WO2018006881A1 - 重组免疫检查点受体及其应用 - Google Patents

重组免疫检查点受体及其应用 Download PDF

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WO2018006881A1
WO2018006881A1 PCT/CN2017/092377 CN2017092377W WO2018006881A1 WO 2018006881 A1 WO2018006881 A1 WO 2018006881A1 CN 2017092377 W CN2017092377 W CN 2017092377W WO 2018006881 A1 WO2018006881 A1 WO 2018006881A1
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nucleic acid
construct
lymphocytes
seq
optionally
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PCT/CN2017/092377
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English (en)
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 recombinant immunological checkpoint receptors and uses thereof, and more particularly, to recombinant receptors, nucleic acids, transgenic lymphocytes, constructs, methods for preparing transgenic lymphocytes, A therapeutic composition for treating cancer and a method for 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 construct.
  • the construct carries a nucleic acid as described above.
  • the construct of the embodiment of the present invention is introduced into the recipient lymphocyte, and the recombinant receptor encoded by the nucleic acid carried by the construct is transmembranely expressed in lymphocytes, and the specific killing effect of the lymphocyte on the tumor cell is remarkably improved.
  • the construct may further comprise at least one of the following additional technical features:
  • the construct further carries a nucleic acid encoding a non-functional EGFR having the nucleotide sequence set forth in SEQ ID NO: 5.
  • 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 construct of the embodiment of the present invention is introduced into the recipient lymphocyte, and the expression of the non-functional EGFR receptor can effectively ensure the targeted killing effect of the lymphocyte on the tumor cell, and if the patient has serious adverse reactions, the lymphocyte can be It is cleared by the anti-EGFR antibody, thereby improving the safety of the construct of the embodiment of the present invention for treating tumor patients.
  • the construct further carries an internal ribosome entry site sequence having the nucleotide sequence set forth in SEQ ID NO: 6, and the internal ribosome
  • the entry site sequence is placed between the nucleic acid described above and the nucleic acid encoding a non-functional EGFR.
  • an internal ribosome entry site sequence allows the initial expression of a nucleic acid encoding a non-functional EGFR to be independent of the 5' cap structure, and the nucleic acid encoding the recombinant receptor is expressed proportionally to the nucleic acid encoding the non-functional EGFR, thereby further facilitating Expression regulation, introduction of the construct of the examples of the present invention into recipient lymphocytes, and the obtained transgenic lymphocytes are more safe to treat.
  • the construct further carries a nucleic acid encoding a linker peptide having the nucleotide sequence shown in SEQ ID NOS: 7 to 10, and the nucleic acid encoding the linker peptide Between the nucleic acid described above and the nucleic acid encoding a non-functional EGFR.
  • the construct of the embodiment of the present invention is introduced into a recipient lymphocyte, the encoded linker peptide is cleaved in the lymphocyte, and the introduction of the linker peptide allows expression of the recombinant receptor and non-function EGFR is expressed in the non-fusion state on the lymphocyte membrane.
  • the construct further carries a first promoter operably linked to a nucleic acid as described above, optionally, the first promoter comprises a CMV selected from the group consisting of EF-1, LTR or RSV promoter.
  • the first promoter described above can independently initiate expression of the first nucleic acid molecule, thereby further facilitating regulation of expression of the corresponding nucleic acid molecule.
  • the inventors have found that the CMV, EF-1, LTR or RSV promoter is capable of efficiently initiating expression of the nucleic acid described above, and the expression efficiency of the aforementioned nucleic acid is remarkably improved.
  • the construct further carries a second promoter operably linked to the nucleic acid encoding a non-functional EGFR, optionally the second promoter comprises From CMV, EF-1, LTR Or the RSV promoter.
  • the second promoter described above can independently initiate expression of a nucleic acid encoding a non-functional EGFR, thereby further facilitating expression regulation of a nucleic acid encoding a non-functional EGFR.
  • the inventors have found that the CMV, EF-1, LTR or RSV promoter is capable of efficiently initiating expression of a nucleic acid encoding a non-functional EGFR, and the expression efficiency of a nucleic acid encoding a non-functional EGFR is significantly improved.
  • the vector of the construct is a retroviral vector, a lentiviral vector or an adeno-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 proposes a construct.
  • the construct carries the following nucleic acid molecule: (1) a nucleic acid molecule encoding a fragment of an immunological checkpoint molecule having the amino acid sequence set forth in SEQ ID NO: 11 or 12, The nucleic acid molecule encoding an immunological checkpoint molecule fragment has the nucleotide sequence set forth in SEQ ID NO: 13 or 14; (2) a nucleic acid molecule encoding an immunostimulatory molecule fragment having SEQ ID NO: The amino acid sequence of 15 or 16, wherein the nucleic acid molecule encoding the immunostimulatory molecule fragment has the nucleotide sequence of SEQ ID NO: 17 or 18; and (3) the nucleic acid molecule encoding the T cell receptor zeta chain, The T cell receptor zeta chain has the amino acid sequence set forth in SEQ ID NO: 19, and the nucleic acid molecule encoding the T cell receptor zeta chain has the
  • the construct of the embodiment of the present invention is introduced into the recipient lymphocyte, and the molecular fragment encoded by the nucleic acid carried by the construct constitutes a fusion receptor protein, and is expressed in a lymphocyte transmembrane, and the lymphocyte is specific to the tumor cell.
  • the sexual killing effect is significantly improved.
  • the above-described construct may further include at least one of the following additional technical features:
  • the construct further carries a nucleic acid molecule encoding a non-functional EGFR having the nucleotide sequence set forth in SEQ ID NO: 5, the non-functional EGFR having The amino acid sequence shown in SEQ ID NO:21.
  • the construct of the embodiment of the present invention is introduced into the recipient lymphocyte, and the expression of the non-functional EGFR receptor can effectively ensure the targeted killing effect of the lymphocyte on the tumor cell, if the patient has serious malaise In response, the lymphocytes can be cleared by the anti-EGFR antibody, thereby improving the safety of the tumor, lymphocyte and the like for treating tumor patients in the examples of the present invention.
  • the construct further carries a nucleic acid molecule of an internal ribosome entry site sequence, the nucleic acid molecule of the internal ribosome entry site sequence having the nucleotide sequence set forth in SEQ ID NO:6, And the nucleic acid molecule encoding an internal ribosome entry site sequence is disposed between the nucleic acid molecule encoding the T cell receptor zeta chain and the nucleic acid molecule encoding the non-functional EGFR.
