CN105384823A - Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof - Google Patents
Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof Download PDFInfo
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Abstract
The invention discloses a chimeric antigen receptor and a gene and a recombinant expression vector thereof, an engineered CD33 targeting NKT cell and an application thereof. The chimeric antigen receptor is CD33ScFv-CD8-CD137-CD3zeta which is formed by connecting CD33ScFv, a hinge domain and a transmembrane domain of CD8, an intracellular signal structural domain of CD137 and an intracellular signal structural domain of CD3zeta in series. By adopting the chimeric antigen receptor CD33ScFv-CD8-CD137-CD3zeta modified NKT cell for treating CD33 positive acute myelogenous leukemia in a progressive stage, drug resistance and chemotherapy resistance caused by combined application of a CD33 monoclonal antibody and chemotherapy can be effectively avoided, and the cell has specific killing activity on leukemia cells.
Description
Technical field
The invention belongs to knubble biological arts, particularly, a kind of Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ in adoptive immunotherapy and gene thereof and recombinant expression vector, the NKT cell (CAR33-NKT cell) of through engineering approaches CD33 targeting and application thereof is related to.
Background technology
Acute myeloid leukemia (acutemyelocyticleukemia, AML) acute leukemia in all non-lymphocytes source is comprised, it is the Clonal malignant proliferative disorders of medullary system initiating cell of hemopoietic system, be a disease group with height heterogeneity, it can be transformed by the hemopoietic progenitor cell malignant change of different steps in normal myeloid cell differentiation and development process.
The initiating cell surface high expression level of the Patients with Acute Myeloid Leukemia of CD33 more than 90%, and CD33 is also expressed in leukemic stem cells surface, and only express low-level CD33 at the normal hemopoietic stem cell of part.In inflammation and immunne response process, the leukocytic function of CD33 controllable, therefore CD33 becomes the promising target for the treatment of acute myeloid leukemia.
At present, when the Patients with Acute Myeloid Leukemia of the treatment of advanced CD33 positive, monoclonal antibody and the combined use of chemotherapy of CD33 are in clinical investigation phase, but, clinical effectiveness shows, the monoclonal antibody of CD33 and combined use of chemotherapy can cause certain resistance and chemoresistance.
Summary of the invention
The resistance caused when the object of the invention is the Patients with Acute Myeloid Leukemia in order to overcome in prior art monoclonal antibody and the combined use of chemotherapy treatment of advanced CD33 positive adopting CD33 and the defect of chemoresistance, a kind of Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ and gene thereof and recombinant expression vector are provided, the NKT cell (CAR33-NKT cell) of through engineering approaches CD33 targeting and application thereof, the NKT cell that Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ modifies is when the acute myeloid leukemia of the treatment of advanced CD33 positive, effectively can avoid the resistance that causes when adopting the monoclonal antibody of CD33 and combined use of chemotherapy and chemoresistance, to leukemia cell, there is certain specific killing active.
The present inventor surprisingly finds under study for action, Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ of the present invention has high efficiency of infection to NKT cell, and the CAR33-NKT cell that obtains has in vitro and kills tumor activity efficiently after infecting, when adopting the acute myeloid leukemia of the CAR33-NKT cell therapy progressive stage CD33 positive simultaneously, effectively can avoid the resistance that causes when adopting monoclonal antibody and the combined use of chemotherapy of CD33 and chemoresistance, to leukemia cell, there is certain specific killing activity.
Therefore, to achieve these goals, first aspect, the invention provides a kind of Chimeric antigen receptor, described Chimeric antigen receptor is CD33ScFv-CD8-CD137-CD3 ζ, by the intracellular signal structural domain of the hinge area (hinge district) of CD33ScFv, CD8 and cross-film district, CD137 and the intracellular signal structural domain of CD3 ζ in series.
Second aspect, the invention provides the gene of above-mentioned Chimeric antigen receptor of encoding.
The third aspect, the invention provides the recombinant expression vector containing said gene.
Fourth aspect, the invention provides a kind of NKT cell of through engineering approaches CD33 targeting, and described NKT cell is the NKT cell that above-mentioned Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ modifies.
5th aspect, the NKT cell that the invention provides above-mentioned through engineering approaches CD33 targeting is for the preparation of the application in the leukemic preparation for the treatment of.
