CN105384826A - Cord blood nucleated cell for expressing chimeric antigen receptor and application of cord blood nucleated cell - Google Patents
Cord blood nucleated cell for expressing chimeric antigen receptor and application of cord blood nucleated cell Download PDFInfo
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Abstract
The invention provides a chimeric antigen receptor (CAR) and relevant nucleic acids, expression vectors, cord blood nucleated cells and injection. According to a cord blood nucleated cell for expressing the chimeric antigen receptor, EGFRvIII positive tumor cells can be efficiently killed by virtue of CAR targeted EGFRvIII antigens; the cord blood nucleated cell can be used for preparing medicines for preventing and treating multiple EGFRvIII positive tumors such as neuroglioma, breast cancer, ovarian cancer, lung cancer and the like.
Description
Technical field
The present invention relates to tumour cell treatment field, more specifically, relating to the transgenic cell treatment field to expressing EGFRvIII positive tumor.
Background technology
Adoptive immunotherapy is by precursor cell that is autologous or allosome anti-tumour effect cell, in vitro with the induction such as IL-2, anti-CD49d McAb, activation and amplification, then transfers to patient, to reach the object for the treatment of and prevention of recurrence.Developed CTL, NKT, gamma delta T, the multiple adoptive immunotherapy such as TIL, CIK, DC-CIK, CAR-T at present, but these adoptive immunotherapy still has larger challenge on effects and side effects.CAR-T immunotherapy is state-of-the-art technology, and transplanted cells can increase nearly thousand times and survival reaches 6 months and continuous expression antigen receptor in vivo.Demonstrate good curative effect clinically, in blood system tumour, have 47 relevant reports, all achieve significant result for the treatment of.But CAR-T does not obtain desirable clinical efficacy on treatment solid tumor.In addition, the tumour specific antigen (TSA) of current discovery is less, the most of target tumor related antigen (TAA) of CAR-T cell therapy, tumor associated antigen is also expressed in other healthy tissues, therefore, CAR-T treatment usually can be attacked normal cell along with the serious cell that misses the target and bring new damage to patient.This treatment usually along with serious effect of missing the target, CAR-T cell challenges normal cell and bring new damage to patient.
EGFRvIII is a kind of saltant type of EGF-R ELISA, this mutant is at cell surface expression, tumour specific antigen (TSA), it can in the glioblastoma multiforme of 50%-60%, the mammary gland of 70%-80% and ovarian cancer and about 16% Expressions in Lung Cancer (Wikstrand etc., CancerRes.55:3140-3148 (1995)), be target site extremely attractive in oncotherapy.
At present, many scholars have prepared the CAR-T cell for EGFRvIII, achieve certain curative effect in vitro, but it does not obtain desirable curative effect in clinical trial in fragmentation test and animal experiment.Reason may have following some, first, malignant cell exist height heterogeneity, the tumour cell in same patient not all expresses this antigen, and the cell killing single type can not effect a radical cure tumour; Secondly, in tumour patient body, T cell is for a long time by the impact of tumour cell escape mechanism, and it may exist certain defect on genetics, and its lethal effect may reduce; Again, tumour patient immunologic hypofunction, although single CAR-T Transplanted cells can kill and wound specific tumor cell, but it may not reconstruction patients immunologic function, improves patient to the immunologic function of tumour.
CAR-T cell therapy also imply that the potential of its treatment solid tumor in blood system tumour good therapeutic action, and many scholars constantly improve to making a breakthrough in treatment of solid tumors CAR-T cell.The present invention uses cord blood nucleate cells as host cell, makes it express CAR by slow-virus transfection, in the Cytotoxicity in vitro of Wild type EGFR vIII positive cell and the positive glioma animal tumor treatment of EGFRvIII, achieve good effect.Cord blood medium size lymphocyte is relatively immature, and antigenicity is more weak, and is also rich in the multiple initiating cells such as hemopoietic stem cell, interstital stem cell, mesenchymal precursor cells and Epithelial precursor cell in bleeding of the umbilicus, may have the lasting curative effect of longer-term in oncotherapy.Cord blood is discarded blood source in addition, does not bear, wide material sources, be easy to Collection and preservation to donor; To be expected to become universal oncotherapy product.
Summary of the invention
First object of the present invention is build a kind of Chimeric antigen receptor (CAR).
Chimeric antigen receptor of the present invention (CAR) is one section of fusion polypeptide, and its aminoacid sequence is SEQIDNO:1; Described CAR comprises antigen recognition structural domain, membrane spaning domain and intracellular T cell intracellular signaling structural domain; Described antigen recognition structural domain comprises the calmodulin binding domain CaM of monoclonal antibody for EGF-R ELISA No. 3 mutant (EGFRvIII) or its variable region or its antigen.
