CN115537398A

CN115537398A - Method for large-scale preparation of GMP-grade mature DC cells

Info

Publication number: CN115537398A
Application number: CN202110739084.0A
Authority: CN
Inventors: 黄英; 李波; 李娜; 胡远; 安婷; 王俊清; 高琰
Original assignee: Shenzhen Jinuoyin Biotechnology Co ltd; Genoimmune Therapeutics Co Ltd
Current assignee: Shenzhen Jinuoyin Biotechnology Co ltd; Genoimmune Therapeutics Co Ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2022-12-30

Abstract

The invention discloses a method for preparing GMP-grade mature DC cells in a large scale. The method comprises the steps of obtaining CD14+ cells through sorting by a monoclonal antibody CD14 magnetic bead forward sorting method, adding rh GM-CSF and rh IL-4, inducing and differentiating into immature DC cells, and then adding a mature induction factor combination and antigen polypeptide stimulation to induce the mature DC cells at GMP level. The DC cell culture period is 5-6 days, the preparation process is simple, and 10 preparations can be prepared by one-time single blood sampling ⁸ ～10 ⁹ Grade DC cells. The phenotype of the prepared DC cell is typically mature, key factors IL-12 and IL-23 can be secreted, the induced polypeptide tetramer CTL is high in proportion, specific IFN-gamma is secreted, immunogenicity is positive, and the DC cell has a remarkable in-vitro killing effect on tumor cells. The prepared GMP-level DC cell can be used for preparing personalized polypeptide vaccine, DC tumor vaccine, induced amplification DC-CTL, DC-CIK, virus specific T cell, tumor new antigen T cell (Neo-T) and the likeCell therapy products are directly returned to patients with specific HLA types for the treatment of solid tumors. The invention has important application value.

Description

Method for large-scale preparation of GMP-grade mature DC cells

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for preparing GMP-grade mature DC cells on a large scale.

Background

DC cells are dendritic cells which are found in blood, tissues and lymph organs and are named after elongated dendrites or extending synapses, are the currently known professional antigen presenting cells with the strongest functions, are also the only antigen presenting cells capable of activating initial T cells in a human body, and have antigen presenting capability far stronger than that of macrophages and B cells. The DC cells are responsible for starting and regulating in the treatment of activating T cell mediated tumor immune cells, the cell number of the DC cells only occupies 0.1 to 0.5 percent of mononuclear cells existing in peripheral blood, and the DC cells are seriously insufficient and are generally differentiated by adopting CD34+ hematopoietic stem cells and the mononuclear cells. The collection of the CD34+ hematopoietic stem cells of bone marrow or umbilical cord blood is difficult, the pollution is easy, and patients are painful; the peripheral blood is convenient to obtain, the preparation is simple, and the DC cell yield is high. Therefore, DC cells are mostly induced by differentiation of peripheral blood-derived monocytes, and CD14+ has been used as the origin of monocytes. The most common type of study in human clinical trials is the use of ex vivo generated monocytes, i.e., CD14+ cell-derived DC cells.

The anti-tumor capacity of DC cells is largely dependent on their maturation status. The DC cells in different differentiation stages have different functions from monocyte to mature DC, the immature DC cells have strong antigen uptake, processing and processing capabilities, the antigen uptake and processing capabilities of the mature DC cells are weakened, and the antigen presentation capability is gradually enhanced. TLRs are ligand-dependent detection modules that play a key role in promoting innate and adaptive immunity, and are also considered "gatekeepers of the immune system". There are 10 different TLRs (TLR 1-10) in humans, monocyte-derived DCs expressing TLR3, TLR4, TLR7, TLR8 and TLR9, TLR-induced DC robust activation, which are used to obtain optimal antigen presentation and T cell activation. IFN-gamma is frequently used in the maturation induction of DC, and IFN-gamma can enhance the production of IL-12p70, is a valid stimulator of DC maturation, and can up-regulate the expression of CD83, CD86 and the like. Compared with the immature state, the mature DC is dendritic and has burred protrusions, large clonal clusters are formed, the phenotype is typical mature, some costimulatory molecules are highly expressed, and some costimulatory factors and chemotactic factors are secreted. The antigen presenting ways comprise endogenous ways and exogenous ways, and some ways also have cross presenting capability. DC cells are responsible for presenting MHC-polypeptide complexes to the surface of T cells and are recognized by TCR, and one DC cell can activate 100 to 3000T cells. DC can handle antigen cross presentation by two pathways: the cytoplasmic pathway and the vacuolar pathway. In the cytosolic pathway antigens are transferred to the cytoplasm and processed on the proteasome before loading into newly formed MHC class I molecules; the vacuolar pathway is less well defined but is thought to occur in the endocytic compartment because the pathway is resistant to proteasome inhibitors, but sensitive to lysosomal proteolytic inhibitors and depends on cathepsin S. Currently, there are many forms of DC that carry tumor antigens, such as DNA, mRNA, polypeptides, tumor lysates, etc.

