CN103483453B - In conjunction with the Chimeric antigen receptor, its composition and purposes of EGFR family proteins - Google Patents

Abstract

The invention belongs to molecular biology and field of immunology, are related to a kind of Chimeric antigen receptor, its composition and the purposes of combination EGFR family proteins.In particular it relates to a kind of Chimeric antigen receptor, including efficiently combining the polypeptide and transmembrane region of EGFR family proteins, wherein the efficient polypeptide in conjunction with EGFR family proteins includes HERIN.The present invention improves the proliferative capacity and lethal effect of T cell under the premise of maintaining T cell killing specificity.

Description

In conjunction with the Chimeric antigen receptor, its composition and purposes of EGFR family proteins

Technical field

The invention belongs to molecular biology and field of immunology, be related to a kind of chimeric antigen of combination EGFR family proteins by Body, its composition and purposes.

Background technology

Tumour adoptive cellular is treated（adoptive cell therapy,ACT）It is by processed self or alloimmune Cell（Mainly autogenous cell）It feeds back to tumor patient, to enhance patient immune function, reaches the method for therapeutic purposes.Currently Tumour ACT progress is rapid, utilizes tumor infiltrating lymphocyte（tumor-infiltrating lymphocyte,TIL）Carry out Adoptive immunotherapy scheme achieves extraordinary clinical effectiveness for melanoma（Science2002;298:850-4）.So And T cell activation needs the stimulation of two signals, i.e. two kinds of T cell activation coherent signals.Wherein, T cell surface TCR-CD3 Complex is combined with Antigenic Peptide-MHC molecule, provides the first signal of T cell activation, determines the killing specificity of T cell；T is thin The costimulatory molecules of cellular surface（Such as CD28）With respective ligand（Such as B7）In conjunction with providing the second signal of T cell activation, promote T Cell activation, proliferation and survival.But the first signal stimulus of tumour cell source（Such as MHC molecule）With second signal ligand（Such as B7）Deng Lack or expression declines, can not effectively provide T cell activation relevant signal, to activate t cell immune response.Thus, it needs Genetic modification is carried out to T cell.Mainly pass through transgenosis TCR at present（T cell receptor）And Chimeric antigen receptor （chimeric antigen receptors,CAR）Two ways realizes the genetic modification of this T cell（Blood 2010; 116:1035-44；Nat Rev Clin Oncol 2011;8:577-85）.

TCR transformations have bigger restricted, and 1. transgenosis TCR chains may occur with the endogenic TCR chains of patient Mispairing is reduced so as to cause the reactive TCR density of T cell surface tumours；2. TCR identifies the antigen of MHC molecule submission, but not Same patient's MHC molecule is different, so the TCR special to all MHC haplotypes must be detached, operability is low；3. big Part TCR cannot identify that carbohydrate or sugared lipid antigen, antigen range of choice are relatively narrow；4. only providing T cell activation relevant first Signal, does not provide second signal, and curative effect is insufficient.Thus, Chimeric antigen receptor CAR receptors are more favored.

Chimeric antigen receptor（CAR）By a scFv single-chain antibody（By antibody VL region amino acid sequences and the areas VH amino acid Sequence is formed by connecting through Linker）, pass through hinge arrangement and cross-film and the born of the same parents derived from TCR complexs or IgE high affinity receptors Interior signal structure connects and composes.The T cell of expression CAR can pass through non-MHC restrictive approaches and antigen-reactive.In addition, with routine The proteantigens that can only be directed to of TCR compare, CAR is not limited to proteantigen, further includes carbohydrate and glycolipid class TAA, and this A little antigens are so easy mutation unlike proteantigen（Curr Opin Immunol 2009;21:215-23；Blood 2010; 116:1035-44；Cancer Res2011;71:3175-81；J Cancer 2011;2:378-82）.

From 1989, since the concept that CAR is put forward for the first time by Eshhar and its colleague, three different hairs have been had been subjected to The exhibition stage.

First generation CAR includes the scFv segments of extracellular specific recognition tumour antigen, and intracellular activation signal is by CD3 ζ or Fc ε The ITAM of RI γ（immunoreceptor tyros ine-based activation motifs）Signal chains are transmitted.But The costimulatory signal of first generation CAR receptor deficiency T cells, causes T cell that can only play moment effect, in vivo existence time It is short, cytokine secretion is few.

Second generation CAR increases the intracellular domain of a costimulatory signal molecule on the basis of first generation CAR, provides Two kinds of signals of T cell activation, including such as CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific Protein tyrosine kinase (LCK), inducible T-cell co-stimulator (ICOS) and DNAX- The structural domains such as activation protein 10 (DAP10) enhance the proliferative capacity of T cell and the secretion work(of cell factor Can, IL-2, IFN-γ and GM-CSF increase, to break through the immunosupress of tumor microenvironment, extend AICD（activation induced cell death,AI CD）.

Third generation CAR increases the intracellular structure of another costimulatory signal molecule on the basis of second generation CAR A two level costimulatory molecules such as 4-1BB is such as merged in domain again between costimulation structure C D28 and I TAM signal chains, is generated The T cell of the CAR of one triple signal, third generation CAR transformations have better effector function and internal time-to-live.

Currently, the T cell of the existing 29 CAR modifications in the U.S. is adopted, therapeutic scheme enters clinical test（Blood 2010; 116:1035-44；Nat Rev Clin Oncol 2011;8:577-85）.It can be seen that first generation CAR only provides T cell work The first signal changed, the second generation is to be merged two signals needed for t cell activation with the CAR of the third generation, by second Signal CD28 or/and 4-1BB Intracellular signals region is directly connected to CD3z molecules, to bypass tumour cell usual second Signal such as B7 etc. lacks the obstacle that caused T cell cannot activate, and after the first signal merges with second signal, substantially increases pair T cell activation, proliferation and killing ability, are allowed to curative effect and increase considerably.But certain risk is brought to clinical treatment therewith, because The normal structure generation that certain a small amount of expression tumor associated antigens can be directed to for T cell after the second generation and third generation CAR modifications is super Strong reaction excessively increases in " waterfall " formula, causes the overactive immune response of normal tissue.There are two inject CAR at present T cell after modification and lead to dead Case report.Its an example application third generation CAR（For Her2）, patient is due to acute Pulmonary edema and it is dead, the reason is that CAR⁺T cell accidentally attack low expression Her2 pulmonary epithelial cells（Mol Ther 2010; 18:843-51）；Another application second generation CAR（For CD19）, the cause of death is complicated, but along with Blood Cytokines Horizontal increases（Hum Gene Ther 2010;21:1039-42；Mol Ther 2010;18:666-8）.

To solve this safety risks, researcher is by the suicides such as HSV-TK, △ Fas, iCasp9, CD20-Rituximab System introduces the CAR modifications of T cell, plays braking action, avoids T cell excessive proliferation（J Cancer 2011;2:378-82； N Engl J Med 2011;365:1673-83）.However, CAR⁺T cell miss the target initiation " waterfall " effect speed very Soon, these Suicide systems may not necessarily play a role in time.Another method is to reduce CAR⁺T cell feed back quantity, however this Kind scheme can be such that treatment curative effect declines.

Therefore, it is highly desirable to be transformed the reaction system of CAR itself, improves the clinic for the tumour ACT that CAR is mediated Safety.

