WO2018137294A1 - 共表达抗msln嵌合抗原受体和无功能egfr的转基因淋巴细胞及其用途 - Google Patents

共表达抗msln嵌合抗原受体和无功能egfr的转基因淋巴细胞及其用途 Download PDF

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WO2018137294A1
WO2018137294A1 PCT/CN2017/081273 CN2017081273W WO2018137294A1 WO 2018137294 A1 WO2018137294 A1 WO 2018137294A1 CN 2017081273 W CN2017081273 W CN 2017081273W WO 2018137294 A1 WO2018137294 A1 WO 2018137294A1
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lymphocyte
lymphocytes
chimeric antigen
cancer
antigen receptor
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French (fr)
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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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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
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Definitions

  • the present invention relates to the field of biomedicine, and in particular to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer, and an augmentation lymph
  • a T lymphocyte a T lymphocyte
  • a lentivirus a transgenic lymphocyte
  • a construct a therapeutic composition for treating cancer
  • an augmentation lymph The method of cell therapy safety.
  • MSLN Mesothelin
  • MMSLN Mesothelin
  • interstitial is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancers and about 50% of lung adenocarcinomas and other cancers such as cholangiocarcinoma, gastric cancer, and intestinal cancer. , esophageal cancer, breast cancer.
  • the interstitial gene encodes a precursor protein of 71KDa, which is then processed into a 31KDa exfoliated fragment and a 40KDa protein fragment.
  • the 31KDa exfoliated fragment is called megakaryocyte promoting factor (MPF), and the 40KDa protein fragment is Known as interstitial, interstitial is immobilized on the cell membrane by the anchoring action of glycosyl-phosphatidylinositol (GPI).
  • MPF megakaryocyte promoting factor
  • GPI glycosyl-phosphatidylinositol
  • mesothelioma is divided into pleural mesothelioma and peritoneal mesothelioma.
  • the pleural mesothelioma is the primary tumor of the pleura, which is limited (mostly benign) and diffuse (both malignant). Divided, malignant mesothelioma is one of the worst tumors in the chest.
  • Peritoneal mesothelioma refers to a tumor that originates in the peritoneal mesothelial cells. Clinical manifestations are not characteristic, common symptoms and signs are: abdominal pain, ascites, abdominal distension and abdominal mass.
  • Interstitial is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancers and about 50% of lung adenocarcinomas and other cancers such as cholangiocarcinoma, gastric cancer, intestinal cancer, esophagus Cancer, breast cancer. Therefore, interstitial represents a highly attractive target in the field of tumor immunotherapy.
  • Non-functional EGFR (epidermal growth factor receptor) lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes wild-type EGFR transmembrane region and intact anti-EGFR antibody The combined sequence, non-functional EGFR can be used as a suicide marker for lymphocytes.
  • the inventors have proposed a construct encoding a non-functional EGFR nucleic acid molecule and a nucleic acid molecule encoding a chimeric antigen receptor and a transgenic lymphocyte formed by the introduction of the construct, the coding chimera
  • the antigen receptor specifically binds to the antigen MSLN. Therefore, the constructs and transgenic lymphocytes proposed by the present invention can be used for immunotherapy of adoptive T cells of tumors, especially mesenchymal positive tumors; the transgenic lymphocytes of the present invention have a strong killing ability for high expression of interstitial tumors. It has weak killing of mesothelial cells with normal MSLN expression level and high safety of immune killing.
  • the invention proposes a T lymphocyte.
  • the T lymphocyte expresses a non-functional EGFR; and a chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region comprising a single-chain antibody a heavy chain variable region and a light chain variable region, the single chain antibody specifically recognizing an antigen MSLN; a transmembrane region, the transmembrane region being linked to the extracellular region, and intercalating into a cell membrane of the T lymphocyte Intracellular region, the intracellular region is linked to the transmembrane region, and the intracellular region comprises an intracellular portion of CD28 or 4-1BB and a CD3 ⁇ chain.
  • Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and intact sequences that bind to anti-EGFR antibodies, and non-functional EGFR can act as lymphocytes. Suicide tag.
  • the T lymphocytes of the embodiments of the present invention have a directed killing effect on tumor cells which highly express MSLN, and have high safety.
  • 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 of SEQ ID NO: 2; and a nucleic acid molecule encoding a non-functional EGFR having the amino acid sequence of SEQ ID NO: 3, the encoding is absent
  • the nucleic acid molecule of functional EGFR has the nucleotide sequence set forth in SEQ ID NO:4.
  • a transgenic lymphocyte obtained by introducing a lentivirus according to an embodiment of the present invention into a lymphocyte has a specific killing ability against a tumor cell, and particularly has a directed killing effect on a tumor cell which highly expresses MSLN, and is safe. High sex.
  • the invention proposes a lentivirus.
  • the lentivirus carries a nucleotide sequence set forth in SEQ ID NO: 5.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the embodiment of the present invention into lymphocytes have the directional killing ability against tumor cells, especially the directed killing effect on tumor cells with high expression of MSLN, and safety. high.
  • the invention provides a transgenic lymphocyte.
  • the lymphocyte cell expresses a non-functional EGFR; and a chimeric antigen receptor is expressed, the chimeric antigen receptor comprising: an extracellular region comprising a heavy chain variable of an antibody a region and a light chain variable region, said antibody being capable of specifically binding to a tumor antigen; a transmembrane region; and an intracellular region comprising an intracellular portion of an immunostimulatory molecule, wherein said antibody is single-stranded
  • the tumor antigen is MSLN.
  • the inventors were surprised to find that expressing non-functional EGFR and expressing chimeric antigen receptors
  • the lymphocytes have specific killing ability against tumor cells, and particularly have a directed killing effect on tumor cells highly expressing MSLN, and have high safety.
  • the above transgenic lymphocytes may further have at least one of the following additional technical features:
  • 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 of the present invention has the effect of positively regulating and enhancing the cellular immune response, so that the effect of the directional killing effect of the transgenic lymphocytes of the embodiment of the present invention on the tumor is further improved;
  • the combination of the expression of the intracellular segment of the immunostimulatory molecule and the expression of the non-functional EGFR allows the transgenic lymphocyte proliferation of the embodiment of the present invention to have a more significant directed killing effect on the tumor and is more safe.
  • the intracellular segment of the immunostimulatory molecule is an intracellular segment of 4-1BB or CD28.
  • the intracellular segment of the immunostimulatory molecule of the chimeric antigen receptor of the transgenic lymphocytes of the present invention is the intracellular portion of CD28 or 4-1BB.
  • the intracellular segment of the immunostimulatory molecule is an intracellular segment of CD28 or 4-1BB, which further enhances the targeted killing effect of the transgenic lymphocytes of the embodiments of the present invention.
  • the non-functional EGFR expressed by the transgenic lymphocytes of the present invention lacks an N-terminal ligand binding region and an intracellular receptor tyrosine kinase activity, but includes a transmembrane region and integrity of wild-type EGFR.
  • the domain that binds to anti-EGFR, non-functional EGFR can be used as a suicide marker for transgenic lymphocytes of the examples of the present invention.
  • the expression of non-functional EGFR, combined with the expression of chimeric antigen receptor can effectively ensure the targeted killing effect of transgenic lymphocytes. If the patient has serious adverse reactions, the transgenic lymphocytes can be cleared by anti-EGFR antibody, which can further The safety of the transgenic lymphocytes of the embodiments of the present invention for treating tumor patients with high expression of MSLN is improved.
  • the lymphocytes are CD3 + T lymphocytes or natural killer cells or natural killer T cells.
  • the above lymphocytes of the embodiments of the present invention express non-functional EGFR, and simultaneously express antigen-specific chimeric antigen receptors, such as MSLN antigen-specific chimeric antigen receptors of the embodiments of the present invention, wherein the lymphocytes have tumors Directional killing effect and higher safety.
  • the invention proposes a construct.
  • the construct comprises: a first nucleic acid molecule encoding a chimeric antigen receptor; and a second nucleic acid molecule encoding a non-functional EGFR.
  • the chimeric antigen receptor and the non-functional EGFR are as described above.
  • the non-functional EGFR and the antigen-specific chimeric antigen receptor can be efficiently expressed, thereby the lymphocyte of the embodiment of the present invention.
  • the cells have a directed killing effect on tumor cells, especially tumor cells that highly express MSLN, and are highly safe.