When the acute myeloid leukemia of the treatment of advanced CD33 positive, Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ of the present invention has high efficiency of infection to NKT cell, and the CAR33-NKT cell obtained after infecting, the i.e. NKT cell of through engineering approaches CD33 targeting, have in vitro and kill tumor activity efficiently, interleukin-22 can not relied in vivo and continue permanent existence, and can specific binding CD33 antigen, the obvious survival time of prolongation immunocyte in patient body, strengthen the ability of immunocyte targets identification acute myeloid leukemia cell surface C D33 antigen, the release cells factor is also strengthened the specific killing of acute myeloid leukemia cell active, and really effectively can avoid the resistance that causes when adopting the monoclonal antibody of CD33 and combined use of chemotherapy and chemoresistance.The NKT cell of through engineering approaches CD33 targeting of the present invention has lower toxic side effect, and patient has good tolerance, and the acute myeloid leukemia for the treatment of advanced CD33 positive provides a kind of selection newly, has good industrial application prospect.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Fig. 1 is the result that flow cytometry is analyzed the NKT cell phenotype of separation and Culture.
Fig. 2 is the electroresis appraisal figure of the restriction enzyme MluI/SalI double digestion fragment of Lentiviral pWPT-CD8-CD137-CD3 ζ of the present invention.
Fig. 3 is the electroresis appraisal figure of the restriction enzyme BamHI/SalI double digestion fragment of Lentiviral pWPT-CD33ScFv-CD8-CD137-CD3 ζ of the present invention.
Fig. 4 is the structural representation of Lentiviral pWPT-CD33ScFv-CD8-CD137-CD3 ζ of the present invention, and wherein, counterclockwise sequence is forward gene sheet degree, is cdna reverse fragment clockwise.
Fig. 5 is that Flow cytometry contains the viral concentration liquid of Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ to the efficiency of infection of NKT cell.
Fig. 6 is the result of NKT cell (CAR33-NKT cell) phenotypic evaluation that Flow cytometry Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ modifies.
Fig. 7 is the cytotoxicity analysis figure of CAR33-NKT cell of the present invention to the lethal effect of human myeloid leukemia cell.
Fig. 8 is that CAR33-NKT cell of the present invention is to body temperature in the Patients with Acute Myeloid Leukemia therapeutic process of the progressive stage CD33 positive and leukocyte count object changing trend diagram.
Fig. 9 is that CAR33-NKT cell of the present invention is to medullary cell change before and after the Patients with Acute Myeloid Leukemia treatment of the progressive stage CD33 positive.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The invention provides a kind of Chimeric antigen receptor, described Chimeric antigen receptor is CD33ScFv-CD8-CD137-CD3 ζ, by the intracellular signal structural domain of the hinge area of CD33ScFv, CD8 and cross-film district, CD137 and the intracellular signal structural domain of CD3 ζ in series.Under preferable case, the aminoacid sequence of Chimeric antigen receptor is as shown in SEQIDNO.1.
The invention provides the gene of above-mentioned Chimeric antigen receptor of encoding.Under preferable case, the nucleotide sequence of the gene of above-mentioned Chimeric antigen receptor of encoding is as shown in SEQIDNO.2.
The invention provides the recombinant expression vector containing said gene.Under preferable case, recombinant expression vector is Lentiviral.For Lentiviral, there is no particular limitation, as long as can with assistant carrier cotransfection packing cell as 293T packing cell, obtain the NKT cell of viral concentration liquid and Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ modification, under preferable case, Lentiviral is pWPT-CD33ScFv-CD8-CD137-CD3 ζ.
For the preparation method of Lentiviral pWPT-CD33ScFv-CD8-CD137-CD3 ζ, there is no particular limitation, the various methods can be able to expected for those skilled in the art, under preferable case, the preparation method of Lentiviral pWPT-CD33ScFv-CD8-CD137-CD3 ζ comprises the following steps:
(1) increase respectively the hinge district of CD8 and cross-film district, the intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 ζ from NKT cell cDNA, and be cloned in carrier pWPT-GFP, builds and obtain pWPT-CD8-CD137-CD3 ζ;
(2) nucleotide sequence of composite coding rat growth hormone signal peptide and CD33ScFv, and be cloned in pWPT-CD8-CD137-CD3 ζ, after sequence verification, obtain the pWPT-CD33ScFv-CD8-CD137-CD3 ζ that sequence is correct.
In step (1), for the method for the hinge district of the CD8 that increases respectively from NKT cell cDNA and cross-film district, the intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 ζ, there is no particular limitation, the various methods can commonly used for this area can be such as RT-PCR method.Wherein, NKT cell by being separated the mononuclearcell in people's venous blood, then can carrying out cultivation and obtaining.
