CN104232578A - Preparation method of polylineage activated killer cells for tumor immunotherapy - Google Patents

Abstract

The invention discloses a preparation method of polylineage activated killer (PAK) cells for tumor immunotherapy. The method comprises the following steps: separating mononuclear cells from peripheral blood of patients with malignant tumor or healthy persons, adding the mononuclear cells of the peripheral blood into anti-TCR gamma delta monoclonal antibodies and IFN-gamma in-vitro induced PAK cells, carrying out in-vitro induction on the obtained objects for 72 hours, and carrying out amplification on the PAK cells in the presence of anti-TCR gamma delta monoclonal antibodies, anti-CD3 monoclonal antibodies and IL-2. The prepared PAK cells are necessary to contain TCR gamma delta<+> cells, CD3<->CD56<+> (NK) cells, CD3<+>CD56<+>(NKT) cells and CD3<+>CD8<+>cells, therefore, the prepared PAK cells have a stronger antitumor effect, and the killing activity of the prepared PAK cells on human lung adenocarcinoma A549 and human lung squamous carcinoma YTMLC is higher than that of cytokine induced killer (cytokine induced killer CIK) cells.

Description

For the preparation method of the polyphyly activated killer of immunotherapy of tumors

Technical field

The present invention relates to the methods for the treatment of of a kind of human cancerous disease, particularly a kind of preparation method being exclusively used in polyphyly activated killer (Polylineage activated killer, the PAK) cell of immunotherapy of tumors.

Background technology

Adoptive cellular treatment (Adoptive cell therapy, ACT) of immunologically competent cell is fed back compared with traditional tumor therapeuticing method in body, can under the prerequisite not damaging body immune system structure and function, direct killing tumour cell; The immunologic function of adjustment and enhancing body simultaneously, thus becoming the important auxiliary treating method of tumor operation, radiation and chemotherapy, is prophylaxis of tumours Preventive, and the life quality improving late tumor patient provides new approach.ACT achieves certain curative effect in the oncotherapies such as malignant melanoma, kidney, lymphoma.

Containing the multiple immune effector cell with tumor cell killing activity in human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC), as TCRgd ⁺t cell, CD3 ^-cD56 ⁺nK cell, CD3 ⁺cD56 ⁺nKT cell and CD3 ⁺cD8 ⁺t cell etc.These effector cells have different identification, the characteristic of killing tumor cell.

1, natural killer (Nature killer, NK) cell gets final product direct killing target cell without the need to antigen presensitization, and do not have " MHC (Major histocompatibility complex; MHC) is restricted ", and tumour cell often lacks the effective expression of MHC I quasi-molecule, therefore, NK cell has important effect in antineoplastic immune.

2, natural killer sample T(Natural killer T, NKT) cell is the T cell subgroup that in immunocyte, has a special sign.NKT cell surface had both expressed T cell surface marker, as CD3, TCR, expressed again the surface marker of NK cell, as CD56, CD122 etc.NKT cell can only identify the specific sugar lipid molecule by CD1d molecule submission, and can not identify by the antigen peptide of MHC molecule submission, what such as alpha-galactosylceramide (α-galactosylceramide, α-GalCer) can limit with CD1d activates NKT cell with the mode of TCR mediation.Activated NKT cell both directly as anti-tumour effect cells play lethal effect, again by activating other immune effector cells, as NK cell, can realize antitumor action indirectly.

3, in the peripheral blood of the mankind, the φt cell receptor (T cell receptor, TCR) of most T cell express alpha β type.Expressing has the T cell of γ δ type TCR only to account for CD3 in peripheral blood ⁺5% ~ 10% of T cell, and the distribution in spleen and lymphoglandula is little, but be distributed in mucosa associated lymphoid tissue (Mucosal associated lymphocytes tissue in large quantities, MALT), as intestines, skin, oesophagus, tracheae, lung and reproductive tract epithelium, it is one of main component of intraepithelial lymphocytes (Intraepithelial lymphocyte, IEL).Research shows that mevalonic acid meta-bolites such as isopentenylpyrophosphate (IPP) can directly act on gdTCR, and then impels gdT cell activation, propagation.The non-classical MHC-I quasi-molecule MICA/MICB of the gdT cell recognition epithelial origin tumor cells expression of activation, this is a kind of approach not needing the MHC molecule non-dependent of angtigen presentation.Therefore, gdT cell may provide a mode selected and make up to identifying and killing and wounding those tumour cells having escaped α β T cell.

