CN105624107A - Expansion method of various lymphocyte subpopulations and application of expansion method - Google Patents
Expansion method of various lymphocyte subpopulations and application of expansion method Download PDFInfo
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Abstract
The invention discloses an expansion method of various lymphocyte subpopulations and application of the expansion method to preparation of an adoptive immunotherapy medicine for cancers. The expansion method comprises the following steps: S1, adding IFN gamma and zoledronic acid into a culture medium of PBMC collected in the blood of a tumor patient, and re-adding an OKT3 antibody and recombinant human interleukin-2 after culture to stimulate PBMC so as to finish preliminary expansion; S2, after finish of preliminary expansion, mixing immune lymphocytes with feeder layer cells, and after adding zoledronic acid, an OKT3 antibody and recombinant human interleukin-2, continually performing expansion. By adoption of the expansion method, after twice expansion, the sum of the immune cells can be 10,000 to 80,000 times by expansion, the expanded immune cells include alpha beta T cells, gamma delta T cells, NKT cells and NK cells, and tumor cells can be directly killed, or a cellular immunotherapy drug is prepared to kill the tumor cells.
Description
Technical field
The invention belongs to biomedical engineering technology field, relate in particular to a kind of method efficiently simultaneously expanding multiple lymphocyte subgroup and the cell amplification test kit prepared.
Background technology
Adoptive immunotherapy is a kind of Therapeutic Method obtaining good result in the recent period in treatment solid tumor and hematologic cancers clinical trial, and becomes the medical research field quickly grown. The method of adoptive immunotherapy includes isolating immune cells, and isolated cells expands, and the immunocyte after amplification is fed back into cancer patient. The example of adoptive immunotherapy successful eradication cancer includes the T cell that application tumor infiltrating lymphocyte (TIL), the T cell modified of ��t cell receptor (TCR) and Chimeric antigen receptor (CAR) are modified. These immunocytes are mainly �� �� T cell. Express alpha and �� chain composition ��t cell receptor. Except �� �� T cell, many other types of immunocyte, the double; two positive NKT cell of such as cytokine-induced killer (CIK) cell, CD3CD56, NK cell positive for CD3 feminine gender CD56 and gamma delta T lymphocytes, it is also possible in the adoptive immunotherapy of cancer.
In above-mentioned cell, CIK cell is the lymphocyte that a kind of external use IFN ��, IL-2 and anti-cd 3 antibodies stimulated for 3 to 4 weeks generated, and is a kind of novel immunologically competent cell. It is firstly added key one step that IFN �� is CIK cell amplification, IFN �� can activate the mononuclear cell being present in PBMC, thering is provided contact dependency factor CD58/LFA-3 and IL-12, these factors are conducive to the cytotoxicity of the lymphocytic Th1 cell phenotype ultimately generated and the CIK cell obtained. IFN-�� can directly suppress the propagation of Th2 cell, thus suppressing the generation of CD4+ cell, is conducive to the generation of CD8+ and TH1 cell. Anti-cd 3 antibodies, such as OKT3, it is provided that an initial mitosis signal, then continued to stimulate T cell propagation and growth as a kind of strong T cell mitogen by IL-2, after the stimulation in 3 to 4 weeks, the immunocyte amplifiable hundreds times of sum, even thousands of times.
The CIK cell produced after amplification contains multiple lymphocyte subgroup. Major part is CD3+CD4+T lymphocyte and CD3+CD8+T lymphocyte. Although CD3+CD56+NKT lymphocyte and CD3-/CD56+NK cell only account for sub-fraction, these immunocytes, particularly NKT cell, there is direct tumor cytotoxicity effect. These cells express NK cell receptor NKG2D (CD314), it is possible to the restrictive mode of non-MHC dissolves tumor cell, carries out Clinic solid tumor adoptive immunotherapy by CIK cell and presents significantly high safety.
Most T lymphocytes belong to �� �� T cell, and their ��t cell receptor is made up of �� and �� chain, sub-fraction T Expressions In Lymphocytes ��t cell receptor gamma and delta heterodimer (�� ��) chain. the activation of �� �� T cell requires over the MHC restriction peptide that antigen processing presents with antigen presenting cell. different from �� �� T cell, gamma delta T cells passes through gamma delta T CR Direct Recognition tumor antigen, microbial pathogens and tumor has the powerful restricted lytic activity of non-MHC-, so gamma delta T cells plays an important role in the immune surveillance of tumor and infection. in blood of human body, gamma delta T cells accounts for the 2-10% of T total lymphocyte count, in order to there be the gamma delta T cells of q.s to carry out adoptive cellular immunotherapy, patient generally requires through excess capacity Leukapheresis to collect a large amount of peripheral blood lymphocytes, singly adopt art technology and can collect 1 �� 10^9 peripheral blood lymphocytes, produce the gamma delta T cells of tens for multiple injection, IPP (isopentenylpyrophosphate) is the native antigen of mankind's gamma delta T cells, have been used for the amplification of gamma delta T cells, synthetic analogues bromohydrin pyrophosphoric acid (BrHPP) and the diphosphonate zoledronic acid (Zometa) of isopentenylpyrophosphate can also optionally activate amplification gamma delta T cells.
