CN108440674A - A kind of Trop-2 specific chimerics antigen receptor cell is prepared and application thereof - Google Patents

A kind of Trop-2 specific chimerics antigen receptor cell is prepared and application thereof Download PDF

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CN108440674A
CN108440674A CN201810401267.XA CN201810401267A CN108440674A CN 108440674 A CN108440674 A CN 108440674A CN 201810401267 A CN201810401267 A CN 201810401267A CN 108440674 A CN108440674 A CN 108440674A
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trop2
antigen receptor
gly
cell
leu
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陈相波
雷鸣
田朋飞
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Hangzhou Rong Biotechnology Co Ltd
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Hangzhou Rong Biotechnology Co Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The invention discloses a kind of Trop2 specific chimerics antigen receptors, it includes signal peptide, antigen binding domain, hinge area, transmembrane region, irritates signal transduction area, the ζ signal transductions areas CD3 and T2A small peptides altogether, wherein signal peptide is CSF2RA signal peptides, antigen binding domain is scFv sequences, hinge area is the areas CD8 α, transmembrane region is CD28 transmembrane regions, costimulatory signal transduces area as CD28 and 4 1BB, and CD3 ζ signal transductions area is CD3 molecule intracellular signal transductions area；Above-mentioned Chimeric antigen receptor is specially RZ 28bbZ, and nucleotide coding sequence is as shown in SEQ ID No.1；The present invention also provides a kind of comprising above-mentioned nucleotide recombinant expression carrier and its host cell, construction method, the application in mammalian cell oncotherapy, Trop2 specific chimerics antigen receptor of the present invention can be by modifying T cell so that CAR T cells efficiently specifically treat oophoroma.

Description

A kind of Trop-2 specific chimerics antigen receptor cell is prepared and application thereof
Technical field
The present invention relates to immunocyte preparation field, in particular it relates to a kind of Trop2 specific chimerics antigen by Body and application thereof.
Technical background
Oophoroma early stage lacks typical clinical manifestation, therefore is usually just found to late stage, five year survival rate Less than 30%, it is the most common cause of the death of feminine proses, seriously threatens the life security of women.Currently, oophoroma Treatment mainly operative treatment is taken to be aided with the conjoint therapy of chemotherapy, but be more than 70% ovarian cancer patients in for the first time treat after answer Hair, and tolerance or resistant function easily are generated to chemotherapy, therefore, there is an urgent need to seek new oophoroma clinical treatment.
Chimeric antigen receptor T cell (chimeric antigen receptor T cell immullomerapy, CAR-T) skill Art is the immunization therapy new method based on antibody target technology, Malignancy therapy field obtain it is breakthrough into Opening up tentative confirmation, it cures the potentiality of tumour, becomes the hot spot in immunotherapy of tumors field in recent years.By CAR-T skills The inspiration that art is applied successfully in Malignancy therapy field, research of the CAR-T technologies in treatment of solid tumors field Expansion in succession, is related to the kinds of tumors type such as celiothelioma, neuroblastoma, liver cancer, cancer of pancreas, colon cancer, wherein CAR-T skills Art achieves significant therapeutic effect in the research for treating glioma, neuroblastoma.
The structure of CAR, which is divided into mainly, extracellular antigen binding domain (single-chain antibody variable region, single chain Fragment Variation, scFv), transmembrane region and intracellular signal area (costimulatory signal and CD3 ζ signals), first generation CAR structures do not contain Costimulatory signal, second generation CAR structures contain there are one costimulatory signal (such as CD28 or 4-1BB), and third generation CAR structures, which also have, goes here and there Two costimulatory signals of connection, the factor of tumor microenvironment can be improved by containing such as IL2 in forth generation CAR molecules.CD28 is total The proliferation efficiency of stimulus signal energy higher cell, when 4-1BB costimulatory signals can preferably maintain the survival of CAR-T cells It is long.CAR-T is made a breakthrough by CD19 target spots in neoplastic hematologic disorder, and effective percentage is studied up to 90% or more in part, however CAR-T is there are no making a breakthrough on solid tumor, so there is an urgent need to new tumor targets, new CAR MOLECULE DESIGNs are to promote The progress in solid tumor field.