Particularly, the method obtaining pWPT-CD8-CD137-CD3 ζ can comprise: the total serum IgE extracting NKT cell, reverse transcription obtains NKT cell cDNA, with the NKT cell cDNA obtained for template, primer P1 (SEQIDNO.11) and P2 (SEQIDNO.12) is utilized to carry out hinge district and cross-film district (SEQIDNO.3) of pcr amplification acquisition CD8 gene; Primer P3 (SEQIDNO.13) and P4 (SEQIDNO.14) is utilized to carry out the intracellular signal structural domain (SEQIDNO.4) of pcr amplification acquisition CD137 gene; Primer P5 (SEQIDNO.15) and P6 (SEQIDNO.16) is utilized to carry out the intracellular signal structural domain (SEQIDNO.5) of pcr amplification acquisition CD3 ζ gene, the PCR primer of acquisition is carried out double digestion respectively, is then connected with the Lentiviral pWPT-GFP after MluI/SalI double digestion.
In step (2), for the method for the nucleotide sequence of composite coding rat growth hormone signal peptide and CD33ScFv, there is no particular limitation, can be the conventional various methods in this area, such as, can be synthesized by full genome synthetic technology.
Particularly, the method obtaining the correct pWPT-CD33ScFv-CD8-CD137-CD3 ζ of sequence can comprise: by the nucleotide sequence (SEQIDNO.8) of full genome synthetic technology composite coding rat growth hormone signal peptide and CD33ScFv fusion gene, be cloned in carrier pGSI, obtain pGSI-CD33ScFv; Then pGSI-CD33ScFv is carried out BamHI/MluI double digestion, the recombinant plasmid pWPT-CD8-CD137-CD3 ζ obtained with the step (1) after BamHI/MluI double digestion is connected, through order-checking qualification, obtain the pWPT-CD33ScFv-CD8-CD137-CD3 ζ that sequence is correct.Wherein, the nucleotide sequence of rat growth hormone signal peptide is as shown in SEQIDNO.6, and CD33ScFv nucleotide sequence is as shown in SEQIDNO.7.
Present invention also offers a kind of NKT cell of through engineering approaches CD33 targeting, described NKT cell is the NKT cell (i.e. CAR33-NKT cell) modified by above-mentioned Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ.
For the preparation method of the NKT cell of through engineering approaches CD33 targeting, there is no particular limitation, the any method can be able to expected for those skilled in the art, under preferable case, the method comprises: packaging carries the slow virus of pWPT-CD33ScFv-CD8-CD137-CD3 ζ encoding gene; Utilize the slow virus infection NKT cell obtained, make NKT cell expressing Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ.
For packing the method for carrying the slow virus of pWPT-CD33ScFv-CD8-CD137-CD3 ζ encoding gene, there is no particular limitation, the various methods can commonly used for those skilled in the art, under preferable case, by Lentiviral pWPT-CD33ScFv-CD8-CD137-CD3 ζ and helper plasmid (as psPAX2, pMD2.G) cotransfection 293T packing cell, viral supernatants is collected during transfection 48-72h, centrifugal, filter, in filtrate, add 5 × PEG6000-NaCl mix, supernatant is abandoned after centrifugal, the aseptic PBS of precipitation 0-4 DEG C of precooling dissolves, obtain viral concentration liquid.
Method for slow virus infection NKT cell is not particularly limited, and the various methods can commonly used for this area, under preferable case, the method comprises: get 1 × 10
7-5 × 10
7individual NKT cell (in the NKT cell that the present invention prepares, CD3
+cD56
+cell accounts for total CD3
+the average ratio >20% of cell), discard old nutrient solution, add the fresh GT-T551 nutrient solution of 2-4mL, then add 200-400 μ L viral concentration liquid, 2-4 μ L1 × 10
-6mg/mL protamine and final concentration are the IL-2 of 300-700U/mL, be placed in 30-37 DEG C, saturated humidity is the CO of 3-6%
2after infecting 12-16h in incubator, abandon nutrient solution, being gone to by cell does not wrap in the culturing bottle of quilt, add the GT-T551 substratum of 20-50mL, add IL-2 that final concentration is 300-700U/mL again, CD3 monoclonal antibody that final concentration is 30-70ng/ml and final concentration be the interleukin 15 of 30-70ng/mL, in 30-37 DEG C, saturated humidity is the CO of 3-6%
2cultivate 12-18h in incubator, obtain the NKT cell that Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ modifies.