In prior art, the widely used a certain kind immunocyte components that is used alone of clinical trial carries out treatment of cancer, and panimmunity cell subsets applies the mode of the different target attacked together in a tumor clinically but without being explored well simultaneously. In theory, the method simultaneously using panimmunity cell subsets can be passed through to attack multiple different tumor target and expand immunoreactive scope, it is possible to can provide the synergism for the treatment of of cancer. In this, widely studied have proven to uniqueness and the important contribution that panimmunity is reacted by gamma delta T cells. Interaction between gamma delta T cells and other immunocytes it is verified that play central role in these immunoreation.
In clinical practice, tumor adoptive cellular immunotherapy is likely to need up to 10^11 immunocyte, so the method for the effective large-scale immune cell expansion of exploitation is the successful key of adoptive immunotherapy. One is called the method for " rapid amplifying program (REP) " and has been used for external a large amount of amplifications of �� �� T cell. Rapid amplifying program is the immune cell expansion scheme of a kind of non-specific (antigen-independent), the program adopts excessive fatal dose gamma irradiation allosome PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) as feeder layer cells, uses anti-cd 3 antibodies (OKT3) and external source recombinant human interleukin (rhIL)-2 simultaneously. This rapid amplifying program may cause to T lymphocyte and expanded 1000 times in 14 days.
Although above-mentioned rapid amplifying program is a kind of conventional method, but its application is subject to material and technical restriction. When clinical practice rapid amplifying program, a T cell would generally stimulate by 200 peripheral blood lymphocytes. So rapid amplifying program needs to collect a large amount of allosome peripheral blood lymphocytes with blood cell separator Large Copacity Leukapheresis from the donor of multiple health. Peripheral blood lymphocytes collected in rapid amplifying scheme is from allosome, it is necessary to through substantial amounts of test in laboratory, to confirm to be in aseptic condition, this is likely to both expensive and time consuming. And, owing to peripheral blood lymphocytes comes from multiple donor, the maintenance effect that T cell is expanded by the PERIPHERAL BLOOD MONONUCLEAR CELL collected and come may show very big diversity between donor's individuality, and large-scale multicenter randomized clinical trial all can be hindered by all these problems to some extent.
Summary of the invention
For this, the technical problem to be solved is in that existing, the method of existing amplification immunocyte is costly and time-consuming and peripheral blood lymphocytes comes from multiple donor, the maintenance effect diversity of T cell is relatively big, thus propose a kind of method that stimulated in vitro peripheral blood efficiently expands multiple lymphocyte subgroup and the cell amplification test kit prepared simultaneously.
For solving above-mentioned technical problem, the technical scheme is that
The present invention provides the amplification method of one kind of multiple lymphocyte subgroups, and it comprises the steps:
S1, the PBMC collected in tumour patient blood culture medium in add IFN �� and zoledronic acid, add OKT3 antibody and recombinant human interleukin--2 after cultivation, stimulate described PBMC, complete lymphocyte immune cell and tentatively expand; Described tumor includes hematological system tumor and solid tumor cell. S2, tentatively expand after, lymphocyte immune cell mixes with feeder layer cells, and continuation expands after adding zoledronic acid, OKT3 antibody and recombinant human interleukin--2.
As preferably, described feeder layer cells is the one in normal pbmc, human erythroleukemia cell strain K562 or human fibroblasts, and the mixed proportion of described feeder layer cells and lymphocyte immune cell is 1:1-300:1.
As preferably, the mixed proportion of described feeder layer cells and lymphocyte immune cell is 50:1-200:1.
As preferably, culture medium described in described step S1 being simultaneously introduced IL-15 and IL-18.
As preferably, described normal pbmc, described human erythroleukemia cell strain K562 and described human fibroblasts process through the �� irradiation of 100G or use ametycin before use.
As preferably, described human erythroleukemia cell's strain K562 cell is the human erythroleukemia cell's strain K562 cell through genetic engineering modified mistake, human erythroleukemia cell's strain K562 cell of described genetic engineering modified mistake is the K562 cell through genetic modification, its can stably express CD64 high-affinity Fc acceptor gene, costimulatory molecules CD86 and molecule CD137L gene.
