CN107586342A - Recombinant immune checkpoint acceptor and its application - Google Patents
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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- C12N15/09—Recombinant DNA-technology
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- C12N15/62—DNA sequences coding for fusion proteins
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Abstract
The present invention proposes recombinant receptor and its application, and the recombinant receptor includes：Cellular immunity checkpoint molecule fragment；Molecules of immunization stimulus fragment；And φt cell receptor zeta chains.The Expressions In Lymphocytes recombinant receptor, it can effectively strengthen specific killing effect of the lymphocyte to tumour cell.
Description
Technical field
The present invention relates to biomedicine field, in particular it relates to recombinant immune checkpoint acceptor and its application, more
In particular it relates to recombinant receptor, nucleic acid, transgenosis lymphocyte, construct, the side of prepare transgenosis lymphocyte
The method of method, the therapeutic combination for the treatment of cancer and raising lymphocyte immunity killing ability.
Background technology
Cancer, because genes within cells mutation causes a kind of disease of uncontrolled cellular proliferation.Turn into human health at present
Significant threat, be one of the main reason for causing human death.The World Health Organization (WHO) is what is delivered《Global cancer report
Accuse 2014》In point out, global cancer patients in 2012 and death are all increasing sharply, and newly-increased cases of cancer has nearly half
Asia is appeared in, wherein most is in first in China, the newly-increased cases of cancer of China.《Chinese tumour Entry year in 2012
Report》Data show that China increases cases of cancer about 3,500,000 newly every year, and it is therefore dead to there are about 2,500,000 people.Therefore, find efficiently special
Cancer treatment method there is great clinical value.
Traditional tumor therapeuticing method mainly includes operation, radiation and chemotherapy, but this several method all has larger office
Sex-limited, such as due to the near-end of cancer cell is invaded or far-end transfer, the tumour metastasis and recurrence rate after surgery excision is higher, and radiotherapy
With chemotherapy serious infringement can be caused for the normal cell especially hemopoietic system and immune system of body itself, therefore for
The patient that metastases have occurred also is difficult to reach preferable late result.Further investigation and biology with tumor cells mechanism
The further development of technology, targeted drug treatment and immunization therapy play more and more big work in the complex treatment of tumour
With.Targeted therapies mainly include monoclonal antibody and (are classified as passive born of the same parents' feedback and tumor vaccine etc. sometimes.Immunotherapy passes through transfer
The immune system of body, tumor microenvironment is antitumor exempts from immunotherapy for enhancing) and small molecule targeted drug, and immunotherapy is main
Including cytokine therapy, immunity inspection point monoclonal antibody, adoptive immunotherapy, so as to control and killing tumor cell, therefore effectively
Rate is high, high specificity, the advantages of better tolerance, is had broad prospects in oncotherapy.
However, the immunotherapy of tumour, still needs further further investigation and exploitation, to strengthen clinical efficacy.
The content of the invention
It is contemplated that at least solves one of technical problem in correlation technique to a certain extent.Therefore, the present invention
One purpose is a kind of method for proposing recombinant receptor and effectively strengthening lymphocyte immunity killing tumor cell using it.
In the first aspect of the present invention, the present invention proposes a kind of recombinant receptor.According to an embodiment of the invention, it is described heavy
Group acceptor includes：Cellular immunity checkpoint molecule fragment；Molecules of immunization stimulus fragment；And φt cell receptor zeta chains.According to this
The embodiment of invention, make the recombinant receptor of the Expressions In Lymphocytes embodiment of the present invention, it is thin to tumour can effectively to strengthen lymphocyte
The specific killing effect of born of the same parents.
According to an embodiment of the invention, above-mentioned recombinant receptor can further include following additional technical feature at least it
One：
According to an embodiment of the invention, cellular immunity checkpoint molecule is PD1.PD1 can with it is special on tumour cell
Property expression PD-L1 or PD-L2 be combined.And then the recombinant receptor of the Expressions In Lymphocytes embodiment of the present invention, it is thin to tumour
Born of the same parents, the target killing of particularly PD-L1 or PD-L2 positive tumor cells further enhance.
According to an embodiment of the invention, the extracellular region of cellular immunity checkpoint molecule fragment including the PD1 and
Optional transmembrane region, the molecules of immunization stimulus fragment include CD28 intracellular region and optional transmembrane region.PD1 extracellular region
With the functional areas being combined with PD-L1 or PD-L2 specific expressed on tumour cell, CD28 intracellular region is exempted from activation
The functional areas of epidemic disease stimulus signal path, and then the recombinant receptor of the lymphocyte cell expression embodiment of the present invention, it is thin to tumour
The targeting fragmentation effect of born of the same parents further improves.
According to an embodiment of the invention, the recombinant receptor includes：(a) extracellular region and transmembrane region of the PD1；And
(b) intracellular region of the CD28, or including：(i) extracellular region of the PD1；And (ii) described CD28 intracellular region and across
Film area.Both combinations remain the work(that PD1 is combined with PD-L1 or PD-L2 specific expressed on tumour cell
Energy area and the functional areas of CD28 activation immunostimulatory signals paths, while whether PD1 transmembrane regions or CD28 transmembrane region,
Recombinant receptor cross-film can expressed, and then the recombinant receptor of the lymphocyte cell expression embodiment of the present invention, it is thin to tumour
The targeting fragmentation effect of born of the same parents further improves.
According to an embodiment of the invention, the φt cell receptor zeta chains are CD3zeta chains.CD3zeta chains specific can swash
Downstream T cell receptor signaling pathways living, and then the recombinant receptor of the lymphocyte cell expression embodiment of the present invention, it is thin to tumour
The fragmentation effect of born of the same parents further improves.
According to an embodiment of the invention, the C-terminal of cellular immunity checkpoint molecule fragment and the molecules of immunization stimulus
The N-terminal of fragment is connected, and the C-terminal of the molecules of immunization stimulus fragment is connected with the N-terminal of the φt cell receptor zeta chains.The present invention
The associated clip of the recombinant receptor of embodiment is advantageous to positioning of the associated clip in cell under the above-mentioned order of connection, and then
It is more beneficial for playing corresponding function-targeting, cross-film, activate immunostimulatory signals path and activating φt cell receptor signal leading to
Road, its killing of targeting to tumour cell ability further improve.
In the second aspect of the present invention, the present invention proposes a kind of recombinant receptor.According to an embodiment of the invention, it is described heavy
Group acceptor has SEQ ID NO:Amino acid sequence shown in 1 or 2.
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTD
KLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTA
HPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVISKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:1)。
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTD
KLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTA
HPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA
YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI
GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:2)。
According to an embodiment of the invention, make the recombinant receptor of the Expressions In Lymphocytes embodiment of the present invention, can effectively strengthen leaching
The specific killing effect of bar cells against tumor cells, particularly PD-L1 or PD-L2 positive tumor cells.
In the third aspect of the present invention, the present invention proposes a kind of nucleic acid.According to an embodiment of the invention, the nucleic acid is compiled
The foregoing recombinant receptor of code, optionally, the nucleic acid has SEQ ID NO：Nucleotide sequence shown in 3 or 4.
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGA
CTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCT
TCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGAC
AAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGG
GCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGG
CCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCC
CACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT
AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA
AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA
GGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC
CCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO：3).
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGA
CTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCT
TCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGAC
AAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGG
GCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGG
CCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCC
CACCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTA
TAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACA
TGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCC
TATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGA
GCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGC
CGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT
GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC
CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO：4).
The nucleic acid of the embodiment of the present invention is imported in recipient lymphocytes, the recombinant receptor of encoded by nucleic acid is in lymphocyte
Mid-span film expression, the lymphocyte improve to the specific killing significant effect of tumour cell.
In the fourth aspect of the present invention, the present invention proposes a kind of construct.According to an embodiment of the invention, the structure
Body carries foregoing nucleic acid.The construct of the embodiment of the present invention is imported in recipient lymphocytes, entrained by construct
The recombinant receptor of encoded by nucleic acid is in lymphocyte mid-span film expression, specific killing effect of the lymphocyte to tumour cell
Significantly improve.
According to an embodiment of the invention, the construct can further include following additional technical feature at least it
One：
According to an embodiment of the invention, the construct further carries coding nonfunctional EGFR nucleic acid, the coding
Nonfunctional EGFR nucleic acid has SEQ ID NO:Nucleotide sequence shown in 5.
ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCACGCCCTGGGAGTCGCAAAGT
CTGTAATGGGATCGGCATCGGCGAGTTCAAGGACAGCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATT
GCACATCTATCAGCGGCGACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTCTG
GACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGCTGATCCAGGCCTGGCCTGAGAA
CAGAACCGATCTGCACGCCTTTGAGAATCTGGAGATCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGG
CCGTGGTGTCTCTGAACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCATCTCC
GGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGGCACCTCTGGCCAGAAGACAAAGAT
CATCTCTAACCGGGGCGAGAATAGCTGCAAGGCAACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTT
GGGGCCCAGAGCCACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGTGTAATCTG
CTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGTGTCACCCCGAGTGCCTGCCTCAGGCCAT
GAACATCACCTGTACAGGCCGCGGCCCCGACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGA
AGACCTGTCCAGCCGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGACACGTGTGCCAC
CTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGAGGGATGCCCAACCAACGGCCCTAAGATCCC
AAGCATCGCCACAGGCATGGTGGGGGCACTGCTGCTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAA
GGTAA(SEQ ID NO:5)。
According to an embodiment of the invention, nonfunctional EGFR acceptors lack N- ends ligand binding domain and intracellular receptor tyrosine
Kinase activity, but the transmembrane region including Wild type EGFR acceptor and the sequence that is completely combined with anti-egfr antibodies, so idle
Energy EGFR acceptors can mark as the suicide of lymphocyte.The construct of the embodiment of the present invention is imported in recipient lymphocytes, nothing
Function EGFR acceptors expression can on the premise of the targeting killing effect to tumour cell of lymphocyte is effectively ensured, if
There is serious adverse reaction in patient, and lymphocyte can be removed by anti-egfr antibodies, and then improve the structure of the embodiment of the present invention
Body treats the security of tumour patient.
According to an embodiment of the invention, the construct further carries internal ribosome entry site sequence, described interior
Portion's ribosomal entry site sequence has SEQ ID NO:Nucleotide sequence shown in 6, and the internal ribosomal entry site
Point sequence is arranged between foregoing nucleic acid and the coding nonfunctional EGFR nucleic acid.
CCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGT
TATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCT
AGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTC
TTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC
AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAA
AGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATC
TGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACG
GGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACC(SEQ ID NO:6)。
The introducing of internal ribosome entry site sequence so that the initiate table of coding nonfunctional EGFR nucleic acid reach independent of
5 ' cap sequences, and encode the proportional expression of nucleic acid of the nucleic acid and coding nonfunctional EGFR of recombinant receptor, so more added with
Beneficial to expression regulation, the construct of the embodiment of the present invention is imported into recipient lymphocytes, the transgenosis lymphocyte obtained
Therapeutic safety is higher.
According to an embodiment of the invention, the construct further carries the nucleic acid of coding connection peptide, the coding connection
The nucleic acid of peptide has SEQ ID NO:Nucleotide sequence shown in 7~10, and it is described coding connection peptide nucleic acid be arranged at before
Between nucleic acid and the nucleic acid for encoding nonfunctional EGFR described in face.
GGCAGCGGCGAGGGCAGGGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCC(SEQ
ID NO:7)。
GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC
(SEQ ID NO:8)。
GGCAGCGGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC
(SEQ ID NO:9)。
GGCAGCGGCGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC
(SEQ ID NO:10)。
According to an embodiment of the invention, the construct of the embodiment of the present invention is imported into recipient lymphocytes, coded company
Connecing peptide can be cut in the lymphocyte, and the introducing for connecting peptide causes expressed recombinant receptor and nonfunctional EGFR
Reached in non-fused state table on Lymphocyte Membrane.
According to an embodiment of the invention, the construct further carries the first promoter, and first promoter is with before
Nucleic acid described in face is operably connected, and optionally, first promoter includes being selected from CMV, EF-1, LTR or RSV startup
Son.Above-mentioned first promoter can independently start the first nucleic acid molecules of expression, and then advantageously in corresponding nucleic developed by molecule
Regulation and control.Inventor has found that CMV, EF-1, LTR or RSV promoters can efficiently start the foregoing nucleic acid of expression, before
The expression efficiency of described nucleic acid significantly improves.
According to an embodiment of the invention, the construct further carries the second promoter, second promoter and institute
The nucleic acid for stating coding nonfunctional EGFR is operably connected, and optionally, second promoter includes being selected from CMV, EF-1, LTR
Or RSV promoters.Above-mentioned second promoter can independently start expression and encode nonfunctional EGFR nucleic acid, so advantageously in
Encode the expression regulation of nonfunctional EGFR nucleic acid.Inventor has found that CMV, EF-1, LTR or RSV promoters can efficiently start
Expression encodes nonfunctional EGFR nucleic acid, and the expression efficiency of coding nonfunctional EGFR nucleic acid significantly improves.
According to an embodiment of the invention, the carrier of the construct is retrovirus vector, slow virus carrier or adenopathy
Malicious related viral vectors.Above-mentioned carrier can realize high efficient expression of the entrained nucleic acid in recipient cell, therapeutic efficiency height.
In the fifth aspect of the present invention, the present invention proposes a kind of construct.According to an embodiment of the invention, the structure
Body carries following nucleic acid molecules:(1) nucleic acid molecules of encoding immune checkpoint molecule fragment, the immunologic test point molecule fragment
With SEQ ID NO：Amino acid sequence shown in 11 or 12, the nucleic acid molecules of encoding immune checkpoint molecule fragment have
SEQ ID NO：Nucleotide sequence shown in 13 or 14；(2) nucleic acid molecules of encoding immune stimulation molecule fragment, the immune thorn
Sharp molecule fragment has SEQ ID NO：Amino acid sequence shown in 15 or 16, the nucleic acid of the encoding immune stimulation molecule fragment
Molecule has SEQ ID NO：Nucleotide sequence shown in 17 or 18；And the nucleic acid of (3) encoding T cell receptor zeta chains point
Son, the φt cell receptor zeta chains have SEQ ID NO：Amino acid sequence shown in 19, the encoding T cell receptor zeta
The nucleic acid molecules of chain have SEQ ID NO：Nucleotide sequence shown in 20.
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTD
KLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTA
HPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI(SEQ ID NO：11).
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTD
KLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTA
H(SEQ ID NO：12).
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGA
CTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCT
TCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGAC
AAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGG
GCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGG
CCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCC
CACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATC(SEQ ID NO：13).
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGA
CTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCT
TCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGAC
AAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGG
GCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGG
CCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCC
CAC(SEQ ID NO：14).
SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO：15).
PSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY
RS(SEQ ID NO：16).
AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTA
CCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO：17).
CCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAG
CTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGA
ACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTAT
CGCTCC(SEQ ID NO：18).
RVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA
EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：19).
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT
AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA
AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA
GGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC
CCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO：20).
The construct of the embodiment of the present invention is imported in recipient lymphocytes, point of the encoded by nucleic acid entrained by construct
Sub-piece forms fusion receptors albumen, and in lymphocyte mid-span film expression, specificity of the lymphocyte to tumour cell
Fragmentation effect significantly improves.
According to an embodiment of the invention, above-mentioned construct can further include following additional technical feature at least it
One：
According to an embodiment of the invention, the construct further carries coding nonfunctional EGFR nucleic acid molecules, described
Coding nonfunctional EGFR nucleic acid molecules have SEQ ID NO:Nucleotide sequence shown in 5, the nonfunctional EGFR have SEQ
ID NO:Amino acid sequence shown in 21.
MALPVTALLLPLALLLHAARPGSRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPL
DPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIIS
GNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNL
LEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCH
LCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR(SEQ ID NO:21)。
As it was previously stated, the construct of the embodiment of the present invention is imported in recipient lymphocytes, the expression of nonfunctional EGFR acceptors
Can be on the premise of the targeting killing effect to tumour cell of lymphocyte be effectively ensured, if patient's appearance is serious bad anti-
Should, lymphocyte can be removed by anti-egfr antibodies, and then improve the treatments such as the construct of the embodiment of the present invention, lymphocyte and swell
The security of knurl patient.
According to an embodiment of the invention, the construct further carries the nucleic acid point of internal ribosome entry site sequence
Son, the nucleic acid molecules of the internal ribosome entry site sequence have SEQ ID NO:Nucleotide sequence shown in 6, and institute
The nucleic acid molecules for stating coding internal ribosome entry site sequence are arranged at the nucleic acid molecules of the encoding T cell receptor zeta chains
Between the nucleic acid molecules of the coding nonfunctional EGFR.As it was previously stated, the introducing of internal ribosome entry site sequence is more
Be advantageous to expression regulation, the Therapeutic safety of the transgenosis lymphocyte obtained is higher.
