CN109836497A - A kind of single-chain antibody of targeting EGFR, Chimeric antigen receptor T cell and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of single-chain antibodies of targeting EGFR, the single-chain antibody of the targeting EGFR includes the amino acid sequence as shown in SEQ ID NO:1, the single-chain antibody of the targeting EGFR is Humanized single chain antibody, the present invention also provides a kind of Chimeric antigen receptor T cells of single-chain antibody including the targeting EGFR, the Chimeric antigen receptor of the targeting EGFR can be with the targeting EGFR of specificity, promote T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific, the Chimeric antigen receptor T cell of targeting EGFR made from Humanized single chain antibody preferably maintains the vigor and lethality of cell simultaneously, and damage is not will cause to normal cell.The present invention also provides a kind of preparation method and application of the Chimeric antigen receptor T cell of targeting EGFR.

Description

A kind of single-chain antibody of targeting EGFR, Chimeric antigen receptor T cell and preparation method thereof And application

Technical field

The present invention relates to field of medical biotechnology, in particular to a kind of single-chain antibody of targeting EGFR, Chimeric antigen receptor T are thin Born of the same parents and its preparation method and application.

Background technique

People's epithelial growth factor receptor (EGFR) is the major receptors of people's epidermal growth factor.Upon activation, EGFR can be sent out Raw homologous dimerization, or heterodimeric occurs with the member of other people epithelial growth factor receptor families, to induce EGFR cell The autophosphorylation of interior structure, and activate downstream passages such as Mitogen-actived protein kinase (MAPK) access etc., and mediate thin Born of the same parents' amplification.Research shows that EGFR is expression or a unconventionality expression in a variety of solid tumor cells such as lung cancer, and in ordinary cells The very faint cell surface antigen of middle expression.The proliferation of EGFR and tumour cell, tumor invasion, transfer and the inhibition of Apoptosis It is related.In cancer, the EGFR of variation obtains the activation of duration, so as to cause the immoderate amplification of cell and eventually leads to cancer Disease.Therefore, EGFR is a cancer specific target spot.

Immune cell therapy is that the method for thoroughly removing cancer cell is uniquely possible in existing science and technology, and treatment tumour has High specificity, almost non-toxic side effect huge advantage the drawbacks of compensating for traditional remedies, at home and abroad have been used to clinic and control Malignant tumour is treated, Chimeric antigen receptor T cell technology (CAR-T) is that current adoptive cell adoptive therapy technology is newest immune One of cell technology, because it can activate self immune system in vivo, routinely targets neoplastic cells are killed, most Achieve the purpose that remove malignant cell and widely paid close attention to and studied eventually.There is not the inosculating antibody of targeting EGFR also at present The preparation and research of original receptor T cell.

Summary of the invention

In view of this, the present invention provides a kind of single-chain antibodies of targeting EGFR, and the list including the targeting E GFR The Chimeric antigen receptor T cell of the targeting EGFR of chain antibody.The Chimeric antigen receptor of the targeting EGFR can be with the target of specificity To EGFR, promote T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific, while humanization list The Chimeric antigen receptor T cell of targeting EGFR made from chain antibody can preferably maintain the vigor of Chimeric antigen receptor T cell And lethality, and not will cause damage to normal cell.The present invention also provides a kind of Chimeric antigen receptor T of targeting EGFR The preparation method and application of cell.

In a first aspect, the present invention provides a kind of single-chain antibody of targeting EGFR, the single-chain antibody packet of the targeting EGFR The amino acid sequence as shown in SEQ ID NO:1 is included, the single-chain antibody of the targeting EGFR is Humanized single chain antibody.

Optionally, the single-chain antibody encoding gene of the targeting EGFR includes the nucleotides sequence as shown in SEQ ID NO:2 Column.

Optionally, the single-chain antibody encoding gene of the targeting EGFR should consider degeneracy base, i.e., such as SEQ ID NO:1 Shown in the encoding gene of amino acid sequence include the nucleotide sequence as shown in SEQ ID NO:2, protection scope should also protect Shield has the nucleotide sequence of base degeneracy matter with SEQ ID NO:2, and the corresponding amino acid sequence of these nucleotide sequences is still It is so SEQ ID NO:1.