As preferably, described normal pbmc is from healthy, without what collect in the blood of the donor of tumor medical history; The human desmocyte like cell that described human fibroblasts behaviour induced multi-potent differentiation of stem cells becomes.
As preferably, in described step S1, described IFN �� concentration is 500-1500IU/ml, and described zoledronic acid concentration is 2-10 ��M, and described OKT3 antibody concentration is 20-100ng/ml, and the concentration of described recombinant human interleukin--2 is 100-600IU/mL.
As preferably, the concentration of described IL-15 is 10ng/ml, and the concentration of described IL-18 is 10ng/ml.
Present invention also offers the application in the adoptive immunotherapy of cancer of the amplification method of described multiple lymphocyte subgroup.
The technique scheme of the present invention has the advantage that compared to existing technology
(1) amplification method of the multiple lymphocyte subgroup of the bright offer of this law, after tentatively having expanded, immunocyte sum is at amplifiable about 10 to 40 times during this period of time, wherein gamma delta T cells expands up to 600 times, the double; two positive NKT cell amplification about 200 times of CD3CD56, cell after usual 50% amplification is gamma delta T cells, and 10% is the double; two positive cell of CD3CD56, and 5% is NK cell; Immunocyte mixes with normal pbmc, after continuing amplification 14 days after adding zoledronic acid, OKT3 antibody and recombinant human interleukin--2, immunocyte sum expands about 2300 times, cell after usual 40% amplification is gamma delta T cells, the cell of 25% is the double; two positive cell of CD3CD56, and the cell of 25% is NK cell. Human erythroleukemia cell's strain K562 mixing with cells of immunocyte and genetic engineering modified mistake, after continuing amplification two weeks after adding zoledronic acid, OKT3 antibody and recombinant human interleukin--2, immunocyte sum expanded at 1400 times to 5500 times the period that co-cultures of 14 days, cell after 40% to 70% amplification is gamma delta T cells, and 6% to 16% is the double; two positive cell of CD3CD56. Immunocyte mixes with human fibroblasts, adding zoledronic acid, after continuing amplification after OKT3 antibody and recombinant human interleukin--2 two weeks, immunocyte sum expands about 1300 to 2200 times, cell after at least 20% amplification is the double; two positive cell of CD3CD56, and 20% is NK cell. Further, no matter adopting which kind of feeder layer cells, the amplification of immunocyte sum (tentatively amplification 1 to two week, adds that feeder layer cells co-cultures two weeks) up to 10000 to 80000 times in 3 to 4 time-of-weeks.
(2) amplification method of multiple lymphocyte subgroup of the present invention, described normal pbmc processes 1h through �� irradiation or the use ametycin of 100G, and uses anti-cd 3 antibodies (OKT3) and external source recombinant human interleukin (rhIL)-2 when allosome PERIPHERAL BLOOD MONONUCLEAR CELL uses simultaneously. Wherein, recombinant human interleukin--2 is the strong T cell mitogen needed for a kind of T cell propagation and growth; Normal pbmc cell as feeder layer expresses Fc-�� I receptor (Fc �� RI), can interact with the Fc section of anti-cd 3 antibodies immunoglobulin (Ig); As a kind of anti-cd 3 antibodies, OKT3 can be tied on ��t cell receptor (the TCR)-CD3-complex on T cell surface, interaction between T cell and PERIPHERAL BLOOD MONONUCLEAR CELL that AntiCD3 McAb-��t cell receptor cross-links and produces by being tied to the antibody Fc of CD3, signal-1 and costimulatory signal-2 can be provided simultaneously, cause that T cell is bred without causing incapability or early apoptosis.
(3) amplification method of multiple lymphocyte subgroup of the present invention, the K562 cell that the K562 cell of genetic engineering modified mistake is genetically modified, energy stably express CD64 high-affinity Fc acceptor gene, costimulatory molecules CD86 and molecule CD137L gene (4-1BBL). CD137L is the part of 4-1BB receptor, and main expression is on the T cell surface of activation. CD137 signal transduction participates in suppressing AICD, compared with CD4+T cell, this can be conducive to the amplification of CD8+T cell, CD137 also can the growth of hypermnesis type cd8 t cell, CD86 is the part of CD28 costimulatory receptor altogether, and CD28 all has expression in the T cell of tranquillization and activation. Being interacted by the Fc of CD64 and OKT3 CD 3-resisting monoclonal antibody part, the first signal (OKT3-CD3) and CD86-CD28 and CD137L-CD137 costimulatory signal can dual signal immune stimulatory cell amplifications simultaneously.