Further preferably, the method of slow virus infection NKT cell also comprises: the final concentration of the NKT cell IL-2 of the slow virus infection above-mentioned cultivation obtained afterwards is that the GT-T551 nutrient solution of 300-700U/mL carries out external evoked, when the density of cell is 80-90%, cell is proceeded in cell culture bags, the final concentration adding IL-2 every 1.5-2.5 days is 300-700U/mL, the final concentration of CD3 monoclonal antibody is 30-70ng/ml, the final concentration of Interleukin-15 is that the fresh GT-T551 nutrient solution of 30-70ng/mL carries out amplification cultivation and is 1 × 10 by cell amplification to total amount
9-2 × 10
9individual cell.Carry out NKT cell infection through the Chimeric antigen receptor of slow virus of the present invention to target CD33 antigen, its efficiency of infection is up to 30%-60%, and the CAR33-NKT cell obtained, its CD3
+cD56
+cell accounts for total CD3
+the ratio of cell is within 15%-40% scope.
The maturation protein aminoacid sequence of the Chimeric antigen receptor of the NKT cell expressing that Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ modifies is as shown in SEQIDNO.1.Wherein, what those skilled in the art should understand that is, Chimeric antigen receptor precursor protein by the intracellular signal structural domain of the hinge district of signal peptide, CD33ScFv, CD8 and cross-film district, CD137 and the intracellular signal structural domain of CD3 ζ in series, in cell, become ripe Chimeric antigen receptor albumen after rough endoplasmic reticulum excision signal peptide after protein translation, secretion exports rear and is positioned on the cytolemma of NKT cell.Gene coded sequence corresponding to the maturation protein aminoacid sequence of this Chimeric antigen receptor is as shown in SEQIDNO.2.The structure that this Chimeric antigen receptor is in series with the intracellular signal structural domain of the hinge district of gene C D8 and cross-film district and CD137 and CD3 ζ is for intracellular signaling structural domain, its aminoacid sequence is as shown in SEQIDNO.9, and corresponding gene coded sequence is as shown in SEQIDNO.10.
Present invention also offers the NKT cell of the through engineering approaches CD33 targeting that aforesaid method prepares.
The NKT cell that present invention also offers through engineering approaches CD33 targeting is for the preparation of the application in the leukemic preparation for the treatment of.Under preferable case, leukemia refers to the acute myeloid leukemia of the progressive stage CD33 positive.
Embodiment
The present invention is further illustrated for following embodiment, but therefore do not limit the present invention.
Experimental technique in following examples, if no special instructions, is this area ordinary method.Experiment material used in following embodiment, if no special instructions, is the purchase from routine biochemistry reagent shop and obtains, wherein:
NKT cell culture medium GT-T551 is purchased from TaKaRa company.
Lymphocyte separation medium is purchased from TBD company.
CD3 monoclonal antibody, recombinant fiber connect albumen (retronectin) all purchased from TaKaRa company.
Recombinant human protein's interferon-γ, rhIL-2, recombinant human interleukin 15 are purchased from protech company.
Total RNA extraction reagent box RNAisoReagent, high-fidelity DNA polymerase (
hSDNAPolymerase), T4DNA ligase enzyme is purchased from TaKaRa company.
RevertAid
tMfirstStrandcDNASynthesisKit is purchased from Fermentas company.
Bgl II, EcoRI, MluI, BamHI, SalI are purchased from Fermentas company.
Sepharose DNA reclaims test kit, common DNA Product Purification Kit, the little extraction reagent kit of plasmid all purchased from Tian Gen biochemical technology company limited.
PWPT-GFP, psPAX2, pMD2.G are all purchased from Addgene company.
PGSI is purchased from Tian Yihuiyuan bio tech ltd, Beijing.
Trans1-T1PhageResistant Competent cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd.
Lipofectamine
tM2000TransfectionReagent transfection reagent is purchased from Invitrogen company.
293T packing cell purchased from American ATCC.
In PEG6000-NaCl, PEG6000 final concentration is 25.5 quality %, NaCl final concentration is that 1.2M, PEG6000 and NaCl are all purchased from Suo Laibao bio tech ltd, Shanghai.
Foetal calf serum is purchased from German PAA company.
Human acute myeloid leukemia cell HL-60 purchased from American ATCC.
CF succinimide ester is purchased from Pu Zhen bio tech ltd, Shanghai.
