Preparation method of immune cell therapy DC-CIK cells and SAFIRE functional human CD3 monoclonal antibody

Summary

SAFIRE is the abbreviation for " Sodium Azide Free Immensely Reduced Endotoxin ". No carrier is added in the production, and the endotoxin level is as low as 0.01 EU/ug or less, which is more suitable for functional and experimental studies in vivo and in vitro.

BioGems SAFIRE Functional Antibody Introduction :

BioGems world's top antibody research and development manufacturing company / brand

SAFIRE functional antibody is an important product line of BioGems flow products, which can be used for activation, blocking and neutralization experiments. SAFIRE is the abbreviation of “Sodium Azide Free Immensely Reduced Endotoxin”, ie “free of sodium azide, pole Low endotoxin." No carrier was added during the production process, and the endotoxin level was as low as 0.01 EU/ug or less, which is more suitable for functional and experimental studies in vivo and in vitro. Among them, BIoGems 05121-25-500 SAFIRE functional CD3 monoclonal antibody is a typical representative of the BioGems brand with subversive quality and price.

Sodium azide, and NaN3, are preservatives that block the mitochondrial electrotransport system and are toxic to most organisms. In the body, azide is similar to cyanide, inhibits cytochrome oxidase and other enzymes, and can block the formation of oxyhemoglobin in the body, has significant antihypertensive effect, and has certain stimulation to eyes and skin. Sex.

Endotoxin can also cause a series of reactions in the body, such as various inflammatory reactions, hypotension, abnormal white blood cell count, intravascular blood coagulation, shock and even death. In vitro, endotoxin can react with CD14 and other receptors. Monocytes and macrophages release cytokines, trigger the complement system, and promote B cell division.

Both can have uncontrollable effects, reduce the specificity of the experiment, and interfere with the accuracy of the results. BioGems' SAFIRE series of functional antibodies do not contain sodium azide, and the level of endotoxin is extremely low, which can fully meet the needs of in vivo and in vitro functional experiments.

BioGems currently has 132 FF functional antibodies, including 49 species, 89 mice, and 8 mice, and the variety of products is increasing.

Catalogue Number :05121-25

Description

The OKT3 monoclonal antibody specifically reacts with the ε chain of the CD3/T lymphocyte antigen receptor complex. The CD3 complex contains γ, δ, and ε chains, and it is part of the TCR complex, expressed by all mature T lymphocytes and by the Thymocyte lineage. CD3 enhances the antigen recognition by signal transduction.

The OKT3 antibody is an immunosuppressive, which has proven to be an effective therapeutic agent in liver, heart, and renal allograft rejection.
Additional Information

Clone: ​​OKT3
Format:SAFIRE Purified
Applications:FC, FA, IHC
Reactivity:Human
Isotype: Mouse IgG2a, kappa
Research Interest:Adaptive Immunity
Cell Type: NK and NKT Cells, T Cells, Treg cells, Th17 cells

Method for preparing DC-CIK cells

background

CIK is an abbreviation of "Cytokine-Induced Killer Cells", which is called "cytokine-induced killer cells" in Chinese. CIK is a group of heterogeneous cells in which mononuclear cells are cultured under the action of CD3 mAb and various cytokines (including IFN-y, IL-2, etc.), and CD3+CD56+ cells are the main effector cells. The powerful anti-tumor activity of T lymphocytes has the non-MHC (major histocompatibility antigen) limiting tumor killing ability of NK cells (natural killer cells). CIK cells have high tumoricidal activity, broad spectrum of tumoricidal activity, low toxicity to normal tissues, and high amplification in vitro. They are currently used immunotherapy cells widely used in clinical practice.

DC is the abbreviation of "Dendritic Cells", which is called "dendritic cells" in Chinese. It is named for its many dendritic or pseudo-protrusions. The DC was discovered in 1973 by the 2011 Nobel Prize winner and Canadian scientist Ralph M. Steinman. It is the most powerful antigen-presenting cell (APC) currently discovered. DC has been shown to be the only APC that can significantly stimulate the proliferation of naive T cells, while other APCs (such as monocyte macrophages, B cells, etc.) can only stimulate activated or memory T cells. DC is the initiator of the body's adaptive T cell immune response and plays an extremely important role in tumor immunity.