Detailed description of the invention
In order to make present disclosure be more likely to be clearly understood, below according to specific embodiments of the invention and in conjunction with accompanying drawing, the present invention is further detailed explanation, wherein:
Fig. 1 is in embodiment 1 after 20 expand, the lymphocyte gate lived in cell FSC-SSC scatterplot;
Fig. 2 is lymphocyte single in embodiment 1 and cell aggregation P2 schematic diagram separately;
Fig. 3 is in embodiment 1 in single lymphocyte, the character of gamma delta T cells and anti-V �� 2-TCR and the CD3 of �� �� T cell;
Fig. 4 is in lymphocyte single in embodiment 1, the character of cd8 t cell and cd4 t cell anti-CD8 �� and CD4;
Fig. 5 is in lymphocyte single in embodiment 1, and single lymphocyte separates NK cell and the schematic diagram of NKT cell;
Fig. 6 is the NKT cell schematic diagram separated in gamma delta T cells in embodiment 1;
Fig. 7 is the NKT cell schematic diagram separated in cd4 t cell in embodiment 1;
Fig. 8 is the NKT cell schematic diagram separated in cd8 t cell in embodiment 1;
Fig. 9 is that in embodiment 2, immunocyte in the PBMC sample of collection in fresh tumour patient blood is tentatively expanded the amplification times of the total lymphocyte count of 14 days by three kinds of distinct methods;
Figure 10 is that in embodiment 2, immunocyte in the PBMC sample of collection in fresh tumour patient blood is tentatively expanded the amplification times of the gamma delta T cells of 14 days by three kinds of distinct methods;
Figure 11 is that in embodiment 2, immunocyte in the PBMC sample of collection in fresh tumour patient blood is tentatively expanded the amplification times of the CD3+CD56+NKT cell of 14 days by three kinds of distinct methods;
Figure 12 is that in embodiment 2, immunocyte in the PBMC sample of collection in freezing tumour patient blood is tentatively expanded the amplification times of the total lymphocyte count of 14 days by three kinds of distinct methods;
Figure 13 is that in embodiment 2, immunocyte in the PBMC sample of collection in freezing tumour patient blood is tentatively expanded the amplification times of the gamma delta T cells of 14 days by three kinds of distinct methods;
Figure 14 is that in embodiment 2, immunocyte in the PBMC sample of collection in freezing tumour patient blood is tentatively expanded the amplification times of the CD3+CD56+NKT cell of 14 days by three kinds of distinct methods;
Figure 15 be in embodiment 3 with or without IL-15 and IL-18 time three kinds of distinct methods the PBMC sample collected in tumour patient blood is tentatively expanded the situation of change of gamma delta T cells of 14 days;
Figure 16 be in embodiment 3 with or without IL-15 and IL-18 time three kinds of distinct methods the PBMC sample collected in tumour patient blood is tentatively expanded the situation of change of CD3+CD56+T cell of 14 days;
Figure 17 be in embodiment 3 with or without IL-15 and IL-18 time three kinds of distinct methods the PBMC sample collected in tumour patient blood is tentatively expanded the situation of change of CD3+CD8+T cell of 14 days;
Figure 18 be in embodiment 3 with or without IL-15 and IL-18 time three kinds of distinct methods the PBMC sample collected in tumour patient blood is tentatively expanded the situation of change of NK cell of 14 days;
Figure 19 be in embodiment 3 with or without IL-15 and IL-18 time three kinds of distinct methods the PBMC sample collected in tumour patient blood is tentatively expanded the CD16 high expressed of 14 days and the situation of change of CD56 high expressing cell;
Figure 20 be in embodiment 3 with or without IL-15 and IL-18 time three kinds of distinct methods the PBMC sample collected in tumour patient blood is tentatively expanded the low expression of CD16 of 14 days and the situation of change of CD56 high expressing cell;
Figure 21 be with amplification after immunocyte co-cultivation under the dissolved cell activity of K562 mankind's marrow series leukemia cell strain;
Figure 22 be with amplification after immunocyte co-cultivation under the dissolved cell activity of Burkitt's lymphoma cell;
Figure 23 adopts the enzyme-linked immunospot assay detection CD19 specific RNA CAR cytotoxic effect schematic diagram to cancerous cell;
Figure 24 adopts the enzyme-linked immunospot assay detection CD19 specific RNA CAR cytotoxic effect block diagram to cancerous cell;
Figure 25 is the immunocyte of the CD19RNACAR modification cytotoxic effect curve chart to Raji cancerous cell in embodiment 4.