CAR-T technologies rely on the specific binding of antibody and antigen, keep the tumour of the accurate targeted expression target antigen of T cell thin Born of the same parents, therefore, suitable target antigen are the key that the applications of CAR-T technologies.Ideal target antigen should have high in tumor tissues Expression and do not express in the normal tissue or the characteristics of low expression.2 (human of human trophocyte cell's surface antigen Trophoblast cell surface antigen 2, Trop-2) it is a kind of I type cross-films by people's TACSTD2 gene codes Glycoprotein.Studies have shown that Trop2 is a kind of tumor correlated albumen, the prognosis of invasion, transfer and disease with tumour cell is close It is related.Trop2 is overexpressed in the kinds of tumor cells such as oophoroma, gastric cancer, colon cancer, prostate, cancer of pancreas, and normal in people It does not express in tissue or low expression, is had potential application in the targeted therapy of tumour.Therefore, this research is chosen Trop2 prepares the CAR-T cells of targeting Trop2 and detects its killing to the Trop2 positive ovarian cancer cells of expression as target spot Wound acts on, and new method is provided for the treatment of oophoroma.
Invention content
The technical problem to be solved by the present invention is to provide a kind of new CAR molecules by targeting Trop2, and prove it Potential application in treatment of solid tumors.Specifically, the present invention provides a kind of Trop2 specific chimerics antigen receptor (CAR), This CAR molecules are by the Trop2 albumen on targets neoplastic cells surface and activate the signal path in T cell downstream, assign T cell Ability of the killing with Trop2 target tumor cells.
In order to solve the above technical problems, the technical solution adopted by the present invention is that：
The first purpose of the invention is to provide a kind of Trop2 specific chimerics antigen receptors comprising signal peptide, antigen Combined area, transmembrane region, irritates signal transduction area, the ζ signal transductions areas CD3 and T2A small peptides at hinge area altogether；Wherein, extracellular cog region For anti-Trop2 antibody sequences.
In order to advanced optimize above-mentioned Trop2 specific chimerics antigen receptor, the technical measures that the present invention is taken also are wrapped It includes：
Preferably, the anti-Trop2 antibody sequences are single-stranded variable region (scFv) sequence of combination Trop2 a kind of, more excellent Selection of land, in an embodiment of the present invention, the antibody sequence are oneself disclosed Trop2 monoclonal antibody HRS from this field, should Trop2 monoclonal antibodies HRS includes light chain variable region (VL) amino acid sequence as shown in SEQ ID NO.3 and such as SEQ ID Heavy chain variable region shown in N0.4 (VH) amino acid sequence.
Further, according to the composition characteristic of scFv, VL and VH sequences are passed through into one section of flexibility linker amino acid sequence (GGGGSGGGGSGGGGS) it connects together, forms scFv sequences.
Further, it is described compared with sequence be the regions CD8hinge, amino acid sequence is as shown in SEQ ID NO.5.
Further, transmembrane region described in the transmembrane region is CD28 transmembrane regions, amino acid sequence such as SEQ ID N0.6 institutes Show.
Further, the intracellular costimulatory signal conducting region is connected in series by CD28,4-1BB, and amino acid sequence is such as Shown in SEQ ID NO.7.
Further, the intracellular signal area is T cell signal transduction active region CD3 ζ, amino acid sequence such as SEQ Shown in ID NO.8.
Further, the structure based on the above Trop2 CAR molecules, the Chimeric antigen receptor of the present invention are CSF2RA-Trop2 scFv-CD8 α-CD28-4-1BB-CD3 ζ (RZ-HRS-28bbZ), structure is as shown in Figure 1, its nucleotide With amino acid sequence as shown in SEQ ID No.1 and SEQ ID NO.2.
Second object of the present invention is to provide a kind of containing the nucleic acid for encoding above-mentioned Trop2 specific chimerics antigen receptor Recombinant expression carrier.