Dendritic cells are powerful tools for enhancing anti-tumor immune responses and are also the core and basis of immune responses. The DC vaccine is proved to be capable of safely and effectively inducing tumor specific immune response, and patients usually have good tolerance, and the DC vaccine loaded with Tumor Associated Antigen (TAA) and personalized polypeptide vaccines, such as PEP-DC vaccine and PPV-DC vaccine, are mainly provided.

Clinically, the traditional Chinese medicine composition is combined with radiotherapy or immune checkpoint molecules, such as CTLA-4 and PD-1, and has obvious curative effects on aspects of melanoma, glioblastoma, colorectal cancer and the like. The first human clinical trials of personalized polypeptide vaccines have shown feasibility, safety and immunotherapeutic activity to target individual tumor mutational features. Recently, the Bedak pharmaceutical industry starts the research and development and clinical research of 2019-nCoV novel coronavirus universal DC vaccines, non-invasive specific genes of antigens are transferred into DC cells, universal DCs are prepared aiming at different HLA types, and the DC vaccines become 'spot' DC products for supply. Tumor immunotherapy mainly focuses on the antitumor activity of CD8+ Cytotoxic T Lymphocytes (CTLs), CTL can directly kill all tumor cell types, DC-CTL recognizes specific antigen peptide presented on the surface of cancer cells by main tissue compatibility through a T Cell Receptor (TCR) to eliminate malignant tumor I/beta-2-microglobulin complex, releases perforin and granzyme B, and kills target cells. The tumor neoantigen predicted by the latest generation sequencing technology and novel bioinformatics tools is generated from somatic mutation of tumors, and DC cells are utilized to activate self (autologous) tumor specific T cells of a patient to treat various solid tumors, such as melanoma, colorectal cancer, lung cancer, pancreatic cancer, glioblastoma and the like, so that the antigen has attractive prospects of high specificity, strong immunogenicity, small toxic and side effects and the like.

At present, the preparation method of the DC cell with GMP grade has the following problems: (1) Most raw materials are derived from bone marrow hematopoietic stem cells, the materials are limited, and the preparation is complex; (2) DC precursor cells, namely monocytes, are sorted by a wall-mounted method, so that the yield is low, impurities are more, and the yield of the monocytes converted into immature DCs is limited; (3) The culture period of the DC cells is long and is not less than 7 days, and a part of preparation methods need half liquid change and have complex processes; (4) Inducing LPS with excessive types of mature factors or toxic and side effects; (5) The preparation amount is small and is at most 10 ⁸ Grade, not more than 10 ⁹ More than grade; (6) The DC cell induction yield is low, and generally induced DCs account for about 10% of PBMCs; (7) The phenotype of the induced DC is poor or difficult to stably control, and the antigen presentation effect of the DC is influenced; (8) The use of fusion peptides or tumor associated antigens, tumor lysates, specific immunogenicity is not strong; meanwhile, the basically adopted antigen mixed loading form cannot eliminate competition among polypeptides; (9) In the preparation process of partial DC cells, fetal bovine serum or non-GMP (good manufacturing practice) level DC culture medium is used, and the clinical use is limited; (10)Most prepared DCs are prepared once and returned once, and even if the DCs are frozen and stored, the survival rate after recovery is low and the stability is poor. In a word, the DC cells prepared by the existing preparation method have low yield, poor purity, low expression of surface marker molecules, weak capacity of inducing and activating T cells and influence the DC cells to play the role of antigen presentation; the polypeptide specificity is low, and the immunogenicity stimulation capability of DC cells is influenced; moreover, the recovery survival rate of the DC cells after cryopreservation is low, the phenotype is changed, and the stability is difficult to control; meanwhile, all raw and auxiliary materials in the preparation process are supplied in non-pharmaceutical grade or GMP grade, and cannot be directly applied to clinic and the like.

