CN103113470B

CN103113470B - Genetically engineered lymphocyte targeting Human EGFR (Epidermal Growth Factor Receptor), preparation method and application of genetically engineered lymphocyte

Info

Publication number: CN103113470B
Application number: CN201310061761.3A
Authority: CN
Inventors: 魏于全; 李炯; 周西坤
Original assignee: Sichuan University
Current assignee: Chengdu weisk biomedical Co.,Ltd.
Priority date: 2013-02-27
Filing date: 2013-02-27
Publication date: 2015-04-22
Anticipated expiration: 2033-02-27
Also published as: CN103113470A

Abstract

The invention relates to the field of genetic engineering, and in particular relates to an anti-EGFR ((Epidermal Growth Factor Receptor) chimeric antigen receptor, and a lymphocyte for expressing the antigen receptor. The technical solution of the invention provides a new effective selection for the anti-tumor technical field. The technical scheme is that a single-chain antibody targeting the EGFR is provided. The single-chain antibody is further designed to be constructed into the chimeric antigen receptor by means of a genetic engineering technology, and the structure of the gained chimeric antigen receptor is anti-EGFR (scFv)-IgG2 (Fc)-CD28-CD3 Zeta. According to the genetically engineered lymphocyte targeting the human EGFR, the chimeric antigen receptor is transfected into the lymphocyte by the nucleofaction technology, and thus the nonspecific lymphocyte can be endowed with the capacity of specifically distinguishing the EGFR tumor antigen and targeting and killing the EGFR over-expression tumor cells. According to the technical scheme, the adoptive cell therapy and the genetic therapy are organically combined, so that remarkable effect of killing the tumor is gained, and a novel effective selection is provided for the field.

Description

Genetically engineered lymphocyte of targeted human EGFR and its production and use

Technical field

The present invention relates to genetically engineered field, be specifically related to the Chimeric antigen receptor of anti-EGFR and the lymphocyte of this antigen receptor can be expressed.

Background technology

EGF-R ELISA (EGFR) is a kind of film surface receptor with tyrosine kinase activity, plays important regulating effect [1,2] in the many physiological processs of cell.EGFR is structurally made up of extracellular region, cross-film district and intracellular region 3 parts, and wherein extracellular region has two halfcystines and enriches district, and intracellular region has typical ATP-binding site and Tyrosylprotein kinase district.EGFR at many malignant cells surface all high expression levels [3], comprising epidermis squama cancer, mammary cancer, ovarian cancer, nonsmall-cell lung cancer, kidney and head-neck malignant tumor etc.Under normal circumstances, EGFR Tyrosylprotein kinase exists with monomeric form, when after specific part and EGFR receptors bind, acceptor is caused to change the dimerization status of activation into by the free state of inactivation, and then activate Tyrosylprotein kinase inherent in born of the same parents, the tyrosine residues of its born of the same parents' inner segment is by autophosphorylation or cross phosphorylation, cause downstream PI3K-AKT, the activation of the signal paths such as MAPK-ERK, its signal transduction pathway mediated is in the propagation of tumour cell, injury repairing, the aspects such as invasion and attack and new vessel formation play an important role, existing numerous results of study confirms that EGFR is the target spot [4-6] of the treating malignant tumor with applications well prospect.

In recent years, the antitumor drug research and development of targeting EGFR family have become a focus.Mainly contain two class medicines to the suppression of EGFR at present: a class is small molecule tyrosine kinase inhibitors, with Gefitinib and Tarceva for representative, these small-molecule drugs achieve good effect clinically.Although these small molecules anti-tumor drugs targetings do not have the cytotoxicity of chemotherapeutics, there is also outstanding resistance problems and the untoward reactions such as effect of missing the target, prolonged application even can cause obvious impact to body.Another kind of is monoclonal antibody, antibody class medicine for EGFR also has three kinds in Discussion on Chinese Listed: Cetuximab, Victibix and Buddhist nun's trastuzumab, these medicines achieve good curative effect in the treatment of the solid tumor such as colorectal carcinoma, nasopharyngeal carcinoma, but because the molecular weight ratio of monoclonal antibody is larger, cause it poor to the penetrance of solid tumor, easily be eliminated in blood, cause its curative effect to be subject to a definite limitation; In addition, the price of monoclonal antibody, concerning very expensive common patient, further limit it and applies in a large number, widely clinically.How while maintenance medicine specific target tropism, can effectively reduce its toxic side effect? investigators are to this has been many-sided exploration.