Further, it is slow virus carrier pCDH-EF1-MCS- that the recombinant expression carrier, which is original recombinant expression carrier, T2A-CopGFP (hereinafter abbreviated as pCDH), collection of illustrative plates is shown in Fig. 2.In the carrier, the expression of CAR molecules is driven by EF1 α promoters Dynamic transcriptional expression, carries mark of the GFP marker gene as lentiviruses transduction.CAR slow virus carriers plasmid packs matter in auxiliary In the presence of grain pSPAX2 and vsv-G envelope plasmids pMD2.G, cotransfection HEK293T cells, you can be packaged as carrying CAR The slow virus of molecule.
Containing the nucleic acid for encoding above-mentioned Trop2 specific chimerics antigen receptor third object of the present invention is to provide a kind of Recombinant expression carrier host cell.
Further, the host cell is T cell or the cell mass containing T cell.
The invention further relates to a kind of construction methods of above-mentioned host cell comprising the construction step of recombinant expression carrier, The packaging step of recombinant expression carrier and the step of recombinant expression carrier is transduceed to host cell.
Finally, the present invention also provides a kind of above-mentioned Trop2 specific chimerics antigen receptor, above-mentioned coding Trop2 specificity The application of the nucleic acid of Chimeric antigen receptor, above-mentioned recombinant expression carrier and host cell in mammal tumor treatment.
Further, mammalian solid tumors include but not limited to, oophoroma tumor, breast cancer, carcinoma of endometrium, black Melanoma, cancer of pancreas, lung cancer, neuroblastoma etc..
More specifically, above application is a kind of Trop2 CAR-T cells, T is made by the transduction of lentivirus mediated Cell carries Trop2 CAR, and to impart the ability that T cell identifies Trop2 molecules, and the lactation for targeting the Trop2 positives is dynamic Object entity tumor.
In one embodiment, by Trop2 CAR-T cells with expression Trop2 tumor cell line OVCAR-3, HO8910, breast cancer cell MCF-7, SKOV3 are incubated altogether, as a result show that Trop2 CAR-T cells being capable of irritating in target cell Lower a large amount of generation IFN-γ；But it cannot be negative with the tumor cell line SKOV3 for expressing a small amount of Trop2 and with Trop2 Cell line A2780, melanoma cells A375 generate IFN-γ when being incubated altogether；The CAR-T cells 19- of non-Trop2 targetings simultaneously 28BBZ can not irritate lower generation IFN-γ in Trop2 positive cells.This embodiment illustrates the good of Trop2 CAR-T cells Good specific target tropism can be irritated in Trop2 and lower be activated T cell killing ability.
In another embodiment, it was demonstrated that in certain effect target ratio (CAR-T cells：Target cell) under, Trop2 CAR-T are thin Born of the same parents are capable of the tumour cell of the effective killer T rop2 positives；But the CD19CAR-T cells for being non-Trop2 targetings can not be effective Kill target cell.The tumour that can effectively crack the Trop2 positives in vitro this embodiment illustrates Trop2 CAR-T is thin Born of the same parents.
In another embodiment, NOD-SCID immune deficiencies are constructed based on Trop2 positive ovarian cancerous cell lines OVCAR-3 Mouse subcutaneous transplantation tumor model.Using the Trop2 CAR-T cell therapies of the present invention tumor-bearing mice, as a result illustrate, Trop2 CAR-T cells can effectively remove Trop2 positive tumor cells, and all survivals at 30 days of the mouse for the treatment of group；But Tumour is constantly grown up in CD19 CAR-T cell 19-28BBZ control groups, and at 30 days under only 10% mouse survival.
Compared with prior art, the invention has the advantages that：
In the application in solid tumor field and unsuccessful, a critically important reason is just the absence of properly current CAR-T technologies Solid tumor target spot.The present invention provides a new target spot Trop2 for targeting solid tumor by CAR-T technologies, which exists It does not express in normal tissue cell or only expresses on a small quantity, and in the solid tumor tissues such as oophoroma, breast cancer and tumor vessel It is highly expressed in endothelial cell.Trop2CAR molecules of the present invention can be by modifying T cell, and makes specific C AR-T The special treatment Trop2 positive entity tumors of cell high-efficient.
