WO2018006880A1 - 重组免疫检查点受体及免疫检查点抑制分子的共表达及应用 - Google Patents

重组免疫检查点受体及免疫检查点抑制分子的共表达及应用 Download PDF

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WO2018006880A1
WO2018006880A1 PCT/CN2017/092376 CN2017092376W WO2018006880A1 WO 2018006880 A1 WO2018006880 A1 WO 2018006880A1 CN 2017092376 W CN2017092376 W CN 2017092376W WO 2018006880 A1 WO2018006880 A1 WO 2018006880A1
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nucleic acid
acid molecule
lymphocytes
recombinant receptor
lymphocyte
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French (fr)
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陈思毅
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生命序有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to co-expression and application of recombinant immunological checkpoint receptors and immunological checkpoint inhibitory molecules, and more particularly, the present invention relates to recombinant receptors, nucleic acids, lentiviruses, transgenic lymphocytes , constructs, methods of preparing transgenic lymphocytes, therapeutic compositions for treating cancer, and methods of increasing lymphocyte immune killing ability.
  • Targeted therapies mainly include monoclonal antibodies (sometimes classified as passive cell transfusion and tumor vaccines, immunotherapy through the immune system of the motivational body, enhanced tumor microenvironment anti-tumor immunotherapy) and small molecule targeted drugs, while immunotherapy It mainly includes cytokine therapy, immunoassay monoclonal antibody, and adoptive immunotherapy to control and kill tumor cells. Therefore, it has the advantages of high efficiency, high specificity and good tolerance, and has broad prospects in cancer therapy.
  • the present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, it is an object of the present invention to provide a recombinant receptor and a method for effectively enhancing lymphocyte immune killing of tumor cells.
  • the invention proposes a recombinant receptor.
  • the recombinant receptor comprises: a cellular immune checkpoint molecule fragment; an immunostimulatory molecule fragment; and a T cell receptor zeta chain.
  • the lymphocytes express the recombinant receptor of the embodiment of the invention, and the specific killing effect of the lymphocytes on the tumor cells can be effectively enhanced.
  • the above recombinant receptor may further comprise at least one of the following additional technical features:
  • the cellular immune checkpoint molecule is PD1.
  • PD1 binds to PD-L1 or PD-L2 specifically expressed on tumor cells.
  • lymphocytes express the recombinant receptor of the present invention, which is further enhanced in targeting killing of tumor cells, particularly PD-L1 or PD-L2 positive tumor cells.
  • the cellular immune checkpoint molecule fragment comprises an extracellular region of the PD1 and optionally a transmembrane region, the immunostimulatory molecule fragment comprising an intracellular region of CD28 and optionally a transmembrane region .
  • the extracellular domain of PD1 has a functional region that binds to PD-L1 or PD-L2 specifically expressed on tumor cells
  • the intracellular region of CD28 has a functional region that activates an immunostimulatory signaling pathway, and thus lymphocyte cell expression is implemented by the present invention.
  • the recombinant receptor has a further enhanced killing effect on tumor cells.
  • the recombinant receptor comprises: (a) an extracellular region and a transmembrane region of the PD1; and (b) an intracellular region of the CD28, or comprising: (i) the PD1 The extracellular region; and (ii) the intracellular and transmembrane regions of the CD28.
  • Both combinations retain the functional domain of PD1 binding to PD-L1 or PD-L2 specifically expressed on tumor cells and the functional domain of CD28-activated immunostimulatory signaling pathway, whether PD1 transmembrane or CD28 In the transmembrane region, the recombinant receptor can be expressed across the membrane, and the lymphocyte cells express the recombinant receptor of the present invention, and the targeted killing effect on the tumor cells is further improved.
  • the T cell receptor zeta chain is a CD3zeta chain.
  • the CD3zeta chain can specifically activate the downstream T cell receptor signaling pathway, and the lymphocyte cells express the recombinant receptor of the embodiment of the present invention, and the killing effect on the tumor cells is further improved.
  • the C-terminus of the cellular immunological checkpoint molecule fragment is linked to the N-terminus of the immunostimulatory molecule fragment, and the C-terminus of the immunostimulatory molecule fragment and the N of the T cell receptor zeta chain Connected to the end.
  • the relevant fragment of the recombinant receptor of the present invention facilitates the localization of the relevant fragment in the cell, thereby facilitating the corresponding functions-targeting, transmembrane, activation of the immune stimulation signaling pathway, and activation of T.
  • the cell receptor signaling pathway further enhances the targeted killing ability of tumor cells.
  • the invention proposes a recombinant receptor.
  • the recombinant receptor has the amino acid sequence set forth in SEQ ID NO: 1 or 2.
  • lymphocytes expressing the recombinant receptor of the present invention can effectively enhance the specific killing effect of lymphocytes on tumor cells, particularly PD-L1 or PD-L2 positive tumor cells.
  • the invention proposes a nucleic acid.
  • the nucleic acid encodes a recombinant receptor as described above, optionally, the nucleic acid has the nucleotide sequence set forth in SEQ ID NO: 3 or 4.
  • the nucleic acid of the embodiment of the present invention is introduced into the recipient lymphocytes, and the recombinant receptor encoded by the nucleic acid is transmembranely expressed in lymphocytes, and the specific killing effect of the lymphocytes on the tumor cells is remarkably improved.
  • the invention proposes a lentivirus.
  • the lentivirus carries the following nucleic acid molecule: (a) a nucleic acid molecule encoding a recombinant receptor as described above, the recombinant receptor having the amino acid sequence set forth in SEQ ID NO: 1 or 2,
  • the nucleic acid molecule encoding the recombinant receptor has the nucleotide sequence shown in SEQ ID NO: 3 or 4;
  • the nucleic acid molecule of the silencing cell immunological checkpoint the nucleotide of the nucleic acid molecule of the silencing cell immunological checkpoint
  • the sequence is at least one selected from the group consisting of SEQ ID NOS: 5-137.
  • the lentivirus of the embodiment of the present invention is introduced into the recipient lymphocyte, and the recombinant receptor described above is transmembranely expressed in the lymphocyte, and the immunological checkpoint molecule on the surface of the lymphocyte membrane is specifically silenced, thereby playing in the recombinant receptor. While enhancing the specific killing function of lymphocytes on tumor cells, the immunosuppressive mechanism is also suppressed.
  • the lentivirus of the embodiment of the present invention is introduced into the recipient lymphocytes, and the specific killing of the lymphocytes by the lymphocytes is strong and effective.
  • the lentivirus may further include at least one of the following additional technical features:
  • the lentivirus may further carry a nucleic acid molecule encoding a non-functional EGFR having the amino acid sequence of SEQ ID NO: 138, the nucleic acid molecule encoding the non-functional EGFR having SEQ ID NO: The nucleotide sequence shown by 139.
  • the non-functional EGFR receptor lacks an N-terminal ligand binding region and intracellular receptor tyrosine kinase activity, but includes a transmembrane region of the wild type EGFR receptor and is fully integrated with an anti-EGFR antibody.
  • the sequence, so the non-functional EGFR receptor can act as a suicide marker for lymphocytes.
  • the lentivirus is introduced into the recipient lymphocytes, and the expression of the non-functional EGFR receptor can effectively ensure the targeted killing effect of the lymphocytes on the tumor cells, and if the patient has serious adverse reactions, the lymphocytes can be It is cleared by the anti-EGFR antibody, thereby improving the safety of the patients with tumors such as lentivirus and lymphocytes in the examples of the present invention.
  • the invention proposes a lentivirus.
  • the lentivirus carries a nucleotide sequence as set forth in SEQ ID NOs: 140-156.
  • a nucleic acid expressing the aforementioned recombinant receptor, a silent immunological checkpoint, and optionally a non-functional EGFR can be introduced into a recipient lymphocyte, thereby realizing the aforementioned recombinant receptor,
  • the invention provides a transgenic lymphocyte.
  • the cellular immune checkpoint of the transgenic lymphocytes is silenced, and the transgenic lymphocytes express the aforementioned recombinant receptor, optionally, the transgenic lymphocytes further express non-functional EGFR.
  • the specific killing of the transgenic lymphocytes of the embodiments of the present invention on tumor cells, especially PD-L1 or PD-L2 positive tumor cells, is strong, effective, and safe.
  • the transgenic lymphocyte may further include at least one of the following additional technical features:
  • the lymphocyte immune checkpoint is independently selected from at least one of CTLA4, PD1, TIM3, BTLA, LAG-3, IRAK-M, SOCS1, A20, CBL-B, preferably,
  • the lymphocyte immune checkpoint is CTLA4, PD1, SOCS1 or CBL-B.
  • the above-mentioned immunological checkpoint molecule exerts an immunosuppressive mechanism in lymphocytes, and the specific silencing of the above-mentioned immunological checkpoint molecule can effectively suppress the immunosuppressive mechanism, and further enhance the specific killing of tumor cells by the transgenic lymphocytes of the embodiments of the present invention. effect.
  • the silencing of the lymphocyte immune checkpoint is achieved by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.
  • shRNA antisense nucleic acid
  • ribozyme dominant negative mutation
  • CRISPR-Cas9 CRISPR-Cpf1
  • zinc finger nuclease zinc finger nuclease.
  • silencing of the lymphocyte immune checkpoint is achieved by shRNA.
  • Silencing lymphocyte cellular immune checkpoints by shRNA is a simple and effective way.
  • the lymphocytes are antigen-specific T lymphocytes, optionally, the lymphocytes are tumor infiltrating T lymphocytes, optionally, the lymphocytes are peripheral blood T lymphocytes, Optionally, the lymphocytes are natural killer T lymphocytes, optionally the lymphocytes are natural killer cells.
  • the antigen-specific T lymphocytes, the tumor infiltrating T lymphocytes, the natural killer T lymphocytes or the natural killer cells according to the embodiments of the present invention can achieve specific immune killing on tumor cells, and has strong specificity, strong killing power and safety. high.
  • the invention proposes a construct.
  • the construct comprises: a first nucleic acid molecule encoding a recombinant receptor as described above; a second nucleic acid molecule, the second nucleic acid molecule silencing a cellular immune checkpoint; And optionally, the construct further comprises: a third nucleic acid molecule encoding a non-functional EGFR, wherein the cellular immune checkpoint, the non-functional EGFR is as defined above.
  • a nucleic acid expressing the aforementioned recombinant receptor, a silent immunological checkpoint, and optionally a non-functional EGFR can be introduced into a recipient lymphocyte, thereby realizing the aforementioned recombinant receptor,
  • the above-described construct may further include at least one of the following additional technical features:
  • the first nucleic acid molecule, the second nucleic acid molecule and optionally the third nucleic acid molecule are arranged to express the recombinant receptor, silencing cellular immunity in lymphocytes as described above Checkpoints and expression of optional non-functional EGFR, and the recombinant receptor is in a non-fused form with the optional non-functional EGFR.
  • the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; a second promoter, the second promoter and The second nucleic acid molecule is operably linked; and optionally a third promoter operably linked to the third nucleic acid molecule.
  • the first, second, and optionally third promoters described above can independently initiate expression of the first, second, and optionally third nucleic acid molecules, respectively, thereby further facilitating regulation of expression of the corresponding nucleic acid molecule.
  • the first promoter, the second promoter, and the third promoter are independently Selected from a U6, H1, CMV, EF-1, LTR or RSV promoter.
  • the inventors have found that the U6, H1, CMV, EF-1, LTR or RSV promoter is capable of efficiently initiating expression of the first, second and optionally third nucleic acid molecules, first, second and optionally third nucleic acid molecules The expression efficiency is significantly improved.
  • the construct comprises a first nucleic acid molecule, a second nucleic acid molecule and a third nucleic acid molecule, and the construct further comprises: an internal ribosome entry site sequence, the internal ribosome entry site A point sequence is disposed between the first nucleic acid molecule and the third nucleic acid molecule, the internal ribosome entry site having the nucleotide sequence set forth in SEQ ID NO:156.
  • the introduction of the internal ribosome entry site sequence allows the initial expression of the third nucleic acid molecule to be independent of the 5' cap structure, and the first and third nucleic acid molecules are expressed proportionally, thereby facilitating expression regulation, and the obtained transgenic lymphocytes
  • the treatment of cells is safer.
  • the construct comprises a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule, and the construct further comprises: a fourth nucleic acid molecule, the fourth nucleic acid molecule being disposed in the A first nucleic acid molecule is interposed between the third nucleic acid molecule and the fourth nucleic acid molecule encodes a linker peptide that is capable of being cleaved in the lymphocyte.
  • the introduction of the fourth linker peptide allows the expressed recombinant receptor as well as the non-functional EGFR to be expressed on the lymphocyte membrane in a non-fusion state.
  • the linker peptide has the amino acid sequence set forth in SEQ ID NOs: 157-160.
  • linker peptide having the amino acid sequence of SEQ ID NOS: 157 to 160, capable of cleavage by itself, and further The efficiency and success rate of expression of the obtained recombinant receptor and non-functional EGFR in the non-fusion state on the lymphocyte membrane were significantly increased.
  • the vector of the construct is a non-pathogenic viral vector.
  • the pathogenic site of the construct vector in the embodiments of the present invention has been modified or mutated, and the pathogenicity of the virus has been lost, thereby further improving the safety of the treatment mediated by the non-pathogenic viral vector according to the embodiment of the present invention. high.
  • 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 above vector can achieve high-efficiency expression of the carried nucleic acid in the recipient cell, and has high therapeutic efficiency.
  • the invention provides a method of preparing the transgenic lymphocytes described above.
  • the method comprises introducing the aforementioned construct or the lentivirus described above into lymphocytes or T lymphocytes.
  • the above-described transgenic lymphocytes can be obtained simply and efficiently, as described above, the obtained transgenic lymphocytes against tumor cells, especially PD-L1 or PD-L2 positive tumors.
  • the specific killing of cells is powerful, effective and safe.
  • the invention provides a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: a construct as described above, a lentivirus as described above, a transgenic lymphocyte as described above, a recombinant receptor as described above or a nucleic acid as described above.
  • the invention provides a method of increasing lymphocyte immune killing ability.
  • the method comprises: silencing a cellular immune checkpoint of said lymphocytes, and subjecting said lymphocytes to a recombinant receptor as previously described.