Adoptive immunotherapy is by tumor mice infusion antineoplastic immune effector cell, makes acceptor obtain or strengthen the methods for the treatment of that antitumor responsing reaction carrys out killing tumor cells.The too drastic immunotherapy of tumour clinically at present mainly adopts two quasi-lymphocytes: a class effector cell is T lymphocyte specific for tumour antigen, comprise tumor infiltrating lymphocyte and cytotoxic T lymphocyte, choose these two kinds of lymphocytic advantages of T to be, they are activated by tumour antigen all, self MHC(MHC can be identified) quasi-molecule antigenic compound, there is the fragment action of high degree of specificity, show obvious curative effect clinically; The cell cultivating acquisition is in addition modified without any transgenosis, and security is also higher.Its shortcoming is that Vitro Culture Techniques system is comparatively complicated, and cell cultures amplification rate is comparatively slow, and cell induction culture cycle is longer, culture success ratio lower [7,8].An other class comprises the killer cell LAK of lymphocyte factor activation and the killer cell CIK of cytokine stimulation, stimulate through lymphokine or cytokine induction after this kind of cell is separated from peripheral blood and obtain, its kill mechanism is that the mode relied on by non-MHC plays antitumor action.LAK kills and wounds to target cell the restriction not having MHC, but its ability increased in vitro is lower and it is not high to kill tumor activity in body yet, and this therapy will use heavy dose of IL-2 in clinical application in addition, usually causes occurring larger side effect [9,10].Current CIK cell treatment technology condition comparative maturity, domestic You Duojia hospital with the clinical treatment carrying out CIK, and achieves good curative effect [11,12].But the lymphocyte that some containing in CIK cell have tumour-specific can not by special a large amount of amplifications, so the outstanding problem that CIK faces is exactly it does not have tomour specific lethal effect.

Along with the maturation of fast development especially genetic engineering technique of biotechnology in modern age, adoptive cellular immunotherapy has welcome new development opportunity.EshharZ. research group utilize genetic engineering technique in last century late nineteen eighties first proposed the method overcoming above-mentioned defect: mosaic t lymphocyte receptor (CAR) technology, the high efficiency of the specificity of monoclonal antibody and cytotoxic T cell is organically melted in order to one by this; With natural TCR(T cell receptor) compared with, the great advantage of CAR be with antigen in conjunction with time can not by the restriction of MHC.CAR gene transfection is entered in T lymphocyte, nonspecific lymphocyte specific recognition tumour antigen can be given and the ability of target killing tumor cell, and can allow this cell when being subject to corresponding antigens and stimulating the release cells factor, show cell killing activity [13,14].

CAR is generally made up of the part such as the outer land (deriving from the scFv of monoclonal antibody) of born of the same parents, constant region for immunoglobulin (IgG Fc), trans-membrane region (TM) and Intracellular signals territory (SD).ScFv is responsible for the specific recognition of tumour specific antigen; IgG Fc makes ScFv keep a natural space conformation, more effective identification antigen, and the dimerization effect mediating CAR; The effect of TM is anchored on cytolemma by CAR; The function in intracellular signal territory is after CAR is in conjunction with tumour antigen, activates intracellular signaling pathways, activated lymphocyte, thus produces the cytotoxicity of the tumour cell identified for CAR.Recently, the lymphocyte through CAR genetic modification obtains good curative effect on the clinical treatment of the kinds of tumors such as lymphoma [15,16], neuroblastoma [17] and metastatic renal cell carcinoma [18].

Also do not use the report of the lymphocyte treatment tumour technical scheme of EGFR target Chimeric antigen receptor technological transformation at present.

Summary of the invention

The object of the invention is for antitumor technical field provides a kind of effective selection newly.

The present invention provide firstly targeting EGFR single-chain antibody, and this single-chain antibody is obtained by the connect light chain of antibody of targeting EGFR and variable region of heavy chain.

Further, described single-chain antibody, aminoacid sequence as shown in SEQ ID No.2.

Present invention also offers the gene of the Chimeric antigen receptor described in coding, its nucleotide sequence is as shown in SEQ ID No.1.

Present invention also offers a kind of Chimeric antigen receptor, this Chimeric antigen receptor splices hIL-2 signal peptide, the single-chain antibody of described targeting EGFR, IgG2-Fc, CD28 cross-film district and CD3 ζ chain by nitrogen end in turn to carbon teminal, and the structure of the Chimeric antigen receptor obtained is anti-EGFR (scFv)-IgG2 (Fc)-CD28-CD3 ζ.

Further, the aminoacid sequence of described Chimeric antigen receptor is as shown in SEQ ID No.4.

Present invention also offers the gene of the Chimeric antigen receptor described in coding, shown in its nucleotide sequence SEQ ID No.3.

Present invention also offers expression vector, contain the encoding gene that also energy express amino acid sequence is the Chimeric antigen receptor shown in SEQ ID No.3.

Further, described expression vector is pmaxCloning plasmid vector.

Present invention also offers the host cell containing described expression vector.

Further, described host cell is lymphocyte.

Present invention also offers described host cell and prepare the purposes in antitumor drug.

Present invention also offers the method for the host cell described in preparation: lymphocyte is entered in described expression vector transfection, thus obtain the genetically engineered lymphocyte expressing Chimeric antigen receptor.

Present invention also offers antitumor drug, be by host cell be prepared from as main active ingredient.

