CN109970869A - It is a kind of target human TNF related apoptosis-inducing ligand death receptor Chimerical receptor ligand and its application - Google Patents

It is a kind of target human TNF related apoptosis-inducing ligand death receptor Chimerical receptor ligand and its application Download PDF

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CN109970869A
CN109970869A CN201910294369.0A CN201910294369A CN109970869A CN 109970869 A CN109970869 A CN 109970869A CN 201910294369 A CN201910294369 A CN 201910294369A CN 109970869 A CN109970869 A CN 109970869A
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王恩秀
汪晨
武国英
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Nanjing Katy Medical Technology Co Ltd
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Abstract

The present invention provides a kind of Chimerical receptor ligands for targeting TRAIL receptor, include death receptor binding domain, transmembrane region and intracellular signal structural domain based on TRAIL.The present invention also provides a kind of T cells that the Chimerical receptor for targeting TRAIL receptor is ligand modified, can specifically combine with the TRAIL receptor (DR4, DR5) of tumor cell surface, and then special lethal effect is generated to tumour cell.The invention further relates to the Chimerical receptor ligands of the targeting TRAIL receptor and its immune effector cell to prepare the application in anti-tumor immunotherapy medicaments.

Description

It is a kind of target human TNF related apoptosis-inducing ligand death receptor Chimerical receptor ligand and its application
Technical field
The invention belongs to biomedical or biopharmaceutical technologies, are related to a kind of being fitted into for targeting human TNF related apoptosis-inducing ligand receptor Receptors ligand and its application.
Background technique
TRAIL (TNF-related apoptosis-inducing ligand, TRAIL) tumor necrosis factor is relevant Apoptosis induction ligand is one of tumor necrosis factor (tumor necrosis factor, TNF) family member.It can specificity Inducing apoptosis of tumour cell and to normal cell nontoxicity, be a kind of neoplasm targeted therapy drug for having very much development potentiality, TRAIL The assignment of genes gene mapping is 1769bp segment in chromosome 3q26.1-26.2, includes 5 exons.Be molecular mass be 33~35kD II type transmembrane protein.Its cDNA long 1050bp encodes 281 amino acid, and 241 amino acid are located at outside after birth, and functional part is 119~241 amino acids residues, the end N- positioned at cytoplasmic domain, without 14 amino acid residues of clear signal peptide. TRAIL by with cell-membrane receptor zygotic induction Apoptosis.Have now been found that 5 kinds of receptors that can be specifically bound with TRAIL: TRAIL-R1 (death receptor 4, DR4), TRAIL-R2 (death receptor 5, DR5) contain apoptosis structure Domain, in conjunction with TRAIL after can induce apoptosis of tumor cells, therefore referred to as death receptor；TRAIL-R3 (decoy receptor1, DcR1) and TRAIL-R4 (decoy receptor 2, DcR2) is imperfect without apoptosis structural domain or apoptosis structural domain, can not induce Apoptosis, therefore referred to as Decoy receptor, and a kind of and lower specific receptor osteoprotegerin of TRAIL affinity (osteoprotegerin,OPG).It is past multiple studies have shown that, normal tissue expression DcR1 and DcR2, in tumor tissues Tend to express DR4, DR5.Therefore, normal cell due to have DcR1, DcR2 and DR4, DR5 competitive binding TRAIL and from it Attack, tumor tissues then due to lack DcR1, DcR2 protection and easily by TRAIL it is apoptosis-induced.So TRAIL is to a variety of swollen Oncocyte has specificity, apoptosis-induced to have certain selectivity, this provides a new strategy for the biological therapy of tumour again.
Chimeric antigen receptor T cell therapy (Chimeric antigen receptor T-cell immunotherapy, CAR-T) become at present one of most promising tumour immunotherapy (J Biomed Biotechnol, 2010, May 5, 2010:956304).Chimeric antigen receptor (CAR) is by a tumor associated antigen combined area or antigen-binding domains, extracellular The structure compositions such as hinge area, transmembrane region and intracellular signal transduction area.The antigen-binding domains of general CAR are by various forms of Antibody composition.
The present invention provides a kind of a kind of Chimerical receptor ligand of novel targeting human TNF related apoptosis-inducing ligand receptor, and it includes intracellular second Conducting structure domain, T2A, extracellular signal peptide, TRAIL receptor binding domain, the first conducting structure intracellular domain, such as intracellular second Conducting structure domain DAP12, T2A, CD8 alpha signal peptide, TRAIL receptor binding domain, the first conducting structure intracellular domain NKp44 (DAP12-T2A-CD8 alpha signal peptide-TRAIL-NKp44).Use the first signal transduction structure intracellular and second signal conduction knot Structure secretes lower level cell factor when conducive to CAR-T cell by TRAIL receptor for stimulating, can guarantee that well clinic is answered Safety.Use TRAIL as TRAIL receptor binding domain, can induced various types of tumors Apoptosis, and protect simultaneously Protecting normal cell has wider array of anticancer spectrum from cell toxicity, and since TRAIL CRL-T uses trail protein As targeted molecular, therefore potential immunogenicity is avoided when being applied in human body.
Summary of the invention
The present invention using the interaction principle between ligand and receptor, match by the Chimerical receptor for providing a kind of targeting human receptor Body (Chimeric Receptor Ligand, CRL), the structure of the Chimeric antigen receptor different from tradition based on antibody scFv fragment It makes, is a kind of structure of completely new, Antibody independent, targeting human receptor similar Chimeric antigen receptor.The present invention provides Targeting human TNF related apoptosis-inducing ligand receptor Chimerical receptor ligand (TRAIL CRL) may include the TRAIL based on natural TRAIL molecule by Body specific binding domains, hinge area or hingeless sequence, transmembrane region and intracellular signal transduction structural domain etc..The present invention is into one Step provides a kind of immunocyte ligand modified by Chimerical receptor, the i.e. T cell of TRAIL receptor chimera receptors ligand modification (Chimeric Receptor Ligand Modified T cell, CRL-T).Targeting human TNF related apoptosis-inducing ligand receptor provided by the invention Chimerical receptor ligand and its immunocyte include two layers of antitumor mechanism, one side TRAIL molecule can be in conjunction with TRAIL receptor It can cause apoptosis of tumor cells access, on the other hand, the combination of TRAIL and TRAIL receptor causes T cell activation and proliferation, hair T cell anti-tumor activity is waved, TRAIL CRL has better target spot specific tumour fragmentation effect, safety and broad spectrum activity；Make Immunotherapy is carried out with based on the natural molecular targeted TRAIL acceptor molecule of human TNF related apoptosis-inducing ligand, heterologous immunogenic will not be generated；? The tumor disease for treating TRAIL receptor positive has high clinical value.
The second object of the present invention is to provide a kind of Chimerical receptor ligand for targeting human TNF related apoptosis-inducing ligand receptor.
The third object of the present invention provides genetic engineering cell of the transformation containing the Chimeric antigen receptor and its application.
The fourth object of the present invention is to provide the Chimerical receptor ligand and its immune effector cell for targeting human TNF related apoptosis-inducing ligand receptor Using.
Targeting human TNF related apoptosis-inducing ligand receptor chimera receptors ligand and its immunological effect are prepared it is yet another object of the invention to provide a kind of The method of cell.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of Chimerical receptor ligand targeting human TNF related apoptosis-inducing ligand receptor includes the second conducting structure intracellular domain, T2A, extracellular signal Peptide, TRAIL receptor binding domain, the first conducting structure intracellular domain.
In embodiments of the invention, the Chimerical receptor ligand of the targeting human TNF related apoptosis-inducing ligand receptor, TRAIL receptor are special Different binding structural domain includes the amino acid sequence that TRAIL receptor is specifically bound shown in SEQ ID NO:5, or is had with it The amino acid sequence of 85%~99% or 90%~99% or 95%~99% identity.