Description of the drawings
Fig. 1 illustrates schematic diagram for Trop2 specific chimerics antigen receptor (CAR) molecular structure：
Fig. 2 illustrates schematic diagram for slow virus carrier pCDH structures：
Fig. 3 is the result schematic diagram that electrophoresis is identified in Trop2 CAR molecule digestions：
Fig. 4 is the expression of results in Western detection Trop-2 CAR plasmids again 293T cells
Fig. 5 A and 5B are the result that flow cytometry and RT-PCR detect different tumor cells expression Trop2 efficiency
Fig. 6 is the lethal effect that CCK methods detect Trop-2 CAR-T cells to different ovarian cancer cells
Fig. 7 is the lethal effect that CCK-8 methods detect TropCAR-T cells to ovarian cancer cell
Fig. 8 is lethal effect of the Trop2 CAR-T cells to different tumour cells
Fig. 9 A and Fig. 9 B are the result schematic diagram of killer T rop2 positive graft tumours in Trop2 CART cell mouse bodies.
Specific implementation
The present invention provides a kind of Trop2 specific chimerics antigen receptor (the CAR molecules for targeting Trop2), and it includes born of the same parents Outer cog region, compared with sequence, transmembrane region and intracellular signal area, wherein extracellular cog region be anti-Trop2 antibody sequences (scFv sequences, SEQ ID NO.3 and SEQ ID NO.4), it is the regions CD8hinge (SEQ ID NO.5) compared with sequence, transmembrane region is CD8 cross-films Area (SEQ ID NO.6), intracellular costimulatory signal area CD28-4-1BB (SEQ ID N0.7), CD3 ζ (SEQ ID NO.8).
The Chimeric antigen receptor of the present invention is specially CSF2RA-Trop2 scFv-CD8 α-CD28-4-1BB-CD3 ζ-T2A, Its nucleotide and amino acid sequence are as shown in SEQ ID No.1 and SEQ ID NO.2.
The present invention provides the above-mentioned CAR molecules of structure to the method in slow virus carrier pCDH in embodiment one.Embodiment Provided in two becomes slow virus by CAR molecules packaging, and T cell of transduceing measures the mode of transfection efficiency.Embodiment three provides Build the ability of Trop2 target cells and CAR-T cell killing Trop2 target cells.It is small that example IV provides CAR-T cells Inhibit the ability of Trop2 positive tumor growths in mouse model.
With reference to the accompanying drawings and examples, the specific implementation mode of the present invention is further described.Following embodiment is only For clearly illustrating technical scheme of the present invention, and not intended to limit the protection scope of the present invention.
Embodiment one
The above-mentioned CAR molecules of structure are present embodiments provided to the method in slow virus carrier pCDH
Trop2 antibody scFvs are built：With anti-Trop-2 Fab and Linker (Gly4Ser) 3 for template, according to In-Fusion PCR principles design V κ, VHAmplimer；Itself and Linker (Gly4Ser) 3 are simultaneously connected into V κ-by amplification V κ, vH segments Linker (Gly4Ser) 3-VH genetic fragments (anti-Trop-2 IgG)；It is bis- using restriction endonuclease Nco I and Xho I Digestion includes the retroviral vector of people CH2CH3 and intracellular signal transduction area CD28,4-1BB, CD3 ζ gene order (SFG.CH2CH3-CD28-4-1BB-CD3ζ)；
HRS-28bbZ vector constructions：Using the method for In-Fusion PCR (table 1) by the inverse of anti-Trop-2 scFv and linearisation Transcription vector connects, and is built into third generation Trop-2 CAR retrovirus expressions plasmid (Trop-2 CAR matter Grain), digestion identification is carried out to recombinant plasmid and sequencing analysis, digestion result are shown in Fig. 3；Use 293fectinTM Transfection Reagent detect it by Trop-2 CAR plasmid transfections to 293T cells by Westem BoIt In the intracellular expression of 293T, Western Blot results are shown in Fig. 4.