DC-CIK, DC and CIK cells, are co-cultured in vitro and then returned to the patient. Strictly speaking, the final effector cells are CIK cells that are activated in vitro by DC. A number of studies have shown that DC and CIK have synergistic effects. After co-incubation, the expression of co-stimulatory molecules on DC surface and antigen-presenting ability are significantly improved, while the proliferative capacity of CIK and cytotoxic activity in vitro and in vivo are also enhanced. -CIK is more effective than separate CIK treatment. Co-culture of tumor antigen-loaded DCs with CIK can stimulate the production of tumor antigen-specific T cells. Such DC-CIK treatment has a specific non-specific dual tumor killing effect, which is more than DC stimulation without tumor antigen loading. Activated CIK is more active and is often used in clinical and scientific research.

Principle of cultivation

Preparation:The product should be stored undiluted at 4°C. Do not freeze. The monoclonal antibody was purified utilizing affinity chromatography. The endotoxin level is determined by LAL test to be less than 0.01 EU/μg of the protein.
Formulation: Phosphate-buffered aqueous solution, ph7.2.

1. DC culture cytokines :

GM-CSF ( granulocyte macrophage colony-stimulating factor ) :

GM-CSF is a hematopoietic growth factor that stimulates the formation of colonies of neutrophils and macrophages in vitro and has the function of promoting the proliferation and development of early red megakaryocytes and eosinophilic progenitor cells. GM-CSF is one of the first cytokines identified to have an effect on DC.

The function of GM-CSF in DC culture is to promote the differentiation of monocytes into large macrophage-like cells, and the expression of MHC class II molecules on the cell surface is enhanced, thereby enhancing the antigen presentation function of cells. In addition, GM-CSF can also promote the survival of DC.

The role of IL-4 in the induction of DCs by monocytes is to inhibit the overgrowth of macrophages, thereby directing monocytes to differentiate into DCs. If IL-4 is not added to the culture system, monocytes will differentiate into macrophages. At the same time, IL-4 also has the ability to reduce the expression of CD14 molecules on the cell surface. A decrease in the expression level of CD14 is an important marker for the differentiation of monocytes into DCs.

The interaction of GM-CSF and IL-4 can differentiate mononuclear cells into immature DCs. At this time, DCs have strong antigen uptake and processing ability, but antigen presentation ability is weak. MHC class I, class II molecules and B7 family molecules (CD80, CD86, etc.) are moderately expressed on the cell surface, but CD14 is not expressed.

TNF-α (tumor necrosis factor-α)
TNF-α can down-regulate the macrocytosis of immature DCs and the expression of surface Fc receptors, so that the class II compartment of MHC class II disappears, but it can up-regulate MHC class I and class II molecules on the cell surface. The expression of B7 family molecules (CD80, CD86, etc.) differentiates immature DCs into mature DCs (mature DC). At this time, DC antigen uptake and processing capacity are significantly weakened, while antigen presentation ability is significantly enhanced and can be strongly activated. T cells.

2. Cytokines and antibodies for CIK culture:
CD3-excited monoclonal antibody:
The first signal for T cell activation comes from receptors on the surface of T cells, ie T cell antigen receptors.
Receptor, TCR) specifically binds to the antigen presented by APC, which is the specific recognition of antigen by T cells.
TCR is a heterodimer composed of two different peptide chains that bind to CD3 molecules via non-covalent bonds on the surface of T cells to form a TCR/CD3 complex. The recognition of specific antigens by TCR causes aggregation of the cytoplasmic tail of the co-receptor CD4 or CD8 molecules on the surface of CD3 and T cells, thereby activating tyrosine kinases linked to the cytoplasmic tail (Lck, Fyn
And ZAP-70, etc., promote tyrosine (Y) phosphorylation in the immunoreceptor tyrosine-based activation motif (ITAM) of the cytoplasmic region of CD3 molecule. Phosphorylated tyrosine (pY) further phosphorylates downstream tyrosine-containing proteins, thereby causing a cascade of kinase activation (phosphatidylinositol pathway or MAP kinase pathway, etc.), ultimately by activating transcription factors In the nucleus, binding to target genes (such as IL-2 and IFN-γ) that regulate T cell proliferation and activation, causing gene expression and transcription, and T cells are thus transferred from a quiescent state to a proliferating and activated state.

