CN106543288A - A kind of application in the T cell preparation of mesothelin Chimeric antigen receptor modification and treatment of pancreatic cancer - Google Patents

A kind of application in the T cell preparation of mesothelin Chimeric antigen receptor modification and treatment of pancreatic cancer Download PDF

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CN106543288A
CN106543288A CN201610936628.1A CN201610936628A CN106543288A CN 106543288 A CN106543288 A CN 106543288A CN 201610936628 A CN201610936628 A CN 201610936628A CN 106543288 A CN106543288 A CN 106543288A
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chimeric antigen
antigen receptor
csf
meso
scfv
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刘明录
吴东颖
万磊
金海峰
冯健海
强邦明
马洪华
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Jinan Xingyi Medical Technology Co Ltd
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Jinan Xingyi Medical Technology Co Ltd
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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Abstract

The invention provides a kind of T cell preparation of mesothelin Chimeric antigen receptor modification and the application on treatment of pancreatic cancer, disclose a kind of Chimeric antigen receptor, especially in conjunction with a granular leukocyte macrophage cluster stimulating factor receptor as pilot sequence Chimeric antigen receptor；The invention further relates to express the T cell of the Chimeric antigen receptor, the T cell can the high expression mesothelin of target killing tumor cell, and the purposes with the medicine that can prepare treatment cancer of pancreas malignant tumor and disease of viral infection.

Description

In a kind of T cell preparation of mesothelin Chimeric antigen receptor modification and treatment of pancreatic cancer Application
Technical field
The present invention relates to biological and new medical technology field, in particular, is that a kind of mesothelin Chimeric antigen receptor is repaiied Application in the T cell preparation of decorations and treatment of pancreatic cancer.
Background technology
Cancer of pancreas (pancreatic carcinoma) is that a kind of grade malignancy is high, all highly difficult digestion of diagnosis and treatment Road malignant tumor, about 90% is the duct adenocarcinoma for originating from glandular tube epithelium.The diagnosis rate of cancer of pancreas early stage is low, and cure rate is extremely low, It is one of worst malignant tumor of prognosis, 5 years survival rates<1%.Cause the transitivity that main causes of death are that cancer of pancreas causes Disease, the infiltration and transfer of tumor are a continuous processes, are mediated by adhesion molecule, make extracellular matrix by protease (extracellular, ECM) degrades and angiogenesis.Cancer of pancreas can betide the head of pancreas, body, afterbody or involve whole pancreas Gland, but, account for the 60%～70% of cancer of pancreas with Diseases In Pancreatic Head at most, person takes second place to betide body of pancreas, and afterbody is most rare.Naked eyes are lower swollen Tumor is rounded or oval, and border diffuses infiltration and closes on pancreatic tissue and be difficult to differentiate.The treatment of cancer of pancreas is well-known A difficult problem, it convenes the natural immune system of human body to build a firm physical barriers around tumor, makes kill tumor cell Immunocyte be unable to penetration barriers.And, pancreatic tumour tissue can be survived under quite deficient blood supply, therefore, pass System chemotherapy (blood circulation administration) is hardly entered inside tumor.
Chimeric antigen receptor (Chimeric antigen receptor, CAR) is a kind of by target cells antigen receptor With the synthesis of receptor of T cell signaling molecule fusion, by increasing capacitance it is possible to increase the targeting of T cell, kill ability and duration of action. In recent years, CAR-T cells achieve surprising breakthrough in the treatment of B cell hematologic system tumor.For example, in clinical practice In, the targeting killing effect and high remission rate (80～90%) of the CAR-T cells Refractory and relapsed acute leukemia cell positive to CD19 Clinical efficacy.The fact that not only reconfirmed the clinical value of immune cell therapy, while also excite scholar for The expansion enthusiasm of entity tumor related antigen and CAR-T cell technologies.But be faced with using CAR-T cells in solid tumor all Difference in many problems, entity tumor and neoplastic hematologic disorder speciality itself determines that this Therapeutic mode must consider its cell again The factors such as the characteristics for the treatment of itself, action rule.For example, solid tumor has height heterogeneity, rank character, metastatic feature, Mainly from CAR-T Therapeutic safeties, the selection of entity tumor target spot, the combined therapy of various CAR-T Mutiple Targets, CAR-T cells Go back to the nest in vivo and activate maintain on regard to CAR-T treatment of solid tumors strategy.