1 GFP of table, Trop2 scFv-CH2CH3-CD28 fragment amplification primers
Embodiment two
This example is the preparation of Trop-2 CAR-T cells
The packaging of CAR slow virus；PCDH Trop-2 CAR-GFP are extracted by the big extraction agent box of QIAGEN endotoxin-free plasmids Plasmid and slow virus auxiliary packaging plasmid pMD2.G and pSPAX2.For 24 hours by 2.5 × 10 before transfection7293 pavings to T75 cells are trained Bottle is supported, 293T culture mediums are changed to 10ml serum free mediums by 1h before transfecting, and use 293fectinTMTransfection Reage transfects 3 plasmids, turns then culture medium for 24 hours and is changed to 15ml complete mediums, cell conditioned medium is collected after turning then 48h Virus liquid, after 0.45 μm of 100kD super filter tube concentrates, -80 DEG C of placement freezes spare viral supernatants.It is detected using qPCR methods Virus titer, viral level of the viral supernatants after being concentrated by ultrafiltration 10 times is more than 1.5 × 109copies/ml。
T cell is transduceed：CD4+/CD8+T cells are detached respectively according to EasySeq kits (STEMCELL) specification, then by T Cell cultivates 3h at 37 DEG C, with complete medium (the RPMI culture mediums containing 10% people's AB serum) and IL2 that viral supernatants are dilute 2 times are released, T cell is incubated overnight in the supernatant, is trained again with X-VIVO15 culture mediums (containing IL2,5% people AB serum) later It supports.With the expression of Flow cytometry GFP to detect the transduction efficiency of CAR molecules after 72h.As illustrated in fig. x, Trop-2 CAR The positive rate of T cell is about 70-80%, the non-targeted control CAR-T cells of CD19 CAR-T cells again.
Embodiment three
The present embodiment is lethal effect of the Trop-2 CAR-T cells in vitro to ovarian cancer cell.
By Flow cytometry ovarian cancer cell OVCAR-3, HO8910, SKOV3, A2780, breast cancer cell MCF-7 and Melanoma cells A375；The expression (table 2) of above-mentioned cell Trop-2 mRNA is detected by RT-PCR.Flow cytometry Show with RT-PCR results：Ovarian cancer cell OVCAR-3, H08910 and breast cancer cell MCF-7 high express Trop-2 antigens, Ovarian cancer cell SKOV3 low expression Trop-2 antigens, ovarian cancer cell A2780, melanoma cells A375 do not express Trop-2 Antigen, as shown in Figure 5 A and 5B.
2 Trop2 and GAPDH fragment amplification primers of table
Tumour cell HO8910, the A2780 for collecting exponential phase, are respectively set 5 density 5 × 105Cell/ml, 2 × 105 Cell/ml, 1 × 105Cell/ml, 5 × 104Cell/ml is added separately to 96 porocyte culture plates with 100 holes μ L/；Wait for 96 holes T cell, Trop-2 CAR-T cells and the CD19 CAR-T cells of activation are collected after plate inner cell is adherent, adjustment cell density is 1×106/ ml is added to 100 holes μ L/ in 96 orifice plates, another to spread individual effect cell hole, places 37 DEG C of 5%CO2It is trained altogether in incubator It supports；Culture plate is taken out after 12h, 1500rpm/min centrifuges 5min, abandons supernatant；CCK-8 solution is diluted 10 times with culture medium, with 100 holes μ L/ add in 96 orifice plates, and 3h is incubated in incubator；Enzyme-linked immunosorbent assay instrument detects absorbance at 450nm.CCK-8 is detected The result shows that：Trop-2 CAR-T cells are to the lethal effect of the positive ovarian cancer cell of Trop-2 expression with effector cell and target Cell proportion (E:T increase) and enhance, and it is related to tumor surface Trop-2 expressions, as shown in Figure 6.