Useful achievement of the present invention: the present invention is creatively utilized the scFv fragment with high-affinity and specific anti-EGFR obtained by ribosomal display technology newly to design and obtains a kind of mosaic antigen receptor.This Chimeric antigen receptor is proceeded to non-virus carrier, achieving the high expression of CAR on human lymphocyte surface by consideration convey dyeing technique, is can identify nonspecific lymphocyte directional transformation Human epidermal growth factor receptor thus mediate the specific lymphocyte tumour cell of process LAN EGFR being carried out to target killing.Wherein the single-chain antibody of particularly preferred anti-EGFR can make mosaic antigen receptor play a role better.The lymphocyte that EGFR target Chimeric antigen receptor is modified, after the tumour cell of specific recognition process LAN EGFR, can play antitumor action by the release of cytokine and CTL effect; Its in vivo can the tumour of remarkable Tumor suppression formed, tumor growth and lung's transfer, and there is no obvious toxic side effect.The present invention devises a kind of genetically engineered lymphocyte newly, provides a kind of safe and effective oncotherapy treatment plan newly.

Accompanying drawing explanation

Fig. 1 is that Chimeric antigen receptor genetic expression detects.Primary antibodie is the rabbit anti-serum of anti-EGFR scFv.Two resist the goat anti-rabbit igg for FITC mark.Mock: do not add plasmid, but the lymphocyte of consideration convey dye; Vehicle: the lymphocyte of consideration convey dye empty plasmid; C, CAR: the lymphocyte of consideration convey dye Chimeric antigen receptor gene plasmid.

Fig. 2 is that after Chimeric antigen receptor gene transfection, lymphocytic tumours killing activity detects.A431 cell is EGFR overexpressing cell, and A2780 cell is EGFR negative cells.

Fig. 3 is that CAR genetic modification lymphocyte is on the impact of the subcutaneous Tumor formation of A549.NS: do not add lymphocyte, an inoculated tumour; Mock: do not add plasmid, but the lymphocyte of consideration convey dye; Vehicle: the lymphocyte of consideration convey dye empty plasmid; C, CAR: the lymphocyte of consideration convey dye Chimeric antigen receptor gene plasmid.Each quasi-lymphocyte described in figure and A549 cell co-inoculation NOD/SCID mouse.Ordinate zou is into knurl mouse percentage, X-coordinate be the postvaccinal time (my god).

Fig. 4 is the impact that CAR genetic modification lymphocyte grows A431 and A2780 subcutaneous tumors.NS: physiological saline treatment group; Mock: do not add plasmid but the lymphocyte treatment group of consideration convey dye; Vehicle: the lymphocyte treatment group of consideration convey dye empty plasmid; C, CAR: the lymphocyte treatment group of consideration convey dye Chimeric antigen receptor gene plasmid.Ordinate zou for becoming knurl mouse tumor volume, X-coordinate be the postvaccinal time (my god).

Fig. 5 is that CAR genetic modification lymphocyte is on the impact of A549 neoplasm lung metastasis.NS: physiological saline treatment group; Mock: do not add plasmid but the lymphocyte treatment group of consideration convey dye; Vehicle: the lymphocyte treatment group of consideration convey dye empty plasmid; C, CAR: the lymphocyte treatment group of consideration convey dye Chimeric antigen receptor gene plasmid.Ordinate zou for becoming knurl mouse survival rate, X-coordinate be the postvaccinal time (my god).

Fig. 6 is that after the treatment of A549 neoplasm lung metastasis mouse, gpt (Alanine Transaminase, ALT) and glutamic-oxal(o)acetic transaminase (Aspartate Transaminase, AST) expression level detect.Mice group is identical with Fig. 5.

Embodiment

The invention provides a kind of Chimeric antigen receptor (CAR) recombination and design thereof, this Chimeric antigen receptor can targeting EGFR.Its specific design mode is splice hIL-2 signal peptide, the single-chain antibody (scFv) of anti-EGFR provided by the present invention, IgG2-Fc, CD28TM-CD3 ζ by nitrogen end in turn to carbon teminal, the structure of the Chimeric antigen receptor obtained is anti-EGFR (scFv)-IgG2 (Fc)-CD28TM-CD3 ζ, and aminoacid sequence is shown in SEQ ID No.4.

The design of CAR provided by the present invention is through repeatedly gropes rear optimization gained, and each fragment of composition Chimeric antigen receptor all plays different functions: hIL-2 signal peptide can be secreted into outside born of the same parents after other assemblies of CAR are expressed; ScFv has the effect of special target in conjunction with EGFR; IgG2-Fc provides suitable space for the stretching, extension of the correct conformation of scFv; CAR is anchored on cytolemma by CD28 cross-film district; CD3 ζ chain can activate intracellular signal, activated lymphocyte after scFv conjugated antigen.

In the present invention CAR consideration convey contaminate lymphocytic condition to be also through repeatedly grope after obtain, thus efficiently the gene transfection of anti-EGFR Chimeric antigen receptor is entered lymphocyte, and make its correction on lymphocytic cytolemma, successfully nonspecific lymphocyte directional transformation is become and can identify Human epidermal growth factor receptor thus mediate the specific lymphocyte cell of high expression level EGFR being carried out to target killing.Genetically engineered flower lymphocyte of the present invention all demonstrates significant antitumor action in testing in vitro and in vivo, and it can use as antitumor drug, has good application prospect.