It can be seen from the above that CD3 molecules play an extremely important role in the transduction of T cell activation signals. Specific binding of CD3-excited mAb to the CD3 molecule on the surface of T cells can cause tyrosine phosphorylation in the ITAM motif of the cytoplasmic region of CD3, which in turn leads to activation of downstream signals of T cell proliferation and activation, thereby enabling T Cell proliferation and activation. In other words, CD3-excited mAb can mimic the recognition and activation process of antigen and TCR/CD3 complex, which leads to the proliferation and activation of T cells, and is therefore an indispensable stimulus in CIK cell culture.
In addition, CD3-excited monoclonal antibodies must pay attention to the clone number when selecting. Studies have shown that only the clone number is OKT-3
CD3-expressing mAbs stimulate the proliferation of T cells in all humans, while CD3-expressing mAbs from other clones only stimulate a subset of human T cells. Therefore, in the case of CIK culture, it is best to use OKT-3 clones to ensure that each patient's T cells can be activated.

IL-2 (Interleukin-2)
IL-2 was originally discovered as T cell growth factor (TCGF) and is the most important cytokine that causes T cell proliferation. IL-2 is both an autocrine cytokine and a paracrine cytokine, which
Specific binding of the IL-2 receptor (IL-2R) on the cell surface promotes T cell activation and enters a cell division state.
In addition, IL-2 stimulates the growth of NK cells and enhances their killing ability. Therefore, it is necessary to add CIK cell culture.
IL-2 to promote proliferation and activation of T cells.

IFN-γ (interferon-γ)
IFN-γ has the effect of up-regulating the expression of IL-2R on the surface of peripheral blood lymphocytes, thus enhancing the sensitivity and intensity of T cells to IL-2 proliferative response. The addition of IFN-γ during the induction of CIK cell formation reduces the amount of IL-2.
The study found that the order of IFN-γ addition is closely related to the cytotoxic activity of CIK. The cytotoxic activity of CIK was significantly increased by adding IFN-γ first, and then adding IL-2 after 24 hours of culture.

IL-1α (interleukin-1α)
IL-1α also mediates up-regulation of IL-2R on the surface of peripheral blood lymphocytes. When IL-1α and IFN-γ are excited
When combined with CD3 monoclonal antibody, the cytotoxic effect of CIK can be significantly improved.

Cell preparation
1. Collection of peripheral blood mononuclear cells
1.1 using a blood cell separator to collect the patient's own peripheral blood mononuclear cells 80 - 100ml;
1.2 Lymphocyte separation solution Density gradient centrifugation further purification of mononuclear cells (PBMC).
1.3 Serum-free medium was washed twice to obtain PBMC with a purity of more than 90%, and the number of cells should be 1-3 x 108.

2. (Optional step) Preparation of tumor antigen The tumor antigen for DC loading may be Tumor-Specific Antigens (TSA) or Tumor-Associated Antigens (TAA), or may be tumor whole cells. antigen. DCs loaded with TSA or TAA have good targeting, but the method has the defects that the tumor-specific antigen or antigen peptide species are determined to be small and the immune attack of a single antigen often fails to kill tumor cells. The use of DCs loaded with tumor whole cell antigens overcomes these deficiencies, as it is not necessary to know that those antigens are TSAs or TAAs of tumor cells, and that multiple tumor antigens in whole antigens can trigger DCs to produce cells against different antigenic determinants. Toxic T lymphocytes (CTL) are cloned to achieve effective killing of tumor cells. There are many methods for tumor cell whole antigen-loaded DC, including loading DC with tumor cell lysate, DC with apoptotic tumor cells, DC with necrotic or dead tumor cells, DC with tumor living cells, and DC with tumor cells. Fusion and so on. At present, it is commonly used in clinical practice to load DC with tumor cell lysate, because the method is simple, rapid and effective. Repeated freezing and thawing is a common method to obtain tumor cell lysate. The specific steps are as follows:
2.1 Surgical removal of tumor specimens, under sterile conditions, remove necrotic tissue and non-tumor tissue adjacent to the tumor;
2.2 Wash 3 times with sterile saline;
2.3 Cut the tumor tissue with a sterile tissue scissors, add RPMI 1640 medium, and grind thoroughly;
2.4 200 mesh sterile mesh filtration after collection of single cell suspension;
2.5 Resuspend the cells in RPMI 1640 medium to 1-2 x 107/ml and place in a 5 ml sterile cryotube;
2.6 The frozen tube was immersed in liquid nitrogen for quick freezing, taken out after 10 min, and then quickly thawed in a 37 ° C water bath for 10 min.
Repeat 3-5 times;
Note: It can also be freeze-thawed 3-5 times at -80 °C / 37 °C.
2.7 Add the tumor lysate to the centrifuge tube, centrifuge at 3000 rpm for 10 min;
2.8 Collect the supernatant, filter and sterilize by 0.22μm filter, and check the protein content and bacteria, fungi and mycoplasma;
Store at 2.9 -80 °C for backup.