Specificity knot can occur with EGFR albumen for the single-chain antibody for the targeting EGFR that first aspect present invention provides It closes, the solid tumor cell for especially expressing tumour cell EGFR has stronger affine activity.

Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells of targeting EGFR, including targeting E GFR's Chimeric antigen receptor CAR-EGFR, the CAR-EGFR include the single-stranded of the sequentially connected targeting EGFR from aminoterminal to c-terminus Antibody, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein the single-chain antibody of the targeting EGFR includes The amino acid sequence as shown in SEQ ID NO:1.

Wherein, described " being sequentially connected with from aminoterminal to c-terminus " specifically: the ammonia of the single-chain antibody of the targeting EGFR The c-terminus of base acid sequence is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, the amino of the extracellular hinge area The c-terminus of acid sequence is connected with the aminoterminal of the amino acid sequence of the transmembrane region, the carboxylic of the amino acid sequence of the transmembrane region Cardinal extremity is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.

The extracellular hinge area described in the present invention is used to promote the EGFR on the single-chain antibody and tumour of the targeting EGFR In conjunction with.

Optionally, the extracellular hinge area include CD8 α hinge area, CD28 hinge area, CD4 hinge area, C D5 hinge area, One of CD134 hinge area, CD137 hinge area, ICOS hinge area or a variety of combinations.

Further alternative, the extracellular hinge area is CD8 α hinge area.

Optionally, the amino acid sequence of the CD8 α hinge area includes the amino acid sequence as shown in SEQ ID NO:6.

Optionally, the encoding gene of the CD8 α hinge area includes the nucleotide sequence as shown in SEQ ID NO:7.

Optionally, the encoding gene of the CD8 α hinge area should consider degeneracy base, i.e., as shown in SEQ ID N O:6 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:7 shown in, protection scope should also protect and SEQ ID NO:7 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:6。

The transmembrane region is used to fix the Chimeric antigen receptor CAR-EGF R of the targeting EGFR in the present invention.

Optionally, the transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region Or a variety of combination.

Further alternative, the transmembrane region is CD8 transmembrane region.

Optionally, the amino acid sequence of the CD8 transmembrane region includes the amino acid sequence as shown in SEQ ID NO:8.

Optionally, the encoding gene of the CD8 transmembrane region includes the nucleotide sequence as shown in SEQ ID NO:9.

Optionally, the encoding gene of the CD8 transmembrane region should consider degeneracy base, i.e., as shown in SEQ ID NO:8 The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:9, and protection scope should also protect and SEQ ID NO:9 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:8。

The intracellular signal area is for providing the signal of T cell activation in the present invention, maintain T cell life span and Activate T cell proliferation signal access.

Optionally, the intracellular signal area includes 4-1BB signaling zone, CD3 ζ signaling zone, ICOS signaling zone, C D27 signal One of area, OX40 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRS F19L signaling zone are more The combination of kind.

Optionally, the intracellular signal area is 4-1BB signaling zone and CD3 ζ signaling zone.

Optionally, the amino acid sequence of the 4-1BB signaling zone includes the amino acid sequence as shown in SEQ ID NO:10.

Optionally, the encoding gene of the 4-1BB signaling zone includes the nucleotide sequence as shown in SEQ ID NO:11.

Optionally, the encoding gene of the 4-1BB signaling zone should consider degeneracy base, i.e., such as SEQ ID N O:10 institute The encoding gene of the amino acid sequence shown includes the nucleotide sequence as shown in SEQ ID NO:11, and protection scope should also be protected There is the nucleotide sequence of base degeneracy matter with SEQ ID NO:11, the corresponding amino acid sequence of these nucleotide sequences is still For SEQ ID NO:10.

Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:12.

Optionally, the encoding gene of the CD3 ζ signaling zone includes the nucleotide sequence as shown in SEQ ID NO:13.

Optionally, the encoding gene of the CD3 ζ signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:12 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:13 shown in, protection scope should also protect and SEQ ID NO:13 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:12。

Optionally, the amino acid sequence of the CAR-EGFR includes the amino acid sequence as shown in SEQ ID NO:3.

Optionally, the encoding gene of the CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:4.