  • the introduction of the internal ribosome entry site sequence is more conducive to expression regulation, and the obtained transgenic lymphocytes are more safe to treat.
  • the construct further carries a nucleic acid encoding a linker peptide having the nucleotide sequence shown in SEQ ID NOS: 7 to 10, the linker peptide having SEQ ID NO The amino acid sequence shown in 22 to 25, and the nucleic acid encoding the linker peptide is disposed between the nucleic acid molecule encoding the T cell receptor zeta chain and the nucleic acid molecule encoding the non-functional EGFR.
  • the construct of the embodiment of the present invention is introduced into a recipient lymphocyte, and the encoded linker peptide can be cleaved in the lymphocyte, and the introduction of the linker peptide enables the recombinant receptor composed of the expressed molecular fragment. It is expressed on the lymphocyte membrane in a non-fused state with non-functional EGFR.
  • the construct further carries a first promoter operably linked to the nucleic acid molecule encoding the immunological checkpoint molecule fragment, optionally, the first promoter
  • the promoter includes a promoter selected from the group consisting of CMV, EF-1, LTR or RSV.
  • the CMV, EF-1, LTR or RSV promoter can efficiently initiate the expression of a nucleic acid molecule encoding an immunological checkpoint molecule fragment, a nucleic acid molecule encoding an immunostimulatory molecule fragment, and a T cell receptor zeta chain.
  • the nucleic acid molecule, the expression efficiency of the nucleic acid molecule fragment described above is significantly improved.
  • the construct further carries a second promoter operably linked to the nucleic acid molecule encoding a non-functional EGFR, optionally the second promoter comprises Selected from the CMV, EF-1, LTR or RSV promoters.
  • the CMV, EF-1, LTR or RSV promoter is capable of efficiently initiating expression of the nucleic acid molecule encoding the non-functional EGFR, and the expression efficiency of the nucleic acid molecule encoding the non-functional EGFR is remarkably improved.
  • the vector of the construct is a retroviral vector, a lentiviral vector or an adeno-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 transgenic lymphocyte.
  • the transgenic lymphocytes express a recombinant receptor as described above, optionally, the transgenic lymphocytes express a non-functional EGFR.
  • the specific killing effect of the transgenic lymphocytes of the embodiments of the present invention on tumor cells is strong and safe.
  • the transgenic lymphocyte may further include at least one of the following additional technical features:
  • 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, tumor infiltrating T lymphocytes, peripheral blood T lymphocytes, natural killer T lymphocytes or natural killer cells according to the embodiments of the present invention, specific immune killing of tumor cells can be achieved, and the safety is high.
  • the invention provides a method of preparing the transgenic lymphocytes described above.
  • the method comprises introducing the construct described above into lymphocytes or T lymphocytes.
  • the invention provides a therapeutic composition for treating cancer.
  • the therapeutic composition comprises 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 invention provides a method of increasing the ability of lymphocytes to treat immune killing.
  • the method comprises: said lymphocytes expressing a recombinant receptor as described above.
  • 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 and non-functional EGFR receptor 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 and non-functional EGFR receptor 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 (srchomology region 2, SH-2) domains, phosphorylated tyrosine residues in SH-2 and zeta chains in ZAP-70
  • ZAP-70 activation further activates the Ras protein, which ultimately activates lymphocytes.
  • 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 joining sequence of the corresponding molecular fragments in the recombinant protein may be: the C-terminus of the molecular fragment of the cellular immunological checkpoint is linked to the N-terminus of the immunostimulatory molecule fragment, and the C-terminus and T of the immunostimulatory molecular fragment are The N-terminus of the cell receptor zeta chain is linked.
  • the inventors have found that the relevant fragments of the recombinant protein 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 the immunostimulatory signaling pathway. As well as activation of the T cell receptor signaling pathway, its ability to target tumor cells is further enhanced.
  • the recombinant receptor has the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • SEQ ID NO: 1 represents the amino acid sequence of the recombinant receptor (PD1-ECD-TM-CD28-ICD-CD3zeta) comprising the extracellular region and transmembrane region of human PD1, the intracellular region of CD28, and the CD3zeta chain
  • SEQ ID NO: 2 represents the amino acid sequence of the recombinant receptor (PD1-ECD-CD28-TM-ICD-CD3zeta) comprising the human PD1 extracellular domain, the CD28 transmembrane domain and the intracellular domain, and the CD3zeta chain.
  • 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.
  • SEQ ID NO: The nucleotide sequence shown in Figure 3 encodes a recombinant receptor (PD1-ECD-TM-CD28-ICD-CD3zeta) comprising the extracellular and transmembrane regions of human PD1, the intracellular region of CD28, and the CD3zeta chain
  • SEQ ID NO:4 The nucleotide sequence shown encodes a recombinant receptor (PD1-ECD-CD28-TM-ICD-CD3zeta) comprising the human PD1 extracellular domain, the CD28 transmembrane domain and the intracellular domain, and the CD3zeta chain.
  • 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 construction.
  • the construct carries a nucleic acid as described above.
  • the construct of the embodiment of the present invention is introduced into the recipient lymphocyte, and the recombinant receptor encoded by the nucleic acid carried by the construct is transmembranely expressed in lymphocytes, and the specific killing effect of the lymphocyte on the tumor cell is remarkably improved.
  • the construct carries the following nucleic acid molecule: (1) a nucleic acid molecule encoding an immunological checkpoint molecule fragment having the amino acid set forth in SEQ ID NO: 11 or 12. a nucleic acid molecule encoding a molecular fragment of an immunological checkpoint having the nucleotide sequence of SEQ ID NO: 13 or 14; wherein the amino acid sequence of SEQ ID NO: 11 is the extracellular domain of human PD1 and transmembrane
  • the amino acid sequence of the region (PD1-ECD-TM), the amino acid sequence shown in SEQ ID NO: 12 is the amino acid sequence of the extracellular region of human PD1 (PD1-ECD);
  • SEQ ID NO: 13 is the encoding for PD1-ECD-TM
  • the nucleotide sequence, SEQ ID NO: 14 is the nucleotide sequence encoding PD1-ECD.