According to the further feature of Chimeric antigen receptor of the present invention (CAR), described antigen recognition structural domain comprises: IgG κ signal peptide, the single chain antibody fragments (scFv) formed with connection peptides connection weight, light chain; The aminoacid sequence of described antigen recognition structural domain is SEQIDNO:2; The aminoacid sequence of described IgG κ signal peptide is SEQIDNO:3; The aminoacid sequence of described single chain antibody fragments (scFv) is SEQIDNO:4.
According to the further feature of Chimeric antigen receptor of the present invention (CAR), described membrane spaning domain comprises: hinge arrangement and cross-film district; The aminoacid sequence of described membrane spaning domain is SEQIDNO:5; The aminoacid sequence of described hinge arrangement is SEQIDNO:6; The aminoacid sequence in described cross-film district is SEQIDNO:7.
According to the further feature of Chimeric antigen receptor of the present invention (CAR), described intracellular T cell intracellular signaling structural domain comprises CD137 and CD247; The aminoacid sequence of described intracellular T cell intracellular signaling structural domain is SEQIDNO:8; The aminoacid sequence of described CD137 is SEQIDNO:9; The aminoacid sequence of described CD247 is SEQIDNO:10.
Present invention also offers a kind of nucleic acid molecule.Described nucleic acid molecule encoding Chimeric antigen receptor of the present invention (CAR).
Present invention also offers a kind of expression vector.Described expression vector contains nucleic acid molecule of the present invention.This carrier can high efficiency transduction cord blood nucleate cells stably express wherein.
The present invention devises the nucleic acid MD1-1-CD8 α-CD137-CD247 (being synthesized by JaRa bio tech ltd, Shanghai) of the Chimeric antigen receptor (CAR) of coding targeting EGFR vIII antigen, the wherein high affine single-chain antibody in conjunction with EGFRvIII (scFv) of MD1-1 coding, it is suddenlyd change by MD1 and is formed.CD8 α coding hinge and transmembrane structure, CD137, CD247 encode T cell activates and intracellular signaling structure.Then, this nucleic acid is connected to Fuw carrier (Fuw empty carrier is so kind as to give by Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health) and constructs Lentiviral Fuw-IgGk-MD1-1-CD8 α-CD137-CD247 and Isotype control carrier Fuw-EGFP thereof, by this carrier transfection 293T cell packaging virus, in one embodiment of the invention, the 293T cell of each 10cm ware can pack titre is 2 × 10
8the virus of TU, virus can be carried out frozen, and after recovery, cell titer will drop to original about 50%.
Present invention also offers a kind of cord blood nucleate cells of expressing Chimeric antigen receptor (CAR).
The cord blood nucleate cells of expression Chimeric antigen receptor (CAR) of the present invention contains nucleic acid molecule of the present invention, or containing carrier of the present invention, described nucleic acid molecule to cord blood nucleate cells, is expressed as Chimeric antigen receptor (CAR) target cell by virus, liposome, electrotransfection or Transposon System transfection.
Preferably, described cord blood nucleate cells derives from human or animal's bleeding of the umbilicus, be be separated by gradient density centrifugal, airflow classification, immunological magnetic bead sorting method the karyocyte group obtained, comprise: T cell, killer cell, hemopoietic stem cell, interstital stem cell, mesenchymal precursor cells and Epithelial precursor cell.
Present invention also offers the purposes of the cord blood nucleate cells of described expression Chimeric antigen receptor (CAR).
The cord blood nucleate cells of expression Chimeric antigen receptor (CAR) of the present invention can be used for the medicine preparing treatment or prevention neurospongioma, mammary cancer, ovarian cancer and lung cancer, this medicine is by CAR targeting EGFR vIII antigen, the cell mass of Efficient killing effect tumour cell, such as target killing EGFRvIII positive tumor cell, and with its for major ingredient prepare a kind of can the multiple EGFRvIII positive tumor of prevention and therapy as the medicine of the tumours such as neurospongioma, mammary cancer, ovarian cancer, lung cancer.
Present invention also offers a kind of cell injection.
Cell injection of the present invention comprises the cord blood nucleate cells of expression Chimeric antigen receptor (CAR) of the present invention.
Accompanying drawing explanation
Fig. 1 display be Lentiviral Fuw-CAR (A) for the Chimeric antigen receptor (CAR) of EGFRvIII antigen and Isotype control carrier Fuw-EGFP (B) structure iron thereof.Wherein Fuw-EGFP is so kind as to give by the biological hospital in Chinese Academy of Sciences Guangzhou and health research institute, and Fuw-CAR is formed for being connected on Fuw empty expression vector by nucleic acid MD1-1-CD8 α-CD137-CD247 (being synthesized by JaRa bio tech ltd, Shanghai).
