CN114957475B (zh) * 2018-09-26 2023-06-20 福州拓新天成生物科技有限公司 抗b7-h3的单克隆抗体及其在细胞治疗中的应用

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