  • the invention provides a method of treating cancer.
  • the method comprises administering to the patient a recombinant receptor as described above, a nucleic acid as described above, a construct as described above or a transgenic lymphocyte as described above.
  • FIG. 1 is a schematic structural view of a lentiviral vector according to an embodiment of the present invention.
  • FIG. 2 is a graph showing the results of anti-EGFR antibody-mediated ADCC killing clearance of lymphocytes co-expressing PD1-CD28-CD3zeta recombinant receptor, non-functional EGFR receptor and shPD1 according to an embodiment of the present invention
  • Figure 3 is a graph showing the results of lymphocyte killing of PD-L1 positive tumor cells co-expressing PD1-CD28-CD3zeta recombinant receptor, non-functional EGFR receptor and shPD1 according to an embodiment of the present invention.
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first” and “second” may include one or more of the features either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
  • the invention provides a recombinant receptor.
  • the recombinant receptor comprises: a cellular immune checkpoint molecule fragment; an immunostimulatory molecule fragment; and a T cell receptor zeta chain.
  • lymphocytes expressing the recombinant receptor of the embodiment of the present invention can effectively enhance the specific killing effect of lymphocytes on tumor cells.
  • the cellular immune checkpoint molecule is PD1.
  • PD1 can bind to PD-L1 or PD-L2 specifically expressed on tumor cells.
  • lymphocytes express the recombinant receptor of the present invention, and lymphocytes specifically target tumor cells under the guidance of PD1. The target for tumor cells is further enhanced.
  • the cellular immunological checkpoint molecule fragment comprises an extracellular region of PDl and optionally a transmembrane region, the immunostimulatory molecule fragment comprising an intracellular region of CD28 and optionally a transmembrane region.
  • the recombinant receptor may comprise: (a) an extracellular region and a transmembrane region of PD1; and (b) an intracellular region of CD28, or comprising: (i) an extracellular region of PD1; And (ii) the intracellular and transmembrane regions of CD28.
  • the extracellular domain of PD1 has a functional region that binds to PD-L1 or PD-L2 specifically expressed on tumor cells
  • the intracellular region of CD28 has a functional region that activates an immunostimulatory signaling pathway, regardless of whether it is a PD1 transmembrane region or
  • the transmembrane region of CD28 can express the recombinant receptor across the membrane, and the lymphocyte cells express the recombinant receptor of the present invention, and the targeted killing effect on the tumor cells is further improved.
  • the T cell receptor zeta chain is a CD3zeta chain.
  • the CD3zeta chain is associated with the T cell receptor (TCR) signaling pathway.
  • TCR T cell receptor
  • the zeta chain can be combined with the same cytoplasm called zeta chain-associated protein 70 (ZAP-70), which has a cytoplasmic A tyrosine kinase (PTK)-active signaling protein containing two SH-2 (src homology region 2, SH-2) domains, phosphorylated tyrosine residues in SH-2 and zeta chains in ZAP-70
  • PTK cytoplasmic A tyrosine kinase
  • the CD3zeta chain can specifically activate the downstream T cell receptor signaling pathway, and then the lymphocytes express the recombinant protein of the present invention, and the killing effect on the tumor cells under the synergistic action of the activation function of the immunostimulatory molecule and the activation of the CD3zeta chain. Further improve.
  • the order of joining the corresponding molecular fragments in the above recombinant receptor may be: cells
  • the C-terminus of the immunological checkpoint molecule fragment is linked to the N-terminus of the immunostimulatory molecule fragment
  • the C-terminus of the immunostimulatory molecule fragment is linked to the N-terminus of the T cell receptor zeta chain.
  • the inventors have found that the relevant fragments of the recombinant receptor of the embodiments of the present invention facilitate the localization of the relevant fragments in the cells under the above-described ligation sequence, thereby facilitating the corresponding functions-targeting, transmembrane, and activation of immunostimulatory signals.
  • the pathway and activation of the T cell receptor signaling pathway further enhance the targeted killing ability of the tumor cells.
  • the recombinant receptor has the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the amino acid sequence represented by SEQ ID NO: 1 is an amino acid comprising the extracellular region and transmembrane region of PD1, the intracellular region of CD28, and the recombinant receptor of CD3zeta (PD1-ECD-TM-CD28-ICD-CD3zeta) sequence.
  • the amino acid sequence shown by SEQ ID NO: 2 is an amino acid sequence including the extracellular region of PD1, the transmembrane region and the intracellular region of CD28, and the recombinant receptor for CD3zeta (PD1-ECD-TM-CD28-ICD-CD3zeta).
  • the recombinant receptor has the above amino acid sequence and is expressed in lymphocytes, which can effectively enhance the specific killing effect of lymphocytes on tumor cells.
  • the invention provides a nucleic acid.
  • the nucleic acid encodes a recombinant receptor as described above, optionally, the nucleic acid has the nucleotide sequence set forth in SEQ ID NO: 3 or 4.
  • the nucleotide sequence shown in SEQ ID NO: 3 encodes a recombinant receptor having the amino acid sequence shown in SEQ ID NO: 1
  • the nucleotide sequence shown in SEQ ID NO: 4 has the sequence of SEQ ID NO: Recombinant receptor showing the amino acid sequence.
  • the nucleic acid of the embodiment of the present invention is introduced into the recipient lymphocytes, and the recombinant receptor encoded by the nucleic acid is transmembranely expressed in lymphocytes, and the specific killing effect of the lymphocytes on the tumor cells is remarkably improved.
  • the invention proposes a lentivirus.
  • the lentivirus carries the following nucleic acid molecule: (a) a nucleic acid molecule encoding a recombinant receptor as described above, the nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 3 or 4; (b) a nucleic acid molecule of a silencing cell immune checkpoint, wherein the nucleotide sequence of the nucleic acid molecule of the silencing cell immunological checkpoint is at least one selected from the group consisting of SEQ ID NOs: 5 to 137, wherein SEQ ID NO: 5 - 16 is a human programmed death receptor 1 (PD1) siRNA nucleotide sequence, SEQ ID NOs: 17 to 32 are human cytotoxic T lymphocyte-associated antigen 4 (CTLA4) siRNA sequences, and SEQ ID NOs: 33-48 are human T cell immunoglobulin mucin molecule 3 (TIM3) siRNA
  • PD1 human programmed death
  • the lentivirus of the embodiment of the present invention is introduced into the recipient lymphocyte, and the recombinant receptor described above is transmembranely expressed in the lymphocyte, and the immunological checkpoint molecule on the surface of the lymphocyte membrane is specifically silenced, thereby playing in the recombinant receptor. While enhancing the specific killing function of lymphocytes on tumor cells, the immunosuppressive mechanism is also suppressed.
  • the lentivirus of the embodiment of the present invention is introduced into the recipient lymphocytes, and the specific killing of the lymphocytes by the lymphocytes is strong and effective.
  • the lentivirus may further carry a nucleic acid molecule encoding a non-functional EGFR.
  • the non-functional EGFR receptor lacks an N-terminal ligand binding region and an intracellular receptor tyrosine kinase activity, but includes a transmembrane region of the wild type EGFR receptor and an intact anti-EGFR antibody Combined sequence.
  • the inventors have found that the above-mentioned non-functional EGFR receptor can be used as a suicide marker of lymphocytes, and the expression of the non-functional EGFR receptor can effectively prevent the targeted killing effect of lymphocytes on tumor cells, if the patient has serious adverse reactions.
  • the lymphocytes can be cleared by the anti-EGFR antibody, thereby improving the safety of the patients with tumors such as lentiviruses, lymphocytes and the like in the embodiments of the present invention.
  • the lentivirus carries a nucleotide sequence represented by SEQ ID NOS: 140 to 155.
  • SEQ ID NO: 140 is a nucleic acid co-expressing the aforementioned recombinant receptor (including the extracellular region and transmembrane region of PD1, the intracellular region of CD28 and the CD3Zeta chain), non-functional EGFR, and silencing cellular immunological checkpoint PD1 Molecules [PD1(ECD-TM)-CD28(IC)-CD3zeta-IRES-tEGFR-pA-U6-shPD1)];
  • SEQ ID NO: 141 is a co-expression of the aforementioned recombinant receptor (including the extracellular domain of PD1 and Transmembrane region, intracellular region of CD28 and CD3Zeta chain), non-functional EGFR and nucleic acid molecule of silencing cellular immunological checkpoint CTLA4 [PD1(ECD-TM)-CD28(
  • a nucleic acid expressing the aforementioned recombinant receptor, a silent immunological checkpoint, and optionally a non-functional EGFR can be introduced into a recipient lymphocyte, thereby realizing the aforementioned recombinant receptor,
  • the high-efficiency expression of the optional non-functional EGFR on the receptor cell membrane achieves the repression of the immunosuppressive mechanism, and the specific killing of the obtained receptor lymphocytes to the tumor cells is strong, effective, and safe.
  • the invention provides a transgenic lymphocyte.
  • the cellular immune checkpoint of the transgenic lymphocyte is silenced, and the transgenic lymphocyte expresses the aforementioned recombination
  • the transgenic lymphocytes further express non-functional EGFR.
  • the specific killing of tumor cells by the transgenic lymphocytes of the embodiments of the present invention is strong, effective, and safe.
  • the selection of the lymphocyte immune checkpoint to be silenced is not particularly limited.
  • the lymphocyte immune checkpoint is independently selected from the group consisting of CTLA4, PD1, TIM3, BTLA, LAG-3. At least one of IRAK-M, SOCS1, A20, and CBL-B.
  • the above-mentioned immunological checkpoint molecule exerts an immunosuppressive mechanism in lymphocytes, and the specific silencing of the above-mentioned immunological checkpoint molecule can effectively suppress the immunosuppressive mechanism, and further enhance the specific killing of tumor cells by the transgenic lymphocytes of the embodiments of the present invention. effect.
  • the manner of silencing the lymphocyte immune checkpoint is also not particularly limited.
  • the lymphocyte immune checkpoint is silenced by shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR -Cas9, at least one of CRISPR-Cpf1 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 RNA and antisense DNA mainly function through translation of mRNA and transcription of gene DNA; antisense nucleic acid prevents ribosome by forming steric hindrance effect by binding to target mRNA Binding to mRNA, on the other hand, binding to mRNA activates endogenous RNase or ribozyme, which in turn degrades mRNA; antisense DNA specifically binds to the regulatory region of the double helix of the gene DNA to form a DNA trimer, or with a DNA coding region Binding, termination of the elongation of the mRNA strand being transcribed; antisense nucleic acids also inhibit processing modifications of post-transcriptional mRNA, such as 5' end capping, 3' end tailing, intermediate splicing, and internal base methylation, etc. Mature mRNA is transported from the nucleus to the cytoplasm. Therefore, antisense RNA is an effective technique for silencing the gene of interest.
  • Ribozyme is a catalytically active RNA molecule that is a biocatalyst that degrades specific mRNA sequences.
  • the ribozyme participates in RNA self-cleavage and processing by catalyzing the hydrolysis of transphosphate and phosphodiester bonds, and general antisense RNA.
  • ribozymes have a relatively stable spatial structure and are not susceptible to RNase attack. More importantly, ribozymes can be detached from the hybridization chain and then re-bound and cleave other mRNA molecules.
  • Dominant negative mutations are those in which certain signal transduction proteins are not only self-functional but also inhibit or block the action of wild-type signal transduction proteins in the same cell, mainly by forming dimers with wild-type proteins.
  • the way to achieve this mutation is toxic and can significantly inhibit or block the action of intracellular target signal transduction proteins.
  • the zinc finger nuclease consists of a DNA recognition domain and a non-specific endonuclease.
  • the DNA recognition domain is composed of a series of Cys2-His2 zinc finger proteins in series (generally 3 to 4). Each zinc finger protein recognizes and binds.
  • One A specific triplet base, zinc finger protein forms a ⁇ - ⁇ - ⁇ secondary structure, wherein the 16 amino acid residues of the ⁇ helix determine the DNA binding specificity of the zinc finger, the skeleton structure is conserved, and the amino acid determining the DNA binding specificity
  • the introduction of sequence changes can obtain new DNA binding specificity, so that different amino acid introduction sequences can be designed for different genes of interest to achieve specific silencing of different genes of interest.
  • CRISPR Clustered regular interspaced short palindromic repeats
  • the CRISPR cluster is a family of specific DNA repeats that are widely found in the genomes of bacteria and archaea.
  • the sequence consists of a leader, multiple short and highly conserved repeats, and multiple spacers (Spacer). )composition.
  • the leader region is generally located upstream of the CRISPR cluster and is a region rich in AT length of 300-500 bp, which is considered to be a promoter sequence of the CRISPR cluster.
  • the repeat sequence region has a length of 21 to 48 bp and contains a palindromic sequence, which can form a hairpin structure.
  • the repeat sequences are separated by a spacer of length 26 to 72 bp.
  • the Spacer region is composed of captured foreign DNA.
  • CRISPR CRISPR-related genes
  • CRISPR is transcribed into a long RNA precursor (Pre RISPR RNA, pre-crRNA) under the control of the leader region, and then processed into a series of short conserved repeats and spacers.
  • the mature crRNA ultimately recognizes and binds to its complementary foreign DNA sequence to exert a cleavage effect.
  • Processing of pre-crRNA is involved by Cas9 in the Cas family. Cas9 contains two unique active sites, RuvC at the amino terminus and HNH in the middle of the protein, which play a role in crRNA maturation and double-strand DNA cleavage.
  • trans-activating crRNA complementary to its repeat sequence is also transcribed, and Cas9 and double-stranded RNA-specific RNase III nuclease are excited to process pre-crRNA.
  • the crRNA, tracrRNA and Cas9 complexes recognize and bind to the complementary sequence of crRNA, then unwind the DNA double strand to form R-loop, which makes the crRNA hybridize with the complementary strand, and the other strand maintains the free single-stranded state.
  • the complementary DNA strand of the crRNA is then cleaved by the HNH active site in Cas9, and the RuvC active site cleaves the non-complementary strand, eventually introducing a DNA double-strand break (DSB).
  • RNA By artificially designing RNA, it is possible to engineer a sgRNA (short guide RNA) sufficient to guide Cas9 to the targeted gene cleavage of DNA.