Mesothelin (Mesothelin) is one kind produced by the mesothelial cell (mesothelial cells) in body cavity The glycoprotein of cell surface glycosylation phosphatidylinositols connection, molecular weight is 40kDa, is the tumor cell surface of Early pancreatic carcinoma Molecular marker.Almost all of Pancreatic Adenocarcinoma all excess generation mesothelins, and a small amount of expression in the normal tissue, are ground in early days Study carefully and show, in all 74 Pancreas cancer patients, have the level of 73 blood samples of patients mesothelins will be apparently higher than Healthy People.The U.S. The research of FredHutchinson DKFZs finds that mesothelin can be recognized and attack as the design target spot of CAR-T cells Pancreatic cancer cell, the research show that the animal of the T cell treatment for receiving the non-cancer protein of identification is put down after they detect cancer of pancreas Survive 54 days, receive the animal the average survival time of CAR (mesothelin)-T cell treatment 96 days, i.e., can extend in cancer of pancreas late period 78% life cycle, the achievement in research are also published in《Cancer Cell》On.As tumour patient is to the immune resistance to of autoantigen By the immunologic escape with tumor cell so that the effect of immunization therapy is limited to, in order to overcome these obstacles, present invention design The specific for tumour antigen T cell of expression Chimeric antigen receptor (CAR-mesothelin), can specifically with expression phase The tumor cell specific for closing antigen (mesothelin) is combined, and reaching directly carries out the purpose of tumor-killing.
The content of the invention
The invention provides one can be with reference to the Chimeric antigen receptor (CAR) of granulocyte-macrophage cluster factor acceptor, table It is used to prepare treatment malignant tumor and disease up to the immunoreactive cell of the Chimeric antigen receptor, and the immunoreactive cell Purposes in the medicine of malicious infectious disease.
The solution of the present invention is：
A kind of detached Chimeric antigen receptor, the Chimeric antigen receptor include the anti-of the tumors such as Humanized anti-human cancer of pancreas It is former --- the single-chain antibody of mesothelin, membrane spaning domain, costimulatory signal conducting region and CD3 ζ signal transduction domains, and combine One granulocyte-macrophage cluster stimulating factor receptor is used as pilot sequence.
Used as preferred technical scheme, the Chimeric antigen receptor includes the anti-of the tumors such as Humanized anti-human cancer of pancreas It is former --- the single-chain antibody of mesothelin, the Hinge areas of CD8 and transmembrane region, the intracellular signal domain of CD28, CD137 and CD3 ζ Nucleotide sequence be connected in turn, and combine a granulocyte-macrophage cluster stimulating factor receptor as pilot sequence.
The present invention also provides a kind of Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ and repaiies The preparation method of the T cell of decorations, by the antigen of the tumors such as Humanized anti-human cancer of pancreas --- the single-chain antibody of mesothelin, CD8 Hinge areas and transmembrane region, the nucleotide sequence of the intracellular signal domain of CD28, CD137 and CD3 ζ are connected in turn, and tie Unification granulocyte-macrophage cluster stimulating factor receptor as pilot sequence, obtain encoding chimeric antigen receptor (GM-CSF)- The fusion gene fragment of scFv (meso)-CD8-CD28-CD137-CD3 ζ, by fusion gene fragment insertion slow viruss expression Carrier, is packaged into the slow viruss of carrying (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ encoding genes, will be described The slow virus infection autologous patient monokaryon for carrying (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ encoding genes is thin The heterogeneous T lymphocytes of born of the same parents' induction, obtain expressing Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137- The T lymphocytes of CD3 ζ.
As preferred technical scheme, described encoding chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8- The fusion gene fragment of CD28-CD137-CD3 ζ is the nucleotide sequence shown in sequence table SEQ ID NO.1.
Used as preferred technical scheme, the CIK of the autologous patient lymphocyte induction is prepared as follows：Take outside autologous patient All blood, separating peripheral blood mononuclear cells, with the culture medium inducing culture 24 hours containing recombinant interferon after, add restructuring white Cytokine, the autologous patient blood plasma induction of OKT-3 and 5% continue culture 24 hours；Every three days multiple proportions liquid feedings, cultivate to 14 days, the positive expression rate of molecular marker CD3+, CD56+ of Flow cytometry CIK cell；When CD3+ positive rates>80%, The double positive rates of CD3+CD56+>20%, it is considered as CIK and induces the CIK for successfully harvesting the induction of autologous patient lymphocyte.
As preferred technical scheme, by the carrying (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ The CIK of the slow virus infection autologous patient lymphocyte induction of encoding gene is operated as follows：Carrying (the GM-CSF)-scFv (meso), after the slow-virus transfection 293T cells of-CD8-CD28-CD137-CD3 ζ encoding genes, the 293T cells release is slow sick Malicious granule, the lentiviral particle infected patient T autologous lymphocytes.
As preferred technical scheme, encoding chimeric antigen receptor (GM-CSF)-scFv (the meso)-CD8-CD28- The fusion gene fragment of CD137-CD3 ζ is produced as follows：Encoding granulocyte-macrophage cluster is stimulated by gene synthesis technology Factor acceptor, the Hinge areas of CD8 and transmembrane region, the nucleotide sequence structure of the intracellular signal domain of CD28, CD137 and CD3 ζ Into fusion fragment (GM-CSF)-CD8-CD28-CD137-CD3 ζ, then by Overlap extension PCR is by scFv (meso) and merges Fragment (GM-CSF)-CD8-CD28-CD137-CD3 ζ are spliced into encoding chimeric antigen receptor (the GM-CSF)-scFv (meso) genetic fragment of-CD8-CD28-CD137-CD3 ζ.
Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ described in claim 3 are repaiied The T cell of decorations is used for the purposes of the medicine for preparing treatment malignant tumor.
The malignant tumor is cancer of pancreas, but is not limited to cancer of pancreas, can also treat mesothelioma and pulmonary carcinoma, mammary gland The tumors such as cancer, ovarian cancer.
As a result of above-mentioned technical proposal, by the antigen of the tumors such as Humanized anti-human cancer of pancreas --- mesothelin it is single-stranded Antibody, the Hinge areas of CD8 and transmembrane region, the nucleotide sequence of the intracellular signal domain of CD28, CD137 and CD3 ζ connect successively Pick up and, and a granulocyte-macrophage cluster stimulating factor receptor is combined as pilot sequence, obtain encoding chimeric antigen and receive The fusion gene fragment of body (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ, the fusion gene fragment is inserted Enter Lentiviral, be packaged into carrying (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ encoding genes Slow viruss, by the slow virus infection of the carrying (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ encoding genes Autologous patient mononuclear cell induction heterogeneous T lymphocytes, obtain express Chimeric antigen receptor (GM-CSF)-scFv (meso)- The T lymphocytes of CD8-CD28-CD137-CD3 ζ, antigen binding domain include a guiding subsequence.Subsequence is placed on gently for guiding The amino terminal of chain variable region.Guiding subsequence in the present invention is human granulocyte-macrophage cluster stimulating factor (GM-CSF) Receptor sequence, granulocyte-macrophage cluster stimulating factor are a kind of single polymer glycoproteins, and its effect is swashed by signal transduction The transcription and translation of living cells, strengthens expression of the recombinant nucleotide sequence in host cell.
In the present invention, the T lymphs of Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ are thin Each module of born of the same parents.
(1) antigen binding domain (scFv)
Antigen binding domain of the Chimeric antigen receptor (CAR) of the present invention there is provided antibody, antigen binding domain it is recognizable and with The specific antigen of special target cells is combined.Specific antigen in the present invention is mesothelin antigen.Antigen binding domain can be one Individual antigen binding site, the present invention use single-stranded variable region (scFv) antibody fragment.
The single-stranded variable region of the present invention includes weight chain variable district, light chain variable district and connexon, is the anti-mesothelin of humanization The single-stranded variable region of antigen.The present invention includes a connexon, and weight chain variable district and the light chain variable district of antigen variable region pass through Connexon connects.Single-stranded variable region sequences are shown in sequence table SEQ ID NO.3.
The funtion part in Chimeric antigen receptor in the present invention, may be in the amino of these functional parts and/or carboxyl End is added with extra aminoacid, and the aminoacid of these extra additions does not disturb the biological work(of Chimeric antigen receptor funtion part Can, for example, target cell is recognized and combined, reach the purpose for the treatment of and prevention.On the contrary, the aminoacid of these extra additions can add The biological function of strong Chimeric antigen receptor.Aminoterminal such as in glutamine adds an arginine, the amino end of threonine End addition L-Tyrosine etc., to increase the affinity of Chimeric antigen receptor and specific antigen (mesothelin).Gln-Asn, Thr- Tyr, Cys-Met, Ala-Ile, Phe-Val, Pro-Gly.
There is " functional variant thereof " in the Chimeric antigen receptor in the present invention, refer to the aminoacid sequence in Chimeric antigen receptor There is change, aminoacid sequence therein addition occurs, deletes or change.In the present invention aminoacid sequence change without interference with Or the biological function of suppression Chimeric antigen receptor, conversely, these functional variant thereofs, in can strengthening the present invention, chimeric antigen is received The biological function of body.Such as conversion A/G-G-C/T-A/T and AGX in sequence, X herein can be C or T.
(2) guide sub (leader)
The antigen binding domain of the present invention includes a guiding subsequence.Guiding subsequence is placed on the amino end of light chain variable district End.Guiding subsequence in the present invention is human granulocyte-macrophage cluster stimulating factor (GM-CSF) receptor sequence, granulocyte- Macrophage cluster stimulating factor is a kind of single polymer glycoprotein, and its effect is the transcription by signal transduction active cell and turns over Translate, strengthen expression of the recombinant nucleotide sequence in host cell.Sequence is shown in sequence table SEQ ID NO.2.