Embodiment 1
Immunocyte subunit cluster analysis: the impact that immunocyte subgroup is expanded by the amplification method in order to study described multiple lymphocyte subgroup, initially sets up a method simultaneously analyzing various immunocyte subgroup with polychrome flow cytometer. the immunocyte of amplification is with antibody staining 6 kinds different, during dyeing, cell is resuspended in the MACS buffer of 100 microlitres, dye 15 minutes at 4 DEG C in the dark with mixtures of antibodies, MACS buffer solution for cleaning is used twice before and after cell dyeing, the antibody used includes: the anti-Vdelta2T cell receptor antibody of FITC labelling, the anti-cd 3 antibodies of APC labelling, the anti-CD 4 antibodies of PE labelling, the anti-CD56 antibody of PE-CyC7 labelling, the anti-CD16 antibody of APC-CyC7 labelling, the anti-CD8a antibody of V450 labelling. also adopt AcuriC6 flow cytometer to carry out monochromatic or double-colored analysis simultaneously, the antibody used includes the anti-Vdelta2T cell receptor antibody of FITC-, APC-anti-CD 3 antibodies and PE-anti-CD 3 antibodies, the anti-CD56 antibody of APC-, result is as shown in figures 1-8: the cell obtained through amplification on the 20th is first marked lymphocyte gate (P1) alive by Fig. 1 on FSC-SSC scatterplot, then single lymphocyte and cell aggregation (P2) are separated, as shown in Figure 2, in single lymphocyte, gamma delta T cells is that anti-V �� 2-TCR is positive and AntiCD3 McAb is positive, and �� �� T cell is anti-V �� 2-TCR feminine gender and the AntiCD3 McAb positive, as shown in Figure 3, in single lymphocyte, the character of cd8 t cell and cd4 t cell anti-CD8 �� and CD4 is as shown in Figure 4, single lymphocyte can continue according to the positive NK cell separated with AntiCD3 McAb feminine gender of anti-CD56, as shown in Figure 5, with the anti-CD56 positive and AntiCD3 McAb positive NKT cell, wherein, NKT cell is from gamma delta T cells, cd4 t cell and cd8 t cell, as shown in figs 6-8.
Embodiment 2
The preliminary amplification of immunocyte: adding concentration in the PBMC culture medium collected in tumour patient blood is the IFN �� of 500-1500IU/ml, the present embodiment is preferably 1000IU/ml, concentration is the zoledronic acid of 2-10 ��M, the present embodiment is preferably 5 ��Ms, the OKT3 antibody that concentration is 20-100ng/ml is added after cultivating one day, described tumor includes hematological system tumor and solid tumor cell, the present embodiment is preferably 50ng/ml and concentration is the recombinant human interleukin--2 of 100-600IU/mL, the present embodiment is preferably 300IU/mL, stimulate peripheral blood lymphocytes 7-14 days, described preliminary amplification is the conventional CIK immune cell expansion method method that is used in combination in conjunction with zoledronic acid (ZOL), referred to as CIK+ZOL, described CIK+ZOL is compared with independent CIK method and ZOL method, add immunocyte sum amplification times, as shown in Figure 9, when being used alone CIK method or ZOL method, immunocyte sum expanded about 20 times in 14 days, when being used in combination, immunocyte sum increases to about 40 times in expanding at 14 days. ZOL method and the method that is used in combination can effectively expand gamma delta T cells (about 600 times) and the double, two positive cell (about 200 times) of CD3CD56, as shown in figs. 10-11, comparatively speaking, CIK method cannot expand gamma delta T cells, and can only the double, two positive cell (about 10 times) of limited amplification CD3CD56. obtain when the above results is the PBMC sample collected in using fresh tumour patient blood, during the PBMC sample collected in using the tumour patient blood of a freezing and defrosting, amplification times is different, but compare with CIK method, with the obvious advantage at the double, two positive cell of amplification gamma delta T cells and CD3CD56 of the method that is used in combination, as shown in figs. 12-14.
Embodiment 3
On the basis of embodiment 2, add the IL-15 that concentration is 10ng/ml and the IL-18 that concentration is 10ng/ml to increase the ratio of the double; two positive cell of CD3CD56 further, with or without in IL-15 and IL-18 situation, the PBMC sample collected in tumour patient blood is tentatively expanded 14 days gamma delta T, CD3+CD56+T, CD3+CD8+T, NK, CD16 high expressed and CD56 high expressed, the low expression of CD16 and CD56 high expressing cell situation of change as shown in figs. 15 to 20, data are shown as meansigma methods �� SD, *, * *: P < 0.05 or 0.01. IL-15 is the cytokine that a kind of structure is similar to IL-2, the activation of scalable T cell and NKT NK cell and propagation, interleukin-18 (IL-18) is belonging to a cytokine of IL-1 family, IL-18 can strengthen T cell and the maturation of NK cell, the generation of cytokine, and cytotoxicity, together with IL-15, IL-18 can stimulate NK cell and T cell to be released to interferon gamma (IFN-��).