In embodiments of the invention, the Chimerical receptor ligand of the targeting human TNF related apoptosis-inducing ligand receptor, intracellular signal structure Domain includes the second conducting structure intracellular domain, T2A, extracellular signal peptide, TRAIL receptor binding domain, the first conducting structure intracellular Domain.
The first signal transduction structural domain intracellular, can be selected from KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4、KIR2DL5A、KIR2DL5B、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、KIR3DL1、KIR3DS1、 KIR3DL2,KIR3DL3,KIR2DP1,KIR3DP1；NKp46,NKp30,NKp44,SLAM,CD48,CD229,2B4,CD84, NTB-A、CRACC、BLAME、、CD2F-10、KLRD1/KLRC2、SIRPB1、PILRB、CLEC5A、TREM1、TREM2、 CD300B, CD300E, SIGLEC-14, SIGLEC-15, SIGLEC-16 and/or NKG2D, preferably NKp44 molecule.
The second conducting structure intracellular domain can be selected from DAP10, DAP12 and/or Fc ε R γ, preferably DAP12 molecule.
In embodiments of the invention, the Chimerical receptor ligand of the targeting human TNF related apoptosis-inducing ligand receptor includes Protein secretion Associated signal peptide sequence can be selected from CD8 alpha signal peptide, GM-CSFR alpha signal peptide or CD4 signal peptide signal peptide, preferably CD8 alpha signal Peptide.
The Chimerical receptor ligand of the targeting human TNF related apoptosis-inducing ligand receptor, may include Linker between extracellular region and transmembrane region Structure, or do not include Linker structure.
The Chimerical receptor ligand of targeting human TNF related apoptosis-inducing ligand receptor of the present invention, may include two or more repetitions The series connection of TRAIL molecule Chimerical receptor ligand or multiple TRAIL receptor binding domains based on TRAIL molecule are serially connected, Or it is connected with the antigen-binding domains based on antibody the multivalence to be formed based on the TRAIL receptor binding domain of TRAIL molecule Target spot specificity Chimerical receptor ligand combination, can be connected by one or more Linker structures between repetitive unit.
In embodiments of the invention, the Chimerical receptor ligand of the targeting human TNF related apoptosis-inducing ligand receptor, it is characterised in that coding Amino acid sequence be or to have with it 85%~99% or 90%~99% or 95%~99% same shown in SEQ ID NO:8 The transformation amino acid sequence of one property.
A kind of nucleic acid molecules, it is characterised in that the core of the Chimerical receptor ligand of coding targeting human TNF related apoptosis-inducing ligand receptor described above Nucleotide sequence.
In embodiments of the invention, the nucleic acid molecules, it is characterised in that nucleotide coding sequence such as SEQ ID Shown in NO:7.
A kind of carrier, it is characterised in that the core comprising encoding the Chimerical receptor ligand of the targeting human TNF related apoptosis-inducing ligand receptor above Acid sequence.
A kind of immune effector cell of genetic engineering transformation, comprising encoding targeting human TNF related apoptosis-inducing ligand receptor of the present invention The gene order of Chimerical receptor ligand.
The preferred T lymphocyte of the immune effector cell, NK cell, NKT cell, macrophage, mescenchymal stem cell, The immunocyte of candidate stem cell, multipotential stem cell or embryonic stem cell culture differentiation, further preferred T lymphocyte.
The immunocyte of the described genetic engineering transformation, it is characterised in that the targeting human TNF related apoptosis-inducing ligand receptor of expression it is chimeric by Body ligand includes TRAIL receptor-specific binding structural domain, transmembrane region and intracellular signal structural domain based on TRAIL molecule.
In embodiments of the invention, the immunocyte of genetic engineering transformation, it is characterised in that the target of expression There is amino acid sequence shown in SEQ ID NO:8 to the Chimerical receptor ligand of human TNF related apoptosis-inducing ligand receptor, or have 85%~99% with it Or 90%~99% or 95%~99% identity amino acid sequence.
The present invention provides a kind of method of Chimerical receptor ligand for preparing targeting human TNF related apoptosis-inducing ligand receptor, and this method includes separation With activate T cell to be finished, then transduceed the T cell with foregoing expression vectors.
It is anti-in preparation that the present invention relates to the Chimerical receptor ligands of the targeting human TNF related apoptosis-inducing ligand receptor and its immune effector cell Application in immunotherapy of tumors drug, the tumour preferably include oophoroma, prostate cancer, cancer of pancreas, kidney, skin Cancer, thymoma, sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, the cancer of the brain, bladder cancer, breast cancer, cervical carcinoma, Colon and rectum Cancer, liver cancer, renal cancer, lymthoma, leukaemia, lung cancer, melanoma, metastasis melanin tumor, celiothelioma, neuroblast Tumor, uterine cancer and any combination thereof.
In a particular embodiment, it is related to the medicinal usage of preparation treatment entity tumor and blood system tumour.
The Chimerical receptor ligand of targeting human TNF related apoptosis-inducing ligand receptor of the present invention is related to preparing anti-tumor drug and cell is exempted from Application in epidemic disease therapy.
Immune effector cell of the present invention is related to preparing the application in anti-tumor drug and cellular immunotherapy.
Beneficial effects of the present invention:
The present invention provides a kind of Chimerical receptor ligands for targeting human TNF related apoptosis-inducing ligand receptor, creatively using TRAIL specificity In conjunction with death receptor: the combination of TRAIL-R1 (DR3), TRAIL-R2 (DR4) as extracellular selectively targeted TRAIL molecule Area specifically identifies kinds of tumors relevant surfaces receptor by ligand/receptor, then will be known by intracellular signal transduction molecule Other signal is transmitted into the cell, immune cell activated it is lethal, and then reduce and eliminate tumour cell.The present invention mentions simultaneously A kind of T cell that the Chimerical receptor for targeting human TNF related apoptosis-inducing ligand receptor is ligand modified has been supplied, it can be specifically dead with tumor cell surface Receptor TRAIL-R1 or TRAIL-R2 combination are died, and then generates special lethal effect to tumour cell.TRAIL CRL-T is shown Better induced various types of tumors Apoptosis out, and protect normal cell from cell toxicity simultaneously, use intracellular first Signal transduction structure and second signal conducting structure are secreted when conducive to TRAIL CRL-T cell by extracellular TRAIL receptor for stimulating Cell factor improves the specificity of targeted therapy.It is provided by the invention targeting human TNF related apoptosis-inducing ligand receptor Chimerical receptor ligand and its Immunocyte includes two layers of antitumor mechanism, and one side TRAIL molecule can cause apoptosis of tumor cells in conjunction with TRAIL receptor Access, on the other hand, the combination of TRAIL and TRAIL receptor cause T cell activation and proliferation, play T cell anti-tumor activity, TRAIL CRL has better target spot specific tumour fragmentation effect, safety and broad spectrum activity；Using based on natural people The molecular targeted TRAIL acceptor molecule of TRAIL carries out immunotherapy, will not generate heterologous immunogenic；In treatment TRAIL receptor sun The tumor disease of property has high clinical value.
Detailed description of the invention
Fig. 1 is the Chimerical receptor ligand structure schematic diagram for the targeting human TNF related apoptosis-inducing ligand that the present invention constructs.
Fig. 2 is that TRAIL CRL-T cell infection slow virus is identified after 7 days by flow cytomery T cell surface
The positive expression rate of trail protein.
Cell proliferative conditions after Fig. 3 TRAIL CRL-T cell infection slow virus, TRAIL CRL-T and NTD cell is in body Outer amplification 7 days, wherein NTD is the T cell of untransfected TRAIL slow virus.
Fig. 4 TRAIL death receptor DR4, DR5 is detected in different tumor cell surface expression situations.
The secretion situation of Fig. 5 TRAIL CRL-T cell IL2 under TRAIL receptor for stimulating, TRAIL CRL-T and target cell It co-cultures for 24 hours.