4, the CD8 of amplification in vitro cultivation ⁺the expression of T cell surface NKG2D is obviously raised, and is blocked, indirectly cytolysis analysis and siRNA study the CD8 confirming purifying by antibody ⁺the cytotoxicity of T cell antitumor cell is the identification and intracellular signaling that are mediated by NKG2D, instead of passes through TCR.Mouse CD8 ⁺cD44 ^hight cell expresses low-level NKG2D acceptor, after IL-2 activates, express high-caliber NK cell receptor NKG2D and 2B4, and has great lethal effect to the homogenic tumour cell of expressing NKG2D part Rae-1.

Immune effector cell prepared by one or more effector cell's stimulants of the many employings of current ACT, the killing activity of the immune effector cell of acquisition usually from some cell masses with particular phenotype, as the CD3 in CIK ⁺cD56 ⁺nKT cell.A very important characteristic of tumour is its " heterogeneity ", therefore, only adopts a kind of effector cell with particular phenotype to be difficult to thoroughly remove tumour cell, its curative effect and suitability poor.

Summary of the invention

The object of this invention is to provide a kind of preparation method of the polyphyly activated killer for immunotherapy of tumors, application anti-tcr γ δ monoclonal antibody, IFN-γ activate TCR γ δ from human peripheral blood single nucleus cell ⁺, CD3 ^-cD56 ⁺, CD3 ⁺cD56 ⁺, CD3 ⁺cD8 ⁺cell also increases in a large number under IL-2, anti-tcr γ δ monoclonal antibody and anti-CD49d McAb exist, and finally obtains polyphyly activated killer (Polylineage activated killer, PAK) cell tumour cell to stronger killing activity.

For a preparation method for the polyphyly activated killer of immunotherapy of tumors, be specially:

Step one, is separated mononuclearcell from Peripheral Blood from Patients with Malignant, is namely the step gathering peripheral blood and be separated PBMC.Heparin, Citric Acid or ethylenediamine tetraacetic acid (EDTA) (EDTA) anti-freezing can be added to prevent from, in blood collection procedure, blood coagulation occurs when gathering malignant tumor patient or healthy human peripheral blood.Be separated PBMC and adopt density gradient centrifugation.

Step 2, peripheral blood mononuclear cell is added anti-tcr γ δ monoclonal antibody and the external evoked PAK cell of IFN-γ, the external evoked time is 72 hours; Liang Ge multiformity subunit TCR α β in TCR/CD3 complex body and TCR γ δ major function be identify, in conjunction with MHC molecule-antigen peptide complex, but cytoplasmic domain is very short.The stimulation of antigen or TCR complex body corresponding antibodies can activated T cell multi-signal pathway, they play cascade (cascade) reaction separately, regulate initial activation step poly-, and signal transduction is entered in nucleus, trigger the some or several approach in heredity in several approach predetermined, and the propagation of inducing T cell and differentiation, thus play its effector function.Research shows that mevalonic acid meta-bolites such as isopentenylpyrophosphate (IPP) or anti-tcr γ δ monoclonal antibody can directly act on gdTCR, and then impels gdT cell activation, propagation.Therefore, employing anti-tcr γ δ monoclonal antibody can be induced effectively, increase TCR γ δ ⁺t cell.IFN-γ can activate CD3 ⁺cD8 ⁺t cell and CD3 ⁺cD56 ⁺nKT cell.Therefore, IFN-γ and anti-tcr γ δ monoclonal antibody Combined culture PBMC is adopted can to activate simultaneously, induce the cell subsets wherein with anti-tumor activity.The preferably 3 days time of induction PAK cell, because this can make TCR γ δ ⁺, CD3 ⁺cD56 ⁺the cells such as NKT are fully activated and advantage pcr.

Step 3, increased under anti-tcr γ δ monoclonal antibody, anti-CD49d McAb and IL-2 exist by PAK cell, the PAK cell prepared should comprise TCR γ δ ⁺cell, CD3 ^-cD56 ⁺(NK) cell, CD3 ⁺cD56 ⁺(NKT) cell and CD3 ⁺cD8 ⁺cell.

PAK is external evoked, increase the serum free medium selecting applicable human lymphocyte to cultivate, as GT-T551, AIM-V, X-Vivo, CellGro.

