CN109136182A - A kind of polarization and the method and composition for expanding CD4+T cell and the application in the tumour for curing expression specificity antigen - Google Patents
A kind of polarization and the method and composition for expanding CD4+T cell and the application in the tumour for curing expression specificity antigen Download PDFInfo
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Abstract
The invention belongs to biological cell fields, it is related to the method and application of a kind of polarization and amplification CD4+T cell, the present invention takes the undifferentiated T cell of the CD4+ of source of people or source of mouse, using t cell activation and transforming growth factor β, interleukin 4, IL-21 and a series of cell factor Co stituations of gamma interferon neutralizing antibody.Polarized T cell in this approach such as 500-1000 times in 14 days, while possessing the lethal and differentiation of self proliferative capacity for cancer cell in the case where a large amount of proliferation.Its character was reported before being different from, and was more than for subgroups such as Th1, Th17 and Th9 to anticancer, and in killing cancer effect.Polarized CD4+T cell subsets in this way has powerful therapeutic effect to the cancer of a variety of expression specificity antigens, including solid and neoplastic hematologic disorder.
Description
Technical field
The invention belongs to biological cell field more particularly to the method and compositions of a kind of polarization and amplification CD4+T cell
And the application in the tumour for curing expression specificity antigen.
Background technique
Immunocyte feedback is the cancer treatment method invented in recent years, the basic principle is that: the cell of feedback can be to host
Cancer cell and cancerous tissue generate specificity aggressiveness or other intracorporal immunocytes of stimulation of host, make its attack
Cancer.
Researcher used various kinds of cell to feed back, including T lymphocyte, macrophage (Macrophage), natural
Kill cell (NK) and natural killer T cells (NKT) etc..Meanwhile many experiments use: the processing of cell listed above and repairing
Decorations (such as Chimeric antigen receptor T cell therapy, CAR-T) or various kinds of cell are used in combination, or using cell and other drugs/thin
Born of the same parents' element/vaccine combination (for example after injection T cell, persistently injecting IL-2).The effect of T lymphocyte be it is leading and can not or
Scarce, other several cells are used alone almost without therapeutic effect.
T cell feeds back therapy, all produces different degrees of therapeutic effect to neoplastic hematologic disorder and solid tumor.CAR-T is treated
Method achieves breakthrough curative effect in treatment B cell acute lymphoblastic leukemia (B cell ALL).Novartis and
The therapeutic scheme Kymriah and Yescarta of Kite Pharma, the former takes with the patient of the chemotherapy failure of B cell ALL
83% cancer remission rate was obtained, the latter has on B cell ALL and B cell lymph cancer (DLBCL) chemotherapy failure patient
51% remission rate.But the defect of these therapies first is that recurrence rate is higher.And once recur, just there is no effective treatments
Means.
For relatively other effects, T cell feeds back therapy, not good enough to solid tumor curative effect.Scientist and doctor attempt to adopt
T cell, the periphery blood T cell and CAR-T cell of DC activation etc. are invaded with cancer, to the melanoma of chemotherapy failure, intestinal cancer, food
Road cancer, lung cancer, many kinds of solids oncotherapy such as glioma, but rarely clinical trial so far, reactivity are more than 50%.It is more
The reactivity of number experiment is only in 10%-20%.
The effect of T cell therapy therapy, is related at least three indexs, first is that T cell is to the lethal of cancer, second is that T is thin
Born of the same parents are directed to the guiding performance of cancer, third is that the survival of T cell itself.And therapy is if you need to success, it is necessary to which specificity is strong, side effect
It is small.Wherein guiding performance then can solve using CAR-T and cancer intrusion T cell.The lethal and viability of T cell, is current
It is badly in need of the property of enhancing.Such as the cytotoxicity cd8 t cell that most of laboratories use at present, after feeding back in patient body, one
It will lose in month across 99%.For another example, for several times in the clinical test of CAR-T, patient dies of side effect-cytokine storm.
T cell has many subgroups, and more known has CD4+T cell, CD8+T cell and NKT cell etc..Wherein
Every kind has the division of subgroup more refined again, and such as CD4+T cell, the subgroup clearly reported by researcher is divided into Th1
(Helper T Cell Type1), Th2, Th9, Th17, Tfh (Follicle B helper T cell) and Treg
(Regulatory T cell)。
Currently, most of researcher is when clinical trial T cell feeds back technology, only focusing on makes using CD4+ or CD8+
With, and have ignored the subgroup more segmented.Such as most clinical trials, using the CD8+T cell of purifying, sub-fraction is real
It tests, uses CD8+T cell and CD4+T cell and injected according to 1:1 ratio.Another fraction experiment, it is thin to use full CD4+T
Born of the same parents.But the specific composition of CD8+ and/or CD4+T cell is deeply paid close attention to almost without clinical trial.