  • SEQ ID NO: 17 is the nucleotide sequence encoding CD28-ICD
  • SEQ ID NO: 18 is the nucleotide sequence encoding CD28-TM-ICD
  • a nucleic acid molecule encoding a T cell receptor zeta chain having the amino acid sequence set forth in SEQ ID NO: 19 the nucleic acid molecule encoding the T cell receptor zeta chain having
  • the construct of the embodiment of the present invention is introduced into the recipient lymphocyte, and the molecular fragment encoded by the nucleic acid carried by the construct constitutes the recombinant receptor, and is expressed in a lymphocyte transmembrane, and the lymphocyte is specific to the tumor cell.
  • the killing effect is significantly improved.
  • the construct further carries a nucleic acid encoding a non-functional EGFR having the nucleotide sequence set forth in SEQ ID NO: 5.
  • 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. Sequence, so non-functional EGFR receptors can be used Suicide markers for lymphocytes.
  • the construct of the embodiment of the present invention is introduced into the recipient lymphocyte, and the expression of the non-functional EGFR receptor can effectively ensure the targeted killing effect of the lymphocyte on the tumor cell, and if the patient has serious adverse reactions, the lymphocyte can be It is cleared by the anti-EGFR antibody, thereby improving the safety of the construct of the embodiment of the present invention for treating tumor patients.
  • the inventors realized that the above recombinant receptor and optionally the non-functional EGFR receptor are independently expressed by at least one of the following methods:
  • the construct further carries an internal ribosome entry site sequence having the sequence set forth in SEQ ID NO:6 a nucleotide sequence, and the internal ribosome entry site sequence is disposed between the nucleic acid described above and the nucleic acid encoding a non-functional EGFR.
  • an internal ribosome entry site sequence allows the initial expression of a nucleic acid encoding a non-functional EGFR to be independent of the 5' cap structure, and the nucleic acid encoding the recombinant receptor is expressed proportionally to the nucleic acid encoding the non-functional EGFR, thereby further facilitating Expression regulation, introduction of the construct of the examples of the present invention into recipient lymphocytes, and the obtained transgenic lymphocytes are more safe to treat.
  • the construct may further further carry a nucleic acid encoding a linker peptide having the nucleotide sequence shown in SEQ ID NOS: 7 to 10, the linker peptide Is a 2A-linked peptide, and the nucleic acid encoding the linker peptide is disposed between the nucleic acid described above and the nucleic acid encoding a non-functional EGFR, wherein the linker peptide encoded by SEQ ID NO: 7 has the SEQ ID NO: 22
  • the amino acid sequence shown in SEQ ID NO: 8 has the amino acid sequence of SEQ ID NO: 13
  • the linker peptide encoded by SEQ ID NO: 9 has the amino acid sequence of SEQ ID NO: 24, SEQ ID NO:
  • the 10-encoding ligation peptide has the amino acid sequence shown in SEQ ID NO: 25.
  • the construct of the embodiment of the present invention is introduced into a recipient lymphocyte, the encoded linker peptide is cleaved in the lymphocyte, and the introduction of the linker peptide allows expression of the recombinant receptor and non-function EGFR is expressed in the non-fusion state on the lymphocyte membrane.
  • the construct may further carry a first promoter operably linked to a nucleic acid as described above, optionally, the first promoter Includes a promoter selected from the group consisting of CMV, EF-1, LTR or RSV.
  • the construct further carries a second promoter operably linked to the nucleic acid encoding a non-functional EGFR, optionally the second promoter comprises From CMV, EF-1, LTR or RSV promoters.
  • the first and second promoters can independently initiate the expression of the recombinant protein and the nucleic acid encoding the non-functional EGFR, thereby further facilitating the expression regulation of the recombinant protein and the nucleic acid encoding the non-functional EGFR.
  • the inventors have found that the CMV, EF-1, LTR or RSV promoter can efficiently initiate the expression of the above recombinant protein, a nucleic acid encoding a non-functional EGFR, and the expression efficiency of the recombinant protein and the nucleic acid encoding the non-functional EGFR is remarkably improved.
  • the non-functional EGFR receptor is efficiently expressed and the above recombinant receptor is efficiently expressed on the transgenic lymphocyte membrane of the present invention, and the non-functional EGFR receptor and the recombinant receptor are expressed in a non-fusion state on the lymphocyte membrane.
  • the targeted killing effect of lymphocytes is more significant, and the safety of immune killing is further improved.
  • the vector of the construct is a retroviral vector, a lentiviral vector or an adeno-associated viral vector.
  • the virus carrier of the embodiment of the invention has a wide range of virus infection during virus packaging and infection, and can infect both terminally differentiated cells and cells in a mitotic phase, and 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 above vector can achieve high-efficiency expression of the carried nucleic acid in the recipient cell, and has high therapeutic efficiency.
  • the inventors in order to construct a lentiviral vector, the inventors inserted a nucleic acid of interest into a viral genome at a position of a certain viral sequence in order to construct a lentiviral vector, thereby producing a replication-defective virus.
  • the inventors further constructed packaging cell lines (containing the gag, pol and env genes, but excluding LTR and packaging components).
  • the inventors introduced a recombinant plasmid containing the gene of interest, together with the lentiviral LTR and the packaging sequence, into a packaging cell line.
  • the packaging sequence allows the recombinant plasmid RNA transcript to be packaged into viral particles which are then secreted into the culture medium.
  • the inventors collected a matrix containing the recombinant lentivirus, selectively concentrated, and used for gene transfer. Slow vectors can infect a variety of cell types, including cleavable cells and non-dividable cells.
  • the lentivirus of the embodiment of the present invention is a complex lentivirus, and in addition to the common lentiviral genes gag, pol and env, other genes having regulatory and structural functions are also included.
  • Lentiviral vectors are well known to those skilled in the art, and lentiviruses include: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors produce a biosafety vector by multiple attenuation of HIV-causing genes, such as deletion of the genes env, vif, vpr, vpu and nef.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and are useful for in vivo and in vitro gene transfer and nucleic acid sequence expression.
  • a suitable host cell together with two or more vectors with packaging functions (gag, pol, env, rev and tat), it is possible to infect non-dividing cells.
  • the targeting of recombinant viruses is achieved by binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
  • the targeting of the recombinant virus confers specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, along with another gene encoding a ligand for the receptor on the particular target cell.
