CN101721684B - Immunowakeup microsystem as well as preparation method and application thereof - Google Patents

Abstract

The invention relates to an immunowakeup microsystem as well as a preparation method and application thereof. The immunowakeup microsystem comprises a cell and an immune microsome, wherein the cell and the immune microsome are crosslinked to form an immune awakening microsystem integral structure by an antibody and an antigen; the cell is a CD4+T cell with a CD4OL expression, and the immune microsome is a microsome of biodegradable material loading recombination rIL-2. The invention simulates the mechanism that bone marrow transplantation (BMT) generates graft versus tumor (GVT) for the first time, constructs the immunowakeup microsystem based on allosome immunocompetence T cells and generates allosome immunoreaction and I type anticancer effect, which are the same with the BMT without the requirement of marrow matching and the serious side effect of graft versus host.

Description

A kind of immunowakeup microsystem

[technical field]

The present invention relates to biological technical field, specifically, is a kind of cell and immune MC immunowakeup microsystem of comprising.

[background technology]

Bone marrow transplantation (BMT) can produce powerful graft antitumor response (GVT), is the present unique method that can cure hematologic cancers and other solid tumors.The application of BMT has three more than ten years historical in the sixth of the twelve Earthly Branches, but anticancer mechanism is also unclear for a long time.BMT treats method for cancer, at first is with the lonizing radiation of high dose and chemicals destruction patient's bone marrow immune system, joins type through bone marrow then, the bone marrow of transplantation donor.Known at present, the method for this BMT " reconstruction immune system " comes down to the reaction of a kind of very effectively antitumor immune, and GVT can remove cancerous cell constantly, disappears until last cancerous cell.

But BMT also exists serious adverse, is called graft versus host disease (GVH).The T cell of donor graft (bone marrow) can be attacked the antigen on host's normal cell, causes the infringement of the multiple target organ of patient, and this infringement usually is a lethal.In recent years, the research of BMT anticancer mechanism has had breakthrough, finds that GVT is a kind of immunoreation, the allosome immunoreation that is induced by donor bone marrow.And this reaction is that donor T is cell-mediated, if emptying T cell is just lost GVH and GVT simultaneously.Find further that in recent years two kinds of reactions of GVH and GVT are same mechanism, the allosome immunoreation that bundles.Find also that in clinical and zoopery successful BMT treatment can be rebuild the mosaic type immune system, can produce the reaction (HVG) that the host repels graft.Though HVG is ostracised graft, produced the reaction (HVT) that the host repels cancerous cell simultaneously, equally with GVT produce the reaction of I type antitumor immune, persistence is removed cancerous cell.In fact, GVH/GVT is two pairs of identical allosome rejectiones with HVG/HVT, and still, HVG only to the variant cell of graft, is repelled it, does not attack host autologous tissue and damages.

Find further that in recent years the allosome immunoreation is not unique, BMT makes immunologic cytotoxicity cell CTL obtain starting and strengthens.Why BMT has powerful anticarcinogenic effect to be because the allosome immunoreation has induced the reaction of I type antitumor immune, and the immunoreactive formation of I type depends on GVH again.Because under the condition that produces GVH, GVH has brought out the secretion of a large amount of I type lymphokines, comprises IFN-γ, GM-CSF, TNF-α etc., suppressed cancer patient II type immunity environment.Reason is that patient's cancerous cell can continuous spontaneous generation II cytokines and the cancerous cell inhibition factor (IL-10 for example; TGF β I); Especially under the condition that high-level cancer antigen exists, the Th1 cell that in normal antitumor response, plays a major role is in the selectivity tolerance status, makes environment disequilibrium in the TH1/TH2 immunity; TH2 immunity environment is preponderated, and causes " dormancy " of immunologic cytotoxicity sexual cell.This is the main mechanism that cancerous cell is escaped immune system attack, claims " immunologic escape ", and this also is a lot of main causes of using the adoptive immunity unsatisfactory curative effect in the past clinically.In fact, the immunologic function of cancer patient is not fully low, not loss of function of immunologic cytotoxicity sexual cell, but be in " dormancy ", " incapability " state, this state can reverse.The allosome immunoreation of BMT finally makes immune system I type antitumor response be restored, and makes immunologic cytotoxicity sexual cell CTL, and NKT sexual cell NK obtains recovery.