(3) transmembrane region (transmembrane domain, TM)
Chimeric antigen receptor (CAR) in the present invention includes the list in a transmembrane region, transmembrane region and Chimeric antigen receptor Chain variable region is connected.Transmembrane region can by the α chains of φt cell receptor, β chains and ζ chains CD8 or CD28, CD3 λ, CD45, CD4CD5, CD9, CD16, CD22, CD33, CD47, CD80, CD86, CD134, CD154 etc. are constituted.The transmembrane region of Chimeric antigen receptor is main It is made up of hydrophobic amino acid, as leucine, proline, Phenylalanine, and tryptophan etc. are constituted.Transmembrane region in the present invention by CD8 α hinge regions (CD8 α Hinge) and CD28 are constituted, CD8 α hinges and CD28 nucleotide sequences be humanization CD8 α hinges and CD28 nucleotide sequences are shown in sequence table SEQ ID NO.4, SEQ ID NO.5..
(4) Intracellular signals area (intracellular domain, ICD)
, comprising an intracellular T cell signaling zone, intracellular region is thin with immunity for Chimeric antigen receptor (CAR) in the present invention The effector functions activation of born of the same parents is relevant.The effector functions of immunocyte include immunocyte to the thin of target cell (tumor cell) The secretion of cellular lysate function and cytokine.Intracellular signals area refers to that the protein molecular in this region can be transduceed effector work( Energy signal, and guides cell to perform some special functions, as making cell released both particle enzyme, ceasma element killing tumor cell, together When also discharge cytokine, as tumor necrosis factor, gamma interferon, interleukin-2 etc..
Intracellular signals area includes CD28, CD137 (4-1BB), and CD3 ζ.CD28, CD137 and CD3 ζ is humanization CD28, CD137 and CD3 ζ.CD28 is the important symbol of T cell costimulation, and CD137, also referred to as 4-1BB strongly can transduce Costimulatory signal strengthens the differentiation of T cell to T cell, extends the life span of T cell.CD3 and TCR produces signal and includes The activation motif (ITAMs) of immunity receptor L-Tyrosine, its major function is the signal transduction for participating in immunocyte.In the present invention The intracellular region gene order of CD28, CD137, CD3 ζ is shown in sequence table SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7.
(6) recombinant expression carrier
Recombinant expression carrier used in the present invention is slow virus carrier pLent-C-GFP.PLent-C-GFP expressing genes Carrier contains original copy (origin of replication), the promoter sequence of a cytomegaloviruses , and restriction endonuclease sites (KpnI, AsiSI etc.), selective resistant gene (anti-ampicillin) and label egg (CMV) In vain (green fluorescent protein).By the polymerase Chimeric antigen receptor (CAR) that even reaction (PCR) synthesizes, i.e. target dna, in T4 Under connection enzyme effect, it is connected with carrier DNA, forms complete vector pLent- (the GM-CSF)-scFv of expression Chimeric antigen receptor (meso)-CD8-CD28-CD137-CD3 ζ are shown in Fig. 1.
(7) host cell
Host cell used by the present invention is a kind of heterogeneous T lymphocytes --- the CIK (killings of cytokine profiles induction Cell, cytokine-induced killer).CIK is that human peripheral blood single nucleus cell is used cytokine profiles in vitro The a group cell that (such as CD 3-resisting monoclonal antibody, IL-2 and IFN-γ etc.) co-cultivation is obtained for a period of time afterwards.In CIK cell Effector lymphocyte (CD3+CD56+) it is few in normal human peripheral blood, only 1%～5%.Relatively with common T lymphocytes, CIK has the advantage that：(1) CIK cell value-added speed is fast；(2) CIK have identification tumor mechanism, to normal cell without Toxic action；(3) kill tumor spectrum wide, can be used for the treatment of the kinds of tumors such as leukemia, lymphoma, pulmonary carcinoma, gastric cancer, intestinal cancer；(4) allusion quotation The personalized biological Therapeutic mode of type, by transgenic T cells receptor (T cell receptors, TCRs) and expression inosculating antibody The gene engineering method such as original receptor (chimeric antigen receptors) improve CIK, and tumor precisely can be killed； (5) as CIK is the autogenous cell of activation, using safety.
(8) application approach and dosage of Jing CAR transfecting T cells
Include the T cell of Chimeric antigen receptor in the present invention, can be autologous T cell, or homologous T cell.In the present invention, T cell used is Autologous T cells.The quantity of T cell used is 0.5x106-1x109/Kg.Generally The dosage of T cell used is 0.5x106-1.0x107/kg。
Description of the drawings
Fusion bases of the Fig. 1 for encoding chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ Because of the design drawing of fragment.
Fig. 2 is slow viruss expression plasmid of the present invention (pLent- (GM-CSF)-scFv (meso)
- CD8-CD28-CD137-CD3 ζ) schematic diagram, wherein, sequence clockwise is positive genetic fragment, is counterclockwise Cdna reverse fragment.
Fig. 3 is the CIK Stereo microscope downward view figures of peripheral blood lymphocyte induction of the present invention.
Fig. 4 is the surface molecular labelling CD3+ of the CIK of peripheral blood lymphocyte of the present invention induction, the streaming of CD56+ expression (B1+B2 quadrants show CD3+ to figure；B2+B4 quadrants show CD56+；B2 quadrants show CD3+, and the double positive rates of CD56+ are 25.1%).