  • the shRNA, the antisense nucleic acid, the ribozyme, the dominant negative mutation, and the CRISPR zinc finger nuclease are effective means for specifically silencing the target gene, and the means for silencing the gene is not particularly limited, and those skilled in the art can
  • the experimental purpose and condition selection such as at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR or zinc finger nuclease used in the embodiments of the present invention, achieve specific silencing of the target gene.
  • the lymphocyte immune 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 siRNA of the examples of the present invention is designed to be homologous to the coding region of PD1, 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. Based on sequence homology in the activated RISC, RISC is directed to the corresponding mRNA; the same nuclease cleavage targets PD1, resulting in specific gene PD1 silencing, inhibiting the expression of the specific gene PD1.
  • RISC Inducible RNA Silencing Complex
  • 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 RNA interference
  • the shRNA of the embodiment of the present invention is continuously produced from a cell, so that the effect thereof is more durable, thereby prolonging the shRNA cycle, and the shRNA used in the embodiment of the present invention has a highly efficient and specific function of silencing the cell immune checkpoint.
  • the successful silencing of the cellular immune checkpoints makes the transgenic lymphocytes significantly resistant to tumor-mediated immunosuppression, and the proliferation and viability of the tumor patients are further improved, and the targeted killing effect on the tumors is more significant.
  • the lymphocytes are antigen-specific T lymphocytes or tumor infiltrating T lymphocytes or natural killer T lymphocytes or natural killer cells.
  • antigen-specific T lymphocytes, tumor infiltrating T lymphocytes, peripheral blood T lymphocytes, natural killer T lymphocytes or natural killer cells can achieve specific immune killing of tumor cells, and have strong specificity. The killing intensity is high and the safety is high.
  • the invention proposes a construct.
  • the construct comprises: a first nucleic acid molecule encoding a recombinant receptor as described above; a second nucleic acid molecule, the second nucleic acid molecule silencing a cellular immune checkpoint; And optionally, the construct further comprises: a third nucleic acid molecule encoding a non-functional EGFR, wherein the cellular immune checkpoint, the non-functional EGFR is as defined above.
  • a nucleic acid expressing the aforementioned recombinant receptor, a silent immunological checkpoint, and optionally a non-functional EGFR can be introduced into a recipient lymphocyte, thereby realizing the aforementioned recombinant receptor,
  • the high-efficiency expression of the optional non-functional EGFR on the receptor cell membrane achieves the repression of the immunosuppressive mechanism, and the specific killing of the obtained receptor lymphocytes to the tumor cells is strong, effective, and safe.
  • the first nucleic acid molecule, the second nucleic acid molecule, and optionally the third nucleic acid molecule are set in the lymphocytes described above to express the recombinant receptor, silence cellular immune checkpoint and expression Optional non-functional EGFR, and the recombinant receptor is in a non-fused form with the optional non-functional EGFR.
  • the obtained lymphocytes are more powerful, more effective, and safer than the specific killing of tumor cells.
  • the inventors independently realize the above-described recombinant receptor, cellular immune checkpoint shRNA, and optionally non-functional EGFR receptor, respectively, by at least one of the following methods, wherein, Expression herein refers to both expression of the receptor and RNA transcription.
  • the construct may further comprise: a first promoter operably linked to the first nucleic acid molecule; a second promoter, the first A second promoter is operably linked to the second nucleic acid molecule; and optionally a third promoter operably linked to the third nucleic acid molecule.
  • the first, second, and optionally third promoters described above can independently initiate expression of the first, second, and optionally third nucleic acid molecules, respectively, thereby further facilitating regulation of expression of the corresponding nucleic acid molecule.
  • the first promoter, the second promoter, and the third promoter are each independently selected from a U6, H1, CMV, EF-1, LTR or RSV promoter.
  • the inventors have found that the U6, H1, CMV, EF-1, LTR or RSV promoter is capable of efficiently initiating expression of the first, second and optionally third nucleic acid molecules, first, second and optionally third nucleic acid molecules The expression efficiency is significantly improved.
  • Internal ribosome entry site sequence When the construct comprises a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule, the construct further comprises: an internal ribosome entry site sequence, the internal A ribosome entry site sequence is disposed between the first nucleic acid molecule and the third nucleic acid molecule.
  • the introduction of the internal ribosome entry site sequence allows the initial expression of the third nucleic acid molecule to be independent of the 5' cap structure, and the first and third nucleic acid molecules are expressed proportionally, thereby facilitating expression regulation, and the obtained transgenic lymphocytes The treatment of cells is safer.
  • Linking peptide when the construct comprises a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule, the construct further comprising: a fourth nucleic acid molecule, the fourth nucleic acid molecule being disposed at the first nucleic acid molecule Between the third nucleic acid molecule and the fourth nucleic acid molecule encodes a linker peptide that is capable of being cleaved in the lymphocyte. The introduction of the fourth linker peptide allows the expressed recombinant receptor as well as the non-functional EGFR to be expressed on the lymphocyte membrane in a non-fusion state.
  • the above-described linker peptide has the amino acid sequence shown by SEQ ID NOS: 157 to 160, and these linker peptides are 2A self-cleaving linker peptides.
  • the 2A linker peptide is found in the foot and mouth disease virus (FMDV), usually an oligopeptide having 19 to 22 amino acids, located between membrane proteins of the picornavirus family.
  • FMDV foot and mouth disease virus
  • the 2A self-cleaving peptide of FMDV virus is capable of self-cleavage, which in turn produces mature viral proteins, which is known as the translational effect.
  • the cleavage site is located between the last glycine at the C-terminus and the first proline of the downstream protein of 2B (-LLNFDLLKLAGDVESNPG ⁇ P-).
  • 2A analogous sequence including porcine virus-1 2A (P2A, sequence as shown in SEQ ID NO: 158), minusa asigna virus 2A (T2A, sequence as shown in SEQ ID NO: 157), equine rhinitis A virus 2A (E2A, sequence is SEQ ID NO: 159), polymorphic polyhedrosis virus (BmCPV 2A) and softening disease virus (BmIFV 2A).
  • the linker peptide showing the amino acid sequence is disposed between the first nucleic acid molecule and the third nucleic acid molecule, and the function of self-cleavage can be well exerted in the recipient cell, and the obtained recombinant receptor and the non-functional EGFR are non-fused state.
  • the efficiency and success rate of expression on the lymphocyte membrane is further significantly increased.
  • the cellular immune checkpoint is efficiently silenced and the non-functional EGFR receptor is efficiently expressed and the above-mentioned recombination is regulated.
  • the body is efficiently expressed on the transgenic lymphocyte membrane of the embodiment of the present invention, and the non-functional EGFR receptor and the recombinant receptor are expressed in the non-fusion state on the lymphocyte membrane, thereby effectively inhibiting the immunological negative regulation of the immune checkpoint and ensuring the recombination.
  • the receptor enhances the biological function of immunity, or effectively achieves the timely removal of transgenic lymphocytes, thereby greatly increasing the survival rate of lymphocytes in the tumor environment, and the targeted killing effect of lymphocytes is more significant, and the safety of immune killing is further improved. improve.
  • the vector of the construct is a non-pathogenic viral vector comprising at least one selected from the group consisting of a retroviral vector, a lentiviral vector or an adeno-associated viral vector.
  • the pathogenic site of the construct vector in the embodiments of the present invention has been modified or mutated, and the pathogenicity of the virus has been lost, thereby further improving the safety of the treatment mediated by the non-pathogenic viral vector according to the embodiment of the present invention. high.
  • 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 can be integrated into the host chromosome or freed from the host. Beyond the chromosomes, a broad spectrum of efficient infection efficiency can be achieved.
  • 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 composite lentivirus, in addition to the common slow
  • the viral genes gag, pol and env also contain other genes with regulatory and structural functions.
  • Lentiviral vectors are well known to those skilled in the art, and lentiviruses include: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors produce a biosafety vector by multiple attenuation of HIV-causing genes, such as deletion of the genes env, vif, vpr, vpu and nef.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and are useful for in vivo and in vitro gene transfer and nucleic acid sequence expression.
  • a suitable host cell together with two or more vectors with packaging functions (gag, pol, env, rev and tat), it is possible to infect non-dividing cells.
  • the targeting of recombinant viruses is achieved by binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
  • the targeting of the recombinant virus confers specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, along with another gene encoding a ligand for the receptor on the particular target cell.
  • the lentiviral vector of the present invention can efficiently transport and co-express shRNA (a transport form of siRNA) which can effectively inhibit the expression of PD1 or CTLA4 or CBL-B.
  • shRNA a transport form of siRNA
  • an adeno-associated viral vector (AAV) of an embodiment of the invention may be constructed using one or more DNAs of a well-known serotype adeno-associated viral vector.
  • AAV adeno-associated viral vector to carry and co-express small hairpin RNA, which can inhibit the expression of genes such as PD1.
  • the embodiment of the present invention also includes a microgene.
  • Microgenes mean the use of a combination (selected nucleotide sequence and operably necessary related linker sequences) to direct expression of the transform, transcription and/or gene product in a host cell in vivo or in vitro.
  • the "operable ligation" sequence is employed to include expression control sequences for a continuous gene of interest, and expression control sequences for trans- or remote control of the gene of interest.
  • vectors of the embodiments of the invention also include conventional control elements that permit transcription, transformation, and/or expression of small hairpin RNA in cell infection with the plasmid vector or in a cellular infection with the viral vector.
  • a large number of expression control sequences may be used.
  • the shRNA expressing promoter is an RNA polymerase promoter.
  • the promoter is a RAN polymerase promoter selected from the group consisting of U6, H1, pol I, pol II and pol III.
  • the promoter is a tissue-specific promoter.
  • the promoter is an inducible promoter.
  • the promoter is selected from a promoter based on the selected vector.
  • the promoter when a lentiviral vector is selected, the promoter is a U6, H1, CMV IE gene, EF-1 ⁇ , ubiquitin C, or phosphoglycerate kinase (PGK) promoter.
  • Other conventional expression control sequences include selectable markers or reporter genes, including 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 inhibit immunological checkpoints) and optionally non-functional EGFR receptors as well as recombinant receptors.
  • shRNA small hairpin RNA
  • the small hairpin RNA carrying the silenced PD1 siRNA and the nucleic acid molecule expressing the optional non-functional EGFR receptor and the viral vector or plasmid expressing the recombinant receptor are combined with the polymer or the plasmid, and the viral vector or plasmid can be combined with the polymer or Other materials to increase its stability or assist in its targeted movement.
  • the invention provides a method of preparing the transgenic lymphocytes described above.
  • the method comprises introducing the aforementioned construct or the lentivirus described above into lymphocytes or T lymphocytes.
  • the above-described transgenic lymphocytes can be obtained simply and efficiently, and as described above, the specific killing of the obtained transgenic lymphocytes to tumor cells is strong, effective, and safe.
  • the invention provides a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: a construct as described above, a lentivirus as described above, a transgenic lymphocyte as described above, a recombinant receptor as described above or a nucleic acid as described above.
  • the therapeutic composition of the embodiments of the invention provided to a patient is preferably applied to a biocompatible solution or an acceptable pharmaceutical carrier.
  • the various therapeutic compositions prepared are suspended or dissolved in a pharmaceutically or physiologically acceptable carrier, such as physiological saline; an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
  • a pharmaceutically or physiologically acceptable carrier such as physiological saline; an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
  • physiological saline such as physiological saline
  • an isotonic saline solution or other relatively obvious formulation of a person skilled in the art.
  • the appropriate carrier will depend to a large extent on the route of administration.
  • Other isotonic sterile injections with water and anhydrous, and sterile suspensions with water and anhydrous are pharmaceutically acceptable carriers.
  • a sufficient number of viral vectors are transduced into targeted T cells and provide sufficient transgenes to silence the immune checkpoints such as PD1 and express optional non-functional EGFR receptors as well as expression-specific recombination body.
  • the dosage of the therapeutic agent depends primarily on the condition of treatment, age, weight, and the health of the patient, which may result in patient variability.
  • Silencing PD1 and other immunological checkpoints and expressing optional non-functional EGFR receptors as well as expressing the above-described recombinant receptors are part of a combination therapy.
  • These viral vectors and anti-tumor T cells for adoptive immunotherapy can be performed alone or in combination with other methods of treating cancer. Under appropriate conditions, one treatment involves the use of one or more drug therapies.
  • the type of cancer to be treated is not particularly limited.
  • the therapeutic composition according to an embodiment of the present invention the specific killing effect on PD-L1 or PD-L2 positive tumor cells is remarkable.
  • the invention provides a method of increasing lymphocyte immune killing ability.
  • the method comprises: silencing a cellular immune checkpoint of said lymphocytes, and subjecting said lymphocytes to a recombinant receptor as previously described.
  • the invention proposes a method of treating cancer.
  • the method comprises administering to the patient a recombinant receptor as described above, a nucleic acid as described above, a construct as described above or a transgenic lymphocyte as described above.
  • the "recombinant receptor" involved in the present invention is a recombinant protein or a fusion protein which is expressed on the membrane of a recipient cell (such as a lymphocyte) and functions as a receptor protein. Binding to extracellular specific signaling molecules activates a series of biochemical reactions in the cell, causing the cells to have a corresponding effect on external stimuli.
  • the aim is to generate a replication-defective lentiviral vector and to collect the lentiviral vector for centrifugation of human T lymphocytes.
  • 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.
  • 15 ⁇ g of lentiviral transgene plasmid, 5 ⁇ g of pVSV-G (VSV glycoprotein expression plasmid), 10 ⁇ g of pCMVR8.74 plasmid (Gag/Pol/Tat/Rev expression plasmid) and 174 ⁇ l of Express-In were added to each plate of cells. The concentration is 1 ⁇ g/ ⁇ l).
  • the supernatant was collected at 24 and 48 hours, respectively, and using an ultracentrifuge at 28,000 rpm (the centrifuge rotor was Beckman SW 32Ti, Centrifugation was carried out for 2 hours under conditions of Beckman Coulter, Brea, CA). Finally, the viral plasmid pellet was resuspended in 0.75 ml of RPMI-1640 medium.
  • Human primary T lymphocytes were isolated from 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 resuspended virus supernatant and Polybrene (concentration of 8 ⁇ g/ml) were added to each well of cells.
