CN107216395B

CN107216395B - A kind of TM4SF1 specific chimerics antigen receptor and its application

Info

Publication number: CN107216395B
Application number: CN201710533614.XA
Authority: CN
Inventors: 郝瑞栋; 刘根桃; 吴国祥
Original assignee: SHANGHAI BIOMED-UNION CO LTD
Current assignee: Keyi Zhejiang Pharmaceutical Technology Co ltd
Priority date: 2017-07-03
Filing date: 2017-07-03
Publication date: 2018-07-20
Anticipated expiration: 2037-07-03
Also published as: CN107216395A

Abstract

The invention discloses a kind of TM4SF1 specific chimerics antigen receptors, including extracellular cog region, hinge area, transmembrane region and intracellular signal area, wherein extracellular cog region is scFv sequences, hinge area is the regions CD8, transmembrane region is any of CD8 transmembrane regions or CD28 transmembrane regions, and intracellular signal area includes at least one of CD28,4 1BB, CD3 ζ；Above-mentioned Chimeric antigen receptor is specially L6 28Z, L6 BBZ and L6 28BBZ, respectively the nucleic acid encode of the sequence shown in SEQ ID NO.12~SEQ ID NO.14；The present invention also provides a kind of recombinant expression carrier comprising above-mentioned nucleic acid and its host cell, construction method, the applications in mammal tumor treatment, TM4SF1 specific chimerics antigen receptor of the present invention can be by modifying T cell, and makes the efficient special treatment entity tumor of specific C AR T cells.

Description

A kind of TM4SF1 specific chimerics antigen receptor and its application

Technical field

The present invention relates to biological immune fields, in particular it relates to a kind of TM4SF1 specific chimerics antigen receptor And its application.

Background technology

Chimeric antigen receptor (CAR) is made of antigen recognizing district, transmembrane region, intracellular signal area, generally passes through viral transduction Or electroporation, so that CAR developed by molecule is formed CAR-T cells on T cell surface.CAR-T cells pass through extracellular antigen recognizing district It identifies tumour antigen, recruits T cell to tumor cell surface, and T cell is activated by intracellular signal area, T cell enforcement is made to kill Wound acts on, with killing tumor cell, to achieve the purpose that treat tumour.The design of CAR molecules according to intracellular signal area not With the development that have passed through for four generations, wherein first generation CAR molecules only include CD3 ζ；Second generation CAR molecules also include in addition to CD3 ζ One costimulating factor, such as CD28, CD137, OX40 etc.；Third generation CAR molecules simultaneously comprising concatenated two costimulations because Son；The cell factor of tumor microenvironment can be improved by containing IL-12 etc. in forth generation CAR molecules.CAR-T is by targeting CD19 points Son makes a breakthrough in neoplastic hematologic disorder direction, and effective percentage has reached 90% during part is studied.But CAR-T is in solid tumor Field is never broken through, so there is an urgent need to new tumor targets, new CAR MOLECULE DESIGNs are to promote it in solid tumor field Application.

TM4SF1 (transmembrane-4L six family member 1) is one and is incorporated on cytoplasma membrane Glycoprotein is highly expressed in kinds cancer cell, including hepatocellular carcinoma, non-small cell lung cancer etc.；TM4SF1 is also highly expressed On the endothelial cell of human cancer blood vessel.There are faint expression in Various Tissues organ simultaneously by TM4SF1.TM4SF1 functions It is that the motility and adhesiveness of kinds of tumor cells can be regulated and controled.It is expressed simultaneously since TM4SF1 is high in kinds of tumor cells It is related with the generation of intratumoral vasculature, so being a potential cancer target.

Invention content

The purpose of the present invention is to overcome the defects in the prior art, provides a kind of new CAR by targeting TM4SF1 Molecule, and prove its potential application in treatment of solid tumors.Specifically, the present invention provides a kind of TM4SF1 specific chimerics Antigen receptor (CAR), this CAR molecules are by the TM4SF1 albumen on targets neoplastic cells surface and activate the letter in T cell downstream Number access assigns ability of the T cell killing with TM4SF1 target tumor cells.

To achieve the above object, the present invention adopts the following technical scheme that：

The first purpose of the invention is to provide a kind of TM4SF1 specific chimerics antigen receptors comprising including extracellular knowledge Other area, hinge area, transmembrane region and intracellular signal area；Wherein, extracellular cog region is anti-TM4SF1 antibody sequences.

In order to advanced optimize above-mentioned TM4SF1 specific chimerics antigen receptor, the technical measures that the present invention is taken also are wrapped It includes：

Preferably, the anti-TM4SF1 antibody sequences are single-stranded variable region (scFv) sequence of combination TM4SF1 a kind of, more Preferably, in an embodiment of the present invention, the antibody sequence be from the published TM4SF1 monoclonal antibodies L6 in this field, TM4SF1 monoclonal antibodies L6 includes heavy chain variable region (VH) amino acid sequence as shown in SEQ ID NO.1 and such as SEQ ID Light chain variable region shown in NO.2 (VL) amino acid sequence.