Optionally, the encoding gene of the CAR-EGFR should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:3 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:4, and protection scope should also protect and SEQ ID NO:4 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:3。

The Chimeric antigen receptor T cell for the targeting EGFR that second aspect of the present invention provides, including the embedding of targeting EG FR Close antigen receptor CAR-EGFR, this receptor for T cell targeted expression EGFR in specific manner tumour cell, in CAR- After EGFR is in conjunction with EGFR, the intracellular signal area of the T cell is activated, and promotes T cell in the amplification of patient's body, and efficiently And the killing tumor cell of specificity.EGFR wide expression in tumour cell, and expressed in ordinary cells it is very faint, therefore The Chimeric antigen receptor T cell of targeting EGFR provided by the invention can specificity combination tumour cell, to tumour cell produce Raw fragmentation effect, not will cause damage to normal cell.

The third aspect, the present invention provides a kind of recombinant viral vector, the recombinant viral vector includes such as second aspect The encoding gene of the CAR-EGFR of the Chimeric antigen receptor T cell of the targeting EGFR.

Optionally, the encoding gene of the CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:4.

Optionally, the viral vectors in the recombinant viral vector includes slow virus carrier, adenovirus vector or reverse transcription Viral vectors.

Further alternative, the viral vectors is slow virus carrier.

The Chimeric antigen receptor T that the recombinant viral vector that third aspect present invention provides can be used for targeting EGFR is thin The preparation of born of the same parents, may advantageously facilitate T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific.

Fourth aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in the third aspect Group viral vectors.

The host cell is used to assemble the recombinant viral vector as described in the third aspect, makes it have infectivity.

Optionally, the host cell may include HEK293T cell, 293 cells, 293T cell, 293FT cell, SW480 cell, u87MG cell, HOS cell, COS1 cell or COS7 cell etc., but not limited to this.

Further alternative, the host cell is HEK293T cell.

5th aspect, the present invention provides a kind of preparation methods of the Chimeric antigen receptor T cell of targeting EGFR, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-EGFR of targeting EGFR is provided, including sequentially from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of connection, the encoding gene of the single-chain antibody of targeting EGFR, C D8 α hinge area encoding gene, CD8 The encoding gene of the encoding gene of transmembrane region, the encoding gene of 4-1BB signaling zone and C D3 ζ signaling zone, wherein the targeting The single-chain antibody of EGFR is Humanized single chain antibody, and the encoding gene of the single-chain antibody of the targeting EGFR includes such as SEQ ID Shown in NO:2；

(2) encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, obtains pWPX LD-CAR-EGFR weight Group plasmid；

(3) it by the pWPXLD-CAR-EGFR recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains To recombinant slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes；

(5) separate and obtain the Chimeric antigen receptor T cell of targeting EGFR.

It is above-mentioned " from 5 ' end to 3 ' end be sequentially connected with " specifically: the coding gene sequence of the signal peptide 3 ' end with it is described 5 ' ends of the encoding gene of the single-chain antibody of targeting EGFR are connected, 3 ' ends of the encoding gene of the single-chain antibody of the targeting EGFR It is connected with 5 ' ends of the encoding gene of the extracellular hinge area, 3 ' ends of the encoding gene of the extracellular hinge area and the cross-film 5 ' ends of the encoding gene in area are connected, 3 ' ends and the encoding gene in the intracellular signal area of the encoding gene of the transmembrane region 5 ' ends are connected.

The signal peptide is for instructing the Chimeric antigen receptor CAR-EGFR expression to cell surface, institute in the present invention Signal peptide is stated to be cut in protein translation maturation by signal peptidase.

Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:14.

Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:15.

Optionally, the encoding gene of the signal peptide should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:14 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:15, and protection scope should also protect and SEQ ID NO:15 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:14。

The extracellular hinge area, transmembrane region, the specific choice in intracellular signal area and corresponding coding gene sequence such as this hair Described in bright second aspect part, which is not described herein again.

Optionally, the coding gene sequence of the CAR-EGFR is as shown in SEQ ID NO:5.

The encoding gene of the CAR-EGFR is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR, and position After the EF1 α of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CA R-EGFR is inserted into pWPXLD carrier When, BamH1 digestion in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-EGFR Site is connected, and 3 ' ends can be added terminator codon (such as TAA) and be connected with EcoR1 restriction enzyme site in pWPXLD carrier.