  • IL-2 Human recombinant interleukin-2
  • T lymphocyte culture medium every 2 to 3 days.
  • the final concentration of IL-2 was 100-IU/ml in T lymphocytes.
  • the density of the cells is maintained at 0.5 x 10 6 to 1 x 10 6 /ml.
  • T lymphocytes are dormant, for example, the cell growth rate is slowed down and the cells become smaller, wherein the cell growth rate and size are assessed by Coulter Counter (purchased from Beckman Coulter), or transduced T lymphocytes.
  • Coulter Counter purchased from Beckman Coulter
  • T lymphocytes can be used for functional analysis.
  • the flow cytometer used in the examples of the present application was BD FACSCanto II (purchased from BD Biosciences), and flow cytometric data was analyzed using FlowJo version 7.2.5 software (purchased from Tree Star, Ashland, OR).
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • the ability of anti-EGFR antibodies to induce cell-dependent lysis of lymphocytes expressing non-functional EGFR receptors was assessed using the 4 hour-51Cr-release method.
  • Human T lymphocytes transduced with a lentiviral vector were used as target cells.
  • 100 ⁇ Ci of Na251CrO4 (available from GE Healthcare Life Sciences, Marlborough, MA) was used to calibrate 2 to 5 x 106 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 105/ml).
  • the calibrated cells were then plated in 96-well plates (5 x 103 cells per well, plus 50 ⁇ l of medium) and 50 ⁇ l of anti-EGFR antibody (purchased from Erbitux, Genentech) was added (final concentration 20 ⁇ g/ml), pre-incubated for 30 minutes under normal temperature conditions. Then, the medium containing the antibody was replaced with a normal medium, thereby detecting the spontaneous release of 51Cr. Triton X-100 was added to a final concentration of 1% to ensure maximum release of 51Cr.
  • 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.
  • the cytotoxic activity of recombinant receptor T cells was evaluated using a 4 - hour 51 chromium release assay.
  • the specific steps are as follows: The target test cells were labeled with 51 Cr at 37 ° C 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.
  • E:T target cell ratios
  • the cells were cultured for 4 hours in a 37 degree incubator. After 4 hours, 30 microliters of the supernatant was taken from each well and placed in a counter 96-well plate for counting analysis.
  • the analytical instrument was a top-level counting NXT micro-scintillator counter (purchased from Packard Bioscience). The number of effector cells in all counting wells was calculated based on the total number of T cells.
  • the labeled target test cells are PD-L1 positive tumor cells.
  • Example 1 Construction of a vector for co-expression of a non-functional EGFR receptor, a shRNA shRNA (shPD1, iPD1) and a PD1-CD28-CD3zeta recombinant receptor
  • the inventors cloned the ⁇ -strand sequence encoding the human PD1 extracellular fragment sequence, the CD28 transmembrane and the intracellular domain and the T cell receptor combination into a lentiviral vector containing the EF-1 promoter.
  • the selected restriction enzymes are double digestion of XbaI and NotI, and double digestion with NotI and XhoI, and the lentivirus expressing recombinant receptor is generated by restriction enzyme digestion, ligation, screening and amplification of the plasmid of interest.
  • the sequence containing IRES and expressing the non-functional EGFR receptor was cloned into the LV-PD1-CD28-CD3 ⁇ vector plasmid and constructed into LV-PD1-CD28-CD3 ⁇ -tEGFR(M) (LV-PD1-CD28z/M).
  • the sequence of shRNA containing U6 and a silent immunological checkpoint, such as the sequence of shPD1 was cloned into LV-PD1-CD28-CD3 ⁇ -tEGFR to form LV-PD1-CD28 -CD3 ⁇ -tEGFR(M)-U6-shPD1 (LV-PD1-CD28z/M/iPD1).
  • Figure 1 is a schematic representation of a lentiviral vector (tEGFR indicates non-functional EGFR (M)), containing a recombinant encoding PD1-CD28-CD3 ⁇
  • the sequence encoding the PD1-CD28-CD3 ⁇ recombinant receptor is Under the promoter of EF-1, the sequence of the shRNA that silences the immune checkpoint, such as the shRNA sequence of the silenced PD1, under the promoter of promoter U6, expresses the sequence of the non-functional EGFR receptor as a separate mRNA transcription unit. Translation begins after the IRES sequence.
  • the process of cloning the ⁇ -strand sequence encoding the human PD1 extracellular fragment and the transmembrane segment sequence, the CD28 intracellular segment and the T cell receptor combination into the lentiviral vector containing the EF-1 promoter is as described above.
  • Anti-EGFR antibodies are effective in killing T lymphocytes that secrete co-expressing non-functional EGFR receptors, recombinant receptors, and shRNAs shPD1 (shPD1, iPD1)
  • 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 cell culture dish containing recombinant fibronectin fragment (FN ch-296; Retronectin) and transduced with lentivirus.
  • the lentiviruses were transduced into LV-PD1-CD28z/M/iPD1, LV-PD1. -CD28z, or no-load (LV-GFP) transduction as previously described.
  • T cells expressing non-functional EGFR receptor 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 (100ng was used. /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.
  • anti-EGFR antibodies can effectively block T lymphocytes that co-express PD1-CD28-CD3 ⁇ recombinant receptor, shPD1 and non-functional EGFR receptors (LV-PD1-CD28z/M/iPD1 transduced T lymphocytes Cells), but anti-EGFR antibodies cannot mediate killing of T lymphocytes expressing only PD1-CD28-CD3 ⁇ recombinant receptor (LV-PD1-CD28z transduced T lymphocytes) and T lymphocytes expressing only GFP (LV-GFP transfection) Guided T lymphocytes), the statistical data represents the mean ⁇ SEM of the three wells.
  • peripheral blood lymphocytes are separated by gradient centrifugation, and the gradient centrifuge is Ficoll-Hypaque.
  • T lymphocytes were incubated with T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, CA) at 5% CO2, 37 ° C for 72 hours.
  • the medium was supplemented with 2 mmol/L glutamine and 10% high temperature.
  • Inactivated fetal bovine serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U/ml penicillin/streptomycin double-antibody RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012).
  • T cells were seeded on a cell culture dish containing recombinant fibronectin fragment (FN ch-296; Retronectin) and transduced with lentivirus.
  • the lentiviruses were transduced into LV-PD1-CD28z/M/iPD1, LV-PD1.
  • - CD28z/M, LV-M, or no-load (LV-GFP) the transduction process is as described above.
  • 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 PD-L1-positive glioma cells.
  • the ratio of target cells was 10:1 or 25:1 or 50:1.
  • the standard method was 4– The hourly 51 chromium release method, wherein the 4 - hour 51 chromium release method is as described above.
  • the results of the test are shown in Figure 3.
  • the results in Figure 3 show that Lentivirus-transduced T lymphocytes (LV-PD1-CD28z/M/iPD1) co-expressing PD1-CD28-CD3 ⁇ receptor, non-functional EGFR receptor and shPD1 Guided T lymphocytes) and T lymphocytes (LV-PD1-CD28z/M transduced T lymphocytes) co-expressing PD1-CD28-CD3 ⁇ receptor and non-functional EGFR receptor have brains that kill PD-L1 + Tumor cell capacity.
  • lentiviral-transduced T lymphocytes (LV-PD1-CD28z/M/iPD1-transduced T lymphocytes) co-expressing PD1-CD28-CD3 ⁇ receptor, non-functional EGFR receptor and shPD1 are more co-expressing PD1-