Invention content

The present inventor passes through in-depth study and performing creative labour, has obtained a kind of Chimeric antigen receptor.It is described chimeric Antigen receptor can combine EGF-R ELISA（Epidermalgrowth factor receptor, EGFR）Family is more A protein member.Specifically, the Chimeric antigen receptor passes through with the EGFR membrane receptor men for combining tumour cell wide expression The polypeptide of the multiple protein member's abilities of race's albumen, to transmit T cell activation coherent signal.In one embodiment of the invention In, relevant first signal of T cell activation is transmitted with it, the T cell activated without antigenic stimulus, specific killing EGFR can be modified The cell that family protein is overexpressed.The relevant second signal of T cell activation is transmitted with it, can identify tumour-specific with another kind The first generation chimeric antibody receptor group Chimeric antigen receptor system in pairs of antigen or tumor associated antigen, it is co-modified to be pierced without antigen The T cell of activation, or the T cell activated through tumour antigen is individually modified, under the premise of maintaining its killing specificity, carry The high proliferative capacity and lethal effect of T cell.Thus provide following inventions：

Summary of the invention

One aspect of the present invention is related to a kind of Chimeric antigen receptor, including efficiently in conjunction with EGFR family proteins polypeptide, Transmembrane region and immunoreceptor tyrosine activating motif peptide fragment and/or costimulatory signal molecule intracellular domain peptide fragment, wherein The efficient polypeptide in conjunction with EGFR family proteins includes HERIN.

According to any one of them Chimeric antigen receptor of the present invention, wherein the transmembrane region is selected from CD28 transmembrane regions, CD8 Transmembrane region, CD3 ζ transmembrane regions, CD134 transmembrane regions, CD137 transmembrane regions, ICOS transmembrane regions and one kind in DAP10 transmembrane regions or It is a variety of.

According to any one of them Chimeric antigen receptor of the present invention, wherein the immunoreceptor tyrosine activating motif peptide Section is CD3 ζ and/or Fc ε RI γ.

According to any one of them Chimeric antigen receptor of the present invention, wherein the costimulatory signal molecule intracellular domain One kind in peptide fragment of the peptide fragment selected from CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS and DAP10 intracellular domain Or it is a variety of.

According to any one of them Chimeric antigen receptor of the present invention, wherein described efficient in conjunction with the more of EGFR family proteins Peptide is single copy or multicopy；Specifically, it is double copies.

According to any one of them Chimeric antigen receptor of the present invention, the efficient polypeptide in conjunction with EGFR family proteins also wraps ScFv-VH containing MUC1 and MUC1 scFv-VL.

Include also signal peptide, and the signal peptide is selected from according to any one of them Chimeric antigen receptor of the present invention SEQ ID NO：17（Signalase 11）Or SEQ ID NO：Peptide fragment shown in 19（Signal peptide 2）.

According to any one of them Chimeric antigen receptor of the present invention, wherein its each section efficiently combines EGFR families egg White polypeptide, transmembrane region, immunoreceptor tyrosine activating motif peptide fragment and/or costimulatory signal molecule intracellular domain peptide fragment, And connected by linker between optional signal peptide, it is preferable that the linker is selected from SEQ ID NO：21 （linker1）、SEQ ID NO：23（linker2）、SEQ ID NO：25（linker3）、SEQ ID NO：27（linker 4）、 Or SEQ ID NO：29（linker 5）Shown in peptide fragment.

It is not limited to theoretical limitation, the present inventor increases chimeric antigen by selecting suitable signal peptide and linker The expression quantity of receptor is conducive to obtain space conformation more preferably Chimeric antigen receptor, to improve the work of Chimeric antigen receptor Property.

According to any one of them Chimeric antigen receptor of the present invention, amino acid sequence is or comprising selected from SEQ ID NO：35、SEQ ID NO：37、SEQ ID NO：39、SEQ ID NO：41、SEQ ID NO：43、SEQ ID NO：45 and SEQ ID NO：Amino acid sequence shown in 49.

Another aspect of the present invention relates to a kind of nucleic acid, encode the Chimeric antigen receptor described in any one of present invention； Specifically, the nucleic acid is or comprising SEQ ID NO：36、SEQ ID NO：38、SEQ ID NO：40、SEQ ID NO：42、 SEQ ID NO：44、SEQ ID NO：46、SEQ ID NO：Nucleotide sequence shown in 50.

A kind of recombinant vector of another aspect of the invention, it includes any one of them nucleic acid of the present invention；Specifically, described Recombinant vector is recombinant eukaryon expression vector or recombinant viral vector；More specifically, the recombinant eukaryon expression vector is recombination pCDNA3.1（+）Carrier；The recombinant viral vector is recombinant retroviral vector；Further specifically, the recombination is inverse Transcription vector is recombination pMXs-IRES-GFP carriers.

Another aspect of the invention is related to a kind of recombinant cell, and it includes any one of them recombinant vectors of the present invention；Tool Body, the recombinant cell is recombination T cell；More specifically, the recombinant cell is recombination Jurkat E6.1 cells.

Another aspect of the invention is related to a kind of T cell, the Chimeric antigen receptor described in any one of expression present invention.

Another aspect of the invention is related to Chimeric antigen receptor described in any one of present invention or nucleic acid or recombinant vector The T cell of genetic modification；Specifically, the T cell is Jurkat E6.1 cells；Specifically, the genetic modification is particle gun Method, transfection, electricity turns or viral transduction.

Another aspect of the invention is related to a kind of composition, and it includes embedding described in any one of one or more present invention Close antigen receptor or nucleic acid or recombinant vector or recombinant cell or T cell；Optionally, also include pharmaceutically acceptable carrier Or auxiliary material.

It is pharmaceutical composition according to any one of them composition of the present invention.

According to any one of them composition of the present invention, it includes two kinds of Chimeric antigen receptors, wherein a kind of chimeric antigen Receptor includes polypeptide, transmembrane region and the immunoreceptor tyrosine activating motif peptide fragment for efficiently combining EGFR family proteins, another Kind Chimeric antigen receptor includes efficiently to combine polypeptide, transmembrane region and the costimulatory signal molecule intracellular knot of EGFR family proteins Structure domain peptide fragment.In one embodiment of the invention, the composition is made of two kinds of Chimeric antigen receptors, wherein a kind of Chimeric antigen receptor includes efficiently to combine polypeptide, transmembrane region and the immunoreceptor tyrosine activating motif of EGFR family proteins Peptide fragment, another Chimeric antigen receptor include efficiently to combine polypeptide, transmembrane region and the costimulatory signal point of EGFR family proteins Sub- intracellular domain peptide fragment.

The present invention separates the first signal with second signal from single CAR, and it is chimeric to construct independent two kinds of dual signal Antigen receptor, the antigen of two kinds of CAR difference tumor cells, two different families, transmits T cell activation relevant two respectively Kind signal, efficiently avoids safety issue.

Two kinds of above-mentioned Chimeric antigen receptors can be expressed in the same carrier, can also distinguish in independent carrier Expression.

According to any one of them composition of the present invention, wherein the transmembrane region be selected from CD28 transmembrane regions, CD8 transmembrane regions, It is one or more in CD3 ζ transmembrane regions, CD134 transmembrane regions, CD137 transmembrane regions, ICOS transmembrane regions and DAP10 transmembrane regions.

According to any one of them composition of the present invention, wherein the immunoreceptor tyrosine activating motif peptide fragment is CD3 ζ and/or Fc ε RI γ.

According to any one of them composition of the present invention, wherein the costimulatory signal molecule intracellular domain peptide fragment choosing From one or more in the peptide fragment of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS and DAP10 intracellular domain.