Further, according to the composition rule of scFv, VH and VL sequences are by one section of flexibility linker amino acid sequence GGGGSGGGGSGGGGS is connected together, and forms scFv sequences.

Preferably, the hinge area is the regions CD8hinge, and amino acid sequence is as shown in SEQ ID NO.3.

Preferably, transmembrane region described in the transmembrane region is any of CD8 transmembrane regions or CD28 transmembrane regions, wherein institute The amino acid sequence of CD8 transmembrane regions is stated as shown in SEQ ID NO.4, the amino acid sequence such as SEQ ID of the CD28 transmembrane regions Shown in NO.5.

Preferably, the intracellular signal area includes at least one of CD28,4-1BB, CD3 ζ；It is highly preferred that the born of the same parents Interior signaling zone is at least two series connection in CD28,4-1BB and CD3 ζ.Wherein, the amino acid sequence of the CD28 such as SEQ Shown in ID NO.6, the amino acid sequence of the 4-1BB is as shown in SEQ ID NO.7, the amino acid sequence such as SEQ of the CD3 ζ Shown in ID NO.8.

Further, the intracellular signal area is T cell signal transduction active region.

It is preferably based on the structure of the above TM4SF1CAR molecules, the Chimeric antigen receptor of the present invention includes L6- Any of 28Z, L6-BBZ, L6-28BBZ, structure are as shown in Figure 1；Wherein, the amino acid sequence of the L6-28Z is such as Shown in SEQ ID NO.9, the amino acid sequence of the L6-BBZ is as shown in SEQ ID NO.10, the amino acid of the L6-28BBZ Sequence is as shown in SEQ ID NO.11.It is understood that also TM4SF1 can be formed according to the other structures that this field is applicable in Chimeric antigen receptor.

Specifically, in different CAR molecules, transmembrane region can be identical, also can be different；Its intracellular signal area can be identical, It also can be different.

Preferably, in a specific embodiment of the invention, if TM4SF1 Chimeric antigen receptors are L6-28Z, transmembrane region For CD8, intracellular signal area is CD28-CD3 ζ；If TM4SF1 Chimeric antigen receptors are L6-BBZ, transmembrane region CD8, Intracellular signal area is 4-1BB-CD3 ζ；If TM4SF1 Chimeric antigen receptors are L6-28BBZ, transmembrane region CD28, intracellular Signaling zone is CD28-4-1BB-CD3 ζ.

Second object of the present invention is to provide a kind of nucleic acid of the above-mentioned TM4SF1 specific chimerics antigen receptor of coding.

Preferably, the nucleotide sequence of the nucleic acid includes sequence shown in SEQ ID NO.12~SEQ ID NO.14.More Specifically, the nucleic acid encode of sequence shown in nucleic acid encode L6-28Z, the SEQ ID NO.13 of sequence shown in SEQ ID NO.12 The nucleic acid encode L6-28BBZ of sequence shown in L6-BBZ, SEQ ID NO.14.

The amino acid sequence and its nucleic acid sequence encoding of CAR molecules are as shown in the table：

Containing the core for encoding above-mentioned TM4SF1 specific chimerics antigen receptor third object of the present invention is to provide a kind of The recombinant expression carrier of acid.

Preferably, the recombinant expression carrier includes slow virus, retrovirus, adenovirus, adeno-associated virus or plasmid Deng；It is highly preferred that in an embodiment of the present invention, original recombinant expression carrier is slow virus.Specifically, institute in this embodiment The carrier used is slow virus carrier pCDH-EF1-MCS-T2A-CopGFP (hereinafter abbreviated as pCDH), and collection of illustrative plates is shown in Fig. 2. In the carrier, the expression of CAR molecules drives transcriptional expression by EF1 promoters, with GFP marker gene as lentiviruses transduction Mark.CAR slow virus carriers plasmid in the presence of assisting packaging plasmid pSPAX2 and VSV-G envelope plasmid pMD2.G, Cotransfection HEK293T cells, you can be packaged as the slow virus with CAR molecules.

Fourth object of the present invention is to provide a kind of containing the core for encoding above-mentioned TM4SF1 specific chimerics antigen receptor The host cell of the recombinant expression carrier of acid.

Preferably, 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 TM4SF1 specific chimerics antigen receptor, above-mentioned coding TM4SF1 is special The application of the nucleic acid of sex-mosaicism antigen receptor, above-mentioned recombinant expression carrier and host cell in mammal tumor treatment.

Preferably, mammalian solid tumors include but not limited to, tumor of kidney, neuroblastoma, gonioma, Osteosarcoma, chondrosarcoma, soft tissue sarcoma, liver neoplasm, thymoma, pulmonary blastoma, pancreatoblastoma, hemangioma etc..

More specifically, above application is a kind of TM4SF1 CAR-T cells, T is made by the transduction of lentivirus mediated Cell carries TM4SF1CAR, to impart the ability that T cell identifies TM4SF1 molecules, and targets the lactation of the TM4SF1 positives Animal carcasses tumour.