Disclosure of Invention

The object of the present invention is to produce GMP-grade mature DC cells.

The invention firstly protects a method for preparing GMP-grade mature DC cells, which sequentially comprises the following steps:

(1) Sorting CD14+ cells from monocytes;

(2) Adding DC cell culture medium containing 1-6% (such as 1-4%, 4-6%, 1%, 2%, 3%, 4%, 5% or 6%) HSA to the above CD14+ cells, adding rh GM-CSF and rh IL-4 to concentrations of 10-120ng/ml (such as 10-50ng/ml, 50-120ng/ml, 10ng/ml, 50ng/ml or 120 ng/ml) and 10-120ng/ml (such as 10-100ng/ml, 100-120ng/ml, 10ng/ml, 100ng/ml or 120 ng/ml) respectively in the system, and culturing for 3-4 days (such as 3 days or 4 days);

(3) Adding rhIFN-gamma, POLYI C, rhTNF-alpha and rhIL-1 beta into the system obtained in the step (2) to obtain an induction system, and culturing for 1 day;

(4) And (4) adding the tumor antigen polypeptide into the system in the step (3), and culturing for 1 day to obtain the GMP-grade mature DC cells.

In the step (1), the mononuclear cells may be mononuclear cells isolated from peripheral blood.

In the step (1), the step of sorting the CD14+ cells from the monocytes may be a step of sorting the CD14+ cells from the monocytes by using a monoclonal antibody CD14 magnetic bead forward sorting method.

The steps of adopting the monoclonal antibody CD14 magnetic bead positive sorting method to sort CD14+ cells from the mononuclear cells are as follows in sequence:

(D1) Taking the PBMC solution, centrifuging to remove the supernatant, and adding buffer for heavy suspension to obtain the heavy suspension.

(D2) Taking the resuspension, adding CD14 magnetic beads (every 10 times) ⁹ 600. Mu.l of CD14 magnetic beads were added to each PBMC), and incubated at 2 ℃ to 8 ℃ for 30min.

(D3) After the step (D2) is completed, 20-50ml of buffer is added for washing, 300g of the mixture is centrifuged for 10min, and then the buffer is added for resuspension to obtain cell suspension.

(D4) And (3) after the step (D3) is finished, adding the cell suspension into the center of the LS sorting column, adding 10ml of buffer into the sorting column, taking down the sorting column, pushing a piston of the sorting column, and pushing the cells on the tube into a collecting tube, namely CD14+ cells.

In the step (2), the DC Cell culture medium can be that of AIM-V, X-VIVO or Cell Genix.

In the step (3), the rhIFN-gamma concentration in the induction system can be 0-50ng/ml (such as 0-5ng/ml, 5-50ng/ml, 0ng/ml, 5ng/ml or 50 ng/ml). The concentration of C may be 1-30 μ g/ml (e.g., 1-5 μ g/ml, 5-30 μ g/ml, 1 μ g/ml, 5 μ g/ml, or 30 μ g/ml). The concentration of rh TNF- α may be 10-50ng/ml (e.g., 10-30ng/ml, 30-50ng/ml, 10ng/ml, 30ng/ml, or 50 ng/ml). The concentration of rh IL-1 β may be 5-40ng/ml (e.g. 5-10ng/ml, 10-40ng/ml, 5ng/ml, 10ng/ml or 40 ng/ml).

In the step (4), after the tumor antigen polypeptide is added, the concentration of the tumor antigen polypeptide in the system can be 1-3 μ g/ml (e.g., 1-2 μ g/ml, 2-3 μ g/ml, 1 μ g/ml, 2 μ g/ml or 3 μ g/ml).

Any one of the above tumor antigen polypeptides can be at least one of antigen polypeptide HLA0201-74, antigen polypeptide HLA0201-13, antigen polypeptide C005-19 and antigen polypeptide HLA 0201-2.