According to an embodiment of the invention, the construct further carries the nucleic acid of coding connection peptide, the coding connection
The nucleic acid of peptide has SEQ ID NO:Nucleotide sequence shown in 7~10, the connection peptide have SEQ ID NO：22~25 institutes
The amino acid sequence shown, and the nucleic acid of the coding connection peptide is arranged at the nucleic acid point of the encoding T cell receptor zeta chains
Between sub and described coding nonfunctional EGFR nucleic acid molecules.
EGRGSLLTCGDVEENPGP(SEQ ID NO：22).
ATNFSLLKQAGDVEENPGP(SEQ ID NO：23).
QCTNYALLKLAGDVESNPGP(SEQ ID NO：24).
VKQTLNFDLLKLAGDVESNPGP(SEQ ID NO：25).
As it was previously stated, the construct of the embodiment of the present invention imported into recipient lymphocytes, coded connection peptide can be
It is cut in the lymphocyte, connects the introducing of peptide so that the recombinant receptor and nonfunctional that expressed molecule fragment is formed
EGFR is reached on Lymphocyte Membrane in non-fused state table.
According to an embodiment of the invention, the construct further carries the first promoter, first promoter and institute
The nucleic acid molecules for stating encoding immune checkpoint molecule fragment are operably connected, and optionally, first promoter includes being selected from
CMV, EF-1, LTR or RSV promoter.As it was previously stated, CMV, EF-1, LTR or RSV promoter efficiently can start before expression
Nucleic acid molecules, the nucleic acid molecules and coding of encoding immune stimulation molecule fragment of described encoding immune checkpoint molecule fragment
The nucleic acid molecules of φt cell receptor zeta chains, the expression efficiency of foregoing nucleic acid molecule fragment significantly improve.
According to an embodiment of the invention, the construct further carries the second promoter, second promoter and institute
The nucleic acid molecules for stating coding nonfunctional EGFR are operably connected, and optionally, second promoter includes being selected from, CMV, EF-
1, LTR or RSV promoters.As it was previously stated, CMV, EF-1, LTR or RSV promoter can efficiently start the described coding nothing of expression
Function EGFR nucleic acid molecules, the expression efficiency of coding nonfunctional EGFR nucleic acid molecules significantly improve.
According to an embodiment of the invention, the carrier of the construct is retrovirus vector, slow virus carrier or adenopathy
Malicious related viral vectors.Above-mentioned carrier can realize high efficient expression of the entrained nucleic acid in recipient cell, therapeutic efficiency height.
In the sixth aspect of the present invention, the present invention proposes a kind of transgenosis lymphocyte.According to an embodiment of the invention,
The foregoing recombinant receptor of transgenosis Expressions In Lymphocytes, optionally, the transgenosis Expressions In Lymphocytes nonfunctional
EGFR.The transgenosis lymphocyte of the embodiment of the present invention is strong, safe to the specific killing effect of tumour cell.
According to an embodiment of the invention, above-mentioned transgenosis lymphocyte can further include following additional technical feature
At least one：
According to an embodiment of the invention, the lymphocyte is antigenspecific T lymphocyte, optionally, the lymph
Cell is tumor-infiltrated T lymphocytes, and optionally, the lymphocyte is periphery blood T lymphocyte, optionally, the lymph
Cell is Natural killer T cells, and optionally, the lymphocyte is NK.According to embodiments of the present invention
Antigenspecific T lymphocyte, tumor-infiltrated T lymphocytes, periphery blood T lymphocyte, Natural killer T cells or nature
Cell is killed, can be achieved to kill the specific immunity of tumour cell, it is safe.
In the seventh aspect of the present invention, the present invention proposes a kind of side for preparing foregoing transgenosis lymphocyte
Method.According to an embodiment of the invention, methods described includes：Foregoing construct is incorporated into lymphocyte or T drenches
Bar cell.Using the above method according to embodiments of the present invention, it is thin can simply, efficiently to obtain foregoing transgenosis lymph
Born of the same parents, as it was previously stated, the transgenosis lymphocyte obtained significantly improves to the specific killing of tumour cell, safety.
In the eighth aspect of the present invention, the present invention proposes a kind of therapeutic combination for treating cancer.According to this hair
Bright embodiment, the therapeutic combination include：Foregoing recombinant receptor, foregoing nucleic acid, foregoing structure
Build body or foregoing transgenosis lymphocyte.Using therapeutic combination according to embodiments of the present invention, can realize pair
The effectively and safely killing of tumour cell.
In the ninth aspect of the present invention, the present invention proposes a kind of side for improving lymphocyte treatment immunologic cytotoxicity ability
Method.According to an embodiment of the invention, methods described includes：Make the foregoing recombinant receptor of the Expressions In Lymphocytes.Utilize
The above method according to embodiments of the present invention, lymphocyte can be effectively improved the specific immunity of tumour cell is killed.
Brief description of the drawings
Fig. 1 is the structural representation of slow virus carrier according to embodiments of the present invention；
Fig. 2 is coexpression PD1-CD28-CD3zeta recombinant receptor and nonfunctional EGFR acceptors according to embodiments of the present invention
Lymphocyte by anti-egfr antibodies mediation ADCC killing remove result figure；And
Fig. 3 is coexpression PD1-CD28-CD3zeta recombinant receptor and nonfunctional EGFR acceptors according to embodiments of the present invention
Lymphocvte Killer PD-L1 positive tumor cells result figure.
Embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for describing purpose, and it is not intended that instruction or hint phase
To importance or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the invention, unless otherwise saying
Bright, " multiple " are meant that two or more.
Recombinant receptor albumen
On the one hand, the present invention proposes a kind of recombinant receptor.According to an embodiment of the invention, the recombinant receptor includes：Carefully
Born of the same parents' immunologic test point molecule fragment；Molecules of immunization stimulus fragment；And φt cell receptor zeta chains.According to an embodiment of the invention,
Make the recombinant receptor of the Expressions In Lymphocytes embodiment of the present invention, can effectively strengthen specific killing of the lymphocyte to tumour cell
Effect.
According to a particular embodiment of the invention, cellular immunity checkpoint molecule is PD1.PD1 can with tumour cell
Specific expressed PD-L1 or PD-L2 is combined, and then, the recombinant receptor of the Expressions In Lymphocytes embodiment of the present invention, lymph is thin
Born of the same parents are under PD1 guiding, selectively targeted tumour cell, and its target to tumour cell further enhances.
According to the still another embodiment of the present invention, the extracellular region of cellular immunity checkpoint molecule fragment including PD1 and
Optional transmembrane region, molecules of immunization stimulus fragment include CD28 intracellular region and optional transmembrane region.For example, according to the present invention
Embodiment, recombinant receptor can include：(a) PD1 extracellular region and transmembrane region；And (b) CD28 intracellular region, or bag
Include：(i) PD1 extracellular region；And (ii) CD28 intracellular region and transmembrane region.PD1 extracellular region has and spy on tumour cell
The functional areas that the PD-L1 or PD-L2 of opposite sex expression are combined, CD28 intracellular region have the work(of activation immunostimulatory signals path
Energy area, while whether PD1 transmembrane regions or CD28 transmembrane region, can express recombinant receptor cross-film, and then lymphocyte
Cell expresses the recombinant receptor of the embodiment of the present invention, and its targeting fragmentation effect to tumour cell further improves.
In addition, according to one more embodiment of the present invention, φt cell receptor zeta chains are CD3zeta chains.CD3zeta chains associate T
Cell receptor (TCR) signal path, CD3zeta chains triggering after, Zeta chains can with same endochylema be referred to as zeta chain GAP-associated protein GAPs
70 (ZAP-70) are combined, and ZAP-70 is the signal protein with EGFR-TK (PTK) activity in a kind of endochylema, contains two
SH-2 (srchomology region 2, SH-2) domain, SH-2 and the junket ammonia of phosphorylation in zeta chains in ZAP-70 molecules
Sour residue is combined, and ZAP-70 activation can further activate Ras albumen, and then final activated lymphocyte.CD3zeta chains can be special
Opposite sex activation downstream T cell receptor signaling pathways, and then the recombinant protein of the Expressions In Lymphocytes embodiment of the present invention, in immune thorn
Under the synergy for swashing molecule activation function fragment and CD3zeta chain activations, its fragmentation effect to tumour cell is entered
One step improves.