Embodiment 4
Immunocyte continuation amplification under the effect of feeder layer cells:
After tentatively having expanded, lymphocyte immune cell mixes with normal pbmc, the mixed proportion of described normal pbmc and described lymphocyte immune cell is 1:1-300:1, it is preferably 50:1-200:1, the present embodiment is preferably 200:1, add zoledronic acid, amplification two weeks is continued after OKT3 antibody and recombinant human interleukin--2, to obtain the effect that immunocyte expands in a large number, in the present embodiment, feeder layer cells is normal pbmc, and described PBMC is from healthy, without what collect in the blood of the donor of tumor medical history; The ametycin that described normal pbmc uses concentration at the front �� irradiation through 100G or use 37 DEG C to be 20 �� g/mL processes 1h. Interleukin II is the strong T cell mitogen needed for a kind of T cell propagation and growth. PBMC expresses Fc-�� I receptor (Fc �� RI), and it can interact with the Fc section of anti-cd 3 antibodies immunoglobulin (Ig). As a kind of anti-cd 3 antibodies, OKT3 can be tied on ��t cell receptor (the TCR)-CD3-complex on T cell surface. Interaction between T cell and PERIPHERAL BLOOD MONONUCLEAR CELL that AntiCD3 McAb-��t cell receptor cross-links and produces by being tied to the antibody Fc of CD3, can provide signal 1 and costimulatory signal-2 simultaneously, causes that T cell is bred without causing incapability or early apoptosis.
Embodiment 5
Immunocyte continuation amplification under the effect of feeder layer cells:
After tentatively having expanded, human erythroleukemia cell's strain K562 mixing with cells of lymphocyte immune cell and genetic engineering modified mistake, described K562 cytogenetics modifies the Zinc finger nuclease technology adopted based on baculovirus transfection, CD64_137L_CD86 fusion gene is integrated into specifically on No. 19 chromosomes of K562 cell, the mixed proportion of described human erythroleukemia cell's strain K562 cell and described lymphocyte immune cell is 1:1-300:1, it is preferably 50:1-200:1, the present embodiment is preferably 100:1, add zoledronic acid, amplification two weeks is continued after OKT3 antibody and recombinant human interleukin--2, the ametycin that described K562 cell uses concentration at the front �� irradiation through 100G or use 37 DEG C to be 20 �� g/mL processes 1h. human erythroleukemia cell's strain K562 cell of genetic engineering modified mistake described in the present embodiment is the K52 cell through genetic modification, its can stably express CD64 high-affinity Fc acceptor gene, costimulatory molecules CD86 and molecule CD137L gene (4-1BBL). CD137L is the part of 4-1BB receptor, and main expression is on the T cell surface of activation. CD137 signal transduction participates in suppressing AICD, and compared with CD4+T cell, this can be conducive to the amplification of CD8+T cell. CD137 also can the growth of hypermnesis type cd8 t cell. CD86 is the part of CD28 costimulatory receptor altogether, and CD28 all has expression in the T cell of tranquillization and activation. being interacted by the Fc of CD64 and OKT3 CD 3-resisting monoclonal antibody part, the first signal (OKT3-CD3) and CD86-CD28 and CD137L-CD137 costimulatory signal dual signal can stimulate T lymphocyte simultaneously.
Embodiment 6
Immunocyte continuation amplification under the effect of feeder layer cells:
After tentatively having expanded, lymphocyte immune cell mixes with human fibroblasts, the mixed proportion of described human fibroblasts and described lymphocyte immune cell is 1:1-300:1, it is preferably 50:1-200:1, the present embodiment is preferably 50:1, add zoledronic acid, after OKT3 antibody and recombinant human interleukin--2, continue amplification two weeks; The ametycin that described human fibroblasts uses concentration at the front �� irradiation through 100G or use 37 DEG C to be 20 �� g/mL processes 1h, the human desmocyte like cell (FLC) that described human fibroblasts behaviour induced multi-potent stem cell (iPS cell) is divided into; The FLC cell generated is further by cell transfecting input SV40-large T antigen. The cell transformation that this antigen can cause, make cellular immortalization (Immortalization), become can the human desmocyte like cell strain of life-time service, then FLC is used as feeder layer cells and comes non-specific (non-antigen dependency) and stimulate and the derivative immunocyte of amplification human peripheral blood.
The amplification method of the multiple lymphocyte subgroup that embodiment 1-6 provides can be used for preparing multiple lymphocyte subgroup amplification kit, and the reagent in described test kit includes the feeder layer cells of effective dose, IFN ��, OKT3 antibody, interleukin II and zoledronic acid. Described feeder layer cells can be the one in PBMC, human erythroleukemia cell strain K562 or human fibroblasts, described in the amplification method such as embodiment 1-6 of described multiple lymphocyte subgroup.