Any of the methods described above may further comprise step (5): after the step (4) is finished, preserving mature DC cells by using a cryopreservation protective agent;

the freezing protective agent comprises compound electrolyte, glucose injection, dextran, human serum albumin and dimethyl sulfoxide.

In the above method, the cryopreservation agent may specifically consist of 15-60% (e.g. 15-30%, 30-60%, 15%, 30% or 60%) (v/v) of compound electrolyte, 5-40% (e.g. 5-25%, 25-40%, 5%, 25% or 40%) of glucose injection (v/v), 0-40% (e.g. 0-20%, 20-40%, 0%, 20% or 40%) of dextran (v/v), 10-60% (e.g. 10-20%, 20-60%, 10%, 20% or 60%) of human albumin (v/v), and 0-15% (e.g. 0-5%, 5-15%, 0%, 5% or 15%) of dimethyl sulfoxide (v/v).

Any of the cryopreservation protective agents described above also belongs to the protection scope of the invention.

The invention also provides a kit which contains any one of the cryopreservation protective agents; the use of the kit may be for preservation of mature DC cells.

The application of any cryopreservation protective agent in the preservation of mature DC cells also belongs to the protection scope of the invention.

Any of the above-described preservation may be liquid nitrogen preservation. The specific preservation steps are as follows: freezing mature DC cells by using a freezing protective agent, transferring a cell freezing tube into a program cooling instrument, and cooling the sample to 4.5 ℃; according to the temperature drop rate of 2 ℃/min until the temperature of the sample is-20 ℃; then the temperature is decreased at a rate of 10 ℃/min until the temperature in the box is-40 ℃; then, the temperature is decreased at a rate of 20 ℃/min until the temperature in the box is-20 ℃; then, the temperature is decreased at a rate of 5 ℃/min until the temperature in the box is-40 ℃; finally, the temperature reduction rate is 20 ℃/min until the temperature in the box is-80 ℃, and the whole program cooling process takes 80-90min; and finally transferring the mixture to a meteorological liquid nitrogen tank at the temperature of 196 ℃ below zero for long-term storage.

Any of the mature DC cells described above can be GMP-grade mature DC cells.

Any of the above cryoprotectants may be directly returned.

The present invention provides a method for large-scale production of GMP-grade mature DC cells. Storing the blood bag of PBMC collected by a hospital apheresis machine in a 2-8 ℃ transport box, arranging a professional cold chain transport supplier to transport to a cell preparation center within 24h at 2-8 ℃, wherein the whole transport process requires no security check, X-ray irradiation is forbidden, and real-time temperature monitoring is carried out. CD14+ cells obtained by sorting through monoclonal antibody CD14 magnetic bead positive sorting method, inducing and differentiating into immature DC cells, and adding mature DC cellsThe inducing factor combination and the antigen polypeptide stimulate to induce mature DC cells with GMP grade. The harvested GMP-grade DC cells are frozen by using the freezing protective agent provided by the invention, are cooled by a program cooling instrument, and are finally transferred to a meteorological liquid nitrogen tank at the temperature of-196 ℃ for long-term storage. Raw materials are easy to obtain in the preparation process, the DC precursor cells obtained by sorting are high in purity and yield after being transported to a cell workshop through a cold chain in the whole process, and the survival rate is close to 100%. The DC cell culture period is 5-6 days, the preparation process is simple, and 10 preparations can be prepared by one-time single blood sampling ⁸ -10 ⁹ GMP-grade DC cells. The cryopreservation protective agent provided by the invention can be used for cryopreservation of the DC in liquid nitrogen for 12 months, has stable quality, and can meet the transfusion requirement of multiple treatment courses. The phenotype of the prepared GMP-level DC cells is typically mature, key factors IL-12 and IL-23 can be secreted, the proportion of induced specific T cells is high, specific IFN-gamma is secreted, immunogenicity is positive, and the DC cells have obvious in-vitro killing effect on tumor cells. The prepared GMP-level DC cell can be used for preparing personalized polypeptide vaccines and DC tumor vaccines, and can be directly returned to a patient body with specific HLA typing for treating solid tumors by inducing and amplifying various cell therapy products such as DC-CTL, DC-CIK, virus specific T cells, tumor neoantigen T cells (Neo-T) and the like. The invention has important application value.

Drawings

FIG. 1 shows the yield and ratio of CD14+ cells sorted by different incubation amounts of magnetic beads.