The acquisition of embodiment one EGFR target CAR full-length gene and the structure of expression plasmid carrier

1 experiment route

CAR gene is made up of scFv, IgG2-Fc, CD28TM-CD3 ζ of SP hIL-2, identification EGFR.Wherein SP hIL-2 sequence is included in upstream amplification primer by the mode that primer synthesizes; EGFR target scFv sequence is gained after anti-EGFR hybrid tumor cell amplification and ribosomal display technical optimization, concrete steps are as follows: prepare anti-EGFR hybridoma, extract total serum IgE, then amplify weight, the light chain gene of anti-EGFR by the mode of RT-PCR respectively, deliver to Invitrogen company and check order.Sequencing result utilizes Jellyfish software analysis, by VBASE2 and GeneBank, alignment is carried out again to the sequence that can correctly translate, and by ribosomal display technology, its specificity and avidity are evolved, the encoding gene (aminoacid sequence is shown in SEQ ID No.2) with the anti-EGFR single-chain antibody of high-affinity obtained.

By designing and synthesizing primer, increase gene order, IgG2Fc section (aminoacid sequence is shown in SEQ IDNo.6) and CD28TM-CD3 ζ gene order (obtained from human peripheral blood mononuclear cell by RT-PCR, aminoacid sequence is shown in SEQ ID No.8) respectively that obtain scFv.Three fragments are after PCR reaction, and three fragments connect by recycling over-lap PCR program.Experiment concrete scheme is as follows:

1) design of primers

(1) primer of amplification scFv:

Upstream primer (F1): SEQ ID No.9

5’-GTGCTAGCTAGC ATGTACAGGATGCA ACTCCTGTCTTGCATTGCACTA AGTCTT GCACT。This is the upstream primer of amplification hIL-2 signal peptide, underscore partial design and the complementation of hIL-2 signal peptide Partial Fragment.This primer also introduces restriction enzyme site NheI.

The upstream primer (F2) of amplification Anti-EGFR scFv: SEQ ID No.10

5 '-GCACTAAGTCTTGCACTTGTCACGAACTCGGCCC gAGGTTCAGCTTCAGCAGTCT g-3 ' wherein, underscore partial design and Anti-EGFR scFv complementary.

The downstream primer (R1) of amplification Anti-EGFR scFv: SEQ ID No.11

5’-ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTC TTGCACTTGTCACG AAcTCGGCCCAGATCCAGTTGCTGCAGTCT-3’。Wherein, underscore partial design is and IgG2Fc complementary, to carry out over-lap PCR.

(2) primer of amplification IgG2Fc:

Upstream primer (F3): SEQ ID No.12

5’- GGGGACCAAGCTGGAAATAAAACCACCTTGCCCAGCAC-3’。Wherein, underscore partial design is and scFv complementary, to carry out over-lap PCR.

Downstream primer (R2): SEQ ID No.13

5’- ACCACCAGCACCCAAAAGGGAATGGCCCTGATTGTGCTGGGGGG-3’。Wherein, underscore partial design and CD28TM-CD3 ζ complementary.

(3) primer of the CD28TM-CD3 ζ sequence that increases:

Upstream primer (F4): SEQ ID No.14

5’- AGAAGAGCCTCTCCCTGTCTCCGGGTAACCCTTTTGGGTGCTGGTGGTG-3’。Underscore part and IgG2Fc fragment complementation.

Downstream primer (R3): SEQ ID No.15

5’-CTAGCTAGCTAGGAAGATCTTCTTAGCGAGGGGGC-3’。Introduce restriction enzyme site Bgl II.

2) PCR reaction

PCR carries out according to TaKaRa company operation instructions, and reaction system is as follows:

Reaction conditions:

94 ° of C, 4min; 94 ° of C, 30sec, 55 ° of C, 30sec, 72 ° of C, 30 ~ 120sec, 30cycles; 72 ° of C, 10min; 10 ° of C insulations.Wherein, the extension time depending on the length of product with the Drawing rate of 1kb/min.

3) over-lap PCR

Three fragments are after PCR reaction, and each overlap of the PCR primer obtained can be complementary, therefore following program can be utilized three fragments to be connected:

(1) overlapping ScFv-IgG2Fc:

Prepare following system:

Reaction conditions:

94 ° of C, 4min; 94 ° of C, 30sec, 55 ° of C, 30sec, 72 ° of C, 90sec, 30cycles; 72 ° of C, 10min; 10 ° of C insulations.

(2) overlapping ScFv-IgG2Fc-CD28TM-CD3 ζ:

In the ScFv-IgG2Fc system that reaction terminates, add CD28TM-CD3 ζ, react as follows: 94 ° of C, 4min; 94 ° of C, 30sec, 55 ° of C, 30sec, 72 ° of C, 90sec, 30cycles; 72 ° of C, 10min; 10 ° of C insulations.