According to any one of them composition of the present invention, wherein the efficient polypeptide in conjunction with EGFR family proteins is single Copy or multicopy；Specifically, it is double copies.

According to any one of them composition of the present invention, the efficient polypeptide in conjunction with EGFR family proteins also includes MUC1 ScFv-VH and MUC1 scFv-VL.

According to any one of them Chimeric antigen receptor of the present invention, amino acid sequence independently be or comprising selected from SEQ ID NO：35、SEQ ID NO：37、SEQ ID NO：39、SEQ ID NO：41、SEQ ID NO：43、SEQ ID NO： 45 and SEQ ID NO：One kind in amino acid sequence shown in 49, two kinds or a variety of.

Another aspect of the invention is related to Chimeric antigen receptor described in any one of present invention or nucleic acid or recombinant vector Or recombinant cell or T cell are in preparing treatment and/prevention and the drug of/adjuvant therapy of malignant tumor or disease of viral infection Purposes；Specifically, the tumour is lung cancer, hepatocellular carcinoma, lymthoma, colon cancer, colorectal cancer, breast cancer, oophoroma, uterine neck Cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, kidney, glioma, melanoma, osteosarcoma, cancer of pancreas or prostate Cancer；Specifically, the virus is AIDS virus（HIV）, hepatitis type B virus（HBV）, Hepatitis C Virus（HCV）, EB disease Poison（Epstein-Barr virus）, papillomavirus（Papillomavirus）, herpesviral（Herpesvirus）Or it is huge Cell virus（cytomegalovirus）.

Another aspect of the invention be related to it is a kind for the treatment of and/or prevention and/or adjuvant therapy of malignant tumor or virus infection The method of property disease, including Chimeric antigen receptor described in any one of using a effective amount of present invention or nucleic acid or recombinant vector Or the step of recombinant cell or T cell；Specifically, the tumour be lung cancer, hepatocellular carcinoma, lymthoma, colon cancer, colorectal cancer, Breast cancer, oophoroma, cervical carcinoma, gastric cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, kidney, glioma, melanoma, bone and flesh Tumor, cancer of pancreas or prostate cancer；Specifically, the virus is AIDS virus（HIV）, hepatitis type B virus（HBV）, the third type Hepatitis virus（HCV）, Epstein-Barr virus（Epstein-Barr virus）, papillomavirus（Papillomavirus）, herpesviral （Herpesvirus）Or cytomegalovirus（cytomegalovirus）.

Detailed description of the invention

As described above, the present invention relates to a kind of Chimeric antigen receptors.More specifically, which passes through tool There is the polypeptide of the EGFR membrane receptor family protein abilities in conjunction with tumour cell wide expression to transmit T cell activation coherent signal.

Term " Chimeric antigen receptor " in the present invention（chimeric antigen receptors,CAR）It is artificial reconstructed The specific molecular (such as antibody) for identifying tumour antigen can be anchored on immunocyte (such as T cell) by receptor, be made immune thin Born of the same parents identify tumour antigen and kill tumour cell.

Term " T cell activation coherent signal " refers to and two signals, i.e. T cell required for T cell activation in the present invention Surface TCR-CD3 complexs are combined with Antigenic Peptide-MHC molecule, are provided the first signal of T cell activation, are determined the killing of T cell Specificity；The costimulatory molecules on T cell surface（Such as CD28）With respective ligand（Such as B7）In conjunction with providing the second of T cell activation Signal promotes T cell activation, proliferation and survival.

Term " immunoreceptor tyrosine activating motif " in the present invention（immunoreceptor tyrosine-based Activation motifs, ITAM）Refer to activated immune cell associated receptor (such as BCR/Ig α/Ig β, TCR/CD3, Fc α R and FcR γ etc.) common to cytoplasmic domain with the aa sequence motifs based on tyrosine residue (tyrosine, Y), feature For:Two tyrosine residues are separated (... YXX [L/V] X 7-11YXX [L/V] ...) by other histidine residues outside about 13, Middle tyrosine is protein kinase phosphorylation site, can be combined with the signaling molecule in signal transduction pathway downstream after being phosphorylated, Lead to the activation of cell.

Term " costimulatory signal molecule " in the present invention（Co-stimulating molecule）It refer to immunocyte surface Some adhesion molecules, such as CD28, CD134/OX40, CD137/4-1BB, CD40, by with its ligand binding, activation is immune The second signal of cell enhances the proliferative capacity of immunocyte and the secreting function of cell factor, extends activating immune cell Time-to-live.

Term " single-chain antibody " in the present invention（single-chain antibody variable region fragment,scFv）Refer to being formed by connecting through Linker by antibody VL region amino acid sequences and VH region amino acid sequences, there is knot Close the antibody fragment of antigenic capacity.

Term " EGFR " refers to human epidermal growth factor acceptor in the present invention（epidermal growth factor receptor）, and referred to as ERBB1 or HER1, family member include EGFR, ERBB2（HER2）、ERBB 3（HER 3）、 ERBB4（HER4）.

In the present invention term " Herin " or " HERIN " refer to mankind Her2 the 8th introne in encode Herstatin 79 amino acid of C-terminal DNA sequence dna.

In one embodiment of the invention, it is HERIN, CD28 transmembrane region, CD3 ζ signals to constitute Chimeric antigen receptor Chain（G1-CAR_EGFR）.

In one embodiment of the invention, it is HERIN, CD28 transmembrane region, CD28 intracellulars to constitute Chimeric antigen receptor Area, CD3 ζ signal chains（G2-CAR_EGFR）.

In one embodiment of the invention, it is HERI N, CD28 transmembrane regions, CD28 intracellulars to constitute Chimeric antigen receptor Area, 4-1BB costimulations peptide fragment, 3 ζ signal chains of CD（G 3-CAR_EGFR）.

In one embodiment of the invention, it is HERIN, CD28 transmembrane region, CD28 intracellulars to constitute antigen Chimerical receptor Area（CAR2a_EGFR）.

In one embodiment of the invention, antigen Chimerical receptor is constituted to pierce altogether for HERIN, CD28 transmembrane region, 4-1BB Kassinin kinin section（CAR2b_EGFR）.

In one embodiment of the invention, it is HERIN, CD28 transmembrane region, CD28 intracellulars to constitute antigen Chimerical receptor Area, 4-1BB costimulation peptide fragments（CAR2c_EGFR）.

In one embodiment of the invention, using CAR2c_EGFRIt is embedding with the first generation for tumor associated antigen MUC1 Close antigen receptor CAR1_MUC1（It is made of ScFv, CD8 transmembrane region of anti-MUC1, CD3 ζ signal chains）Common modification is pierced without antigen Sharp T cell strain.

In one embodiment of the invention, using CAR2a_EGFROr CAR2b_EGFRIt is separately cultured in modification liver cancer tissue Til cell.

In one embodiment of the invention, using CAR1_MUC1With CAR2c_EGFRThe T cell Cytotoxicity in vitro A431 of modification Tumour cell.

In one embodiment of the invention, using CAR2a_EGFROr CAR2b_EGFRTil cell Cytotoxicity in vitro after modification SMMC-7721 liver cancer cells.

In the present invention,

Term " effective quantity " refers to that can realize to treat, prevent, mitigate and/or alleviate disease of the present invention in subject Or the dosage of illness.

The term as used herein " composition " mean include comprising specified amount each specified ingredient product, and directly or Any product generated indirectly from the combination of each specified ingredient of specified amount.

Term " composition ", can also refer to pharmaceutical composition, can be used for realizing in subject and treats, prevents, subtracting Light and/or alleviation disease of the present invention or illness.