That Fig. 2 shows is the nucleic acid electrophoresis figure that EcoR1 and BamH1 double digestion identifies slow virus plasmid, and wherein 1-6 swimming lane is respectively six Fuw-CAR Plasmid samples enzymes and cuts rear and protoplasm grain electrophorogram, and 7-8 is after Fuw-EGFP enzyme is cut and protoplasm grain electrophorogram.Show that the carrier size that the present embodiment builds conforms to design.
What Fig. 3 showed is the transfection efficiency using PEI transfection 293T cell packaging slow virus, and transfection efficiency is more than 90%.
Fig. 4 display be used alone the using method of t cell activation factor CD3 and concentration and T cell cultivation effect graph of a relation: result shows directly adds 80ng/mlCD3 when activating in the medium, and cell proliferation is the fastest.
Fig. 5 display be EGFP expression efficiency after Fuw-CAR and Isotype control Fuw-EGFP slow virus infection cord blood nucleate cells.
Fig. 6 display be the cord blood nucleate cells streaming qualification figure of transfection, in cell mass, major part is CD3 positive cell.
What Fig. 7 showed is from specimens, extract genome EGFRvIII express overview, and 1 swimming lane is positive, and 2 swimming lanes are negative, and positive is applicable to cell therapy of the present invention.
What Fig. 8 showed is that the present invention expresses the cell of CAR and Isotype control cell is positive to the EGFRvIII be separated from specimens and the kill capability of negative glial oncocyte.The cell of expressing CAR has significant lethal effect to EGFRvIII positive neurons glioma cell, and increases along with the increase of cell consumption.Compared with the control significant difference is not had to EGFRvIII negative glial cytotoxic effect ability.
What Fig. 9 showed is that the present invention expresses the cell of CAR and Isotype control cell is positive to the EGFRvIII be separated from specimens and the kill capability of negative breast cancer cells.The cell of expressing CAR has significant lethal effect to EGFRvIII positive breast cancer cells, and increases along with the increase of cell consumption.Compared with the control significant difference is not had to EGFRvIII negative breast cancer cells kill capability.
What Figure 10 showed is that the present invention expresses the cell of CAR and Isotype control cell is positive to the EGFRvIII be separated from specimens and the kill capability of negative ovarian cancer cell.The cell of expressing CAR has significant lethal effect to EGFRvIII positive ovarian cancer cells, and increases along with the increase of cell consumption.Compared with the control significant difference is not had to the negative ovarian cancer cell kill capability of EGFRvIII.
Figure 11 display be to express the expression amount of INF-γ in supernatant after the cord blood nucleate cells of CAR and Isotype control cell thereof and the EGFRvIII positive and negative neuroglial cytoma, breast cancer cell, ovarian cancer cell Dual culture.After expressing the cell of CAR and EGFRvIII positive cell Dual culture, the expression of INF-γ is significantly higher than and EGFRvIII negative cells Dual culture, and its Isotype control cell and EGFRvIII are positive and after negative cells Dual culture the expression of its INF-γ lower, there are no significant difference.
What Figure 12 showed is that the cord blood nucleate cells of expression CAR is on the impact of human glioma transplanted tumor immunodeficient mouse model.Transplant the growth of cord blood nucleate cells to immunodeficient mouse transplanted tumor expressing CAR and there is remarkable restraining effect.At the 10th day transplanted cells, within about 10 days afterwards, its tumor growth rate is significantly slack-off, its volume is reduction trend backward again, shows that the cord blood nucleate cells of expressing CAR has good Tumor suppression growth result and can effectively reduce killing tumor cell, reduces gross tumor volume.Transplant its Isotype control groups of cells and the still sustainable growth of PBS group tumour, there is no significant difference between the two.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Below in embodiment as the experiment of unreceipted actual conditions, then conveniently condition is carried out, and has clearly stated Reagent Company's specification sheets in an embodiment and be, then to specifications suggestion condition carry out.
Embodiment 1: the design & formulation of expressing the embedding antigen receptor of the present invention and expression EGFP homotype vector plasmid thereof.
1.CAR structure design
Chimeric antigen receptor of the present invention (CAR), it comprises the antigen-binding domains of people's antibody EGFRvIII, extracellular hinge domain, membrane spaning domain and intracellular T cell intracellular signaling structural domain.
Chimeric antigen receptor (CAR) is artificial constructed fusion rotein or polypeptide, they utilize the antigen-binding matter of monoclonal antibody to change T cell to the specificity of selected target and reactive ability in the mode that non-MHC limits, and walk around the main mechanism of tumor escape thus.