In one embodiment, by the tumor cell line Calu-3, Calu- of TM4SF1 CAR-T cells and Expression of TM 4SF1 6-TM4SF1 is incubated altogether, as a result shows that TM4SF1 CAR-T cells can largely generate IFN-γ under the stimulation of target cell； But IFN-γ cannot be generated when the cell line Calu-6 with TM4SF1 feminine genders is incubated altogether；Non- TM4SF1 targetings simultaneously CAR-T cells 19-28BBZ can not generate IFN-γ under the stimulation of TM4SF1 positive cells.This embodiment illustrates TM4SF1 The good specific target tropism of CAR-T cells can activate T cell killing ability under TM4SF1 stimulations.

In another embodiment, it was demonstrated that in certain effect target ratio (CAR-T cells：Target cell) under, TM4SF1CAR-T is thin Born of the same parents are capable of the tumour cell of the effective killer T M4SF1 positives；But the CAR-T cells 19-28BBZ for being non-TM4SF1 targetings cannot Enough effectively killing target cells.This embodiment illustrates TM4SF1 CAR-T can effectively crack the TM4SF1 positives in vitro Tumour cell.

In another embodiment, NOD-SCID Immune deficient mices are constructed based on TM4SF1 positive lung cancer cells system Calu-3 Subcutaneous transplantation tumor model.The tumor-bearing mice is treated using TM4SF1 CAR-T cells L6-28BBZ of the present invention, is as a result illustrated, TM4SF1 CAR-T cells can effectively remove TM4SF1 positive tumor cells, and the mouse for the treatment of group was all deposited at 30 days It is living；But tumour is constantly grown up in control 19-28BBZ groups, and at 30 days under only 20% 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 TM4SF1 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 lung cancer, the solid tumor tissues such as liver cancer and tumor vascular endothelium It is highly expressed in cell, the high expression especially on tumor stem cell.TM4SF1CAR molecules of the present invention can be by repairing Adorn T cell, and the treatment TM4SF1 positive entity tumors for keeping specific C AR-T cell high-efficients special.

Description of the drawings

Fig. 1 illustrates schematic diagram for TM4SF1 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 TM4SF1CAR molecule digestions；

Fig. 4 is the result schematic diagram of Flow cytometry TM4SF1 transduction T cell transduction efficiencies；

Fig. 5 is the schematic diagram of TM4SF1 expressions in different tumor cell lines；

Fig. 6 is the schematic diagram of TM4SF1 CAR-T cell IFN-γ secretion situations；

Fig. 7 A and Fig. 7 B are the result schematic diagram of TM4SF1 CAR-T cells in vitro killer T M4SF1 target cells；

Fig. 8 A and Fig. 8 B are the result signal of killer T M4SF1 positive graft tumours in TM4SF1 CAR-T cell mouse bodies Figure.

Specific implementation mode

The present invention provides a kind of TM4SF1 specific chimerics antigen receptor (the CAR molecules for targeting TM4SF1), it includes Extracellular cog region, hinge area, transmembrane region and intracellular signal area, wherein extracellular cog region is anti-TM4SF1 antibody sequences (scFv sequences Row, SEQ ID NO.1 and SEQ ID NO.2), hinge area is the regions CD8hinge (SEQ ID NO.3), transmembrane region CD8 Any of transmembrane region (SEQ ID NO.4) or CD28 transmembrane regions (SEQ ID NO.5), intracellular signal area include CD28 (SEQ ID NO.6), 4-1BB (SEQ ID NO.7), at least one of CD3 ζ (SEQ ID NO.8).

The present invention Chimeric antigen receptor be specially L6-28Z (SEQ ID NO.9), L6-BBZ (SEQ ID NO.10) and L6-28BBZ (SEQ ID NO.11), the respectively nucleic acid encode of the sequence shown in SEQ ID NO.12~SEQ ID NO.14.

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 TM4SF1 target cells and CAR-T cell killing TM4SF1 target cells.Example IV provides CAR-T cells Inhibit the ability of TM4SF1 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.

Structure TM4SF1CAR molecules have been classified as L6- according to the difference in CAR molecular signals region through this embodiment Tri- kinds of 28Z, L6-BBZ, L6-28BBZ：CD28-CD3 ζ is in the intracellular signal area used in wherein L6-28Z；It is used in L6-BBZ Intracellular signal area be 4-1BB-CD3 ζ；And the intracellular signal area employed in L6-28BBZ is CD28-4-1BB-CD3 ζ, Molecular structure ideograph is as shown in Figure 1.CAR molecule constructions as above slow virus carrier has been arrived by common molecular cloning approach In pCDH, the structure of slow virus carrier pCDH is as shown in Figure 2.