Optionally, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T Cell.

The envelope plasmid PMD2G encodes vesicular stomatitis virus glycoprotein capsid, the vesicular stomatitis virus sugar egg White capsid assists recombinant slow virus to adhere to cell membrane, and keeps the infectivity of recombinant slow virus.

Recombinant slow virus of the present invention can further contain the envelope protein from other viruses.For example, as this Kind protein, is preferably the virus enveloped protein for carrying out self-infection human cell.To this protein, there is no particular limitation, can example The amphophilic virus hand epithelium albumen etc. for enumerating retrovirus, can be used for example from mouse leukemia virus (MuMLV) 4070A plants of envelope protein.Alternatively, it is also possible to use the envelope protein from MuMLV 10Al.In addition, as herpetoviridae Albumen, it can be cited for example that, gB, gD, gH, gp85 albumen of herpes simplex virus, gp350, gp220 albumen of Epstein-Barr virus Deng.As the albumen of Hepadna Virus section, the S protein etc. of hepatitis B virus can be included.The envelope protein can also be morbilli It is formed after viral glycoprotein and other single chain antibody fusions.

The packaging of recombinant slow virus is generallyd use transient transfection or is packed using cell line.It may be used as wrapping when transient transfection Human cell's strain that dress cell uses, for example including 293 cells, 293T cell, 293FT cell, 293LTV cell, 293EBNA Cell and other clones separated from 293 cells；SW480 cell, u87MG cell, HOS cell, C8166 cell, MT-4 are thin Born of the same parents, Molt-4 cell, HeLa cell, HT1080 cell, TE671 cell etc..It can also be using the cell strain from monkey, example Such as, COS1 cell, COS7 cell, CV-1 cell, BMT10 cell etc..Moreover, the calcium phosphate and PEI transfection reagent that generally use, It is also well used there are also some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin.

The packaging of recombinant slow virus also uses some slow virus package cell lines, such as most common Env glycoprotein of use, Stable cell lines caused by VSVG albumen or HIV-1gag-pol albumen.

For the sake of security, the slow virus carrier system of large-scale use is all the method using split gene group, i.e., will The assignment of genes gene mapping of different miscellaneous functions is played in different plasmids.There are four pUC pUCs (encoding gag-pol gene, Rev base at present Cause, VSVG gene, SIN metastatic gene be located at four different plasmids), three pUC pUCs (eliminate coding Rev gene Plasmid, gag-pol gene uses the codon of the preferences in people's cell in gag-pol plasmid) and two pUC pUCs are (slowly Auxiliary gene necessary to viral vectors is packed is located on the same plasmid, these auxiliary genes are single gene orders；Separately One is then transgenosis plasmid).Also the slow virus packaging system for having more than four pUC pUCs is using.

Optionally, in step (4), the CD3 positive t lymphocytes are to separate to obtain from source of people peripheral blood mononuclear cells ?.

Optionally, the source of people peripheral blood mononuclear cells derives from autologous vein blood, autologous bone marrow, Cord blood and placenta Blood etc..

Fresh peripheral that is further alternative, being acquired after cancer patient's operation one month, after chemicotherapy one month Blood or marrow.

Specifically, the acquisition process of the CD3 positive t lymphocytes is as follows: into peripheral blood mononuclear cells by certain CD3/CD28 immunomagnetic beads are added in ratio, after being incubated for a period of time, are put into magnet and are screened, it is coated to obtain immunomagnetic beads CD3 positive t lymphocytes after removing magnetic bead, obtain CD3 positive t lymphocytes.

6th aspect, the present invention provides a kind of single-chain antibodies of targeting EGFR as described in relation to the first aspect, such as second party Described in face or the Chimeric antigen receptor T cell of targeting EGFR made from the preparation method as described in terms of the 5th, such as third party Recombinant viral vector described in face or the host cell as described in fourth aspect are preparation prevention, diagnosing and treating malignant tumour Application in drug.

The application specifically: provide a kind of kit, the kit includes targeting EGFR described in first aspect Single-chain antibody, targeting EGFR as described in second aspect Chimeric antigen receptor T cell, the recombination disease as described in the third aspect One of poisonous carrier, host cell as described in fourth aspect are a variety of.