FIG. 2 shows the amount of IFN-. Gamma.secreted by CTL cells stimulated by different polypeptide-loaded forms.

FIG. 3 shows the content of tetramer of antigen polypeptide and the amount of IFN-. Gamma.secretion in different forms of loading polypeptide to stimulate activation of CD8+ cells into CTL cells

FIG. 4 shows recovery yield and survival rate of DC cells frozen 30 days after three different cell freezing protective agents are used.

FIG. 5 shows the sorting yields and purities of three batches of CD14+ cells.

FIG. 6 shows the expression of three batches of mature DC cell surface marker molecules.

FIG. 7 shows the IL-12 and IL-23 secretion amounts of culture supernatants of mature DC cells.

FIG. 8 shows the yield and viability of three batches of mature DC cells.

FIG. 9 shows the IFN-. Gamma.secretion from T cells stimulated by three batches of mature DC cells.

FIG. 10 shows the tumor cell killing activity of T cells induced by mature DC cells (DC cells activated into CTL cells by CD8+ cells after two rounds of stimulation).

FIG. 11 shows the survival rate and DC phenotype of DC cells after thawing after 0-12 months of cryopreservation using the cryoprotectant prepared by the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Buffer in the following examples

PBS/EDTA Buffer, MILTENYI, cat # 200-070-029.

Example 1 optimization of the method for increasing the sorting yield of CD14+ cells from apheresis sources

1. Isolation of monocytes from peripheral blood

1. Will contain 2.2X 10 ⁹ -6.5×10 ⁹ Blood was collected from individual PBMCs, mixed well and transferred to centrifuge tubes.

2. And (3) after the step 1 is finished, centrifuging at room temperature and 700g for 15min, collecting a lower red blood cell layer, adding a DPBS solution, and blowing, beating and uniformly mixing.

3. And (3) adding the solution obtained in the step (2) to the upper layer of the Ficoll monocyte separation solution, centrifuging for 30min at 800g, sucking out the middle cloudy leucocyte layer cells, washing and centrifuging, and collecting precipitated cells.

4. The precipitated cells collected in step 3 were resuspended in a buffer containing 1% HSA to obtain a PBMC solution.

2. Sorting CD14+ cells by monoclonal antibody CD14 magnetic bead positive sorting method

1. The PBMC solution obtained in step one (4) was sieved, centrifuged to remove the supernatant, and 20ml of 1% HSA-containing buffer was added for resuspension to obtain a resuspension solution.

2. After the step 1 is finished, adding CD14 magnetic beads into the resuspension, and incubating for 30min at the temperature of 2-8 ℃.

Group A: every 10 th ⁹ 300. Mu.l of CD14 magnetic beads were added to each PBMC

Group B: every 10 th ⁹ Add 600. Mu.l of CD14 magnetic beads to each PBMC

Group C: every 10 th ⁹ 900. Mu.l of CD14 magnetic beads were added to each PBMC

3. After completion of step 2, 20ml of 1% HSA-containing buffer wash was added, centrifuged at 300g for 10min, and then 1% HSA-containing buffer resuspension was added to obtain a cell suspension.

4. After step 3, the cell suspension was added to the midpoint of the LS sorting column (LS Columns, mltenyi, cat # 130-042-401), after which 10ml buffer was added to the column, the column was removed, the column plunger was pushed down, and the cells in the tube were pushed into the collection tube, i.e., CD14+ cells.

The forward sorted CD14+ cells were collected to obtain the yield and purity (i.e. ratio) of CD14+ cells.

CD14+ yield (%) = (number of CD14+ cells/number of PBMCs) × 100%

The CD14+ ratio was detected by Beckmann Cytoflex flow cytometer PE-CD14 labeling, i.e., the sample CD14+ ratio was read through a gate-round on the flow analyzer minus the difference in the isotype control CD14+ ratio (TEST% -ISO%).

The results are shown in FIG. 1. The results showed that each 10 ⁹ The PBMC were incubated with 600. Mu.l of CD14 magnetic beads and the yield and purity were highest.

Example 2 optimization of the different antigenic polypeptide Loading modalities

1. Isolation of monocytes from peripheral blood

The same procedure as in example 1.