L6-28BBZ vector constructions：(Shanghai is difficult to understand lucky in chemical synthesis L6-28BBZ nucleic acid fragments to pUC57 carriers first Biological Co., Ltd), obtain L6-28BBZ segments using NotI/XbaI double digestions.NotI/XbaI double digestion slow virus carriers PCDH, gel recycling 7KB sizes or so DNA fragmentation after electrophoresis.L6-28BBZ and carrier pCDH is used into T4DNA ligases, room Temperature connection 1-2 hours, then converts stbl3 competent cells.Inoculation single bacterium drops down onto LB amplification cultivations within second day, and extracts matter Grain.Whether correct using plasmid obtained by NcoI digestion verifications, digestion result is shown in Fig. 3, the second swimming lane.Send correct plasmid to sequencing, Whether correct further verify its sequence.It leaves correct plasmid and is named as pCDH-L6-28BBZ progress next step experiments.

L6-28Z vector constructions：The chemical synthesis hinge area of L6-28Z, transmembrane region and intracellular region sequence pUC57- first 28Z (Shanghai lucky biological Co., Ltd's synthesis difficult to understand).It is bis- that pUC57-28Z and pCDH-L6-28BBZ are carried out to BamHI/NotI respectively Digestion, and electrophoresis recycles.By pCDH-L6-28BBZ and 28Z T4DNA ligases, room temperature connects 1-2 hours, then converts Stbl3 competent cells.Inoculation single bacterium drops down onto LB amplification cultivations within second day, and extracts plasmid.Obtained by NcoI digestion verifications Whether plasmid is correct, and digestion result is shown in Fig. 3, third swimming lane.It send correct plasmid to sequencing, whether just further verifies its sequence Really.It leaves correct plasmid and is named as pCDH-L6-28Z progress next step experiments.

L6-BBZ vector constructions：Use primer BBF (5 '-AAACGGGGCAGAAAGA AACTCC TGT-3 ', SEQ ID NO.17) and CD3ZR (5 '-GCGAGGGGGCAGGGCCTGCAT GTGA-3 ', SEQ ID NO.18), using L6-28BBZ as template Carry out PCR amplification BBZ signal region sequences；With primer CAR F (5 '-ATGGCCTTACCAGTGACCGCCTTGC-3 ', SEQ ID NO.19) and CD8TMR (5 '-GCAGTAAAGGGTGATAACCAGTGAC-3 ', SEQ ID NO.20), expand by template of L6-28Z Increase L6scFv, CD8 hinge areas and transmembrane domain.It is complete L6- that bridging PCR is carried out later by front and back two parts sequence assembly BBZ, primer be CAR F2 (5 '-ctagctagcATGGCCTTACCAGTGACCGCCTTGC-3 ', SEQ ID NO.21) and Both CD3ZR2 (5 '-cgggatccGCGAGGGGGCAGGGCCTGCATGTGA-3 ', SEQ ID NO.22) are used and are divided respectively Not carry out NotI/XbaI double digestions, and electrophoresis recycle.It is used in combination T4DNA ligases, room temperature to connect 1-2 hours, then converts Stbl3 competent cells.Inoculation single bacterium drops down onto LB amplification cultivations within second day, and extracts plasmid.Obtained by NcoI digestion verifications Whether plasmid is correct, and digestion result is shown in Fig. 3, the 4th swimming lane.It send correct plasmid to sequencing, whether just further verifies its sequence Really.It leaves correct plasmid and is named as pCDH-L6-BBZ progress next step experiments.

Embodiment two

The present embodiment is the structure of TM4SF1 CAR-T cells.

The packaging of CAR slow virus：Plasmid kit is carried greatly using QIAGEN endotoxin-frees first extracts embodiment one respectively Slow virus plasmid pCDH-L6-28Z, pCDH-L6-BBZ, pCDH-L6-28BBZ, pCDH-19-28BBZ of structure are (non-targeted TM4SF1 compares CAR molecules plasmid) and slow virus system auxiliary packaging plasmid pMD2.G and pSPAX2.It is incited somebody to action in the day before transfection 2.5 × 10E7 293T are spread into T75 culture bottles.293T cell culture mediums are changed to 10ml free serum cultures in first 1 hour by transfection Base.Using calcium phosphate precipitation by plasmid co-transfection to 293T cells, cell culture medium was changed to 15ml in 24 hours after transfection Complete medium DMEM+10%FBS.Cell conditioned medium is collected, and 15ml fresh complete mediums are added within 48 hours after transfection.72 is small Shi Zaici collects cell conditioned medium, discards cell.The cell conditioned medium collected is used into 0.45um membrane filtrations, 5000g is centrifuged later 1min is to remove impurity.Subsequent 40000g is centrifuged 2 hours, precipitate virus, and virus is resuspended using 0.1ml PBS.Place -80 DEG C of jellies It deposits spare.