Experimental example
1. when test adopts three kinds of feeder layer cells in embodiment 4-6, immunocyte subgroup distribution situation and immunocyte subgroup amplification times, result is such as shown in table 1-2, wherein, table 1 is immunocyte subgroup distribution table, table 2 is immunocyte subgroup amplification times table, and result shows: immunocyte sum can be made to expand about 2300 times the period that co-cultures of 14 days with normal pbmc feeder layer cells co-culture method; Cell after usual 40% amplification is gamma delta T cells, and the cell of 25% is the double; two positive cell of CD3CD56, and the cell of 25% is NK cell. The method co-cultured with K562 engineering cell can make immunocyte sum expand in the scope of 1400 times to 5500 times the period that co-cultures of 14 days; Cell after 40% to 70% amplification is gamma delta T cells, and 6% to 16% is the double; two positive cell of CD3CD56, and NK cell amplification is inconspicuous. Immunocyte sum can be made to expand about 1300 to 2200 times the period that co-cultures of 14 days with human fibroblasts co-culture method; Cell after at least 20% amplification is the double; two positive cell of CD3CD56, and 20% is NK cell, but this method is little to gamma delta T cells amplification effect, and after amplification, only the cell of 8% to 9% is gamma delta T cells.
Table 1
Table 2
2. the direct tumor cytotoxicity effect of the immunocyte after amplification: use the immunocyte after the amplification of K562 feeder layer cells to co-culture with effect/target (EtoT) ratio specified with blood cell, carry out Delphi Asia (DELFIA) cytotoxicity analysis after jointly hatching 3 hours to measure, measure and use DELFIAEuTDA cell reagent box: first with Fluorescence Increasing part (BATDA) solution labelling target cell 15 minutes in 37 DEG C, after a large amount of cleanings, Eddy diffusion cell mass, adjustment cell density mixes to 5xE4 cells/ml and with effect immunocyte, ready effect and target cell are inoculated in 96U shape bottom outlet plate, the usage rate scope of effector lymphocyte and target cell (E:T) is from 40:1 to 1:1, it is set to matched group (only with the addition of target cell), experimental group (target cell adds 10 Al lysis buffer) and blank group (not adding cell), after 37 DEG C of common hatchings 2��3 hours, after centrifugal for culture plate, 20 microlitre supernatants are proceeded in flat underside carefully and adds 200 microlitre europium solution, before using Victor3 reading numerical values, mixed solution is put under room temperature vibrate 15 minutes, and use equation below to calculate killing rate: % specificity release=[experiment release (counting)-spontaneous release (counting)]/[maximum release (counting)-spontaneous release (counting)] �� 100, described target cell adopts K562 mankind's marrow series leukemia cell strain and DaudiBurkitt's lymphoma cell, cell measures tumor carcinoma cells lytic activity after training altogether 3 hours, result and effector lymphocyte used and target ration be as shown in fig. 21-22: uses the immunocyte that the amplification of described method obtains can By Direct Pyrolysis mankind's marrow series leukemia cell strain K562 cell and DaudiBurkitt's lymphoma cell.
3. after amplification, immunocyte is used for CAR immunization therapy:
The essence of CAR treatment technology is gene therapy, and action principle is to utilize technique for gene engineering, modifies immunocyte and makes it express mosaic antigen receptor (CAR) to identify tumor surface specific molecular. CAR gene for preparing CAR immunocyte includes tumor cells identification territory, extracellular, cell membrane spacer domain, and intracellular signal transduction domain, and well-designed CART cell can rely on the effective specific killing cancer cell of its targeting advantage.
First, build a kind of CD19 specific RNA CAR, the sequential coding that when CAR builds, first one DNA fragmentation of synthetic contains includes the single chain antibody fragments (FMC63scFv) of anti-CD19, CD8 membrane spaning domain, stimulates territory CD27 and CD3 �� domain altogether, as shown in table 3.
DNA fragmentation two ends are associated with SphI and the SalI in two digestion sites. After digestive enzyme processes, this DNA fragmentation inserts pFastBac plasmid. The expression CMV of CAR and T7 promoter control. In order to improve the stability after mRNA cell transfecting, to CAR both sides, ' UTR and 3'UTR couples with beta globin 5. In vitro before mRNA synthesis, PCR is adopted to prepare the template transcribed, one couple of PCR primers starts the end to beta globin 3'UTR from CMV promoter, downstream PCR primer contains the 120NT of synthesis, can be used for preparing polyadenylic acid (polyA), mRNA synthesis uses the mMessagemMachineT7 test kit of Invitrogen.