CAR of the present invention has antigen-specific to Urogastron receptor variant III (EGFRvII1).EGFRvIII is the variant of EGF-R ELISA (EGFR), is the most general in several EGFR sudden change found in human glioma, and expresses in the glioblastoma multiforme (GBM) of about 30% to about 50%.The expression of EGFRvIII is derived to be eliminated EGFR exon 2-7 and causes disappearance in the gene of the exons 1 of encoding sequence and the connection of 8 to reset.EGFRvIII by the tumor cells expression of various cancers, such as: glioblastoma, mammary cancer, ovarian cancer and nonsmall-cell lung cancer; Head and neck squamous cell carcinoma; Medulloblastoma, colorectal cancer, prostate cancer and the dirty cancer of wing etc.It is believed that antigen one specific reaction by causing for EGFRvIII, CAR of the present invention can target and destroy and express the tumour cell of EGFRvIII, and reduce or eliminate tumour, Promote immunity cell, to the infiltration of tumor locus, strengthens and extend antitumor reaction.Because EGFRvIII does not express in normal tissue, CAR of the present invention avoids destroying normal tissue and cell substantially.
Be called MD1-1 in conjunction with the scFv of EGFRvIIL specifically described in the present invention, its sequence is disclosed in patent CN01807175.9, and described document is incorporated herein by reference.MD1-1 is that the CDR3 heavy chain of MD1 and light chain mutant come, and is connected, have higher bonding force to EGFRvIII between variable region of light chain and variable region of heavy chain by joint peptide.Its aminoacid sequence comprises SEQIDNO:4, consisting of or consisting essentially of.
Antigen-binding domains described in the present invention comprises leader sequence IgG κ.Described leader sequence can be positioned at the aminoterminal of variable region of light chain.Its aminoacid sequence comprises SEQIDNO:3, consisting of or consisting essentially of.
The hinge domain that CAR described in the present invention comprises, membrane spaning domain comprise CD8 α, and its aminoacid sequence includes SEQIDNO:5, consisting of or consisting essentially of.
In the born of the same parents that CAR described in the present invention comprises, T cell intracellular signaling structural domain includes CD137 and CD3 ζ.CD137 effectively will can irritate intracellular signaling to T cell altogether, thus promotes differentiation and strengthen the lymphocytic long-term surviving of T.CD3 ζ and TCR combines to produce signal, and containing the activation motifs (ITAM) based on the cruel propylhomoserin of immunity receptor.Its aminoacid sequence comprises SEQIDNO:9 (CD137), SEQIDNO:10 (CD3 ζ), consisting essentially of or consisting of.
2. the structure of Lentiviral Fuw-GFP and Fuw-CAR.
Entrust JaRa bio tech ltd, Shanghai synthetic gene sequence BamHI-IgG κ-MD1-1-CD8 α-CD137-CD247-EcoR1, obtain carrier pUC57-IgG κ-MD1-1-CD8 α-CD137-CD247 (hereinafter referred to as CAR).Lentiviral Fuw-EGFP is built by this laboratory and preserves.
Fig. 1 display be Lentiviral Fuw-CAR (A) for the Chimeric antigen receptor (CAR) of EGFRvIII antigen and Isotype control carrier Fuw-EGFP (B) structure iron thereof.Wherein Fuw-EGFP is so kind as to give by the biological hospital in Chinese Academy of Sciences Guangzhou and health research institute, and Fuw-CAR is formed for being connected on Fuw empty expression vector by nucleic acid MD1-1-CD8 α-CD137-CD247 (being synthesized by JaRa bio tech ltd, Shanghai).
Respectively double digestion is carried out to plasmid Fuw-GFP and pUC57-CAR with BamHI and EcoRI, after agarose gel electrophoresis, glue reclaims the large fragment of Fuw-GFP and the size of the pUC57-CAR fragment at about 1400bp, after using SolutionI to connect, junction fragment is converted into competence intestinal bacteria, qualification result correct (Fig. 2) after amplification, thus obtain Lentiviral Fuw-GFP and Fuw-CAR.
3. plasmid transfection 293T packs slow virus
By 293T passage good for growth conditions to 10cm culture dish, cell proliferation is waited to change fresh culture to when being about 80-90% and carrying out transfection.