At present, bone marrow transplantation is used allosome BMT clinically usually, by the donor of the close coupling of HLA bone marrow is provided.Because the derived from bone marrow of coupling is limited, treatment facility requires high, medical expense is expensive, has limited BMT being widely used clinically.

[summary of the invention]

The objective of the invention is, a kind of immunowakeup microsystem is provided.

One purpose more of the present invention is that a kind of method for preparing of immunowakeup microsystem is provided.

One purpose more of the present invention is that a kind of application of immunowakeup microsystem is provided.

For realizing above-mentioned purpose; The technical scheme that the present invention takes is: a kind of immunowakeup microsystem; Comprise cell and immune microsome; Cell becomes the immunowakeup microsystem overall structure with immune microsome through antibody-antigen crosslinking, and described cell is to have the CD4+T cell that CD40L expresses, and immune microsome is the microsome of Biodegradable material bag load-carrying group rIL-2.

Described immune microsome has been the surface adsorption microsome of anti-Fc monoclonal antibody.

Described immunowakeup microsystem is the allosome CD4+T cell of HLA mispairing crosslinked the forming of microsome of anti-Fc monoclonal antibody of having adsorbed mAb-CD3/mAb-CD28 and surface adsorption.

Described Biodegradable material is selected from: a kind of in lactic acid Polyethylene Glycol (PLG), polylactic acid (PLA), polycaprolactone (PCL), the polylactic acid diethanol acid copolymer (PLGA).

The ratio of described cell number and immune microsome number is 1:10.

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is: a kind of method for preparing of immunowakeup microsystem may further comprise the steps:

A, immune MC preparation: contain in the anti-Fc monoclonal antibody buffer solution adding in the aaerosol solution of Biodegradable material, will contain the rIL-2 microsphere and join in the above-mentioned solution;

B, the activation of CD4+ cells in vitro: adding mAb-CD3 and mAb-CD28 monoclonal antibody are cultivated in serum-free medium in the CD4+ cell solution of purification;

C, immune microsome and CD4+ cell are crosslinked: immune microsome and the external activatory CD4+ mixing with cells of step B that steps A is prepared;

D, the mixed liquor cultivation that step C is made, amplification, liquid nitrogen are preserved.

The ratio of immune microsome and CD4+ cell number of crosslinks is 1:10 among the said step C.

For realizing above-mentioned the 3rd purpose, the technical scheme that the present invention takes is:

The application of a kind of immunowakeup microsystem in preparation treatment solid tumor and hematologic cancers medicine; Described immunowakeup microsystem comprises cell and immune microsome; Cell becomes the immunowakeup microsystem overall structure with immune microsome through antibody-antigen crosslinking; Described cell is to have the CD4+T cell that CD40L expresses, and immune microsome is the microsome of Biodegradable material bag load-carrying group rIL-2.

Described immune microsome has been the surface adsorption microsome of anti-Fc monoclonal antibody, described cell is the allosome resting stage CD4+T cell of HLA mispairing.

Described immunowakeup microsystem is the allosome CD4+T cell of HLA mispairing crosslinked the forming of microsome of anti-Fc monoclonal antibody of having adsorbed mAb-CD3/mAb-CD28 and surface adsorption.

The invention has the advantages that:

1, the present invention simulates the mechanism that BMT produces GVT first; " immunity wakes up " micro-system that structure is the basis with allosome immunocompetence T cell (" ImmunoWakeUp " Micro System; IMS); Produce allosome immunoreation identical and I type anticarcinogenic effect,, wake the immunologic cytotoxicity sexual cell that is in " resting state " up with " immunity wakes up " (ImmunoWakeUp) new mechanism with BMT.The present invention does not need bone marrow, do not need HLA to join type, has avoided risk and the serious adverse of the GVH that bone marrow transplantation brings, makes it to be widely used in the patient of leukemia and many other cancers.