Term " subject " can refer to patient or other receive the present composition to treat, prevent, mitigate and/or delay Solve the animal of disease or illness of the present invention, especially mammal, such as people, dog, monkey, ox, horse etc..

Term " disease and/or illness " refers to a kind of physical condition of the subject, the physical condition and institute of the present invention It states disease and/or illness is related.

The actual dose that each active constituent in pharmaceutical composition of the present invention can be changed is horizontal, to obtain capable of being effectively directed to tool Body patient, composition and administering mode obtain required therapeutic response.Dosage level must be according to concrete activity, administration route, institute The severity of the patient's condition and the patient's condition of patient to be treated and medical history are treated to select.But the way of this field is agent It measures since less than required therapeutic effect is obtained desired level, dosage is gradually increased, until obtaining required effect.

Advantageous effect of the invention

Compared with the CAR method of modifying of existing T cell, the present invention has the advantages that：

The present invention will be chimeric with combining the polypeptide of tumour cell wide expression such as EGFR family members albumen ability to introduce Antigen receptor transmits relevant first signal of T cell activation with it, can modify the T cell activated without antigenic stimulus, specifically kill Hinder the cell of EGFR overexpressions.The relevant second signal of T cell activation is transmitted with it, it can be anti-with another kind identification tumour-specific Former or tumor associated antigen first generation chimeric antibody receptor group Chimeric antigen receptor system in pairs, it is co-modified without antigenic stimulus The T cell of activation is ensureing safety（As shown in embodiment 6-8, relative to the third generation CAR of MUC1, to MUC1 low expressions The non-specific killing of cell apparent weaken）Under the premise of, improve the proliferative capacity and lethal effect of T cell；Or it is used for The T cell that activate through tumour antigen is modified, under the premise of maintaining its killing specific, the proliferative capacity of T cell is improved and kills Wound acts on.

Description of the drawings

Fig. 1：The tactic pattern figure of CAR.SP indicates that signal peptide, SP1 indicate that signalase 11, SP2 indicate signal peptide 2；L1–L5 Linker 1-Linker 5 are indicated respectively;CD28TM indicates CD28 transmembrane regions；CD28IR indicates CD28 intracellular regions；CD28 is indicated Or include CD28 transmembrane regions and intracellular region.

Fig. 2：CAR1_MUC1CAR2_EGFRExpression vector structure chart（Abbreviation pcDNA 3.1-CAR1:2）.

Fig. 3：A-B, the Jurka t cells through the CAR modifications of EGFR family specificities（Jurkat^G1-eCAR、Jurkat^G2-eCAR、 Jurkat^G3-eCAR）Contact the proliferative conditions after A431 or MCF7 tumour cells.

Fig. 4：Jurkat cell through the CAR modifications of EGFR family specificities（Jurkat^G1-eCAR、Jurkat^G2-eCAR、 Jurkat^G3-eCAR）After being co-cultured with A431 or MCF7 tumour cells, the secretory volume of INFr γ.

Fig. 5：A-B, the Jurka t cells through the CAR modifications of EGFR family specificities（Jurkat^G1-eCAR、Jurkat^G2-eCAR、 Jurkat^G3-eCAR）To the lethal effect of A431 or MCF7 tumour cells.

Fig. 6：A-C, the Jurkat cell modified through double CAR（Jurkat^mCAR1eCAR2）And the specific list CAR modifications of MUC1 Jurka t cells（Jurkat^mCAR1、Jurkat^G3-mCAR1) contact the proliferative conditions after A431, MCF7, U-2OS tumour cell.

Fig. 7：The Jurkat cell modified through double CAR（Jurkat^mCAR1eCAR2）And the specific list CAR modifications of MUC1 Jurka t cells（Jurka t^mCAR1、Jurkat^G3-mCAR1）After being co-cultured with A431, MCF7, U-2OS tumour cell, INFr γ Secretory volume.

Fig. 8：A-C, the Jurkat cell modified through double CAR（Jurkat^mCAR1eCAR2）And the specific list CAR modifications of MUC1 Jurkat cell（Jurka t^mCAR1、Jurkat^G3-mCAR1）To the lethal effect of A431, MCF7, U-2OS tumour cell.

Fig. 9：Proliferative conditions after CAR modifies front and back TI L cells contact SMMC-7721.

Figure 10：Lethal effect of the front and back til cell to SMMC-7721 is modified through CAR.

Specific implementation mode

Embodiment of the present invention is described in detail below in conjunction with embodiment.Those skilled in the art will manage Solution, the following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Technology or condition person, according to technology or condition described in document in the art（It is yellow such as with reference to works such as J. Pehanorm Brookers What training hall etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press）, corresponding bibliography or said according to product Bright book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.

Embodiment 1：The synthesis of CAR expression cassettes and the structure of expression vector

According to the amino acid sequence and coded sequence for constituting CAR each components, be spliced into the amino acid sequence that entirely merges with Coding DNA expression cassette, wherein constituting CAR2a_EGFRAnd CAR2b_EGFRPeptide fragment include：

The amino acid residue sequence of HERIN is：

GTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPVSVGRGPDPDAHVAVDLSRY EG(SEQ ID NO：1).

The coded sequence of HERIN is：

GGTACCCACTCACTGCCCCCGAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGT CCTATCCTTCCTCAGACCCTCTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAA GTGTCCCTATATCCCCTGTCAGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTAT GAAGGC(SEQ ID NO：2).

CD28 transmembrane regions（CD28TM）Amino acid residue sequence is:

PFWVLVVVGGVLACYSLLVTVAFIIFWVRS(SEQ ID NO：3).

CD28 transmembrane regions（CD28TM）Coded sequence be:

CCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTT CTGGGTGAGGAGT(SEQ ID NO：4).

CD28 intracellular regions（CD28IR）Amino acid residue sequence is:

KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO：5).

CD28 intracellular regions（CD28IR）Coded sequence be:

AAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCA GCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO：6).

CD28 transmembrane regions and intracellular region（CD28）Amino acid residue sequence is:

PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO：7).

CD28 transmembrane regions and intracellular region（CD28）Coded sequence be:

CCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTT CTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCC GCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO：8).

The amino acid residue sequence of CD3 ζ is：

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：9).

The coded sequence of CD3 ζ is：

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA GGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC CCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO：10).

4-1BB costimulatory signals domain peptide fragment（41BB）Amino acid residue sequence be:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO：11).

4-1BB costimulatory signals domain peptide fragment（41BB）Coded sequence be:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGA TGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO：12).

MUC1s cFv-VH（VH_MUC1）Amino acid residue sequence be：EVQLQQSGGGLVQPGGSMKLSCVASGFTFSN YWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWG QGTTVTVSS(SEQ ID NO：13).

MUC1 scFv-VH（VH_MUC1）Coded sequence be：

GAGGTCCAGCTGCAGCAGTCAGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGG ATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTA GATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCC AAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTC CTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO：14).

MUC1 scFv-VL（VL_MUC1）Amino acid residue sequence be：

DIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTI TGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE(SEQ ID NO：15).

MUC1s cFv-VL（VL_MUC1）Coded sequence be：

GATATCGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTAC TGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTG GTACCAACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATC ACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGG AACCAAACTGACTGTCCTAGGATCCGAG(SEQ ID NO：16).

The amino acid residue sequence of signalase 11 is：

MEFWLSWVFLVAILKGVQC(SEQ ID NO：17).