Get 15mL centrifuge tube, add 1mLOpti-MEM, then add 8 μ g plasmid Fuw-EGFP/Fuw-CAR, 2.5 μ g envelope plasmid pMD2G, 5.5 μ g packaging plasmid PSPAX2, shake up standing 5 minutes.Add 64 μ gPEI transfection reagents, firmly shake up, leave standstill 12-15 minute.Mentioned reagent is dropped to 293T cell surface, change liquid after shaking up 37 DEG C of cultivation 12h, then observe luciferase expression (Fig. 3) after cultivating 36h and gather in the crops supernatant, remove cell debris with 0.45 μm of frit, ultracentrifugation 25000rpm, 2.5h.Abandon supernatant, add the resuspended virus of T cell substratum, filtration sterilization-80 DEG C preservation.
4. slow virus titer determination
1 × 10 is diluted to after being counted by 293T cell dissociation good for growth conditions
5/ ml, adds 96 orifice plates, 100 μ L/ holes, for each virus prepares 10 holes.Prepare 10 1.5mLEP pipes, often pipe adds 90 μ L nutrient solutions, adds 10 μ L virus stock solution useds in first pipe, after mixing, draws 10 μ L and adds second pipe mixing.The rest may be inferred, is ten extent of dilution (10-10
-8).Drawing original substratum in 96 orifice plates, adding the virus liquid containing having diluted.Liquid feeding 100 μ L after 24h.Observations after transfection 48h also calculates titre: observations under fluorescent microscope, and counts that latter two has the fluorocyte clone number of fluorescence.Be assumed to be X and Y, then the content (μ l) of the virus liquid in titre (TU/ml)=(X+Y × 10) × 1000/2/X hole.
In one embodiment of the invention, each 10cm ware 293T cell packaging virus titre is about 2 × 10
8tU, after cryopreservation resuscitation, titre is about 1 × 10
8tU.
Embodiment 2: recombinant virus infection cord blood nucleate cells.
1. the condition being used alone CD3mAb activation Cord Blood Mononuclear Cell is groped
Activated lymphocyte method conventional is at present CD3/CD28 magnetic bead activation method, but this method introduces magnetic bead in cell, and later separation magnetic bead complex operation, adds the risk of pollution, and magnetic bead price is high, is unfavorable for that clinical-scale uses.Therefore, we have groped only to use CD3dmAb to activate the method for cord blood nucleate cells, and by the screening to its working concentration and using method, when being finally defined as directly adding the CD3mAb of 80ng/mL in substratum, cord blood nucleate cells propagation is comparatively fast shown in Fig. 4.
2. slow virus infection Cord Blood Mononuclear Cell and amplification thereof
From Healthy People bleeding of the umbilicus, karyocyte is separated with Percoll gradient centrifugation.With 1 × 10
6gT-T551 (Takara) the T lymphocytes culture medium that the density of cell/mL adds containing 300IU/mlIL-2 (Beijing Shuan Lu pharmaceutical Co. Ltd) is cultivated, and add the blood plasma of the same bleeding of the umbilicus of 5%, and the CD3mAb of 80ng/mL (Wei Ke bio tech ltd, Shanghai) activated T cell stimulates cultivation 48h, then cord blood nucleate cells is infected with the above-mentioned recombinant slow virus of MOI=5, metainfective cell changes liquid in second day, goes down to posterity every 2-3 days.
Harvested cell about cell cultures to 10 day, cell can increase 30-80 doubly, and still can increase after cell reactivation.Isotype control cell high expression level green fluorescent protein (Fig. 5) is can be observed under fluorescent microscope.And by various types of cells (T cell, NK cell, CIK cell etc.) ratio and CAR expression efficiency (Fig. 6) in the cord blood nucleate cells of flow cytometer detection technical Analysis transfection, visible most cells is CD3 positive t lymphocytes, include part CIK and a small amount of NK cell, also have in addition (data do not show) such as the hemopoietic stem cells of the CD34 positive.
Embodiment 3: the foundation of glioma cell line and EGFRvIII detection of expression thereof
1. neuroglial cytoma, breast cancer cell and ovarian cancer cell separation and Culture with go down to posterity
Get in art without necrosis, without cystis degeneration's sample, immerse DMEM/F12 fresh-keeping, carried out being separated, cultivating in 30 minutes.Remove necrotic tissue around, DMEM/F12 rinses 3 times, tissue block is cut into diameter 0.5mm size, pasteur pipet blows and beats into single cell suspension 70 μm of aperture sieve net filtrations repeatedly, centrifugal 5 minutes of 200g, reservation 1ml substratum in proportion 1:5 adds 37 DEG C of preheating erythrocyte cracked liquids effect 2 minutes, and centrifugal 5 minutes of 200g, abandons supernatant.Perfect medium re-suspended cell, the blue dyeing counting viable cell of placenta, with 2 × 10
5cell/mL is inoculated in culturing bottle, cultivates in containing the DMEM/F12 of 10% foetal calf serum.