Finally, according to an embodiment of the invention, the order of connection of corresponding molecule fragment can be in above-mentioned recombinant protein：Cell
The C-terminal of immunologic test point molecule fragment is connected with the N-terminal of molecules of immunization stimulus fragment, and the C-terminal and T of molecules of immunization stimulus fragment are thin
The N-terminal of born of the same parents' acceptor zeta chains is connected.Inventor has found that the associated clip of the recombinant protein of the embodiment of the present invention is suitable in above-mentioned connection
Under sequence, be advantageous to positioning of the associated clip in cell, so be more beneficial for playing corresponding function-targeting, cross-film, activation are exempted from
Epidemic disease stimulus signal path and activation φt cell receptor signal path, its killing of targeting to tumour cell ability further carry
It is high.
Specifically, according to an embodiment of the invention, recombinant receptor has SEQ ID NO：Amino acid sequence shown in 1 or 2.
Wherein, SEQ ID NO：1 represents the recombinant receptor comprising people PD1 extracellular regions and transmembrane region, CD28 intracellular regions and CD3zeta chains
(PD1-ECD-TM-CD28-ICD-CD3zeta) amino acid sequence；SEQ ID NO：2 represent comprising people PD1 extracellular regions, CD28
The amino acid sequence of the recombinant receptor of transmembrane region and intracellular region and CD3zeta chains (PD1-ECD-CD28-TM-ICD-CD3zeta)
Row.According to an embodiment of the invention, recombinant receptor has above-mentioned amino acid sequence, makes its expression in lymphocyte, can be effective
Strengthen specific killing effect of the lymphocyte to tumour cell.
Nucleic acid
On the other hand, the present invention proposes a kind of nucleic acid.According to an embodiment of the invention, nucleic acid coding is foregoing
Recombinant receptor, optionally, the nucleic acid have SEQ ID NO：Nucleotide sequence shown in 3 or 4.Wherein, SEQ ID NO：3 institutes
The nucleotide sequence coded recombinant receptor comprising people PD1 extracellular regions and transmembrane region, CD28 intracellular regions and CD3zeta chains shown
(PD1-ECD-TM-CD28-ICD-CD3zeta), SEQ ID NO：Shown in 4 it is nucleotide sequence coded comprising people PD1 extracellular regions,
The recombinant receptor of CD28 transmembrane regions and intracellular region and CD3zeta chains (PD1-ECD-CD28-TM-ICD-CD3zeta).This is sent out
The nucleic acid of bright embodiment is imported in recipient lymphocytes, and the recombinant receptor of encoded by nucleic acid, should in lymphocyte mid-span film expression
Lymphocyte improves to the specific killing significant effect of tumour cell.
Construct
On the other hand, the present invention proposes a kind of structure.According to an embodiment of the invention, the construct carries above institute
The nucleic acid stated.The construct of the embodiment of the present invention is imported in recipient lymphocytes, the encoded by nucleic acid entrained by construct
Recombinant receptor improves in lymphocyte mid-span film expression, the lymphocyte to the specific killing significant effect of tumour cell.
Or according to an embodiment of the invention, the construct carries following nucleic acid molecules:(1) encoding immune checkpoint
The nucleic acid molecules of molecule fragment, the immunologic test point molecule fragment have SEQ ID NO：Amino acid sequence shown in 11 or 12
Row, the nucleic acid molecules of encoding immune checkpoint molecule fragment have SEQ ID NO：Nucleotide sequence shown in 13 or 14；
Wherein, SEQ ID NO：Amino acid sequence shown in 11 is people PD1 extracellular regions and the amino acid sequence (PD1-ECD- of transmembrane region
TM), SEQ ID NO：Amino acid sequence shown in 12 is the amino acid sequence (PD1-ECD) of people's PD1 extracellular regions；SEQ ID NO：
13 be encode PD1-ECD-TM nucleotide sequence, SEQ ID NO：14 be the nucleotide sequence for encoding PD1-ECD.(2) encode
The nucleic acid molecules of molecules of immunization stimulus fragment, the molecules of immunization stimulus fragment have SEQ ID NO：Amino shown in 15 or 16
Acid sequence, the nucleic acid molecules of the encoding immune stimulation molecule fragment have SEQ ID NO：Nucleotides sequence shown in 17 or 18
Row, wherein, SEQ ID NO：Amino acid sequence shown in 15 is the amino acid sequence (CD28-ICD) of people's CD28 intracellular regions, SEQ
ID NO：Amino acid sequence shown in 16 is the amino acid sequence (CD28-TM-ICD) of people CD28 transmembrane regions and intracellular region, SEQ
ID NO：17 be encode CD28-ICD nucleotide sequence, SEQ ID NO：18 be the nucleotide sequence for encoding CD28-TM-ICD；
And the nucleic acid molecules of (3) encoding T cell receptor zeta chains, the φt cell receptor zeta chains have SEQ ID NO：Shown in 19
Amino acid sequence, the nucleic acid molecules of the encoding T cell receptor zeta chains have SEQ ID NO：Nucleotides sequence shown in 20
Row.The construct of the embodiment of the present invention is imported in recipient lymphocytes, the molecule piece of the encoded by nucleic acid entrained by construct
The above-mentioned recombinant receptors of Duan Zucheng, and the specific killing of tumour cell is imitated in lymphocyte mid-span film expression, the lymphocyte
Fruit significantly improves.
According to a particular embodiment of the invention, the construct further carries coding nonfunctional EGFR nucleic acid, described
Coding nonfunctional EGFR nucleic acid has SEQ ID NO:Nucleotide sequence shown in 5.According to an embodiment of the invention, nonfunctional
EGFR acceptors lack N- ends ligand binding domain and intracellular receptor tyrosine kinase activity, but including Wild type EGFR acceptor across
Film area and the sequence completely combined with anti-egfr antibodies, so nonfunctional EGFR acceptors can be as the suicide mark of lymphocyte
Note.The construct of the embodiment of the present invention is imported in recipient lymphocytes, and leaching can be effectively ensured in the expression of nonfunctional EGFR acceptors
On the premise of the targeting killing effect to tumour cell of bar cell, if serious adverse reaction occurs in patient, lymphocyte can
Removed by anti-egfr antibodies, and then improve the security of the construct treatment tumour patient of the embodiment of the present invention.
Wherein, inventor is to realize above-mentioned recombinant receptor and optional nonfunctional at least one of in the following way
EGFR acceptors are separately expressed：
Carry internal ribosome entry site sequence (IRES)：According to an embodiment of the invention, the construct is further
Internal ribosome entry site sequence is carried, the internal ribosome entry site sequence has SEQ ID NO:Core shown in 6
Nucleotide sequence, and the internal ribosome entry site sequence is arranged at foregoing nucleic acid and the coding nonfunctional
Between EGFR nucleic acid.The introducing of internal ribosome entry site sequence so that the initiate table of coding nonfunctional EGFR nucleic acid
Up to independent of 5 ' cap sequences, and the proportional expression of nucleic acid of the nucleic acid and coding nonfunctional EGFR of recombinant receptor is encoded, entered
And advantageously in expression regulation, the construct of the embodiment of the present invention is imported into recipient lymphocytes, the transgenosis leaching obtained
The Therapeutic safety of bar cell is higher.
Connect peptide：According to an embodiment of the invention, the construct can also further carry the nucleic acid of coding connection peptide,
The nucleic acid of the coding connection peptide has SEQ ID NO:Nucleotide sequence shown in 7~10, the connection peptide are 2A connection peptides, and
And the nucleic acid of the coding connection peptide is arranged between foregoing nucleic acid and the coding nonfunctional EGFR nucleic acid, its
In, SEQ ID NO:The connection peptide of 7 codings has SEQ ID NO：Amino acid sequence shown in 22, SEQ ID NO:8 codings
Connection peptide has SEQ ID NO：Amino acid sequence shown in 23, SEQ ID NO:The connection peptide of 9 codings has SEQ ID NO：
Amino acid sequence shown in 24, SEQ ID NO:The connection peptide of 10 codings has SEQ ID NO：Amino acid sequence shown in 25.
According to an embodiment of the invention, the construct of the embodiment of the present invention is imported into recipient lymphocytes, coded connection peptide can
It is cut in the lymphocyte, the introducing for connecting peptide causes expressed recombinant receptor and nonfunctional EGFR in non-fused
State table is reached on Lymphocyte Membrane.