Summary of the invention
In order to solve the above technical problems, the present invention provides the method and composition of a kind of polarization and amplification CD4+T cell
And the application in the tumour for curing expression specificity antigen, a kind of novel T cell subgroup can be induced；Abductive approach letter
Single, easy to operate, the subgroup of generation is also clearly distinguishable from existing T cell in gene expression and character, the novel T induced
Lymphocyte has powerful lethality and scavenging to the solid and neoplastic hematologic disorder that present specific antigen, while possessing and depositing for a long time
Living, resisting fatigue will not be divided into control T cell, cytokine storm (side effect is low) and cancerous tissue will not be caused to lead
Tropism etc..This novel T cell subgroup has the internal survival ability of stem cell and strength.
The present invention provides the method for solving a kind of polarization and amplification CD4+T cell of the above technical problem: including following step
It is rapid:
(1) CD4+T cell, CD4+ or CD62L+T after the CD4+T cell of acquisition purifying: isolating and purifying or induction culturing
Cell；
(2) active cell: the cell obtained in activation step (1) in 0-24 hours in special culture medium；
Activiation method is Dynabeads, and antiCD3 antibody adds antiCD28 antibody, more by DC presented by cells specificity
Peptide, Concanavalin A reagent or Brefeldin A Cocktail (phorbol 12-myristate-13-acetate,
Ionomycin adds Brefeldin A) reagent or similar reagents.
(3) culture and cell Proliferation: cell is centrifuged, removes supernatant, and be incubated at and be aided with 10% by third day upon activation
Fetal calf serum, antibiotic (Antibiotic-Antimycotic (1X)-Thermo Fisher Scientific) and 100U/mL
In RPMI-1640 (being referred to as RPMI afterwards) culture medium of interleukin 2；Stimulation increment and induction differentiation；
Quick proliferation can be presented in T cell after being activated, and the cell number doubling time was less than 24 hours.Whole proliferation time
Big appointment continues 14 days.The cell doubling time is a standard of measure of cell value-added speed.Time is shorter, and cell Proliferation is got over
Fastly.Cell is proliferated daily with 2-10 times of speed in the present invention, and proliferation continues >=14 days.Protection polarization proliferation stimulation at least 7
It, the cell characteristics after at most 14 days.
(4) cell in activation latter stage and detection are taken.
T cell in activation latter stage is finished product, should be detected to its effect, including experiment in vitro and internal reality
It tests.
Cell origin in the step (1) includes the peripheral blood for being not limited to source of people, Cord blood, the blood in immune organ
Cell and blood precursor, cancer invade the peripheral blood of lymphocyte (including in situ tumor and metastatic tumour) and source of mouse and are immunized
Intraorganic haemocyte, for example obtained from spleen or lymph node.
Initial CD4+T cell includes the T cell of all expression CD4 surface antigens.Including but not limited to, human peripheral,
Cord blood, cancer intrusion cell and other CD4+T cells operated in advance.Can be first from marrow, the sources such as Cord blood obtain
Candidate stem cell, the T cells precursor such as lymphoblast, and induce differentiation to CD4+T cell.
The culture medium of active cell is aided in the culture medium of 10% fetal calf serum for RPMI and is added in the step (2)
100ng/ml transforming growth factor β, 100ng/mL interleukin 4,100ng/mL IL-21,100ng/mL
Gamma interferon neutralizing antibody or identical reagent, various concentration；Culture medium is that RPMI is aided with 10% tire ox blood in the step (3)
Clearly+antibiotic and 100u/ml interleukin 2.The cell induced in this way, has lethality in patient body, to cancer defeated time.
T cell is activated using immunomagnetic beads (Dynabeads) in the step (2), immunomagnetic beads and cell proportion are 1:
1 or similar proportion.
There are also carry out modification step to CD4+T cell before the step (3)；Such as to T cell transduce Chimeric antigen receptor or
Other cell factors.Modification step described in prioritization scheme can be completed for -96 hours 4 hours after CD4+T cell is activated；More
Modification step described in scheme is advanced optimized to complete in 24 hours after CD4+T cell is activated.