The primary cell of firm separation starts adherent in about 4 hours, and within 24 hours, trail, within 48 hours, full extension is opened, and the cell of original cuiture starts propagation on the 3rd day, within the 6th day, can go down to posterity.Go down to posterity after 1-2 generation, Growth of Cells is accelerated, and about 3-4 days goes down to posterity once.The 7-8 that goes down to posterity is slack-off for rear primary cell growth, about 7-8 days goes down to posterity once, part cell shrinkage, rear appearance is old and feeble, dead, and now also bottle is cultivated again, adhere to changing liquid once in every 3 days, after 1-2 week, cell starts propagation, then within every 3 days, changes a not good liquor, passes a generation weekly, accelerate to the cellular-restoring growth of the 15th generation, about 3-4 days goes down to posterity once.
Cell growth curve is that in order to hide the adaptive phase, from the 3rd day cell, propagation was accelerated and entered logarithmic phase, within the 8th day, peaked, entered plateau later in after shape S inoculates 1-2 days.Population doubling time is about 60h.Cloning efficiency is about 4%.
2. tumour cell EGFRvIII expression identification
Synthetic primer (Shanghai Jierui Biology Engineering Co., Ltd)
EGFRvIII-R：5’-GATCCAGAGGAGGAGTATGTGTG-3’
EGFRvIII-F：5’-GTCCAGTATTGATCGGGAGAGC-3’
RNA isolation kit extracts neuroglial cytoma RNA (operation is shown in that day root total RNA from animal tissues extracts test kit (DP431) working instructions in detail), and reverse transcription becomes cDNA, and (FormentasRevertAid is shown in operation in detail
tMfirstStrandcDNASynthesiskit (K1622) working instructions).Carry out PCR with above-mentioned primer and cDNA, its reaction system is as follows:
Response procedures following (32 circulation):
PCR primer is carried out agarose gel electrophoresis qualification, and EGFRvIII positive cell has band at about 700bp and/or about 2800bp, and EGFRvIII negative cells has band, as Fig. 7 without band or about 2800bp.
Therefore, the present invention can set up the detection method whether a kind of cancer patients of detection is applicable to treating with expression Chimeric antigen receptor cord blood nucleate cells of the present invention: extract specimens DNA (or RNA reverse transcription becomes cDNA), PCR is carried out as template design primer, then detect in tissue whether express EGFRvIII by agarose gel electrophoresis method, if be positive, then the medicine being suitable for adopting cord blood nucleate cells of the present invention to prepare is treated.
Embodiment 4: express the cord blood nucleate cells of CAR to the killing effect in vitro of Wild type EGFR vIII positive tumor cell
Get the positive and negative tumor cells of the EGFRvIII growing to logarithmic phase, with the trysinization of 0.25%, be collected in 15ml centrifuge tube respectively, add after washing with DMEM in high glucose substratum that cell culture medium is resuspended and to adjust cell density be 10
5cell/mL.Be seeded to 96 orifice plates, 100uL/ hole, cultivate 24 hours.
By the following design (cord blood nucleate cells (A1:1:5, A2:1:10, A3:1:20, A4:1:40) of A:EGFRvIII+ cell+expression CAR; B:EGFRvIII+ cell+Isotype control cell (B1:1:5, B2:1:10, B3:1:20, B4:1:40); C:EGFRvIII+ cell; D: the cord blood nucleate cells of expressing CAR; E: Isotype control cell; F: substratum; The cord blood nucleate cells (G1:1:5, G2:1:10, G3:1:20, G4:1:40) of G:EGFRvIII-cell+expression CAR; H:EGFRvIII+ cell+Isotype control cell (H1:1:5, H2:1:10, H3:1:20, H4:1:40); I:EGFRvIII-cell); Add the porose substratum amount adjustment of rear institute consistent, Dual culture 20 hours.
Every hole adds 10uLCCK-8 reagent, mixing.Continuation cultivation in microplate reader, measured OD value after 1-4 hour under 450nm and 630nm dual wavelength.
The cord blood nucleate cells of expressing CAR kills and wounds EGFRvIII+ cell efficiency=[OD (A)-OD (D)]/[OD (C)-OD (F)] × 100%.
Isotype control cell killing EGFRvIII+ cell efficiency=[OD (B)-OD (E)]/[OD (C)-OD (F)] × 100%.
The cord blood nucleate cells of expressing CAR kills and wounds EGFRvIII-cell efficiency=[OD (G)-OD (D)]/[OD (I)-OD (F)] × 100%.
Isotype control cell killing EGFRvIII-cell efficiency=[OD (H)-OD (E)]/[OD (I)-OD (F)] × 100%.