(3) increase CAR fragment:

After completing second time overlap, in original system, add primers F 1 and each 1 μ L of R3, add Pyrobest ^tMdNAPolymerase0.25 μ L, reacts as follows, amplification CAR fragment: 94 ° of C, 4min; 94 ° of C, 30sec, 55 ° of C, 30sec, 72 ° of C, 120sec, 30cycles; 72 ° of C, 10min; 10 ° of C insulations.

3) structure of pmax-CAR plasmid vector is completed

(1) enzyme cuts the PCR primer of pmaxCloning and CAR fragment.

It is as follows that enzyme cuts system:

Double digestion pmaxCloning:

Double digestion CAR:

(2) pmax and CAR gene is connected:

After enzyme cuts end, utilize T4 ligase enzyme to carry out the connection of fragment and carrier, system is as follows:

After having connected, transform DH5 α competent cell, the solid culture being applied to kalamycin resistance is dull and stereotyped, carries out colony screening.After growing clone, in the liquid nutrient medium enlarged culturing of kalamycin resistance, and extracting plasmid.After extracting plasmid, utilize NheI/BglII enzyme to cut qualification, the correct invitrogen company that send checks order.

2 experimental results

Through PCR reaction, complete the amplification of each fragment of CAR gene first respectively.Then carry out over-lap PCR reaction, obtain and expect the object fragment of molecular size range, the total length of CAR gene fragment is 2004bp.Agarose gel electrophoresis shows this fragments molecules amount at DNA Marker2kb place.

After glue reclaims this fragment, after utilizing double digestion (NheI and BglII), be connected with pmaxCloning plasmid vector, be configured to pmax-CAR plasmid vector.After successful connection, design corresponding primer, CAR gene order in pmax-CAR plasmid sequence is checked order.Compared with theoretical CAR gene order by sequencing result and find, the sequence of the two is identical.

The lymphocytic transfection and expression of embodiment two detects

1 experiment route

1) lymphocytic cultivation

(1) lymphocyte is separated by density gradient centrifugation and obtains from 5 Volunteer Blood Donor peripheral bloods: in X-VIVO15 substratum (Lonza company, article No.: 04-744Q) in add human plasma and rhIL-2(Shandong Quan Gang medicine company after deactivation, spring is strange, 2000000 IU/ prop up) to final concentration be 3% and 300IU/mL, in this, as lymphocytic conventional medium.

(2) lymphocytic activation amplification: add in lymphocytes culture medium mouse-anti people CD3 and CD28 antibody (BDBioscience, CD3 antibody article No.: 555337, CD28 antibody article No.: 55725) respectively to final concentration be 1 μ g/mL.Dilute separating obtained lymphocyte with this substratum, make its cell density be 3 × 10 ⁶.37 DEG C, 5%CO ₂cultivate.After 72h, replace medium to lymphocytic conventional medium.

2) lymphocytic transfection

(1) collect the lymphocyte cultivated, Trypan Blue carries out viable count, makes the quantity of transfectional cell be 5 ~ 10 × 10 ⁶.

(2) add lymphocyte electrotransfection liquid 100 μ L and pmax-CAR plasmid 2 ~ 5 μ g to be transfected (being no more than 10 μ L), fully mix, cell suspension is added in electric revolving cup.Precaution: after adding electrotransfection liquid, operation should as quickly as possible, can not make the time of lymphocyte in transfection liquid more than 20min.

(3) inserted in Nucleofector II consideration convey instrument (Lonza company, article No.: AAB-1001) by electric revolving cup, select procedure T20/T23, carries out electrotransfection.Wherein, the efficiency of T20 program transfection is a little less than T23, and the mortality ratio of cell after electricity turns is lower.T23 then efficiency is higher, but larger to the loss of cell.

(4) after transfection terminates, take out electric revolving cup, add the lymphocytes culture medium of 500 μ L preheatings, draw in cell to 6 orifice plate and cultivate.After 6h, change fresh culture.

(5) trypan blue stain living cells counting after transfection 16h, detects the experiment that expression or carry out is correlated with.

3) expression of flow cytometer detection CAR on Lymphocyte Membrane

After lymphocyte nuclear transfection 16h, detect the expression amount of Transfected cells CAR on cytolemma with homemade anti-EGFR (ScFv) rabbit polyvalent antibody.

(1) lymphocyte of cell and normal cultivation after step experiment consideration convey on collected by centrifugation, abandons supernatant.PBS phosphoric acid buffer cleans 2 times, cell counting, and often in pipe, cell is about 2 × 10 ⁵individual;

(2) add the anti-EGFR ScFv rabbit polyvalent antibody (100 times of dilutions) after 200 μ L dilutions, hatch 60min for 4 DEG C;

(3) the centrifugal 3min of 1500rpm, abandons supernatant, adds 200 μ L PBS and cleans.Repeat twice.