The modification step is the encoding gene for being carrier by DNA or RNA to one section of CD4+T cell transfecting or transduction；It is excellent
Transfection described in change scheme or the encoding gene transduceed, packet expand but are not limited to inserted type antigen receptor, T cell receptor sequence or its
His cytokine or cell surface receptor；The method of the transfection or transduction includes but is not limited to use retrovirus, slow virus
Or transposons.
The method of the transfection or transduction successively includes carrying out according to sequence production DNA plasmid step, using these plasmids
The package step of retrovirus, obtain host T cell go forward side by side line activating and polarisation steps, after polarization 24 hours using virus
Transduction protocol is carried out to T cell, and continues to expand T cell in aforementioned manners, until reaching required T cell number or T
Cell stops amplification.
Using when can by cell by venous re-transfusion into host；It selects while assisting with other cancer therapies, including
But it is not limited to cytokine injections；It selects cytokine to inject some as therapy, other therapies can also be cooperated.
Obtained cell should detect in this way, be divided into vitro detection and vivo detection；
The experiment in vivo includes:
(a) it is ground as receptor using the T cell of TCR transgenic mice as donor using the mouse of source of mouse tumor model
New subgroup (Thscm) subgroup described in this patent is studied carefully in the intracorporal survival of tumor-carrying mouse, character and differentiation；
(b) several source of mouse cancer-TCR transgenic donor cell models are used, study new subgroup donorcells to a variety of, table
Up to the solid of specific antigen and the lethal effect of neoplastic hematologic disorder cancer cell, and observe the time-to-live of mouse.
The experiment in vitro includes: the gene expression that new subgroup is measured using real-time fluorescence quantitative PCR, and with other
Some CD4+ subgroups are compared；CD4+, the cell of CD62L+, induction differentiation are specially extracted from the splenocyte of C57B6
As Th1, Th17 and new subgroup (Thscm), then the RNA of the RNeasy Kit extraction cell using Qiagen, use Thermo
RNA reverse transcription is cDNA template by the SuperScript III of Fisher Scientific, and with the primer provided in annex
Do realtime fluorescent quantitative PCR experiment.
New subgroup CD4+T cell has reported the CD4+T cell subgroup for anticancer therapy with other, such as Th1,
Th17, Th9 have significant difference.We are named as Thscm subgroup (T helper stem cell memory), because of new Asia
Group's cell has compared other above-mentioned CD4+T cell subsets, there is stronger embryonal.
Cell in the present invention to the preparation of top method can be applied to a kind of pharmaceutical composition, which can be used for
Cure the application in the tumour of expression specificity antigen；The tumour is solid tumor or neoplastic hematologic disorder.
Enrichment or the lymphocyte polarization to be enriched with are the Asia Thscm by the T lymphocyte that receptor is acquired in the present invention
Group may be selected to make other changes to cell in activation, such as to T cell transduction Chimeric antigen receptor or other cells
The factor.Latter stage is expanded, quality testing, including but not limited to bacterium, fungi, the inspection of mycoplasma and bacterial endotoxin are carried out to cell
It surveys.
There is the solid and neoplastic hematologic disorder of expression specificity antigen strong lethal cell subset in the present invention.Meanwhile
This subgroup T cell has that viability is strong after feeding back in host, Small side effects, self-regeneration, and antifatigueization resists unfavorable differentiation
(being such as divided into the regulatory T cells for reducing cell invasiveness, Treg) and enhancing cancer guiding performance and other effects.In receptor for swollen
In the case where oncocyte, cell mass has cancer cell powerful lethal.
In the present invention Thscm cell transplantation enter cancer human or animal can be to realize tumour control.Such as by Thscm cell
It is injected into venous patient, for example Thscm cell is injected into patient's cancerous tissue.Thscm cell is in homologous, tumour carrying organism
It is interior that there is cancerous tissue guidance type and powerful tumoricidal.
Thscm cell long-term surviving in homologous organisms body, resisting fatigue in the present invention, will not make a variation for regulation type T it is thin
Born of the same parents and cause cytokine storm.
This polarized CD4+T cell subsets of method in the present invention, to cancer, especially advanced stage solid and neoplastic hematologic disorder
There is powerful therapeutic effect.The undifferentiated T cell of the CD4+ of source of people or source of mouse is taken, using t cell activation and IL-4, IL-21, TGF
β, a series of anti-cell factor Co stituations of INF gamma antibodies.Polarized T cell in this approach, in the case where a large amount of proliferation, such as
500-1000 times in 14 days, while possessing the lethal and differentiation of self proliferative capacity for cancer cell.Its character is different from it
Before reported, for subgroups such as Th1, Th17 and Th9 to anticancer, and the treating cancer the effect of on be more than.