The killing activity of the cord blood nucleate cells must not expressing Chimeric antigen receptor according to above detection method and negative cells positive to EGFRvIII, it the results are shown in Figure 8 (neuroglial cytomas), Fig. 9 (breast cancer cell), Figure 10 (ovarian cancer cell).As seen from the figure, the cord blood nucleate cells reaching Chimeric antigen receptor all has higher killing activity to three of the EGFRvIII positive kinds of tumour cells, and increases with the increase of cell usage quantity, has significant difference compared with the control.And it is to the killing activity of EGFRvIII negative cells, and difference is not remarkable compared with the control.
Embodiment 5:ELISA method detects the secretion of cytokine IFN-γ after cell of the present invention and target cell Dual culture
By EGFRvIII, positive and negative glial oncocyte, breast cancer cell and ovarian cancer cell are respectively with 2 × 10
5individual/hole adds in 2 24 well culture plates cultivates, 37 DEG C, 5%CO
2cultivate in incubator.Next day, every hole added 2 × 10 respectively after tumour cell is adherent
6the cord blood nucleate cells of individual expression CAR or and Isotype control cell, continue incubator Dual culture 24 hours.Get supernatant 500ul, the centrifugal 10min of 4000r/min, remove particle and polymkeric substance.The emission levels of IFN – γ is detected by human gamma-interferon (IFN-γ) ELISA detection kit (Yi Feng bio tech ltd, Shanghai).Concrete operation step is as follows:
With standard dilutions dilution standard product, make concentration be followed successively by 800,400,200,100,50,25pg/mL.Place the aluminium foil bag of 20 minutes from room temperature and take out micropore enzyme plate, after residue sealing, put back to 4 DEG C.Rotation standard sample wells and sample aperture, add the standard substance 50uL of different concns in standard sample wells.Respective markers is carried out in each hole of sample to be tested.3 multiple holes established by often kind of sample, first add sample to be tested 10uL, then add Sample dilution 40ul.Add the detection antibody of 100ul HRP mark to standard orifice and the every hole of sample aperture, seal reacting hole, put 37 DEG C of water-bath about 1h.Abandon most liquid, every Kong Zhongman washings, leave standstill 1 minute, again abandon most liquid, so repeat to wash plate 5 times.Every hole adds substrate A, each 50ul of B, in 15 minutes, measures each hole OD value at 450nm wavelength place.In Excel table, take standard concentration as X-coordinate, corresponding OD value is ordinate zou, draws out the linear regression curves of standard substance, calculates the concentration value of IFN-γ in sample by curvilinear equation.Its result is as Figure 11, after expressing the Cord Blood Mononuclear Cell of CAR and EGFRvIII positive cell Dual culture, the expression of INF-γ is significantly higher than and EGFRvIII negative cells Dual culture, and its Isotype control cell and EGFRvIII are positive and after negative cells Dual culture the expression of its INF-γ lower, there are no significant difference.
Embodiment 6: express the cord blood nucleate cells of Chimeric antigen receptor to the impact of human glioma transplanted tumor immunodeficient mouse model
1. set up immunodeficient mouse human glioma Transplanted tumor model
15ml centrifuge tube is collected in positive for the EGFRvIII growing to logarithmic phase and negative cells digestion, with DMEM washing, abandons supernatant.Adding the resuspended adjustment cell density of PBS is 5 × 10
7/ ml.With the tincture of iodine and the northern skin of ethanol disinfection immunodeficient mouse, it is subcutaneous that extraction 100ul cell is inoculated in right side of mice skin of back, oppresses pin hole for a moment after pulling out pin, sends SPF receptacle back to and raise after inoculation.After inoculation, every day observes mouse diet, and defecation and mental status are also weighed, and observes each injection point with or without red and swollen ulceration and with the length of vernier caliper measurement lump and width.Gross tumor volume assessment calculates by V=0.5 × a × b2 (a is length, and b is width).
2. cell infusion
When gross tumor volume reaches about 500mm
3time, nude mice is divided into 3 groups at random by often organizing 5.From tail vein transplantation cell or PBS in Mice Body.A group transplanting 1 × 10
7express the cord blood nucleate cells of Chimeric antigen receptor, B group transplants the EGFP compared with control cells of equal amts, and C group transplants PBS.After infusion, continue to observe mouse, weigh and measure assessment gross tumor volume, observing 2 months altogether.When gross tumor volume reaches 2000mm
3time, put to death mouse.Add up each group of data, draw a diagram.