3. CIK cell culture and identification
3.1 The PBMC obtained in step 1 was adjusted to a cell concentration of 2 x 106/ml with serum-free medium and placed in a culture flask;
3.2 Incubate for 2 h at 37 ° C in a 5% CO 2 incubator to allow monocytes to adhere;
3.3 collecting suspended cells, adjusting the cell concentration to 1-2 x 106/ml with serum-free medium;
3.4 adding 1,000 U/ml recombinant human IFN-γ culture;
3.5 After 24 h, 50 ng/ml CD3 monoclonal antibody and 300 U/ml recombinant human IL-2 were added to stimulate the growth and proliferation of CIK cells;
Note: 100 U/ml recombinant human IL-1α can also be added at this time.
3.6 Change the liquid or expand the bottle once every 3 days and add the recombinant human IL-2 300 U/ml;
3.7 On the 7th day of culture, CIK cells were harvested, and the number should be 1x 109 or more.
3.8 CIK Cell Quality Control:
3.8.1 Trypan blue staining to detect cell viability: live cells should be above 80%;
3.8.2 The expression of CD3, CD8 and CD56 on the cell surface was detected by flow cytometry, and the proportion of CD3+CD56+ cells was significantly increased.

4. Culture and identification of DC cells
4.1 The remaining adherent cells (mainly CD14+ monocytes) in step 3.2 were added to serum-free medium containing recombinant human GM-CSF 500-1,000 U/ml and recombinant human IL-4 500 U/ml at 37 °C. Cultured in a 5% CO2 incubator to induce differentiation of monocytes into DC cells;
4.2 Change the liquid once every 3d and supplement the cytokines;
4.3 (optional step) On the 5th day of culture, the tumor antigen 50 μg/ml obtained in step 2 was added to carry out antigen loading on DC;
Note: This step is omitted if the antigen load is not applied to the DC.
4.4 On the 6th day of culture, recombinant human TNF-α (500 U/ml) was added to induce DC cell maturation;
4.5 On the 7th or 8th day of culture, DC cells should be harvested in an amount of 1×106 or more;
4.6 DC quality inspection:
4.6.1 Trypan blue staining to detect cell viability: live cells should be above 80%;
4.6.2 Flow cytometry detects the expression of HLA-DR, CD83 and CD86 molecules on the surface of DC cells to determine whether DCs are mature.

5. Preparation and quality inspection of DC-CIK cells
5.1 Collect the DC cells and CIK cells obtained in steps 4 and 3, and co-culture in a ratio of 1:10 (number ratio), and add recombinant human IL-2 (300 U/ml) to the serum-free medium;
5.2 Change the amount once every 3 days and add recombinant human IL-2 (300 U/ml).
5.3 Collect cells on the 7th day, the number of cells should reach 1×1010 or more;
5.4 Quality inspection of DC-CIK cells:
5.4.1 Trypan blue staining test: live cells should be above 80%;
5.4.2 Flow cytometry to detect the expression of CD3, CD8, CD56 and other molecules on the cell surface: the proportion of CD3+CD56+ cells should be above 20%.
5.4.3 Cell killing experiment: DC-CIK cells are used as effector cells, tumor cells (which may be primary tumor cells or tumor cell lines) as target cells, and effector cells and target cells are 10:1 (number ratio). The ratio was added to a 96-well U-shaped plate containing 1 x 104 target cells per well, with a final volume of 200 μl and 3 replicate wells. After incubation for 4 h, the culture supernatant was taken, and the killing rate of the effector cells against the target cells was detected by a lactate dehydrogenase (LDH) kit.
5.4.4 Before harvesting the cells, take a small amount of culture for bacterial and fungal culture, and test for mycoplasma and chlamydia.
And endotoxin (standard: negative for pathogen detection, endotoxin <5 Eu).

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