Beneficial effects of the present invention:

(1) EGFR wide expression in tumour cell, and very faint increasing with tumour cell is expressed in ordinary cells Grow, tumor invasion, transfer and the inhibition of Apoptosis it is related, therefore it is provided by the invention targeting E GFR Chimeric antigen receptor T Cell can specificity combination tumour cell, the tumour cell of the targeted expression E GFR of specificity, promote T cell in patient Intracorporal amplification generates fragmentation effect to tumour cell, not will cause damage to normal cell；

(2) single-chain antibody of targeting EGFR is Humanized single chain antibody, and the Chimeric antigen receptor T of targeting EGFR obtained is thin Born of the same parents preferably maintain the vigor and lethality of cell.

Detailed description of the invention

Fig. 1 is the plasmid map of pWPXLd-CAR-EGFR recombinant plasmid provided in an embodiment of the present invention.

Fig. 2 is the positive rate of the Chimeric antigen receptor T cell of targeting EGFR provided in an embodiment of the present invention；(a) is in Fig. 2 Negative control group, (b) is the experimental group of the Chimeric antigen receptor T cell of targeting EGFR provided in an embodiment of the present invention in Fig. 2.

Fig. 3 is the tumor cell in vitro killing of the Chimeric antigen receptor T cell of targeting EGFR provided in an embodiment of the present invention Effect picture.

Fig. 4 is that the Chimeric antigen receptor T cell of targeting EGFR provided in an embodiment of the present invention treats the effect of mice with tumor Figure.

Specific embodiment

The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as Protection scope of the present invention.

Embodiment one

A kind of preparation method of the Chimeric antigen receptor T cell of targeting EGFR, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-EGFR of targeting EGFR is prepared

Prepare respectively signal peptide, the single-chain antibody of targeting EGFR, CD8 α hinge area, CD8 transmembrane region, 4-1BB signaling zone and The encoding gene of CD3 ζ signaling zone, the encoding gene of the signal peptide is as shown in SEQ ID NO:15, the list of the targeting EGFR The encoding gene of chain antibody as shown in SEQ ID NO:2, the encoding gene of the CD8 α hinge area as shown in SEQ ID NO:7, The encoding gene of the CD8 transmembrane region is as shown in SEQ ID NO:9, the encoding gene of the 4-1BB signaling zone such as SEQ ID Shown in NO:11, the encoding gene of the C D3 ζ signaling zone is as shown in SEQ ID NO:13.

By the method for PCR by above-mentioned signal peptide, the single-chain antibody of targeting EGFR, CD8 α hinge area, CD8 transmembrane region, 4- 1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the chimeric of targeting EGFR The encoding gene of antigen receptor CAR-EGFR, the encoding gene of the CAR-EGFR is as shown in SEQ ID NO:5, wherein the target It is Humanized single chain antibody to the single-chain antibody of EGFR.

(2) pWPXLd-CAR-EGFR recombinant plasmid is constructed

The encoding gene of CAR-EGFR is inserted between BamH1 the and EcoR1 restriction enzyme site of pWPXLD carrier, and After pWPXLD carrier EF1 α, using EF1 α as promoter.When the encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, institute 5 ' end additions initiation codon (such as ATG) for stating the encoding gene of CAR-EGFR and BamH1 restriction enzyme site phase in pWPXLD carrier Even, 3 ' ends are also connected added with terminator codon with EcoR1 restriction enzyme site in (such as TAA) pWPXLD carrier.Then it is transferred to large intestine Bacillus competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and survey Sequence identification meets target fragment size and sequence, successfully constructs pWPXLd-CAR-E GFR recombinant plasmid, is as shown in Figure 1 PWPXLd-CAR-EGFR recombinant plasmid.

(3) recombinant slow virus constructs

PWPXLd-CAR-EGFR recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into training The HEK293T cell supported.48h harvest is protected in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration It deposits；Supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration merge with the viral supernatants of 48h harvest and are added together It in ultracentrifugation pipe, is put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature are controlled at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and virus is added Liquid is saved, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence method after being centrifuged 5min Measuring titre, virus is according to 100 μ l, and 2 × 10⁸A/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains recombinant slow virus.