Signalase 11 coded sequence is：

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGT(SEQ ID NO： 18)。

The amino acid residue sequence of signal peptide 2 is：

MEAPAQLLFLLLLWLPDTTG(SEQ ID NO：19).

2 coded sequence of signal peptide is：

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(SEQ ID NO：20).

The amino acid residue sequence of Linker1 is:

EPKSCDKTHTCPPCPAPE(SEQ ID NO：21).

The coded sequence of Linker1 is:

GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA(SEQ ID NO：22).

The amino acid residue sequence of Linker2 is：

GGGGSGGGGSGGGGS(SEQ ID NO：23).

The coded sequence of Linker2 is：

GGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCG(SEQ ID NO：24).

The amino acid residue sequence of Linker3 is:

GGGGGGGGG(SEQ ID NO：25).

The coded sequence of Linker3 is:

GGTGGAGGTGGAGGTGGAGGTGGAGGT(SEQ ID NO：26).

The amino acid residue sequence of Linker4 is:

GGSGSGGSGSGGSGS(SEQ ID NO：27).

The coded sequence of Linker4 is:

GGTGGTTCTGGTTCTGGCGGCTCCGGTTCCGGTGGATCCGGCTCT(SEQ ID NO：28).

The amino acid residue sequence of Linker5 is:

PKLEEGEFSEARVDIVLTQSP(SEQ ID NO：29).

The coded sequence of Linker5 is:

CCCAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGATATCGTTCTCACTCAATCTCCA(SEQ ID NO：30).

CD8 transmembrane region peptide fragments（CD8TM）Amino acid residue sequence be:

YIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO：31).

CD8 transmembrane region peptide fragments（CD8TM）Coded sequence be:

TACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO：32).

The amino acid residue sequence of Furin-2A is:

RAKRAPVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO：33).

The coded sequence of Furin-2A is:

CGTGCTAAACGAGCTCCTGTTAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTCGAGTCCAACCC TGGGCCC(SEQ ID NO：34).

G1-CAR_EGFRIt is made of successively signalase 11-HERIN-Linker1-CD28TM-Linker2-CD3 ζ fusions（See figure 1）, amino acid sequence is：

MEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPV SVGRGPDPDAHVAVDLSRYEGEPKSCDKTHTCPPCPAPEPFWVLVVVGGVLACYSLLVTVAFIIFWVRSGGGGSGGG GSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：35).

G1-CAR_EGFRCoded sequence be:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAATCTTG TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTGGTGGAGGCGGTTCAGGCGGAGGT GGCAGCGGCGGTGGCGGGTCGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCC TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGC CACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO：36).

G2-CAR_EGFRIt is merged successively by signalase 11-HERIN-Linker1-CD28TM-CD28IR--Linker2-CD3 ζ It constitutes（See Fig. 1）, amino acid sequence is：

MEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPV SVGRGPDPDAHVAVDLSRYEGEPKSCDKTHTCPPCPAPEPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHS DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR(SEQ ID NO：37).

G2-CAR_EGFRCoded sequence be:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAATCTTG TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGT GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTT CGCAGCCTATCGCTCCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGAGAGTGAAGTTCAGCA GGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCC CTGCCCCCTCGC(SEQ ID NO：38).

G3-CAR_EGFRSuccessively by signal peptide-HERIN-Linker1-CD28TM-CD28IR--Linker2-4-1BB- Linker3-CD3 ζ fusions are constituted（See Fig. 1）：

MEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPV SVGRGPDPDAHVAVDLSRYEGEPKSCDKTHTCPPCPAPEPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHS DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGGSGGGGSGGGGSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELGGGGGGGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：39).

G3-CAR_EGFRCoded sequence be：

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAATCTTG TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGT GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTT CGCAGCCTATCGCTCCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGAAACGGGGCAGAAAGA AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGGGTGGAGGTGGAGGTGGAGGTGGAGGTAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGT ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCC TGCCCCCTCGC(SEQ ID NO：40).

CAR2a_EGFRIt is made of successively signalase 11-HERIN-Linker2-CD28 fusions（See Fig. 1）, amino acid sequence For：

MEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPV SVGRGPDPDAHVAVDLSRYEGGGGGSGGGGSGGGGSPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO：41).

CAR2a_EGFRCoded sequence be：

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGGTGGAGGCGGTTC AGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATG AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTA TCGCTCC(SEQ ID NO：42).

CAR2b_EGFRIt is made of successively signalase 11-HERIN-Linker2-CD28TM-Linker3-41BB fusions（See figure 1）, amino acid sequence is：

MEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPV SVGRGPDPDAHVAVDLSRYEGGGGGSGGGGSGGGGSPFWVLVVVGGVLACYSLLVTVAFIIFWVRSGGGGGGGGGKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO：43).

CAR2b_EGFRCoded sequence be:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGGTGGAGGCGGTTC AGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTGGTGGAGGTGGAGGTGGAGGTGGAGGTAAACGG GGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTG TAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO：44).

CAR2c_EGFRIt is made of successively signalase 11-HERIN-Linker2-CD28-Linker3-41BB fusions（See Fig. 1）, Its amino acid sequence is：

MEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPV SVGRGPDPDAHVAVDLSRYEGGGGGSGGGGSGGGGSPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGGGGGGGKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC EL(SEQ ID NO：45).

CAR2c_EGFRCoded sequence be:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGGTGGAGGCGGTTC AGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATG AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTA TCGCTCCGGTGGAGGTGGAGGTGGAGGTGGAGGTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCAT TTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGT GAACTG (SEQ ID NO：46).

CAR1_MUC1Successively by signal peptide 2-VH_MUC1-Linker4-VL_MUC1- Linker1-CD8TM-Linker5-CD3 ζ melt It closes and constitutes（See Fig. 1）, amino acid sequence is：

MEAPAQLLFLLLLWLPDTTGEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNN YATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGSGSGGSGSGGSGSDI VVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITG AQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEEPKSCDKTHTCPPCPAPEYIWAPLAGTCGVLLLSLVITLYCPKL EEGEFSEARVDIVLTQSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：47).

CAR1_MUC1Coded sequence be:

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAGGTCCAGCTGCAGCA GTCAGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACT ACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAAT TATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCT GCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGGTGGTTCTGGTTCTGGCGGCTCCGGTTCCGGTGGATCCGGCTCTGATATC GTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGC TGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCA ACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGG GCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAA ACTGACTGTCCTAGGATCCGAGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAT ACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCCCCAAGCTT GAAGAAGGTGAATTTTCAGAAGCACGCGTAGATATCGTTCTCACTCAATCTCCAAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGC(SEQ ID NO：48).

CAR1_MUC1CAR2_EGFRBy CAR1_MUC1With CAR2c_EGFRIt is connected and composed through Furin-2A, amino acid sequence is：

MEAPAQLLFLLLLWLPDTTGEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNN YATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGSGSGGSGSGGSGSDI VVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITG AQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEEPKSCDKTHTCPPCPAPEYIWAPLAGTCGVLLLSLVITLYCPKL EEGEFSEARVDIVLTQSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRAPVKQTLNFDLLKLAGDVES NPGPEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPI SPVSVGRGPDPDAHVAVDLSRYEGGGGGSGGGGSGGGGSPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHS DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGGGGGGGKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL (SEQ ID NO：49).