  • the CD28-CD3 purinoceptor and non-functional EGFR receptor T lymphocytes (LV-PD1-CD28z/M transduced T lymphocytes) have the ability to kill PD-L1 + brain tumor cells more strongly.
  • Lentiviral-transduced T lymphocytes (LV-M transduced lymphocytes) expressing no functional EGFR receptor or T lymphocytes transduced with lentiviral transduction (control LV-GFP transduced T lymphocytes) PD-L1 + brain tumor cells have no obvious killing effect.

Abstract

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，该转基因淋巴细胞的细胞免疫检查点被沉默以及表达重组受体，该重组受体包括：细胞免疫检查点分子片段；免疫刺激分子片段；以及T细胞受体zeta链。

Description

重组免疫检查点受体及免疫检查点抑制分子的共表达及应用
优先权信息
本申请请求2016年07月08日向中国国家知识产权局提交的、专利申请号为201610537696.0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，本发明涉及重组免疫检查点受体及免疫检查点抑制分子的共表达及应用，更具体地，本发明涉及重组受体、核酸、慢病毒、转基因淋巴细胞、构建体、制备转基因淋巴细胞的方法、治疗癌症的治疗组合物以及提高淋巴细胞免疫杀伤能力的方法。
背景技术
癌症，由于细胞内基因突变导致细胞增殖失控的一种疾病。目前已成为人类健康的重大威胁，是导致人类死亡的主要原因之一。世界卫生组织(WHO)在发表的《全球癌症报告2014》中指出，2012年全球癌症患者和死亡病例都在迅速增加，而新增癌症病例有近一半出现在亚洲，其中大部分在中国，中国新增癌症病例高居第一位。《2012年中国肿瘤登记年报》数据显示，中国每年新增癌症病例约350万，约有250万人因此死亡。因此，寻找高效特异的癌症治疗方法具有重大的临床价值。
传统的肿瘤治疗方法主要包括手术、放疗和化疗，但这几种方法都具有较大的局限性，比如由于癌细胞的近端入侵或远端转移，手术切除后的肿瘤转移复发率较高，而放疗和化疗对于机体自身的正常细胞尤其是造血系统和免疫系统会造成严重的损害，因此对于已发生肿瘤转移的患者也很难达到较好的远期疗效。随着肿瘤分子机制的深入研究和生物技术的进一步发展，靶向药物治疗和免疫治疗在肿瘤的综合治疗中发挥着愈来愈大的作用。靶向疗法主要包括单克隆抗体(有时归为被动胞回输和肿瘤疫苗等。免疫疗法通过调动机体的免疫系统，增强肿瘤微环境抗肿瘤免免疫疗法)和小分子靶向药物，而免疫疗法主要包括细胞因子疗法、免疫检验点单抗、过继免疫疗法，从而控制和杀伤肿瘤细胞，因此有效率高，特异性强，耐受性好的优点，在肿瘤治疗中具有广阔的前景。
然而，肿瘤的免疫疗法，仍有待进一步深入研究和开发，来增强临床疗效。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出一种重组受体及利用其有效增强淋巴细胞免疫杀伤肿瘤细胞的方法。
在本发明的第一方面，本发明提出了一种重组受体。根据本发明的实施例，所述重组受体包括：细胞免疫检查点分子片段；免疫刺激分子片段；以及T细胞受体zeta链。根据本发明 的实施例，使淋巴细胞表达本发明实施例的重组受体，可有效增强淋巴细胞对肿瘤细胞的特异性杀伤效果。
根据本发明的实施例，上述重组受体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，所述细胞免疫检查点分子为PD1。PD1可与肿瘤细胞上特异性表达的PD-L1或PD-L2相结合。进而，淋巴细胞表达本发明实施例的重组受体，其对肿瘤细胞，尤其是PD-L1或PD-L2阳性肿瘤细胞的靶向杀伤性进一步增强。
根据本发明的实施例，所述细胞免疫检查点分子片段包括所述PD1的胞外区以及任选的跨膜区，所述免疫刺激分子片段包括CD28的胞内区以及任选的跨膜区。PD1的胞外区具有与肿瘤细胞上特异性表达的PD-L1或PD-L2相结合的功能区，CD28的胞内区具有激活免疫刺激信号通路的功能区，进而淋巴细胞细胞表达本发明实施例的重组受体，其对肿瘤细胞的靶向性杀伤效果进一步提高。
根据本发明的实施例，所述重组受体包括：(a)所述PD1的胞外区和跨膜区；以及(b)所述CD28的胞内区，或者包括：(i)所述PD1的胞外区；以及(ii)所述CD28的胞内区和跨膜区。这两种组合方式均保留了PD1与肿瘤细胞上特异性表达的PD-L1或PD-L2相结合的功能区以及CD28激活免疫刺激信号通路的功能区，同时不论是PD1跨膜区还是CD28的跨膜区，均可使重组受体跨膜表达，进而淋巴细胞细胞表达本发明实施例的重组受体，其对肿瘤细胞的靶向性杀伤效果进一步提高。
根据本发明的实施例，所述T细胞受体zeta链为CD3zeta链。CD3zeta链可特异性激活下游T细胞受体信号通路，进而淋巴细胞细胞表达本发明实施例的重组受体，其对肿瘤细胞的杀伤效果进一步提高。
根据本发明的实施例，所述细胞免疫检查点分子片段的C端与所述免疫刺激分子片段的N端相连，所述免疫刺激分子片段的C端与所述T细胞受体zeta链的N端相连。本发明实施例的重组受体的相关片段在上述连接顺序下，有利于相关片段在细胞中的定位，进而更有利于发挥相应的功能-靶向、跨膜、激活免疫刺激信号通路以及激活T细胞受体信号通路，其对肿瘤细胞的靶向性杀伤能力进一步提高。
在本发明的第二方面，本发明提出了一种重组受体。根据本发明的实施例，所述重组受体具有SEQ ID NO：1或2所示的氨基酸序列。
Figure PCTCN2017092376-appb-000001
Figure PCTCN2017092376-appb-000002
根据本发明的实施例，使淋巴细胞表达本发明实施例的重组受体，可有效增强淋巴细胞对肿瘤细胞，特别是PD-L1或PD-L2阳性肿瘤细胞的特异性杀伤效果。
在本发明的第三方面，本发明提出了一种核酸。根据本发明的实施例，所述核酸编码前面所述的重组受体，任选地，所述核酸具有SEQ ID NO：3或4所示的核苷酸序列。
Figure PCTCN2017092376-appb-000003
Figure PCTCN2017092376-appb-000004
将本发明实施例的核酸导入受体淋巴细胞中，核酸所编码的重组受体在淋巴细胞中跨膜表达，该淋巴细胞对肿瘤细胞的特异性杀伤效果显著提高。
在本发明的第四方面，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：(a)编码前面所述重组受体的核酸分子，所述重组受体具有SEQ ID NO：1或2所示的氨基酸序列，所述编码重组受体的核酸分子具有SEQ ID NO：3或4所示的核苷酸序列；(b)沉默细胞免疫检查点的核酸分子，所述沉默细胞免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：5～137的至少之一。
Figure PCTCN2017092376-appb-000005
Figure PCTCN2017092376-appb-000006
Figure PCTCN2017092376-appb-000007
Figure PCTCN2017092376-appb-000008
Figure PCTCN2017092376-appb-000009
Figure PCTCN2017092376-appb-000010
将本发明实施例的慢病毒导入受体淋巴细胞中，前面所述的重组受体在淋巴细胞中跨膜表达，同时淋巴细胞膜表面的免疫检查点分子被特异性沉默，进而在重组受体发挥加强淋巴细胞对肿瘤细胞的特异性杀伤功能的同时，免疫抑制机制也被阻遏。将本发明实施例的慢病毒导入受体淋巴细胞中，淋巴细胞对肿瘤细胞的特异性杀伤强大而有效。
根据本发明的实施例，上述慢病毒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，上述慢病毒还可以进一步携带编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：138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：139所示的核苷酸序列。
Figure PCTCN2017092376-appb-000011
Figure PCTCN2017092376-appb-000012
根据本发明的实施例，无功能EGFR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受体的跨膜区和完整的与抗EGFR抗体结合的序列，所以无功能EGFR受体可作为淋巴细胞的自杀标记。本发明实施例的慢病毒导入受体淋巴细胞中，无功能EGFR受体的表达可在有效保证淋巴细胞的对肿瘤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，淋巴细胞可被抗EGFR抗体清除，进而提高了本发明实施例的慢病毒、淋巴细胞等治疗肿瘤病人的安全性。
在本发明的第五方面，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：140～156所示的核苷酸序列。
Figure PCTCN2017092376-appb-000013
Figure PCTCN2017092376-appb-000014
Figure PCTCN2017092376-appb-000015
Figure PCTCN2017092376-appb-000016
Figure PCTCN2017092376-appb-000017
Figure PCTCN2017092376-appb-000018
Figure PCTCN2017092376-appb-000019
Figure PCTCN2017092376-appb-000020
Figure PCTCN2017092376-appb-000021
Figure PCTCN2017092376-appb-000022
Figure PCTCN2017092376-appb-000023
Figure PCTCN2017092376-appb-000024
Figure PCTCN2017092376-appb-000025
Figure PCTCN2017092376-appb-000026
Figure PCTCN2017092376-appb-000027
Figure PCTCN2017092376-appb-000028
Figure PCTCN2017092376-appb-000029
Figure PCTCN2017092376-appb-000030
Figure PCTCN2017092376-appb-000031
Figure PCTCN2017092376-appb-000032
Figure PCTCN2017092376-appb-000033
Figure PCTCN2017092376-appb-000034
Figure PCTCN2017092376-appb-000035
利用根据本发明实施例的上述慢病毒，可将表达前面所述重组受体、沉默免疫检查点以及任选的表达无功能EGFR的核酸导入受体淋巴细胞，进而实现前面所述重组受体、任选的无功能EGFR在受体细胞膜上的高效表达，实现免疫抑制机制的阻遏，所获得的受体淋巴细胞的对肿瘤细胞，尤其是PD-L1或PD-L2阳性肿瘤细胞的特异性杀伤强大、有效、安全。
在本发明的第六方面，本发明提出了一种转基因淋巴细胞。根据本发明的实施例，所述转基因淋巴细胞的细胞免疫检查点被沉默，并且所述转基因淋巴细胞表达前面所述的重组受体，任选地，所述转基因淋巴细胞进一步表达无功能EGFR。本发明实施例的转基因淋巴细胞对肿瘤细胞，尤其是PD-L1或PD-L2阳性肿瘤细胞的特异性杀伤强大、有效、安全。
根据本发明的实施例，上述转基因淋巴细胞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，所述淋巴细胞细胞免疫检查点独立地选自CTLA4、PD1、TIM3、BTLA、LAG-3、IRAK-M、SOCS1、A20、CBL-B的至少之一，优选地，所述淋巴细胞细胞免疫检查点为CTLA4、PD1、SOCS1或CBL-B。上述免疫检查点分子在淋巴细胞中发挥着免疫抑制机制，上述免疫检查点分子的特异性沉默，可有效阻遏免疫抑制机制，进一步提高了本发明实施例的转基因淋巴细胞对肿瘤细胞的特异性杀伤效果。
根据本发明的实施例，所述淋巴细胞细胞免疫检查点被沉默是通过shRNA、反义核酸、核酶、显性负突变、CRISPR-Cas9，CRISPR-Cpf1和锌指核酸酶至少之一实现的。发明人通过实验发现，上述方式的任意一种或几种均能够沉默细胞免疫检查点，进而免疫抑制机制被显著阻遏。
根据本发明的实施例，所述淋巴细胞细胞免疫检查点被沉默是通过shRNA实现的。通过shRNA沉默淋巴细胞细胞免疫检查点是一种简便、有效方式。
根据本发明的实施例，所述淋巴细胞是抗原特异性T淋巴细胞，任选地，所述淋巴细胞是肿瘤浸润T淋巴细胞，任选地，所述淋巴细胞是外周血T淋巴细胞，任选地，所述淋巴细胞是自然杀伤T淋巴细胞，任选地，所述淋巴细胞是自然杀伤细胞。根据本发明实施例的抗原特异性T淋巴细胞、肿瘤浸润T淋巴细胞、自然杀伤T淋巴细胞或自然杀伤细胞，可实现对肿瘤细胞的特异性免疫杀伤，特异性强、杀伤力度大、安全性高。
在本发明的第七方面，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前面所述的重组受体；第二核酸分子，所述第二核酸分子沉默细胞免疫检查点；以及任选地，所述构建体进一步包括：第三核酸分子，所述第三核酸分子编码无功能EGFR，其中，所述细胞免疫检查点、所述无功能EGFR是如前面所定义的。利用根据本发明实施例的上述构建体，可将表达前面所述重组受体、沉默免疫检查点以及任选的表达无功能EGFR的核酸导入受体淋巴细胞，进而实现前面所述重组受体、任选的无功能EGFR在受体细胞膜上的高效表达，实现免疫抑制机制的阻遏，所获得的受体淋巴细胞的对肿瘤细胞，尤其是PD-L1或PD-L2阳性肿瘤细胞的特异性杀伤强大、有效、安全。
根据本发明的实施例，上述构建体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，所述第一核酸分子、所述第二核酸分子和任选的所述第三核酸分子被设置在前面所述的淋巴细胞中表达所述重组受体、沉默细胞免疫检查点和表达任选的无功能EGFR，并且所述重组受体与任选的所述无功能EGFR呈非融合形式。利用根据本发明实施例的上述构建体导入前面所述的淋巴细胞，所获得的淋巴细胞对肿瘤细胞的特异性杀伤更强大、更有效、更安全。
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；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；以及任选的第三启动子，所述任选的第三启动子与所述第三核酸分子可操作地连接。上述第一、第二以及任选的第三启动子可分别独立地启动表达第一、第二以及任选的第三核酸分子，进而更加有利于相应核酸分子表达的调控。
根据本方明的实施例，所述第一启动子、所述第二启动子、所述第三启动子分别独立地 选自U6，H1，CMV，EF-1，LTR或RSV启动子。发明人发现，U6，H1，CMV，EF-1，LTR或RSV启动子能够高效启动表达第一、第二以及任选的第三核酸分子，第一、第二以及任选的第三核酸分子的表达效率显著提高。
根据本发明的实施例，所述构建体包括第一核酸分子、第二核酸分子和第三核酸分子，并且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：156所示的核苷酸序列。
Figure PCTCN2017092376-appb-000036
内部核糖体进入位点序列的引入，使得第三核酸分子的起始表达不依赖5’帽子结构，并且第一和第三核酸分子成比例表达，进而更加有利于表达调控，所获得的转基因淋巴细胞的治疗安全性更高。
根据本发明的实施例，所述构建体包括第一核酸分子、第二核酸分子和第三核酸分子，并且所述构建体进一步包括:第四核酸分子，所述第四核酸分子设置在所述第一核酸分子与所述第三核酸分子之间，并且所述第四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。第四连接肽的引入使得所表达的重组受体以及无功能EGFR呈非融合状态表达在淋巴细胞膜上。
根据本发明的实施例，所述连接肽具有SEQ ID NO：157～160所示的氨基酸序列。
Figure PCTCN2017092376-appb-000037