  • 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.
  • the embodiment of the present invention also includes a microgene.
  • Microgene means using a combination (Selected nucleotide sequences and operably necessary related linker sequences) to direct expression of the transform, transcription and/or gene product in host cells in vivo or in vitro.
  • the "operable ligation" sequence is employed to include expression control sequences for a continuous gene of interest, and expression control sequences for trans- or remote control of the gene of interest.
  • the carrier of an embodiment of the invention further includes conventional control elements.
  • a large number of expression control sequences may be used.
  • the promoter is a tissue-specific promoter.
  • the promoter is an inducible promoter.
  • the promoter is selected from a promoter based on the selected vector.
  • the promoter when a lentiviral vector is selected, the promoter is the CMV IE gene, EF-1 ⁇ , ubiquitin C, or phosphoglycerate kinase (PGK) promoter.
  • Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding geneticin, hygromycin, ampicillin or puromycin resistance.
  • Other components of the carrier include an origin of replication.
  • the inventors constructed viral vectors that co-express an optional non-functional EGFR receptor as well as a recombinant receptor.
  • the nucleic acid molecule expressing the optional non-functional EGFR receptor and the viral vector or plasmid expressing the recombinant receptor are complexed in the embodiments of the present invention, and the viral vector or plasmid may be combined with a polymer or other material to increase the stability thereof. Or assist with its targeted movement.
  • the invention provides a transgenic lymphocyte.
  • the transgenic lymphocytes express a recombinant receptor as described above, optionally, the transgenic lymphocytes express a non-functional EGFR.
  • the specific killing effect of the transgenic lymphocytes of the embodiments of the present invention on tumor cells is strong and safe.
  • the lymphocytes are antigen-specific T lymphocytes, tumor infiltrating T lymphocytes, peripheral blood T lymphocytes, natural killer T lymphocytes, and natural killer cells.
  • the antigen-specific T lymphocytes, tumor infiltrating T lymphocytes, peripheral blood T lymphocytes, natural killer T lymphocytes or natural killer cells according to the embodiments of the present invention, specific immune killing of tumor cells can be achieved, and the safety is high.
  • the invention provides a method of making the transgenic lymphocytes described above.
  • the method comprises introducing the construct described above into lymphocytes or T lymphocytes.
  • the invention provides a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: a construct 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 a transgene of sufficient strength to express an optional non-functional EGFR receptor and to express a unique recombinant receptor.
  • the dosage of the therapeutic agent depends primarily on the condition of treatment, age, weight, and the health of the patient, which may result in patient variability.
  • the type of the cancer is not particularly limited, and the specific killing effect of the pharmaceutical composition according to the embodiment of the present invention on PD-L1 positive tumor cells is remarkable.
  • the invention provides a method of increasing lymphocyte immune killing ability.
  • the method comprises: the recombinant receptor described above in the lymphocyte table.
  • 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.
  • 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).
  • 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 are then plated in 96-well plates (5 x 103 per well) One cell, 50 ⁇ l of medium was added, and 50 ⁇ l of anti-EGFR antibody (purchased from Erbitux, Genentech) (final concentration of 20 ⁇ g/ml) was added, and pre-incubated for 30 minutes at room temperature. The antibody-containing medium was then added. The spontaneous release of 51Cr was detected by switching to a common medium. Triton X-100 was added to a final concentration of 1% to ensure maximum release of 51Cr.
  • human PBMC effector cells
  • 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: Target test cells were labeled with 51 Cr at 37 degrees Celsius for 1 hour. After labeling, the cells were rinsed with RPMI medium containing 10% fetal bovine serum (FCS). After rinsing, the cells were resuspended in the same medium, and the concentration of the resuspended cells was 1 ⁇ 10 5 /ml. After transduction, T cells were added to the target test cell suspension at different target cell ratios (E:T), and the cells were seeded in 96-wells at a volume of 200 microliters per well.
  • 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 co-expressing a non-functional EGFR receptor 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.
  • Figure 1 is a schematic representation of a lentiviral vector (where E represents the extracellular domain; tEGFR represents a non-functional EGFR), comprises a sequence encoding a PD1-CD28-CD3 ⁇ recombinant receptor, an IRES, and a non-functional EGFR receptor sequence.
  • the sequence encoding the PD1-CD28-CD3 ⁇ recombinant receptor is under the promoter of EF-1, and the sequence expressing the non-functional EGFR receptor is translated as a single mRNA transcription unit from 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 antibody effectively kills T lymphocytes that co-express non-functional EGFR receptors and recombinant receptors cell
  • 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) 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).
  • FCS Inactivated fetal bovine serum
  • T cells were seeded on a recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were transduced into LV-PD1-CD28-CD3 ⁇ -tEGFR, LV-PD1, respectively.
  • FN ch-296; Retronectin fibronectin fragment
  • lentiviruses were transduced into LV-PD1-CD28-CD3 ⁇ -tEGFR, LV-PD1, respectively.
  • - CD28-CD3 ⁇ or no-load (LV-GFP) transduction process 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 antibody can effectively block T lymphocytes co-expressing PD1-CD28-CD3 ⁇ recombinant receptor and non-functional EGFR receptor, but anti-EGFR antibody can not induce killing and only express PD1-CD28-CD3 ⁇ Recombinant receptor T lymphocytes, anti-EGFR antibodies do not mediate the killing of nap lentivirus-transduced T lymphocytes, and the statistical data represent 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 recombinant cultured fibronectin fragment (FN ch-296; Retronectin) cell culture dish and transduced with lentivirus, and the lentiviruses were transduced into LV-PD1-CD28-CD3 ⁇ -tEGFR, LV-PD1, respectively.
  • FN ch-296; Retronectin fibronectin fragment
  • lentiviruses were transduced into LV-PD1-CD28-CD3 ⁇ -tEGFR, LV-PD1, respectively.
  • - CD28-tEGFR structure shown in Figure 1
  • LV-tEGFR structure shown in Figure 1
  • no-load LV-GFP
  • 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 test results are shown in Figure 3.
  • the results in Figure 3 show that Lentivirus-transduced T lymphocytes (LV-PD1-CD28-CD3 ⁇ -tEGFR T lymphocytes) co-expressing PD1-CD28-CD3 ⁇ receptor and non-functional EGFR receptor ) Brain tumor cells capable of significantly killing PD-L1 + .