2, the technical scheme of the simulation BMT that (ImmunoWakeUp) names with " immunity wakes up " of the present invention has following two significant feature: first; Immune micro-system with manual method makes up can be started allosome immunoreation HVG/HVT; Can induce a large amount of I cytokines; Reverse the abnormal immune state of Th1/Th2 in the body, overcome immunologic tolerance; The second, the immune micro-system that makes up with manual method can swash the in vivo immune system of self, sets up the reaction of I type antitumor immune, strengthens the function of antigen presentation and identification, wakes the immunologic cytotoxicity sexual cell that is in dormancy up, improves the immunologic cytotoxicity function.

[description of drawings]

Fig. 1: IMS microscopically instance: the CD4+-CD40L express cell is crosslinked with the made a price reduction microsome that has wrapped up rIL-2 through mAbCD3/mAbCD28, forms IMS.The 1st, the CD4+-CD40L express cell, the 2nd, the microsome of parcel rIL-2, the 3rd, CD4+-CD40L express cell and microsome are crosslinked through mAb-CD3/mAb-CD28.

Fig. 2: CD40L expresses dynamically figure behind the CD45RA+ cell activation: the CD4+ cell is with CD45RA-mAb-FITC and biotin-CD40L-mAb labelling and PE dyeing; Do dynamic analysis in 0-24 hour with FACScalibur cell streaming appearance (Becton Dickinson), the result representes with average candle power density (MFI) positive staining percentage of cells.Show among the figure that the CD40L expression begins from 1.0h, 8-12h arrives the peak, more than the continuity 24h.

Fig. 3: behind the resting stage CD4+T cell activation, free IFN-γ growing amount is measured with the ELISA method in the 1-6 days medium supernatants, the pg/ml of unit.Cell Only is not activating cell contrast in the legend; MAbCD3/mAbCD28 is an IFN-γ growing amount in the 1-6 days supernatant of culture medium in the crosslinked back of CD4+T cell and CD3/CD28 monoclonal antibody; IMS is the crosslinked whole micro-system of CD4+T cell and immune microsome 1-6 days IFN-γ growing amounts in culture medium.

Fig. 4: C57BL/6 mouse entity tumor OVA model is accepted the IMS injection of quadratic B alb/c mouse spleen lymphocyte preparation, after IMS injects three days for the second time, measures the lymphocyte toxic effect with 6 hours Cr51 method for releasing of standard.E/T Ratio is imitated the target ratio and is respectively 200:1,100:1,50:1 among the figure.Control is a normal mouse in the legend; Cell Only uses the CD4+-CD40L cell therapy separately; IMS is an IMS treatment group.

Fig. 5: Balb/c mouse B cell leukemia model is accepted the IMS injection of secondary C57BL/6 mouse spleen lymphocyte preparation, after IMS injects three days for the second time, measures the lymphocyte toxic effect with 6 hours Cr51 method for releasing of standard.E/T Ratio is imitated the target ratio and is respectively 200:1,100:1,50:1 among the figure.Control is a normal mouse in the legend; Cell Only uses the CD4+-CD40L cell therapy separately; IMS is an IMS treatment group.

[specific embodiment]

Below in conjunction with accompanying drawing to a kind of immunowakeup microsystem provided by the invention and its production and application the specific embodiment elaborate.

Embodiment 1

A kind of method for preparing of immunowakeup microsystem

One, immune MC preparation

1, bag carries MC preparation:

Lactic acid Polyethylene Glycol (PLG poly (D, L-lactide-co-glycolide)), RG503BoehringerIngelheim Mr2400050:50 (w/w); 6%w/v; Add bovine serum albumin (RIA level Sigma) contain 1% and the rIL-2 (Roche) that is dissolved in buffer in advance add dichloromethane, middling speed stirs into emulsion (w/o); Add emulsifying agent polyvinyl alcohol 10%PVAw/v rapidly; The ice bath high speed stirs and became emulsion (w/o/w) in 30 minutes, and stirring at low speed is spent the night under the room temperature, with organic solvent evaporation.Microsphere solidifies the centrifugal collection in back, distilled water washing three times, and vacuum drying, yield is greater than 70%.Microsphere, is got one and is placed on the microscope slide in distilled water through ultrasonic dispersing, measures its particle diameter with the particle size distribution appearance.