B16 (melanoma) is being carried, Lewis Lung (lung cancer), E0771 (breast cancer), MC38 (colon cancer), RM-1
In the mouse model of (prostate cancer), PANC02 (cancer of pancreas) cell and Raji (B cell lymphoma), with few quantity,
250,000/ mouse can be treated to be developed to the tumour in advanced stage.
Detailed description of the invention
The method that Fig. 1 is Delta-Delta Ct in the present invention carries out result analysis chart
Fig. 2 be the present invention in cell survive in Mice Body sum scheme
Fig. 3 and Fig. 4 is the streaming for the cell fatigue surface antigen that donorcells are expressed in Mice Body inner surface in the present invention
Cytometric Analysis figure
Fig. 5 is cell in the present invention in the intracorporal IFN γ staining analysis figure of mouse
Fig. 6 be the present invention in cell the intracorporal FoxP3 of mouse staining analysis figure
Fig. 7 is CD3+ in the present invention, ratio comparison diagram of the CD4+ cell in cancer microenvironment
Fig. 8-Figure 13 is the effect contrast figure that various solids and neoplastic hematologic disorder are treated in the present invention
Specific embodiment
With reference to the accompanying drawing and the method for the present invention is further described in specific embodiment:
Embodiment 1
(1) it obtains the CD4+T cell of the source of people of purifying: by peripheral blood, carrying out the processing of Ficoll density gradient, obtain survival
Cell, and remove red blood cell.Separation system is magnetized using the Pan Human CD4+T cell that Miltenyi is produced, is obtained pure
CD4+T cell after change.
(2) activation of T cell: from the T cell obtained in step 1, in culture medium, (RPMI is aided with the training of 10% fetal calf serum
It supports and 100ng/ml TGF β, 100ng/mL IL-4,100ng/mL IL-21 and 100ng/mL anti-IFN γ is added in base；With
Under it is identical) in the Dynabeads produced with Thermal Fisher activate.
(3) culture and cell Proliferation: cell is centrifuged, removes supernatant, and be incubated at and be aided with 10% by third day upon activation
Fetal calf serum, in the RPMI culture medium of Antibiotic-Antimycotic and 100U/mL IL-2；Stimulation increment and induction point
Change；
Quick proliferation can be presented in T cell after being activated, and the cell number doubling time was less than 24 hours.Whole proliferation time
Big appointment continues 14 days.The cell doubling time is a standard of measure of cell value-added speed.Time is shorter, and cell Proliferation is got over
Fastly.Cell is proliferated daily with 2-10 times of speed in the present invention, and proliferation continues >=14 days.Protection polarization proliferation stimulation at least 7
It, the cell characteristics after at most 14 days.
(4) it takes the cell in activation latter stage and detects its biological safety, including possible bacterium, fungi and mycoplasma sense
Dye.By the cell cryopreservation of harvest.
Embodiment 2
In other contents such as embodiment 1, the method for polarization and amplification CD4+T cell.Increase the Transduction protocol to T cell.
(1) such as embodiment 1, the CD4+T cell of the source of people of purifying is obtained from Type B leukemia patient peripheral blood.In order to more preferable
Final result, CD4+, the T cell of CD62+ can be sorted, because initial cell purity is higher, the cell purity after induction is higher.
(2) as embodiment 1 is activated by CD4+T cell in culture medium with Dynabeads
(3) using the method recorded in other documents, the retrovirus for the source of people T cell that can transduce, transduction are wrapped up
Sequence is the Chimeric antigen receptor molecule of CAR019.[United States Patent (USP) US 9,328,156B2Seq8]
(4) culture and cell Proliferation: cell is centrifuged, removes supernatant, and be incubated at and be aided with 10% by third day upon activation
In the RPMI culture medium of fetal calf serum, antibiotic and 100U/mL IL-2.Culture achieves the goal to cell number always or cell stops
Only it is proliferated.
(5) cell induced in this way, defeated time in patient body, has lethality to Type B leukaemia.