  • Lentivirus-transduced T lymphocytes (LV-PD1-CD28-tEGFR T lymphocytes) co-expressing PD1-CD28 receptor (without ligated CD3zeta chain fragment) and non-functional EGFR receptor have no significant killing of PD-L1 + brain Tumor cell function.
  • Lentiviral-transduced T lymphocytes (LV-tEGFR T lymphocytes) expressing no functional EGFR receptor or T lymphocytes transduced with lentiviral (control LV-GFP T lymphocytes) to PD-L1 + brain Tumor cells have no obvious killing effect.

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Abstract

一种重组受体及其应用，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其应用
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201610538767.9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其应用，更具体地，本发明涉及重组受体、核酸、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，特别是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 PCTCN2017092377-appb-000001
Figure PCTCN2017092377-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 PCTCN2017092377-appb-000003
Figure PCTCN2017092377-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，本发明提出了一种构建体。根据本发明的实施例，所述构建体携带前面所述的核酸。将本发明实施例的构建体导入受体淋巴细胞中，构建体所携带的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。
根据本发明的实施例，所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，所述构建体进一步携带编码无功能EGFR的核酸，所述编码无功能EGFR的核酸具有SEQ ID NO:5所示的核苷酸序列。
Figure PCTCN2017092377-appb-000005
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。本发明实施例的构建体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构建体治疗肿瘤病人的安全性。
根据本发明的实施例，所述构建体进一步携带内部核糖体进入位点序列，所述内部核糖体进入位点序列具有SEQ ID NO:6所示的核苷酸序列，并且所述内部核糖体进入位点序列设置于前面所述的核酸和所述编码无功能EGFR的核酸之间。
Figure PCTCN2017092377-appb-000006
Figure PCTCN2017092377-appb-000007
内部核糖体进入位点序列的引入，使得编码无功能EGFR的核酸的起始表达不依赖5’帽子结构，并且编码重组受体的核酸和编码无功能EGFR的核酸成比例表达，进而更加有利于表达调控，将本发明实施例的构建体导入受体淋巴细胞，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，所述构建体进一步携带编码连接肽的核酸，所述编码连接肽的核酸具有SEQ ID NO:7～10所示的核苷酸序列，并且所述编码连接肽的核酸设置于前面所述的核酸和所述编码无功能EGFR的核酸之间。
Figure PCTCN2017092377-appb-000008
根据本发明的实施例，将本发明实施例的构建体导入受体淋巴细胞，所编码的连接肽能够在所述淋巴细胞中被切割，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，所述构建体进一步携带第一启动子，所述第一启动子与前面所述的核酸可操作地连接，任选地，所述第一启动子包括选自CMV，EF-1，LTR或RSV启动子。上述第一启动子可独立地启动表达第一核酸分子，进而更加有利于相应核酸分子表达的调控。发明人发现，CMV，EF-1，LTR或RSV启动子能够高效启动表达前面所述的核酸，前面所述的核酸的表达效率显著提高。
根据本发明的实施例，所述构建体进一步携带第二启动子，所述第二启动子与所述编码无功能EGFR的核酸可操作地连接，任选地，所述第二启动子包括选自CMV，EF-1，LTR 或RSV启动子。上述第二启动子可独立地启动表达编码无功能EGFR的核酸，进而更加有利于编码无功能EGFR的核酸的表达调控。发明人发现，CMV，EF-1，LTR或RSV启动子能够高效启动表达编码无功能EGFR的核酸，编码无功能EGFR的核酸的表达效率显著提高。
根据本发明的实施例，所述构建体的载体为反转录病毒载体、慢病毒载体或腺病毒相关病毒载体。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。
在本发明的第五方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体携带下列核酸分子:(1)编码免疫检查点分子片段的核酸分子，所述免疫检查点分子片段具有SEQ ID NO：11或12所示的氨基酸序列，所述编码免疫检查点分子片段的核酸分子具有SEQ ID NO：13或14所示的核苷酸序列；(2)编码免疫刺激分子片段的核酸分子，所述免疫刺激分子片段具有SEQ ID NO：15或16所示的氨基酸序列，所述编码免疫刺激分子片段的核酸分子具有SEQ ID NO：17或18所示的核苷酸序列；以及(3)编码T细胞受体zeta链的核酸分子，所述T细胞受体zeta链具有SEQ ID NO：19所示的氨基酸序列，所述编码T细胞受体zeta链的核酸分子具有SEQ ID NO：20所示的核苷酸序列。
Figure PCTCN2017092377-appb-000009
Figure PCTCN2017092377-appb-000010
Figure PCTCN2017092377-appb-000011
将本发明实施例的构建体导入受体淋巴细胞中，构建体所携带的核酸所编码的分子片段组成融合受体蛋白，并在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。
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，所述构建体进一步携带编码无功能EGFR的核酸分子，所述编码无功能EGFR的核酸分子具有SEQ ID NO:5所示的核苷酸序列，所述无功能EGFR具有SEQ ID NO:21所示的氨基酸序列。
Figure PCTCN2017092377-appb-000012
如前所述，本发明实施例的构建体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构建体、淋巴细胞等治疗肿瘤病人的安全性。