2, microsome surface protein absorption:

Contain 10mg PLG bag and carry to add in the MC aaerosol solution and contain the anti-Fc monoclonal antibody of 0.1mg 1ml buffer solution, spend the night 4 ℃ of low speed joltings.It is subsequent use to add stabilizing agent (mannitol and sucrose) back lyophilizing in the suspension.

3, rIL-2 in vitro discharges:

Get 50ul and (contain 10 approximately ⁶The rIL-2 bag carries microsome) join in the permeable membrane sleeve pipe, be immersed in vibration release in 37 ℃ of constant temperature oscillators in the 5ml distilled water after the sleeve pipe closed at both ends.Collect distilled water sample 5ml every day, detect medicine box (R&D System), measure wherein rIL-2 content, and add the fresh distilled water of 5ml, be the sample of next day with lymphokine Elisa.

Two, the preparation of cell: under the sterile closed condition of GMP, prepare:

1, White Blood Cells Concentrate (layering of PBMC blood canescence) is provided by the blood station, center, does not need HLA to join type.Clean secondary with no Ca++Mg++Hack ' s solution centrifugal, yield is greater than 80%.

2, leukocyte is used the selection of Dynal beads magnetic bead positivity: leukocyte at first uses anti--CD4-Biotin monoclonal antibody to encapsulate, and the cell that encapsulates is that the magnetic bead that Biotin encapsulates combines, and the applied magnetic filter adsorbs magnetic bead then, and the CD4+ cell obtains separating.Keep whole process in airtight gnotobasis, to carry out.In this process, about 6 * 10 ¹⁰Leukocyte separable 5 * 10 ⁹The CD4+ cell.

3, cell marking analysis: the CD4+ cell mainly is (> 70%) the resting stage cell (surface marker: CD4+, CD45RA+, CD62LH1) with fraction memory cell (< 30%) (surface marker: CD4+, CD45RO+, CD62L10).

4, CD4+ cells in vitro activation process: the CD4+ cell of purification is through the crosslinked activation of mAb-CD3/mAb-CD28.This process is with 10 ⁷/ ml cell 10ml adds mAb-CD3 and each 10ug/ml of mAb-CD28 monoclonal antibody, and in serum-free medium, 4 ℃ were shaken 30 minutes, cleans cell then and removes remaining antibody, aligns cell to 10 again ⁷In serum-free medium, cultivate.

Three, prepare immunowakeup microsystem

1, cross-linking process: use the microsome that encapsulates with anti-Fc monoclonal antibody in advance,, cultivated 30 minutes for 4 ℃ with the bonded mixing with cells of mAb-CD3/mAb-CD28, the ratio of 1 cell: 1-2 microgranule.

2, cell culture: it is 10 that cell concentration is aligned again ⁶/ ml cell 100ml is at CO ₂In the incubator, 37 ℃ of static cultivations two days are not shaken.

3, added mAb-CD3 and each 10ug/ml of mAb-CD28 monoclonal antibody on the 3rd day once more, and microsome, the ratio of 1 cell: 1-2 microgranule, 37 ℃ of static overnight incubation are not shaken.

4, cell can be from initial 10 ⁸/ 100ml increases 10 ⁹, with 10 ⁸Packing keeps cell survival and tool activity with frozen culture medium, and liquid nitrogen is preserved.The cell recovery of thawing in 24 hours before use.

5, activatory CD4+ cell carries out venoclysis through monoclonal antibody and crosslinked immune MC IMS form, or intradermal injection.Infusion solution can be normal saline or 5% glucosan, adds the 1-2% human serum albumin.The ultimate density of cell infusion is 10 ⁷-10 ⁸/ ml.Infusion velocity can be selected 50-100ml/ hour.

6, the cell of infusion should be expressed: IFN γ, CD40L, IL-2, IL-15, FasL, TNF-α, TNF-β.The cell of infusion should not expressed: CTLA-4, IL4, IL10, TGF-β.Lymphokine is measured with presentation markup standard medicine box.