  • 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 disposed in the lymphocytes described above to express the chimeric antigen receptor and express non-functional EGFR, and Chimeric antigen receptor It is in a non-fused form with the non-functional EGFR.
  • the lymphocytes of the first nucleic acid molecule and the second nucleic acid molecule are successfully set, and the lymphocyte surface successfully expresses non-functional EGFR, and the antigen-specific chimeric antigen receptor is successfully expressed on the lymphocyte surface.
  • the MSLN-specific chimeric antigen receptor according to the embodiment of the present invention, and the chimeric antigen receptor and the non-functional EGFR are in a non-fused form on the lymphocyte membrane, and the lymphocytes of the embodiments of the present invention have specific tumor killing. The effect is more secure.
  • 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.
  • effectively expressing the non-functional EGFR thereby effectively ensuring the targeted killing effect of the lymphocytes of the embodiments of the present invention on tumors, especially the targeted killing of tumor cells with high expression of MSLN, and ensuring the safety of immune killing. .
  • the first promoter and the second promoter are each independently selected from the group consisting of CMV, EF-1, LTR, RSV promoters.
  • the above promoter of the embodiment of the invention has the characteristics of high activation efficiency and strong specificity, thereby ensuring high-efficiency expression of non-functional EGFR and high-efficiency expression of chimeric antigen receptor, thereby efficiently ensuring the present invention.
  • the construct further comprises: an internal ribosome entry site sequence, the internal ribosome entry site sequence being disposed between the first nucleic acid molecule and the third nucleic acid molecule,
  • the internal ribosome entry site has the nucleotide sequence set forth in SEQ ID NO: 6.
  • an internal ribosome entry site sequence allows the first nucleic acid molecule and the second nucleic acid molecule to be expressed independently, respectively.
  • the introduction of an internal ribosome entry site sequence ensures the biological action of the chimeric antigen receptor antigen targeting and the high expression of non-functional EGFR, thereby enabling lymphocytes of the embodiments of the present invention to tumor Directional kill The wound effect is more pronounced, and lymphocytes are safer for tumor killing.
  • the construct further comprises: a third nucleic acid molecule disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encoding a linker peptide,
  • the linker peptide is capable of being cleaved in the lymphocytes.
  • the introduction of a third nucleic acid molecule encoding a linker peptide allows expression of a non-functional EGFR and a chimeric antigen receptor in a non-fusion state on the lymphocyte membrane, thereby further ensuring the biological effects of non-functional EGFR and chimeric antigen receptors,
  • the lymphocytes of the embodiments of the present invention have a more specific tumor killing effect and are more safe.
  • the linker peptide has the amino acid sequence set forth in SEQ ID NO: 7.
  • GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 7).
  • the amino acid sequence shown by SEQ ID NO: 7 is the 2A peptide of Hand, Foot and Mouth Virus (a small RNA virus).
  • the introduction of the linker peptide allows the non-functional EGFR and chimeric antigen receptor to be expressed in a non-fusion state on the lymphocyte membrane.
  • the introduction of the linker peptide ensures the biological effects of the non-functional EGFR and the chimeric antigen receptor, and the lymphocytes of the embodiments of the present invention have a more specific tumor killing effect and are more safe.
  • 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 construct in lymphocytes, thereby greatly increasing the expression of non-functional EGFR and the high expression of chimeric antigen receptors in lymphocytes, making lymphocytes
  • the targeting effect is further enhanced and the safety is further improved.
  • the viral vector comprises at least one selected from the group consisting of a retroviral vector, a lentiviral vector or an adenovirus-associated viral vector.
  • the virus carrier of the embodiment of the invention has a wide range of virus infection during virus packaging and infection, and can infect both terminally differentiated cells and cells in a mitotic phase, and the genome can be integrated into the host chromosome or free.
  • the 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 lymphocytes or T lymphocytes to achieve expression of lymphocytes expressing non-functional EGFR and chimeric antigen receptors, thereby producing transgenic lymphocytes or T prepared by the preparation method of the present invention.
  • Lymphocytes have a targeted killing effect on tumor cells, especially tumor cells that express MSLN, and are safer.
  • the invention provides a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: the above construct, lentivirus, T lymphocyte or transgenic lymphocyte.
  • the composition of any of the above therapeutic compositions can achieve the expression of non-functional EGFR of transgenic lymphocytes or T lymphocytes.
  • the chimeric antigen receptor is highly expressed in transgenic lymphocytes or T lymphocytes, so that the obtained transgenic lymphocytes or T lymphocytes have a targeted killing effect on tumor cells, and the therapeutic composition for treating cancer of the present invention has The targeted killing effect on tumor cells, especially the targeted killing effect on tumor cells with high expression of MSLN, and high safety.
  • the above therapeutic composition may further comprise at least one of the following additional technical features:
  • the cancer comprises at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.
  • the above tumor cells have high specific expression of MSLN
  • the therapeutic composition of the present invention can express lymphocyte cells on the surface of non-functional EGFR and express antigen-specific chimeric antigen receptors, such as the MSLN antigen of the present invention.
  • the sexual chimeric antigen receptor, the obtained lymphocyte or T lymphocyte has targeted killing of tumor cells which express MSLN high, and has high safety.
  • the invention provides a method of improving the safety of lymphocyte therapy, the lymphocyte carrying a chimeric antigen receptor, characterized in that the method comprises: expressing the lymphocyte without Functional EGFR, said non-functional EGFR, said lymphocyte, said chimeric antigen receptor as previously described.
  • Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and intact sequences that bind to anti-EGFR antibodies, and non-functional EGFR can act as lymphocytes. Suicide tag.
  • lymphocytes of the embodiments of the present invention are used for the treatment of tumor cells with high expression of MSLN, if the patient develops a serious adverse reaction, the lymphocytes of the embodiments of the present invention can be cleared by the anti-EGFR antibody, thereby improving the lymph of the embodiment of the present invention.
  • the invention provides a method of treating cancer.
  • the method comprises: administering to a cancer patient a construct as described above, a lentivirus as described above, a T lymphocyte as described above or a transgenic lymphocyte as described above, wherein The antigen receptor specifically binds to the tumor antigen MSLN.
  • the method for treating cancer according to the embodiment of the invention can effectively achieve targeted killing of tumor cells of cancer patients, in particular, has targeted killing effect on tumor cells with high expression of MSLN, thereby effectively treating cancer, and the therapeutic effect is good and High security.
  • the above method for treating cancer may further comprise at least one of the following additional technical features:
  • the method comprises: isolating lymphocytes from a cancer patient; introducing the aforementioned construct, or the lentivirus described above, into the lymphocytes to obtain transgenic lymphocytes, the transgene Lymphocytes express a chimeric antigen receptor; and the transgenic lymphocytes are administered to the cancer patient.
  • the method for treating cancer according to the embodiment of the invention can further effectively achieve targeted killing of tumor cells of cancer patients, especially having targeted killing effect on tumor cells with high expression of MSLN, thereby further effectively treating cancer, and the therapeutic effect Good and safe.
  • the cancer comprises at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.
  • the method for treating cancer according to an embodiment of the present invention enables lymphocyte cells to express a chimeric antigen receptor, such as the MSLN antigen-specific chimeric antigen receptor of the present invention, and the obtained lymphocyte or T lymphocyte has specific expression to MSLN.
  • a chimeric antigen receptor such as the MSLN antigen-specific chimeric antigen receptor of the present invention
  • FIG. 1 is a schematic view showing the structure of a lentiviral vector which co-expresses a chimeric antigen receptor against MSLN and a non-functional EGFR according to an embodiment of the present invention
  • FIG. 2 is a graph showing the results of anti-EGFRL-mediated ADCC killing clearance of co-expressing anti-MSLN chimeric antigen receptor and non-functional EGFR lymphocytes according to an embodiment of the present invention.
  • the invention provides a T lymphocyte or transgenic lymphocyte.
  • a T lymphocyte according to an embodiment of the present invention expresses a non-functional EGFR; and a chimeric antigen receptor is expressed, wherein the chimeric antigen receptor comprises: an extracellular region, and the extracellular region includes the weight of the single-chain antibody The chain variable region and the light chain variable region, the single-chain antibody specifically recognizes the antigen MSLN; the transmembrane region, the transmembrane region is linked to the extracellular region, and is embedded in the cell membrane of the T lymphocyte; the intracellular region, the intracellular region Linked to the transmembrane region, and the intracellular region includes the intracellular portion of CD28 or 4-1BB and the CD3 ⁇ chain.
  • Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and intact sequences that bind to anti-EGFR antibodies, and non-functional EGFR can act as lymphocytes. Suicide tag.
  • the T lymphocyte or the transgenic lymphocyte of the embodiment of the invention expresses the chimeric antigen receptor specific to the MSLN antigen, and the T lymphocyte or the transgenic lymphocyte of the embodiment of the invention has the killing ability against the specific tumor cell, especially has a high Specific killing of tumor cells expressing MSLN; T lymphocytes or transgenic lymphocyte cells of the present invention express non-functional EGFR in combination with a chimeric antigen receptor specific for MSLN antigen, T lymphocytes or transgenes of the present invention Lymphocyte killing is safe.
  • the non-functional EGFR of the present invention lacks an N-terminal ligand binding region and an intracellular receptor tyrosine kinase activity, but includes a transmembrane region of wild-type EGFR and an intact anti-antibody.
  • the sequence of EGFR antibody binding, non-functional EGFR can be used as a suicide marker for lymphocytes. Lymphocytes expressing non-functional EGFR can be cleared in vivo by anti-EGFR antibodies.
  • the T lymphocytes or transgenic lymphocytes of the embodiments of the present invention express non-functional EGFR, and under the premise of ensuring the targeted killing effect of the transgenic lymphocytes, if the patient has serious adverse reactions, the transgenic drenching
  • the ba cells can be cleared by the anti-EGFR antibody, which can further improve the safety of the transgenic lymphocytes or T lymphocytes of the embodiments of the present invention for treating tumor patients with high expression of MSLN.
  • 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, and further enhance the targeted killing effect of the transgenic lymphocytes on the targeted tumor cells.
  • the binding antigen of the above antibody is MSLN. Therefore, the transgenic lymphocytes of the embodiments of the present invention have a directional killing effect on the cells expressing the antigen MSLN, and the specific binding effect of the antigen-antibody is stronger, further improving the orientation of the transgenic lymphocytes of the present invention to the MSLN antigen-expressing tumor cells. Killing effect.
  • 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 has a positive regulation and enhances the cellular immune response, so that the effect of transgenic lymphocytes on the targeted killing of tumors with high expression of MSLN is further enhanced, and the expression of the intracellular segment of the immunostimulatory molecule is combined.
  • the expression of functional EGFR makes the immune killing effect of transgenic lymphocytes more safe and effective.
  • 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 above lymphocytes express non-functional EGFR and express chimeric antigen receptors, so that the lymphocyte immune killing effect on tumor cells is safer and more effective.
  • 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 of SEQ ID NO: 2; and a nucleic acid molecule encoding a non-functional EGFR having the amino acid sequence of SEQ ID NO: 3, which encodes a non-functional EGFR
  • the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:4.
  • a lentivirus or a construct of the embodiment of the present invention is introduced into a transgenic lymphocyte obtained from a lymphocyte, and a cell surface thereof expresses a non-functional EGFR, and a chimeric antigen receptor anti-MSLN is expressed on the cell surface thereof. Therefore, the transgenic lymphocytes of the embodiments of the present invention have significant directional killing ability against tumor cells, and the safety of the immunological killing is remarkably improved, and the specific killing effect of the transgenic lymphocytes of the embodiments of the present invention on the tumor cells with high expression of MSLN is particularly Significant.
  • the lentivirus or construct of the embodiment of the invention carries the SEQ ID NO: 5 Nucleotide sequence.
  • SEQ ID NO: 5 shows the nucleotide sequence (MSLN CAR/tEGFR) co-expressing the anti-MSLN chimeric antigen receptor and non-functional EGFR.
  • the transgenic lymphocytes obtained by introducing the lentivirus of the present invention into lymphocytes express the expression of non-functional EGFR and anti-MSLN chimeric antigen receptor, so that the transgenic lymphocytes have directional killing ability against tumors.
  • the immune killing safety is high, especially for tumor cells with high expression of MSLN, and the specific killing safety for tumor cells with high expression of MSLN is high.
  • the inventors realize that the above-described cell chimeric antigen receptor and non-functional EGFR are independently expressed by at least one of the following methods:
  • the internal ribosome entry site sequence of the present invention is set between a nucleic acid molecule encoding a chimeric antigen receptor and a nucleic acid molecule expressing a non-functional EGFR, and an internal ribosome entry site
  • the dot has the nucleotide sequence shown by SEQ ID NO: 6.
  • the internal ribosome entry site is usually located in the 5' untranslated region (UTR) of the RNA viral genome, so that the translation of one viral protein can be independent of the 5' cap structure, and the other protein usually initiates translation by the 5' hat structure.
  • the expression of the two genes before and after IRES is usually proportional.
  • an internal ribosome entry site sequence allows expression of a nucleic acid molecule encoding a chimeric antigen receptor independently of a nucleic acid molecule encoding a non-functional EGFR.
  • the internal ribosome entry site sequence effectively ensures the high expression of the chimeric antigen receptor and the non-functional EGFR, so that the specific killing effect of lymphocytes on the tumor is further improved, and the immune killing effect is further improved. Security is further improved.
  • a third nucleic acid molecule, a third nucleic acid molecule of an embodiment of the invention is disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a linker peptide that is capable of being cleaved in the lymphocyte.
  • the linker peptide has the amino acid sequence set forth in SEQ ID NO: 7.
  • the amino acid sequence shown by SEQ ID NO: 7 is the 2A peptide of Hand, Foot and Mouth Virus (a small RNA virus).
  • the introduction of the third nucleic acid molecule allows the non-functional EGFR and the chimeric antigen receptor to be expressed in a non-fusion state on the lymphocyte membrane, thereby ensuring the biological effects of the non-functional EGFR and the chimeric antigen receptor, and its specificity is more Strong tumor killing effect and higher safety.
  • a first promoter of an embodiment of the invention is operably linked to a nucleic acid molecule encoding a chimeric antigen receptor; and a second promoter is operably linked to a nucleic acid molecule expressing a non-functional EGFR.
  • the first promoter and the second promoter employed are independently selected from the group consisting of CMV, EF-1, LTR, RSV promoter, introduction of the first and second promoters such that the chimeric antigen is encoded
  • the nucleic acid molecule of the receptor and the nucleic acid molecule expressing the non-functional EGFR are independently expressed, thereby ensuring the high expression of the chimeric antigen receptor, the lymphocyte targeting effect is stronger, and the specific killing effect on the tumor is further improved, and the immunity is further enhanced. The safety of killing is further improved.
  • the cell-free functional EGFR is efficiently expressed and the chimeric antigen receptor is efficiently expressed on the transgenic lymphocyte membrane of the embodiment of the present invention, thereby ensuring The biological role of the chimeric antigen receptor effectively realizes the timely removal of transgenic lymphocytes, thereby making the targeted killing effect of lymphocytes more significant, and the safety of immune killing is further improved.
  • the vector of the construct of the embodiment of the present invention is a non-pathogenic viral vector.
  • the non-pathogenic viral vector greatly enhances the replication and amplification efficiency of the construct in lymphocytes, and further enhances the lymphocyte targeting effect of the embodiment of the invention, further enhances the killing effect on the tumor cells, and further improves the safety of the immune killing. improve.
  • 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, whereby the non-functional EGFR is highly expressed and the chimeric antigen receptor is highly expressed in lymphocytes, and the lymphocyte target of the present invention is targeted.
  • the effect is further enhanced, the killing effect on tumor cells is more significant, and the immune killing safety of lymphocytes is further improved.
  • the inventors in order to construct a lentiviral vector, the inventors inserted a nucleic acid of interest into a viral genome at a position of a certain viral sequence in order to construct a lentiviral vector, thereby producing a replication-defective virus.
  • the inventors further constructed packaging cell lines (containing the gag, pol and env genes, but excluding LTR and packaging components).
  • the inventors introduced a recombinant plasmid containing the gene of interest, together with the lentiviral LTR and the packaging sequence, into a packaging cell line.
  • the packaging sequence allows the recombinant plasmid RNA transcript to be packaged into viral particles which are then secreted into the culture medium.
  • the inventors collected a matrix containing the recombinant lentivirus, selectively concentrated, and used for gene transfer. Slow vectors can infect a variety of cell types, including cleavable cells and non-dividable cells.