Promoter：According to an embodiment of the invention, the construct can also further carry the first promoter, and described
One promoter is operably connected with foregoing nucleic acid, and optionally, first promoter includes being selected from CMV, EF-1,
LTR or RSV promoters.According to an embodiment of the invention, the construct further carries the second promoter, and described second starts
The sub nucleic acid with the coding nonfunctional EGFR is operably connected, and optionally, second promoter includes being selected from CMV, EF-
1, LTR or RSV promoters.Above-mentioned first, second promoter can independently start the above-mentioned recombinant protein of expression, coding nonfunctional
EGFR nucleic acid, and then advantageously in recombinant protein, the expression regulation of coding nonfunctional EGFR nucleic acid.Inventor's discovery,
CMV, EF-1, LTR or RSV promoter, which can efficiently start, expresses above-mentioned recombinant protein, coding nonfunctional EGFR nucleic acid, restructuring
Albumen, the expression efficiency for the nucleic acid for encoding nonfunctional EGFR significantly improve.
Pass through above-mentioned internal ribosome entry site sequence or first, second promoter or the nucleic acid point of coding connection peptide
The introducing of son so that nonfunctional EGFR acceptors are efficiently expressed and above-mentioned recombinant receptor is efficiently expressed in the embodiment of the present invention
Transgenosis Lymphocyte Membrane on, and nonfunctional EGFR acceptors and recombinant receptor are reached in Lymphocyte Membrane in non-fused state table
On, so as to ensure that the immune biological action of recombinant protein enhancing, or effectively realize the timely clear of transgenosis lymphocyte
Remove, so that the targeting killing effect of lymphocyte is more notable, the security of immunologic cytotoxicity further improves.
According to an embodiment of the invention, the carrier of the construct is retrovirus vector, slow virus carrier or adenopathy
Malicious related viral vectors.For the viral carrier of the embodiment of the present invention in virus packaging and course of infection, virus-infected area is wide
It is general, terminally differentiated cells can be both infected, the cell in division stage can be infected again, can both be incorporated into host chromosome, can be swum again
From outside host chromosome, so wide spectrum can be realized and efficient efficiency of infection.Above-mentioned carrier can realize that entrained nucleic acid exists
The high efficient expression of recipient cell, therapeutic efficiency are high.
According to a particular embodiment of the invention, exemplified by building a slow virus carrier, inventor is slow in order to build one
Viral vector, in the position of some virus sequences, purpose nucleic acid is inserted into viral genome, so as to produce replication defective
Virus.In order to produce virion, inventor and then build package cell line and (include gag, pol and env genes, but do not include LTR
With packaging composition).Recombinant plasmid containing target gene together with slow virus LTR and packaging sequence, is concomitantly introduced into bag by inventor
Fill in cell line.Packaging sequence allows recombinant plasmid rna transcription product to be wrapped into virion, is then secreted into culture
In base.And then inventor collects the matrix for including recombinant slow virus, selectively concentrates, and be used for gene transfer.Slow carrier
It can infect various kinds of cell type, including can somatoblast and can not somatoblast.
In addition, according to an embodiment of the invention, the slow virus of the embodiment of the present invention is compound slow virus, except common slow
Viral gene gag, pol and env, also include other genes of regulation and control and structure function.Slow virus carrier is art technology
Known to personnel, slow virus includes：Human immunodeficiency virus HIV -1, HIV -2 and simian immunodeficiency virus SIV.Slow disease
Poisonous carrier is produced by Multiple decrements AIDS virus Disease-causing gene, such as all deletes gene env, vif, vpr, vpu and nef,
Slow virus carrier is set to form biological safe type carrier.Recombined lentivirus vector can infect Unseparated Cell, while can be used for body
The transfer of interior and outer-gene and nucleotide sequence expression.Such as：In suitable host cell, and with packaging function (gag,
Pol, env, rev and tat) two or more carriers together, Unseparated Cell can be infected.The targeting of recombinant virus,
It is to be realized by antibody or particular ligand (targeting particular cell types acceptor) and the combination of memebrane protein.Meanwhile recombinate disease
The targeting of poison is encoded specific by inserting an ordered sequence (including regulatory region) into viral vector together with another
The gene of the part of acceptor on target cell, carrier is set to be provided with specific targeting.Various useful slow virus carriers, and respectively
Carrier caused by kind method and operation etc., for changing the expression of cell.According to an embodiment of the invention, the embodiment of the present invention
The DNA structures of one or more known serum type gland association viral vectors can be used in gland association viral vector (AAV).
In addition, according to an embodiment of the invention, the embodiment of the present invention also includes micro- gene.Micro- gene means with combination
(selected nucleotide sequence and exercisable necessary relevant connection sequence) is instructed to convert, transcribed and/or gene outcome exists
Expression in inner or in vitro host cell.The expression for including continuous target gene using " exercisable connection " sequence controls
Sequence, and act on trans or far distance controlled target gene expression control sequence.
In addition, the carrier of the embodiment of the present invention also includes conventional control element.Substantial amounts of expression control sequence (including it is natural
, inducible and/or particular organization promoter) it is likely to be used.According to an embodiment of the invention, promoter is organizing specific
Type promoter.According to an embodiment of the invention, promoter is inducible promoter.According to an embodiment of the invention, promoter is
Selected from the promoter based on selected carrier.According to an embodiment of the invention, when selecting slow virus carrier, promoter is CMV IE
Gene, EF-1 α, ubiquitin C, or phosphoglycerokinase (PGK) promoter.Other conventional expression control sequences include it is optional mark or
The nucleotide sequence of reporter gene, including encoding geneticin, hygromycin, ampicillin or Puromycine resistance etc..Carry
The other assemblies of body include replication orgin.
What the technology of carrier construction was well known to those skilled in the art, these technologies include conventional cloning techniques.
According to an embodiment of the invention, inventor, which constructs, co-expresses optional nonfunctional EGFR acceptors and recombinant receptor
Viral vector.The transport of the embodiment of the present invention express optional nonfunctional EGFR acceptors nucleic acid molecules and expression restructuring by
The viral vector or plasmid of body be it is compound, this viral vector or plasmid can conjugated polymer or other materials it is stable to increase its
Property, or assist its targeting motion.
Transgenosis lymphocyte
On the other hand, the present invention proposes a kind of transgenosis lymphocyte.According to an embodiment of the invention, the transgenosis
The foregoing recombinant receptor of Expressions In Lymphocytes, optionally, the transgenosis Expressions In Lymphocytes nonfunctional EGFR.The present invention
The transgenosis lymphocyte of embodiment is strong, safe to the specific killing effect of tumour cell.
According to a particular embodiment of the invention, the lymphocyte is antigenspecific T lymphocyte, tumor-infiltrated T leaching
Bar cell, periphery blood T lymphocyte, Natural killer T cells, NK.Antigen according to embodiments of the present invention
T lymphocyte specific, tumor-infiltrated T lymphocytes, periphery blood T lymphocyte, Natural killer T cells or NKT
Cell, it can be achieved to kill the specific immunity of tumour cell, it is safe.
The method of prepare transgenosis lymphocyte
On the other hand, the present invention proposes a kind of method for preparing foregoing transgenosis lymphocyte.According to this hair
Bright embodiment, methods described include：Foregoing construct is incorporated into lymphocyte or T lymphocytes.Utilize
The above method according to embodiments of the present invention, foregoing transgenosis lymphocyte can be simply, efficiently obtained, such as preceding institute
State, the transgenosis lymphocyte obtained significantly improves to the specific killing of tumour cell, safety.
Therapeutic combination for treating cancer
Another further aspect, the present invention propose a kind of therapeutic combination for treating cancer.According to an embodiment of the invention,
The therapeutic combination includes：Foregoing construct, foregoing transgenosis lymphocyte, it is foregoing restructuring by
Body or foregoing nucleic acid.Using therapeutic combination according to embodiments of the present invention, can realize has to tumour cell
Effect, safely kill.
According to an embodiment of the invention, there is provided to the therapeutic combination of the embodiment of the present invention of patient, be preferably applied to
Bio-compatible solution or acceptable pharmacy delivery vehicle.Various therapeutic combinations as preparation are suspended or are dissolved in medicine
Upper or physiologically acceptable carrier, such as physiological saline；Isotonic salting liquid or other people's being proficient in the knowledge of is obvious
In formula.Appropriate carrier depends greatly on method of administration.Other have water and anhydrous isotonic sterile injection liquid and
There are water and anhydrous sterile suspensions, be pharmaceutically acceptable carrier.
According to an embodiment of the invention, sufficient amount of viral vector is transduceed in targeting T-cells, and is provided sufficiently strong
The transgenosis of degree, express optional nonfunctional EGFR acceptors and the distinctive recombinant receptor of expression.The dosage of therapeutic reagent is main
Depending on treating situation, age, body weight, the health degree of patient, so as to cause the variability of patient.