Fig. 5 is slow viruss expression plasmid of the present invention (pLent- (GM-CSF)-scFv (meso)-CD8-CD28- CD137-CD3 ζ) transfection 293T cell bright field figures.
Fig. 6 is observation slow viruss expression plasmid (pLent- (GM-CSF)-scFv (meso)-CD8- under the green fluorescence visual field CD28-CD137-CD3 ζ) transfection 293T cells transfection efficiency figure.
Fig. 7 is all of Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ of the invention The T cell Stereo microscope downward view figure of modification.
Fig. 8 is green fluorescence basis of microscopic observation Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28- The T cell expression efficiency figure of CD137-CD3 ζ modifications.
Fig. 9 is flow cytometry Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 The efficiency of the expression CAR of the T cell of ζ modifications is 16.5%.
Specific embodiment
In order that technological means, creation characteristic, reached purpose and effect that the present invention is realized are easy to understand, tie below Specific embodiment is closed, the present invention is expanded on further.
Embodiment of the present invention is described in detail below in conjunction with embodiment, those skilled in the art will manage Solution, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted concrete bar in embodiment Part person, the condition advised according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, being can With by city available from conventional products.
The present invention compares the periphery hemolymph of the peripheral blood lymphocyte through Chimeric antigen receptor transfection and non-transfection Cell is co-cultured with several tumor cells, which includes the tumor cell of mesothelin secretion, the secretion situation of IFN-γ.
Embodiment one：Will be fusion gene fragment (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ insertions slow Virus expression carrier pLent-C-GFP.
The CAR modules of Anti-meso are illustrated to see Fig. 1 (complete nucleic-acid sequences are shown in annex SEQ ID NO.1).
The each sequence of modules of CAR of Anti-meso
(1) GM-CSF signals peptide nucleic acid(PNA) artificial sequence (SEQ ID NO.2)
(2)Anti–mesothelin monoclonal antibody single chain Fv antibody(scFv) Nucleic acid artificial sequence (SEQ ID NO.3)
(3) CD8 α Hinge areas nucleic acid artificial sequence (SEQ ID NO.4)
(4) CD28 transmembrane regions and intracellular region nucleic acid artificial sequence (SEQ ID NO.5)
(5) CD137 intracellular regions nucleic acid artificial sequence (SEQ ID NO.6)
(6) CD3 ζ intracellular regions nucleic acid artificial sequence (SEQ ID NO.7)
Press respectively the nucleic acid artificial sequence of signal peptide, the nucleic acid artificial sequence of Anti-meso, the nucleic acid artificial sequence of CD8, The nucleic acid artificial sequence of linker, the nucleic acid artificial sequence of CD28TM, the nucleic acid artificial sequence of CD28ICD, the nucleic acid people of CD137 Operation row, the nucleic acid artificial sequence of CD3 ζ, commission Sangon Biotech (Shanghai) Co., Ltd. synthesize its whole expression cassette, insert PLent-C-GFP carriers (Invitrogen) KpnI-AsiSI sites (see Fig. 2), is transformed into E.coli (DH5 α), and Jing is just sequenced After really, extracted and plasmid purification using the plasmid purification kit of Qiagen companies, obtain the high-quality of each recombinant expression carrier Plasmid.
Embodiment 2：The T of Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ modifications is thin The preparation of born of the same parents
(1) heterogeneous T cell --- the preparation of CIK
75ml autologous patient peripheral bloods are taken, with TBD sample rate separating liquids (biological purchased from Tianjin Hao oceans China Tech), is separated outer All blood mononuclear cells.(it is purchased from the culture medium of the recombinant interferon alpha-2a containing 1000IU/ml (being purchased from the pharmacy of the Shenyang three lives) CORNING companies, 88-551-CM) after 24 hours, the recombinant interleukin 2 of addition 1000IU/ml (is purchased from Shen to inducing culture Positive three lives pharmacy), the induction of the autologous patient blood plasma of the OKT-3 of 50ng/ml and 5% continue culture 24 hours.Every three days multiple proportions Liquid feeding, cultivated to the 14th day, the positive expression rate of CD3+, CD56+ in Flow cytometry CIK cell (CD3-FITC, CD16/CD56-PE antibody is purchased from BECKMAN companies, A07735).CD3+ positive rates>The double positive rates of 80%, CD3+CD56+> 20%, it is considered as CIK and induces successfully (see Fig. 3, Fig. 4), and leave and take the CIK and treats viral infection.
