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-CAREGFR).
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-CAREGFR).
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-CAREGFR).
In one embodiment of the invention, it is HERIN, CD28 transmembrane region, CD28 intracellulars to constitute antigen Chimerical receptor
Area(CAR2aEGFR).
In one embodiment of the invention, antigen Chimerical receptor is constituted to pierce altogether for HERIN, CD28 transmembrane region, 4-1BB
Kassinin kinin section(CAR2bEGFR).
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(CAR2cEGFR).
In one embodiment of the invention, using CAR2cEGFRIt is embedding with the first generation for tumor associated antigen MUC1
Close antigen receptor CAR1MUC1(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 CAR2aEGFROr CAR2bEGFRIt is separately cultured in modification liver cancer tissue
Til cell.
In one embodiment of the invention, using CAR1MUC1With CAR2cEGFRThe T cell Cytotoxicity in vitro A431 of modification
Tumour cell.
In one embodiment of the invention, using CAR2aEGFROr CAR2bEGFRTil 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:CAR1MUC1CAR2EGFRExpression 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(JurkatG1-eCAR、JurkatG2-eCAR、
JurkatG3-eCAR)Contact the proliferative conditions after A431 or MCF7 tumour cells.
Fig. 4:Jurkat cell through the CAR modifications of EGFR family specificities(JurkatG1-eCAR、JurkatG2-eCAR、
JurkatG3-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(JurkatG1-eCAR、JurkatG2-eCAR、
JurkatG3-eCAR)To the lethal effect of A431 or MCF7 tumour cells.
Fig. 6:A-C, the Jurkat cell modified through double CAR(JurkatmCAR1eCAR2)And the specific list CAR modifications of MUC1
Jurka t cells(JurkatmCAR1、JurkatG3-mCAR1) contact the proliferative conditions after A431, MCF7, U-2OS tumour cell.
Fig. 7:The Jurkat cell modified through double CAR(JurkatmCAR1eCAR2)And the specific list CAR modifications of MUC1
Jurka t cells(Jurka tmCAR1、JurkatG3-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(JurkatmCAR1eCAR2)And the specific list CAR modifications of MUC1
Jurkat cell(Jurka tmCAR1、JurkatG3-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 CAR2aEGFRAnd CAR2bEGFRPeptide 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(VHMUC1)Amino acid residue sequence be:EVQLQQSGGGLVQPGGSMKLSCVASGFTFSN
YWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWG
QGTTVTVSS(SEQ ID NO:13).
MUC1 scFv-VH(VHMUC1)Coded sequence be:
GAGGTCCAGCTGCAGCAGTCAGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGG
ATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTA
GATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCC
AAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTC
CTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:14).
MUC1 scFv-VL(VLMUC1)Amino acid residue sequence be:
DIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTI
TGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE(SEQ ID NO:15).
MUC1s cFv-VL(VLMUC1)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-CAREGFRIt 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-CAREGFRCoded sequence be:
ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC
GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT
CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC
AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAATCTTG
TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG
CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTGGTGGAGGCGGTTCAGGCGGAGGT
GGCAGCGGCGGTGGCGGGTCGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA
GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA
TGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCC
TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGC
CACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:36).
G2-CAREGFRIt 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-CAREGFRCoded sequence be:
ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC
GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT
CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC
AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAATCTTG
TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG
CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGT
GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTT
CGCAGCCTATCGCTCCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGAGAGTGAAGTTCAGCA
GGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG
TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG
CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG
GCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCC
CTGCCCCCTCGC(SEQ ID NO:38).
G3-CAREGFRSuccessively 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-CAREGFRCoded sequence be:
ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC
GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT
CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC
AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAATCTTG
TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG
CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGT
GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTT
CGCAGCCTATCGCTCCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGAAACGGGGCAGAAAGA
AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA
TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGGGTGGAGGTGGAGGTGGAGGTGGAGGTAGAGTGAAGTTCAGCAG
GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGT
ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC
CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG
CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCC
TGCCCCCTCGC(SEQ ID NO:40).
CAR2aEGFRIt is made of successively signalase 11-HERIN-Linker2-CD28 fusions(See Fig. 1), amino acid sequence
For:
MEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPV
SVGRGPDPDAHVAVDLSRYEGGGGGSGGGGSGGGGSPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM
NMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:41).
CAR2aEGFRCoded sequence be:
ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC
GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT
CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC
AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGGTGGAGGCGGTTC
AGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA
GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATG
AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTA
TCGCTCC(SEQ ID NO:42).
CAR2bEGFRIt 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).
CAR2bEGFRCoded sequence be:
ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC
GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT
CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC
AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGGTGGAGGCGGTTC
AGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA
GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTGGTGGAGGTGGAGGTGGAGGTGGAGGTAAACGG
GGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTG
TAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:44).
CAR2cEGFRIt 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).
CAR2cEGFRCoded sequence be:
ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGGTACCCACTCACTGCCCCC
GAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCT
CTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTC
AGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGGTGGAGGCGGTTC
AGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA
GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATG
AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTA
TCGCTCCGGTGGAGGTGGAGGTGGAGGTGGAGGTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCAT
TTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGT
GAACTG (SEQ ID NO:46).
CAR1MUC1Successively by signal peptide 2-VHMUC1-Linker4-VLMUC1- 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).