(4) add the goat anti-rabbit igg antibody of FITC mark, flick mixing, hatch 30min for 4 DEG C;

(5) the centrifugal 3min of 1500rpm, abandons supernatant, adds 200 μ L PBS; Repeat twice; Machine testing on sample.

2 experimental results

In Lonza company on the basis of System specification sheets, pmax-GFP plasmid is utilized to be optimized lymphocyte consideration convey condition further from 3 aspects such as consideration convey program (T-20 and T-23), consideration convey cell and plasmid quantity (2 ~ 5 μ g).Experimental result finds, in T-23 consideration convey program, each consideration convey cup, add pmax-GFP plasmid 3 μ g and lymphocyte 8 × 10 ⁶transfection conditions under, the transfection efficiency that can reach and the optimum balance of cell survival.

Subsequently, the condition after this optimization is utilized to carry out the lymphocyte transfection of object carrier.After lymphocyte electrotransfection, with Flow cytometry its whether can correction on cytolemma.Fig. 1 result shows, and after pmax-CAR plasmid consideration convey dye lymphocyte 16h, has the CAR of 32.05% can express on film.

Embodiment three CAR genetic modification lymphocytes in vitro killing activity detects

This experiment adopts citotoxicity detection kit (Promega company CytoTox non-RadioactiveCytotoxicityAssay Kit, article No. G1780) carry out the detection of CAR genetic modification lymphocytes in vitro killing activity.

1 experiment route

1) lymphocyte after transfection 16h and people's epidermis squamous cell carcinoma system A431 and abortion syndrome A2780 is collected, cell counting;

2) in 96 orifice plates, 4000 tumour cells are added, as target cell; In Target cell wells in Xiao answer Xi Bao ︰ target cell be 10,20 and 40 ratio add effector cell.Each group is done 3 secondary orifices; Each group of target cell, effector cell all reserve the release that spontaneous serum lactic dehydrogenase (Lactate Dehydrogenase, LDH) is detected in 3 holes; 250G is centrifugal, 4min; 37 DEG C, 5%CO ₂incubator hatches 6h;

3) Lysis Solution is added in the Target cell wells of maximum release group, cracking 3min; Add chromogenic substrate SubstrateMix, lucifuge hatches 30min;

4) add termination reagent Stop Solution, mixing, places 5min; 490nm wavelength reads absorbance; The calculation formula of target cell killing-efficiency is: (experiment reading numerical values-effector cell's Spontaneous release values-target cell Spontaneous release values)/(maximum releasing value-effector cell's Spontaneous release values) × 100%.

2 experimental results

For the killing tumor cell verifying whether the lymphocyte of CAR modification can be special, citotoxicity detection kit (Cytotox96Non-Radioactive Cytotoxicity Assay, Promega) is utilized to measure the lymphocytic killing activity of transfection pmax-CAR.Experimental result shows, the lymphocyte of idle running and the unloaded lymphocyte of transfection, to target cell EGFR not expression cell line A2780 and EGFR overexpressing cell strain A431 all there is no obvious killing activity, and the lymphocyte of expressing CAR can produce special cytotoxicity to A431, and increase along with the increase of percentage of lymphocyte (Fig. 2).More than experiment shows, what the lymphocyte that CAR modifies can be stronger kills and wounds EGFR process LAN tumour cell.

Embodiment four CAR genetic modification lymphocyte activity in vivo detects

1 experiment route

1) Tumor formation experiment

Collect the A431 tumour cell of logarithmic phase, the centrifugal 3min of 1500rpm, the substratum of cell precipitation serum-free, antibiotic-free washs 1 time, and adjusting cell concn after counting cells quantity is 2 × 10 ⁷/ mL.Collect Mock, Vehicle and CAR lymphocyte after transfection 16h, cell counting, concentration is adjusted to 2 × 10 ⁸/ mL.According to the ratio mixing that Lin Ba Xi Bao ︰ tumour cell is 10 ︰ 1, namely collect 1 × 10 ⁶individual tumour cell, adds 1 × 10 ⁷individual lymphocyte, the volume of final cell suspension is 100 μ L.Cell is finally resuspended in X-VIVO serum free medium, delivers to animal housing under aseptic condition, seeds cells into right side of mice dorsal sc respectively.By tumor cell inoculation after subcutaneous, observe animal Tumor formation every day.Every 3 days length by vernier caliper measurement tumour and wide, calculate gross tumor volume.

2) subcutaneous tumors experiment

Collect A431 and the A2780 tumour cell of logarithmic phase, after counting cells quantity, adjust cell concn, every right side of mice dorsal sc inoculation 5 × 10 ⁶individual cell.Treat that gross tumor volume reaches 200 ~ 300mm ³time, collect Mock, Vehicle and CAR lymphocyte after transfection 16h, cell counting, concentration is adjusted to 2 × 10 ⁸/ mL, Tail Vein injection Mouse 100 μ L/ only, injected once every one day, three times totally.Every 3 days length by vernier caliper measurement tumour and wide, calculate gross tumor volume.