3. experimental result: transplant the growth of cord blood nucleate cells to immunodeficient mouse transplanted tumor expressing CAR and there is remarkable restraining effect (Figure 12).At the 10th day transplanted cells, within about 10 days afterwards, its tumor growth rate is significantly slack-off, its volume is reduction trend backward again, shows that the cord blood nucleate cells of expressing CAR has good Tumor suppression growth result and can effectively reduce killing tumor cell, reduces gross tumor volume.Transplant its Isotype control groups of cells and the still sustainable growth of PBS group tumour, there is no significant difference between the two.
Embodiment 7: the preparation of cell injection
From bleeding of the umbilicus, cord blood nucleate cells is isolated by density gradient centrifugation method, use erythrocyte cracked liquid cracking wherein to use serum-free T lymphocytes culture medium GT-T551 (Takara) to add IL-2 after red corpuscle to cultivate, activate with independent CD3mAb, avoid immunomagnetic beads activate tedious steps and magnetic bead pollute, can be safer for clinical.
Activate latter 2nd day, with the virus infected cell packed (see embodiment 1 and 2), after infecting, the visible compared with control cells of 48-72h expresses green fluorescence.Within every 2-3 days, can carry out enlarged culturing, within 10-14 days, harvested cell quality examination salable product are frozen.
Add normal saline solution after recovery washing during use and make the cord blood nucleate cells suspension of expressing Chimeric antigen receptor.
Cell injection described in the present embodiment comprises the cord blood nucleate cells of expression Chimeric antigen receptor (CAR) of the present invention, can be used for preparation treatment or prevention neurospongioma, mammary cancer, ovarian cancer and lung cancer.
Claims (10)
1. a Chimeric antigen receptor (CAR), is characterized in that: described CAR is one section of fusion polypeptide, and its aminoacid sequence is SEQIDNO:1; Described CAR comprises antigen recognition structural domain, membrane spaning domain and intracellular T cell intracellular signaling structural domain; Described antigen recognition structural domain comprises the calmodulin binding domain CaM of monoclonal antibody for EGF-R ELISA No. 3 mutant (EGFRvIII) or its variable region or its antigen.
2. Chimeric antigen receptor according to claim 1 (CAR), is characterized in that, described antigen recognition structural domain comprises: IgG κ signal peptide, the single chain antibody fragments (scFv) formed with connection peptides connection weight, light chain; The aminoacid sequence of described antigen recognition structural domain is SEQIDNO:2; The aminoacid sequence of described IgG κ signal peptide is SEQIDNO:3; The aminoacid sequence of described single chain antibody fragments (scFv) is SEQIDNO:4.
3. Chimeric antigen receptor according to claim 1 (CAR), is characterized in that, described membrane spaning domain comprises: hinge arrangement and cross-film district; The aminoacid sequence of described membrane spaning domain is SEQIDNO:5; The aminoacid sequence of described hinge arrangement is SEQIDNO:6; The aminoacid sequence in described cross-film district is SEQIDNO:7.
4. the cord blood nucleate cells of expression Chimeric antigen receptor (CAR) according to claim 1, is characterized in that: described intracellular T cell intracellular signaling structural domain comprises CD137 and CD247; The aminoacid sequence of described intracellular T cell intracellular signaling structural domain is SEQIDNO:8; The aminoacid sequence of described CD137 is SEQIDNO:9; The aminoacid sequence of described CD247 is SEQIDNO:10.
5. a nucleic acid molecule, is characterized in that: described nucleic acid molecule encoding Chimeric antigen receptor according to claim 1 (CAR).
6. an expression vector, is characterized in that: described expression vector contains nucleic acid molecule according to claim 5.
7. express the cord blood nucleate cells of Chimeric antigen receptor (CAR) for one kind, it is characterized in that: it contains nucleic acid molecule according to claim 5, or containing carrier according to claim 6, described nucleic acid molecule to cord blood nucleate cells, is expressed as Chimeric antigen receptor (CAR) target cell by virus-mediated, liposome-mediated, the method transfection such as electrotransfection or Transposon System.
8. the cord blood nucleate cells of expression Chimeric antigen receptor (CAR) according to claim 7, it is characterized in that: described cord blood nucleate cells derives from human or animal's bleeding of the umbilicus, be be separated by gradient density centrifugal, airflow classification, immunological magnetic bead sorting method the karyocyte group obtained, comprise: T cell, killer cell, hemopoietic stem cell, interstital stem cell, mesenchymal precursor cells and Epithelial precursor cell.
9. the cord blood nucleate cells of expressing Chimeric antigen receptor (CAR) is as claimed in claim 7 for the preparation of the purposes of the medicine for the treatment of or prevention neurospongioma, mammary cancer, ovarian cancer and lung cancer.
10. a cell injection, is characterized in that, comprises the cord blood nucleate cells of expressing Chimeric antigen receptor (CAR) as claimed in claim 7.
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