Table 3
Then detect whether to modify the immunocyte of K562 feeder layer cells used above amplification with this RNACAR, thus improving they cytotoxic effects to cancerous cell positive for CD19, adopt the immunocyte after electroporation technology transfection amplification, when using mGFPRNACAR electroporation assessment transfection efficiency, the immunocyte of about 80% transfects successfully, mGFPRNACAR is a kind of control vector of CD19 specific RNA CAR, CD19 strand variable fragment (scFv) is replaced with GFP, so None-identified expresses the cancerous cell that CD19 is positive during structure. When CD19 positive human EBV B lymph matricyte system LCL, DaudiBurkitt's lymphoma cell converted and RajiBurkitt lymphoma cell co-culture with the CD19 specific RNA CAR immunocyte transplanted, enzyme-linked immunospot assay detection finds that CD19 specific RNA CAR transplants the cytotoxicity that greatly strengthen immunocyte, as shown in figs. 23-24. Delphi sub-(DELFIA)
Cytotoxicity further demonstrate that the cytotoxicity of enhancing, the immunocyte that CD19 specific RNA CAR transplants when E:T ratio is 20:1 100% can kill Raji tumor carcinoma cells, the comparison immunocyte that mGFPCAR transplants kills the Rajixb less than 80%, as shown in figure 25.
Obviously, above-described embodiment is only for clearly demonstrating example, and is not the restriction to embodiment. For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description. Here without also cannot all of embodiment be given exhaustive. And the apparent change thus extended out or variation are still among the protection domain of the invention.
Claims (11)
1. the amplification method of one kind of multiple lymphocyte subgroups, it is characterised in that comprise the steps:
S1, the PBMC collected in tumour patient blood culture medium in add IFN �� and zoledronic acid, add OKT3 antibody and recombinant human interleukin--2 after cultivation, stimulate described PBMC, complete lymphocyte immune cell and tentatively expand; Described tumor includes hematological system tumor and solid tumor cell. S2, tentatively expand after, lymphocyte immune cell mixes with feeder layer cells, and continuation expands after adding zoledronic acid, OKT3 antibody and recombinant human interleukin--2.
2. the amplification method of multiple lymphocyte subgroup according to claim 1, it is characterized in that, described feeder layer cells is the one in normal pbmc, human erythroleukemia cell strain K562 or human fibroblasts, and the mixed proportion of described feeder layer cells and lymphocyte immune cell is 1:1-300:1.
3. the amplification method of multiple lymphocyte subgroup according to claim 2, it is characterised in that the mixed proportion of described feeder layer cells and lymphocyte immune cell is 50:1-200:1.
4. the amplification method of the multiple lymphocyte subgroup according to any one of claim 1-3, it is characterised in that be simultaneously introduced IL-15 and IL-18 in culture medium described in described step S1.
5. the amplification method of multiple lymphocyte subgroup according to claim 4, it is characterized in that, described normal pbmc, described human erythroleukemia cell strain K562 and described human fibroblasts process through the �� irradiation of 100G or use ametycin before use.
6. the amplification method of multiple lymphocyte subgroup according to claim 5, it is characterized in that, described human erythroleukemia cell's strain K562 cell is the human erythroleukemia cell's strain K562 cell through genetic engineering modified mistake, human erythroleukemia cell's strain K562 cell of described genetic engineering modified mistake is the K52 cell through genetic modification, its can stably express CD64 high-affinity Fc acceptor gene, costimulatory molecules CD86 and molecule CD137L gene.
7. the amplification method of multiple lymphocyte subgroup according to claim 6, it is characterised in that described normal pbmc is from healthy, without what collect in the blood of the donor of tumor medical history; The human desmocyte like cell that described human fibroblasts behaviour induced multi-potent differentiation of stem cells becomes.
8. the amplification method of the multiple lymphocyte subgroup according to any one of claim 5-7, it is characterized in that, in described step S1, described IFN �� concentration is 500-1500IU/ml, described zoledronic acid concentration is 2-10 ��M, described OKT3 antibody concentration is 20-100ng/ml, and the concentration of described recombinant human interleukin--2 is 100-600IU/mL.
9. the amplification method of multiple lymphocyte subgroup according to claim 8, it is characterised in that the concentration of described IL-15 is 10ng/ml, the concentration of described IL-18 is 10ng/ml.
10. the amplification method of the multiple lymphocyte subgroup described in any one of claim 1-9 application in the adoptive immunotherapy medicine of preparation cancer.
11. the application that the amplification method of multiple lymphocyte subgroup as claimed in claim 10 is in preparing CD19 specific RNA CAR cellular immunization medicine.
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