  • the lentivirus of the embodiment of the present invention is a complex lentivirus, and in addition to the common lentiviral genes gag, pol and env, other genes having regulatory and structural functions are also included.
  • Lentiviral vectors are well known to those skilled in the art, and lentiviruses include: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors produce a biosafety vector by multiple attenuation of HIV-causing genes, such as deletion of the genes env, vif, vpr, vpu and nef.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and are useful for in vivo and in vitro gene transfer and nucleic acid sequence expression.
  • a suitable host cell together with two or more vectors with packaging functions (gag, pol, env, rev and tat), it is possible to infect non-dividing cells.
  • the targeting of recombinant viruses is achieved by binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
  • the targeting of the recombinant virus confers specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, along with another gene encoding a ligand for the receptor on the particular target cell.
  • a variety of useful lentiviral vectors, as well as vectors produced by various methods and procedures, are used to alter the expression of cells.
  • 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 a nucleotide molecule that co-expresses a chimeric antigen receptor and a non-functional EGFR.
  • 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.
  • the vectors of the embodiments of the present invention further include conventional control elements which permit transcription and transformation of mRNA expression in cell infection with the plasmid vector or/and cell infection together with the viral vector.
  • a large number of expression control sequences may be used.
  • the promoter is the RAN polymerase promoter of 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 the 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.
  • Techniques for constructing vectors are well known to those skilled in the art and include conventional cloning techniques such as the polymerase chain reaction used in the examples of the invention and any suitable method for providing the desired nucleotide sequence.
  • the inventors constructed a viral vector that co-expresses non-functional EGFR as well as a chimeric antigen receptor (CAR).
  • the nucleic acid molecule expressing the non-functional EGFR 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 the stability thereof, or Assist in its targeted movement.
  • the invention provides a method of preparing a T lymphocyte or a transgenic lymphocyte as described above.
  • the method comprises introducing the construct described above or the lentivirus described above into lymphocytes or T lymphocytes.
  • the mode of introduction can be introduced in a manner selected from the group consisting of electroporation or viral infection of host cells.
  • the construct or lentivirus of the embodiment of the present invention is successfully introduced into the above lymphocyte or T lymphocyte, and the expression of the chimeric antigen receptor against the antigen MSLN and the expression of the non-functional EGFR are realized, thereby causing the obtained lymphocyte or T lymphocyte. It has a targeted killing effect on tumor cells, especially tumor cells with high expression of MSLN, and the safety of immune killing is high.
  • the invention provides a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: the above construct, the above lentivirus, the above T lymphocyte or the above transgenic lymphocyte.
  • the composition of any of the above therapeutic compositions can achieve high expression of antigen-based MSLN chimeric antigen receptor in transgenic lymphocytes or T lymphocytes and non-functional EGFR in transgenic lymphocytes or T lymphocytes The expression of the surface, so that the obtained transgenic lymphocytes or T lymphocytes have a targeted killing effect on tumor cells which express MSLN high, and the safety of immune killing is high.
  • 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 a transgene of sufficient strength to express non-functional EGFR and express a unique MSLN chimeric antigen receptor.
  • the dosage of the therapeutic agent depends primarily on the condition of treatment, age, weight, and the health of the patient, which may result in patient variability.
  • These methods of expressing non-functional EGFR and expressing a unique receptor for the antigen MSLN chimeric antigen are part of a combination therapy.
  • These viral vectors and anti-tumor T cells for adoptive immunotherapy can be performed alone or in combination with other methods of treating cancer. Under appropriate conditions, one treatment involves the use of one or more drug therapies.
  • the cancer comprises at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.
  • the expression of non-functional EGFR is combined with the high expression of chimeric antigen receptor in transgenic lymphocytes or T lymphocytes, so that the obtained lymphocytes or T lymphocytes have a targeted killing effect on tumor cells, especially the above for high expression of MSLN.
  • the killing effect of tumor cells is more remarkable, and the immune killing effect on the above tumor cells with high expression of MSLN is safer and more effective.
  • the invention provides a method of increasing the safety of lymphocyte therapy, the lymphocyte carrying a chimeric antigen receptor, characterized in that the method comprises: expressing the lymphocyte without Functional EGFR, said non-functional EGFR, said lymphocyte, said chimeric antigen receptor as previously described.
  • Non-functional EGFR lacks N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of wild-type EGFR and intact sequences that bind to anti-EGFR antibodies, and non-functional EGFR can act as lymphocytes. Suicide tag.
  • lymphocytes of the embodiment of the present invention are used for treating tumor cells having high expression of MSLN, if the patient develops a serious adverse reaction, the lymphocytes of the embodiment of the present invention can be cleared by the anti-EGFR antibody, thereby further improving the lymphocytes of the embodiment of the present invention. Treatment of cancer patients with high expression of MSLN is safe.
  • the invention provides a method of treating cancer.
  • the method comprises: administering to a cancer patient a construct as described above, a lentivirus as described above, a T lymphocyte as described above or a transgenic lymphocyte as described above, wherein The antigen receptor specifically binds to the tumor antigen MSLN.
  • the method for treating cancer according to the embodiment of the invention can effectively achieve targeted killing of tumor cells of cancer patients, in particular, has targeted killing effect on tumor cells with high expression of MSLN, thereby effectively treating cancer, and the therapeutic effect is good and High security.
  • the method comprises: isolating lymphocytes from a cancer patient; introducing the aforementioned construct, or the lentivirus described above, into the lymphocytes to obtain transgenic lymphocytes,
  • the transgenic lymphocytes express a chimeric antigen receptor; and the transgenic lymphocytes are administered to the cancer patient.
  • the method for treating cancer according to the embodiment of the invention can further effectively achieve targeted killing of tumor cells of cancer patients, especially having targeted killing effect on tumor cells with high expression of MSLN, thereby further effectively treating cancer, and the therapeutic effect Good and safe.
  • the cancer includes at least one selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.
  • the method for treating cancer according to an embodiment of the present invention enables lymphocyte cells to express a chimeric antigen receptor, such as the MSLN antigen-specific chimeric antigen receptor of the present invention, and the obtained lymphocyte or T lymphocyte has specific expression to MSLN.
  • a chimeric antigen receptor such as the MSLN antigen-specific chimeric antigen receptor of the present invention
  • a lentiviral vector having a replication defect is produced, and the lentiviral vector is collected by centrifugation for transduction of human T lymphocytes.
  • the following is a brief introduction to the experimental procedure for the generation and collection of lentiviral vectors: 293T cells are plated in cell culture dishes with a bottom area of 150-cm 2 and using Express-In according to the instructions (purchased from Open Biosystems/Thermo Scientific, Waltham) , MA) Virus transduction of 293T cells.
  • lentiviral transgenic plasmid 15 ⁇ g of lentiviral transgenic plasmid, 5 ⁇ g of pVSV-G (VSV glycoprotein expression plasmid), 10 ⁇ g of pCMVR8.74 plasmid (Gag/Pol/Tat/Rev expression plasmid) and 174 pico of Express-In to each plate. (concentration is 1 ⁇ g / pico).
  • the supernatant was collected at 24 hours and 48 hours, respectively, and centrifuged for 2 hours using an ultracentrifuge at 28,000 rpm (the centrifuge rotor was Beckman SW 32Ti, available from Beckman Coulter, Brea, CA).
  • the virus plasmid was precipitated in 0.75 ml of RPMI-1640 medium. The line rehangs.
  • 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.
  • 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.
  • T lymphocytes at a planned time point can be used for functional analysis.
  • the flow cytometer used in the examples of the present application was BD FACSCanto II (purchased from BD Biosciences), and flow cytometric data was analyzed using FlowJo version 7.2.5 software (purchased from Tree Star, Ashland, OR).
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • the ability of anti-EGFR antibodies to induce cell-dependent lysis of lymphocytes expressing non-functional EGFR was assessed using the 4 hour- 51 Cr-release method.
  • Human T lymphocytes transduced with a lentiviral vector were used as target cells.
  • 100 ⁇ Ci Na 2 51 CrO 4 (available from GE Healthcare Life Sciences, Marlborough, MA) was used to calibrate 2 to 5 x 10 6 target cells under the conditions of shaking for 1 hour at 37 ° C with shaking. The cells were washed three times with PBS and resuspended in medium (cell density was 1 x 10 5 /ml).