Annexin V-RPE test kit purchased from American BD company.
All primers synthesize by Tian Yihuiyuan bio tech ltd, Beijing.
The preparation of embodiment 1NKT cell
(1) people's venous blood is got in the valve tube containing heparin.Adopt lymphocyte separation medium, be separated by density gradient centrifugation method and obtain mononuclearcell (PBMCs).
(2), after PBMCs washes three times, adopting the NKT cell culture medium GT-T551 of the Human autologous serum containing 0.6 volume % to adjust final concentration of cells is 2 × 10
6individual cell/mL; Cell is inoculated in advance through 75cm that final concentration is the retronectin bag quilt of 10 μ g/mL
2in Tissue Culture Flask.Then in substratum, add the recombination human interleukin-15 of rhIL-2,50ng/mlCD3 monoclonal antibody and the 50ng/mL that final concentration is 500U/mL, 37 DEG C, saturated humidity is the CO of 5%
2cultivate in incubator.
(3) cultivate the 4th day, be transferred to by cell and do not wrap in the culturing bottle of quilt, within every 2 days, add NKT cell culture medium GT-T551 according to Growth of Cells quantity, controlling cell concn is 1 × 10
8individual cell/mL, and the rhIL-2 adding that final concentration is 500U/ml; Be cultured to the 12nd day, obtain NKT cell, flow cytometry is analyzed NKT cell phenotype.The results are shown in Figure 1, wherein CD3
+: 95.04%; CD3
+cD8
+: 90.99%; CD3
+cD56
+: 24.12%; CD8
+cD56
+: 24.63%.
The structure of embodiment 2 Lentiviral pWPT-CD33ScFv-CD8-CD137-CD3 ζ
(1) preparation of NKT cell cDNA
Centrifugation embodiment 1 cultivates the NKT cell obtained, and extract the total serum IgE of cell with total RNA extraction reagent box RNAisoReagent ,-80 DEG C save backup.The total serum IgE Reverse Transcriptase kit RevertAid extracted
tMfirstStrandcDNASynthesisKit reverse transcription obtains NKT cell cDNA, and-20 DEG C save backup.
(2) preparation of slow virus plasmid pWPT-CD8-CD137-CD3 ζ
Design and synthesize following primer sequence (wherein, underscore is labeled as protection base, and square frame is restriction enzyme site):
With NKT cell cDNA in step (1) for template, pcr amplification is carried out with primer P1 and P2, obtain hinge district and the cross-film district of the CD8 of long 287bp, nucleotide sequence is as shown in SEQIDNO.3, and two ends are respectively containing MluI and Bgl II restriction enzyme site and protection base; Carry out pcr amplification with primer P3 and P4, obtain the CD137 intracellular signal structural domain of long 146bp, nucleotide sequence is as shown in SEQIDNO.4, and two ends are contained Bgl II and EcoRI restriction enzyme site respectively and protected base; Carry out pcr amplification with primer P5 and P6, obtain the intracellular signal structural domain of the CD3 ζ of long 359bp, nucleotide sequence is as shown in SEQIDNO.5, and two ends are respectively containing EcoRI and SalI restriction enzyme site and protection base.Each step pcr amplification reaction system is identical, for the CD137 intracellular signal structural domain that increases, carries out pcr amplification, the reference of PCR reaction conditions
the specification sheets of HSDNAPolymerase, reaction system (50 μ L) is as follows:
Distilled water: 32.5 μ L
5 × reaction buffer:10 μ L
DNTP mixture (often kind of 2.5mM): 4 μ L
P3(10mM):1μL
P4(10mM):1μL
NKT cell cDNA (200ng/ul): 1 μ L
HSDNAPolymerase：0.5μL
By above-mentioned PCR primer with 1% sepharose be separated, with sepharose DNA reclaim test kit carry out DNA fragmentation recovery.Carry out double digestion reaction respectively after obtaining fragment, the common DNA Product Purification Kit of digestion products reclaims for subsequent use.