根据本发明的实施例，所述构建体进一步携带内部核糖体进入位点序列的核酸分子，所述内部核糖体进入位点序列的核酸分子具有SEQ ID NO:6所示的核苷酸序列，并且所述编码内部核糖体进入位点序列的核酸分子设置于所述编码T细胞受体zeta链的核酸分子和所述编码无功能EGFR的核酸分子之间。如前所述，内部核糖体进入位点序列的引入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，所述构建体进一步携带编码连接肽的核酸，所述编码连接肽的核酸具有SEQ ID NO:7～10所示的核苷酸序列，所述连接肽具有SEQ ID NO：22～25所示的氨基酸序列，并且所述编码连接肽的核酸设置于所述编码T细胞受体zeta链的核酸分子和所述编码无功能EGFR的核酸分子之间。
Figure PCTCN2017092377-appb-000013
Figure PCTCN2017092377-appb-000014
如前所述，将本发明实施例的构建体导入受体淋巴细胞，所编码的连接肽能够在所述淋巴细胞中被切割，连接肽的引入使得所表达的分子片段所组成的重组受体与无功能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，所述构建体进一步携带第一启动子，所述第一启动子与所述编码免疫检查点分子片段的核酸分子可操作地连接，任选地，所述第一启动子包括选自CMV，EF-1，LTR或RSV启动子。如前所述，CMV，EF-1，LTR或RSV启动子能够高效启动表达前面所述的编码免疫检查点分子片段的核酸分子、编码免疫刺激分子片段的核酸分子以及编码T细胞受体zeta链的核酸分子，前面所述的核酸分子片段的表达效率显著提高。
根据本发明的实施例，所述构建体进一步携带第二启动子，所述第二启动子与所述编码无功能EGFR的核酸分子可操作地连接，任选地，所述第二启动子包括选自，CMV，EF-1，LTR或RSV启动子。如前所述，CMV，EF-1，LTR或RSV启动子能够高效启动表达所述的编码无功能EGFR的核酸分子，编码无功能EGFR的核酸分子的表达效率显著提高。
根据本发明的实施例，所述构建体的载体为反转录病毒载体、慢病毒载体或腺病毒相关病毒载体。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表达前面所述的重组受体，任选地，所述转基因淋巴细胞表达无功能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效果强、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，所述淋巴细胞为抗原特异性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、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，安全性高。
在本发明的第七方面，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显著提高、安全。
在本发明的第八方面，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组合物，能够实现对肿瘤细胞的有效、安全地杀伤。
在本发明的第九方面，本发明提出了一种提高淋巴细胞治疗免疫杀伤能力的方法。根据本发明的实施例，所述方法包括：使所述淋巴细胞表达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的特异性免疫杀伤。
在本发明的第十方面，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重组受体和无功能EGFR受体的淋巴细胞被抗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重组受体和无功能EGFR受体的淋巴细胞杀伤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。
重组受体蛋白
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(srchomology 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，上述重组蛋白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蛋白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表示包含人PD1胞外区和跨膜区、CD28胞内区以及CD3zeta链的重组受体(PD1-ECD-TM-CD28-ICD-CD3zeta)的氨基酸序列；SEQ ID NO：2表示包含人PD1胞外区、CD28跨膜区和胞内区以及CD3zeta链的重组受体(PD1-ECD-CD28-TM-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包含人PD1胞外区和跨膜区、CD28胞内区以及CD3zeta链的重组受体(PD1-ECD-TM-CD28-ICD-CD3zeta)，SEQ ID NO：4所示的核苷酸序列编码包含人PD1胞外区、CD28跨膜区和胞内区以及CD3zeta链的重组受体(PD1-ECD-CD28-TM-ICD-CD3zeta)。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。
构建体
另一方面，本发明提出了一种构建。根据本发明的实施例，所述构建体携带前面所述的核酸。将本发明实施例的构建体导入受体淋巴细胞中，构建体所携带的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。
或者，根据本发明的实施例，所述构建体携带下列核酸分子:(1)编码免疫检查点分子片段的核酸分子，所述免疫检查点分子片段具有SEQ ID NO：11或12所示的氨基酸序列，所述编码免疫检查点分子片段的核酸分子具有SEQ ID NO：13或14所示的核苷酸序列；其中，SEQ ID NO：11所示的氨基酸序列是人PD1胞外区和跨膜区的氨基酸序列(PD1-ECD-TM)，SEQ ID NO：12所示的氨基酸序列是人PD1胞外区的氨基酸序列(PD1-ECD)；SEQ ID NO：13是编码PD1-ECD-TM的核苷酸序列，SEQ ID NO：14是编码PD1-ECD的核苷酸序列。(2)编码免疫刺激分子片段的核酸分子，所述免疫刺激分子片段具有SEQ ID NO：15或16所示的氨基酸序列，所述编码免疫刺激分子片段的核酸分子具有SEQ ID NO：17或18所示的核苷酸序列，其中，SEQ ID NO：15所示的氨基酸序列是人CD28胞内区的氨基酸序列(CD28-ICD)，SEQ ID NO：16所示的氨基酸序列是人CD28跨膜区和胞内区的氨基酸序列(CD28-TM-ICD)，SEQ ID NO：17是编码CD28-ICD的核苷酸序列，SEQ ID NO：18是编码CD28-TM-ICD的核苷酸序列；以及(3)编码T细胞受体zeta链的核酸分子，所述T细胞受体zeta链具有SEQ ID NO：19所示的氨基酸序列，所述编码T细胞受体zeta链的核酸分子具有SEQ ID NO：20所示的核苷酸序列。将本发明实施例的构建体导入受体淋巴细胞中，构建体所携带的核酸所编码的分子片段组成上述重组受体，并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。
根据本发明的具体实施例，所述构建体进一步携带编码无功能EGFR的核酸，所述编码无功能EGFR的核酸具有SEQ ID NO:5所示的核苷酸序列。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。本发明实施例的构建体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构建体治疗肿瘤病人的安全性。
其中，发明人是通过如下方式的至少之一实现上述重组受体以及任选的无功能EGFR受体分别独立地表达的：