Tumour cell OVCAR-3, HO8910, SKOV3, A2780, MCF7 of exponential phase are collected, CCK methods detect E:T is 20: Trop-2 cells are to the lethal effects of different ovarian cancer cells when 1, as a contrast with T cell group and CD19 CAR-T groups, CCK-8 Method kit test method is same as above.The results are shown in Figure 7, Trop-2 CAR-T cells to OVCAR-3, HO8910, SKOV3, The killing-efficiency of MCF-7 is apparently higher than CD19 CAR-T and T cell group (P ＜ 0.05), and CD19 CAR-T cells and T cell group Killing-efficiency no significant difference (P ＞ 0.05)；Trop-2 is to the killing-efficiency of A2780 boxes A375, with CD19 CAR-T groups of cells and T cell group difference are not statistically significant (P ＞ 0.05).
It is thin using IFN-γ and IL2 ELISA kits (eBioscience, USA) detection Trop-2 CAR-T cell killing targets Cell factor IFN-γ, IL-2 secretion variations when born of the same parents.Collect E:T is 20:Supernatant is co-cultured when 1, and 3 multiple holes are set, it is horizontal It to doubling dilution, is added in the IL2/IFN- γ ELISA Plates being coated with by kit specification with 100 holes μ L/, 4 DEG C of closings Overnight, supernatant is abandoned, PBST is washed 4 times, and the 100 μ L of detection antibody of biotin labeling are added per hole, is incubated at room temperature 1h；PBST is washed 5 times, the 100 μ L of Avidin of HRP labels are added per hole, are incubated at room temperature 30min；PBST wash 7 times, with 100 holes μ L/ be added 1 × TMB developing solutions, are protected from light 15min；1M H are added with 50 holes μ L/3PO4Or 2N H2SO4Terminate reaction；Enzyme-linked immunosorbent assay instrument Detect absorbance at 450nm.ELISA testing results show：The gonad cell of Trop-2 CAR-T cells and expression Trop-2 After OVCAR-3, HO8910, SKOV3 co-culture 48h, cell factor IFN-γ, IF-2 secretory volumes obviously increase compared with control group in supernatant Add (P ＜ 0.01), as shown in Figure 8.
Example IV
The present embodiment verification CAR-T cells inhibit the ability of Trop-2 positive tumor growths in mouse model.
5~7 week old NOD-SCID immune deficiencies sub-cutaneous injections 4~6 × 106OVCAR-3 ovarian cancer cell lines allow tumour to give birth to It is 8-12 days long, with vernier caliper measurement tumor size, multiplied with tumour extreme length extreme length vertical direction length obtain with mm2For the tumor size of unit.When tumor size reaches 20-30mm2When, pass through intravenous injection 4~6 × 106Trop-2 CAR-T cells and CD19CAR-T cells measure tumor size in every 3 days later, when diameter of tumor reaches 1.5cm, put to death small Mouse.The present embodiment result as shown in fig. 9 a and fig. 9b, injection Trop-2 CAR-T cells after 6 days tumour start to become smaller, arrive 15- Tumour is substantially completely removed within 18 days, and all mouse survived by the 30th day.However the non-targeted Trop-2 of control CD19 CAR-T cells can not be such that tumour cell reduces, the survival rate only 20% of mouse at 30 days.
This example demonstrates that the CAR-T cells of targeting Trop-2 of the invention can effectively remove mouse Trop-1 positive tumors Tumour in Transplanted tumor model.
Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention be not restricted to The specific embodiment of upper description.To those skilled in the art, any equivalent modifications that the present invention is carried out and replacement All among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and modification, all It should cover within the scope of the invention.