Lentiviral pWPT-GFP MluI/SalI double digestion, digestion products is separated through the sepharose of 1%, reclaim test kit with sepharose DNA and reclaim large carrier segments, then be connected by T4DNA ligase enzyme with CD8, CD137, CD3 ζ fragment reclaimed before, connect product conversion Trans1-T1PhageResistant Competent cell, picking mono-clonal after 37 DEG C of cultivation 16h, extracts plasmid with the little extraction reagent kit of plasmid after 250rpm cultivates 12h by 37 DEG C.The plasmid extracted is through restriction enzyme MluI and the qualification of SalI double digestion, and qualification electrophorogram is shown in Fig. 2, wherein, and 1 swimming lane: DNA molecular amount mark D2000; 2 swimming lanes: the endonuclease bamhi (835bp) of plasmid pWPT-GFP; 3 swimming lanes: the endonuclease bamhi (756bp) of plasmid pWPT-CD8-CD137-CD3 ζ.Tian Yihuiyuan bio tech ltd, Beijing is sent to check order to the fusion gene fragment inserted the correct plasmid of qualification.By recombinant plasmid called after pWPT-CD8-CD137-CD3 ζ correct for sequencing result.
(3) preparation of slow virus plasmid pWPT-CD33ScFv-CD8-CD137-CD3 ζ
The nucleotide sequence of full genome composite coding rat growth hormone signal peptide and CD33ScFv fusion gene, sequence is as shown in SEQIDNO.8, synthesized by Tian Yihuiyuan bio tech ltd, Beijing, its 5 ' end is containing BamHI, kozak sequence, 3 ' end is containing MluI restriction enzyme site, by foregoing fusion gene clone in plasmid pGSI, called after pGSI-CD33ScFv.Plasmid is through BamHI/MluI double digestion, and digestion products is separated through 1% sepharose, reclaims test kit recovery object fragment for subsequent use with sepharose DNA.
PWPT-CD8-CD137-CD3 ζ plasmid is cut through restriction enzyme BamHI/MluI enzyme, and digestion products is separated through 1% sepharose, reclaims test kit recovery carrier segments for subsequent use with sepharose DNA.Then be connected by T4DNA ligase enzyme with the DNA fragmentation containing rat growth hormone signal peptide and CD33ScFv reclaimed, concrete grammar is shown in specification sheets.Product conversion Trans1-T1PhageResistant Competent cell will be connected, picking mono-clonal after 37 DEG C of cultivation 16h, 37 DEG C, after 250rpm cultivates 12h, extract plasmid with the little extraction reagent kit of plasmid.Extract plasmid through restriction enzyme BamHI/SalI double digestion qualification, qualification result as shown in Figure 3, wherein, M1:DNA molecular weight marker D2000; 1 swimming lane: the endonuclease bamhi (1572bp) of plasmid pWPT-CD33ScFv-CD8-CD137-CD3 ζ; 2 swimming lanes: the endonuclease bamhi (774bp) of plasmid pWPT-CD8-CD137-CD3 ζ; 3 swimming lanes: the endonuclease bamhi (853bp) of plasmid pWPT-GFP; M2:DNA molecular weight marker D15000.Tian Yihuiyuan bio tech ltd, Beijing is sent to check order to the fusion gene fragment inserted the correct plasmid of qualification.By recombinant plasmid called after pWPT-CD33ScFv-CD8-CD137-CD3 ζ correct for sequencing result, its structural representation as shown in Figure 4, comprising rat growth hormone signal peptide (nucleotide sequence is as shown in SEQIDNO.6), anti-CD 33 single-chain antibody (nucleotide sequence is as shown in SEQIDNO.7), the intracellular signal structural domain of the hinge district of CD8 and the intracellular signal structural domain of cross-film district and CD137 and CD3 ζ, wherein, the structure that this Chimeric antigen receptor is in series with the intracellular signal structural domain of the hinge district of gene C D8 and cross-film district and CD137 and CD3 ζ is for intracellular signaling structural domain, its aminoacid sequence is as shown in SEQIDNO.9, corresponding gene coded sequence is as shown in SEQIDNO.10.
The preparation of the NKT cell that embodiment 3 Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ modifies
(1) packaging of slow virus and concentrated
Measure the concentration of slow virus expression plasmid pWPT-CD33ScFv-CD8-CD137-CD3 ζ and helper plasmid psPAX2, pMD2.G respectively with spectrophotometer, three kinds of plasmids are with the mass ratio Lipofectamine of 4:2:1
tM2000TransfectionReagent transfection reagent cotransfection 293T packing cell.Collect viral supernatants respectively when transfection 48h, 72h in 50mLEP pipe, 4 DEG C, the centrifugal 10min of 2000g, shift the supernatant that obtains for twice extremely in new EP pipe, with 4.5 μm of frit viral supernatants; The viral supernatants filtered and 5 × PEG6000-NaCl mix according to the volume ratio of 4:1,4 DEG C of standing 2h, then 4 DEG C, the centrifugal 20min of 10000g, abandon supernatant, the aseptic PBS of 4 DEG C of precoolings of precipitation 1mL dissolves, and obtains the viral concentration liquid of Chimeric antigen receptor, carry out packing by every pipe 100 μ L ,-80 DEG C save backup.