3) Lung metastases Inhibition test

Collect the A549 cell of logarithmic phase, the centrifugal 3min of 1500rpm, the substratum of cell precipitation serum-free, antibiotic-free washs 1 time, and adjusting cell concn after counting cells quantity is 2 × 10 ⁷/ mL, delivers to animal housing under aseptic condition.Respectively A549 cell suspension tail vein injection is entered Nod/SCID mouse (buying from Beijing HFK Bio-Technology Co., Ltd.), every only inoculation 100 μ L.Within 3 days, be divided into 4 groups: PBS control group at random after inoculation; Mock lymphocyte group; Vehicle lymphocyte group; CAR lymphocyte treatment group; Injection tumour cell after the 3rd, 6,9,12,15 and 18 day, tail vein injection 2 × 10 ⁶the individual lymphocyte through genetic modification, the PBS of control group injection same volume.The lifetime of observation experiment animal, within every 3 days, measure a Mouse Weight; After injecting 24h the last time, put to death mouse and separation of serum.Serum sample is sent National Chengdu Center For Safety Evaluation of Drugs's biochemical analysis room, full automatic biochemical apparatus is utilized to measure serum glutamic pyruvic transminase (Alanineaminotransferase, and the level of glutamic-oxal(o)acetic transaminase (Aspartate aminotransferase, AST) ALT).

2 experimental results

By lymphocyte and the A549 tumour cell of expressing CAR in the ratio of 10 ︰ 1 for carrying out subcutaneous vaccination Nod/SCID mouse (buying from Beijing HFK Bio-Technology Co., Ltd.), the Tumor formation of observation tumour.After inoculation in 60 days, NS group, Mock cell and Vehicle cell are total to separately inoculation experiments mouse and all become knurl.And the one-tenth knurl time adding the lymphocyte group experimental mouse of transfection CAR gene is obviously delayed, within the 60th day, still have the non-bearing tumor of the mouse of 60% after inoculation.These data show, the lymphocyte of CAR genetic modification can significantly suppress the tumour of A549 tumour cell to form (Fig. 3).Subcutaneous vaccination A431 tumour, the lymphocyte of three tail vein injection transfection CAR genes, result demonstrates significant function of tumor inhibition; And CAR genetic modification lymphocyte to A2780 tumor model without any result for the treatment of, this result shows that CAR genetic modification lymphocyte has extraordinary EGFR targeting specific and anti-tumor activity (Fig. 4).

Further, the present invention establishes A549 Lung metastases model.According to previous bibliographical information, take the mode of multiple dosing, to expect the result for the treatment of obtained.After Modling model the 3rd, 6,9,12,15 and 18 days input lymphocytes, assess its impact on metastases.Experimental result shows, input CAR lymphocytic NOD/SCID C.B-17 mouse, its lifetime obviously extends.And it is all dead in mouse in other experimental group after inoculation 27 to 65 days.This shows that the lymphocyte of transfection CAR gene can effectively shift by Tumor suppression, extends the existence life-span (Fig. 5) of experiment mice.In order to detect the lymphocytic security of genetic modification, have rated its system toxicity to NOD-SCID mouse.Serum ALT, AST level to reflect that whether and the conventional Testing index of degree of injury hepatocellular injury sensitively.Result shows, ALT with the AST level of genetic modification lymphocyte treatment group mouse does not have difference (Fig. 6) with comparing of control group.

Reference

[1]Baselga J,Arteaga CL(2005).Critical update and emerging trends in epidermal growth factor receptortargeting in cancer Journal of Clinical Oncology23,2445.

[2]Reid A,Vidal L,Shaw H,de Bono J(2007).Dual inhibition of ErbB1(EGFR/HER1)and ErbB2(HER2/neu)European Journal ofCancer43,481-489.

[3]Yarden Y(2001).The EGFR family and its ligands in human cancer.signalling mechanisms and therapeuticopportunities European Journal ofCancer37Suppl4,S3-8.

[4]Howe LR,Brown PH(2011).Targeting the HER/EGFR/ErbB family to prevent breast cancer Cancer Prev Res(Phila)4,1149-1157.

[5]Troiani T，Martinelli E,Capasso A,Morgillo F,Orditura M,De Vita F,Ciardiello F(2012).Targeting EGFR inPancreatic Cancer Treatment Curr Drug Targets.

[6]Sharafinski ME,Ferris RL,Ferrone S,Grandis JR(2010).Epidermal growth factor receptor targeted therapyof squamous cell carcinoma ofthehead andneckHeadNeck32,1412-1421.

[7]Aguilar LK,Guzik BW，Aguilar-Cordova E(2011).Cytotoxic immunotherapy strategies for cancer:mechanisms and clinical development J Cell Biochem112,1969-1977.

[8]Shirabe K,Motomura T，Muto J,Toshima T，Matono R,Mano Y，Takeishi K,Ijichi H,Harada N,Uchiyama H,et al.(2010).Tumor-infiltrating lymphocytes and hepatocellular carcinoma:pathology and clinicalmanagement Int J Clin Oncol15,552-558.