2. Sorting CD14+ cells by monoclonal antibody CD14 magnetic bead forward sorting

The same procedure as the second step in example 1 was followed using the optimal 600. Mu.l of CD14 magnetic beads.

3. Inoculating CD14+ cells, adding mature inducing factor combination, loading tumor antigen polypeptide, inducing in vitro to differentiate into mature DC cells

1. Taking the CD14+ cells prepared in step two, adding DC cell culture medium containing 1% HSA, adding rh GM-CSF and rh IL-4 to final concentrations of 50ng/ml and 100ng/ml, respectively, 37 deg.C, 5% ₂ Culturing for 3-4 days.

The DC Cell culture medium can be AIM-V, X-VIVO or Cell Genix.

2. After the step 1 is completed, adding rhIFN-gamma, POLYI C, rhTNF-alpha and rhIL-1 beta into the system to obtain an induction system; in the induction system, the concentration of rhIFN-gamma is 5ng/ml, the concentration of poly I is 5 mu g/ml, the concentration of rh TNF-alpha is 10ng/ml, and the concentration of rh IL-1 beta is 10ng/ml.

rhIFN-gamma is a product of Shanghai Puxin biotech, catalog number GMP-106-06.

POLYI C is a product of INVIVVOGEN, catalog number vac-pic.

rh TNF- α is a product of Shanghai Puxin biotech, catalog number GMP-103-01.

rh IL-1 β is a product of Shanghai Puxin Biotech, inc., under the catalog number GMP-101-01B.

3. After completion of step 2, the induction system was taken and treated at 37 ℃ with 5% CO ₂ The culture was carried out for 1 day.

4. After the step 3 is completed, adding the antigen polypeptide HLA0201-74 (with the number of HLA0201-74 and the amino acid sequence of KLMGIVYKV) and the antigen polypeptide HLA0201-13 (with the number of HLA0201-13 and the amino acid sequence of SLDWWAFGV) into the system to obtain a system 1; in System 1, antigen polypeptide HLA0201-74 and antigen polypeptide HLA0201-13 each had a final concentration of 2. Mu.g/ml.

Adding antigen polypeptide HLA0201-74 into the system to obtain a system 2; in the system 2, the final concentration of the antigen polypeptide HLA0201-74 is 2. Mu.g/ml.

Adding antigen polypeptide HLA0201-13 into the system to obtain a system 3; in the system 3, the final concentration of the antigen polypeptide HLA0201-13 is 2. Mu.g/ml.

Adding antigen polypeptide C005-19 (numbered as C005-19, and amino acid sequence is VTFHIPFEV) into the system to obtain system 4; in the system 4, the final concentration of the antigenic polypeptide C005-19 was 2. Mu.g/ml.

Adding antigen polypeptide HLA0201-2 (number is HLA0201-2, amino acid sequence is AVGSYVYSV) into the system to obtain system 5; in the system 5, the final concentration of the antigen polypeptide HLA0201-2 is 2. Mu.g/ml.

Adding antigen polypeptide HLA0201-74, antigen polypeptide HLA0201-13, antigen polypeptide C005-19 and antigen polypeptide HLA0201-2 into the system to obtain a system 6; in the system 6, the final concentrations of the antigen polypeptide HLA0201-74, the antigen polypeptide HLA0201-13, the antigen polypeptide C005-19 and the antigen polypeptide HLA0201-2 are 2 mu g/ml.

5. After completion of step 4, the system (System 1, system 2, system 3, system 4, system 5 or System 6) was taken, and the CO was calculated at 37 ℃ and 5% ₂ After 1 day of culture, mature DC cells were obtained.

4. Specific T cell IFN-gamma secretion amount stimulated by different polypeptide loading forms

1. The ELISPOT, also called enzyme-linked immunospot assay, is characterized in that target cells are activated by a stimulating source and effectively secrete target cytokines, the cytokines secreted by the cells in culture, such as IFN-gamma, are captured by antibodies, the cytokines are expressed in an enzyme-linked spot color development mode, and the secretion activity of the cytokines is judged according to the number of the developed spots. IFN-gamma is produced by CD4+ helper T cells and CD8+ T Cells (CTL) when stimulated by antigen presented by major histocompatibility antigen complex (MHC), and in the research of tumor immunotherapy, an ELISPOT method is generally used for detecting the expression activity of the IFN-gamma, and the strength of antigen-specific T cell response is characterized by the expression quantity of the IFN-gamma.