According to the method described above, utilize slow virus expression plasmid pWPT-GFP and helper plasmid psPAX2, pMD2.G cotransfection 293T packing cell, collect viral supernatants, concentrated, obtain the slow virus concentrated solution of expressing GFP green fluorescent protein.
(2) amplification cultivation of slow virus infection NKT cell and infected cell
Example 1 at 75cm
2cultivate in culturing bottle 1 × 10
7individual NKT cell, discards old nutrient solution, add 2mL fresh NKT cell culture medium GT-T551, viral concentration liquid that 200 μ L steps (1) obtain, 2 μ L1 × 10
-6mg/mL protamine, final concentration is the rhIL-2 of 500U/mL, is placed in 37 DEG C, saturated humidity is the CO of 5%
2infect after 12 hours in incubator, abandon nutrient solution.With the slow virus concentrated solution of expressing GFP green fluorescent protein, NKT cell is synchronously infected (the NKT cell obtained is called CART-GFP cell), for calculating the efficiency of infection of this virus simultaneously.Metainfective cell is gone to the 75cm without CD3 and retronectin bag quilt
2in culturing bottle, add the NKT cell culture medium GT-T551 of 20mL, add rhIL-2 that final concentration is 500U/mL again, CD3 monoclonal antibody that final concentration is 50ng/ml and final concentration be the recombinant human interleukin 15 of 50ng/mL, in 37 DEG C, saturated humidity is the CO of 5%
2cultivate 18h in incubator, the NKT cell obtained is called CAR33-NKT cell.CAR33-T cell (preparation method's reference literature of T cell: YajingZhang is prepared by identical method, etal.AutologousCIKCellImmunotherapyinPatientswithRenalCe llCarcinomaafterRadicalNephrectomy.ClinicalStudy, the preparation method of 2.4 part CIK cell in 2013).With the efficiency of infection of this virus of Flow cytometry, as shown in Figure 5, the efficiency of infection of CAR33-NKT cell is 44.79% to result.
(3) external evoked amplification CAR33-NKT cell mass
The NKT cell culture medium GT-T551 being 500U/mL by the final concentration of the NKT cell rhIL-2 after above-mentioned cultivation carries out external evoked, when the density of cell is 85%, cell is proceeded in cell culture bags, the final concentration adding rhIL-2 every 2 days is that the fresh NKT cell culture medium GT-T551 that the final concentration of 500U/mL, CD3 monoclonal antibody is 50ng/ml, the final concentration of recombinant human interleukin 15 is 50ng/mL carries out amplification cultivation, treats that cell amplification is 1.5 × 10 to total amount
9after about individual cell, adopt flow cytometer to identify the cell colony infected, cell phenotype generally reaches CD3 positive cell ratio > 85%; CD3CD8 positive cell ratio >70%; The two positive cell ratio >15% of CD3CD56, the results are shown in Figure 6, CD3
+: 95.37%; CD3
+cD8
+: 70.09%; CD3
+cD56
+: 20.32%; CD8
+cD56
+: 16.85%.
Embodiment 4CAR33-NKT cell is to the cytotoxicity analysis of human acute myeloid leukemia killing functions of immunocytes
The CAR33-NKT cell of preparation in Example 3 respectively, the NKT cell cultivated in CAR33-T cell and embodiment 1 is inoculated in 96 orifice plates, dye with CF succinimide ester (CFSE), with human acute myeloid leukemia cell HL-60 to imitate target ratio (killer cell: target cell) 5:1, 10:1, 20:1, the ratio of 40:1 carries out Dual culture, after the Dual culture of 24h, cell annexin V-RPE test kit is dyeed, arrange control group is do not add the HL-60 cell that immunocyte kills and wounds process simultaneously, and cell annexin V-RPE test kit is dyeed.Flow cytometry detects apoptosis, and apoptotic amount is according to formulae discovery below: apoptosis rate=(contrast-sample)/contrast × 100%), contrast the cell survival number killing and wounding process for not adding immunocyte; Sample is the cell survival number that the immunocyte adding corresponding effect target ratio (killer cell: target cell) kills and wounds process, sees Fig. 7.The NKT cell that Chimeric antigen receptor CD33ScFv-CD8-CD137-CD3 ζ modifies has specific killing activity to human acute myeloid leukemia cell, and the specific killing activity of CAR33-NKT cell is obviously better than CAR33-T cell.