T cell is transduceed：It is taken a blood sample using conventional method and detaches PBMC, thin in vain containing 5% people AB serum and 300IU/ml In the X-VIVO15 culture mediums of born of the same parents' interleukin -2, AntiCD3 McAb/CD28 monoclonal antibodies stimulation is used in combination.Start culture 48 hours later, it will Activating T cell is suspended in the X-VIVO15 culture mediums containing protamine sulfate and 300IU/IL-2, and corresponding slow virus is added (L6-28Z, L6-BBZ, L6-28BBZ or 19-28BBZ).Then T cell is cultivated 3 hours at 37 DEG C.Then it is trained with RPMI It supports+10% fetal calf serum of base and IL-2 and supernatant is diluted 2 times, T cell is incubated overnight in diluted supernatant, is then returned Return culture in X-VIVO15 culture mediums (containing IL-2,5% people AB serum).Flow cytometry GFP tables are utilized after 72 hours Up to detect CAR molecule transduction efficiencies.As shown in figure 4, observe L6-28Z, L6-BBZ, L6-28BBZ, 19-28BBZ is thin in T The positive rate of born of the same parents is about in 60%-70%, non-targeted control CAR-T cells of the 19-28BBZ as TM4SF1 CAR-T cells.

Embodiment three

The present embodiment is the measurement of TM4SF1 CAR-T cell activity.

The identification of TM4SF1 CAR-T target cells and structure：Calu-6, the lung carcinoma cells such as Calu-3 are had detected using qPCR The expression of TM4SF1 is fastened, as shown in figure 5, the high Expression of TM 4SF1 genes of Calu-3 cell lines, and Calu-6 is hardly expressed TM4SF1 genes.It using the slow virus carrier of coding TM4SF1 genes, is screened using puromycin, structure obtains high expression The Calu-6 cell lines of TM4SF1 genes.The TM4SF1 expressions of Calu-6-TM4SF1 cell lines are shown in Fig. 5.According to Fig. 5 results It is found that Calu-3, Calu-6-TM4SF1 cell line are TM4SF1 positive expressions, and Calu-6 cell lines TM4SF1 is negative.

IFN-γ secretion is tested：By L6-28Z, L6-BBZ, L6-28BBZ in 96 orifice plates, tetra- kinds of CAR-T of 19-28BBZ are thin Born of the same parents (effector cell) are with target cell with 5:1 effect target is than mixing.It cultivates 24 hours at 37 DEG C, is examined later using standard ELISA assay Survey the expression of IFN-γ.The results are shown in Figure 6, when the CAR-T cells and Calu-3 or Calu-6- of three kinds of TM4SF1 targetings When TM4SF1 is co-cultured, CAR-T cells can a large amount of secretion of gamma-IFN, and the secretory volume of L6-28BBZ is higher than L6-28Z and L6- 28BBZ, illustrate third generation CAR structures L6-28BBZ targeting TM4SF1 activity better than second generation CAR structures L6-28Z with And L6-28BBZ.And the Calu-6 cells of three kinds of CAR molecules of TM4SF1 and low expression TM4SF1 be incubated altogether be cannot big volume production Raw IFN-γ illustrates that CAR-T cells constructed by the present invention have good targeting specific.And as a contrast, it is non-targeted The 19-28BBZ CAR-T cells of TM4SF1 do not generate a large amount of IFN-γ, also illustrate that CAR-T cells constructed by the present invention have There is good targeting specific.

Example IV

The present embodiment verifies the ability of TM4SF1 CAR-T cell body killer T M4SF1 target cells.

Target cell is dyed with 5-carboxyfluorescein succinimido (CFSE), respectively the CAR-T in Example two The Calu-3 of cell and the TM4SF1 positives, Calu-6-TM4SF1 are co-cultured with following effect target than mixing：0.5:1,1:1,2:1, 5:1.After 4 hours co-culture, cell PI kits is dyed, while control group is set, target cell is added in control group But effect CAR-T cells are not added simultaneously.Cell killing is detected using flow cytometry.As a result see Fig. 7 A and Fig. 7 B. TM4SF1 CAR-T cells can effectively kill Calu-3 and Calu-6-TM4SF1 cells, but the 19-28BBZ compareed CAR-T cells can not effectively kill both target cells.The L6-28BBZ killer T M4SF1 positive lung cancers of third generation CAR structures The activity of cell is better than the L6-28Z and L6-28BBZ of second generation CAR structures.

This example demonstrates that the CAR-T cells of tri- kinds of CAR molecule constructions of L6-28BBZ, L6-28Z, L6-BBZ can be effective The lung carcinoma cell of the killer T M4SF1 positives.

Embodiment five

The present embodiment verification CAR-T cells inhibit the ability of TM4SF1 positive tumor growths in mouse model.

Using 6 week old NOD-SCID Immune deficient mices, it is subcutaneously injected 5 × 10⁶Calu-3 lung cancer cell lines allow tumour to give birth to It is 5-15 days long, tumor size is measured with caliper, the length that extreme length vertical direction is multiplied with tumour extreme length obtains a mm² For the tumor size of unit.When tumor size reaches 20-30mm²When, pass through intravenous injection 5 × 10⁶TM4SF1 CAR-T cells L6-28BBZ or 19-28BBZ measures tumor size in every 3 days later, when diameter of tumor reaches 1.5cm, puts to death mouse.