Sequence table
<110>The Hangzhou bio tech ltd Rong Ze
<120>A kind of Trop-2 specific chimerics antigen receptor cell is prepared and application thereof
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tccgaggtgc agctgttgga gtctggggga ggcttggtac agcctggggg gtccctgaga 120
ctctcctgtg cagcctctgg attcaccttt agcggttatt atatgagctg ggtccgccag 180
gctccaggga aggggctgga gtgggtctca ttgatctctc atggtggtgg taatatatat 240
tacgctgatt ctgtaaaagg tcggttcacc atctccagag acaattccaa gaacacgctg 300
tatctgcaaa tgaacagcct gagagccgag gacacggccg tgtattactg tgcgagattt 360
ccgcttgagt tcgactactg gggccagggt acactggtca ccgtgagctc aggtggaggc 420
ggttcaggcg gaggtggatc cggcggtggc ggatcgcagt ctgtgctgac tcagccaccc 480
tcagcgtctg ggacccccgg gcagagggtc accatctctt gtactggctc ttcatctaat 540
attggcagta atgctgtctc ctggtaccag cagctcccag gaacggcccc caaactcctc 600
atctatgatg ataataagcg gccaagcggg gtccttgacc gattctctgg ctccaagtct 660
ggcacctcag cctccctggc catcagtggg ctccggtccg aagatgaggc tgattattac 720
tgtggtactt gggatgatag cctgagtggt tatgtcttcg gcggaggcac caagctgacg 780
gtcctagaat tcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatttt tgggtgctgg tggtggttgg tggagtcctg 960
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc 1140
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1200
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1260
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1320
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1380
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1440
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1500
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1560
gacgcccttc acatgcaggc cctgccccct cgcggatccg cggccgctga gggcagagga 1620
agtcttctaa catgcggtga cgtggaggag aatcccggcc ct 1662
<210> 2
<211> 554
<212> PRT
<213>The mankind (human)
<400> 2
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Ser Gly Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Val Ser Leu Ile Ser His Gly Gly Gly Asn Ile Tyr
65 70 75 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
85 90 95
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Phe Pro Leu Glu Phe Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro
145 150 155 160
Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly
165 170 175
Ser Ser Ser Asn Ile Gly Ser Asn Ala Val Ser Trp Tyr Gln Gln Leu
180 185 190
Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp Asp Asn Lys Arg Pro
195 200 205
Ser Gly Val Leu Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala
210 215 220
Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr
225 230 235 240
Cys Gly Thr Trp Asp Asp Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly
245 250 255
Thr Lys Leu Thr Val Leu Glu Phe Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu
305 310 315 320
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
325 330 335
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
340 345 350
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
355 360 365
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu
370 375 380
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
385 390 395 400
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
405 410 415
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
420 425 430
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
435 440 445
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
450 455 460
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
465 470 475 480
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
485 490 495
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
500 505 510
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
515 520 525
Pro Pro Arg Gly Ser Ala Ala Ala Glu Gly Arg Gly Ser Leu Leu Thr
530 535 540
Cys Gly Asp Val Glu Glu Asn Pro Gly Pro
545 550
<210> 3
<211> 116
<212> PRT
<213>The mankind (human)
<400> 3
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Leu Ile Ser His Gly Gly Gly Asn Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Pro Leu Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 4
<211> 110
<212> PRT
<213>The mankind (human)
<400> 4
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn
20 25 30
Ala Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Asp Asp Asn Lys Arg Pro Ser Gly Val Leu Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu
85 90 95
Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 5
<211> 45
<212> PRT
<213>The mankind (human)
<400> 5
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 6
<211> 16
<212> PRT
<213>The mankind (human)
<400> 6
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
1 5 10 15
<210> 7
<211> 90
<212> PRT
<213>The mankind (human)
<400> 7
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
1 5 10 15
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
20 25 30
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40 45
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
50 55 60
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
65 70 75 80
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
85 90
<210> 8
<211> 112
<212> PRT
<213>The mankind (human)
<400> 8
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110

Claims (12)

1. a kind of Trop2 specific chimerics antigen receptor CSF2RA-Trop2scFv-CD8 α-CD28-4-1BB-CD3 ζ-T2A, Be characterized in that, the Chimeric antigen receptor by anti-human Trop2 monoclonal antibodies light chain and heavy chain variable region hTrop2scFv, CSF2RA Antigenic Peptides, people CD8 α hinge areas, people CD28 transmembrane regions, people's CD28 and 4-1BB costimulatory signal conducting region, people CD3 ζ letters Number active region and T2A small peptide structures in series are constituted；The nucleotide and amino acid sequence of the Chimeric antigen receptor such as sequence SEQ Shown in ID No.1 and SEQ ID No.2.