It is therapeutic alliance to express optional nonfunctional EGFR acceptors and distinctive these methods of above-mentioned recombinant receptor of expression
A part.These viral vectors and the antitumor T cell for adoptive immunotherapy, can be by individually or with reference to other treatment
The method of cancer performs together.Under suitable conditions, treatment method including the use of one or more medicinal treatments.
According to an embodiment of the invention, the type of the cancer is not particularly limited, medicine according to embodiments of the present invention
Specific killing significant effect of the composition to PD-L1 positive tumor cells.
The method for improving lymphocyte immunity killing ability
The present invention last in terms of, the present invention propose it is a kind of improve lymphocyte immunity killing ability method.Root
According to embodiments of the invention, this method includes：The foregoing recombinant receptor of lymphocyte table.Using according to of the invention real
The above method of example is applied, lymphocyte can be effectively improved the specific immunity of tumour cell is killed.
It should be noted that " recombinant receptor " involved in the present invention is recombinant protein or fusion protein, the restructuring by
Body surface is reached on the film of recipient cell (such as lymphocyte), plays the function of receptor protein, can be with extracellular single-minded signal point
Son combines and then series of biochemical reactions in active cell, cell is produced corresponding effect to environmental stimuli.
The solution of the present invention is explained below in conjunction with embodiment.
It will be understood to those of skill in the art that the following examples are merely to illustrate the present invention, and it should not be regarded as limiting this
The scope of invention.Unreceipted particular technique or condition in embodiment, according to the technology or bar described by document in the art
Part (such as write with reference to J. Pehanorm Brookers etc., what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press)
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can be by acquisition purchased in market
Conventional products.
Used cell line and basic experiment technology are as described below in the examples below：
The generation of slow virus and the transduction of human T lymphocyte
Purpose is to produce the slow virus carrier of replication defective, and slow virus carrier is collected by centrifugation for human T lymphocyte
Transduction.
The generation of slow virus carrier, the experimentation collected is briefly described below：293T cells are layered on into floor space is
In the Tissue Culture Dish of 150- square centimeters, and according to specification, (Open Biosystems/ are purchased from using Express-In
Thermo Scientific, Waltham, MA) viral transduction is carried out to 293T cells.Often disk cell adds 15 μ g slow virus
PVSV-G (VSV P-glycoprotein expressions plasmid), the 10 μ g pCMVR8.74 plasmids (Gag/Pol/Tat/Rev of transgenosis plasmid, 5 μ g
Expression plasmid) and 174 μ l Express-In (concentration is 1 μ g/ μ l).Supernatant was collected respectively at 24 hours and 48 hours, and is made
With ultracentrifuge 28,000 rpm (centrifuge rotor is Beckman SW 32Ti, purchased from Beckman Coulter,
Brea, CA) under conditions of centrifuge 2 hours.Weight finally is carried out to virus particle precipitation with 0.75ml RPMI-1640 culture mediums
It is outstanding.
The primary T lymphocytes of people are separated from Volunteer donor.Human T lymphocyte is cultivated in RPMI-1640 culture mediums
And use AntiCD3 McAb and the CD28 coated pearl of monoclonal antibody (being purchased from Invitrogen, Carlsbad, CA) to carry out stimulation and swash
It is living.18~24 hours after human T lymphocyte's activation, T lymphocytes are transduceed using the method for spin-inoculation, transduceed
Process is as described below：In 24- orifice plates, 0.5 × 10 is covered with per hole6T lymphocytes, the upper of 0.75ml is added into every hole cell
State the viral supernatants and Polybrene of resuspension (concentration is 8 μ g/ml).The mixed liquor of cell and virus particle is in desk centrifuge
(it is purchased from Sorvall ST 40；Thermo Scientific) in centrifuge, centrifugal condition is room temperature, 2500rpm, and the time is 90 points
Clock.People's recombination leukocyte mesonium-2 (IL-2；Purchased from Novartis, Basel, Switzerland) every 2~3 days addition T lymphs
In cell culture fluid, IL-2 final concentration of 100-IU/ml, in T lymphocyte incubations, the density for keeping cell is
0.5×106~1 × 106/ml.Once dormancy occur in the T lymphocytes transduceed, such as vitro growth rates are slack-off and cell becomes
It is small, wherein, vitro growth rates and size are assessed by Coulter Counter (being purchased from Beckman Coulter), or
On the time point that some is planned, T lymphocytes can be used to do work and can analyze the T lymphocytes transduceed.
Flow cytometer used (is purchased from BD for BD FACSCanto II in embodiments herein
Biosciences), and flow cytometric analysis data using FlowJo version 7.2.5 softwares (be purchased from Tree Star,
Ashland, OR) analyzed.
The CDCC (ADCC) of antibody dependent cellular mediation
In the examples below, anti-EGFR-antibodies induced expression nonfunctional EGFR was assessed using 4 hours -51Cr- method for releasing
The ability of the cell dependent antibody cracking of the lymphocyte of acceptor.The human T-lymphocyte of slow virus carrier of having been transduceed is used as
Target cell.100 μ Ci Na251CrO4 (being purchased from GE Healthcare Life Sciences, Marlborough, MA) demarcation 2
The target cells of~5x 106, demarcation condition are that concussion is incubated 1 hour at 37 DEG C.Cell uses PBS rinses three times, and uses culture medium
It is resuspended (cell density is 1x 105/ml).Then, the cell being calibrated is layered in 96- orifice plates (to be covered with 5 × 103 thin per hole
Born of the same parents, added with 50 μ l culture mediums), and add 50 μ l anti-EGFR-antibodies (being purchased from Erbitux, Genentech) (final concentration of 20 μ
G/ml), 30 minutes of preculture then change the culture medium containing antibody into ordinary culture medium under normal temperature condition, thus examine
Survey 51Cr spontaneous release.Final concentration of 1% Triton X-100 are added to ensure 51Cr maximum burst size.In following tool
During body is implemented, human PBMC (effector cell) adds in orifice plate (per 5 × 105, hole cell) and by cell in 37 DEG C of overnight incubations.
Second day, cell conditioned medium is collected, and determines 51Cr release with this using gamma counter calculating cpm.Cytotoxicity ratio is used
Below equation calculates：% Specific lytics=(experiment release cpm data-spontaneous release cpm data)/(maximum release cpm numbers
According to-spontaneous release cpm data) * 100, wherein, maximum release cpm data, which pass through, to be added Triton X-100 and realizes in target cell
, spontaneous release cpm data measure under conditions of no anti-egfr antibodies and effector cell.
Chromium release experiment
Apply for 4-hour in embodiment51The cytotoxic activity of chromium method for releasing analysis and evaluation recombinant receptor T cell.Specific steps
It is as follows：Target detection cell is used51Cr mark 1 hour under 37 degrees Celsius.After mark, with containing 10% hyclone (FCS)
RPMI culture medium rinse cells.After rinse, cell is resuspended in identical culture medium, the concentration that cell is resuspended is 1 × 105/
ml.T cell is with different effect target cell ratio (E after transduction：T) add in target detection cell suspending liquid, and cell kind is existed
It it is 200 microlitres per pore volume in 96- holes.Cell is cultivated 4 hours in 37 degree of incubators.After 4 hours, taken out from every hole
The 96- microwell plates that 30 microlitres of supernatant is put in counter carry out analysis of accounts.Analytical instrument is the micro- scinticountings of top counting NXT
Device (is purchased from Packard Bioscience).The number of effector cell is calculated based on T cell sum in all counting holes.
Labeled target detection cell is PD-L1 positive tumor cells.
The carrier of the structure of embodiment 1 coexpression nonfunctional EGFR acceptors and PD1-CD28-CD3zeta recombinant receptors
In the present embodiment, inventor will encode someone's PD1 extracellular segments sequence, CD28 wear film and intracellular section and T cell by
ζ-chain-ordering of body combination is cloned on the slow virus carrier containing EF-1 promoters (lentiviral vector), is cloned
Cheng Zhong, the restricted digestion of selection are XbaI and NotI double digestions, and NotI and XhoI double digestions, pass through digestion, connection, sieve
Choosing and the amplification of purpose plasmid, the slow virus plasmid (LV-PD1-CD28-CD3 ζ) of generation expression recombinant receptor.Comprising IRES and
The sequence of expression nonfunctional EGFR acceptors is cloned into LV-PD1-CD28-CD3 ζ vector plasmids, is built into LV-PD1-CD28-
CD3ζ-tEGFR.Fig. 1 is schematic diagram (the wherein E expression extracellular fragments of slow virus carrier；TEGFR represents nonfunctional EGFR), comprising
Encode the sequence of PD1-CD28-CD3 ζ recombinant receptors, IRES and coding nonfunctional EGFR receptor sequences.Encode PD1-CD28-
The sequence of CD3 ζ recombinant receptors expresses the sequence of nonfunctional EGFR acceptors as a list under promoter EF-1 startup regulation and control
Only mRNA transcriptional units after the IRES sequences translate.