Embodiment 3
(1) CD4+, the cell of CD62L+ are extracted from the splenocyte of C57B6 mouse.Using mouse spleen or lymph node, obtain
After taking, 40 microns of sieves are ground and crossed, cell suspending liquid is obtained.Cell suspending liquid or mouse blood are split using common red blood cell
Liquid processing is solved, and is cleaned twice with phosphate buffer.The Pan Mouse that cell is counted, and is produced using Miltenyi
CD4+T cell magnetizes separation system, obtains CD4+T cell after purification.CD4+, the T cell of CD62+, because initially can be sorted
Cell purity is higher, and the cell purity after induction is higher.
(2) according to previously described method and the method delivered, induce differentiation into for Th1, Th17 and Thscm. (by
In it is presumed that new subgroup may have certain common point with Th17 subgroup, we mainly compare it with Th17 subgroup
Compared with.) using Qiagen RNeasy Kit extract cell RNA.And with Thermo Fisher Scientific's
RNA reverse transcription is cDNA template by SuperScript III.And real-time fluorescence quantitative PCR reality is done with the primer provided in following table
It tests.
This detection uses gene GAPDH and ACTB as House Keeping Gene, and uses Delta-Delta Ct
Method carry out interpretation of result, the result is shown in Figure 1., it is apparent that faciation ratio Th17 subgroup in new Asia has stronger embryonal.Together
When, new subgroup and Th1 subgroup have more obvious difference.New subgroup is different from subgroup Th9, and being embodied in it and comparing Th9 has more
Strong stem cell properties, as the expression quantity of the stem cell factor SELL (CD62) of Th9 subgroup, IL7R (CD127) etc. are lower than Th17
Subgroup, therefore further below new subgroup.Meanwhile correlation factor such as CD44 is activated, Th9 subgroup is higher than new subgroup.
Embodiment 4
Content in other contents such as embodiment 3, wherein donor mice can be wildtype type, is also possible to TCR and turns
Genotype.
Embodiment 5
After Thscm cell transplantation enters in Mice Body in the present invention, compared to other CD4+T cell subsets, there is stronger deposit
Live time and stronger fatigue resistance.Side effect simultaneously is also smaller.
(1) homologous Murine cancer models, source of mouse B16 melanoma and TRP-1 transgenic T cells are used.It need to be using same
The donor and receptor in source, but expression of some cell-surface factors in the two need to be different.Such as, C57B6 CD45.2 is selected
When mouse is as receptor, TRP-1CD45.1 mouse T cell should be selected as donor, to be distinguish on flow cytometer.
From TRP-1CD45.1 Transgenic mice spleen extract CD4+, CD62L+ cell, and according to method provided above and other
It is reported that the method crossed is lured and is divided into, and Th1, Th17, Th9 and Thscm subgroup, and by same number of cell, pass through
The mode of tail vein injection is input in the Mice Body for carrying B16 tumour (when diameter of tumor reaches 10mm), and when specific
Between point take blood or put to death mouse, the T cell of the T cell and mouse itself to injection studies.
(2) in the 15th and 30 day execution mouse of cell injection, and the spleen and draining lymph node needle cell of mouse are extracted,
To CD45.1, CD3, CD4, PD-1, Lag-3, the dyeing of the cell surface antigens such as KLRG-1, CD244.The CD4+T cell of input
CD45.1+ total number of cells/(CD3+, CD4+) total number of cells can be calculated by following in survival rate.It is total to obtain cell survival
Number.See that Fig. 2, conclusion, new Asia faciation compare Th1, Th17 and Th9 cell has the longer time-to-live in host.
(3) cell preparation and the injection mouse of above-mentioned experiment are repeated, and in the 10th day execution mouse.Its tumour of digestion process
Tissue becomes single cell suspension, then anti-to the cell fatigue surface of wherein donorcells (CD45.1+) cell surface expression
Original carries out flow cytometry analysis (see Fig. 3, Fig. 4).It as a result is the fatigueization surface antigen of Thscm cell, including PD-1,
Lag-3, KLRG-1 and CD244, expression quantity, lower than Th17 and Th9 and well below Th1.It can thus be concluded that going out, Thscm subgroup
There is the characteristic of resisting fatigue.
(4) cell preparation and the injection mouse of above-mentioned experiment are repeated, and in the 10th day execution mouse.Its tumour of digestion process
Tissue becomes single cell suspension, to its surface and intracellular antigen CD4 5.1, CD3, CD4, CD8, IFN γ, FoxP3 into
Row dyeing.By the dyeing of IFN γ to cell, it can be seen that, Thscm is different from tradition Th1 and Th9, be do not secrete IFN γ (see
Fig. 5).Due to it has been proved that IFN γ is the main inducing of cytokine storm and a variety of side effects, it is known that, Thscm is being fed back
After in vivo, caused a variety of side effects can be slighter.