  • the calibrated cells were then plated in 96-well plates (5 x 10 3 cells per well, plus 50 pico of medium) and 50 pf of anti-EGFR antibody (purchased from Erbitux, Genentech) ( The final concentration was 20 ⁇ g/ml, and preculture was carried out for 30 minutes under normal temperature conditions. Then, the medium containing the antibody was changed to a normal medium, thereby detecting the spontaneous release of 51 Cr. Triton X-100 was added to a final concentration of 1% to ensure maximum release of 51 Cr.
  • the human PBMC effector cells
  • % specific lysis (experimental release cpm data - spontaneous release of cpm data) / (maximum release cpm data - spontaneous release of cpm data) * 100, wherein the maximum release cpm data was added through the target cells
  • the spontaneous release of cpm data by Triton X-100 was measured in the absence of anti-EGFR antibodies and effector cells.
  • anti-MSLN CAR T lymphocytes The cytotoxic activity of anti-MSLN chimeric antigen receptor T cells (anti-MSLN CAR T lymphocytes) was evaluated in the Examples using a 4 - hour 51 chromium release assay. The specific steps are as follows: Target test cells were labeled with 51 Cr at 37 degrees Celsius for 1 hour. After labeling, the cells were rinsed with RPMI medium containing 10% fetal bovine serum (FCS). After rinsing, the cells were resuspended in the same medium, and the concentration of the resuspended cells was 1 ⁇ 10 5 /ml.
  • FCS fetal bovine serum
  • T cells were added to the target test cell suspension at different target cell ratios (E:T), and the cells were seeded in 96-wells at a volume of 200 picograms per well.
  • the cells were cultured for 4 hours in a 37 degree incubator. After 4 hours, 30 picofs of the supernatant was taken from each well and placed in a counter 96-well plate for counting analysis.
  • the analytical instrument was a top-level counting NXT micro-scintillator counter (purchased from Packard Bioscience). The number of effector cells in all counting wells was calculated based on the total number of T cells.
  • the target test cell to be labeled is MSLN + MSTO-211H (human pleural mesothelioma cells (from ATCC)).
  • the inventors cloned the sequence encoding the single-chain antibody against human MSLN, the 4-1BB intracellular domain and the T cell receptor combined ⁇ -strand sequence into a lentiviral vector containing the EF-1 promoter ( On the lentiviral vector), during the cloning process, the restriction enzyme digestion is the double digestion of XbaI and NotI, and the double digestion of NotI and XhoI, and the expression of anti-MSLN is generated by restriction enzyme digestion, ligation, screening and amplification of the plasmid of interest.
  • Anti-receptor lentiviral plasmid (LV-MSLN CAR).
  • Figure 1 is a schematic representation of a lentiviral vector comprising a sequence encoding an anti-MSLN chimeric antigen receptor, an IRES, and a coding non-functional EGFR sequence.
  • the sequence of the anti-MSLN chimeric antigen receptor is expressed under the initiation of the promoter EF-1, and the sequence expressing the non-functional EGFR is translated as a single mRNA transcription unit from the IRES sequence.
  • Anti-EGFR antibody effectively kills T lymphocytes that co-express non-functional EGFR and anti-MSLN chimeric antigen receptors
  • peripheral blood lymphocytes are taken from an unnamed blood donor. Peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. T lymphocytes were incubated with T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) for 72 hours at 5% CO 2 at 37 ° C. The medium was supplemented with 2 mmol/L glutamine, 10%. High temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U/ml penicillin/streptomycin double antibody in RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012).
  • FCS High temperature inactivated fetal calf serum
  • T cells were seeded on a recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were LV-MSLN CAR/tEGFR, LV-MSLN CAR or empty.
  • the loading (LV-GFP) transduction process is as described in Example 1. T cells expressing non-functional EGFR after transfection were stained with anti-EGFR antibody, and then separated by flow cytometry (FACS).
  • T cells were cultured in RPMI-1640 medium and recombinant human IL-2 factor (100 ng/ml). ; purchased from R&D Systems) for induction amplification for 7-10 days and then as target cells for the experiment.
  • the inventors measured the killing effect of anti-EGFR antibody-differentiated T cells transduced with different lentiviruses by ADCC assay using a standard 4 - hour 51 chromium release method, 4 - hour 51 chromium release method as in Example 1. Said. The result is shown in Figure 2.
  • the anti-EGFR antibody is effective to mediate the killing of T lymphocytes co-expressing the anti-MSLN chimeric antigen receptor and non-functional EGFR, but the anti-EGFR antibody cannot mediate killing and express only the anti-MSLN chimeric antigen receptor.
  • T lymphocytes, anti-EGFR antibodies were not able to mediate empty-loaded lentiviral transduced T lymphocytes, and the statistical data represented the mean ⁇ SEM of the three wells.
  • Example 4 Co-expressed T lymphocyte tumor cell lysis ability of non-functional EGFR and anti-MSLN chimeric antigen receptor.
  • peripheral blood lymphocytes are taken from an unnamed blood donor. Peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. T lymphocytes were incubated with T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) for 72 hours at 5% CO 2 at 37 ° C. The medium was supplemented with 2 mmol/L glutamine, 10%. High temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U/ml penicillin/streptomycin double antibody in RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012).
  • FCS High temperature inactivated fetal calf serum
  • T cells were seeded on a recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were LV-MSLN CAR, LV-tEGFR, or empty ( The 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 transduced with different lentiviruses on MSLN-positive MSTO-211H tumor cells.
  • the ratio of target cells was 50:1 or 25:1 or 1:1, and the standard method was 4–hour 51 chrome. Release method, 4 - hour 51 chromium release method as described in Example 1.
  • the results showed that co-expression of anti-MSLN chimeric antigen receptor and non-functional EGFR lentiviral transduced T lymphocytes (effector cells) killed MSLN positive MSTO-211H tumor cells (target cells).
  • Non-functional EGFR lentiviral transduced T lymphocytes (LV-tEGFR T lymphocytes) or empty lentiviral transduced T lymphocytes (control LV-GFP T lymphocytes) had no significant killing effect on MSLN positive tumor cells.