Embodiment 2

With IMS treatment solid tumor and leukemia animal model test

One, in mice OVA tumor experiment, 6-10 mice C57BL/6 in age in week is as host receptor, and Balb/c is as donor CD4+ cell preparation IMS.The EG-7 cell 10 that OVA expresses ⁶Intradermal injects the C57BL/6 mice respectively, and the posterior tuberosity body reached the 5-7mm begin treatment in five days.In the B cell leukemia experiment, 6-10 mice Balb/c in age in week is as host receptor, and C57BL/6 is as donor CD4+ cell preparation IMS.BCL2 cell 10E6 injects in the Balb/c mice body, and spleen is extended to original two times after the week, and sees PBLC destruction is arranged and dissolve Jie's begin treatment.Donor CD4+ cell prepares IMS by splenocyte separation, activation and crosslinked immune microsome by embodiment 1 step.

Two, survival rate: inject the IMS of 10E5 in all posterior veins, after two weeks once more Intradermal inject the IMS of 10E5, normal saline compares group.Check survival rate after 90 days.

Three, lethal cell function: IMS injected the back the 5th day in the second time, and the effector lymphocyte is gathered by the DLN lymph node, and target cell is the strain of corresponding mouse tumor model cell, and the Cr51 labelling is measured the lethal cell function of CTL.Method discharges by 6 hours Cr51 of standard to be measured, and presses normalized form and calculates.

Four, immunodot test is measured IFN γ express cell: IMS injected the back the 5th day in the second time, treatment posterior tuberosity model mice splenocyte and the preceding tumor model mice splenocyte 10 of treatment ⁵Raji cell assay Raji IFN-γ express cell is pressed ELISPOT medicine box (R&D System) method and is measured, and the specificity speckle forms cell (SFC) counting.

Five, principle and result

1, the cell of IMS:

Formerly on the research work basis to allosome resting stage CD4+T cell receptor structure, life cycle and activation process, the cell that IMS of the present invention contains has used the express cell at the activatory allosome CD4+-CD40L of external short-term (CD154).Competent cell is in the GMP laboratory, the blood donor CD4+ resting stage T cell of using the HLA mispairing through the TCR receptor identification aglucon CD3 and stimulate the signal short-term activation of aglucon CD28 to form altogether.The present invention is with mAbCD3/mAbCD28 and resting stage CD4+T co-culture of cells starting activation, and the cell after the activation has CD40L expresses, and secretion I cytokines IFN-γ and TNF-α, final differentiating into T h1 cell.CD40L can produce the inflammatory starting process, is that allosome, HLA are unmatched based on this cell to the host, and under the condition that IFN-γ exists, stimulation of host produces the allosome immunoreation by the host immune system mediation rapidly, causes the HVG/HVT reaction.

CD40L is the thing that induces of the strongest Th1 reaction; The CD4+T cell that CD40L expresses is stimulation of host generation allosome immunoreation rapidly not only; Also make the CD40L on host self the CD4+T cell express also rise through the CD40L that expresses; Its result causes the CD4+-CD40L express cell of host's autoimmune system to increase in vivo, has greatly strengthened the interaction of CD40L-CD40, finally is divided into a large amount of host's self Th1 cell.Because the APC that is expressed in the host that CD40 continues widely; DC; On the cells such as NK, and CD40L can discern the target cell with CD40 expression, so produced effect widely: make the T cell produce the cytotoxic activity and a large amount of I cytokines of generation (IFN γ of the anticancer cell of high special; TNF-α, IL-2); Suppress CD4+CD25+ and regulate T cell (Treg); Activation NK cell; Improve CTL and separate the carcinolysis effect of leading; Even can directly act on the cancerous cell that CD40 expresses, the anticancer growth also causes apoptosis.

On the other hand because CD40L and IFN-γ are sophisticated two signals of formation host DC, the CD4+-CD40L cytositimulation maturation of host DC.Sophisticated DC (DC1) can produce a large amount of IL-12, and the two further starts host's self I type antitumor HVT reaction.It is now know that, and IL-12 has play a part crucial in activating the reaction of I type antitumor immune.IL-12 can not only promote resting stage CD4+T cell differentiation to become the excretory Th1 cell of IFN-γ; Equally also can strengthen the molten cytosis and the natural killer cell activity of CD8+CTL mediation, also suppress the effect (32) that CD4+CD25+ regulates T cell (Treg) simultaneously.Clinical research finds that the enhancing of DC1 and IL-12 level is directly linked to the survival condition that cancer does not have recurrence.