(5) by the dyeing to FoxP3 it is found that Th17 cell is in cancer microenvironment, meeting be partially converted into Treg, and
Thscm cell does not have this variation (see Fig. 6).
(6) finally, by comparison CD3+, ratio of the CD4+ cell in cancer microenvironment can show that Thscm subgroup T is thin
Born of the same parents' quantity highest in cancer microenvironment (see Fig. 7).It is found that Thscm has optimal cancer guiding performance.
Embodiment 6
After Thscm cell transplantation enters in Mice Body in the present invention, compared to other CD4+T cell subsets, for cancer
Lethal stronger, the duration is also at most.
(1) in order to verify the interaction of CD4+T cell and CD8+T cell, on the other hand, also for verifying Thscm subgroup pair
Other solids and neoplastic hematologic disorder it is lethal, B16 (melanoma) tumour cell and other several solid tumors need to be used thin
Born of the same parents and B lymphocytic cancer cell.Except other several tumour cells of B16, i.e. Lewis Lung (lung cancer), E0771 (breast cancer),
MC38 (colon cancer), RM-1 (prostate cancer), PANC02 (cancer of pancreas) cell and Raji (B lymph cancer) cell.It is needed before use
By molecular biotechnology in transgenic Ovalbumin (Lewis Lung, E0771, MC38, RM-1 and PANC02) or
Luciferase(Raji).It, must be using TRP-1CD45.1+ transgenic mice T cell as donor when using B16 tumour.Using
When other Ovalbumin transgenic tumors, OT-II should be accordingly used, the T cell of CD45.1+ mouse is as donor.Using Raji
When (Luciferase transgenosis) tumour, source of people T cell and immunodeficient mouse should be used as receptor, and upon activation to T
The Chimeric antigen receptor molecule of cell progress CAR019.[9,328,156 B2 Seq8 of United States Patent (USP) US]
(2) it in order to imitate Terminal cancer, and divide the diversified patient of technology adaptation e.g. can only in tumour
The patient for separating out a small amount of cancer intrusion T cell, need to use more extreme experiment condition.1,000,000B16 melanoma, or
The cancer cell of other Ovalbumin transgenosis, it is subcutaneous to be injected at mouse, to form tumour.During tumour formation, need
(CD4+ is separated from the spleen of donor mice.CD62L+) cell and according to above and other report described in, be induced to differentiate into Th1,
Th17, Th9 and Thscm subgroup.It need to coordinate growth of cancers time and T cell differentiation and proliferation time, so that, in the length of cancer cell
Diameter starts to treat when reaching 10mm, i.e., to the removing of host immune cell, (every mouse peritoneal injects 2mg
) and the tail vein injection of T cell 24 hours of injection (cyclophosphamide after) cyclophosphamide.It is treating
Before, again to mice group to ensure that the average external volume of the mouse tumor of each group is closest.Injection measurement is
0.25million Th1, Th17, Th9 or Thscm. note that the injection volume of 0.25million well below mainstream academic paper
The donor T-cells injection volume of report.It should be time point with every -7 days on the 5th, to the tumor size of all groups of mouse after tumor injection
It measures.Vernier caliper equal length metering outfit can be used in measurement, and passes through formula v=1/2* long diameter * (section diameter) ^2
To calculate the volume of tumour.
(3) when using Luciferase Raji B lymphocytic cancer cell, tail vein injection (1,000,000 should be carried out to mouse
Cell/mouse).And every 5 days, in-vivo imaging is carried out to measure the extent of growth of lymph cancer to mouse.In every mouse strong light
Degree starts to treat when reaching 1000000 unit, and treatment method is the same as above-mentioned solid tumor method.Then every 5 days, body is carried out to mouse
Interior imaging.
(4) experimental result is, Thscm subgroup and was reported in the past in terms of treat various solids and neoplastic hematologic disorder
Th1, Th17 and Th9 subgroup are compared, and best therapeutic effect (Fig. 8-Figure 13) is achieved.When being treated using Thscm subgroup,
In neoplastic hematologic disorder and many kinds of solids tumor model, 100% cure rate is realized, and in observation in tens days to several hundred days
Between in, no relapse.In contrast, such effect is unable to reach using the T cell of other subgroups.Even if there is the case where healing,
Also much it is not achieved 100%.It proves simultaneously, many kinds of solids and neoplastic hematologic disorder tool of the Thscm subgroup for expression specificity antigen
Have strong lethal.