(4) preparation of the Chimeric antigen receptor T cell of targeting EGFR

A) separation of PBMC (peripheral blood mononuclear cells)

PBMC is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..Preferably derive from cancer patient's hand The fresh peripheral blood or marrow acquired after art one month, after chemicotherapy one month.

Extract patient blood, sample presentation to blood separating chamber；Peripheral blood mononuclear cells is acquired, is taken after Ficoll centrifuge separation Intermediate layer cell；After PBS is washed, PBMC is obtained.

B) immunomagnetic beads antigenspecific T lymphocyte

Above-mentioned PBMC is taken, the basal medium for being free of serum is added, is made into cell suspension；Ratio in magnetic bead and cell is 3:1, is added CD3/CD28 immunomagnetic beads, and room temperature incubates 1-2h；The cell for being incubated for magnetic bead is screened using magnet, is put into magnetic Iron is separated；PBS washing obtains CD3 positive t lymphocytes after removing immunomagnetic beads.

C) virus transfection method prepares antigenspecific T lymphocyte

The above-mentioned CD3 positive t lymphocytes obtained by magnetic activated cell seperation are taken, are added and CD3 positive cell number phase The recombinant slow virus for the virus titer answered is cultivated.

The 3rd day of culture carries out cell count and changes liquid, and adjustment cell concentration is 1 × 10⁶A/mL is inoculated with, culture；Training Cell state is observed in feeding the 5th day, if cell density increases, diluting cells concentration is 1 × 10⁶A/mL detects cell Activity continues to cultivate.Amplification cultivation collected cell, and obtained the Chimeric antigen receptor T cell of targeting EGFR by the 9-11 days, and It is stored in and feeds back in dedicated cells frozen storing liquid.

Effect example

In order to assess the Chimeric antigen receptor T cell effect of targeting EGFR prepared by the above method described in the invention, Carry out following effect example.

Effect example one: the positive rate of the Chimeric antigen receptor T cell of targeting EGFR prepared by the assessment present invention

The Chimeric antigen receptor T cell (experimental group) of targeting EGFR will be prepared by the method for the present invention and without the T of preparation Lymphocyte (negative control group), using its positive rate of flow cytomery, as a result as shown in Fig. 2, wherein (a) is in Fig. 2 Negative control group, i.e., without the T cell of preparation, (b) is experimental group in Fig. 2, targeting EGFR as produced by the present invention it is chimeric Antigen receptor T cell.(a) can be obtained compared with (b) in Fig. 2, the Chimeric antigen receptor T of targeting EGFR prepared by the present invention The positive rate of cell is 46.5%.

Effect example two: the tumor cell in vitro killing situation of the Chimeric antigen receptor T cell of targeting EGFR is assessed

It will be by the Chimeric antigen receptor T cell (experimental group) of targeting EGFR made from the method for the present invention and without preparation The Vitro Tumor fragmentation effect of T lymphocyte (negative control group) is compared, specific: in vitro by effector cell's (targeting The Chimeric antigen receptor T cell of EGFR or T lymphocyte without preparation) with target cell (HT1080 cell) quantity ratio be 1: 10,1:3,1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Under co-cultured, after incubation 15-18 hours, collect Cell carries out streaming dyeing, detects cell killing situation, as a result as shown in Figure 3.As can be seen from Figure 3, process is of the present invention Method preparation targeting EGFR Chimeric antigen receptor T cell tumor-killing power 20% or more, even up to 75%, far Much higher than negative control group, therefore the Chimeric antigen receptor T cell of the targeting EGFR through the method for the present invention preparation has strong swell Tumor kills ability.

Effect example three: the mouse interior tumor cell killing feelings of the Chimeric antigen receptor T cell of targeting EGFR are assessed Condition

By the Chimeric antigen receptor T cell (experimental group) of the targeting EGFR by the method for the present invention preparation, without the T of preparation Lymphocyte (negative control group) and physiological saline (blank control group), it is quiet to every mouse tail in mouse tumor model Arteries and veins injection 1 × 10⁶A HT1080 cell (n=9), obtains the survivorship curve of mouse, as a result as shown in Figure 4.As can be seen from Figure 4 The Chimeric antigen receptor T cell of targeting EGFR by this method preparation is also higher than mouse survival rate when cultivating 65 days 50%, considerably beyond negative control group and blank control group.Fig. 4's the result shows that by this method preparation targeting EGFR it is embedding Conjunction antigen receptor T cell is dead caused by capable of preferably protecting mice against because of tumour.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Sequence table