[9]Ueno M(1998).Lymphokine-activated killer cells induced in vivo in mice showing IL-2toxicity havecytoplasmic granules containing perforin and its hemolytic activity Immunopharmacology39,75-82.

[10]Vetto JT， Papa MZ,Lotze MT,Chang AE,Rosenberg SA(1987).Reduction of toxicity of interleukin-2andlymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol5,496-503.

[11]Li H,Wang C,Yu J,Cao S,Wei F,Zhang W，HanY，Ren XB(2009).Dendritic cell-activated cytokine-inducedkiller cells enhance the anti-tumor effect of chemotherapy on non-small cell lung cancer in p atients aftersurgery Cytotherapy11,1076-1083.

[12] Zhao Ming, Wu Peihong, Zeng Yixin, Xia Jianchuan, Zhang Fujun, Xian Lijian, Zhang Yuping, Zhou Kun, Fan Weijun, Zhang Liang, et al. (2006). the random research Chinese Medical Journal 86,1823-1828. of the sequential associating radio-frequency (RF) ablation of transcatheter hepatic arterial chemoembolization and cytokine induced kill cell treatment hepatocellular carcinoma

[13]Gross G， Waks T， Eshhar Z(1989).Expression of immunoglobulin-T-cell receptor chimeric molecules asfunctional receptors with antibody-type specificity Proc Natl Acad Sci USA86,10024-10028.

[14]Gross G， Gorochov G，Waks T，Eshhar Z(1989).Generation of effector T cells expressing chimeric T cellreceptor with antibody type-specificity TransplantProc21,127-130.

[15]Till BG，Jensen MC,Wang J,Chen EY，Wood BL,Greisman HA,Qian X,James SE,Raubitschek A,FormanSJ,et al.(2008).Adoptive immunotherapy fbr indolent non-Hodgkin lymphoma and mantle cell lymphomausing genetically modified autologous CD20-specific T cellsBlood112,2261-2271.

[16]Kochenderfer JN,Wilson WH,Janik JE,Dudley ME,Stetler-Stevenson M,Feldman SA,Maric I,Raffeld M,Nathan DA,Lanier BJ,et al.(2010).Eradication of B-lineage cells and regression of lymphoma in a patienttreated with autologous T cells genetically engineered to recognize CD19Blood116,4099-4102.

[17]Pule MA,Savoldo B,Myers GD,Rossig C,Russell HV，Dotti G，Huls MH,LiuE,Gee AP，Mei Z,et al.(2008).Virus-specific T cells engineered to coexpress tumor-specific receptors:persistence and antitumor activity inindividuals with neuroblastoma NatMed14,1264-1270.

[18]Lamers CH,Sleijfer S,Vulto AG，Kruit WH,Kliffen M,Debets R,Gratama JW，Stoter G，Oosterwijk E(2006).Treatment of metastatic renalcell carcinoma with autologous T-lymphocytes genetically retargeted againstcarbonic anhydrase IX:first clinic al experience JClinOncol24,e20-22.

Claims

1. the single-chain antibody of targeting EGFR, it is characterized in that this single-chain antibody is obtained by the connect light chain of antibody of targeting EGFR and variable region of heavy chain, the aminoacid sequence of described targeting EGFR single-chain antibody is as shown in SEQ ID No.2.

2. the gene of the single-chain antibody of coding targeting EGFR according to claim 1, is characterized in that: its nucleotide sequence is as shown in SEQ ID No.1.

3. a Chimeric antigen receptor, is characterized in that: this Chimeric antigen receptor splices hIL-2 signal peptide, the single-chain antibody of targeting EGFR according to claim 1, IgG2-Fc, CD28 cross-film district and CD3 ζ chain by nitrogen end in turn to carbon teminal; The structure of the Chimeric antigen receptor obtained is anti-EGFR (scFv)-IgG2 (Fc)-CD28-CD3 ζ, and aminoacid sequence is as shown in SEQID No.4.

4. the gene of Chimeric antigen receptor according to claim 3 of encoding, is characterized in that: its nucleotide sequence is as shown in SEQ IDNo.3.

5. expression vector, contains the encoding gene that also energy express amino acid sequence is the Chimeric antigen receptor shown in SEQ ID No.4.

6. expression vector according to claim 5, is characterized in that described expression vector is pmaxCloning plasmid vector.

7. the host cell containing expression vector according to claim 5.

8. host cell according to claim 7, is characterized in that: described host cell is lymphocyte.

9. the host cell described in any one of claim 7 or 8 is preparing the purposes in antitumor drug.

10. prepare the method for host cell according to claim 8, it is characterized in that: lymphocyte is entered in the expression vector transfection described in any one of claim 5 or 6, thus obtain the genetically engineered lymphocyte expressing Chimeric antigen receptor.

11. antitumor drugs are prepared from as one of activeconstituents by the host cell described in any one of claim 7 or 8.