携带内部核糖体进入位点序列(IRES)：根据本发明的实施例，所述构建体进一步携带内部核糖体进入位点序列，所述内部核糖体进入位点序列具有SEQ ID NO:6所示的核苷酸序列，并且所述内部核糖体进入位点序列设置于前面所述的核酸和所述编码无功能EGFR的核酸之间。内部核糖体进入位点序列的引入，使得编码无功能EGFR的核酸的起始表达不依赖5’帽子结构，并且编码重组受体的核酸和编码无功能EGFR的核酸成比例表达，进而更加有利于表达调控，将本发明实施例的构建体导入受体淋巴细胞，所获得的转基因淋巴细胞的治疗安全性更高。
连接肽：根据本发明的实施例，所述构建体还可以进一步携带编码连接肽的核酸，所述编码连接肽的核酸具有SEQ ID NO:7～10所示的核苷酸序列，该连接肽是2A连接肽，并且所述编码连接肽的核酸设置于前面所述的核酸和所述编码无功能EGFR的核酸之间，其中，SEQ ID NO:7编码的连接肽具有SEQ ID NO：22所示的氨基酸序列，SEQ ID NO:8编码的连接肽具有SEQ ID NO：23所示的氨基酸序列，SEQ ID NO:9编码的连接肽具有SEQ ID NO：24所示的氨基酸序列，SEQ ID NO:10编码的连接肽具有SEQ ID NO：25所示的氨基酸序列。根据本发明的实施例，将本发明实施例的构建体导入受体淋巴细胞，所编码的连接肽能够在所述淋巴细胞中被切割，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。
启动子：根据本发明的实施例，所述构建体还可以进一步携带第一启动子，所述第一启动子与前面所述的核酸可操作地连接，任选地，所述第一启动子包括选自CMV，EF-1，LTR或RSV启动子。根据本发明的实施例，所述构建体进一步携带第二启动子，所述第二启动子与所述编码无功能EGFR的核酸可操作地连接，任选地，所述第二启动子包括选自CMV，EF-1，LTR或RSV启动子。上述第一、第二启动子可独立地启动表达上述重组蛋白、编码无功能EGFR的核酸，进而更加有利于重组蛋白、编码无功能EGFR的核酸的表达调控。发明人发现，CMV，EF-1，LTR或RSV启动子能够高效启动表达上述重组蛋白、编码无功能EGFR的核酸，重组蛋白、编码无功能EGFR的核酸的表达效率显著提高。
通过上述内部核糖体进入位点序列、或第一、第二启动子或编码连接肽的核酸分子 的引入，使得无功能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，从而保证了重组蛋白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，从而使得淋巴细胞的靶向杀伤作用更加显著，免疫杀伤的安全性进一步提高。
根据本发明的实施例，所述构建体的载体为反转录病毒载体、慢病毒载体或腺病毒相关病毒载体。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。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。
根据本发明的具体实施例，以构建一个慢病毒载体为例，发明人为了构建一个慢病毒载体，在某些病毒序列的位置，将目的核酸插入到病毒基因组中，从而产生复制缺陷的病毒。为了产生病毒体，发明人进而构建包装细胞系(包含gag，pol和env基因，但不包括LTR和包装成分)。发明人将含有目的基因的重组质粒，连同慢病毒LTR和包装序列，一起引入包装细胞系中。包装序列允许重组质粒RNA转录产物被包装到病毒颗粒中，然后被分泌到培养基中。进而发明人收集包含重组慢病毒的基质，有选择性地浓缩，并用于基因转移。慢载体可以感染多种细胞类型，包括可分裂细胞和不可分裂细胞。
另外，根据本发明的实施例，本发明实施例的慢病毒是复合慢病毒，除了常见的慢病毒基因gag，pol和env，还包含有调控和结构功能的其他基因。慢病毒载体是本领域技术人员所熟知的，慢病毒包括：人类免疫缺陷病毒HIV–1，HIV–2和猿猴免疫缺陷病毒SIV。慢病毒载体通过多重衰减艾滋病毒致病基因产生，例如全部删除基因env，vif，vpr，vpu和nef，使慢病毒载体形成生物安全型载体。重组慢病毒载体能够感染非分裂细胞，同时可用于体内和体外基因转移和核酸序列表达。例如：在合适的宿主细胞中，和带有包装功能(gag，pol，env，rev和tat)的两个或更多的载体一起，能够感染非分裂细胞。重组病毒的靶向性，是通过抗体或特定配体(靶向特定细胞类型受体)与膜蛋白的结合来实现的。同时，重组病毒的靶向性通过插入一个有效序列(包括调控区域)到病毒载体中，连同另一个编码了特定靶细胞上的受体的配体的基因，使载体具有了特定的靶向。各种有用的慢病毒载体，以及各种方法和操作等产生的载体，用于改变细胞的表达。根据本发明的实施例，本发明实施例的腺关联病毒载体(AAV)可使用一种或多种为人熟知的血清类型腺关联病毒载体的DNA构建。
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。大量的表达控制序列(包括天然的，可诱导和/或特定组织的启动子)可能被使用。根据本发明的实施例，启动子为组织特异型启动子。根据本发明的实施例，启动子为诱导型启动子。根据本发明的实施例，启动子为选自基于所选载体的启动子。根据本发明的实施例，当选择慢病毒载体时，启动子为CMV IE基因，EF-1α，泛素C，或磷酸甘油激酶(PGK)启动子。其他常规表达控制序列包括可选标记或报告基因，包括编码遗传霉素，潮霉素，氨苄青霉素或嘌呤霉素耐药性等的核苷酸序列。载体的其他组件包括复制起点。
构建载体的技术为本领域技术人员所熟知的，这些技术包括常规克隆技术。
根据本发明的实施例，发明人构建了共表达任选的无功能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。
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表达前面所述的重组受体，任选地，所述转基因淋巴细胞表达无功能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效果强、安全。
根据本发明的具体实施例，所述淋巴细胞为抗原特异性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、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，安全性高。
制备转基因淋巴细胞的方法
另一方面，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显著提高、安全。
用于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，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。
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，所述癌症的类型不受特别限制，根据本发明实施例的药物组合物对PD-L1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：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
在本发明再一方面，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)。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。图1是慢病毒载体的示意图(其中E表示胞外段；tEGFR表示无功能EGFR)，包含编码PD1-CD28-CD3ζ重组受体的序列，IRES、及编码无功能EGFR受体序列。编码PD1-CD28-CD3ζ重组受体的序列在启动子EF-1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受体和重组受体的T淋巴 细胞