CAR1_MUC1CAR2_EGFRCoded sequence be：

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAGGTCCAGCTGCAGCA GTCAGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACT ACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAAT TATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCT GCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGGTGGTTCTGGTTCTGGCGGCTCCGGTTCCGGTGGATCCGGCTCTGATATC GTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGC TGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCA ACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGG GCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAA ACTGACTGTCCTAGGATCCGAGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAT ACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCCCCAAGCTT GAAGAAGGTGAATTTTCAGAAGCACGCGTAGATATCGTTCTCACTCAATCTCCAAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCCGTGCTAAACGAGCTCCTGTTAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTCGAGTCC AACCCTGGGCCCGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTC ACTGCCCCCGAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCC TCAGACCCTCTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATA TCCCCTGTCAGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGGTGG AGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGT GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTT CGCAGCCTATCGCTCCGGTGGAGGTGGAGGTGGAGGTGGAGGTAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAA GGAGGATGTGAACTG(SEQ ID NO:50)。

Compare G 3-CAR_MUC1Successively by signal peptide 2-VH_MUC1-Linker4-VL_MUC1-Linker1-CD28-Linker3- 41BB-Linker5-CD3 ζ fusions are constituted（See Fig. 1）, amino acid sequence is：

MEAPAQLLFLLLLWLPDTTGEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNN YATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGSGSGGSGSGGSGSDI VVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITG AQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEEPKSCDKTHTCPPCPAPEPFWVLVVVGGVLACYSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGGGGGGGKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELPKLEEGEFSEARVDIVLTQSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：51).

G3-CAR_MUC1Coded sequence be：

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAGGTCCAGCTGCAGCA GTCAGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACT ACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAAT TATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCT GCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGGTGGTTCTGGTTCTGGCGGCTCCGGTTCCGGTGGATCCGGCTCTGATATC GTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGC TGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCA ACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGG GCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAA ACTGACTGTCCTAGGATCCGAGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAC CCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC TGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCG CAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGGTGGAGGTGGAGGTGGAGGTGGAG GTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA GATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCCCAAGCTTGAAGAAGGTGAATTTTC AGAAGCACGCGTAGATATCGTTCTCACTCAATCTCCAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACC AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGT GGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACC AGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO： 52)。

G1-CAR is pressed respectively_EGFRDNA encoding sequence (SEQ ID NO：36)、G2-CAR_EGFRDNA encoding sequence (SEQ ID NO：38)、G3-CAR_EGFRDNA encoding sequence (SEQ ID NO：40), CAR2a_EGFRDNA encoding sequence (SEQ ID NO：42)、CAR2b_EGFRDNA encoding sequence (SEQ ID NO：44)、CAR2c_EGFRDNA encoding sequence (SEQ ID NO： 46)、CAR1_MUC1DNA encoding sequence (SEQ ID NO：48)、CAR1_MUC1CAR2_EGFRCoded sequence (SEQ ID NO：50)、 G3-CAR_MUC1Coded sequence (SEQ ID NO：52), student on commission's workBioengineering（Shanghai）It is entire that Co., Ltd synthesizes it Expression cassette is inserted into pCDNA3.1（+）Carrier（Invitrogen）The sites EcoRI-XbaI（See Fig. 2）, it is transformed into E.coli (DH5 α）, after being sequenced correctly, using the plasmid purification kit extraction of Qiagen companies and plasmid purification, obtain each recombinant expression and carry The high-quality plasmid of body.

Embodiment 2：The genetic modification of T cell strain

The high-quality plasmid of each recombinant expression carrier of acquisition will be built and purified in embodiment 1, is utilized Lipofectamine 2000（Invitrogen）It is transfected respectively to Jurkat E6.1（T lymphocyte strains, it is typical purchased from the U.S. Object collection, ATCC）.After 2 days, the Jurkat E6.1 cells after transfection are transferred to the RPMI 1640 with neomycin and are trained It supports in base, and by limiting dilution assay by Cell-cloned.Through screening in 21 days, established respectively with neomycin resistance, through G1- CAR_EGFR、G2-CAR_EGFR、G3-CAR_EGFR、CAR1_MUC1、CAR1_MUC1CAR2_EGFR、G3-CAR_MUC1The Jurkat of genetic modification E6.1 cell strains Jurkat^G1-eCAR、Jurkat ^G2-eCAR、Jurkat^G3-eCAR、Jurkat^mCAR1、Jurkat^mCAR1eCAR2And Jurkat^G3-mCAR。

Embodiment 3：T cell strain proliferative conditions measure after EGFR family specificity CAR genetic modifications

By Jurkat^G1-eCAR、Jurkat^G2-eCAR、Jurkat^G3-eCARAnd unmodified JurkatE6.1 cells（5× 10⁵/ hole, 1640 culture mediums of RPMI, IL-2 containing 20U/ml）, it is separately added into A431, MCF7 cell overlay through radiation treatment （It is purchased from ATCC）6 orifice plates（5×10⁵/ hole）In, respectively on day 3, the 7th day, the Jurkat cell of suspension is counted Number.The result shows that after the A431 cells of the contact EGFR positives, Jurkat^G2-eCAR、Jurkat^G3-eCARCan largely it be proliferated, and Jurkat^G1-eCARSubstantially it is not proliferated；After the MCF7 cells for contacting EGFR family protein weakly positives, Jurkat^G1-eCAR、Jurkat^{G2 -eCAR}、Jurkat^G3-eCARIt is not proliferated substantially（See Fig. 3 A-B）.

Embodiment 4：T cell strain IFN γ secretory volume measures after EGFR family specificity CAR genetic modifications

By Jurkat^G1-eCAR、Jurkat^G2-eCAR、Jurkat^G3-eCARAnd unmodified JurkatE6.1 cells（5× 10⁵/ hole）In 24 orifice plates with A431, MCF7 cell（It is purchased from ATCC, 1 × 10⁵/ hole）It co-cultures, supernatant is collected after 72 hours, Pass through the ELI SA detection kits of IFN γ（BD Biosciences）Detect the secretory volume of IFN γ.The result shows that with EGFR After positive A431 cells co-culture, Jurkat^G2-eCAR、Jurkat^G3-eCARIFN γ can be largely secreted, secretory volume is higher than Jurkat^G1-eCAR；After being co-cultured with the MCF7 cells of EGFR family protein weakly positives, Jurkat^G1-eCAR、Jurkat^G2-eCAR、 Jurkat^G3-eCARDo not secrete IFN γ substantially.

Embodiment 5：The killing effect in vitro of the T cell strain after EGFR family specificity CAR genetic modifications measures

By different effect target ratios（50:1,25:1,5:1,1:1）, will be by Jurkat^G1-eCAR、Jurkat^G2-eCAR、Jurkat^{G3 -eCAR}And unmodified Jurkat E6.1 cells are co-cultured with A431, MCF7, are examined using LDH lactic dehydrogenases-cytotoxicity Survey assay kit（LDH-Cytotoxicity Assay Kit, Biovision）After detecting distinct methods genetic modification Cytotoxicity in vitro ability of the Jurkat E6.1 cells to different type tumour cell.Method is as follows：Target cell spreads 96 orifice plates（5× 10³/ hole）If the spontaneous LDH releases of culture medium background, volume correction, target cell, target cell maximum LDH releases, effector cell are certainly It sends out LDH and discharges control wells, treatment group hole, every group of 3 hole of repetition, the final volume in each hole is identical and no less than 100 μ L.250g is centrifuged 4min, at 37 DEG C, 5%CO2 is incubated at least 4h.10 × lysate, body is added to target cell maximum release aperture in 45min before centrifugation The lysate of equivalent is added in product correction hole.It centrifuges again, is shifted in 50 μ L supernatants to 96 new orifice plates from every hole, add 50 μ L Substrate solution, room temperature, which is protected from light, is incubated 30min.50 μ L terminate liquids are added per hole, D490 is measured in 1h.Cytotoxicity（%）=[（D is real - D culture medium backgrounds of verifying hole）-（The spontaneous LDH release apertures-D culture mediums background hole of D effector cell）-（The spontaneous LDH of D target cells is released Discharge hole-D culture medium backgrounds hole）]/[（D target cell maximum LDH release aperture-D volume corrections hole）-（The spontaneous LDH releases of D target cells Hole-D culture medium backgrounds hole）]×100%.