In addition, the ζ that coding someone PD1 extracellular segments and cross-film section sequence, CD28 intracellulars section and φt cell receptor are combined-
The process that chain-ordering is cloned into the slow virus carrier containing EF-1 promoters is as described above.
The anti-egfr antibodies of embodiment 2 can effectively kill the T lymphs for removing coexpression nonfunctional EGFR acceptors and recombinant receptor
Cell
In the present embodiment, PBLC is derived from blank blood donor.PBLC by gradient from
The heart is separated, Gradient Centrifuge Ficoll-Hypaque.T lymphocytes and t cell activation factor magnetic bead CD3/CD28 (purchases
From Invitrogen, Carlsbad, CA) culture 72 hours is incubated under 5%CO2,37 degrees Celsius, culture medium is added with 2mmol/
L glutamine, the hyclone (FCS) (being purchased from Sigma-Aldrich Co.) of 10% high-temperature inactivation and 100U/ml mould
The dual anti-RPMI culture mediums 1640 of element/streptomysin (are purchased from Invitrogen Gibco Cat.no.12633-012).Activation culture
After 72 hours, with washing lotion rinse cell, magnetic bead is washed away.T cell kind is being covered with restructuring CH-296 (FN ch-296；
Retronectin) on Tissue Culture Dish, and with lentiviruses transduction, transduction slow virus be respectively LV-PD1-CD28-CD3 ζ-
TEGFR, LV-PD1-CD28-CD3 ζ or zero load (LV-GFP) transductive process are as previously described.Nonfunctional EGFR acceptors are expressed after transduction
T cell dyed with anti-egfr antibodies after, then fluidic cell cell (FACS) separate, T cell culture is in RPMI- after separation
In 1640 culture mediums and with the recombinant human IL-2 factors (100ng/ml；Purchased from R＆D Systems) carry out induced amplification 7-10 days,
Then the target cell as experiment.Inventor is situated between different to different slow virus of having transduceed with ADCC detection methods measurement anti-egfr antibodies
T cell lethal effect, measuring method uses the chromium method for releasing of 4-hour of standard 51, the chromium method for releasing such as embodiment 1 of 4-hour 51
It is described.As a result it is as shown in Figure 2.The different killing coexpression PD1-CD28-CD3 ζ restructuring as shown in Fig. 2 anti-egfr antibodies can effectively be situated between
The T lymphocytes of acceptor and nonfunctional EGFR acceptors, but anti-egfr antibodies can not be situated between different killing only express PD1-CD28-CD3 ζ weight
The T lymphocytes of group acceptor, anti-egfr antibodies can not be situated between the T lymphocytes of different killing zero load lentiviruses transduction, statistics generation
Average value ± the SEM in three holes of table.
Embodiment 3 co-expresses the T lymphocytic tumours cells of nonfunctional EGFR acceptors and PD1-CD28-CD3 ζ recombinant receptors
Solvability
In the present embodiment, PBLC is separated by gradient centrifugation, Gradient Centrifuge Ficoll-
Hypaque.T lymphocytes are with t cell activation factor magnetic bead CD3/CD28 (being purchased from Invitrogen, Carlsbad, CA) 5%
CO2, culture 72 hours is incubated under 37 degrees Celsius, culture medium is added with 2mmol/L glutamine, the tire ox blood of 10% high-temperature inactivation
The dual anti-RPMI culture mediums 1640 of (FCS) (being purchased from Sigma-Aldrich Co.) and 100U/ml penicillin/streptomycin (purchase clearly
From Invitrogen Gibco Cat.no.12633-012).After activation culture 72 hours, with washing lotion rinse cell, magnetic bead is washed
Go.T cell kind is being covered with restructuring CH-296 (FN ch-296；Retronectin) on Tissue Culture Dish, and with slowly
Viral transduction, transduction slow virus is respectively LV-PD1-CD28-CD3 ζ-tEGFR, LV-PD1-CD28-tEGFR (structure such as Fig. 1 institutes
Show), LV-tEGFR (structure is as shown in Figure 1), or it is unloaded (LV-GFP), transductive process is as previously described.T cell culture after transduction
In RPMI-1640 culture mediums and with the recombinant human IL-2 factors (100ng/ml；Purchased from R＆D Systems) carry out induced amplification
7-10 days, then carry out functional test experiment.Inventor's measurement has been transduceed the T cell brain positive to PD-L1 of different slow virus
The lethal effect of glioma cell, effect target ration is 10：1 or 25:1 or 50:1, measuring method uses 4-hour of standard 51
Chromium method for releasing, wherein, the chromium method for releasing of 4-hour 51 is as previously described.
Test result is as shown in figure 3, Fig. 3 results are shown：Co-express PD1-CD28-CD3 ζ acceptors and nonfunctional EGFR acceptors
The T lymphocytes (LV-PD1-CD28-CD3 ζ-tEGFR T lymphocytes) of lentiviruses transduction can significantly kill PD-L1+'s
Tumor cells.The slow virus for co-expressing PD1-CD28 acceptors (not connecting CD3zeta chains fragment) and nonfunctional EGFR acceptors turns
The T lymphocytes (LV-PD1-CD28-tEGFR T lymphocytes) led are without obvious killing PD-L1+Tumor cells effect.Only table
T lymphocytes (LV-tEGFR T lymphocytes) or unloaded lentiviruses transduction up to the lentiviruses transduction of nonfunctional EGFR acceptors
T lymphocytes (control LV-GFP T lymphocytes) are to PD-L1+Tumor cells are without obvious lethal effect.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with office
Combined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this area
Art personnel can be tied the different embodiments or example and the feature of different embodiments or example described in this specification
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Claims (21)
- A kind of 1. recombinant receptor, it is characterised in that including：Immunologic test point molecule fragment；Molecules of immunization stimulus fragment；Andφt cell receptor zeta chains.
- 2. recombinant receptor according to claim 1, it is characterised in that the immunologic test point molecule is PD1.
- 3. recombinant receptor according to claim 2, it is characterised in that the immunologic test point molecule fragment includes PD1's Extracellular region and optional transmembrane region, the molecules of immunization stimulus fragment include CD28 intracellular region and optional transmembrane region.
- 4. recombinant receptor according to claim 3, it is characterised in that including：(a) extracellular region and transmembrane region of the PD1；And(b) intracellular region of the CD28,Or including：(i) extracellular region of the PD1；And(ii) intracellular region and transmembrane region of the CD28.
- 5. recombinant receptor according to claim 1, it is characterised in that the φt cell receptor zeta chains are CD3zeta chains.
- 6. recombinant receptor according to claim 1, it is characterised in that the C-terminal of the immunologic test point molecule fragment and institute The N-terminal for stating molecules of immunization stimulus fragment is connected, the C-terminal of the molecules of immunization stimulus fragment and the N of the φt cell receptor zeta chains End is connected.
- 7. a kind of recombinant receptor, it is characterised in that the recombinant receptor has SEQ ID NO：Amino acid sequence shown in 1 or 2.
- A kind of 8. nucleic acid, it is characterised in that the recombinant receptor described in the nucleic acid coding any one of claim 1~7,Optionally, the nucleic acid has SEQ ID NO：Nucleotide sequence shown in 3 or 4.
- 9. a kind of construct, it is characterised in that the construct carries the nucleic acid described in claim 8.
- 10. construct according to claim 9, it is characterised in that the construct further carries coding nonfunctional EGFR nucleic acid, the nucleic acid of the coding nonfunctional EGFR have SEQ ID NO：Nucleotide sequence shown in 5.
- 11. construct according to claim 10, it is characterised in that the construct further carries internal ribosome and entered Angle of striking sequence, the internal ribosome entry site sequence have SEQ ID NO：Nucleotide sequence shown in 6, and it is described Internal ribosome entry site sequence be arranged at nucleic acid described in claim 8 and the coding nonfunctional EGFR nucleic acid it Between.