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Abstract

提供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，该转基因淋巴细胞表达无功能EGFR以及表达嵌合抗原受体，该嵌合抗原受体包括：胞外区，该胞外区包括单链抗体的重链可变区和轻链可变区，该单链抗体特异性识别抗原MSLN；跨膜区，该跨膜区与该胞外区相连，并且嵌入到该淋巴细胞的细胞膜中；胞内区，该胞内区与该跨膜区相连，并且该胞内区包括CD28或4-1BB的胞内段以及CD3ζ链。

Description

共表达抗MSLN嵌合抗原受体和无功能EGFR的转基因淋巴细胞及其用途
优先权信息
本申请请求2017年01月25日向中国国家知识产权局提交的、专利申请号为201710056385.7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。
技术领域
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和一种提高淋巴细胞治疗安全性的方法。
背景技术
间质素(mesothelin，MSLN)是一种分化抗原，它在人类正常组织的表达仅限于的胸膜、心包膜和腹膜衬里的间皮细胞。然而，间质素却在多种人类癌症组织中高表达，包括几乎所有的间皮瘤和胰腺癌和约70％的卵巢癌和约50％的肺腺癌以及其他癌症，例如胆管癌，胃癌，肠癌，食管癌，乳腺癌。间质素基因编码71KDa的前体蛋白，前体蛋白继而被加工成31KDa的脱落片段和40KDa的蛋白片段，31KDa的脱落片段被称为巨核细胞促进因子(MPF)，而40KDa的蛋白片段即被称为间质素，间质素通过糖基磷脂酰肌醇(glycosyl-phosphatidylinositol，GPI)的锚定作用固定在细胞膜上。
以间皮瘤为例，间皮瘤有胸膜间皮瘤和腹膜间皮瘤之分，胸膜间皮瘤是胸膜原发肿瘤，有局限型(多为良性)和弥漫型(都是恶性)之分，其中弥漫型恶性间皮瘤是胸部预后最坏的肿瘤之一。腹膜间皮瘤是指原发于腹膜间皮细胞的肿瘤。临床表现不具有特征性，常见的症状和体征有：腹痛、腹水、腹胀及腹部包块等。恶性胸膜间皮瘤的治疗，目前仍然没有有效的根治方法。治疗方法上，有姑息性治疗、外科治疗、化学治疗及放射治疗等，一般认为对于肿瘤相对局限的I期病人，主张做根治的胸膜肺切除术。对于Ⅱ、Ⅲ、Ⅳ期病人，根治性手术已经没有意义了，只有施行姑息性手术。事实上，多数病人到疾病明确诊断时，已处于II期以上。迅速增长的胸水常导致患者严重的呼吸困难，姑息性手术只能暂时提高这些晚期病人的生活质量，而无法根治。
由此可见，开发针对间质素高表达肿瘤的治疗方法尤为迫切。
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：
间质素却在多种人类癌症组织中高表达，包括几乎所有的间皮瘤和胰腺癌和约70％的卵巢癌和约50％的肺腺癌以及其他癌症，例如胆管癌，胃癌，肠癌，食管癌，乳腺癌。因此，间质素代表着肿瘤免疫治疗领域中一个有着巨大吸引力的靶点。
无功能EGFR(epidermal growth factor receptor，表皮生长因子受体)缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。
基于上述发现，发明人提出了一种编码无功能EGFR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MSLN。因此，本发明提出的构建体和转基因淋巴细胞可用于肿瘤，尤其是间质素阳性肿瘤的过继T细胞的免疫治疗；本发明所提出转基因淋巴细胞对高表达间质素肿瘤的特异杀伤能力强，对正常MSLN表达水平的间皮细胞具有较弱的杀伤，并且免疫杀伤安全性高。
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淋巴细胞表达无功能EGFR；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MSLN；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。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明实施例的T淋巴细胞具有对高表达MSLN的肿瘤细胞的定向杀伤作用，且安全性高。
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；以及编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：3所示的氨基酸序列，所述编码无功能EGFR的核酸分子具有SEQ ID NO：4所示的核苷酸序列。
Figure PCTCN2017081273-appb-000001
Figure PCTCN2017081273-appb-000002
Figure PCTCN2017081273-appb-000003
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，其具有对肿瘤细胞的特意性杀伤能力尤其具有对高表达MSLN的肿瘤细胞的定向杀伤作用，且安全性高。
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：5所示的核苷酸序列。
Figure PCTCN2017081273-appb-000004
Figure PCTCN2017081273-appb-000005
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，具有对肿瘤细胞的定向杀伤能力，尤其具有对高表达MSLN的肿瘤细胞的定向杀伤作用，并且安全性高。
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，所述淋巴细胞细胞表达无功能EGFR；以及表达嵌合抗原受体，所述嵌合抗原受体包括：胞外区，所述胞外区包括抗体的重链可变区和轻链可变区，所述抗体能够与肿瘤抗原特异性结合；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，其中，所述抗体为单链抗体，所述肿瘤抗原为MSLN。发明人惊奇的发现，表达无功能EGFR和表达嵌合抗原受体 的淋巴细胞具有对肿瘤细胞的特异性杀伤能力，尤其具有对高表达MSLN的肿瘤细胞的定向杀伤作用，且安全性高。
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对肿瘤的定向杀伤作用效果进一步提高；本发明实施例的免疫共刺激分子胞内段的表达以及无功能EGFR的表达的联合，使得本发明实施例的转基因淋巴细胞增殖对肿瘤的具有更加显著的定向杀伤作用，且更加安全。
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，本发明实施例的转基因淋巴细胞表达的无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR结合的结构域，无功能EGFR可作为本发明实施例的转基因淋巴细胞的自杀标记。无功能EGFR的表达，联合嵌合抗原受体的表达可在有效保证转基因淋巴细胞的靶向杀伤作用的前提下，如果病人出现严重不良反应，转基因淋巴细胞可被抗EGFR抗体清除，进而可进一步提高本发明实施例的转基因淋巴细胞治疗高表达MSLN的肿瘤病人的安全性。
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表达无功能EGFR，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MSLN抗原特异性的嵌合抗原受体，上述淋巴细胞具有对肿瘤的定向杀伤作用，且安全性更高。
在本发明的第五方面，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，所述第二核酸分子编码无功能EGFR。其中，所述嵌合抗原受体、所述无功能EGFR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，可高效表达无功能EGFR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具有对肿瘤细胞，尤其对高表达MSLN的肿瘤细胞的具有定向杀伤作用，且安全性高。
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表达所述嵌合抗原受体和表达无功能EGFR，并且所述嵌合抗原受体 与所述无功能EGFR呈非融合形式。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，其淋巴细胞表面成功表达无功能EGFR，同时在淋巴细胞表面成功表达了抗原特异性嵌合抗原受体，如本发明实施例的MSLN特异性的嵌合抗原受体，且嵌合抗原受体与无功能EGFR在淋巴细胞膜上呈非融合形式，本发明实施例的淋巴细胞具有特异性强的肿瘤杀伤效果，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表达了无功能EGFR，从而有效保证了本发明实施例的淋巴细胞的对肿瘤的靶向杀伤作用，尤其是对高表达MSLN的肿瘤细胞的定向杀伤，且保证了免疫杀伤的安全性。
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，从而保证了无功能EGFR的高效表达和嵌合抗原受体的高效表达，从而高效保证了本发明实施例的淋巴细胞对肿瘤的定向杀伤效果和杀伤安全性。
根据本发明的实施例，所述构建体进一步包括：内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第三核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：6所示的核苷酸序列。
Figure PCTCN2017081273-appb-000006
内部核糖体进入位点序列的引入使得第一核酸分子和第二核酸分子分别独立的表达。根据本发明的实施例，内部核糖体进入位点序列的引入保证了嵌合抗原受体抗原靶向性的生物学作用和无功能EGFR的高效表达，进而使得本发明实施例的淋巴细胞对肿瘤的定向杀 伤效果更加显著，淋巴细胞对肿瘤杀伤的安全性更高。
根据本发明的实施例，所述构建体进一步包括：第三核酸分子，设置在所述第一核酸分子与所述第二核酸分子之间，并且所述第三核酸分子编码连接肽，所述连接肽能够在所述淋巴细胞中被切割。编码连接肽的第三核酸分子的引入使得无功能EGFR和嵌合抗原受体成非融合状态的形式表达在淋巴细胞膜上，进而进一步保证了无功能EGFR和嵌合抗原受体的生物学作用，本发明实施例的淋巴细胞具有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，所述连接肽具有SEQ ID NO：7所示的氨基酸序列。
GSGATNFSLLKQAGDVEENPGP(SEQ ID NO：7)。
SEQ ID NO：7所示的氨基酸序列是手足口病毒(一种小RNA病毒)的2A肽段。连接肽的引入使得无功能EGFR和嵌合抗原受体成非融合状态表达在淋巴细胞膜上。根据本发明的实施例，连接肽的引入保证了无功能EGFR和嵌合抗原受体的生物学作用，本发明实施例的淋巴细胞具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，从而大大提高了无功能EGFR的表达和嵌合抗原受体在淋巴细胞中的高效表达，使得淋巴细胞的靶向作用进一步增强，安全性进一步提高。
根据本发明的实施例，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，无功能EGFR在淋巴细胞中高效表达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靶向作用进一步增强，对肿瘤细胞，尤其是高表达MSLN的肿瘤细胞的定向杀伤作用更加显著，淋巴细胞的杀伤作用安全性更高。
在本发明的第六方面，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，实现了淋巴细胞表达无功能EGFR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淋巴细胞具有对肿瘤细胞，尤其具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，且安全性更高。
在本发明的第七方面，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淋巴细胞无功能EGFR的表达和 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淋巴细胞具有对肿瘤细胞的靶向杀伤作用，本发明实施例的治疗癌症的治疗组合物具有对肿瘤细胞的靶向杀伤作用，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，且安全性高。