The result shows that Jurkat^G1-eCAR、Jurkat^G2-eCAR、Jurkat^G3-eCARIt can effectively kill the EGFR positives A431 tumour cells, lethal effect Jurkat^G1-eCAR<Jurkat^G2-eCAR<Jurkat^G3-eCARTo EGFR family protein weakly positives The lethal effect of MCF7, Jurkat^G1-eCAR、Jurkat^G2-eCAR、Jurkat^G3-eCARIt is similar to unmodified Jurkat cell, Substantially it does not kill（See Fig. 5 A-B）.

Embodiment 6：T cell strain proliferative conditions through double CAR modifications and the specific list CAR modifications of MUC1 measure

By Jurkat^mCAR1、Jurkat^mCAR1eCAR2、Jurkat^G3-mCARAnd unmodified JurkatE6.1 cells（5× 10⁵/ hole, 1640 culture mediums of RPMI, IL-2 containing 20U/ml）, it is separately added into and overlays the A431 through radiation treatment（People's epidermal carcinoma is thin Born of the same parents）、MCF7（Human breast cancer cell）、U-2OS（Human osteosarcoma cell）（Purchased from ATCC）6 orifice plates（5×10⁵/ hole）In, respectively On day 3, the 7th day, the Jurkat cell of suspension is counted.The result shows that the A431 of contact MUC1 and the bis- positives of EGFR After cell, Jurkat^mCAR1eCAR2It can largely be proliferated, proliferation times are higher than Jurkat^G3-mCAR；Contact that MUC1 is high positive and EGFR house After the MCF7 cells of race's albumen weakly positive, Jurkat^mCAR1eCAR2Proliferation times and Jurkat^mCAR1It is similar, slightly below Jurkat^G3-mCAR；After the U-2OS cells for contacting MUC1 weakly positives, EGFR weakly positives, Jurkat^mCAR1eCAR2Substantially it is not proliferated, and Jurkat^G3-mCARIt remains to be proliferated（See Fig. 6 A-C）.

Embodiment 7：T cell strain IFN γ secretory volume through double CAR modifications and the specific list CAR modifications of MUC1 measures

By Jurkat^mCAR1、Jurkat^mCAR1eCAR2、Jurkat^G3-mCARAnd unmodified JurkatE6.1 cells（5× 10⁵/ hole）In 24 orifice plates with A431, MCF7, U-2OS（It is purchased from ATCC, 1 × 10⁵/ hole）It co-cultures, is collected after 72 hours Clearly, pass through the ELISA detection kit of IFN γ（BD Biosciences）Detect the secretory volume of IFN γ.The result shows that with After MUC1 and the A431 cells of the bis- positives of EGFR co-culture, Jurkat^mCAR1eCAR2I FN γ can be largely secreted, secretory volume is higher than Jurkat^G3-mCAR；After high positive and EGFR family protein weakly positives MCF7 cells co-culture with MUC1, Jurkat^mCAR1eCAR2's IFN γ secretory volume and Jurkat^mCAR1It is similar, it is less than Jurkat^G3-mCAR；With the U-2OS cells of MUC1 weakly positives, EGFR weakly positives After co-cultivation, Jurkat^mCAR1eCAR2Substantially IFN γ is not secreted, and Jurkat^G3-mCARIt remains to secrete more IFN γ（See Fig. 7）.

Embodiment 8：The killing effect in vitro of T cell strain through double CAR modifications and the specific list CAR modifications of MUC1 measures

By different effect target ratios（50:1,25:1,5:1,1:1）, by Jurkat^mCAR1、Jurkat^mCAR1eCAR2、Jurkat^{G3 -mCAR}And unmodified Jurkat E6.1 cells are co-cultured with A431, MCF7, U-2OS, using LDH lactic dehydrogenases-cell Toxicity detection assay kit（LDH-Cytotoxicity Assay Kit, Biovision）Detect distinct methods genetic modification Jurkat E6.1 cells afterwards are to the Cytotoxicity in vitro ability of different type tumour cell, and method is the same as embodiment 5.

The result shows that Jurkat^mCAR1eCAR2The A431 tumour cells of MUC1 and the bis- positives of EGFR can effectively be killed；It is right MUC1 is high positive and the lethal effect and Jurkat of the MCF7 of EGFR family protein weakly positives^mCAR1It is similar, it is less than Jurkat^{G3 -mCAR}；The U-2OS cells of MUC1 weakly positives, EGFR weakly positives are not killed substantially（See Fig. 8 A-C）.

Embodiment 9：Liver cancer tissue source TIL's is separately cultured

The liver cancer sample that fresh cut is removed is collected, is aseptically handled immediately.The specific method is as follows：Remove liver cancer mark Normal structure around this and necrotic zone remove the small tissue blocks that size is 1-2mm3,24 orifice plates from the different zones of sample Each hole place one piece.Add 2mL complete mediums per hole（GT-T551 culture mediums containing 10%FBS）With 3000 IU/mL's IL-2.24 orifice plates are placed in 37 DEG C, are cultivated in 5% CO2 incubators.Half is carried out for all holes and is measured within the 5-6 days after culture starting Change liquid.Later, according to TIL growing states, primary half amount was carried out every 1-2 days and changes liquid.Once TIL is covered in hole, and all patches Parietal cell has removed, and just collects each TIL covered in hole.

Then, 1 × 10⁶TIL is resuspended in complete medium containing 150mL, 30ng/mL anti-cd 3 antibodies, is no less than 200 times The irradiated feeder cells of TIL（PBMC from 3 different Healthy Peoples）In the T175 culture bottles of 6000IU/mL IL-2, Culture bottle is cultivated vertically.Culture was to the 5th day, and 65% liquid is changed to new complete medium and IL-2 in bottle.It cultivates to the 7th day, Cell suspension in 2 T175 culture bottles is transferred in cell culture bags, and 300mL complete mediums and IL-2 is added.From the 6th day Start, a Trypan Blue was carried out every 1 day and is counted, cell density is controlled extremely by the way that new complete medium and IL-2 is added 0.5-2×10⁶/mL.Cell is collected in the 14th day of amplification.