在本实施例中，外周血淋巴细胞取自不记名供血者。外周血淋巴细胞通过梯度离心进行分离，梯度离心机为Ficoll-Hypaque。T淋巴细胞与T细胞激活因子磁珠CD3/CD28(购自Invitrogen，Carlsbad，CA)在5％CO2、37摄氏度下孵育培养72小时，培养基是加有2mmol/L谷氨酰胺，10％高温灭活的胎牛血清(FCS)(购自Sigma-Aldrich Co.)和100U/ml的青霉素/链霉素双抗的RPMI培养基1640(购自Invitrogen Gibco Cat.no.12633-012)。激活培养72小时后，用洗液润洗细胞，将磁珠洗去。将T细胞种在铺有重组纤连蛋白片段(FN ch-296；Retronectin)细胞培养皿上，并用慢病毒转导，转导慢病毒分别为LV-PD1-CD28-CD3ζ-tEGFR，LV-PD1-CD28-CD3ζ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重组受体和无功能EGFR受体的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，抗EGFR抗体不能介异杀伤空载慢病毒转导的T淋巴细胞，统计数据代表三个孔的平均值±SEM。
实施例3共表达无功能EGFR受体和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-CD28-CD3ζ-tEGFR，LV-PD1-CD28-tEGFR(结构如图1所示)，LV-tEGFR(结构如图1所示)，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受体和无功能EGFR受体的慢病毒转导的T淋巴细胞(LV-PD1-CD28-CD3ζ-tEGFR T淋巴细胞)能够显著杀死PD-L1+的脑瘤细胞。共表达PD1-CD28受体(没有连接CD3zeta链片段)和无功能EGFR受体的慢病毒转导的T淋巴细胞(LV-PD1-CD28-tEGFR T淋巴细胞)无明显杀伤PD-L1+的脑瘤细胞作用。仅表达无功能EGFR受体的慢病毒转导的T淋巴细胞(LV-tEGFR T淋巴细胞)或空载慢病毒转导的T淋巴细胞(对照LV-GFP T淋巴细胞)对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 (22)

  1. 一种重组受体，其特征在于，包括：
    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CD3zeta链。
  6. 根据权利要求1所述的重组受体，其特征在于，所述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. 一种构建体，其特征在于，所述构建体携带权利要求8所述的核酸。
  10. 根据权利要求9所述的构建体，其特征在于，所述构建体进一步携带编码无功能EGFR的核酸，所述编码无功能EGFR的核酸具有SEQ ID NO：5所示的核苷酸序列。
  11. 根据权利要求10所述的构建体，其特征在于，所述构建体进一步携带内部核糖体进入位点序列，所述内部核糖体进入位点序列具有SEQ ID NO：6所示的核苷酸序列，并且所述内部核糖体进入位点序列设置于权利要求8所述的核酸和所述编码无功能EGFR的核酸之间。
  12. 根据权利要求10所述的构建体，其特征在于，所述构建体进一步携带编码连接肽的核酸，所述编码连接肽的核酸具有SEQ ID NO：7～10所示的核苷酸序列，并且所述编码连接肽的核酸设置于权利要求8所述的核酸和所述编码无功能EGFR的核酸之间。
  13. 根据权利要求9所述的构建体，其特征在于，所述构建体进一步携带第一启动子，所述第一启动子与权利要求8所述的核酸可操作地连接，
    任选地，所述第一启动子包括选自CMV，EF-1，LTR或RSV启动子。
  14. 根据权利要求10所述的构建体，其特征在于，所述构建体进一步携带第二启动子，所述第二启动子与所述编码无功能EGFR的核酸可操作地连接，
    任选地，所述第二启动子包括选自CMV，EF-1，LTR或RSV启动子。
  15. 根据权利要求9所述的构建体，其特征在于，所述构建体的载体为反转录病毒载体、慢病毒载体或腺病毒相关病毒载体。
  16. 一种构建体，其特征在于，所述构建体携带下列核酸分子:
    (1)编码免疫检查点分子片段的核酸分子，所述免疫检查点分子片段具有SEQ ID NO：11或12所示的氨基酸序列，所述编码免疫检查点分子片段的核酸分子具有SEQ ID NO：13或14所示的核苷酸序列；
    (2)编码免疫刺激分子片段的核酸分子，所述免疫刺激分子片段具有SEQ ID NO：15或16所示的氨基酸序列，所述编码免疫刺激分子片段的核酸分子具有SEQ ID NO：17或18所示的核苷酸序列；以及
    (3)编码T细胞受体zeta链的核酸分子，所述T细胞受体zeta链具有SEQ ID NO：19所示的氨基酸序列，所述编码T细胞受体zeta链的核酸分子具有SEQ ID NO：20所示的核苷酸序列，
    任选地，所述构建体进一步携带编码无功能EGFR的核酸分子，所述编码无功能EGFR的核酸分子具有SEQ ID NO：5所示的核苷酸序列，所述无功能EGFR具有SEQ ID NO：21所示的氨基酸序列，
    任选地，所述构建体进一步携带内部核糖体进入位点序列的核酸分子，所述内部核糖体进入位点序列的核酸分子具有SEQ ID NO：6所示的核苷酸序列，并且所述编码内部核糖体进入位点序列的核酸分子设置于所述编码T细胞受体zeta链的核酸分子和所述编码无功能EGFR的核酸分子之间，
    任选地，所述构建体进一步携带编码连接肽的核酸，所述编码连接肽的核酸具有SEQ ID NO：7～10所示的核苷酸序列，所述连接肽具有SEQ ID NO：22～25所示的氨基酸序列，并且所述编码连接肽的核酸设置于所述编码T细胞受体zeta链的核酸分子和所述编码无功能EGFR的核酸分子之间，
    任选地，所述构建体进一步携带第一启动子，所述第一启动子与所述编码免疫检查点分子片段的核酸分子可操作地连接，
    任选地，所述第一启动子包括选自CMV，EF-1，LTR或RSV启动子，
    任选地，所述构建体进一步携带第二启动子，所述第二启动子与所述编码无功能EGFR的核酸分子可操作地连接，
    任选地，所述第二启动子包括选自CMV，EF-1，LTR或RSV启动子，
    任选地，所述构建体的载体为反转录病毒载体、慢病毒载体或腺病毒相关病毒载体。
  17. 一种转基因淋巴细胞，其特征在于，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表达无功能EGFR。
  18. 根据权利要求17所述的转基因淋巴细胞，其特征在于，所述淋巴细胞为抗原特异性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。
  19. 一种制备权利要求17～18任一项所述的转基因淋巴细胞的方法，其特征在于，包括：
    将权利要求9～16任一项所述的构建体引入到淋巴细胞中或者T淋巴细胞。
  20. 一种用于治疗癌症的治疗组合物，其特征在于，包括：
    权利要求1～7任一项所述的重组受体、权利要求8所述的核酸、权利要求9～16任一项所述的构建体或者权利要求17～18任一项所述的转基因淋巴细胞。
  21. 一种提高淋巴细胞治疗免疫杀伤能力的方法，其特征在于，包括：
    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。
  22. 一种治疗癌症的方法，其特征在于，包括：
    给患者给予权利要求1～7任一项所述的重组受体、权利要求8所述的核酸、权利要求9～16任一项所述的构建体或者权利要求17～18任一项所述的转基因淋巴细胞。
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