Embodiment 5CAR33-NKT cell is to the result for the treatment of of the Patients with Acute Myeloid Leukemia of the progressive stage CD33 positive
Get 5 × 10
8the NKT cell (i.e. CAR33-NKT cell) that individual CD33ScFv-CD8-CD137-CD3 ζ modifies, after 100ml normal saline dilution, continuous three days venous re-transfusion are to producing the Patients with Acute Myeloid Leukemia of the progressive stage CD33 positive of resistance and chemoresistance (before utilizing CAR33-NKT cell of the present invention to carry out target immunotherapy to the monoclonal antibody of CD33 and combined use of chemotherapy, through repeatedly treating (as radiotherapy, chemotherapy, monoclonal antibody targeted therapy and other symptomatic treatments etc.), but all without obvious curative effects) in body, after feedback, the treatment situation of patient is assessed.
Fig. 8 is after analysis on hemogram CAR33-NKT cell is fed back to and produces the Patients with Acute Myeloid Leukemia of the progressive stage CD33 positive of resistance and chemoresistance to the monoclonal antibody of CD33 and combined use of chemotherapy, its body temperature and the change of leukocyte count object.Result shows, after target immunocyte (CAR33-NKT cell) treatment, the number of white blood cells of patient and patient temperature are negative correlation, namely when patient's number of white blood cells declines, patient temperature raises, illustrate that CAR33-NKT cell can kill and wound the white corpuscle produced the monoclonal antibody of CD33 and combined use of chemotherapy in the Patients with Acute Myeloid Leukemia body of the progressive stage CD33 positive of resistance and chemoresistance, and then release inflammatory factor, cause fervescence, that is, CAR33-NKT cell can kill and wound the marrow series leukemia cell produced the monoclonal antibody of CD33 and combined use of chemotherapy in the peripheral blood of the Patients with Acute Myeloid Leukemia of the progressive stage CD33 positive of resistance and chemoresistance.
Fig. 9 be marrow Wright-Giemsa staining examine CAR33-NKT cell be fed back to the progressive stage CD33 positive of resistance and chemoresistance produces on the monoclonal antibody of CD33 and combined use of chemotherapy Patients with Acute Myeloid Leukemia body in after medullary cell change (namely immune cell therapy affects the original Blast counts object of marrow), result shows, target immune cell therapy after one month in marrow original juvenile cell obviously reduce, and observe the appearance of smudge cells and dissolved cell.Illustrating that CAR33-NKT cell can play lethal effect to producing marrow series leukemia cell in the marrow of the Patients with Acute Myeloid Leukemia of the progressive stage CD33 positive of resistance and chemoresistance to the monoclonal antibody of CD33 and combined use of chemotherapy, the disease of aforementioned Patients with Acute Myeloid Leukemia can be made to alleviate to some extent.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. a Chimeric antigen receptor, it is characterized in that, described Chimeric antigen receptor is CD33ScFv-CD8-CD137-CD3 ζ, by the intracellular signal structural domain of the hinge area of CD33ScFv, CD8 and cross-film district, CD137 and the intracellular signal structural domain of CD3 ζ in series.
2. Chimeric antigen receptor according to claim 1, wherein, the aminoacid sequence of described Chimeric antigen receptor is as shown in SEQIDNO.1.
3. the gene of the Chimeric antigen receptor of coding described in claim 1 or 2.
4. gene according to claim 3, wherein, the nucleotide sequence of described gene is as shown in SEQIDNO.2.
5. the recombinant expression vector containing the gene described in claim 3 or 4.
6. recombinant expression vector according to claim 5, wherein, described recombinant expression vector is Lentiviral.
7. recombinant expression vector according to claim 6, wherein, described Lentiviral is pWPT-CD33ScFv-CD8-CD137-CD3 ζ.
8. a NKT cell for through engineering approaches CD33 targeting, is characterized in that, described NKT cell is the NKT cell modified by the Chimeric antigen receptor described in claim 1 or 2.
9. through engineering approaches CD33 targeting according to claim 8 NKT cell for the preparation for the treatment of leukemic preparation in application.
10. application according to claim 9, wherein, described leukemia refers to the acute myeloid leukemia of the progressive stage CD33 positive.
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