- 12. construct according to claim 10, it is characterised in that the construct further carries coding connection peptide Nucleic acid, the nucleic acid of the coding connection peptide have SEQ ID NO：Nucleotide sequence shown in 7~10, and the coding connection The nucleic acid of peptide is arranged between the nucleic acid described in claim 8 and the coding nonfunctional EGFR nucleic acid.
- 13. construct according to claim 9, it is characterised in that the construct further carries the first promoter, institute The first promoter is stated to be operably connected with the nucleic acid described in claim 8,Optionally, first promoter includes being selected from CMV, EF-1, LTR or RSV promoters.
- 14. construct according to claim 10, it is characterised in that the construct further carries the second promoter, Second promoter is operably connected with the nucleic acid of the coding nonfunctional EGFR,Optionally, second promoter includes being selected from CMV, EF-1, LTR or RSV promoters.
- 15. construct according to claim 9, it is characterised in that the carrier of the construct be retrovirus vector, Slow virus carrier or adeno-associated virus (AAV) carrier.
- 16. a kind of construct, it is characterised in that the construct carries following nucleic acid molecules:(1) nucleic acid molecules of encoding immune checkpoint molecule fragment, the immunologic test point molecule fragment have SEQ ID NO： Amino acid sequence shown in 11 or 12, the nucleic acid molecules of encoding immune checkpoint molecule fragment have SEQ ID NO：13 or Nucleotide sequence shown in 14；(2) nucleic acid molecules of encoding immune stimulation molecule fragment, the molecules of immunization stimulus fragment have SEQ ID NO：15 or Amino acid sequence shown in 16, the nucleic acid molecules of the encoding immune stimulation molecule fragment have SEQ ID NO：Shown in 17 or 18 Nucleotide sequence；And(3) nucleic acid molecules of encoding T cell receptor zeta chains, the φt cell receptor zeta chains have SEQ ID NO：Shown in 19 Amino acid sequence, the nucleic acid molecules of the encoding T cell receptor zeta chains have SEQ ID NO：Nucleotides sequence shown in 20 Row,Optionally, the construct further carries coding nonfunctional EGFR nucleic acid molecules, the coding nonfunctional EGFR's Nucleic acid molecules have SEQ ID NO：Nucleotide sequence shown in 5, the nonfunctional EGFR have SEQ ID NO：Shown in 21 Amino acid sequence,Optionally, the construct further carries the nucleic acid molecules of internal ribosome entry site sequence, the internal ribosomal The nucleic acid molecules of body entry site sequence have SEQ ID NO：Nucleotide sequence shown in 6, and the coding internal ribosomal The nucleic acid molecules of body entry site sequence be arranged at the encoding T cell receptor zeta chains nucleic acid molecules and it is described coding it is idle Between energy EGFR nucleic acid molecules,Optionally, the construct further carries the nucleic acid of coding connection peptide, and the nucleic acid of the coding connection peptide has SEQ ID NO：Nucleotide sequence shown in 7~10, the connection peptide have SEQ ID NO：Amino acid sequence shown in 22~25, and And the nucleic acid of the coding connection peptide is arranged at the nucleic acid molecules of the encoding T cell receptor zeta chains and the coding nonfunctional Between EGFR nucleic acid molecules,Optionally, the construct further carries the first promoter, first promoter and the encoding immune checkpoint The nucleic acid molecules of molecule fragment are operably connected,Optionally, first promoter includes being selected from CMV, EF-1, LTR or RSV promoters,Optionally, the construct further carries the second promoter, second promoter and the coding nonfunctional EGFR Nucleic acid molecules be operably connected,Optionally, second promoter includes being selected from CMV, EF-1, LTR or RSV promoters,Optionally, the carrier of the construct is retrovirus vector, slow virus carrier or adeno-associated virus (AAV) carrier.
- A kind of 17. transgenosis lymphocyte, it is characterised in that any one of described transgenosis Expressions In Lymphocytes claim 1~7 Described recombinant receptor,Optionally, the transgenosis Expressions In Lymphocytes nonfunctional EGFR.
- 18. transgenosis lymphocyte according to claim 17, it is characterised in that the lymphocyte is antigentic specificity T lymphocytes,Optionally, the lymphocyte is tumor-infiltrated T lymphocytes,Optionally, the lymphocyte is periphery blood T lymphocyte,Optionally, the lymphocyte is Natural killer T cells,Optionally, the lymphocyte is NK.
- A kind of 19. method for preparing the transgenosis lymphocyte described in any one of claim 17~18, it is characterised in that bag Include：Construct described in any one of claim 9~16 is incorporated into lymphocyte or T lymphocytes.
- A kind of 20. therapeutic combination for treating cancer, it is characterised in that including：Any one of nucleic acid described in recombinant receptor, claim 8, claim 9~16 described in any one of claim 1~7 Transgenosis lymphocyte described in described construct or any one of claim 17~18.
- A kind of 21. method for improving lymphocyte treatment immunologic cytotoxicity ability, it is characterised in that including：Make the recombinant receptor described in any one of Expressions In Lymphocytes claim 1~7.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610538767.9A CN107586342A (en) | 2016-07-08 | 2016-07-08 | Recombinant immune checkpoint acceptor and its application |
| PCT/CN2017/092377 WO2018006881A1 (en) | 2016-07-08 | 2017-07-10 | Recombinant immune-checkpoint receptor and application thereof |
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| CN201610538767.9A CN107586342A (en) | 2016-07-08 | 2016-07-08 | Recombinant immune checkpoint acceptor and its application |
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| BR112018006237A2 (en) | 2015-09-29 | 2018-10-09 | Celgene Corp | pd-1 binding proteins and methods of using them |
| KR102257154B1 (en) | 2016-09-19 | 2021-05-28 | 셀진 코포레이션 | Methods of treating immune diseases using PD-1 binding protein |
| US10766958B2 (en) | 2016-09-19 | 2020-09-08 | Celgene Corporation | Methods of treating vitiligo using PD-1 binding antibodies |
| WO2018127918A1 (en) | 2017-01-05 | 2018-07-12 | Kahr Medical Ltd. | A sirp alpha-cd70 fusion protein and methods of use thereof |
| US11566060B2 (en) | 2017-01-05 | 2023-01-31 | Kahr Medical Ltd. | PD1-CD70 fusion protein and methods of use thereof |
| US11702458B2 (en) | 2017-01-05 | 2023-07-18 | Kahr Medical Ltd. | PD1-41BBL fusion protein and methods of use thereof |
| HRP20220230T1 (en) | 2017-01-05 | 2022-04-29 | Kahr Medical Ltd. | A sirp1 alpha-41bbl fusion protein and methods of use thereof |
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| CN104114233A (en) * | 2011-07-29 | 2014-10-22 | 宾夕法尼亚大学董事会 | Switch co-stimulatory receptors |
| WO2016025880A1 (en) * | 2014-08-14 | 2016-02-18 | Novartis Ag | Treatment of cancer using gfr alpha-4 chimeric antigen receptor |
| CN105392888A (en) * | 2013-03-16 | 2016-03-09 | 诺华股份有限公司 | Treatment of cancer using humanized anti-cd19 chimeric antigen receptor |
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| CN104769103B (en) * | 2012-09-04 | 2018-06-08 | 塞勒克提斯公司 | Multichain Chimeric antigen receptor and its purposes |
| CN103965361B (en) * | 2013-02-06 | 2018-10-30 | 上海细胞治疗工程技术研究中心集团有限公司 | A kind of chimeric molecule converter of T cell signal and application thereof |
| MX2017001011A (en) * | 2014-07-21 | 2018-05-28 | Novartis Ag | Treatment of cancer using humanized anti-bcma chimeric antigen receptor. |
| CA2964958A1 (en) * | 2014-10-31 | 2016-05-06 | The Trustees Of The University Of Pennsylvania | Methods and compositions for modified t cells |
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- 2016-07-08 CN CN201610538767.9A patent/CN107586342A/en not_active Withdrawn
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- 2017-07-10 WO PCT/CN2017/092377 patent/WO2018006881A1/en active Application Filing
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| CN104114233A (en) * | 2011-07-29 | 2014-10-22 | 宾夕法尼亚大学董事会 | Switch co-stimulatory receptors |
| CN105392888A (en) * | 2013-03-16 | 2016-03-09 | 诺华股份有限公司 | Treatment of cancer using humanized anti-cd19 chimeric antigen receptor |
| WO2016025880A1 (en) * | 2014-08-14 | 2016-02-18 | Novartis Ag | Treatment of cancer using gfr alpha-4 chimeric antigen receptor |
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