(2) slow viruss packaging plasmid liposome transfection 239T cells
Cell culture enables second day cell to grow to 70-80% completely in six orifice plates.To change in six orifice plates before transfection The fresh medium of 2ml, by LipoFiterTMLipofectamine (being purchased from Chinese Hang Seng thing) is mixed, (GM-CSF)-scFv (meso) tri- kinds of plasmids of-CD8-CD28-CD137-CD3 ζ and helper plasmid psPAX2, pMDNA2G are with 4:3:1 ratio is by 4.0 μ The DNA of g is dissolved in the DMEM culture medium (purchased from Life Technologies, Inc. of the U.S.) of 100 μ L, while the LipoFiter of 12 μ LTMIt is molten In the DMEM culture medium of 88 μ L, 5 minutes are stored at room temperature.DNA and LipoFiter by more than in two stepsTMMixing, incubation at room temperature 20 minutes.By LipoFiterTMDuring-DNA mixture adds a hole of six orifice plates, eight words wave mixing, after cultivating 6 hours, go Except LipoFiterTM- DNA culture fluid, adds fresh culture to continue culture.After transfection 48 hours, fluorescence microscope 239T In cell, the expression (see Fig. 5, Fig. 6) of GFP, determines expression plasmid pLent- (GM-CSF)-scFv (meso)-CD8- The transfection efficiency of CD28-CD137-CD3 ζ.To contain in virulent cells and supernatant suction EP pipes, 4 DEG C, 2000g centrifugations 10min, is transferred in new EP pipes, and after 4.5 μm of filters are filtered, -80 DEG C preserve.
(3) slow virus infection T cell and infection after T cell amplification cultivation
2ml virus liquids are taken out from -80 DEG C, the polybrene (being purchased from Sigma companies) of final concentration of 8 μ g/ml is added, with the disease Venom resuspended 1 × 106The T cell of individual above-mentioned induction.Cell suspension is added in 1 hole of 6 orifice plates, make virion number with T cell number ratio is about 3:1,1000g, 32 DEG C, it is centrifuged 90 minutes.37 DEG C, 5% CO2After cultivating 8 hours in incubator, receive Collection cell, rejoins virus liquid and polybrene, 1000g, 32 DEG C, and recentrifuge is after 90 minutes, 37 DEG C, 5% CO2Incubator It is middle to continue culture, multiple infection is so repeated, the efficiency of infection of T cell is improved.2ml culture supernatants are abandoned in suction, add 2ml's Fresh CORNING culture medium, continues amplification culture, cultivates 17 days to cell amplification to enough consumptions.
(4) immunofluorescence microscopy Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137- Expression of the CD3 ζ in T cell, detects (GM-CSF)-scFv (meso)-CD8-CD28-CD137- using Flow Cytometry Expression efficiencies of the CD3 ζ in T cell.
With the resuspended T cell of 100 μ L normal saline, obtained cell suspension makes cell smear, uses fluorescence microscope antigen Expression efficiencies of receptor (GM-CSF)-scFv (the meso)-CD8-CD28-CD137-CD3 ζ in T cell (see Fig. 7, Fig. 8)
2mL infection cells are taken out from culture bottle, using flow cytomery infection cell FITC (isothiocyanate) The positive expression rate (see Fig. 9) of infection cell in passage.
Embodiment 5：The killing effect in vitro of T cell strain after genetic modification is determined
By different effect target ratios (50:1,25:1,5:1,1:1), modified cell and unmodified T cell and A431, A431-H9, HAY are co-cultured, using LDH lactic acid dehydrogenases-cytotoxicity detection and analysis test kit (LDH-Cytotoxicity Assay Kit, Biovision) detect it is genetically modified after Cytotoxicity in vitro ability of the T cell to different type tumor 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, the spontaneous LDH of effector lymphocyte discharge control wells, treatment group hole, per group of 3 hole of repetition, the final volume in each hole It is identical and be no less than 100 μ L.250g is centrifuged 4min, and at 37 DEG C, 5%CO2 is incubated at least 4h.45min before centrifugation, to target cell Maximum release aperture adds 10 × lysate, volume correction hole to add the lysate of equivalent.Recentrifuge, from 0 μ L of every hole transferase 45 Clear to add 50 μ L substrate solutions into 96 new orifice plates, room temperature lucifuge is incubated 30min.50 μ L terminate liquids are added per hole, in 1h Determine D490.Cytotoxicity (%)=[(D experimental port-D culture medium backgrounds hole)-(spontaneous LDH release aperture-D cultures of D effector lymphocyte Base background hole)-(the spontaneous LDH release aperture-D culture medium background hole of D target cells)]/[(D target cells maximum LDH release aperture-D volumes Correction hole)-(the spontaneous LDH release aperture-D culture medium background hole of D target cells)] × 100%.
Embodiment 6：Expression Chimeric antigen receptor T cell clinical application method：
Present invention also offers include the application process of Chimeric antigen receptor T cell, including parenteral application, such as local Tumor injection, oral cavity, nasal cavity, subcutaneous, vein, tremulous pulse, intramuscular, Intradermal, in intraperitoneal, thoracic cavity etc..In specific environment, It is not limited to using a kind of Therapeutic Method, generally selects suitable, effective therapy approach.But it is now generally used for tumour patient Therapeutic Method be local tumor injection and venous re-transfusion.