Serum in the perfect medium that the immune cells being usually used in now external preparation treatment tumour is cultivated comprises people AB serum, though business-like normal human AB serum is through detections such as HBV, HCV and HIV, but still likely propagate other diseases, security is low; And there is differences between batches, not easily Quality Control, quality can not ensure.The use of autoserum or blood plasma can avoid the problems referred to above, and the inventive method preferably uses autologous plasma, and substratum contains autologous plasma and adopts in step one autologous plasma being separated peripheral blood and obtaining.The a large amount of plasma proteinss contained in blood plasma can provide necessary nutrition for the growth of PAK cell.The blood products of not deactivation complement has hemolytic action, usually adopts 56 ° of C, 30 minutes deactivation complements.Complement loses activity, and does not just have destruction to cell.Therefore, autologous plasma is preferably hatched 30 minutes with complement in deactivation blood plasma through 56 ° of C water-baths by the inventive method.

TCR γ δ is comprised in PAK cell prepared by the present invention ⁺cell, CD3 ^-cD56 ⁺nK cell, CD3 ⁺cD56 ⁺nKT cell and CD3 ⁺cD8 ⁺t cell.CD3 ⁺cD56 ⁺nKT cell is the main component of CIK cell.Therefore, compared with CIK cell, PAK cell will have stronger anti-tumour effect.PAK cell prepared by the present invention to the killing activity of human lung adenocarcinoma A549 and people's lung squamous cancer YTMLC all higher than CIK cell.

Accompanying drawing explanation

Fig. 1 vitro culture PAK cell of the present invention 14th day TCR γ δ ⁺cell phenotype detected result.

Fig. 2 vitro culture PAK cell of the present invention 14th day CD3 ^-cD56 ⁺and CD3 ⁺cD56 ⁺cell phenotype detected result.

Fig. 3 vitro culture PAK cell of the present invention 14th day CD3 ⁺cD8 ⁺cell phenotype detected result.

Fig. 4 vitro culture PAK cell of the present invention and CIK cell are to the killing activity detected result of human pulmonary epithelial cells.

Fig. 5 vitro culture PAK cell of the present invention and CIK cell are to the killing activity detected result of people's lung squamous cancer YTMLC cell.

The PAK cell of external evoked 1 day of Fig. 6 the present invention and acquisition in 3 days is to the killing activity detected result of human pulmonary epithelial cells.

The PAK cell of external evoked 1 day of Fig. 7 the present invention and acquisition in 3 days is to the killing activity detected result of people's lung squamous cancer YTMLC cell.

Embodiment

For a preparation method for the polyphyly activated killer of immunotherapy of tumors, be specially:

Step one, is separated mononuclearcell from malignant tumor patient or healthy human peripheral blood:

Peripheral blood mononuclear cell is separated: gather human peripheral 50mL under sterile state.Blood collection uses 50mL syringe, and antithrombotics adopts heparin, and working concentration is that every milliliter of peripheral blood adds 15IU heparin, and heparin solution and blood volume are than being 1:9.Anticoagulation is injected 50mL centrifuge tube, centrifugal 10 minutes of 400g; Careful absorption upper plasma is in 50mL centrifuge tube, and residue anticoagulation adds physiological saline by 1:1 and fully mixing; Get 50mL centrifuge tube, add human peripheral lymphocyte parting liquid (Tianjin Hao ocean), carefully add dilution peripheral blood on lymphocyte separation medium upper strata, parting liquid and peripheral blood volume ratio are 1:2; Centrifugal 20 minutes of 400g; Careful absorption tunica albuginea confluent monolayer cells, in 50mL centrifuge tube, adds the physiological saline of 2 times of volumes, centrifugal 10 minutes of 400g; Carefully sop up supernatant liquor, with Fresh human lymphocyte substratum (GT-T551, the precious biotechnology in Dalian) suspendible cell again; Cell concn is also adjusted to 1 × 10 by trypan blue staining counting cells concentration ⁶cell/mL.

Prepare containing autologous plasma human lymphocyte substratum: the blood plasma that above-mentioned separation obtains is put into 56 ° of C water-baths and hatch 30 minutes; Get 500mL human lymphocyte substratum (GT-T551), add the blood plasma of deactivation complement, final concentration is 1 ~ 5%.