The allosome immunoreation that above-mentioned CD4+T cell of being expressed by CD40L starts and the foundation of I type antitumor response are consistent with the HVG/HVT mechanism of allosome BMT.

2, the immune microsome of IMS:

More existing immunotherapies and cell therapy usually after cell gets in the body, because body fluid has changed the environment that the cell of adopting is grown in vivo, cause the change of cell function, have lost expected effect.Therefore; The present invention is except using starting allosome immunoreation of allosome immunocyte and the anticancer chain reaction of I type; The strategy of simulation BMT also must be after allosome CD4+-CD40L express cell gets in the body; The functional status that can keep cell, and after cell is repelled by HVG, still can further keep the state of activation and strengthen the reaction of I type antitumor immune.The present invention has prepared a kind of immune microsome of recombinant il-2 (rIL-2) that contains and has been used for crosslinked activatory CD4+-CD40L express cell.The immunity microsome uses biodegradable lactic acid Polyethylene Glycol, and (poly-DL-lactide-co-glycolide, PLG) polymer material bag has carried the rIL-2 core material, has the function of slow release core material to cell micro-environment.And in the microsome surface adsorption anti-Fc monoclonal antibody be used for crosslinked cell.Can make a price reduction PLG polymer material has good biocompatibility, can make a price reduction into monomer by biology, can make a price reduction material price reduction fully in 10-15 days, and safety and stability is used for human body by the FDA approval.

Immunity microsome bag has carried rIL-2, forms the microsome of 5-15 micron, and this microsome can slowly discharge rIL-2, can continue to discharge 5-10 days according to different experimental conditions, and the IL-2 that keeps more than 75% is active.Immunity microsome surface adsorption anti-Fc monoclonal antibody.Therefore, this microsome can keep the lasting release of core material in vivo, and keeps the crosslinking active of the anti-Fc monoclonal antibody of surface adsorption.After anti-Fc and the bonded mAbCD3/mAbCD28 of CD4+T cell are crosslinked, form behind the 48h and have the cell that CD40L and IFN-γ express, formation IMS overall structure (Fig. 1).The CD4+ cell of being expressed by activatory CD40L has carried rIL-2 immunity microsome with bag and has made up the IMS that forms, in getting into body after, rIL-2 can be in the closely release of cell.This situation is different fully with the situation of cell suspension in containing the rIL-2 culture fluid, and cell can highly increase in vivo, and reaches the state of activation and the functional status of height.Because the early stage expression of CD40L needs the CD28 signal, and the expression of later stage CD40L needs the environment of IL-2,, and improve the secretion of INF-γ so rIL-2 makes activatory CD4+T cell have lasting CD40L to express.The component of this IMS and construction method are extremely important to simulation BMT starting HVG/HVT.

TCR signal (anti-CD3) is not sufficient to induce the secretion of CD4+T cell proliferation and IL-2 of stopping separately; The assistance stimulating factor or the anti-CD28 that add simultaneously from APC can cause effective activation, and the mAb-CD3/mAb-CD28 monoclonal anti physical ability that is fixed on the solid phase is more effectively transmitted activation signals entering cell (36).Therefore, the present invention at first uses mAb-CD3/mAb-CD28 monoclonal antibody co-activation CD4+T cell in culture medium, and the made a price reduction polymer microgranule that has carried rIL-2 with bag then is crosslinked through the anti-Fc monoclonal antibody of surface adsorption.This structure of IMS provides CD4+T cell solid phase activated condition, has strengthened cell proliferation and activation, accelerates the expression and the continuous release of lymphokine.After IMS formed, CD40L expressed beginning, 6 hours peak (Fig. 2) at 2 hours.IFN γ began to produce expression after 4 hours, growing amount straight line rising (Fig. 3) in external 3 days.Therefore, make up and characteristic according to IMS, the present invention has adopted the external activation of CD4+T cell short-term in the preparation of IMS, the scheme of amplification in the body.