The present embodiment result is as shown in Figure 8 A and 8 B, injection L6-28BBZ CAR-T cells after 6 days tumour start to become It is small, tumour was substantially completely understood by 15-18 days, and all mouse survived by the 30th day.However control is non-targeted The 19-28BBZ CAR-T cells of TM4SF1 can not be such that tumour cell reduces, and at 30 days, the survival rate of mouse was only 20%.

This example demonstrates that the CAR-T cells of targeting TM4SF1 of the invention can effectively remove the mouse TM4SF1 positives Tumour in tumour transplatation tumor model.

Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention is not intended to limit In particular embodiments described above.To those skilled in the art, any to the equivalent modifications that carry out of the present invention and to replace In generation, is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and repair Change, all should be contained within the scope of the invention.

SEQUENCE LISTING

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<223>L6-28Z amino acid sequences, CAR, people's source protein

<400> 9

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 Ala Ser Gln Ile Val Leu Ser Gln Ser Pro Ala

20 25 30

Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Leu Thr Cys Arg Ala

35 40 45

Ser Ser Ser Val Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser

50 55 60

Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val

65 70 75 80

Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Ala

85 90 95

Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Trp Asn Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly

145 150 155 160

Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn

165 170 175

Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp

180 185 190

Met Gly Trp Ile Asn Thr Tyr Thr Gly Gln Pro Thr Tyr Ala Asp Asp

195 200 205

Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Tyr Thr Ala

210 215 220

Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe

225 230 235 240

Cys Ala Arg Phe Ser Tyr Gly Asn Ser Arg Tyr Ala Asp Tyr Trp Gly

245 250 255

Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Ser Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

305 310 315 320

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

325 330 335

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

340 345 350

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

355 360 365

Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 10

<211> 490

<212> PRT

<213> Artificial Sequence

<220>

<223>L6-BBZ amino acid sequences, CAR, people's source protein

<400> 10

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 Ala Ser Gln Ile Val Leu Ser Gln Ser Pro Ala

20 25 30

Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Leu Thr Cys Arg Ala

35 40 45

Ser Ser Ser Val Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser

50 55 60

Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val

65 70 75 80

Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Ala

85 90 95

Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Trp Asn Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly

145 150 155 160

Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn

165 170 175

Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp

180 185 190

Met Gly Trp Ile Asn Thr Tyr Thr Gly Gln Pro Thr Tyr Ala Asp Asp

195 200 205

Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Tyr Thr Ala

210 215 220

Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe

225 230 235 240

Cys Ala Arg Phe Ser Tyr Gly Asn Ser Arg Tyr Ala Asp Tyr Trp Gly

245 250 255

Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Ser Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

305 310 315 320

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

325 330 335

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

340 345 350

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

355 360 365

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 11

<211> 534

<212> PRT

<213> Artificial Sequence

<220>

<223>L6-28BBZ amino acid sequences, CAR, people's source protein

<400> 11

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 Ala Ser Gln Ile Val Leu Ser Gln Ser Pro Ala

20 25 30

Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Leu Thr Cys Arg Ala

35 40 45

Ser Ser Ser Val Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser

50 55 60

Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val

65 70 75 80

Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Ala

85 90 95

Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Trp Asn Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly

145 150 155 160

Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn

165 170 175

Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp

180 185 190

Met Gly Trp Ile Asn Thr Tyr Thr Gly Gln Pro Thr Tyr Ala Asp Asp

195 200 205

Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Tyr Thr Ala

210 215 220

Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe

225 230 235 240

Cys Ala Arg Phe Ser Tyr Gly Asn Ser Arg Tyr Ala Asp Tyr Trp Gly

245 250 255

Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Ser Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly

305 310 315 320

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

325 330 335

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

340 345 350

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

355 360 365

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg

370 375 380

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

385 390 395 400

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

405 410 415

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

420 425 430

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

435 440 445

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

450 455 460

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

465 470 475 480

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

485 490 495

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

500 505 510

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

515 520 525

Gln Ala Leu Pro Pro Arg

530

<210> 12

<211> 1464

<212> DNA

<213> Artificial Sequence

<220>

<223>The code nucleic acid of L6-28Z, people's source nucleic acid sequence

<400> 12

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggctagcc aaattgttct ctcccagtct ccagcaatcc tgtctgcatc tccaggggag 120

aaggtcacat tgacttgcag ggccagctca agtgtaagtt tcatgaactg gtaccagcag 180

aagccaggat cctcccccaa accctggatt tatgccacat ccaatttggc ttctggagtc 240

cctggtcgct tcagtggcag tgggtctggg acctcttact ctctcgcaat cagcagagtg 300

gaggctgaag atgctgccac ttattactgc cagcagtgga atagtaaccc actcacgttc 360

ggtgctggga ccaagctgga gctgaaacga ggtggtggtg gtagcggcgg cggcggctct 420

ggtggtggcg gatcccagat ccagttggtg cagtctggac ctgagctgaa gaagcctgga 480

gagacagtca agatctcctg caaggcttct gggtatacct tcacaaacta tggaatgaac 540

tgggtgaagc aggctccagg aaagggttta aagtggatgg gctggataaa cacctacact 600

ggacagccaa catatgctga tgacttcaag ggacggtttg ccttctcttt ggaaacctct 660

gcctacactg cctatttgca gatcaacaac ctcaaaaatg aggacatggc tacatatttc 720

tgtgcaagat ttagctatgg taactcacgt tacgctgact actggggcca aggcaccact 780

ctcacagtct cctcaggatc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcag taagaggagc 1020

aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag 1080

cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccag agtgaagttc 1140

agcaggagcg cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc 1200

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgc 1464

<210> 13

<211> 1470

<212> DNA

<213> Artificial Sequence

<220>

<223>The code nucleic acid of L6-BBZ, people's source nucleic acid sequence

<400> 13

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggctagcc aaattgttct ctcccagtct ccagcaatcc tgtctgcatc tccaggggag 120

aaggtcacat tgacttgcag ggccagctca agtgtaagtt tcatgaactg gtaccagcag 180

aagccaggat cctcccccaa accctggatt tatgccacat ccaatttggc ttctggagtc 240

cctggtcgct tcagtggcag tgggtctggg acctcttact ctctcgcaat cagcagagtg 300

gaggctgaag atgctgccac ttattactgc cagcagtgga atagtaaccc actcacgttc 360

ggtgctggga ccaagctgga gctgaaacga ggtggtggtg gtagcggcgg cggcggctct 420

ggtggtggcg gatcccagat ccagttggtg cagtctggac ctgagctgaa gaagcctgga 480

gagacagtca agatctcctg caaggcttct gggtatacct tcacaaacta tggaatgaac 540

tgggtgaagc aggctccagg aaagggttta aagtggatgg gctggataaa cacctacact 600

ggacagccaa catatgctga tgacttcaag ggacggtttg ccttctcttt ggaaacctct 660

gcctacactg cctatttgca gatcaacaac ctcaaaaatg aggacatggc tacatatttc 720

tgtgcaagat ttagctatgg taactcacgt tacgctgact actggggcca aggcaccact 780

ctcacagtct cctcaggatc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140

aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1200

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440

gcccttcaca tgcaggccct gccccctcgc 1470

<210> 14

<211> 1602

<212> DNA

<213> Artificial Sequence

<220>

<223>The code nucleic acid of L6-28BBZ, people's source nucleic acid sequence

<400> 14

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggctagcc aaattgttct ctcccagtct ccagcaatcc tgtctgcatc tccaggggag 120

aaggtcacat tgacttgcag ggccagctca agtgtaagtt tcatgaactg gtaccagcag 180

aagccaggat cctcccccaa accctggatt tatgccacat ccaatttggc ttctggagtc 240

cctggtcgct tcagtggcag tgggtctggg acctcttact ctctcgcaat cagcagagtg 300

gaggctgaag atgctgccac ttattactgc cagcagtgga atagtaaccc actcacgttc 360

ggtgctggga ccaagctgga gctgaaacga ggtggtggtg gtagcggcgg cggcggctct 420

ggtggtggcg gatcccagat ccagttggtg cagtctggac ctgagctgaa gaagcctgga 480

gagacagtca agatctcctg caaggcttct gggtatacct tcacaaacta tggaatgaac 540

tgggtgaagc aggctccagg aaagggttta aagtggatgg gctggataaa cacctacact 600

ggacagccaa catatgctga tgacttcaag ggacggtttg ccttctcttt ggaaacctct 660

gcctacactg cctatttgca gatcaacaac ctcaaaaatg aggacatggc tacatatttc 720

tgtgcaagat ttagctatgg taactcacgt tacgctgact actggggcca aggcaccact 780

ctcacagtct cctcaggatc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgattttt gggtgctggt ggtggttggt 960

ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1020

agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080

cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1200

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1260

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 1320

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1380

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1440

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1500

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1560

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 1602

<210> 15

<211> 532

<212> PRT

<213> Artificial Sequence

<220>

<223>19-28BBZ amino acid sequences, people's source protein

<400> 15

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 Ala Ser Gln Val Gln Leu Gln Gln Ser Gly Pro

20 25 30

Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His

50 55 60

Gly Lys Ser Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala

65 70 75 80

Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp

85 90 95

Lys Ser Ser Ser Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu

100 105 110

Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu

145 150 155 160

Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val

165 170 175

Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr

180 185 190

Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser

195 200 205

Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala

225 230 235 240

Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr Tyr Gly Ala

245 250 255

Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val

305 310 315 320

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

325 330 335

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

340 345 350

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

355 360 365

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys

370 375 380

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

385 390 395 400

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

405 410 415

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

420 425 430

Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

435 440 445

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

450 455 460

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

465 470 475 480

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

485 490 495

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

500 505 510

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

515 520 525

Leu Pro Pro Arg

530

<210> 16

<211> 1596

<212> DNA

<213> Artificial Sequence

<220>

<223>The code nucleic acid of 19-28BBZ, people's source nucleic acid sequence

<400> 16

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggctagcc aggtacaact gcagcagtct gggcctgagc tggagaagcc tggcgcttca 120