2. Trop2 specific chimerics antigen receptor according to claim 1, which is characterized in that the anti-Trop2 antibody sequence It is classified as a kind of single-stranded variable region sequences of combination Trop2.
3. Trop2 specific chimerics antigen receptor according to claim 1, which is characterized in that the anti-Trop2 antibody sequence It is classified as Trop2 monoclonal antibodies HRS comprising the heavy chain variable amino acid sequence as shown in SEQ ID NO.3 and such as SEQ Chain variable region amino acid sequence shown in ID NO.4, the chain variable region amino acid sequence and heavy chain variable amino acid Sequence is connected by one section of linker amino acid sequence for GGGGSGGGGSGGGGS.
4. Trop2 specific chimerics antigen receptor according to claim 1, which is characterized in that described to be compared with sequence The regions CD8hinge, amino acid sequence is as shown in SEQ ID.5.
5. Trop2 specific chimerics antigen receptor according to claim 1, which is characterized in that the transmembrane region is CD28 Transmembrane region, amino acid sequence is as shown in SEQ ID NO.6.
6. Trop2 specific chimerics antigen receptor according to claim 1, which is characterized in that the costimulatory signal passes It is that CD28 and 4-1BB is composed in series to lead area, and amino acid sequence is as shown in SEQ ID NO.7.
7. Trop2 specific chimerics antigen receptor according to claim 1, which is characterized in that the CD3 ζ signal transductions The amino acid sequence in area is as shown in SEQ ID N0.8.
8. a kind of nucleic acid of coding such as Trop2 specific chimerics antigen receptor according to any one of claims 1 to 7.
9. a kind of recombinant expression carrier containing nucleic acid as described in any of claims 8.
10. a kind of host cell containing recombinant expression carrier as claimed in claim 9.
11. a kind of construction method of host cell as described in any of claims 10, which is characterized in that including recombinating table It transduces to the step of host cell up to the construction step of carrier, the packaging step of recombinant expression carrier and by recombinant expression carrier Suddenly.
12. Trop2 specific chimerics antigen receptor according to any one of claims 1 to 7, any one of claim 8~9 The nucleic acid, the recombinant expression carrier and host cell according to any one of claims 10 described in claim 9 are in mammal Application in oncotherapy.
CN201810401267.XA 2018-04-28 2018-04-28 A kind of Trop-2 specific chimerics antigen receptor cell is prepared and application thereof Pending CN108440674A (en)

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CN113423726A (en) * 2019-01-22 2021-09-21 英研生物(英国)有限公司 Receptor providing targeted co-stimulation for adoptive cell therapy
CN110317822A (en) * 2019-07-19 2019-10-11 英威福赛生物技术有限公司 TROP2 Chimeric antigen receptor, its T cell and its preparation method and application
CN110317822B (en) * 2019-07-19 2020-06-05 英威福赛生物技术有限公司 TROP2 chimeric antigen receptor, T cell thereof, and preparation method and application thereof
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WO2022261301A1 (en) 2021-06-11 2022-12-15 Gilead Sciences, Inc. Combination mcl-1 inhibitors with anti-cancer agents
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WO2023273595A1 (en) * 2021-06-30 2023-01-05 益科思特(北京)医药科技发展有限公司 Antibody binding to trop2, bispecific antibody targeting trop2 and cd3, preparation methods therefor and uses thereof
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WO2023201268A1 (en) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Combination therapy for treating tumor antigen expressing cancers
WO2023232746A1 (en) * 2022-05-30 2023-12-07 Mediterranea Theranostic S.R.L. Anti-activated trop-2 chimeric antigen receptor constructs for use in cancer therapy
WO2023241493A1 (en) * 2022-06-14 2023-12-21 上海恒润达生生物科技股份有限公司 Antibody specifically binding to trop2 or antigen-binding fragment thereof, preparation method therefor and use thereof

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