The secretion situation of Fig. 6 TRAIL CRL-T cell IFN-γ under TRAIL receptor for stimulating, TRAIL CRL-T and target Cell co-cultures for 24 hours.
The secretion situation of Fig. 7 TRAIL CRL-T cell TNF under TRAIL receptor for stimulating, TRAIL CRL-T and target cell It co-cultures for 24 hours.
The secretion situation of Fig. 8 TRAIL CRL-T cell IL6 under TRAIL receptor for stimulating, TRAIL CRL-T and target cell It co-cultures for 24 hours.
For Fig. 9 TRAIL CRL-T cell to antigen positive cell strain lethal effect, TRAIL CRL-T is total with target cell respectively Culture killing 8h.
Specific embodiment
The present invention provides a kind of a kind of Chimerical receptor ligand of novel targeting human TNF related apoptosis-inducing ligand death receptor, and it includes intracellular Second conducting structure domain, T2A, extracellular signal peptide, TRAIL receptor binding domain, the first conducting structure intracellular domain, below with reference to Specific embodiment, the present invention is further explained.
" the Chimerical receptor ligand of TRAIL death receptor " as described herein is with specific bond death receptor DR4, DR5 Ability.
The term as used herein " intracellular signal structural domain " refer to can transfer cell effector function signal and guide carefully The protein structure region of born of the same parents' execution specific function.Intracellular signal structural domain may include the first signal transduction structural domain, second Signal transduction structural domain and/or transmembrane domain.
The Chimerical receptor ligand of targeting human TNF related apoptosis-inducing ligand death receptor of the invention includes extracellular signal peptide structure, such as CD8 α Signal peptide, 4-1BB signal peptide, GM-CSFR alpha signal peptide or CD4 signal peptide, preferably CD8 alpha signal peptide.
" identity " (identity) of the term as used herein amino acid sequence can be used interchangeably with " similitude ", be referred to Be between amino acid sequence by sequence alignment program such as BLAST determine similarity degree.The side of amino acid alignment Method and software are well known to the skilled person.It can be by carrying out one or several (examples to known amino acid sequence Such as 1-15, such as 2,3,5,8,10 or 12) substitution of amino acid residue, deletion and/or addition and obtain modified ammonia Base acid sequence.For example, by conventional protein engineering means (such as conservative substitution etc.), to SEQ ID NO of the present invention: TRAIL receptor binding domain shown in 6 is transformed, and can obtain has at least 85% (such as 85% with SEQ ID NO:6 ~99% or 90%~99% or 95%~99%) sequence identity, and there is essentially identical TRAIL receptor integrated structure The variant sequence thereof in domain.
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.It is not specified in embodiment specific The experimental method of condition, usually according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker etc. write) Described in condition or according to the normal condition proposed by manufacturer.Test material as used in the following examples, such as without special theory It is bright, it is to be commercially available from routine biochemistry reagent shop.
Embodiment 1, Chimeric antigen receptor preparation
The present invention provides a kind of Chimerical receptor ligand for targeting human TNF related apoptosis-inducing ligand death receptor.Chimeric antigen of the invention Receptor is suitable by the second conducting structure intracellular domain-T2A- extracellular signal peptide-TRAIL binding structural domain-the first conducting structure intracellular domain Sequence is composed in series.
DAP12-T2A-CD8 alpha signal peptide-TRAIL receptor binding domain-NKp44
1, the gene order of the Chimerical receptor ligand of human TNF related apoptosis-inducing ligand death receptor is targeted
Design successively contains natural kill activated receptor (abbreviation DAP12), T2A, CD8 alpha signal peptide, TRAIL integrated structure Domain, nature cell toxin receptor (abbreviation NKp44), structure are as shown in Figure 1.The wherein amino acid sequence of DAP12 such as SEQ ID Shown in NO:1, the amino acid sequence of T2A is as shown in SEQ ID NO:2, the amino acid sequence of CD8 alpha signal peptide such as SEQ ID NO:3 Shown, the amino acid sequence of NKp44 is as shown in SEQ ID NO:4, the nucleotide sequence of TRAIL receptor binding domain such as SEQ Shown in ID NO:5, amino acid sequence is as shown in SEQ ID NO:6.
2, the slow virus carrier of the Chimerical receptor ligand of building expression TRAIL death receptor
PELNS-DAP12-T2A-CD8 α-scfv-NKp44 (plasmid is saved by Nanjing Ka Ti medical science and technology Co., Ltd, or Person is according to document (Enxiu Wang et al.Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based,Multichain,Chimeric Immunoreceptors.2015,Cancer Immunology Research, 3 (7): method disclosed in 815) is constructed, TRAIL receptor binding domain gene chemical synthesis by Sangon Biotech (Shanghai) Co., Ltd. synthesizes and provides pUC57-TRAIL plasmid, by plasmid pUC57-TRAIL and PELNS-DAP12-T2A-CD8 α-scfv-NKp44 segment passes through BamHI, NheI double digestion (being purchased from Takara company), digestion It reacts by specification to carry out, be reconnected after digestion, obtain the slow virus carrier pELNS-DAP12- of expression Chimeric antigen receptor T2A-CD8 alpha signal peptide-TRAIL binding structural domain-NKp44 (abbreviation TRAIL CRL).5 μ L slow virus carriers are converted into large intestine bar (Nanjing An Jieyou Biotechnology Co., Ltd is purchased from) in bacterium Trans-I competent cell, picking Dan Ke after 37 DEG C of culture 16h Grand, the monoclonal of picking is taken out after cultivating 12h under the conditions of 37 DEG C with plasmid extraction kit (purchased from Beijing Quan Shi genome company) Upgrading grain, specific method are shown in specification.
3, slow virus is packed
The present embodiment packs slow virus and uses calcium phosphate method, the specific steps are as follows:
(1) 293T cell passes on every other day
Each T150 cell bottle plantation 5 × 106A cell.After 48 hours, cell number should reach million/bottle of 20-25.
(2) 293T cell spreads bottle
A) by taking 1 T150 Tissue Culture Flask as an example, cell is gently washed twice with 1 × PBS of about 15ml
B) 3ml0.25% pancreatin -2.21mM EDTA is added
C) cell detachment is waited until, the DMEM culture medium that 12ml 10% (wt) FBS (being purchased from Gibico) is added (is purchased from Corning) into the cell to have fallen off
D) it collects and cell is transferred to sterile centrifugation tube, 1000rpm is centrifuged 10 minutes
E) supernatant is sopped up, precipitating is resuspended in the DMEM culture solution of 10ml 10% (wt) FBS.
F) cell count calculates 12 × 10 according to cell concentration6Volume required for a cell
G) the DMEM culture solution of cell and 10% (wt) FBS of 25ml are merged, is put into T150 cell bottle, jog makes It obtains cell and is evenly distributed to 37 DEG C of cell bottle bottom, overnight incubation in 5%CO2 incubator.
(3) cell transfecting
Cell is observed, cell density reaches about 80%-90%, can start to transfect at this time
A) 30-60 minutes before transfection, culture solution is softly sopped up.
B) it mixes Plasmid DNA and calcium chloride solution needs 28ug pRSV.rev (to be purchased from for one T150 bottles Invitrogen company), 28ug pGAG-Pol (is purchased from Invitrogen company), and 11ug pVSVG is (public purchased from Invitrogen Department), 23ug recombinant slow virus expression plasmid TRAIL CRL is added in 1.5ml calcium chloride solution, is mixed.
C) 1.5ml BBS solution is added in 15ml sterile centrifugation tube, DNA- calcium chloride solution is mixed with 1ml pipette tips After be added drop-wise in BBS solution, rapidly mix 15-20 under, be incubated at room temperature 25-30 minutes.
D) with 5ml pipette that DNA- calcium chloride-BBS mixture (being purchased from the green skies Bioisystech Co., Ltd in Shanghai) is equal It is even to be added dropwise in T150 bottles.It is cultivated in 37 DEG C of cell incubators containing 5% carbon dioxide, 6h changes liquid.