Step 2, peripheral blood mononuclear cell is added anti-tcr γ δ monoclonal antibody and the external evoked PAK cell of IFN-γ, the external evoked time is 72 hours:

1, antibody bag is by the preparation of culturing bottle, draw anti-tcr γ δ (clone B1, U.S. BD Biosciences) and/or anti-CD49d McAb (Orthoclone OKT3, U.S. Johnson & Johnson (J & J) company) be 2 μ g/ml antibody-solutions to compound concentration in physiological saline, 5ml antibody-solutions is joined 75cm ²in culturing bottle (U.S. Corning), jiggle culturing bottle and solution is uniformly distributed in culturing bottle bottom surface.Culturing bottle is put in 4 ° of C refrigerators.From refrigerator, take out culturing bottle after 24 hours, carefully sop up antibody-solutions in culturing bottle.3 times are washed with human lymphocyte substratum before using.

2, PAK cell is external evoked, the peripheral blood mononuclear cell of fresh separated is joined the 75cm of anti-tcr γ δ monoclonal antibody bag quilt ²in culturing bottle, add recombinanthumanifn-γ's (Shanghai Kai Mao biological medicine) by 1000IU/mL; Culturing bottle is put into 37 ° of C, 5%CO ₂, cultivate 3 days in saturated humidity incubator.

Step 3, increases PAK cell, the PAK cell being induced to the 3rd day is transferred to the 75cm of anti-tcr γ δ and anti-CD49d McAb bag quilt under anti-tcr γ δ monoclonal antibody, anti-CD49d McAb and IL-2 exist ²in culturing bottle, add recombinant human il-2's (bio-pharmaceuticals of Fourth Ring, Beijing) by 1000IU/mL; Culturing bottle is put into 37 ° of C, 5%CO ₂, cultivate in saturated humidity incubator, within every 2-3 days, add fresh cultures and recombinant human il-2 and PAK cell be transferred to 640cm ²continue to cultivate in culture bag (GT-T610 (A), the precious biotechnology in Dalian).In time being cultured to the 14th day, can 0.5 × 10 be obtained ⁹~ 1 × 10 ¹⁰pAK cell.The PAK cell prepared should comprise TCR γ δ ⁺cell, CD3 ^-cD56 ⁺(NK) cell, CD3 ⁺cD56 ⁺(NKT) cell and CD3 ⁺cD8 ⁺cell.

CD3 molecule is the important symbol of T cell, by γ, δ, ε, ζ and η five peptide species chain form, their cytoplasmic domain, all containing ITAM, has the function of conduction TCR signal, makes T cell activation.CD3 molecule and TCR are combined with non covalent bond and form TCR-CD3 mixture, and its major function is that the activation signals produced after TCR is combined with antigen is delivered in cell, inducing T cell activation.Therefore, anti-CD49d McAb is that current various antineoplastic immune cell prepares most important a kind of stimulating factor.The inventive method adopts anti-CD49d McAb to carry out a large amount of amplification to PAK cell equally can for the cell quantity of clinical treatment to obtain.

Through repeatedly testing, represent with the streaming histogram that gamma delta T CR in PAK cell expresses, X-axis is the cell of PE-TCR γ anti-δ mark, and Y-axis is cell counting.In this histogram, dashed region represents the PAK cell that isotype control Ab marks, and be that baseline delimit C door with dashed region, black region represents the PAK cell that PE-TCR γ anti-δ marks, and C door inner cell is TCR γ δ ⁺cell.According to TCR γ δ in PAK cell prepared by embodiment of the present invention ⁺cell proportion is higher than 80%.

Represent to show the streaming scatter diagram that in PAK cell, CD3 and CD56 expresses, wherein X-axis is the cell of FITC-CD3 antibody labeling, and Y-axis is the cell of PE-CD56 antibody labeling.PAK cell is divided into four regions by this scatter diagram, and wherein upper left (K1) shows CD3 ^-cD56 ⁺nK cell, upper right portion (K2) shows CD3 ⁺cD56 ⁺nKT cell.According to CD3 in PAK cell prepared by embodiment of the present invention ^-cD56 ⁺nK cell proportion is higher than 10%; CD3 ⁺cD56 ⁺nKT cell proportion is higher than 60%.

Separately represent to show the streaming scatter diagram that in PAK cell, CD3 and CD8 expresses, X-axis is the cell of ECD-CD3 antibody labeling, and Y-axis is the cell of PE-Cy5-CD8 antibody labeling.PAK cell is divided into four regions by this scatter diagram, and wherein upper right portion (S2) shows CD3 ⁺cD8 ⁺t cell, according to CD3 in PAK cell prepared by embodiment of the present invention ⁺cD8 ⁺t cell ratio is higher than 60%.