The purpose of the present invention is to give animal or patient by the Chimeric antigen receptor (CAR) that doses are invented, and is passed through After certain hour, enough treatments or prophylactic response to be caused.For example, give the Chimeric antigen receptor in the present invention to be enough to With antigen binding, after certain hour, such as two hours or longer can be detected, and reach prevention or therapeutical effect.From It can be 12 hours that the application of Chimeric antigen receptor reaches prevention or the time of therapeutic purposes, or 24 hours, or it is longer.Give The dosage of Chimeric antigen receptor can be according to the efficiency of Chimeric antigen receptor in the present invention, the health of patient itself and treatment Depending on age of patient, body weight etc..
The Efficiency testing of Chimeric antigen receptor is included by giving receiving containing chimeric antigen for doses in the present invention After the T cell of body, the secretion situation of IFN-γ.By the T cell for giving the various dose that patient contains Chimeric antigen receptor, According to the different reaction condition of patient, the initial amount and total effective dose of medicinal application are determined.
Present invention additionally comprises the chemicalses of lymphocyte suppression are carried out before including the T cell application of Chimeric antigen receptor Treatment, radiotherapy etc..Commonly used chemicalses are cyclophosphamide and fludarabine, and its common dose is respectively 60mg/ Kg and daily 25mg/m2, but concrete application dosage will also be according to the general physical condition for treating effect to be obtained and patient And the body weight of patient is determined.
Include the T cell of Chimeric antigen receptor in the present invention, can be autologous T cell, or homologous T cell.In the present invention, T cell used is Autologous T cells.The quantity of T cell used is 0.5x106-1x109/Kg.Generally The dosage of T cell used is 0.5x106-1.0x107/kg。
As the Chimeric antigen receptor in the present invention is the Chimeric antigen receptor for mesothelin specific antigen, due to Skin element can be expressed in many cancers, and institute can use many cancers with the inventive method.Including ovarian cancer, cancer of pancreas, lung Cancer (adenocarcinoma of lung), the esophageal carcinoma, gastric cancer, synovial sarcoma and mesothelioma etc..
Treatment and prevention mentioned in the present invention, do not refer to 100% or fully treat or prevent, and refer to different The treatment or prevention of degree, i.e., after medicinal application, can have potential treatment or preventive effect.
The invention provides Chimeric antigen receptor, nucleotide, recombinant expression carrier, host cell and its in treatment and pre- Application during anti-.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to disclosed all teachings, various modifications and replacement can be carried out to those details, these changes are in the guarantor of the present invention Within the scope of shield.The four corner of the present invention is given by claims and its any equivalent.
Sequence table
<110>Shandong Xing Rui bio tech ltd
<120>A kind of application in the T cell preparation of mesothelin Chimeric antigen receptor modification and treatment of pancreatic cancer
<130> 2016
<160> 1
<170> PatentIn version 3.5
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atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ctttctgctc 60
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ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180
castgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240
ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300
acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360
tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420
gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480
gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540
atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600
ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660
cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcutc gctgcagccg 720
gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780
gggaccaagc tggagatcaa acgtgcggcc gcattcgtgc cggtcttcct gccagcaagc 840
ccaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc 900
tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac acgagggggc 960
tggacctcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt ggggtccttc 1020
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agtggtggag gcggttcagg cggaggtggc agcggcggtg gcggatcgga catccagatg 420
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aaactcctga tctataaggc ctctagttta gccagtgggg ccccatcaag gttcagcggc 600
agtggatctg ggacagattt cactctcacc atcagcagcc tgcagcctga tgattttgca 660
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cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180
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Claims (9)

1. a kind of Chimeric antigen receptor, it is characterised in that：The Chimeric antigen receptor includes the tumors such as Humanized anti-human cancer of pancreas Antigen --- the single-chain antibody of mesothelin, membrane spaning domain, costimulatory signal conducting region and CD3 ζ signal transduction domains, and With reference to a granulocyte-macrophage cluster stimulating factor receptor as pilot sequence.
2. a kind of Chimeric antigen receptor as claimed in claim 1, it is characterised in that：The Chimeric antigen receptor includes humanization The single-chain antibody of the antigen of the tumors such as anti-human cancer of pancreas --- mesothelin, the Hinge areas of CD8 and transmembrane region, CD28, CD137 and The nucleotide sequence of the intracellular signal domain of CD3 ζ is connected in turn, and stimulates with reference to a granulocyte-macrophage cluster Factor acceptor is used as pilot sequence.