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，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。上述肿瘤细胞具有MSLN的特异性高表达，本发明实施例的治疗组合物可使淋巴细胞细胞表面表达无功能EGFR和高效表达抗原特异性嵌合抗原受体，如本发明实施例的MSLN抗原特异性嵌合抗原受体，所得淋巴细胞或T淋巴细胞具有对高表达MSLN的肿瘤细胞的靶向杀伤，且安全性高。
在本发明的第八方面，本发明提出了一种提高淋巴细胞治疗安全性的方法，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：使所述淋巴细胞表达无功能EGFR，所述无功能EGFR、所述淋巴细胞、所述嵌合抗原受体如前所述。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明实施例的淋巴细胞在用于治疗治疗高表达MSLN的肿瘤细胞时，如果病人出现严重不良反应，本发明实施例的淋巴细胞可被抗EGFR抗体清除，进而可提高本发明实施例的淋巴细胞治疗高表达MSLN的肿瘤病人的安全性。
在本发明的第九方面，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MSLN。利用本发明实施例的治疗癌症的方法，可有效实现对癌症患者肿瘤细胞的靶向杀伤，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，进而能够有效治疗癌症，治疗效果好且安全性高。
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表达嵌合抗原受体；以及为所述癌症患者给药所述转基因淋巴细胞。利用本发明实施例的治疗癌症的方法，可进一步有效实现对癌症患者肿瘤细胞的靶向杀伤，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，进而能够进一步有效治疗癌症，治疗效果好且安全性高。
根据本发明的实施例，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。本发明实施例的治疗癌症的方法可使淋巴细胞细胞表达嵌合抗原受体，如本发明实施例的MSLN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MSLN特异性表达的间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌或乳腺癌的肿瘤细胞的靶向杀伤。
附图说明
图1是根据本发明实施例的共表达抗MSLN的嵌合抗原受体和无功能EGFR的慢病毒载体的结构示意图；以及
图2是根据本发明实施例的共表达抗MSLN的嵌合抗原受体和无功能EGFR的淋巴细胞被抗EGFR抗体-介导ADCC杀伤清除的结果图。
具体实施方式
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淋巴细胞表达无功能EGFR；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MSLN；跨膜区，跨膜区与胞外区相连，并且嵌入到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。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明实施例的T淋巴细胞或转基因淋巴细胞表达MSLN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具有对特异性肿瘤细胞的杀伤能力，尤其具有对高表达MSLN的肿瘤细胞的特异性杀伤；本发明实施例的T淋巴细胞或转基因淋巴细胞细胞表达无功能EGFR联合表达MSLN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杀伤安全性高。
另外，根据本发明的实施例，本发明实施例的无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。表达无功能EGFR的淋巴细胞可被抗EGFR抗体在体内清除。从而，本发明实施例的T淋巴细胞或转基因淋巴细胞表达无功能EGFR，在保证转基因淋巴细胞的靶向杀伤作用的前提下，如果病人出现严重不良反应，转基因淋 巴细胞可被抗EGFR抗体清除，进而可进一步提高本发明实施例的转基因淋巴细胞或T淋巴细胞治疗高表达MSLN的肿瘤病人的安全性。
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，进一步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MSLN。因此本发明实施例的转基因淋巴细胞针对表达抗原MSLN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，进一步提高了本发明实施例的转基因淋巴细胞对MSLN抗原表达肿瘤细胞的定向杀伤作用。
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对高表达MSLN的肿瘤的定向杀伤作用效果进一步提高，免疫共刺激分子胞内段的表达联合无功能EGFR的表达，使得转基因淋巴细胞的免疫杀伤作用更加安全有效。
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。上述淋巴细胞表达无功能EGFR和表达嵌合抗原受体，使得上述淋巴细胞的对肿瘤细胞免疫杀伤作用更加安全有效。
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；以及编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：3所示的氨基酸序列，所述编码无功能EGFR的核酸分子具有SEQ ID NO：4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，其细胞表面表达无功能EGFR，同时在其细胞表面表达抗MSLN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对肿瘤细胞的定向杀伤能力，且免疫杀伤安全性显著提高，本发明实施例的转基因淋巴细胞对高表达MSLN的肿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：5所示的 核苷酸序列。SEQ ID NO：5表示的是共表达抗MSLN嵌合抗原受体和无功能EGFR的核苷酸序列(MSLN CAR/tEGFR)。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，其表达无功能EGFR以及抗MSLN的嵌合抗原受体表达，使得转基因淋巴细胞对肿瘤的定向杀伤能力，免疫杀伤安全性高，尤其对高表达MSLN的肿瘤细胞具有特异性杀伤效果，对高表达MSLN的肿瘤细胞的特异性杀伤安全性高。
根据本发明的实施例，发明人是通过如下方式的至少之一实现上述细胞嵌合抗原受体以及无功能EGFR分别独立地表达的：。
内部核糖体进入位点序列(IRES)，本发明实施例的内部核糖体进入位点序列设置在编码嵌合抗原受体的核酸分子与表达无功能EGFR的核酸分子之间，内部核糖体进入位点具有SEQ ID NO：6所示的核苷酸序列。内部核糖体进入位点通常位于RNA病毒基因组的5’非翻译区(UTR)，这样一个病毒蛋白的翻译就可以不依赖于5’帽子结构，另一个蛋白通常靠5’帽子结构起始翻译，IRES前后的两个基因的表达通常是成比例的。内部核糖体进入位点序列的引入使得编码嵌合抗原受体的核酸分子与编码无功能EGFR的核酸分子分别独立的表达。根据本发明的实施例，本发明实施例采用内部核糖体进入位点序列有效保证了嵌合抗原受体和无功能EGFR的高效表达，使得淋巴细胞对肿瘤的特异性杀伤效果进一步提高，免疫杀伤安全性进一步提高。
第三核酸分子，本发明实施例的第三核酸分子设置在第一核酸分子与第二核酸分子之间，并且第三核酸分子编码连接肽，连接肽能够在淋巴细胞中被切割。根据本发明的实施例，连接肽具有SEQ ID NO：7所示的氨基酸序列。SEQ ID NO：7所示的氨基酸序列是手足口病毒(一种小RNA病毒)的2A肽段。第三核酸分子的引入使得无功能性EGFR和嵌合抗原受体成非融合状态表达在淋巴细胞膜上，从而保证了的无功能EGFR和嵌合抗原受体的生物学作用，其具特异性更强的肿瘤杀伤效果，安全性更高。
启动子：本发明实施例的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；以及第二启动子与表达无功能EGFR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、第二启动子独立地选自CMV，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表达无功能EGFR的核酸分子分别独立的表达，从而保证了嵌合抗原受体的高效表达，淋巴细胞的靶向作用更强，对肿瘤的特异性杀伤作用进一步提高，免疫杀伤的安全性也进一步提高。
通过上述内部核糖体进入位点序列或第一、第二启动子的引入，使得细胞无功能EGFR高效地表达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，从而保证了嵌合抗原受体的生物学作用，有效实现了转基因淋巴细胞的及时清除，从而使得淋巴细胞的靶向杀伤作用更加显著，免疫杀伤的安全性进一步提高。
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靶向作用进一步增强，对肿瘤细胞的杀伤作用进一步提高，免疫杀伤安全性进一步提高。
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，从而无功能EGFR被高效表达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靶向作用进一步增强，对肿瘤细胞的杀伤作用更加显著，淋巴细胞的免疫杀伤安全性进一步提高。
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，以此携带共表达嵌合抗原受体和无功能EGFR的核苷酸分子。
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mRNA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，启动子为选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无功能EGFR以及嵌合抗原受体(CAR)的病毒载体。本发明实施例的表达无功能EGFR的核酸分子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MSLN的嵌合抗原受体的表达和无功能EGFR的表达，从而使得所得淋巴细胞或T淋巴细胞具有对肿瘤细胞，尤其是高表达MSLN的肿瘤细胞的靶向杀伤作用，免疫杀伤的安全性高。
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MSLN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无功能EGFR在转基因淋巴细胞或T淋巴细胞表 面的表达，从而使得所得转基因淋巴细胞或T淋巴细胞具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，免疫杀伤的安全性高。
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，表达无功能EGFR和表达特有的MSLN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。
表达无功能EGFR以及表达特有的针对抗原MSLN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，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。无功能EGFR的表达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淋巴细胞具有对肿瘤细胞的靶向杀伤作用，尤其具有对高表达MSLN的上述肿瘤细胞的杀伤作用更加显著，对高表达MSLN的上述肿瘤细胞的免疫杀伤作用更加安全有效。
提高淋巴细胞治疗安全性的方法
在本发明的另一方面，本发明提出了一种提高淋巴细胞治疗安全性的方法，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：使所述淋巴细胞表达无功能EGFR，所述无功能EGFR、所述淋巴细胞、所述嵌合抗原受体如前所述。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明实施例的淋巴细胞在用于治疗高表达MSLN的肿瘤细胞时，如果病人出现严重不良反应，本发明实施例的淋巴细胞可被抗EGFR抗体清除，进而可提高本发明实施例的淋巴细胞治疗高表达MSLN的肿瘤病人的安全性。
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，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MSLN。利 用本发明实施例的治疗癌症的方法，可有效实现对癌症患者肿瘤细胞的靶向杀伤，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，进而能够有效治疗癌症，治疗效果好且安全性高。