具有SEQ ID NO：157～160所示的氨基酸序列的连接肽，能够自身切割断裂，进而所 获得的重组受体以及无功能EGFR呈非融合状态表达在淋巴细胞膜上的效率和成功率显著提高。
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。本发明实施例中的构建体载体的致病位点经过修饰或突变，已丧失病毒的致病性，进而在根据本发明实施例的非致病性病毒载体介导下的治疗的安全性更高。
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。上述载体可实现所携带核酸在受体细胞的高效表达，治疗效率高。
在本发明的第八方面，本发明提出了一种制备前面所述的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。利用根据本发明实施例的上述方法，可简便、高效地获得前面所述的转基因淋巴细胞，如前所述，所获得的转基因淋巴细胞对肿瘤细胞，尤其是PD-L1或PD-L2阳性肿瘤细胞的特异性杀伤强大、有效、安全。
在本发明的第九方面，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：前面所述的构建体、前面所述的慢病毒、前面所述的转基因淋巴细胞、前面所述的重组受体或者前面所述的核酸。利用根据本发明实施例的治疗组合物，能够实现对肿瘤细胞，尤其是PD-L1或PD-L2阳性肿瘤细胞的特异、强大、有效、安全地杀伤。
在本发明的第十方面，本发明提出了一种提高淋巴细胞免疫杀伤能力的方法。根据本发明的实施例，所述方法包括：使所述淋巴细胞的细胞免疫检查点被沉默，以及使所述淋巴细胞表前面所述的重组受体。利用根据本发明实施例上述方法，能够有效提高淋巴细胞对肿瘤细胞，尤其是PD-L1或PD-L2阳性肿瘤细胞的特异性免疫杀伤。
在本发明的第十一方面，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：给患者给予前面所述的重组受体、前面所述的核酸、前面所述的构建体或者前面所述的转基因淋巴细胞。利用根据本发明实施例的治疗方法，能够实现对肿瘤病人体内的肿瘤细胞的有效、安全地杀伤。
附图说明
图1是根据本发明实施例慢病毒载体的结构示意图；
图2是根据本发明实施例的共表达PD1-CD28-CD3zeta重组受体、无功能EGFR受体和shPD1的淋巴细胞被抗EGFR抗体介导ADCC杀伤清除的结果图；以及
图3是根据本发明实施例的共表达PD1-CD28-CD3zeta重组受体、无功能EGFR受体和shPD1的淋巴细胞杀伤PD-L1阳性肿瘤细胞的结果图。
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，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
重组受体
一方面，本发明提出了一种重组受体。根据本发明的实施例，该重组受体包括：细胞免疫检查点分子片段；免疫刺激分子片段；以及T细胞受体zeta链。根据本发明的实施例，使淋巴细胞表达本发明实施例的重组受体，可有效增强淋巴细胞对肿瘤细胞的特异性杀伤效果。
根据本发明的具体实施例，所述细胞免疫检查点分子为PD1。PD1可与肿瘤细胞上特异性表达的PD-L1或PD-L2相结合，进而，淋巴细胞表达本发明实施例的重组受体，淋巴细胞在PD1的引导下，特异性靶向肿瘤细胞，其对肿瘤细胞的靶标性进一步增强。
根据本发明的再一具体实施例，细胞免疫检查点分子片段包括PD1的胞外区以及任选的跨膜区，免疫刺激分子片段包括CD28的胞内区以及任选的跨膜区。例如，根据本发明的实施例，重组受体可以包括：(a)PD1的胞外区和跨膜区；以及(b)CD28的胞内区，或者包括：(i)PD1的胞外区；以及(ii)CD28的胞内区和跨膜区。PD1的胞外区具有与肿瘤细胞上特异性表达的PD-L1或PD-L2相结合的功能区，CD28的胞内区具有激活免疫刺激信号通路的功能区，同时不论是PD1跨膜区还是CD28的跨膜区，均可使重组受体跨膜表达，进而淋巴细胞细胞表达本发明实施例的重组受体，其对肿瘤细胞的靶向性杀伤效果进一步提高。
另外，根据本发明的再一实施例，T细胞受体zeta链为CD3zeta链。CD3zeta链关联T细胞受体(TCR)信号通路，CD3zeta链触发后，Zeta链可以同一胞浆内称为zeta链相关蛋白70(ZAP-70)相结合，ZAP-70为一种胞浆内具有酪氨酸激酶(PTK)活性的信号蛋白，含有两个SH-2(src homology region 2，SH-2)结构域，ZAP-70分子中SH-2与zeta链中磷酸化的酪氨酸残基相结合，ZAP-70激活可进一步激活Ras蛋白，进而最终活化淋巴细胞。CD3zeta链可特异性激活下游T细胞受体信号通路，进而淋巴细胞表达本发明实施例的重组蛋白，在免疫刺激分子活化功能片段以及CD3zeta链活化作用的协同作用下，其对肿瘤细胞的杀伤效果进一步提高。
最后，根据本发明的实施例，上述重组受体中相应分子片段的连接顺序可为：细胞 免疫检查点分子片段的C端与免疫刺激分子片段的N端相连，免疫刺激分子片段的C端与T细胞受体zeta链的N端相连。发明人发现，本发明实施例的重组受体的相关片段在上述连接顺序下，有利于相关片段在细胞中的定位，进而更有利于发挥相应的功能-靶向、跨膜、激活免疫刺激信号通路以及激活T细胞受体信号通路，其对肿瘤细胞的靶向性杀伤能力进一步提高。
具体地，根据本发明的实施例，重组受体具有SEQ ID NO：1或2所示的氨基酸序列。其中，SEQ ID NO：1所示的氨基酸序列是包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3zeta的重组受体(PD1-ECD-TM-CD28-ICD-CD3zeta)的氨基酸序列。SEQ ID NO：2所示的氨基酸序列是包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3zeta的重组受体(PD1-ECD-TM-CD28-ICD-CD3zeta)的氨基酸序列。根据本发明的实施例，重组受体具有上述氨基酸序列，使其表达在淋巴细胞中，可有效增强淋巴细胞对肿瘤细胞的特异性杀伤效果。
核酸
另一方面，本发明提出了一种核酸。根据本发明的实施例，该核酸编码前面所述的重组受体，任选地，所述核酸具有SEQ ID NO：3或4所示的核苷酸序列。其中，SEQ ID NO：3所示的核苷酸序列编码具有SEQ ID NO：1所示氨基酸序列的重组受体，SEQ ID NO：4所示的核苷酸序列编码具有SEQ ID NO：2所示氨基酸序列的重组受体。将本发明实施例的核酸导入受体淋巴细胞中，核酸所编码的重组受体在淋巴细胞中跨膜表达，该淋巴细胞对肿瘤细胞的特异性杀伤效果显著提高。
慢病毒
另一方面，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：(a)编码前面所述重组受体的核酸分子，所述核酸分子具有SEQ ID NO：3或4所示的核苷酸序列；(b)沉默细胞免疫检查点的核酸分子，所述沉默细胞免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：5～137的至少之一，其中，SEQ ID NO：5～16是人类程序性死亡受体1(PD1)siRNA核苷酸序列，SEQ ID NO：17～32是人类细胞毒T淋巴细胞相关抗原4(CTLA4)siRNA序列，SEQ ID NO：33～48是人类T细胞免疫球蛋白粘蛋白分子3(TIM3)siRNA序列，SEQ ID NO：49～59是人类T淋巴细胞衰减因子(BTLA)siRNA序列，SEQ ID NO：60～70是人类淋巴细胞活化基因3蛋白(LAG1)siRNA序列，SEQ ID NO：71～87是人类IRAK-M(白细胞介素-1受体相关激酶3)siRNA核苷酸序列，SEQ ID NO：88～98是人类SOCS1(细胞因子信号转导抑制因子1)siRNA序列，SEQ ID NO：99～118是人类A20(肿瘤坏死因子-α诱导蛋白A20)siRNA序列，SEQ ID NO：119～137是人类CBL-B(E3泛素蛋白连接酶CBL-B) siRNA序列。将本发明实施例的慢病毒导入受体淋巴细胞中，前面所述的重组受体在淋巴细胞中跨膜表达，同时淋巴细胞膜表面的免疫检查点分子被特异性沉默，进而在重组受体发挥加强淋巴细胞对肿瘤细胞的特异性杀伤功能的同时，免疫抑制机制也被阻遏。将本发明实施例的慢病毒导入受体淋巴细胞中，淋巴细胞对肿瘤细胞的特异性杀伤强大而有效。
另外，根据本发明的实施例，上述慢病毒还可以进一步携带编码无功能EGFR的核酸分子。根据本发明的具体实施例，无功能EGFR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受体的跨膜区和完整的与抗EGFR抗体结合的序列。发明人发现，上述无功能EGFR受体可作为淋巴细胞的自杀标记，无功能EGFR受体的表达可在有效保证淋巴细胞的对肿瘤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，淋巴细胞可被抗EGFR抗体清除，进而提高了本发明实施例的慢病毒、淋巴细胞等治疗肿瘤病人的安全性。
具体地，所述慢病毒携带含有SEQ ID NO：140～155所示的核苷酸序列。其中，SEQ ID NO：140是共表达前面所述重组受体(包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3Zeta链)、无功能EGFR和沉默细胞免疫检查点PD1的核酸分子【PD1(ECD-TM)-CD28(IC)-CD3zeta-IRES-tEGFR-pA-U6-shPD1)】；SEQ ID NO：141是共表达前面所述重组受体(包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3Zeta链)、无功能EGFR和沉默细胞免疫检查点CTLA4的核酸分子【PD1(ECD-TM)-CD28(IC)-CD3zeta-IRES-tEGFR-pA-U6-sh CTLA4)】；SEQ ID NO：142是共表达前面所述重组受体(包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3Zeta链)、无功能EGFR和沉默细胞免疫检查点CBL-B的核酸分子【PD1(ECD-TM)-CD28(IC)-CD3zeta-IRES-tEGFR-pA-U6-sh CBL-B)】；SEQ ID NO：143是共表达前面所述重组受体(包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3Zeta链)、无功能EGFR和沉默细胞免疫检查点SOCS1的核酸分子【PD1(ECD-TM)-CD28(IC)-CD3zeta-IRES-tEGFR-pA-U6-sh SOCS1)】；SEQ ID NO：144是共表达前面所述重组受体(包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3Zeta链)、无功能EGFR和沉默细胞免疫检查点PD1的核酸分子【PD1(ECD)-CD28(TM-IC)-CD3zeta-IRES-tEGFR-pA-U6-sh PD1)】；SEQ ID NO：145是共表达前面所述重组受体(包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3Zeta链)、无功能EGFR和沉默细胞免疫检查点CTLA4的核酸分子【PD1(ECD)-CD28(TM-IC)-CD3zeta-IRES-tEGFR-pA-U6-sh CTLA4)】；SEQ ID NO：146是共表达前面所述重组受体(包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3Zeta链)、无 功能EGFR和沉默细胞免疫检查点CBL-B的核酸分子【PD1(ECD)-CD28(TM-IC)-CD3zeta-IRES-tEGFR-pA-U6-sh CBL-B)】；SEQ ID NO：147是共表达前面所述重组受体(包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3Zeta链)、无功能EGFR和沉默细胞免疫检查点SOCS1的核酸分子【PD1(ECD)-CD28(TM-IC)-CD3zeta-IRES-tEGFR-pA-U6-shSOCS1)】；SEQ ID NO：148是共表达前面所述重组受体(包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3Zeta链)和沉默细胞免疫检查点PD1的核酸分子【PD1(ECD-TM)-CD28(IC)-CD3zeta-pA-U6-shPD1)】；SEQ ID NO：149是共表达前面所述重组受体(包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3Zeta链)和沉默细胞免疫检查点CTLA4的核酸分子【PD1(ECD-TM)-CD28(IC)-CD3zeta-pA-U6-sh CTLA4)】；SEQ ID NO：150是共表达前面所述重组受体(包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3Zeta链)和沉默细胞免疫检查点CBL-B的核酸分子【PD1(ECD-TM)-CD28(IC)-CD3zeta-pA-U6-sh CBL-B)】；SEQ ID NO：151是共表达前面所述重组受体(包括PD1的胞外区和跨膜区、CD28的胞内区以及CD3Zeta链)和沉默细胞免疫检查点SOCS1的核酸分子【PD1(ECD-TM)-CD28(IC)-CD3zeta-pA-U6-sh SOCS1)】；SEQ ID NO：152是共表达前面所述重组受体(包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3Zeta链)和沉默细胞免疫检查点PD1的核酸分子【PD1(ECD)-CD28(TM-IC)-CD3zeta-pA-U6-sh PD1)】；SEQ ID NO：153是共表达前面所述重组受体(包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3Zeta链)和沉默细胞免疫检查点CTLA4的核酸分子【PD1(ECD)-CD28(TM-IC)-CD3zeta-pA-U6-sh CTLA4)】；SEQ ID NO：154是共表达前面所述重组受体(包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3Zeta链)和沉默细胞免疫检查点CBL-B的核酸分子【PD1(ECD)-CD28(TM-IC)-CD3zeta-pA-U6-sh CBL-B)】；SEQ ID NO：155是共表达前面所述重组受体(包括PD1的胞外区、CD28的跨膜区和胞内区以及CD3Zeta链)和沉默细胞免疫检查点SOCS1的核酸分子【PD1(ECD)-CD28(TM-IC)-CD3zeta-pA-U6-shSOCS1)】。利用根据本发明实施例的上述慢病毒，可将表达前面所述重组受体、沉默免疫检查点以及任选的表达无功能EGFR的核酸导入受体淋巴细胞，进而实现前面所述重组受体、任选的无功能EGFR在受体细胞膜上的高效表达，实现免疫抑制机制的阻遏，所获得的受体淋巴细胞的对肿瘤细胞的特异性杀伤强大、有效、安全。
转基因淋巴细胞
另一方面，本发明提出了一种转基因淋巴细胞。根据本发明的实施例，所述转基因淋巴细胞的细胞免疫检查点被沉默，并且所述转基因淋巴细胞表达前面所述的重组受 体，任选地，所述转基因淋巴细胞进一步表达无功能EGFR。本发明实施例的转基因淋巴细胞对肿瘤细胞的特异性杀伤强大、有效、安全。
需要说明的是，待沉默的淋巴细胞免疫检查点的选择不受特别限制，根据本发明的实施例，所述淋巴细胞细胞免疫检查点独立地选自CTLA4、PD1、TIM3、BTLA、LAG-3、IRAK-M、SOCS1、A20、CBL-B的至少之一。上述免疫检查点分子在淋巴细胞中发挥着免疫抑制机制，上述免疫检查点分子的特异性沉默，可有效阻遏免疫抑制机制，进一步提高了本发明实施例的转基因淋巴细胞对肿瘤细胞的特异性杀伤效果。
另外，沉默淋巴细胞免疫检查点的方式也不受特别限制，根据本发明的实施例，所述淋巴细胞细胞免疫检查点被沉默可通过shRNA、反义核酸、核酶、显性负突变、CRISPR-Cas9，CRISPR-Cpf1和锌指核酸酶至少之一实现的。
小发夹RNA或短发夹RNA(shRNA)是siRNA(小干扰RNA)的导入形式，siRNA是一种小RNA分子(由21～25个核苷酸组成)，由Dicer(RNAaseⅢ家族中对双链RNA具有特异性剪切作用的酶)加工而成；siRNA在RNA沉默通路中起中心作用，对特定信使RNA(mRNA)进行降解，为转录水平后调控。
反义核酸包括反义RNA和反义DNA，反义RNA是指能和mRNA完全互补的一段小分子RNA或寡聚核苷酸片段，反义DNA是指能与基因DNA双链中的有义链互补结合的短小DNA分子，反义RNA和反义DNA主要是通过mRNA的翻译和基因DNA的转录而发挥作用的；反义核酸一方面通过与靶mRNA结合形成空间位阻效应，阻止核糖体与mRNA结合，另一方面其与mRNA结合后激活内源性RNA酶或核酶，进而降解mRNA；反义DNA与基因DNA双螺旋的调控区特异结合形成DNA三聚体，或与DNA编码区结合，终止正在转录的mRNA链的延长；反义核酸还可抑制转录后mRNA的加工修饰，如5'端加帽、3'端加尾、中间剪接和内部碱基甲基化等，并阻止成熟mRNA由细胞核向细胞浆内运输，因此，反义RNA是一种有效的沉默目的基因的技术。