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cagccaccga agcgccttat ctcactggtg tcgaagctgg attcgggagt gcccgatcgc 600

ttctccggct cgggatcggg tactgacttc accctcacta tctcctcgct tcaagcagag 660

gacgtggccg tctactactg ctggcaggga acccactttc cgggaacctt cggcggaggg 720

acgaaagtgg agatcaag 738

<210> 3

<211> 469

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe Asn Ile Glu Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys Tyr Gly Pro Ile Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser Ile Asn Thr Val Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Asp Ser

130 135 140

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

145 150 155 160

Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln

165 170 175

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

180 185 190

Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

210 215 220

Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

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

260 265 270

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

275 280 285

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ala Leu Pro Pro Arg

465

<210> 4

<211> 1407

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gagattcagc tcgtgcaatc gggagcggaa gtcaagaagc caggagagtc cttgcggatc 60

tcatgcaagg gtagcggctt taacatcgag gattactaca tccactgggt gaggcagatg 120

ccggggaagg gactcgaatg gatgggacgg atcgacccag aaaacgacga aactaagtac 180

ggtccgatct tccaaggcca tgtgactatt agcgccgata cttcaatcaa taccgtgtat 240

ctgcaatggt cctcattgaa agcctcagat accgcgatgt actactgtgc tttcagagga 300

ggggtctact ggggacaggg aactaccgtg actgtctcgt ccggcggagg cgggtcagga 360

ggtggcggca gcggaggagg agggtccggc ggaggtgggt ccgacgtcgt gatgacccag 420

agccctgaca gcctggcagt gagcctgggc gaaagagcta ccattaactg caaatcgtcg 480

cagagcctgc tggactcgga cggaaaaacg tacctcaatt ggctgcagca aaagcctggc 540

cagccaccga agcgccttat ctcactggtg tcgaagctgg attcgggagt gcccgatcgc 600

ttctccggct cgggatcggg tactgacttc accctcacta tctcctcgct tcaagcagag 660

gacgtggccg tctactactg ctggcaggga acccactttc cgggaacctt cggcggaggg 720

acgaaagtgg agatcaagac cactacccca gcaccgaggc cacccacccc ggctcctacc 780

atcgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc tggtggggcc 840

gtgcataccc ggggtcttga cttcgcctgc gatatctaca tttgggcccc tctggctggt 900

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 960

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1020

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1080

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1140

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1200

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1260

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1320

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1380

cttcacatgc aggccctgcc gcctcgg 1407

<210> 5

<211> 1467

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gccctccctg tcaccgccct gctgcttccg ctggctcttc tgctccacgc cgctcggccc 60