3, IMS simulation BMT has started HVG identical with BMT and HVT, and consequently cancerous cell and allosome immunocyte are ostracised simultaneously, has prevented the reaction that variant cell is attacked the host.

The HVG reaction helps allosome immunocyte withdrawing rapidly after accomplishing " inflammatory starting process " among the IMS greatly, thereby has avoided the generation of GVHD fully.The allosome immunologically competent cell of IMS has finally caused the release of a large amount of I cytokines; Reversed the immunosuppressant environment; Set up I type antitumor immune reaction, activation host's cellular immunization such as CTL precursor and NK, reach " immunity wakes up " effect of removing cancerous cell.

The present invention is unique to be used by the microsome and the crosslinked structure of the cell IMS that can make a price reduction the rIL-2 that the polymer bag carries.This construction method not only keeps activatory cell differentiation to become the Th1 hypotype to produce the IFN-γ of capacity, and because rIL-2 constantly closely discharges to cell from microsome, the microenvironment that rIL-2 forms makes the Th1 cell that the expression of the CD40L in later stage arranged.This structure makes it to adopt cell at the external activatory cultural method that carries out 3 days short-terms, and is different with external 12-14 days long-term cultivation, avoided because of bringing contamination of heavy and saved manpower and financial resources in external activation and amplification long-term cultivation.The present invention obtains just can import in the body for the first time with the form of IMS after CD40L expresses at allosome CD4+T cell.The CD4+-CD40L express cell that injects mainly is distributed in draining lymph node, and amplification, activation DC, and the I type anamnestic response to variant cell is set up in the immunoreation of starting allosome.When IMS imports in the body once more, cause then and recall immunoreation and tardy property hypersusceptibility (DTH) that promote the development of I type antitumor immune reaction, gram is multiple " immunologic tolerance " make " wake up " immunocyte discern cancer antigen again.Whole immune process is very fast; Mat woven of fine bamboo strips secondary IMS imports back five days and just can detect body endolymph cell IFN-γ express cell quantity rising (table 1) with immunodot test (EliSpot); The main effects that the anamnestic reaction of I type produces is described; And the development that causes I type antitumor immune to react; The lethal effect of immunocyte is strengthened greatly (Fig. 4,5).Finally make the tumor animal survival rate improve (table 2).

Table 1. mice OVA and mouse B cell leukemia model are measured IFN γ express cell with immunodot test after IMS injects 5 days for the second time, every group of 5 mices, and the result forms cell (SFC) counting for the specificity speckle.

IFN?γ?spot-formingcells	OVA	BCL2
			Before?Treatment	13±5	14±6
After?Treatment	86±28	67±14

Table 2. mice OVA and mouse B cell leukemia model are accepted secondary IMS injection, treat back 90 days the survival number of every group of 10 mices with IMS.

I.V.Infusion+I.D.	OVA	BCL2
			Normal?Saline	0	0
Cell?only	2	1
			IMS	8	4

In recent years the GVT result of study is shown that the immunogenicity of cancerous cell has equally also determined the efficient of GVT.IMS makes that to have a cell that CD40L expresses crosslinked with the CD40 that host DC expresses, and (MHCclassII, CD58 CD80/CD86), have improved cancer and assisted antigenic expression, the effect that kills and wounds cancerous cell of accelerating effect cell in rise " the helper cell factor ".While IMS activation natural immunity cell, like the lethal effect of NK cell and mononuclear phagocyte, also the activating T cell surface molecular like FasL, TRAIL, TWEAK and TNFR, acts on cancerous cell and causes apoptosis.The final differentiating into T h1 of activatory CD4+-CD40L cell cell, the GM-CSF of generation and IFN-γ can raise the MHC developed by molecule of cancerous cell, promote the identification of allosome immunoreation CTL immunocyte to cancerous cell.The variation of the lethal effect of the effector molecule of these rises, " the helper cell factor " and immunocyte is regarded as the evidence that IMS simulates BMT in vivo in the present invention, is IMS cell CD40L and host cell CD40 results of interaction.