According to the present invention, by induction under IFN-γ, anti-tcr γ δ monoclonal antibody, anti-CD49d McAb and IL-2 existence, amplification human peripheral blood single nucleus cell, PAK cell can be obtained.TCR γ δ is comprised in this PAK cell ⁺cell, CD3 ^-cD56 ⁺nK cell, CD3 ⁺cD56 ⁺nKT cell and CD3 ⁺cD8 ⁺t cell.CD3 ⁺cD56 ⁺nKT cell is the main component of CIK cell.Therefore, compared with CIK cell, PAK cell will have stronger anti-tumour effect.Show the detected result of vitro culture PAK cell killing tumour cell after testing.Imitate target than 20:1,10:1,5:1 time, PAK cell prepared by the present invention to the killing activity of human lung adenocarcinoma A549 and people's lung squamous cancer YTMLC all higher than CIK cell.

During vitro culture the 14th day, the detected result of the PAK cell killing tumour cell obtained respectively for 1 day and 3 days is induced to show, when imitating target than 20:1,10:1,5:1, the PAK cell of preparation in external evoked 3 days to human lung adenocarcinoma A549 and people's lung squamous cancer YTMLC killing activity higher than the PAK cell of induction 1 day preparation.

When being cultured to the 14th day, collect PAK cell.Collector's lung cell A549 and YTMLC in addition.Serum lactic dehydrogenase (Lactate dehydrogenase, LDH) method for releasing (U.S. Promega) detects PAK cell killing activity of tumor cells.

Collect Secondary Culture and the good target cell of growth conditions: human lung adenocarcinoma cell line A549 and CH27 YTMLC RPMI-1640 substratum wash twice, are resuspended in the RPMI-1640 substratum containing 5%FBS.Adjustment cell concn is 2 × 10 ⁵cell/mL, joins in 96 orifice plates at the bottom of U-shaped by cell according to 50 μ L/ holes, and centrifugal 3 minutes of 300g, puts 37 C, 5%CO ₂, cultivate in saturated humidity incubator.

Collect the PAK cell that the middle growth conditions prepared of the present invention is good, it is 4 × 10 that the RPMI-1640 substratum be resuspended in after washing containing 5%FBS adjusts cell concn by effect target respectively than 20:1,10:1,5:1 ⁶cell/mL, 2 × 10 ⁶cell/mL, 1 × 10 ⁶cell/mL, joins cell in Target cell wells according to 50 μ L/ holes.

The spontaneous release aperture of laying effect cell, every hole adds 4 × 10 ⁶cell/mL, 2 × 10 ⁶cell/mL, 1 × 10 ⁶cell/mL effector cell and each 50 μ L of nutrient solution.

If the spontaneous release aperture of target cell, every hole adds 2 × 10 ⁵cell/mL target cell and each 50 μ L of nutrient solution.

If the maximum release aperture of target cell, every hole adds 2 × 10 ⁵cell/mL target cell and each 50 μ L of nutrient solution.

Above-mentionedly everyly all establish three multiple holes, in 37 C, 5%CO ₂cultivate 4h in incubator, then by 96 well culture plates with the centrifugal 4min of 300g, every hole is drawn in supernatant 50 μ L to flat 96 well culture plates (enzyme plate), add LDH matrix liquid 50 μ L simultaneously, room temperature, lucifuge hatch 30min, and every hole adds stop buffer 50 μ L, and sample injector removes bubble; Microplate reader 490nm place measures optical density value (Optical density, OD).

Killing activity calculates

In sum, the PAK cell that prepared by the present invention will have stronger anti-tumour effect compared with CIK cell.To the killing activity of human lung adenocarcinoma A549 and people's lung squamous cancer YTMLC all higher than CIK cell.

Claims (1)

1., for a preparation method for polyphyly activated killer (Polylineage activated killer, the PAK) cell of immunotherapy of tumors, it is characterized in that: preparation method is specially:

Step one, is separated mononuclearcell from malignant tumor patient or healthy human peripheral blood;

Step 2, peripheral blood mononuclear cell is added anti-tcr γ δ monoclonal antibody and the external evoked PAK cell of IFN-γ, the external evoked time is 72 hours;

Step 3, increased under anti-tcr γ δ monoclonal antibody, anti-CD49d McAb and IL-2 exist by PAK cell, the PAK cell prepared should comprise TCR γ δ ⁺cell, CD3 ^-cD56 ⁺(NK) cell, CD3 ⁺cD56 ⁺(NKT) cell and CD3 ⁺cD8 ⁺cell.