2.DC Mix: and equivalently mixing mature DC cells obtained by the system 2, the system 3, the system 4 and the system 5 in the third step, and then co-culturing the mature DC cells and CD8+ cells according to a certain proportion to stimulate the CD8+ T cells to be expanded into CTL cells. Polypeptide Mix: and (3) co-culturing the mature DC cells obtained from the system 6 in the third step and CD8+ cells according to a certain proportion, and stimulating the CD8+ T cells to expand into CTL cells.

3. Taking T2-A02:01 cells in logarithmic growth phase, counting viable cells, centrifuging (1000rpm, 5 min), discarding supernatant, suspending cells by IMDM culture medium, adjusting cell density to 2 × 10 ⁵ one/mL. The method comprises the following three groups: the negative group is T2 cells not loaded with antigen polypeptide; the positive group is PHA-loaded T2 cells; the test group is T2 cells loaded with antigen polypeptide (the T2 cells can be respectively loaded with single polypeptide or polypeptide mixture), and the loading time is 3.5h.

4. After the loading time is over, the T2 cells and the CTL cells are respectively washed by centrifugation and repeated for 2 times, and the T2 cells and a round of CTL cells of each group are co-cultured in Human IFN-gamma ELISPOT according to the proportion of 2 ^PRO And (3) a 96-well plate (MABTECH), 5000 or 10000T cells are put in each well, the incubation time is 18-24h, the antibody is added and the plate is developed on the next day, after the plate bottom is dried, an IFN-gamma spot counting mode is selected on a fluorescence enzyme-linked immunosorbent Assay (AID), and scanning counting is carried out. By substituting the following formula, the amount of IFN-. Gamma.secreted by CTL cells stimulated by a single polypeptide or a mixture of polypeptides can be calculated.

IFN-gamma secretion (IFN-gamma expression positive cell number/10) ⁶ Viable cells) = { (number of test sample plaques-number of negative control plaques)/number of T-cell wells } × 10 ⁶

The results are shown in FIG. 2. The IFN-gamma secretion amount of one round of T cells stimulated by the DCMix loading form is remarkably higher than that stimulated by the polypeptide Mix loading form, wherein the specific immunogenicity of the HLA0201-74 polypeptide is far higher than that of other antigen polypeptides. Compared with the polypeptide Mix loaded form, the DC Mix loaded form reduces the competitive relationship between the polypeptides, and can activate more antigen-specific T cells.

5. T cell antigen polypeptide tetramer content and IFN-gamma secretion amount stimulated by different polypeptide loading forms

1. The MHC-tetramer is composed of four monomer molecules formed by combining Major Histocompatibility Complex (MHC) and antigen peptide through a biotin-streptavidin system, and the content of antigen-specific CTL cells is directly detected by a flow cytometer through enhancing the affinity of a T cell receptor and the peptide MHC-complex.

2. And (3) co-culturing the mature DC cells obtained in the system 1 in the third step with CD8+ cells, and stimulating the CD8+ T cells to expand into CTL cells. Mature DC cells obtained from the system 2 and the system 3 in the third step are mixed in equal amount, and then are co-cultured with CD8+ cells to stimulate the expansion of CD8+ T cells into CTL cells.

3. APC is adopted to mark antigen polypeptide HLA0201-74 and antigen polypeptide HLA0201-13 to prepare MHC-Tetramer, PE anti-human CD8 is added for co-staining, and a Beckmann flow cytometer is utilized to detect the antigen polypeptide specificity proportion of T cells, namely the proportion of Tetramer + CD8+ CTL cells, and the result is shown in figure 3.

Proportion of Tetramer + CD8+ CTL cells (%) = Test (%) -UV (%)

4. By using Elispot counting, T2 cells not loaded with the antigen polypeptide are used as a negative group, PHA-loaded T2 cells are used as a positive group, T2 cells loaded with the antigen polypeptide are used as a test group, the T2 cells and a round of CTL cells are respectively incubated for 18-24h according to a ratio of 2.