根据本发明的具体实施例，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表达嵌合抗原受体；以及为所述癌症患者给药所述转基因淋巴细胞。利用本发明实施例的治疗癌症的方法，可进一步有效实现对癌症患者肿瘤细胞的靶向杀伤，尤其是具有对高表达MSLN的肿瘤细胞的靶向杀伤作用，进而能够进一步有效治疗癌症，治疗效果好且安全性高。
具体地，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。本发明实施例的治疗癌症的方法可使淋巴细胞细胞表达嵌合抗原受体，如本发明实施例的MSLN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MSLN特异性表达的间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌或乳腺癌的肿瘤细胞的靶向杀伤。
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15微克的慢病毒转基因质粒、5μg的pVSV-G(VSV糖蛋白表达质粒)、10微克的pCMVR8.74质粒(Gag/Pol/Tat/Rev表达质粒)和174微微的Express-In(浓度为1微克/微微)。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8微克/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的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。
抗体依赖性细胞介导的细胞毒作用(ADCC)
在有关ADCC实施例中，采用4小时-51Cr-释放法评估抗-EGFR抗体诱导表达无功能EGFR的淋巴细胞的细胞依赖性裂解的能力。被转导了慢病毒载体的人类T淋巴细胞被用作靶细胞。100μCi Na2 51CrO4(购自GE Healthcare Life Sciences，Marlborough，MA)标定2～5x 106靶细胞，标定条件是37℃下震荡孵育1小时。细胞采用PBS润洗三次，并且用培养基重悬(细胞密度是1x 105/ml)。继而，被标定的细胞铺在96-孔板中(每孔铺有5×103个细胞，加有50微微培养基)，并加入50微微的抗-EGFR抗体(购自Erbitux，Genentech)(终浓度为20微克/ml)，在常温条件下预培养30分钟.继而将含有抗体的培养基换成普通培养基，由此来检测51Cr的自发释放。加入终浓度为1％的Triton X-100以保证51Cr的最大释放量。在以下有关ADCC实施中，人PBMC(效应细胞)加入孔板中(每孔5×105个细胞)并将细胞在37℃培养过夜。第二天，收集细胞上清，并利用γ计数器计算cpm以此来确定51Cr的释放。细胞毒性比例用以下公式计算：％特异性裂解＝(实验释放cpm数据-自发释放cpm数据)/(最大释放cpm数据-自发释放cpm数据)*100，其中，最大释放cpm数据通过靶细胞中加入Triton X-100实现的，自发释放cpm数据是在没有抗EGFR抗体和效应细胞的条件下测量的。
铬释放实验
实施例中应用4–小时51铬释放法分析评估抗MSLN嵌合抗原受体T细胞(抗MSLN 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微微。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微微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MSLN+MSTO-211H(人胸膜间皮瘤细胞，human pleural mesothelioma cells(来自ATCC))。
实施例2构建共表达无功能EGFR和抗MSLN嵌合抗原受体的载体
本实施例中，发明人将编码有抗人MSLN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MSLN嵌合抗原受体的慢病毒质粒(LV-MSLN CAR)。包含合成IRES和表达无功能EGFR的序列被克隆进LV-MSLN CAR载体质粒，构建成LV-MSLN CAR/tEGFR。图1是慢病毒载体的示意图，包含编码抗MSLN嵌合抗原受体的序列，IRES、及编码无功能EGFR序列。抗MSLN嵌合抗原受体的序列在启动子EF-1的启动调控下，表达无功能EGFR的序列作为一个单独的mRNA转录单元从IRES序列后开始翻译。
实施例3抗EGFR抗体可有效杀伤清除共表达无功能EGFR和抗MSLN嵌合抗原受体的T淋巴细胞
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-MSLN CAR/tEGFR，LV-MSLN CAR或空载(LV-GFP)转导过程如实施例1所述。转导后表达无功能EGFR的T细胞用抗EGFR抗体染色后，然后流式细胞细胞(FACS)分离，分离后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，然后作为实验的靶细胞。发明人用ADCC检测法测量抗EGFR抗体介异的对转导了不同慢病毒的T细胞的杀伤作用，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2所示。如图2所示，抗EGFR抗体可有效介异杀伤共表达抗MSLN嵌合抗原受体和无功能EGFR的T淋巴细胞，但抗EGFR抗体不能介异杀伤只表达抗MSLN嵌合抗原受体的T淋巴细胞，抗EGFR抗体不能介异杀伤空载慢病毒转导的T淋巴细胞，统计数据代表三个孔的平均值±SEM。
实施例4共表达无功能EGFR和抗MSLN嵌合抗原受体的T淋巴细胞肿瘤细胞溶解能力。
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-MSLN CAR，LV-tEGFR，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细胞对MSLN阳性MSTO-211H肿瘤细胞的杀伤作用，效靶细胞比例是50：1或25:1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。结果显示，共表达抗MSLN嵌合抗原受体和无功能EGFR慢病毒转导的T淋巴细胞(效应细胞)可杀死MSLN阳性MSTO-211H肿瘤细胞(靶细胞)。无功能EGFR慢病毒转导的T淋巴细胞(LV-tEGFR T淋巴细胞)或空载慢病毒转导的T淋巴细胞(对照LV-GFP T淋巴细胞)对MSLN阳性肿瘤细胞无明显杀伤作用。
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 (25)

  1. 一种T淋巴细胞，其特征在于，所述T淋巴细胞表达无功能EGFR；
    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MSLN；
    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。
  2. 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
    编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：3所示的氨基酸序列，所述编码无功能EGFR的核酸分子具有SEQ ID NO：4所示的核苷酸序列。
  3. 一种慢病毒，其特征在于，所述慢病毒携带含有SEQ ID NO：5所示的核苷酸序列。
  4. 一种转基因淋巴细胞，其特征在于，所述淋巴细胞细胞表达无功能EGFR；以及表达嵌合抗原受体，所述嵌合抗原受体包括：
    胞外区，所述胞外区包括抗体的重链可变区和轻链可变区，所述抗体能够与肿瘤抗原特异性结合；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，
    其中，所述抗体为单链抗体，所述肿瘤抗原为MSLN。
  5. 根据权利要求4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。
  6. 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是4-1BB或CD28的胞内段。
  7. 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。
  8. 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。
  9. 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。
  10. 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，所述第二核酸分子编码无功能EGFR，
    其中，所述嵌合抗原受体、所述无功能EGFR是如权利要求2、4～9任一项中所定义的。
  11. 根据权利要求10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4～9任一项所述的淋巴细胞中表达所述嵌合抗原受体和表达无功能EGFR，并且所述嵌合抗原受体与所述无功能EGFR呈非融合形式。
  12. 根据权利要求10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。
  13. 根据权利要求12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CMV，EF-1，LTR或RSV启动子。
  14. 根据权利要求10所述的构建体，其特征在于，进一步包括：
    内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：6所示的核苷酸序列。
  15. 根据权利要求10所述的构建体，其特征在于，进一步包括:
    第三核酸分子，设置在所述第一核酸分子与所述第二核酸分子之间，并且所述第三核酸分子编码连接肽，所述连接肽能够在所述淋巴细胞中被切割。
  16. 根据权利要求15所述的构建体，其特征在于，所述连接肽具有SEQ ID NO：7所示的氨基酸序列。
  17. 根据权利要求10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。
  18. 根据权利要求17所述的构建体，其特征在于，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。
  19. 一种制备权利要求1所述的T淋巴细胞或者权利要求4～9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10～18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中或者T淋巴细胞。
  20. 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10～18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4～9任一项所述的转基因淋巴细胞。
  21. 根据权利要求20所述的治疗组合物，其特征在于，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。
  22. 一种提高淋巴细胞治疗安全性的方法，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：
    使所述淋巴细胞表达无功能EGFR，
    所述无功能EGFR、所述淋巴细胞、所述嵌合抗原受体如权利要求2、4～9任一项中所定义的。
  23. 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10～18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4～9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MSLN。
  24. 根据权利要求23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10～18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表达嵌合抗原受体；以及
    为所述癌症患者给药所述转基因淋巴细胞。
  25. 根据权利要求24所述的方法，其特征在于，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。
PCT/CN2017/081273 2017-01-25 2017-04-20 共表达抗msln嵌合抗原受体和无功能egfr的转基因淋巴细胞及其用途 WO2018137294A1 (zh)

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