核酶是具有催化功能的RNA分子，是生物催化剂，可降解特异的mRNA序列，核酶通过催化转磷酸酯和磷酸二酯键水解反应参与RNA自身剪切、加工过程，与一般的反义RNA相比，核酶具有较稳定的空间结构，不易受到RNA酶的攻击，更重要的是，核酶在切断mRNA后，又可从杂交链上解脱下来，重新结合和切割其它的mRNA分子。
显性负性突变是指某些信号转导蛋白突变后不仅自身无功能，还能抑制或阻断同一细胞内的野生型信号转导蛋白的作用，其主要通过和野生型蛋白形成二聚物的方式实现，这种突变毒性作用大，能显著抑制或阻断细胞内目标信号转导蛋白的作用。
锌指核酸酶由一个DNA识别域和一个非特异性核酸内切酶构成，DNA识别域是由一系列Cys2-His2锌指蛋白串联组成(一般3～4个)，每个锌指蛋白识别并结合一 个特异的三联体碱基，锌指蛋白形成α-β-β二级结构，其中α螺旋的16氨基酸残基决定锌指的DNA结合特异性，骨架结构保守，对决定DNA结合特异性的氨基酸引入序列的改变可以获得新的DNA结合特异性，从而可以针对不同的目的基因设计不同的氨基酸引入序列，实现不同目的基因的特异性沉默。
CRISPR(Clustered regularly interspaced short palindromic repeats规律成簇间隔短回文重复)，是一种基因编辑器，是细菌用以保护自身对抗病毒的一个系统。它可以用来删除、添加、激活或抑制其他生物体的目标基因，这些目标基因包括人细胞内的目标基因。
CRISPR簇是一个广泛存在于细菌和古生菌基因组中的特殊DNA重复序列家族，其序列由一个前导区(Leader)、多个短而高度保守的重复序列(Repeat)和多个间隔区(Spacer)组成。前导区一般位于CRISPR簇上游，是富含AT长度为300～500bp的区域，被认为可能是CRISPR簇的启动子序列。重复序列区长度为21～48bp，含有回文序列，可形成发卡结构。重复序列之间被长度为26～72bp的间隔区隔开。Spacer区域由俘获的外源DNA组成，当含有同样序列的外源DNA入侵时，可被细菌机体识别，并进行剪切使之表达沉默，达到保护自身安全的目的。通过对CRISPR簇的侧翼序列分析发现，在其附近存在一个多态性家族基因。该家族编码的蛋白质均含有可与核酸发生作用的功能域(具有核酸酶、解旋酶、整合酶和聚合酶等活性)，并且与CRISPR区域共同发挥作用，因此被命名为CRISPR关联基因(CRISPR associated)，缩写为Cas。目前发现的Cas包括Cas1～Cas10等多种类型。Cas基因与CRISPR共同进化，共同构成一个高度保守的系统。当细菌抵御噬菌体等外源DNA入侵时，在前导区的调控下，CRISPR被转录为长的RNA前体(Pre RISPR RNA，pre-crRNA)，然后加工成一系列短的含有保守重复序列和间隔区的成熟crRNA，最终识别并结合到与其互补的外源DNA序列上发挥剪切作用。pre-crRNA的加工由Cas家族中的Cas9参与。Cas9含有在氨基末端的RuvC和蛋白质中部的HNH2个独特的活性位点，在crRNA成熟和双链DNA剪切中发挥作用。pre-crRNA转录的同时，与其重复序列互补的反式激活crRNA(Trans-activating crRNA，tracrRNA)也转录出来，并且激发Cas9和双链RNA特异性RNase III核酸酶对pre-crRNA进行加工。加工成熟后，crRNA、tracrRNA和Cas9组成复合体，识别并结合于crRNA互补的序列，然后解开DNA双链，形成R-loop，使crRNA与互补链杂交，另一条链保持游离的单链状态，然后由Cas9中的HNH活性位点剪切crRNA的互补DNA链，RuvC活性位点剪切非互补链，最终引入DNA双链断裂(DSB)。通过人工设计RNA，可以改造形成具有引导作用的sgRNA(short guide RNA)，足以引导Cas9对DNA的定点目标基因切割。
综上所述，shRNA、反义核酸、核酶、显性负突变、CRISPR锌指核酸酶为特异性沉默目标基因的有效手段，沉默基因的手段不受特别限制，本领域技术人员可根据具体的实验目的和条件选择，如本发明实施例所采用的shRNA、反义核酸、核酶、显性负突变，CRISPR或锌指核酸酶的至少之一，实现目的基因的特异性沉默。根据本发明的实施例，淋巴细胞细胞免疫检查点被沉默优选采用shRNA实现。ShRNA所携带的siRNA分子通常是一个长度在10和30之间的碱基对的双重区域。本发明实施例的PD1siRNA被设计为同源于PD1的编码区域，通过mRNA的降解来抑制基因表达。siRNA关联于被称为诱导RNA沉默复合物(RISC)的多重蛋白复合物，在此期间正义链被酶裂解。被激活的RISC中基于序列同源性，指引RISC到对应的mRNA；相同的核酸酶切割靶向PD1，产生特定基因PD1沉默，抑制特定基因PD1的表达。siRNA以shRNA的形式导入细胞(shRNA包含大约18-23的核苷酸siRNA序列，后跟一个9-15长度的核苷酸环和一个siRNA序列的反向补充)，shRNA的设计较好的避免了在3’UTR细胞基因中的匹配点；确保了适当的链选择。一个单一siRNA分子可被重复应用于多靶向mRNA分子的分裂。RNAi(RNA干扰)可通过引入合成siRNA的方式被诱导。根据本发明的实施例，本发明实施例的shRNA不断产自细胞内，因此其效果更加持久，从而延长shRNA周期，本发明实施例采用的shRNA具有高效、特异性的沉默细胞免疫检查点的作用，细胞免疫检查点的成功沉默，使得转基因淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的特性，在肿瘤病人体内的增殖和生存能力得到进一步提高，对肿瘤的定向杀伤作用效果更加显著。
具体地，根据本发明的实施例，所述淋巴细胞是抗原特异性T淋巴细胞或肿瘤浸润T淋巴细胞或自然杀伤T淋巴细胞或自然杀伤细胞。根据本发明实施例的抗原特异性T淋巴细胞、肿瘤浸润T淋巴细胞、外周血T淋巴细胞、自然杀伤T淋巴细胞或自然杀伤细胞，可实现对肿瘤细胞的特异性免疫杀伤，特异性强、杀伤力度大、安全性高。
构建体
另一方面，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前面所述的重组受体；第二核酸分子，所述第二核酸分子沉默细胞免疫检查点；以及任选地，所述构建体进一步包括：第三核酸分子，所述第三核酸分子编码无功能EGFR，其中，所述细胞免疫检查点、所述无功能EGFR是如前面所定义的。利用根据本发明实施例的上述构建体，可将表达前面所述重组受体、沉默免疫检查点以及任选的表达无功能EGFR的核酸导入受体淋巴细胞，进而实现前面所述重组受体、任选的无功能EGFR在受体细胞膜上的高效表达，实现免疫抑制机制的阻遏，所获得的受体淋巴细胞的对肿瘤细胞的特异性杀伤强大、有效、安全。
具体地，所述第一核酸分子、所述第二核酸分子和任选的所述第三核酸分子被设置在前面所述的淋巴细胞中表达所述重组受体、沉默细胞免疫检查点和表达任选的无功能EGFR，并且所述重组受体与任选的所述无功能EGFR呈非融合形式。利用根据本发明实施例的上述构建体导入前面所述的淋巴细胞，所获得的淋巴细胞对肿瘤细胞的特异性杀伤更强大、更有效、更安全。
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重组受体、细胞免疫检查点shRNA以及任选的无功能EGFR受体分别独立地表达的，其中，需要说明的是，此处的表达既指受体的表达又指RNA转录。
启动子：根据本发明具体的实施例，所述构建体可进一步包括：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；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；以及任选的第三启动子，所述任选的第三启动子与所述第三核酸分子可操作地连接。上述第一、第二以及任选的第三启动子可分别独立地启动表达第一、第二以及任选的第三核酸分子，进而更加有利于相应核酸分子表达的调控。
其中，根据本方明的实施例，所述第一启动子、所述第二启动子、所述第三启动子分别独立地选自U6，H1，CMV，EF-1，LTR或RSV启动子。发明人发现，U6，H1，CMV，EF-1，LTR或RSV启动子能够高效启动表达第一、第二以及任选的第三核酸分子，第一、第二以及任选的第三核酸分子的表达效率显著提高。
内部核糖体进入位点序列(IRES)：当所述构建体包括第一核酸分子、第二核酸分子和第三核酸分子，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。内部核糖体进入位点序列的引入，使得第三核酸分子的起始表达不依赖5’帽子结构，并且第一和第三核酸分子成比例表达，进而更加有利于表达调控，所获得的转基因淋巴细胞的治疗安全性更高。
连接肽：当所述构建体包括第一核酸分子、第二核酸分子和第三核酸分子，所述构建体进一步包括:第四核酸分子，所述第四核酸分子设置在所述第一核酸分子与所述第三核酸分子之间，并且所述第四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。第四连接肽的引入使得所表达的重组受体以及无功能EGFR呈非融合状态表达在淋巴细胞膜上。
根据本发明的实施例，上述连接肽具有SEQ ID NO：157～160所示的氨基酸序列，这些连接肽为2A自切割连接肽。2A连接肽在口足病病毒(FMDV)中被发现，通常为具有19～22个氨基酸的寡肽，定位在小核糖核酸病毒家族的膜蛋白之间。FMDV病毒的2A自切割肽能够自切割，进而产生成熟病毒蛋白，这就是所熟知的翻译效应“停止前 进”或“停止搬运”。切割位点位于C-末端的最后一个甘氨酸和2B下游蛋白的第一个脯氨酸之间(-LLNFDLLKLAGDVESNPG↓P-)。目前，已经成功在其他病毒mRNA分子发现了2A类似序列，包括猪捷申病毒-1 2A(P2A，序列如SEQ ID NO：158所示)，thosea asigna病毒2A(T2A，序列如SEQ ID NO：157所示)，马鼻炎A病毒2A(E2A，序列如SEQ ID NO：159)，质型多角体病毒(BmCPV 2A)和软化病病毒(BmIFV 2A)。发明人通过筛选实验发现，具有自身切割能力的SEQ ID NO：157～160所示氨基酸序列的连接肽设置在第一核酸分子与第三核酸分子之间，其自身切割的功能在受体细胞中能够得到很好地发挥，获得的重组受体以及无功能EGFR呈非融合状态表达在淋巴细胞膜上的效率和成功率进一步显著提高。
通过上述内部核糖体进入位点序列、或第一、第二、第三启动子或第三核酸分子的引入，使得细胞免疫检查点被高效沉默和无功能EGFR受体高效地表达以及上述重组受体高效地表达在本发明实施例的转基因淋巴细胞膜上，并且无功能EGFR受体和重组受体呈非融合状态表达在淋巴细胞膜上，从而高效抑制了免疫检查点的免疫负调控和保证了重组受体增强免疫的生物学作用，或有效实现了转基因淋巴细胞的及时清除，从而使得淋巴细胞在肿瘤环境中的成活率大大提高，淋巴细胞的靶向杀伤作用更加显著，免疫杀伤的安全性进一步提高。
另外，根据本发明的具体实施例，所述构建体的载体是非致病性病毒载体，所述病毒载体包括选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体的至少之一。本发明实施例中的构建体载体的致病位点经过修饰或突变，已丧失病毒的致病性，进而在根据本发明实施例的非致病性病毒载体介导下的治疗的安全性更高。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，进而可实现广谱而高效的感染效率。
根据本发明的具体实施例，以构建一个慢病毒载体为例，发明人为了构建一个慢病毒载体，在某些病毒序列的位置，将目的核酸插入到病毒基因组中，从而产生复制缺陷的病毒。为了产生病毒体，发明人进而构建包装细胞系(包含gag，pol和env基因，但不包括LTR和包装成分)。发明人将含有目的基因的重组质粒，连同慢病毒LTR和包装序列，一起引入包装细胞系中。包装序列允许重组质粒RNA转录产物被包装到病毒颗粒中，然后被分泌到培养基中。进而发明人收集包含重组慢病毒的基质，有选择性地浓缩，并用于基因转移。慢载体可以感染多种细胞类型，包括可分裂细胞和不可分裂细胞。
另外，根据本发明的实施例，本发明实施例的慢病毒是复合慢病毒，除了常见的慢 病毒基因gag，pol和env，还包含有调控和结构功能的其他基因。慢病毒载体是本领域技术人员所熟知的，慢病毒包括：人类免疫缺陷病毒HIV–1，HIV–2和猿猴免疫缺陷病毒SIV。慢病毒载体通过多重衰减艾滋病毒致病基因产生，例如全部删除基因env，vif，vpr，vpu和nef，使慢病毒载体形成生物安全型载体。重组慢病毒载体能够感染非分裂细胞，同时可用于体内和体外基因转移和核酸序列表达。例如：在合适的宿主细胞中，和带有包装功能(gag，pol，env，rev和tat)的两个或更多的载体一起，能够感染非分裂细胞。重组病毒的靶向性，是通过抗体或特定配体(靶向特定细胞类型受体)与膜蛋白的结合来实现的。同时，重组病毒的靶向性通过插入一个有效序列(包括调控区域)到病毒载体中，连同另一个编码了特定靶细胞上的受体的配体的基因，使载体具有了特定的靶向。各种有用的慢病毒载体，以及各种方法和操作等产生的载体，用于改变细胞的表达。根据本发明的实施例，本发明实施例的慢病毒载体可有效运送和共表达shRNA(siRNA的转运形式)，该小shRNA可以有效抑制PD1或CTLA4或CBL-B的表达。
根据本发明的实施例，本发明实施例的腺关联病毒载体(AAV)可使用一种或多种为人熟知的血清类型腺关联病毒载体的DNA构建。本领域技术人员构建一个合适的腺关联病毒载体，以此携带和共表达小发夹RNA，该小发夹RNA可以抑制PD1等基因的表达。
另外，根据本发明的实施例，本发明实施例的也包含微基因。微基因意味着用组合(选定的核苷酸序列和可操作的必要的相关连接序列)来指导转化、转录和/或基因产物在体内或体外的宿主细胞中的表达。应用“可操作的连接”序列包含连续目的基因的表达控制序列，和作用于反式或远距离控制目的基因的表达控制序列。
另外，本发明实施例的载体还包括常规控制元素，在和质粒载体一起的细胞转染或/和病毒载体一起的细胞感染中，这些元素允许转录、转化和/或小发夹RNA的表达。大量的表达控制序列(包括天然的，可诱导和/或特定组织的启动子)可能被使用。根据本发明的实施例，表达shRNA的启动子为RNA聚合酶启动子。同时，根据本发明的实施例，启动子为选自U6，H1，pol I，pol II and pol III的RAN聚合酶启动子。根据本发明的实施例，启动子为组织特异型启动子。根据本发明的实施例，启动子为诱导型启动子。根据本发明的实施例，启动子为选自基于所选载体的启动子。根据本发明的实施例，当选择慢病毒载体时，启动子为U6，H1，CMV IE基因，EF-1α，泛素C，或磷酸甘油激酶(PGK)启动子。其他常规表达控制序列包括可选标记或报告基因，包括编码遗传霉素，潮霉素，氨苄青霉素或嘌呤霉素耐药性等的核苷酸序列。载体的其他组件包括复制起点。
构建载体的技术为本领域技术人员所熟知的，这些技术包括常规克隆技术，例如在本发明实施例中所使用的shRNA、聚合酶链反应和任何适当的提供所需的核苷酸序列的方法。
根据本发明的实施例，发明人构建了共表达小发夹RNA(shRNA)(用来抑制免疫检查点)和任选的无功能EGFR受体以及重组受体的病毒载体。本发明实施例的运送沉默PD1siRNA的小发夹RNA和表达任选的无功能EGFR受体的核酸分子以及表达重组受体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
再一方面，本发明提出了一种制备前面所述的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。利用根据本发明实施例的上述方法，可简便、高效地获得前面所述的转基因淋巴细胞，如前所述，所获得的转基因淋巴细胞对肿瘤细胞的特异性杀伤强大、有效、安全。
治疗癌症的治疗组合物
再一方面，本发明提出了一种用于治疗癌症的治疗组合物。根据本发明的实施例，所述治疗组合物包括：前面所述的构建体、前面所述的慢病毒、前面所述的转基因淋巴细胞、前面所述的重组受体或者前面所述的核酸。利用根据本发明实施例的治疗组合物，能够实现对肿瘤细胞的特异、强大、有效、安全地杀伤。
根据本发明的实施例，提供给患者的本发明实施例的治疗组合物，较好的应用于生物兼容溶液或可接受的药学运载载体。作为准备的各种治疗组合物被悬浮或溶解在医药上或生理上可接受的载体，如生理盐水；等渗的盐溶液或其他精于此道的人的比较明显的配方中。适当的载体在很大程度上取决于给药途径。其他有水和无水的等渗无菌注射液和有水和无水的无菌悬浮液，是医药上可接受的载体。
根据本发明的实施例，足够数量的病毒载体被转导入靶向T细胞中，并提供足够强度的转基因，沉默PD1等免疫检查点和表达任选的无功能EGFR受体以及表达特有的重组受体。治疗试剂的剂量主要取决于治疗状况，年龄，体重，病人的健康程度，从而可能造成病人的变异性。