gagattcagc tcgtgcaatc gggagcggaa gtcaagaagc caggagagtc cttgcggatc 120

tcatgcaagg gtagcggctt taacatcgag gattactaca tccactgggt gaggcagatg 180

ccggggaagg gactcgaatg gatgggacgg atcgacccag aaaacgacga aactaagtac 240

ggtccgatct tccaaggcca tgtgactatt agcgccgata cttcaatcaa taccgtgtat 300

ctgcaatggt cctcattgaa agcctcagat accgcgatgt actactgtgc tttcagagga 360

ggggtctact ggggacaggg aactaccgtg actgtctcgt ccggcggagg cgggtcagga 420

ggtggcggca gcggaggagg agggtccggc ggaggtgggt ccgacgtcgt gatgacccag 480

agccctgaca gcctggcagt gagcctgggc gaaagagcta ccattaactg caaatcgtcg 540

cagagcctgc tggactcgga cggaaaaacg tacctcaatt ggctgcagca aaagcctggc 600

cagccaccga agcgccttat ctcactggtg tcgaagctgg attcgggagt gcccgatcgc 660

ttctccggct cgggatcggg tactgacttc accctcacta tctcctcgct tcaagcagag 720

gacgtggccg tctactactg ctggcaggga acccactttc cgggaacctt cggcggaggg 780

acgaaagtgg agatcaagac cactacccca gcaccgaggc cacccacccc ggctcctacc 840

atcgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc tggtggggcc 900

gtgcataccc ggggtcttga cttcgcctgc gatatctaca tttgggcccc tctggctggt 960

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 1020

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1140

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1200

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1260

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1320

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1380

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1440

cttcacatgc aggccctgcc gcctcgg 1467

<210> 6

<211> 46

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

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 Ile

35 40 45

<210> 7

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgatatc 138

<210> 8

<211> 26

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

1 5 10 15

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

20 25

<210> 9

<211> 78

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

tacatttggg cccctctggc tggtacttgc ggggtcctgc tgctttcact cgtgatcact 60

ctttactgta agcgcggt 78

<210> 10

<211> 39

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

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

20 25 30

Glu Glu Gly Gly Cys Glu Leu

35

<210> 11

<211> 117

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 60

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactg 117

<210> 12

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

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

<210> 13

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300

tatgacgctc ttcacatgca ggccctgccg cctcgg 336

<210> 14

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His

1 5 10 15

Ala Ala Arg Pro

20

<210> 15

<211> 60

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

gccctccctg tcaccgccct gctgcttccg ctggctcttc tgctccacgc cgctcggccc 60

Claims (10)

1. a kind of single-chain antibody of targeting EGFR, which is characterized in that the single-chain antibody of the targeting EGFR includes such as SEQ ID Amino acid sequence shown in NO:1, the single-chain antibody of the targeting EGFR are Humanized single chain antibody.

2. the single-chain antibody of targeting EGFR as described in claim 1, which is characterized in that the single-chain antibody of the targeting EGFR is compiled Code gene includes the nucleotide sequence as shown in SEQ ID NO:2.

3. a kind of Chimeric antigen receptor T cell of targeting EGFR, which is characterized in that the Chimeric antigen receptor including targeting EGFR CAR-EGFR, the CAR-EGFR include the single-chain antibody that targeting EGFR is sequentially connected with from aminoterminal to c-terminus, extracellular hinge Area, transmembrane region and intracellular signal area amino acid sequence, wherein the single-chain antibody of the targeting EGFR includes such as SEQ ID NO:1 Shown in amino acid sequence.

4. the Chimeric antigen receptor T cell of targeting EGFR as claimed in claim 3, which is characterized in that the CAR-EGFR's Amino acid sequence includes the amino acid sequence as shown in SEQ ID NO:3.

5. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes such as any one of claim 3 or 4 institute The encoding gene of the CAR-EGFR of the Chimeric antigen receptor T cell for the targeting EGFR stated.

6. recombinant viral vector as claimed in claim 5, which is characterized in that the encoding gene of the CAR-EGFR includes such as Nucleotide sequence shown in SEQ ID NO:4.

7. a kind of host cell, which is characterized in that the host cell includes such as the described in any item recombinations of claim 5 or 6 Viral vectors.

8. a kind of preparation method of the Chimeric antigen receptor T cell of targeting EGFR characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-EGFR of targeting EGFR is provided, including is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of signal peptide, the encoding gene of single-chain antibody of targeting EGFR, the encoding gene of CD8 α hinge area, CD8 cross-film The encoding gene of the encoding gene in area, the encoding gene of 4-1BB signaling zone and CD3 ζ signaling zone, wherein the targeting EGFR Single-chain antibody is Humanized single chain antibody, and the encoding gene of the single-chain antibody of the targeting EGFR includes such as SEQ ID NO:2 institute Show；

(2) encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, obtains pWPXLD-CAR-EGFR recombination matter Grain；

(3) by the pWPXLD-CAR-EGFR recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, weight is obtained Group slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes；

(5) separate and obtain the Chimeric antigen receptor T cell of targeting EGFR.

9. the preparation method of the Chimeric antigen receptor T cell of targeting EGFR as claimed in claim 8, which is characterized in that described Envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T cell.

10. it is a kind of such as the single-chain antibody of the described in any item targeting EGFRs of claim 1-2, such as any one of claim 3-4 institute The Chimeric antigen receptor T cell of targeting EGFR made from preparation method state or as described in claim 8-9 or as right is wanted Ask the described in any item recombinant viral vectors of 5-6 or host cell as claimed in claim 7 in preparation prevention, diagnosing and treating Application in the drug of malignant tumour.