gtgaagatat cctgcaaggc ttctggttac tcattcactg gctacaccat gaactgggtg 180

aagcagagcc atggaaagag ccttgagtgg attggactta ttactcctta caatggtgct 240

tctagctaca accagaagtt caggggcaag gccacattaa ctgtagacaa gtcatccagc 300

acagcctaca tggacctcct cagtctgaca tctgaagact ctgcagtcta tttctgtgca 360

agggggggtt acgacgggag gggttttgac tactggggcc aagggaccac ggtcaccgtc 420

tcctcaggtg gaggcggttc aggcggcggt ggctctagcg gtggcggatc ggacatcgag 480

ctcactcagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 540

agtgccagct caagtgtaag ttacatgcac tggtaccagc agaagtcagg cacctccccc 600

aaaagatgga tttatgacac atccaaactg gcttctggag tcccaggtcg cttcagtggc 660

agtgggtctg gaaactctta ctctctcaca atcagcagcg tggaggctga agatgatgca 720

acttattact gccagcagtg gagtaagcac cctctcacgt acggtgctgg gacaaagttg 780

gaaatcaaag gatccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat ttttgggtgc tggtggtggt tggtggagtc 960

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 1020

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 1080

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccaaacgg 1140

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1200

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1260

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1320

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1380

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1440

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1500

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1560

tacgacgccc ttcacatgca ggccctgccc cctcgc 1596

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223>Primer BBF

<400> 17

aaacggggca gaaagaaact cctgt 25

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223>Primer CD3ZR

<400> 18

gcgagggggc agggcctgca tgtga 25

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223>Primer CAR F

<400> 19

atggccttac cagtgaccgc cttgc 25

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223>Primer CD8TMR

<400> 20

gcagtaaagg gtgataacca gtgac 25

<210> 21

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223>Primer CAR F2

<400> 21

ctagctagca tggccttacc agtgaccgcc ttgc 34

<210> 22

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223>Primer CD3ZR2

<400> 22

cgggatccgc gagggggcag ggcctgcatg tga 33

Claims

1. a kind of TM4SF1 specific chimerics antigen receptor, which is characterized in that including extracellular cog region, hinge area, transmembrane region and Intracellular signal area；Wherein, extracellular cog region is anti-TM4SF1 antibody sequences；

Wherein, the anti-TM4SF1 antibody sequences are TM4SF1 monoclonal antibodies L6 comprising as shown in SEQ ID NO.1 Heavy chain variable amino acid sequence and the chain variable region amino acid sequence as shown in SEQ ID NO.2, the light chain variable region Amino acid sequence and heavy chain variable amino acid sequence are connected by one section of linker amino acid sequence for GGGGSGGGGSGGGGS It connects.

2. TM4SF1 specific chimerics antigen receptor according to claim 1, which is characterized in that the hinge area is CD8 The regions hinge.

3. TM4SF1 specific chimerics antigen receptor according to claim 2, which is characterized in that the areas the CD8 hinge The amino acid sequence in domain is as shown in SEQ ID NO.3.

4. TM4SF1 specific chimerics antigen receptor according to claim 1, which is characterized in that the transmembrane region is CD8 Any of transmembrane region or CD28 transmembrane regions.

5. TM4SF1 specific chimerics antigen receptor according to claim 4, which is characterized in that the CD8 transmembrane regions Amino acid sequence is as shown in SEQ ID NO.4, and the amino acid sequence of the CD28 transmembrane regions is as shown in SEQ ID NO.5.

6. TM4SF1 specific chimerics antigen receptor according to claim 1, which is characterized in that the intracellular signal area packet Containing at least one of CD28,4-1BB, CD3 ζ.

7. TM4SF1 specific chimerics antigen receptor according to claim 6, which is characterized in that the amino acid of the CD28 Sequence is as shown in SEQ ID NO.6, and the amino acid sequence of the 4-1BB is as shown in SEQ ID NO.7, the amino acid of the CD3 ζ Sequence is as shown in SEQ ID NO.8.

8. TM4SF1 specific chimerics antigen receptor according to claim 1, which is characterized in that the Chimeric antigen receptor packet Include at least one of amino acid sequence as shown in NO.9~11 SEQ ID.

9. a kind of nucleic acid of coding such as TM4SF1 specific chimerics antigen receptor according to any one of claims 1 to 8.

10. nucleic acid according to claim 9, which is characterized in that the nucleotide sequence of the nucleic acid includes SEQ ID At least one of nucleotide sequence shown in NO.12~SEQ ID NO.14.

11. a kind of recombinant expression carrier containing the nucleic acid as described in any one of claim 9~10.

12. a kind of host cell containing recombinant expression carrier as claimed in claim 11.

13. a kind of construction method of host cell as described in any of claims 12, 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.

14. the TM4SF1 specific chimeric antigen receptors described in any one of claim 1-8, any one of claim 9-10 The nucleic acid, the recombinant expression carrier described in claim 11 and the host cell described in claim 12 are preparing lactation Application in animal tumor medicine.