E) liquid is changed after 6h.Culture plate is shaked gently for several times with some calcium phosphate precipitations that sufficiently suspend, and it is heavy to suck phosphoric acid calcium The culture solution in shallow lake is added the DMEM culture solution of 5% fresh (wt) FBS of 20ml, continues to cultivate.
(4) viral supernatants are collected for the first time
A) the 293T cells and supernatant that the previous day transfects is collected into centrifuge tube, 1000rpm is centrifuged 5 minutes, label, temporarily It is stored in 4 DEG C of refrigerators.
B) the DMEM culture medium of 20ml 5% (wt) FBS preheated in advance is added in cell bottle, 37 DEG C of cell incubators Continue overnight incubation.
(5) vial supernatant (48h/ the 4th day) is collected second.
(6) supernatant is filtered
The supernatant collected twice is concentrated in together, removes cell fragment with 0.45 μm of membrane filtration.
(7) viral concentration
4 DEG C, 12000-24000rpm centrifuged overnight
(8) virus storage
After centrifugation, whole supernatants are toppled over, the DMEM culture medium that fresh 5% (wt) FBS is added is resuspended, viral packing is carried out, It is spare to deposit in -80 DEG C of refrigerators rapidly
(9) slow virus titer determination
A) virus infection 293T cell
293T cell is spread into 24 orifice plates before infection, takes and has purified 200 μ L of concentrating virus and be added in 293T cell, for 24 hours Liquid is changed with the DMEM culture medium containing 10%FBS (wt) afterwards, is centrifuged 5min under the conditions of 1200r/min after infecting 72h to collect carefully Born of the same parents extract genome.
B) genome is extracted
Genome extraction agent box is to operate purchased from Beijing Quan Shi genome company by kit specification
C) qPCR measures virus titer
Reaction system is as follows: 12.5 μ L of Probe qPCR Mix (is purchased from Takara), and 0.5 μ L of upstream primer is (by Nanjing gold Si Rui synthesis), 0.5 μ L of downstream primer (being synthesized by Nanjing Jin Sirui), 1 μ L of probe (being synthesized by Nanjing Jin Sirui), 2 μ L of template, 8.5 μ L of aqua sterilisa, reaction system are 25 μ L, and reaction condition is arranged to specifications, after reaction, with analysis software number According to according to standard curve calculating virus titer.Calculated result shows that virus titer is 1 × 107TU/ml。
Embodiment 2, virus infection T cell
1, the separation activation and virus infection of T cell
(1) separation of human peripheral blood mononuclear cell
Peripheral blood about 10ml, room temperature (18-25 DEG C) natural subsidence about 30min are acquired with the heparin tube containing anti-coagulants, is received Collect upper plasma, the upper plasma of collection is centrifuged 10min under the conditions of 5000r/min, 1:1 is added to lymphocyte by volume In separating liquid (being purchased from the ocean Tianjin Hao biological products science and technology limited Company), gradient centrifugation, 3000r/min, centrifugation 30min, after centrifugation, centrifuge tube is by upper lower leaf: first layer is plasma layer；The second layer is lymphocyte tunica albuginea layer；Third layer For transparent separation liquid layer；4th layer of red blood cell layer.Draw lymphocyte tunica albuginea layer, and washed 2 times with PBS, be centrifuged twice with 1500r/min is centrifuged 10min, and cell is resuspended in PBS, and it is complete that 5% autologous plasma+300IU/ml recombinant human il-2+KBM581 is added Culture medium culture human peripheral blood mononuclear cell.
(2) slow-virus infection T lymphocyte
With containing the 5% freshly prepd single core of autologous plasma+300IU/ml recombinant human il-2's+KBM581 complete medium culture Cell PBMC, IL-2 are purchased from R&D Systems, and KBM581 is purchased from Corning, and addition CD3/CD28Dynabeads is immune within the 0th day Magnetic bead (being purchased from invitrogen) activating T cell, progress slow-virus infection in first 3 days are added the corresponding slow virus of 0.25MOI and carry Culture medium is changed to containing 5% autologous plasma+300IU/ml after 48h as blank control by body, the T lymphocyte being uninfected by Recombinant human il-2's+KBM581 complete medium continues culture 7-9 days.
2, in T cell CAR positive rate detection
By the T cell of the virus infection of culture to the 7th day, 1200r/min is centrifuged 5min, and it is thin to collect to abandon supernatant to the greatest extent Born of the same parents are resuspended cell with the PBS solution containing volume fraction 1%FBS, and are 1 × 10 by cell adjustment density5A/ml is added PE-anti-human CD253,4 DEG C of incubation 20min, PBS solution washing 2 times, flow cytometer is detected, as the result is shown By culture in 7 days, the positive rate of TRAIL CRL-T cell: 24% (Fig. 2).
The influence of embodiment 3, virus infection TRAIL CRL-T cell by cell proliferation
After the complete T cell of each group virus infection, by T cell 5% autologous plasma+300IU/ml recombined human containing volume fraction IL-2+KBM581 complete medium counts primary for every 1-2 days.Then T lymphocyte growing state is observed, as a result such as Fig. 3 institute Show.The result shows that cell is still capable of forming typical proliferating clones group after the virus of infection expression TRAIL CRL-T, lead to It crosses and cell is counted, the visible TRAIL CRL-T proliferation of cell Proliferation curve is drawn, than T cell (Fig. 3 of uninfecting virus Middle T cell group) proliferative capacity is slightly weak.
Embodiment 4, the cytokine secretion for detecting virus infection TRAIL CRL-T cell
(1) method that cytokines measurement uses ELISA is carried out using R&D company kit.
(2) dilution of standard items: preparing 1ml centrifuge tube 7, and first standard is added in each centrifuge tube in number consecutively number Then 500 μ L of product dilution takes 500 μ L of original content standard items to be added to 1 in the centrifuge tube for the number of finishing, mixes well, then It takes 500 μ L to be added in second centrifuge tube in the centrifuge tube, mixes well；Take 500 μ L that third is added in the centrifuge tube again In centrifuge tube, mix well；It takes 500 μ L to be added in the 4th centrifuge tube in the centrifuge tube again, mixes well；Again this from It takes 500 μ L to be added in the 5th centrifuge tube in heart pipe, mixes well；Take 500 μ L that the 6th centrifugation is added in the centrifuge tube again Guan Zhong is mixed well；It takes 500 μ L to be added in the 7th centrifuge tube in the centrifuge tube again, mixes well.
(3) it is marked with quasi- sample wells on enzyme mark coating plate, sequentially adds standard items 100 the μ L, each concentration 2-3 of various concentration A parallel hole.
(4) be loaded: blank well is respectively set, and (blank control wells are replaced with water, the antibody of enzyme marking reagent and biotin labeling Operation is as before), sample to be tested hole, 100 μ L of product is first loaded in sample to be tested hole on enzyme mark coating plate, sample is added on enzyme by sample-adding Target hole bottom, does not touch hole wall as far as possible, shakes gently mixing
(5) it is incubated for: being placed at room temperature for and be incubated for 2h
(6) it washs: discarding liquid, dry, every hole adds 200 μ L cleaning solutions, discards after static 30s, be so repeated 3 times, and claps It is dry
(7) add antibody: 100 μ L detection antibody being added on enzyme mark coating plate
(8) it is incubated for: biconditional operation (5)
(9) it washs: biconditional operation (6)
(10) label: 100 μ L horseradish peroxidase-labeled Streptavidins are added in every hole
(11) it is incubated for: being protected from light and be placed at room temperature for incubation 20min
(12) it washs: biconditional operation (6)
(13) develop the color: 100 μ L of developing solution is added in every hole, and gently concussion mixes, and is protected from light and is placed at room temperature for incubation 20min
(14) terminate: 50 μ L of terminate liquid is added in every hole, terminates reaction
(15) measure: with blank value school zero, 450nm wavelength sequentially measures the absorbance (OD value) in each hole, measures Ying Jia Enter and is carried out in 15min after terminate liquid.