沉默PD1等免疫检查点和表达任选的无功能EGFR受体以及表达特有的上述重组受体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，所治疗癌症的类型不受特别限制。利用根据本发明实施例的治疗组合物，对PD-L1或PD-L2阳性肿瘤细胞的特异性杀伤效果显著。
提高淋巴细胞免疫杀伤能力的方法
在本发明再一方面，本发明提出了一种提高淋巴细胞免疫杀伤能力的方法。根据本发明的实施例，该方法包括：使所述淋巴细胞的细胞免疫检查点被沉默，以及使所述淋巴细胞表前面所述的重组受体。利用根据本发明实施例的上述方法，能够有效提高淋巴细胞对肿瘤细胞的特异性免疫杀伤。
治疗癌症的方法
在本发明的最后一方面，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：给患者给予前面所述的重组受体、前面所述的核酸、前面所述的构建体或者前面所述的转基因淋巴细胞。利用根据本发明实施例的治疗方法，能够实现对肿瘤病人体内的肿瘤细胞的有效、安全地杀伤。
需要说明的是，本发明中所涉及的“重组受体”为重组蛋白或融合蛋白，该重组受体表达在受体细胞(如淋巴细胞)的膜上，发挥受体蛋白的功能，即能与细胞外专一信号分子结合进而激活细胞内一系列生物化学反应，使细胞对外界刺激产生相应的效应。
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。
在以下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
目的是产生复制缺陷的慢病毒载体，并将慢病毒载体离心收集用于人T淋巴细胞的转导。
下面简要介绍慢病毒载体的产生、收集的实验过程：将293T细胞铺在底面积为150-平方厘米的细胞培养皿中，并根据说明书，使用Express-In(购自Open Biosystems/Thermo Scientific，Waltham，MA)对293T细胞进行病毒转导。每盘细胞加入15μg的慢病毒转基因质粒、5 μg的pVSV-G(VSV糖蛋白表达质粒)、10μg的pCMVR8.74质粒(Gag/Pol/Tat/Rev表达质粒)和174 μl的Express-In(浓度为1 μg/μl)。分别于24小时和48小时收集上清，并使用超速离心机在28，000 rpm(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0.75 ml的RPMI-1640培养基对病毒质粒沉淀进行重悬。
从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0.75 ml的上述重悬的病毒上清和Polybrene(浓度为8 μg/ml)。细胞和病毒质粒的混合液在台式离心机(购自Sorvall ST 40；Thermo Scientific)中离心，离心条件是室温，2500rpm，时间为90分钟。人重组白细胞介素-2(IL-2；购自Novartis，Basel，Switzerland)每隔2～3天加入T淋巴细胞培养液中，IL-2的终浓度为100-IU/ml，在T淋巴细胞培养过程中，保持细胞的密度为0.5×106～1×106/ml。一旦被转导的T淋巴细胞出现休眠，例如细胞生长速度变慢和细胞变小，其中，细胞生长速度和大小是通过Coulter Counter(购自Beckman Coulter)评估的，或被转导的T淋巴细胞在某个计划的时间点上，T淋巴细胞即可用来做功能分析。
本申请的实施例中所用的流式细胞仪为BD FACSCanto II(购自BD Biosciences)，并且流式细胞分析数据使用FlowJo version 7.2.5软件(购自Tree Star，Ashland，OR)进行分析。
抗体依赖性细胞介导的细胞毒作用(ADCC)
在以下实施例中，采用4小时-51Cr-释放法评估抗-EGFR抗体诱导表达无功能EGFR受体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 Na251CrO4(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50μl培养基)，并加入50μl的抗-EGFR抗体(购自Erbitux，Genentech)(终浓度为20μg/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。在以下具体实施中，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铬释放法分析评估重组受体T细胞的细胞毒活性。具体步骤如 下：目标测试细胞用51Cr在37摄氏度下标记1小时。标记后，用含有10％胎牛血清(FCS)的RPMI培养基润洗细胞。润洗后，将细胞重悬在相同的培养基中，重悬细胞的浓度是1×105/ml。转导后T细胞以不同的效靶细胞比值(E：T)加入目标测试细胞悬浮液中，并将细胞种在96-孔中，每孔体积是200微升。将细胞在37度培养箱中培养4小时。4小时后，从每孔中取出30微升的上清放于计数器的96-微孔板进行计数分析。分析仪器是顶级计数NXT微闪烁计数器(购自Packard Bioscience)。所有计数孔中效应细胞的数目是基于T细胞总数来计算的。被标记的目标测试细胞是PD-L1阳性肿瘤细胞。
实施例1构建共表达无功能EGFR受体、沉默PD1的shRNA(shPD1，iPD1)和PD1-CD28-CD3zeta重组受体的载体
本实施例中，发明人将编码有人PD1胞外片段序列、CD28穿膜及胞内段和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，生成表达重组受体的慢病毒质粒(LV-PD1-CD28-CD3ζ(LV-PD1-CD28z)。包含IRES和表达无功能EGFR受体的序列被克隆进LV-PD1-CD28-CD3ζ载体质粒，构建成LV-PD1-CD28-CD3ζ-tEGFR(M)(LV-PD1-CD28z/M)。包含U6和沉默免疫检查点的shRNA的序列，如shPD1的序列被克隆进LV-PD1-CD28-CD3ζ-tEGFR，构成LV-PD1-CD28-CD3ζ-tEGFR(M)-U6-shPD1(LV-PD1-CD28z/M/iPD1)。图1是慢病毒载体的示意图(tEGFR表示无功能EGFR(M))，包含编码PD1-CD28-CD3ζ重组受体的序列、IRES、编码无功能EGFR受体序列、U6及沉默PD1、CTLA4、Cbl-B或SOCS1的shRNA，。编码PD1-CD28-CD3ζ重组受体的序列在启动子EF-1的启动调控下，沉默免疫检查点的shRNA的序列，如沉默PD1的shRNA序列在启动子U6的启动调控下，表达无功能EGFR受体的序列作为一个单独的mRNA转录单元从IRES序列后开始翻译。
另外，将编码有人PD1胞外片段和跨膜段序列、CD28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的过程如上所述。
实施例2抗EGFR抗体可有效杀伤清除共表达无功能EGFR受体、重组受体和沉默PD1的shRNA(shPD1，iPD1)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-PD1-CD28z/M/iPD1，LV-PD1-CD28z，或空载(LV-GFP)转导过程如前所述。转导后表达无功能EGFR受体的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PD1-CD28-CD3ζ重组受体、shPD1和无功能EGFR受体的T淋巴细胞(LV-PD1-CD28z/M/iPD1转导T淋巴细胞)，但抗EGFR抗体不能介异杀伤只表达PD1-CD28-CD3ζ重组受体的T淋巴细胞(LV-PD1-CD28z转导T淋巴细胞)和只表达GFP的T淋巴细胞(LV-GFP转导T淋巴细胞)，统计数据代表三个孔的平均值±SEM。
实施例3共表达无功能EGFR受体、shPD1和PD1-CD28-CD3ζ重组受体的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-PD1-CD28z/M/iPD1，LV-PD1-CD28z/M，LV-M，或空载(LV-GFP)，转导过程如前所述。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PD-L1阳性的脑胶质瘤细胞的杀伤作用，效靶细胞比例是10：1或25：1或50:1，测量方法采用标准4–小时51铬释放法，其中，4–小时51铬释放法如前所述。
测试结果如图3所示，图3结果显示：共表达PD1-CD28-CD3ζ受体、无功能EGFR受体和shPD1的慢病毒转导的T淋巴细胞(LV-PD1-CD28z/M/iPD1转导的T淋巴细胞)及共表达PD1-CD28-CD3ζ受体和无功能EGFR受体的T淋巴细胞(LV-PD1-CD28z/M转导的T淋巴细胞)都有杀伤PD-L1+的脑瘤细胞能力。但是，共表达PD1-CD28-CD3ζ受体、无功能EGFR受体和shPD1的慢病毒转导的T淋巴细胞(LV-PD1-CD28z/M/iPD1转导的T淋 巴细胞)比共表达PD1-CD28-CD3ζ受体和无功能EGFR受体的T淋巴细胞(LV-PD1-CD28z/M转导的T淋巴细胞)具有更强杀伤PD-L1+的脑瘤细胞的能力。仅表达无功能EGFR受体的慢病毒转导的T淋巴细胞(LV-M转导的淋巴细胞)或空载慢病毒转导的T淋巴细胞(对照LV-GFP转导的T淋巴细胞)对PD-L1+脑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 (28)

  1. 一种重组受体，其特征在于，包括：
    细胞免疫检查点分子片段；
    免疫刺激分子片段；以及
    T细胞受体zeta链。
  2. 根据权利要求1所述的重组受体，其特征在于，所述细胞免疫检查点分子为PD1。
  3. 根据权利要求2所述的重组受体，其特征在于，所述细胞免疫检查点分子片段包括PD1的胞外区以及任选的跨膜区，所述免疫刺激分子片段包括CD28的胞内区以及任选的跨膜区。
  4. 根据权利要求3所述的重组受体，其特征在于，包括：
    (a)所述PD1的胞外区和跨膜区；以及
    (b)所述CD28的胞内区，
    或者包括：
    (i)所述PD1的胞外区；以及
    (ii)所述CD28的胞内区和跨膜区。
  5. 根据权利要求1所述的重组受体，其特征在于，所述T细胞受体zeta链为CD3 zeta链。
  6. 根据权利要求1所述的重组受体，其特征在于，所述细胞免疫检查点分子片段的C端与所述免疫刺激分子片段的N端相连，所述免疫刺激分子片段的C端与所述T细胞受体zeta链的N端相连。
  7. 一种重组受体，其特征在于，所述重组受体具有SEQ ID NO：1或2所示的氨基酸序列。
  8. 一种核酸，其特征在于，所述核酸编码权利要求1～7任一项所述的重组受体，
    任选地，所述核酸具有SEQ ID NO：3或4所示的核苷酸序列。
  9. 一种慢病毒，其特征在于，所述慢病毒携带下列核酸分子：
    (a)编码权利要求1～7任一项所述重组受体的核酸分子，所述重组受体具有SEQ ID NO：1或2所示的氨基酸序列，所述编码重组受体的核酸分子具有SEQ ID NO：3或4所示的核苷酸序列；
    (b)沉默细胞免疫检查点的核酸分子，所述沉默细胞免疫检查点的核酸分子的核苷酸序列为选自SEQ ID NO：5～137的至少之一；
    任选地，进一步携带编码无功能EGFR的核酸分子，所述无功能EGFR具有SEQ ID NO：138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：139所示的核苷酸序列。
  10. 一种慢病毒，其特征在于，所述慢病毒携带含有SEQ ID NO：140～155所示的核苷 酸序列。
  11. 一种转基因淋巴细胞，其特征在于，所述转基因淋巴细胞的细胞免疫检查点被沉默，并且所述转基因淋巴细胞表达权利要求1～7任一项所述的重组受体，
    任选地，所述转基因淋巴细胞进一步表达无功能EGFR。
  12. 根据权利要求11所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点独立地选自CTLA4、PD1、TIM3、BTLA、LAG-3、IRAK-M、SOCS1、A20、CBL-B的至少之一；
    优选地，所述淋巴细胞细胞免疫检查点为CTLA4、PD1、SOCS1或CBL-B。
  13. 根据权利要求12所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点被沉默是通过shRNA、反义核酸、核酶、显性负突变、CRISPR-Cas9，CRISPR-Cpf1，和锌指核酸酶至少之一实现的。
  14. 根据权利要求13所述的转基因淋巴细胞，其特征在于，所述淋巴细胞细胞免疫检查点被沉默是通过shRNA实现的。
  15. 根据权利要求11所述的转基因淋巴细胞，其特征在于，所述淋巴细胞是抗原特异性T淋巴细胞，
    任选地，所述淋巴细胞是肿瘤浸润T淋巴细胞，
    任选地，所述淋巴细胞是外周血T淋巴细胞，
    任选地，所述淋巴细胞是自然杀伤T淋巴细胞，
    任选地，所述淋巴细胞是自然杀伤细胞。
  16. 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权利要求1～7任一项所述的重组受体；
    第二核酸分子，所述第二核酸分子沉默细胞免疫检查点；以及
    任选地，所述构建体进一步包括：第三核酸分子，所述第三核酸分子编码无功能EGFR，
    其中，所述细胞免疫检查点、所述无功能EGFR是如权利要求9、12任一项中所定义的。
  17. 根据权利要求16所述的构建体，其特征在于，所述第一核酸分子、所述第二核酸分子和任选的所述第三核酸分子被设置在权利要求11～15任一项所述的淋巴细胞中表达所述重组受体、沉默细胞免疫检查点和表达任选的无功能EGFR，并且所述重组受体与任选的所述无功能EGFR呈非融合形式。
  18. 根据权利要求16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；
    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；以及
    任选的第三启动子，所述任选的第三启动子与所述第三核酸分子可操作地连接。
  19. 根据权利要求18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、所述第三启动子分别独立地选自U6，H1，CMV，EF-1，LTR或RSV启动子。
  20. 根据权利要求16所述的构建体，其特征在于，所述构建体包括第一核酸分子、第 二核酸分子和第三核酸分子，并且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：156所示的核苷酸序列。
  21. 根据权利要求16所述的构建体，其特征在于，所述构建体包括第一核酸分子、第二核酸分子和第三核酸分子，并且所述构建体进一步包括:
    第四核酸分子，所述第四核酸分子设置在所述第一核酸分子与所述第三核酸分子之间，并且所述第四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。
  22. 根据权利要求21所述的构建体，其特征在于，所述连接肽具有SEQ ID NO：157～160所示的氨基酸序列。
  23. 根据权利要求16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。
  24. 根据权利要求23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。
  25. 一种制备权利要求11～15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16～24任一项所述的构建体或者权利要求9～10任一项所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。
  26. 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16～24任一项所述的构建体、权利要求9～10任一项所述的慢病毒、权利要求11～15任一项所述的转基因淋巴细胞、权利要求1～7任一项所述的重组受体或者权利要求8所述的核酸。
  27. 一种提高淋巴细胞免疫杀伤能力的方法，其特征在于，包括：
    使所述淋巴细胞的细胞免疫检查点被沉默，以及
    使所述淋巴细胞表达权利要求1～7任一项所述的重组受体。
  28. 一种治疗癌症的方法，其特征在于，包括：
    给予癌症患者权利要求16～24任一项所述的构建体、权利要求9～10任一项所述的慢病毒、权利要求11～15任一项所述的转基因淋巴细胞、权利要求1～7任一项所述的重组受体或者权利要求8所述的核酸。
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