CAR1MUC1Coded sequence be:
ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAGGTCCAGCTGCAGCA
GTCAGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACT
ACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAAT
TATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCT
GCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACTGGGGCC
AAGGGACCACGGTCACCGTCTCCTCAGGTGGTTCTGGTTCTGGCGGCTCCGGTTCCGGTGGATCCGGCTCTGATATC
GTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGC
TGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCA
ACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGG
GCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAA
ACTGACTGTCCTAGGATCCGAGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAT
ACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCCCCAAGCTT
GAAGAAGGTGAATTTTCAGAAGCACGCGTAGATATCGTTCTCACTCAATCTCCAAGAGTGAAGTTCAGCAGGAGCGC
AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG
TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC
AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG
GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC
CTCGC(SEQ ID NO:48).
CAR1MUC1CAR2EGFRBy CAR1MUC1With CAR2cEGFRIt 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).
CAR1MUC1CAR2EGFRCoded 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-CARMUC1Successively by signal peptide 2-VHMUC1-Linker4-VLMUC1-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-CARMUC1Coded 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 respectivelyEGFRDNA encoding sequence (SEQ ID NO:36)、G2-CAREGFRDNA encoding sequence (SEQ
ID NO:38)、G3-CAREGFRDNA encoding sequence (SEQ ID NO:40), CAR2aEGFRDNA encoding sequence (SEQ ID
NO:42)、CAR2bEGFRDNA encoding sequence (SEQ ID NO:44)、CAR2cEGFRDNA encoding sequence (SEQ ID NO:
46)、CAR1MUC1DNA encoding sequence (SEQ ID NO:48)、CAR1MUC1CAR2EGFRCoded sequence (SEQ ID NO:50)、
G3-CARMUC1Coded 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-
CAREGFR、G2-CAREGFR、G3-CAREGFR、CAR1MUC1、CAR1MUC1CAR2EGFR、G3-CARMUC1The Jurkat of genetic modification
E6.1 cell strains JurkatG1-eCAR、Jurkat G2-eCAR、JurkatG3-eCAR、JurkatmCAR1、JurkatmCAR1eCAR2And
JurkatG3-mCAR。
Embodiment 3:T cell strain proliferative conditions measure after EGFR family specificity CAR genetic modifications
By JurkatG1-eCAR、JurkatG2-eCAR、JurkatG3-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, JurkatG2-eCAR、JurkatG3-eCARCan largely it be proliferated, and
JurkatG1-eCARSubstantially it is not proliferated;After the MCF7 cells for contacting EGFR family protein weakly positives, JurkatG1-eCAR、JurkatG2 -eCAR、JurkatG3-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 JurkatG1-eCAR、JurkatG2-eCAR、JurkatG3-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, JurkatG2-eCAR、JurkatG3-eCARIFN γ can be largely secreted, secretory volume is higher than
JurkatG1-eCAR;After being co-cultured with the MCF7 cells of EGFR family protein weakly positives, JurkatG1-eCAR、JurkatG2-eCAR、
JurkatG3-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 JurkatG1-eCAR、JurkatG2-eCAR、JurkatG3 -eCARAnd 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 JurkatG1-eCAR、JurkatG2-eCAR、JurkatG3-eCARIt can effectively kill the EGFR positives
A431 tumour cells, lethal effect JurkatG1-eCAR<JurkatG2-eCAR<JurkatG3-eCARTo EGFR family protein weakly positives
The lethal effect of MCF7, JurkatG1-eCAR、JurkatG2-eCAR、JurkatG3-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 JurkatmCAR1、JurkatmCAR1eCAR2、JurkatG3-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, JurkatmCAR1eCAR2It can largely be proliferated, proliferation times are higher than JurkatG3-mCAR;Contact that MUC1 is high positive and EGFR house
After the MCF7 cells of race's albumen weakly positive, JurkatmCAR1eCAR2Proliferation times and JurkatmCAR1It is similar, slightly below
JurkatG3-mCAR;After the U-2OS cells for contacting MUC1 weakly positives, EGFR weakly positives, JurkatmCAR1eCAR2Substantially it is not proliferated, and
JurkatG3-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 JurkatmCAR1、JurkatmCAR1eCAR2、JurkatG3-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, JurkatmCAR1eCAR2I FN γ can be largely secreted, secretory volume is higher than
JurkatG3-mCAR;After high positive and EGFR family protein weakly positives MCF7 cells co-culture with MUC1, JurkatmCAR1eCAR2's
IFN γ secretory volume and JurkatmCAR1It is similar, it is less than JurkatG3-mCAR;With the U-2OS cells of MUC1 weakly positives, EGFR weakly positives
After co-cultivation, JurkatmCAR1eCAR2Substantially IFN γ is not secreted, and JurkatG3-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 JurkatmCAR1、JurkatmCAR1eCAR2、JurkatG3 -mCARAnd 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 JurkatmCAR1eCAR2The 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 positivesmCAR1It is similar, it is less than JurkatG3 -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 CAR2aEGFRCoded sequence(SEQ ID NO:42)、CAR2bEGFRCoded 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 carriedEGFR、CAR2bEGFRThe 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 respectivelyCAR2aWith TILCAR2b。
Embodiment 11:The proliferative conditions of til cell measure after genetic modification
By TILCAR2a、TILCAR2bAnd 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 modificationCAR2aIt 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 TILCAR2a、TILCAR2bAnd 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 TILCAR2aSMMC-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.