IMS simulation BMT of the present invention produces HVT with " immunity wakes up " mechanism, meanwhile; Also has another important mechanism of BMT; Can improve cancer and assist the expression of antigen and MHC, strengthen the immunogenicity of cancerous cell, more help immunity identification; Strengthen the removing of cancerous cell, play a part very important preventing cancer return and raising survival rate.

4, the method for amplification in structure that IMS of the present invention is unique and the activation of short-term cell in vitro, the body; Make IMS can start HVG/HVT in vivo rapidly; Reverse immune microenvironment and tilt, wake the lethal effect that the immunologic cytotoxicity sexual cell has been strengthened immunocyte up, treatment back survival rate is improved to Th1.Merit attention, these results likewise show on the animal model of solid tumor and hematologic cancers.Therefore, the clinical scope of application of the present invention can not only substitute the BMT application on hematologic cancers such as leukemia at present, also possibly expand many solid tumors to.Obviously, the application of IMS has been avoided allosome BMT and has been brought out GVHD, has also avoided BMT Secondary cases viral infection.Simultaneously, can expect, can improve the existence of curative effect lotus tumor and reach and not have recurrence recovery from illness state if unite other cancer treatment methods.

The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; Can also make some improvement and replenish, these improvement and replenish and also should be regarded as protection scope of the present invention.

Claims (10)

1. the application of immunowakeup microsystem in preparation treatment solid tumor and hematologic cancers medicine; Described immunowakeup microsystem is a kind of cell and immune MC product of comprising; Cell becomes the immunowakeup microsystem overall structure with immune microsome through antibody-antigen crosslinking; Described cell is to have CD40L allosome CD4+T cell that express, the HLA mispairing, and described immune microsome is the microsome of Biodegradable material bag load-carrying group rIL-2.

2. application according to claim 1 is characterized in that: described immune microsome has been the surface adsorption microsome of anti-Fc monoclonal antibody.

3. application according to claim 2 is characterized in that: described immunowakeup microsystem is the allosome CD4+T cell of HLA mispairing crosslinked the forming of microsome of anti-Fc monoclonal antibody of having adsorbed mAb-CD3/mAb-CD28 and surface adsorption.

4. immunowakeup microsystem; Said immunowakeup microsystem is meant a kind of product; Comprise cell and immune microsome; Cell becomes the immunowakeup microsystem overall structure with immune microsome through antibody-antigen crosslinking, it is characterized in that: described cell is to have CD40L allosome CD4+T cell that express, the HLA mispairing, and described immune microsome is the microsome of Biodegradable material bag load-carrying group rIL-2.

5. immunowakeup microsystem according to claim 4 is characterized in that: described immune microsome has been the surface adsorption microsome of anti-Fc monoclonal antibody.

6. immunowakeup microsystem according to claim 5 is characterized in that: described immunowakeup microsystem is the allosome CD4+T cell of HLA mispairing crosslinked the forming of microsome of anti-Fc monoclonal antibody of having adsorbed mAb-CD3/mAb-CD28 and surface adsorption.

7. immunowakeup microsystem according to claim 6 is characterized in that: described Biodegradable material is selected from: a kind of in lactic acid Polyethylene Glycol, polylactic acid, polycaprolactone, the polylactic acid diethanol acid copolymer.

8. immunowakeup microsystem according to claim 7 is characterized in that: the ratio of cell number and immune microsome number is 1: 10.

9. the method for preparing of an immunowakeup microsystem as claimed in claim 4 may further comprise the steps:

A, immune MC preparation: contain in the anti-Fc monoclonal antibody buffer solution adding in the aaerosol solution of Biodegradable material, will contain the rIL-2 microsphere and join in the above-mentioned solution;

The allosome CD4+ cells in vitro activation of B, HLA mispairing: adding mAb-CD3 and mAb-CD28 monoclonal antibody are cultivated in serum-free medium in the allosome CD4+ cell solution of the HLA mispairing of purification;

C, immune microsome and CD4+ cell are crosslinked: immune microsome and the external activatory CD4+ mixing with cells of step B that steps A is prepared;

D, the mixed liquor cultivation that step C is made, amplification, liquid nitrogen are preserved.

10. method for preparing according to claim 9 is characterized in that: the ratio of immune microsome and CD4+ cell number of crosslinks is 1: 10 among the said step C.

Images