In conclusion the present invention takes the undifferentiated T cell of the CD4+ of source of people or source of mouse, given birth to using t cell activation and conversion
Long factor-beta (TGF β), interleukin 4 (IL-4), IL-21 (IL-21) and gamma interferon neutralizing antibody
A series of (anti-IFN γ) cell factor Co stituations.Polarized T cell in this approach, in the case where a large amount of proliferation, such as
500-1000 times in 14 days, while possessing the lethal and differentiation of self proliferative capacity for cancer cell.Its character is different from it
Before reported, for subgroups such as Th1, Th17 and Th9 to anticancer, and the treatment of cancer the effect of on be more than.With
This polarized CD4+T cell subsets of method, there is the cancer of a variety of expression specificity antigens, including solid and neoplastic hematologic disorder
Powerful therapeutic effect.
Basic principles and main features and advantages of the present invention of the invention, above-described embodiment has been shown and described above
It is merely illustrated the principles of the invention with described in specification, without departing from the spirit and scope of the present invention, the present invention
It will also have various changes and improvements, these changes and improvements are fallen in scope of the claimed invention.The present invention claims
The range of protection is defined by the appending claims and its equivalent thereof.
Claims (10)
1. a kind of method of polarization and amplification CD4+T cell: the following steps are included:
CD4+T cell, CD4+ or CD62L+ T cell after the CD4+T cell of acquisition purifying: isolating and purifying or induction culturing；
(2) active cell: the cell obtained in activation step (1) in 0-24 hours in the medium；
(3) culture and cell Proliferation: cell is centrifuged, removes supernatant by third day upon activation, and is incubated at and is aided with 10% tire ox
In the culture medium of serum, antibiotic and 100U/mL interleukin 2；Stimulation increment and induction differentiation；
(4) cell in activation latter stage and detection are taken.
2. a kind of method of polarization and amplification CD4+T cell according to claim 1, it is characterised in that: the step (1)
In cell origin include the peripheral blood for being not limited to source of people, Cord blood, haemocyte and blood precursor in immune organ, cancer
Disease invades the haemocyte in lymphocyte and the peripheral blood and immune organ of source of mouse.
3. a kind of method of polarization and amplification CD4+T cell according to claim 1, it is characterised in that: the step (2)
The culture medium of middle active cell be RPMI-1640 be aided in the culture medium of 10% fetal calf serum be added 100ng/ml conversion growth because
Sub- β, 100ng/mL interleukin 4, the gamma interferon neutralizing antibody of 100ng/mL IL-21,100ng/mL,
Or identical reagent, various concentration；In the step (3) culture medium be RPMI-1640 be aided with 10% fetal calf serum+antibiotic and
100 u/ml interleukin 2s.
4. a kind of method of polarization and amplification CD4+T cell according to claim 1, it is characterised in that: the step (2)
Middle to activate T cell using immunomagnetic beads, immunomagnetic beads and cell proportion are 1:1 or similar proportion.
5. a kind of method of polarization and amplification CD4+T cell according to claim 1, it is characterised in that: the step (3)
It is preceding that there are also carry out modification step to CD4+T cell；Modification step described in prioritization scheme can after CD4+T cell is activated 4-
It completes within 96 hours；Modification step described in further prioritization scheme is completed in 24 hours after CD4+T cell is activated.
6. a kind of method of polarization and amplification CD4+T cell according to claim 5, it is characterised in that: the modification step
The rapid encoding gene to one section of CD4+T cell transfecting or transduction by DNA or RNA to be carrier；Described in prioritization scheme transfection or
The encoding gene of transduction, including but not limited to inserted type antigen receptor, T cell receptor sequence or other cytokines or cell surface
Receptor；The method of the transfection or transduction includes but is not limited to use retrovirus, slow virus or transposons.
7. it is according to claim 6 it is a kind of polarization and amplification CD4+T cell method, it is characterised in that: it is described transfection or
The method of transduction successively includes that the package step of retrovirus is carried out according to sequence production DNA plasmid step, using these plasmids
Suddenly, obtain host T cell go forward side by side line activating and polarisation steps, transduceed to T cell using viral after polarization 4-96 hour
Step, and continue to expand T cell in aforementioned manners, until the T cell number or T cell that reach required stop amplification.