It selects antigenic expression to have discrepant target cell and the co-cultivation of TRAIL CRL-T cell, detects TRAIL CRL-T is generated response effect secretion IL2, IFN-γ, TNF-α, IL-6 level by antigenic stimulus, and antigen target cell selects A549 And LN229, show that TRAIL CRL-T is being gone out IL2, IFN-γ by TRAIL receptor for stimulating when institute specific secretion with this TNF, IL6 are horizontal, as a result reflect that TRAIL CRL-T cell produces response for the target cell of high expression TRAIL receptor and makees With.TRAIL CRL-T cell significantly secretes IL2, IFN-γ, TNF, IL6 (Fig. 5-when co-culturing with positive target cell A549 8), show that TRAIL CRL-T cell has response effect for the tumour cell of high expression TRAIL receptor.
Embodiment 5, the assessment of virus infection TRAIL CRL-T cells in vitro fragmentation effect
(1) targeting TRAIL receptor killing: culture target cell HeLa, HepG2, MCF-7, Ln229, U87 (TRAIL receptor sun Property) (Fig. 4), 293T (TRAIL receptor negative) and effector cell TRAIL CRL-T；
(3) target cell and effector cell are collected, 1500rpm/min is centrifuged 5min, abandons supernatant
(4) target cell and effector cell is resuspended with 10%FBS+1640 complete medium
(5) real-time cell analysis system (RTCA) is utilized, in aerial addition 50 μ L, 1640 culture medium of E-Plate16
(6) baseline is detected using RTCA, determines that selected holes contact is normal
(7) setting effect target ratio is 0:1,1:1,5:1,10:1
(8) E-Plate16 is taken out, according to effect target ratio, uniformly mixed 100 μ L of target cell suspension is added in every hole, makes Every hole kind cell number is 104cells/100μL。
(9) E-Plate16 is placed in incubator, with 37 DEG C, under the conditions of 5%CO2, is placed overnight
(10) second days, E-Plate16 is taken out, the 50 corresponding effector cells of μ L are added, calculated and effector cell 8h is added Killing rate afterwards.
(11)
(12) testing result is as shown in Figure 9.TRAIL CRL-T positive cell lethal effect is significant, hence it is evident that be higher than TRAIL by Body feminine gender group.The result shows that Novel chimeric antigen receptor target TRAIL CRL-T has, very strong wide spectrum is anti-to swell killing experiments in vitro Tumor activity.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims limited range of the present invention.
The present invention provides a kind of Chimerical receptor ligands for targeting TRAIL receptor, include the death receptor knot based on TRAIL It closes structural domain, include the second conducting structure intracellular domain, T2A, extracellular signal peptide, the first conducting structure intracellular domain.The present invention also mentions Supplied a kind of ligand modified T cell of Chimerical receptor for targeting TRAIL receptor, can specifically with tumor cell surface TRAIL receptor (DR4, DR5) combines, and then special lethal effect is generated to tumour cell.The invention further relates to the targetings The Chimerical receptor ligand and its immune effector cell of TRAIL receptor are preparing the application in anti-tumor immunotherapy medicaments.
SEQUENCE LISTING
<110>Nanjing Ka Ti medical science and technology Co., Ltd
<120>a kind of Chimerical receptor ligand for targeting TRAIL death receptor and its application
<130> 2019
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 113
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<213>artificial sequence (artificial sequence)
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Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu
1 5 10 15
Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp
20 25 30
Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met
35 40 45
Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu
50 55 60
Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg
65 70 75 80
Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly
85 90 95
Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr
100 105 110
Lys
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Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 3
<211> 21
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<213>artificial sequence (artificial sequence)
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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 4
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Thr Ala Gly Ala Arg Gln Ala Pro Glu Ser Pro Ser Thr Ile Pro Val
1 5 10 15
Pro Ser Gln Pro Gln Asn Ser Thr Leu Arg Pro Gly Pro Ala Ala Pro
20 25 30
Ile Ala Leu Val Pro Val Phe Cys Gly Leu Leu Val Ala Lys Ser Leu
35 40 45
Val Leu Ser Ala Leu Leu Val Trp Trp Gly Asp Ile Trp Trp Lys Thr
50 55 60
Met Met Glu Leu Arg Ser Leu Asp Thr Gln Lys Ala Thr Cys His Leu
65 70 75 80
Gln Gln Val Thr Asp Leu Pro Trp Thr Ser Val Ser Ser Pro Val Glu
85 90 95
Arg Glu Ile Leu Tyr His Thr Val Ala Arg Thr Lys Ile Ser Asp Asp
100 105 110
Asp Asp Glu His Thr Leu
115
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Gly Ala Gly Cys Thr Gly Ala Ala Gly Cys Ala Gly Ala Thr Gly Cys
1 5 10 15
Ala Gly Gly Ala Cys Ala Ala Gly Thr Ala Cys Thr Cys Cys Ala Ala
20 25 30
Ala Ala Gly Thr Gly Gly Cys Ala Thr Thr Gly Cys Thr Thr Gly Thr
35 40 45
Thr Thr Cys Thr Thr Ala Ala Ala Ala Gly Ala Ala Gly Ala Thr Gly
50 55 60
Ala Cys Ala Gly Thr Thr Ala Thr Thr Gly Gly Gly Ala Cys Cys Cys
65 70 75 80
Cys Ala Ala Thr Gly Ala Cys Gly Ala Ala Gly Ala Gly Ala Gly Thr
85 90 95
Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Cys Cys Thr Gly Cys Thr
100 105 110
Gly Gly Cys Ala Ala Gly Thr Cys Ala Ala Gly Thr Gly Gly Cys Ala
115 120 125
Ala Cys Thr Cys Cys Gly Thr Cys Ala Gly Cys Thr Cys Gly Thr Thr
130 135 140
Ala Gly Ala Ala Ala Gly Ala Thr Gly Ala Thr Thr Thr Thr Gly Ala
145 150 155 160
Gly Ala Ala Cys Cys Thr Cys Thr Gly Ala Gly Gly Ala Ala Ala Cys
165 170 175
Cys Ala Thr Thr Thr Cys Thr Ala Cys Ala Gly Thr Thr Cys Ala Ala
180 185 190
Gly Ala Ala Ala Ala Gly Cys Ala Ala Cys Ala Ala Ala Ala Thr Ala
195 200 205
Thr Thr Thr Cys Thr Cys Cys Cys Cys Thr Ala Gly Thr Gly Ala Gly
210 215 220