Embodiment 10：The genetic modification of TIL

By CAR2a_EGFRCoded sequence（SEQ ID NO：42）、CAR2b_EGFRCoded sequence（SEQ ID NO：44）It inserts Enter retrovirus package carrier pMXs-IRES-GFP（Purchased from Cell BioLabs）In the sites EcoRI-XhoI, it is transformed into E.coli（DH5α）, after being sequenced correctly, use the plasmid purification kit extraction of Qiagen companies and plasmid purification.In 175- cm²Incasing cells Amphotropic is cultivated in flasks（Purchased from Cell BioLabs, cell number is about 1-2 × 10⁷）, utilize Lipofec tamine 2000（Invitrogen）By high-quality plasmid transfection after purification in.After 3 days, collect containing ill The cell culture medium of malicious particle centrifuges 10min with 4000g, collects supernatant, and 0.45 μm of filter filtering, the liquid of filtration is used in combination It being placed in 40mL ultracentrifugation pipes, 4 DEG C, 25000r/min is centrifuged 20 minutes, and viral pellet is resuspended using 500ul ice PBS liquid, point CAR2a Huo get not carried_EGFR、CAR2b_EGFRThe recombinant retrovirus of expression cassette.Then, at twice（100 μ L every time）It will be viral Suspension and 2 × 10⁶Til cell co-culture, collect metainfective til cell TIL respectively^CAR2aWith TIL^CAR2b。

Embodiment 11：The proliferative conditions of til cell measure after genetic modification

By TIL^CAR2a、TIL^CAR2bAnd unmodified til cell（5×10⁵/ hole, GT-T551 culture medium bases contain 3000U/ ml IL-2）, it is separately added into and overlays the SMMC-7721 through radiation treatment（Purchased from Shanghai life science institute biochemistry and carefully Born of the same parents' biological study institute）6 orifice plates（5×10⁵/ hole）In, respectively on day 3, the 7th day, the til cell of suspension is counted Number.The result shows that the TIL after modification^CAR2aIt can be largely proliferated after contact SMMC-7721 cells, and unmodified til cell then base This is not proliferated（See Fig. 9）.

Embodiment 12：The killing effect in vitro of til cell measures after genetic modification

By different effect target ratios（50:1,25:1,5:1,1:1）, by TIL^CAR2a、TIL^CAR2bAnd unmodified til cell It is co-cultured with SMMC-7721, kit is tested and analyzed using LDH lactic dehydrogenases-cytotoxicity（LDH-Cytotoxicity Assay Kit, Biovision）Til cell before and after detection genetic modification is to the Cytotoxicity in vitro ability of SMMC-7721 cells, side Method is the same as embodiment 5.The result shows that genetically modified TIL^CAR2aSMMC-7721 tumour cells can be effectively killed, lethal effect is aobvious It writes and is higher than unmodified TI L cells（See Figure 10）.

Although the specific implementation mode of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change the guarantor in the present invention Within the scope of shield.The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims (28)

1. a kind of Chimeric antigen receptor, including efficiently combining polypeptide, transmembrane region and the immunity receptor junket ammonia of EGFR family proteins Acid activation motif peptide fragment and/or costimulatory signal molecule intracellular domain peptide fragment, wherein the efficient combination EGFR family proteins Polypeptide be HERIN, and the amino acid sequence of the HERIN such as SEQ ID NO:Shown in 1.

2. Chimeric antigen receptor according to claim 1, wherein the transmembrane region be CD28 transmembrane regions, CD8 transmembrane regions, It is one or more in CD3 ζ transmembrane regions, CD134 transmembrane regions, CD137 transmembrane regions, ICOS transmembrane regions and DAP10 transmembrane regions.

3. Chimeric antigen receptor according to claim 1, wherein the immunoreceptor tyrosine activating motif peptide fragment is CD3 ζ and/or Fc ε RI γ.

4. Chimeric antigen receptor according to claim 1, wherein the costimulatory signal molecule intracellular domain peptide fragment choosing From one or more in the peptide fragment of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS and DAP10 intracellular domain.

5. Chimeric antigen receptor according to claim 1, wherein the efficient polypeptide in conjunction with EGFR family proteins is single Copy or multicopy.

6. Chimeric antigen receptor according to claim 5, wherein the efficient polypeptide in conjunction with EGFR family proteins is double Copy.

7. Chimeric antigen receptor according to claim 1, amino acid sequence is selected from SEQ ID NO：35、SEQ ID NO：37、SEQ ID NO：39、SEQ ID NO：41、SEQ ID NO：43、SEQ ID NO：45 and SEQ ID NO：Shown in 49 Amino acid sequence.

8. a kind of nucleic acid encodes the Chimeric antigen receptor described in any claim in claim 1 to 7.

9. nucleic acid according to claim 8, wherein the nucleic acid is SEQ ID NO：36、SEQ ID NO：38、SEQ ID NO：40、SEQ ID NO：42、SEQ ID NO：44、SEQ ID NO：46 or SEQ ID NO：Nucleotide sequence shown in 50.

10. a kind of recombinant vector, it includes the nucleic acid described in claim 8 or 9.

11. recombinant vector according to claim 10, wherein the recombinant vector is recombinant eukaryon expression vector or recombination Viral vectors.

12. recombinant vector according to claim 11, wherein the recombinant eukaryon expression vector is recombination pCDNA3.1 (+) carrier.

13. recombinant vector according to claim 11, wherein the recombinant viral vector is recombinant retroviral vector Or recombined lentivirus vector.

14. recombinant vector according to claim 13, wherein the recombinant retroviral vector is recombination pMXs- IRES-GFP carriers.

15. a kind of recombinant cell, it includes the recombinant vectors described in any claim in claim 10 to 14.

16. recombinant cell according to claim 15, wherein the recombinant cell is recombination T cell.

17. recombinant cell according to claim 15, wherein the recombinant cell is recombination Jurkat E6.1 cells.

18. a kind of T cell expresses the Chimeric antigen receptor described in any claim in claim 1-7.

19. by described in claim 8 or 9 nucleic acid or claim 10 to 14 in recombinant vector described in any claim lose Pass the T cell of modification.

20. the T cell of genetic modification according to claim 19, wherein the T cell is Jurkat E6.1 cells.

21. the T cell of genetic modification according to claim 19, wherein the genetic modification be particle bombardment, transfection, Electricity turns or viral transduction.

22. a kind of composition, it includes one or more：

Chimeric antigen receptor in claim 1 to 7 described in any claim,

Recombinant vector in nucleic acid or claim 10 to 14 described in claim 8 or 9 described in any claim,

Recombinant cell in claim 15 to 17 described in any claim, or

T cell in claim 18 to 21 described in any claim.

23. composition according to claim 22 also includes pharmaceutically acceptable carrier or auxiliary material.

24. the composition according to claim 22 or 23, it includes two kinds of Chimeric antigen receptors, wherein a kind of inosculating antibody Original receptor includes polypeptide, transmembrane region and the immunoreceptor tyrosine activating motif peptide fragment for efficiently combining EGFR family proteins, separately A kind of Chimeric antigen receptor includes efficiently to combine polypeptide, transmembrane region and the costimulatory signal molecule intracellular knot of EGFR family proteins Structure domain peptide fragment.

25. any one in (1)-chosen from the followings (5) item is preparing treatment or is preventing malignant tumour or viral infection Purposes in the drug of disease：

(1) Chimeric antigen receptor in claim 1 to 7 described in any claim,

(2) nucleic acid described in claim 8 or 9,

(3) recombinant vector in claim 10 to 14 described in any claim,

(4) recombinant cell in claim 15 to 17 described in any claim, and

(5) T cell in claim 18 to 21 described in any claim.

26. purposes according to claim 25, wherein the malignant tumour is lung cancer, hepatocellular carcinoma, lymthoma, colon Cancer, colorectal cancer, breast cancer, oophoroma, cervical carcinoma, gastric cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, kidney, glioma, black Plain tumor, osteosarcoma, cancer of pancreas or prostate cancer.

27. purposes according to claim 25, wherein it is described virus be HIV, hepatitis type B virus, Hepatitis C Virus, Epstein-Barr virus, papillomavirus, herpesviral or cytomegalovirus.

28. the purposes according to any claim in claim 25 to 27, wherein the treatment is auxiliary treatment.