8. the detection method of a kind of polarization and amplification CD4+T cell according to claim 1, it is characterised in that: described thin
Born of the same parents are vitro detection and vivo detection；
The experiment in vivo includes:
(a) using the mouse of source of mouse tumor model as receptor, using the T cell of TCR transgenic mice as donor, detection is new
Subgroup Thscm is in the intracorporal survival of tumor-carrying mouse, character and differentiation；
(b) several source of mouse cancer-TCR transgenic donor cell models are used, study new subgroup donorcells to a variety of, expression spy
The solid of Specific Antigen and the lethal effect of neoplastic hematologic disorder cancer cell, and observe the time-to-live of mouse；
The experiment in vitro is included: the gene expression for being measured new subgroup using real-time fluorescence quantitative PCR, and had with other
CD4+ subgroup be compared；CD4+ is specially extracted from the splenocyte of C57B6, the cell of CD62L+ induces differentiation into
For Th1, Th17 and the new subgroup Thscm, then the RNA of the RNeasy Kit extraction cell using Qiagen, Thermo is used
RNA reverse transcription is cDNA template by the SuperScript III of Fisher Scientific, and with the primer provided in annex
Do realtime fluorescent quantitative PCR experiment.
9. a kind of pharmaceutical composition, it is characterised in that: combination contains the cell prepared using method in claim 1-8.
10. a kind of application of pharmaceutical composition according to claim 9, it is characterised in that: the pharmaceutical composition is curing table
Application up in the tumour of specific antigen；The tumour is solid tumor or neoplastic hematologic disorder.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109439627A (en) * | 2018-09-17 | 2019-03-08 | 田野 | A kind of polarization and the method and composition for expanding CD4+T cell and the application in the tumour for curing expression specificity antigen |
| CN109913412A (en) * | 2019-03-05 | 2019-06-21 | 上海鑫湾生物科技有限公司 | Compositions, media and methods for inducing and/or expanding TSCM in vitro |
| CN118255684A (en) * | 2024-03-14 | 2024-06-28 | 广州医科大学 | A compound that enhances the differentiation of CD4+T cells into Th9 cells |
| CN118995604A (en) * | 2024-10-23 | 2024-11-22 | 浙江大学 | In-vitro treatment method for improving differentiation efficiency of Th9 cells |
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| CN101603030B (en) * | 2009-07-14 | 2011-05-25 | 吕凌 | In vitro amplification method for regulatory T cells |
| WO2012063875A1 (en) * | 2010-11-11 | 2012-05-18 | シスメックス株式会社 | Markers for detecting human follicular helper t cells and method for detecting human follicular helper t cells |
| CA3003334A1 (en) * | 2015-10-28 | 2017-05-04 | Life Technologies As | Selective expansion of different subpopulations of t cells by the alteration of cell surfacing signals and signal ratio |
| CN107083360B (en) * | 2017-05-23 | 2019-07-05 | 华中科技大学同济医学院附属同济医院 | A method for inducing and expanding human antigen non-specific regulatory T cells in vitro |
| CN107254440A (en) * | 2017-07-10 | 2017-10-17 | 湖南未名三胞转化医学科技有限公司 | CD4 positive TH17T cell culture processes |
| CN109136182A (en) * | 2018-09-17 | 2019-01-04 | 鲁勇 | A kind of polarization and the method and composition for expanding CD4+T cell and the application in the tumour for curing expression specificity antigen |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439627A (en) * | 2018-09-17 | 2019-03-08 | 田野 | A kind of polarization and the method and composition for expanding CD4+T cell and the application in the tumour for curing expression specificity antigen |
| CN109439627B (en) * | 2018-09-17 | 2021-10-01 | 田野 | Method and composition for polarizing and amplifying CD4+ T cells and application of composition in curing tumors expressing specific antigens |
| CN109913412A (en) * | 2019-03-05 | 2019-06-21 | 上海鑫湾生物科技有限公司 | Compositions, media and methods for inducing and/or expanding TSCM in vitro |
| CN109913412B (en) * | 2019-03-05 | 2021-05-11 | 上海鑫湾生物科技有限公司 | In vitro induction and/or amplification of TSCMCompositions, media and methods of |
| CN118255684A (en) * | 2024-03-14 | 2024-06-28 | 广州医科大学 | A compound that enhances the differentiation of CD4+T cells into Th9 cells |
| CN118995604A (en) * | 2024-10-23 | 2024-11-22 | 浙江大学 | In-vitro treatment method for improving differentiation efficiency of Th9 cells |
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