Ala Gly Ala Ala Ala Gly Ala Gly Gly Thr Cys Cys Thr Cys Ala Gly
225 230 235 240
Ala Gly Ala Gly Thr Ala Gly Cys Ala Gly Cys Thr Cys Ala Cys Ala
245 250 255
Thr Ala Ala Cys Thr Gly Gly Gly Ala Cys Cys Ala Gly Ala Gly Gly
260 265 270
Ala Ala Gly Ala Ala Gly Cys Ala Ala Cys Ala Cys Ala Thr Thr Gly
275 280 285
Thr Cys Thr Thr Cys Thr Cys Cys Ala Ala Ala Cys Thr Cys Cys Ala
290 295 300
Ala Gly Ala Ala Thr Gly Ala Ala Ala Ala Gly Gly Cys Thr Cys Thr
305 310 315 320
Gly Gly Gly Cys Cys Gly Cys Ala Ala Ala Ala Thr Ala Ala Ala Cys
325 330 335
Thr Cys Cys Thr Gly Gly Gly Ala Ala Thr Cys Ala Thr Cys Ala Ala
340 345 350
Gly Gly Ala Gly Thr Gly Gly Gly Cys Ala Thr Thr Cys Ala Thr Thr
355 360 365
Cys Cys Thr Gly Ala Gly Cys Ala Ala Cys Thr Thr Gly Cys Ala Cys
370 375 380
Thr Thr Gly Ala Gly Gly Ala Ala Thr Gly Gly Thr Gly Ala Ala Cys
385 390 395 400
Thr Gly Gly Thr Cys Ala Thr Cys Cys Ala Thr Gly Ala Ala Ala Ala
405 410 415
Ala Gly Gly Gly Thr Thr Thr Thr Ala Cys Thr Ala Cys Ala Thr Cys
420 425 430
Thr Ala Thr Thr Cys Cys Cys Ala Ala Ala Cys Ala Thr Ala Cys Thr
435 440 445
Thr Thr Cys Gly Ala Thr Thr Thr Cys Ala Gly Gly Ala Gly Gly Ala
450 455 460
Ala Ala Thr Ala Ala Ala Ala Gly Ala Ala Ala Ala Cys Ala Cys Ala
465 470 475 480
Ala Ala Gly Ala Ala Cys Gly Ala Cys Ala Ala Ala Cys Ala Ala Ala
485 490 495
Thr Gly Gly Thr Cys Cys Ala Ala Thr Ala Thr Ala Thr Thr Thr Ala
500 505 510
Cys Ala Ala Ala Thr Ala Cys Ala Cys Ala Ala Gly Thr Thr Ala Thr
515 520 525
Cys Cys Thr Gly Ala Cys Cys Cys Thr Ala Thr Ala Thr Thr Gly Thr
530 535 540
Thr Gly Ala Thr Gly Ala Ala Ala Ala Gly Thr Gly Cys Thr Ala Gly
545 550 555 560
Ala Ala Ala Thr Ala Gly Thr Thr Gly Thr Thr Gly Gly Thr Cys Thr
565 570 575
Ala Ala Ala Gly Ala Thr Gly Cys Ala Gly Ala Ala Thr Ala Thr Gly
580 585 590
Gly Ala Cys Thr Cys Thr Ala Thr Thr Cys Cys Ala Thr Cys Thr Ala
595 600 605
Thr Cys Ala Ala Gly Gly Gly Gly Gly Ala Ala Thr Ala Thr Thr Thr
610 615 620
Gly Ala Gly Cys Thr Thr Ala Ala Gly Gly Ala Ala Ala Ala Thr Gly
625 630 635 640
Ala Cys Ala Gly Ala Ala Thr Thr Thr Thr Thr Gly Thr Thr Thr Cys
645 650 655
Thr Gly Thr Ala Ala Cys Ala Ala Ala Thr Gly Ala Gly Cys Ala Cys
660 665 670
Thr Thr Gly Ala Thr Ala Gly Ala Cys Ala Thr Gly Gly Ala Cys Cys
675 680 685
Ala Thr Gly Ala Ala Gly Cys Cys Ala Gly Thr Thr Thr Thr Thr Thr
690 695 700
Thr Gly Gly Gly Gly Cys Cys Thr Thr Thr Thr Thr Ala Gly Thr Thr
705 710 715 720
Gly Gly Cys
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Glu Leu Lys Gln Met Gln Asp Lys Tyr Ser Lys Ser Gly Ile Ala Cys
1 5 10 15
Phe Leu Lys Glu Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu Glu Ser
20 25 30
Met Asn Ser Pro Cys Trp Gln Val Lys Trp Gln Leu Arg Gln Leu Val
35 40 45
Arg Lys Met Ile Leu Arg Thr Ser Glu Glu Thr Ile Ser Thr Val Gln
50 55 60
Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln
65 70 75 80
Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu
85 90 95
Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn
100 105 110
Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His
115 120 125
Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile
130 135 140
Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr
145 150 155 160
Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr
165 170 175
Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser
180 185 190
Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe
195 200 205
Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His
210 215 220
Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val
225 230 235 240
Gly
<210> 7
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<213>artificial sequence (artificial sequence)
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Ala Thr Gly Gly Gly Gly Gly Gly Ala Cys Thr Thr Gly Ala Ala Cys
1 5 10 15
Cys Cys Thr Gly Cys Ala Gly Cys Ala Gly Gly Thr Thr Cys Cys Thr
20 25 30
Gly Cys Thr Cys Cys Thr Gly Cys Cys Thr Cys Thr Cys Cys Thr Gly
35 40 45
Cys Thr Gly Gly Cys Thr Gly Thr Ala Ala Gly Thr Gly Gly Thr Cys
50 55 60
Thr Cys Cys Gly Thr Cys Cys Thr Gly Thr Cys Cys Ala Gly Gly Thr
65 70 75 80
Cys Cys Ala Gly Gly Cys Cys Cys Ala Gly Ala Gly Cys Gly Ala Thr
85 90 95
Thr Gly Cys Ala Gly Thr Thr Gly Cys Thr Cys Thr Ala Cys Gly Gly
100 105 110
Thr Gly Ala Gly Cys Cys Cys Gly Gly Gly Cys Gly Thr Gly Cys Thr
115 120 125
Gly Gly Cys Ala Gly Gly Gly Ala Thr Cys Gly Thr Gly Ala Thr Gly
130 135 140
Gly Gly Ala Gly Ala Cys Cys Thr Gly Gly Thr Gly Cys Thr Gly Ala
145 150 155 160
Cys Ala Gly Thr Gly Cys Thr Cys Ala Thr Thr Gly Cys Cys Cys Thr
165 170 175
Gly Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Thr Cys Cys Thr Gly
180 185 190
Gly Gly Cys Cys Gly Gly Cys Thr Gly Gly Thr Cys Cys Cys Thr Cys
195 200 205
Gly Gly Gly Gly Gly Cys Gly Ala Gly Gly Gly Gly Cys Thr Gly Cys
210 215 220
Gly Gly Ala Gly Gly Cys Ala Gly Cys Gly Ala Cys Cys Cys Gly Gly
225 230 235 240
Ala Ala Ala Cys Ala Gly Cys Gly Thr Ala Thr Cys Ala Cys Thr Gly
245 250 255
Ala Gly Ala Cys Cys Gly Ala Gly Thr Cys Gly Cys Cys Thr Thr Ala
260 265 270
Thr Cys Ala Gly Gly Ala Gly Cys Thr Cys Cys Ala Gly Gly Gly Thr
275 280 285
Cys Ala Gly Ala Gly Gly Thr Cys Gly Gly Ala Thr Gly Thr Cys Thr
290 295 300
Ala Cys Ala Gly Cys Gly Ala Cys Cys Thr Cys Ala Ala Cys Ala Cys
305 310 315 320
Ala Cys Ala Gly Ala Gly Gly Cys Cys Gly Thr Ala Thr Thr Ala Cys
325 330 335
Ala Ala Ala Gly Thr Cys Gly Ala Gly Gly Gly Cys Gly Gly Cys Gly
340 345 350
Gly Ala Gly Ala Gly Gly Gly Cys Ala Gly Ala Gly Gly Ala Ala Gly
355 360 365
Thr Cys Thr Thr Cys Thr Ala Ala Cys Ala Thr Gly Cys Gly Gly Thr
370 375 380
Gly Ala Cys Gly Thr Gly Gly Ala Gly Gly Ala Gly Ala Ala Thr Cys
385 390 395 400
Cys Cys Gly Gly Cys Cys Cys Thr Ala Gly Gly Ala Thr Gly Gly Cys
405 410 415
Cys Thr Thr Ala Cys Cys Ala Gly Thr Gly Ala Cys Cys Gly Cys Cys
420 425 430
Thr Thr Gly Cys Thr Cys Cys Thr Gly Cys Cys Gly Cys Thr Gly Gly
435 440 445
Cys Cys Thr Thr Gly Cys Thr Gly Cys Thr Cys Cys Ala Cys Gly Cys
450 455 460
Cys Gly Cys Cys Ala Gly Gly Cys Cys Gly Gly Gly Ala Thr Cys Cys
465 470 475 480
Gly Ala Gly Cys Thr Gly Ala Ala Gly Cys Ala Gly Ala Thr Gly Cys
485 490 495
Ala Gly Gly Ala Cys Ala Ala Gly Thr Ala Cys Thr Cys Cys Ala Ala