3. one kind prepares the T cell of Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ modifications Method, it is characterised in that：By the antigen of the tumors such as Humanized anti-human cancer of pancreas --- the single-chain antibody of mesothelin, CD8 Hinge areas and transmembrane region, the nucleotide sequence of the intracellular signal domain of CD28, CD137 and CD3 ζ are connected in turn, and tie Unification granulocyte-macrophage cluster stimulating factor receptor as pilot sequence, obtain encoding chimeric antigen receptor (GM-CSF)- The fusion gene fragment of scFv (meso)-CD8-CD28-CD137-CD3 ζ, by fusion gene fragment insertion slow viruss expression Carrier, is packaged into the slow viruss of carrying (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ encoding genes, will be described The slow virus infection autologous patient monokaryon for carrying (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ encoding genes is thin The heterogeneous T lymphocytes of born of the same parents' induction, obtain expressing Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137- The T lymphocytes of CD3 ζ.
4. it is as claimed in claim 3 to prepare Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 The method of the T lymphocytes of ζ, it is characterised in that：Described encoding chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8- The fusion gene fragment of CD28-CD137-CD3 ζ is the nucleotide sequence shown in sequence table SEQ .ID.NO.1.
5. it is as claimed in claim 3 to prepare Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 The method of the T lymphocytes (CIK) of ζ, it is characterised in that the CIK of the autologous patient lymphocyte induction is prepared as follows：Take trouble Person's autologous peripheral blood, separating peripheral blood mononuclear cells, with the culture medium inducing culture 24 hours containing recombinant interferon after, plus The autologous patient blood plasma induction for entering recombinant interleukin, OKT-3 and 5% continues culture 24 hours；Every three days multiple proportions liquid feedings, Cultivate to the 14th day, the positive expression rate of molecular marker CD3+, CD56+ of Flow cytometry CIK cell；When CD3+ it is positive Rate>The double positive rates of 80%, CD3+CD56+>20%, it is considered as CIK and induces the CIK for successfully harvesting the induction of autologous patient lymphocyte.
6. it is as claimed in claim 3 to prepare Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 The method of the T lymphocytes of ζ, it is characterised in that by the carrying (GM-CSF)-scFv (meso)-CD8-CD28-CD137- The CIK of the slow virus infection autologous patient lymphocyte induction of CD3 ζ encoding genes is operated as follows：The carrying (GM-CSF)- After the slow-virus transfection 293T cells of scFv (meso)-CD8-CD28-CD137-CD3 ζ encoding genes, the 293T cells release Lentiviral particle, the lentiviral particle infected patient T autologous lymphocytes.
7. it is as claimed in claim 3 to prepare Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 The method of the T lymphocytes of ζ, it is characterised in that encoding chimeric antigen receptor (GM-CSF)-scFv (the meso)-CD8- The fusion gene fragment of CD28-CD137-CD3 ζ is produced as follows：By gene synthesis technology by encoding granulocyte-macrophage cluster Stimulating factor receptor, the Hinge areas of CD8 and transmembrane region, the nucleotides sequence of the intracellular signal domain of CD28, CD137 and CD3 ζ Row constitute fusion fragment (GM-CSF)-CD8-CD28-CD137-CD3 ζ, then by Overlap extension PCR is by scFv-meso and melts Close fragment (GM-CSF)-CD8-CD28-CD137-CD3 ζ and be spliced into encoding chimeric antigen receptor (the GM-CSF)-scFv (meso) genetic fragment of-CD8-CD28-CD137-CD3 ζ.
8. a kind of medicine for the treatment of cancer, it is characterised in that：Containing the Chimeric antigen receptor (GM-CSF) described in claim 3- The T cell of scFv (meso)-CD8-CD28-CD137-CD3 ζ modifications.
9. Chimeric antigen receptor (GM-CSF)-scFv (meso)-CD8-CD28-CD137-CD3 ζ modifications described in claim 3 T cell be used for prepare treatment malignant tumor medicine purposes.
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CN106544365A (en) * 2016-10-25 2017-03-29 山东兴瑞生物科技有限公司 A kind of preparation method and application of the CIK of the anti-CD19 Chimeric antigen receptors modification of people
CN107475276A (en) * 2017-09-05 2017-12-15 深圳大学 The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation are modified
CN107699591A (en) * 2017-05-09 2018-02-16 山东兴瑞生物科技有限公司 A kind of knockout PD 1 T cell preparation method and applications
CN109097396A (en) * 2018-09-10 2018-12-28 上海细胞治疗工程技术研究中心集团有限公司 A method of preparing the CAR-T cell of targeting mesothelin
WO2019020088A1 (en) * 2017-07-27 2019-01-31 上海细胞治疗研究院 Chimeric antigen receptor-modified t cell targeting mesothelin and use thereof
WO2019063018A1 (en) * 2017-09-30 2019-04-04 亘喜生物科技（上海）有限公司 Engineered immune cell having suicide gene switch and targeting human mesothelin
WO2019128996A1 (en) * 2017-12-28 2019-07-04 上海细胞治疗研究院 Mesothelin-specific car-t cell expressing cd47 antibody, and use thereof
CN110055269A (en) * 2019-03-20 2019-07-26 英威福赛生物技术有限公司 People's mesothelin Chimeric antigen receptor, its T cell and its preparation method and application
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