500 505 510
Ala Ala Gly Thr Gly Gly Cys Ala Thr Thr Gly Cys Thr Thr Gly Thr
515 520 525
Thr Thr Cys Thr Thr Ala Ala Ala Ala Gly Ala Ala Gly Ala Thr Gly
530 535 540
Ala Cys Ala Gly Thr Thr Ala Thr Thr Gly Gly Gly Ala Cys Cys Cys
545 550 555 560
Cys Ala Ala Thr Gly Ala Cys Gly Ala Ala Gly Ala Gly Ala Gly Thr
565 570 575
Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Cys Cys Thr Gly Cys Thr
580 585 590
Gly Gly Cys Ala Ala Gly Thr Cys Ala Ala Gly Thr Gly Gly Cys Ala
595 600 605
Ala Cys Thr Cys Cys Gly Thr Cys Ala Gly Cys Thr Cys Gly Thr Thr
610 615 620
Ala Gly Ala Ala Ala Gly Ala Thr Gly Ala Thr Thr Thr Thr Gly Ala
625 630 635 640
Gly Ala Ala Cys Cys Thr Cys Thr Gly Ala Gly Gly Ala Ala Ala Cys
645 650 655
Cys Ala Thr Thr Thr Cys Thr Ala Cys Ala Gly Thr Thr Cys Ala Ala
660 665 670
Gly Ala Ala Ala Ala Gly Cys Ala Ala Cys Ala Ala Ala Ala Thr Ala
675 680 685
Thr Thr Thr Cys Thr Cys Cys Cys Cys Thr Ala Gly Thr Gly Ala Gly
690 695 700
Ala Gly Ala Ala Ala Gly Ala Gly Gly Thr Cys Cys Thr Cys Ala Gly
705 710 715 720
Ala Gly Ala Gly Thr Ala Gly Cys Ala Gly Cys Thr Cys Ala Cys Ala
725 730 735
Thr Ala Ala Cys Thr Gly Gly Gly Ala Cys Cys Ala Gly Ala Gly Gly
740 745 750
Ala Ala Gly Ala Ala Gly Cys Ala Ala Cys Ala Cys Ala Thr Thr Gly
755 760 765
Thr Cys Thr Thr Cys Thr Cys Cys Ala Ala Ala Cys Thr Cys Cys Ala
770 775 780
Ala Gly Ala Ala Thr Gly Ala Ala Ala Ala Gly Gly Cys Thr Cys Thr
785 790 795 800
Gly Gly Gly Cys Cys Gly Cys Ala Ala Ala Ala Thr Ala Ala Ala Cys
805 810 815
Thr Cys Cys Thr Gly Gly Gly Ala Ala Thr Cys Ala Thr Cys Ala Ala
820 825 830
Gly Gly Ala Gly Thr Gly Gly Gly Cys Ala Thr Thr Cys Ala Thr Thr
835 840 845
Cys Cys Thr Gly Ala Gly Cys Ala Ala Cys Thr Thr Gly Cys Ala Cys
850 855 860
Thr Thr Gly Ala Gly Gly Ala Ala Thr Gly Gly Thr Gly Ala Ala Cys
865 870 875 880
Thr Gly Gly Thr Cys Ala Thr Cys Cys Ala Thr Gly Ala Ala Ala Ala
885 890 895
Ala Gly Gly Gly Thr Thr Thr Thr Ala Cys Thr Ala Cys Ala Thr Cys
900 905 910
Thr Ala Thr Thr Cys Cys Cys Ala Ala Ala Cys Ala Thr Ala Cys Thr
915 920 925
Thr Thr Cys Gly Ala Thr Thr Thr Cys Ala Gly Gly Ala Gly Gly Ala
930 935 940
Ala Ala Thr Ala Ala Ala Ala Gly Ala Ala Ala Ala Cys Ala Cys Ala
945 950 955 960
Ala Ala Gly Ala Ala Cys Gly Ala Cys Ala Ala Ala Cys Ala Ala Ala
965 970 975
Thr Gly Gly Thr Cys Cys Ala Ala Thr Ala Thr Ala Thr Thr Thr Ala
980 985 990
Cys Ala Ala Ala Thr Ala Cys Ala Cys Ala Ala Gly Thr Thr Ala Thr
995 1000 1005
Cys Cys Thr Gly Ala Cys Cys Cys Thr Ala Thr Ala Thr Thr Gly
1010 1015 1020
Thr Thr Gly Ala Thr Gly Ala Ala Ala Ala Gly Thr Gly Cys Thr
1025 1030 1035
Ala Gly Ala Ala Ala Thr Ala Gly Thr Thr Gly Thr Thr Gly Gly
1040 1045 1050
Thr Cys Thr Ala Ala Ala Gly Ala Thr Gly Cys Ala Gly Ala Ala
1055 1060 1065
Thr Ala Thr Gly Gly Ala Cys Thr Cys Thr Ala Thr Thr Cys Cys
1070 1075 1080
Ala Thr Cys Thr Ala Thr Cys Ala Ala Gly Gly Gly Gly Gly Ala
1085 1090 1095
Ala Thr Ala Thr Thr Thr Gly Ala Gly Cys Thr Thr Ala Ala Gly
1100 1105 1110
Gly Ala Ala Ala Ala Thr Gly Ala Cys Ala Gly Ala Ala Thr Thr
1115 1120 1125
Thr Thr Thr Gly Thr Thr Thr Cys Thr Gly Thr Ala Ala Cys Ala
1130 1135 1140
Ala Ala Thr Gly Ala Gly Cys Ala Cys Thr Thr Gly Ala Thr Ala
1145 1150 1155
Gly Ala Cys Ala Thr Gly Gly Ala Cys Cys Ala Thr Gly Ala Ala
1160 1165 1170
Gly Cys Cys Ala Gly Thr Thr Thr Thr Thr Thr Thr Gly Gly Gly
1175 1180 1185
Gly Cys Cys Thr Thr Thr Thr Thr Ala Gly Thr Thr Gly Gly Cys
1190 1195 1200
Gly Cys Thr Ala Gly Cys Gly Gly Thr Gly Gly Cys Gly Gly Ala
1205 1210 1215
Gly Gly Thr Thr Cys Thr Gly Gly Ala Gly Gly Thr Gly Gly Gly
1220 1225 1230
Gly Gly Thr Thr Cys Cys Ala Cys Thr Gly Cys Ala Gly Gly Ala
1235 1240 1245
Gly Cys Cys Ala Gly Ala Cys Ala Ala Gly Cys Cys Cys Cys Thr
1250 1255 1260
Gly Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Thr Ala Cys Cys
1265 1270 1275
Ala Thr Cys Cys Cys Thr Gly Thr Cys Cys Cys Thr Thr Cys Ala
1280 1285 1290
Cys Ala Gly Cys Cys Ala Cys Ala Gly Ala Ala Cys Thr Cys Cys
1295 1300 1305
Ala Cys Gly Cys Thr Cys Cys Gly Cys Cys Cys Thr Gly Gly Cys
1310 1315 1320
Cys Cys Thr Gly Cys Ala Gly Cys Cys Cys Cys Cys Ala Thr Thr
1325 1330 1335
Gly Cys Cys Cys Thr Gly Gly Thr Gly Cys Cys Thr Gly Thr Gly
1340 1345 1350
Thr Thr Cys Thr Gly Thr Gly Gly Ala Cys Thr Cys Cys Thr Cys
1355 1360 1365
Gly Thr Ala Gly Cys Cys Ala Ala Gly Ala Gly Cys Cys Thr Gly
1370 1375 1380
Gly Thr Gly Cys Thr Gly Thr Cys Ala Gly Cys Cys Cys Thr Gly
1385 1390 1395
Cys Thr Cys Gly Thr Cys Thr Gly Gly Thr Gly Gly Gly Gly Gly
1400 1405 1410
Gly Ala Cys Ala Thr Ala Thr Gly Gly Thr Gly Gly Ala Ala Ala
1415 1420 1425
Ala Cys Cys Ala Thr Gly Ala Thr Gly Gly Ala Gly Cys Thr Cys
1430 1435 1440
Ala Gly Gly Ala Gly Cys Cys Thr Gly Gly Ala Thr Ala Cys Cys
1445 1450 1455
Cys Ala Ala Ala Ala Ala Gly Cys Cys Ala Cys Cys Thr Gly Cys
1460 1465 1470
Cys Ala Cys Cys Thr Thr Cys Ala Ala Cys Ala Gly Gly Thr Cys
1475 1480 1485
Ala Cys Gly Gly Ala Cys Cys Thr Thr Cys Cys Cys Thr Gly Gly
1490 1495 1500
Ala Cys Cys Thr Cys Ala Gly Thr Thr Thr Cys Cys Thr Cys Ala
1505 1510 1515
Cys Cys Thr Gly Thr Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala
1520 1525 1530
Ala Thr Ala Thr Thr Ala Thr Ala Thr Cys Ala Cys Ala Cys Thr
1535 1540 1545
Gly Thr Thr Gly Cys Ala Ala Gly Gly Ala Cys Thr Ala Ala Gly
1550 1555 1560
Ala Thr Ala Ala Gly Cys Gly Ala Thr Gly Ala Thr Gly Ala Thr
1565 1570 1575
Gly Ala Thr Gly Ala Ala Cys Ala Cys Ala Cys Thr Thr Thr Gly
1580 1585 1590
Thr Gly Ala
1595
<210> 8
<211> 531
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 8
Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu
1 5 10 15
Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp
20 25 30
Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met
35 40 45
Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu
50 55 60
Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg
65 70 75 80
Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly
85 90 95
Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr
100 105 110
Lys Val Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly
115 120 125
Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala
130 135 140
Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser
145 150 155 160
Glu Leu Lys Gln Met Gln Asp Lys Tyr Ser Lys Ser Gly Ile Ala Cys
165 170 175
Phe Leu Lys Glu Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu Glu Ser
180 185 190
Met Asn Ser Pro Cys Trp Gln Val Lys Trp Gln Leu Arg Gln Leu Val
195 200 205
Arg Lys Met Ile Leu Arg Thr Ser Glu Glu Thr Ile Ser Thr Val Gln
210 215 220
Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln
225 230 235 240
Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu
245 250 255
Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn
260 265 270
Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His
275 280 285
Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile
290 295 300
Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr
305 310 315 320
Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr
325 330 335
Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser
340 345 350
Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe
355 360 365
Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His
370 375 380
Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val
385 390 395 400
Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ala Gly
405 410 415
Ala Arg Gln Ala Pro Glu Ser Pro Ser Thr Ile Pro Val Pro Ser Gln
420 425 430
Pro Gln Asn Ser Thr Leu Arg Pro Gly Pro Ala Ala Pro Ile Ala Leu
435 440 445
Val Pro Val Phe Cys Gly Leu Leu Val Ala Lys Ser Leu Val Leu Ser
450 455 460
Ala Leu Leu Val Trp Trp Gly Asp Ile Trp Trp Lys Thr Met Met Glu
465 470 475 480
Leu Arg Ser Leu Asp Thr Gln Lys Ala Thr Cys His Leu Gln Gln Val
485 490 495
Thr Asp Leu Pro Trp Thr Ser Val Ser Ser Pro Val Glu Arg Glu Ile
500 505 510
Leu Tyr His Thr Val Ala Arg Thr Lys Ile Ser Asp Asp Asp Asp Glu
515 520 525
His Thr Leu
530

Claims (10)

1. a kind of Chimerical receptor ligand for targeting TRAIL death receptor, includes: the death receptor based on TRAIL molecule combines knot Structure domain, the second conducting structure intracellular domain, T2A, extracellular signal peptide, the first conducting structure intracellular domain.
2. the Chimerical receptor ligand of targeting TRAIL death receptor according to claim 1, wherein death receptor combines knot Structure domain includes at least 200 continuous amino acid residues of SEQ ID NO:6, or has 85%~99% or 90%~99% with it Or 95%~99% identity transformation amino acid sequence.
3. the Chimerical receptor ligand of targeting TRAIL death receptor according to claim 1 or 2, wherein the Chimerical receptor Ligand includes one or more death receptor binding domains based on TRAIL molecule.The signal peptide includes CD8 alpha signal Peptide, GM-CSFR alpha signal peptide or CD4 signal peptide, preferably CD8 alpha signal peptide, amino acid sequence is as shown in SEQ ID NO:3；It is described First signal transduction structural domain intracellular include KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、KIR3DL1、KIR3DS1、KIR3DL2、KIR3DL3、 KIR2DP1,KIR3DP1；NKp46,NKp30,NKp44,SLAM,CD48,CD229,2B4,CD84,NTB-A,CRACC, BLAME、、CD2F-10、KLRD1/KLRC2、SIRPB1、PILRB、CLEC5A、TREM1、TREM2、CD300B、CD300E、 SIGLEC-14, SIGLEC-15, SIGLEC-16 and/or NKG2D, preferred first signal transduction structural domain intracellular are NKp44, Amino acid sequence is as shown in SEQ ID NO:4, and the second conducting structure intracellular domain is selected from DAP10, DAP12 and/or Fc ε R γ, preferably The second conducting structure intracellular domain DAP12, amino acid sequence is as shown in SEQ ID NO:1.Second conducting structure intracellular domain passes through T2A connects with extracellular signal peptide and TRAIL receptor binding domain, and the T2A amino acid sequence is as shown in SEQ ID NO:2.
4. the Chimerical receptor ligand of described in any item targeting TRAIL death receptors according to claim 1~3, it is characterised in that Amino acid sequence be SEQ ID NO:8 shown in, or with its have 95%~99% identity amino acid sequence.
5. a kind of nucleic acid molecules, it is characterised in that any one of coding Claims 1 to 4 is described to target the embedding of TRAIL death receptor Close the nucleotide sequence of receptors ligand, it is characterised in that nucleotide coding sequence is as shown in SEQ ID NO:7.
6. a kind of carrier, it is characterised in that include the nucleic acid molecules described in claim 5.
7. a kind of immunocyte of genetic engineering transformation, it is characterised in that include the nucleic acid molecules described in claim 5.
8. the immunocyte of genetic engineering transformation according to claim 7, it is characterised in that comprising as claimed in claim 6 Carrier, it is characterised in that it is thin to can be selected from T lymphocyte, NK cell, NKT cell, macrophage, mescenchymal stem cell, Hematopoietic Stem The immunocyte of born of the same parents, multipotential stem cell or embryonic stem cell culture differentiation, further preferred T lymphocyte, it is characterised in that table The Chimerical receptor ligand of the targeting TRAIL death receptor reached includes extracellular signal peptide, the death receptor combination based on TRAIL molecule Structural domain, the second conducting structure intracellular domain, T2A, extracellular signal peptide, the first conducting structure intracellular domain, it is characterised in that expression Target TRAIL death receptor Chimerical receptor ligand have SEQ ID NO:8 shown in amino acid sequence, or with its have 95%~ The amino acid sequence of 99% identity.
9. a kind of method, it is characterised in that targeting TRAIL death receptor is chimeric described in any one of preparation Claims 1 to 5 Receptors ligand.
10. prepared by immunocyte described in the described in any item Chimeric antigen receptors of Claims 1 to 5 or claim 6~9 For the application in immunotherapy of tumors drug.
CN201910294369.0A 2019-04-12 2019-04-12 It is a kind of target human TNF related apoptosis-inducing ligand death receptor Chimerical receptor ligand and its application Pending CN109970869A (en)

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Application publication date: 20190705