The results are shown in FIG. 3, where DC Mix is the content of specific T cells obtained by stimulation of CD8+ cells with equal amounts of DC induced by system 2 and system 3; polypeptide Mix is the specific T cell content obtained by stimulation of CD8+ cells by DC induced by system 1. The results show that the ratio of Tetramer + CD8+ CTL cells and IFN- γ secretion amount stimulated by Mix form of single polypeptide loaded DC are significantly higher than those stimulated by polypeptide Mix loaded form (fig. 3 only shows the results of HLA0201-74, and there is no significant difference between the result groups of HLA0201-13, and therefore not shown).

The results of combining MHC-peptide tetramer and ELISPOT show that the MIX form of single polypeptide loaded DC is superior to the MIX form of polypeptide, the former can reduce the competitive relationship among polypeptides, can be effectively identified by TCR, and can activate more proportion of antigen-specific CTL cells, thereby improving the antigen presentation capability of DC.

And selecting equal amount of DC respectively induced by the system 2 and the system 3 in the third step to mix for subsequent T cell stimulation experiments.

Example 3 optimization of immune cell cryopreservation protectant with direct reinfusion

1. Preparation of immune cell freezing protective agent capable of being directly returned (hereinafter referred to as freezing protective agent)

The inventor of the invention prepares the cryopreservation protective agent through a large number of experiments.

The cryopreservation protective agent consists of 30% (v/v) of compound electrolyte, 25% (m/v) of glucose injection, 20% (m/v) of dextran, 20% (m/v) of human serum albumin and 5% (v/v) of dimethyl sulfoxide.

2. Comparison of three cryopreservation protectants

Freezing and storing protective agent A: over one bearing (three-life organism).

Freezing and storing protective agent B:

(BioLife)。

freezing and storing protective agent C: the preparation method comprises the first step.

Mature DC cells are preserved at ultralow temperature for 30 days by using a cryopreservation protective agent (a cryopreservation protective agent, B cryopreservation protective agent or C cryopreservation protective agent), and then are revived at 37 ℃. Detecting the recovery yield and the recovery activity rate within 2 hours at the temperature of 25 +/-2 ℃.

DC cell recovery yield = (cell density 2h after recovery/cell density before cryopreservation) × 100%

Survival rate of DC cells = (number of viable cells recovered for 2 h/total number of cells recovered for 2 h) × 100%

The results are shown in FIG. 4. The result shows that compared with the cryopreservation protective agent A or B, the recovery yield and recovery survival rate of DC cells cryopreserved by the cryopreservation protective agent C are obviously improved. Meanwhile, compared with the cryopreservation protective agent A or B, the cryopreservation protective agent C is a medicinal reagent, so that the DMSO ratio is lower, and the clinical use safety is higher.

Example 4: establishment of method for large-scale preparation of GMP-grade mature DC cells

Based on the above results, the present invention established a method for large scale production of GMP-grade mature DC cells comprising the steps of:

1. isolation of monocytes from peripheral blood

1. Will contain 2.2X 10 ⁹ -6.5×10 ⁹ Blood from individual PBMC was collected, mixed and transferred to centrifuge tubes.

2. And (3) after the step 1 is finished, centrifuging at room temperature for 15min at 700g, collecting a lower red blood cell layer, adding a DPBS solution, and blowing and uniformly mixing.

4. The precipitated cells collected in step 3 were taken and resuspended in a buffer containing 1% HSA to obtain a PBMC solution.

1. And (3) sieving the PBMC solution obtained in the first step through a screen, centrifuging to remove supernatant, and adding 20ml of buffer for resuspension to obtain a resuspension solution.

2. After completion of step 1, the resuspension was taken and CD14 beads (every 10) were added ⁹ 600. Mu.l of CD14 magnetic beads were added to each PBMC), and incubated at 2 ℃ to 8 ℃ for 30min.

3. And (3) after the step 2 is finished, adding 20-50ml of buffer for washing, centrifuging for 10min at 300g, and then adding the buffer for heavy suspension to obtain cell suspension.

4. And 3, after the step 3 is completed, adding the cell suspension into the center of the LS sorting column, after the step is completed, adding 10ml of buffer into the sorting column, taking down the sorting column, pushing a piston of the sorting column, and pushing the cells on the tube into a collecting tube, namely the CD14+ cells.

3. Inoculating CD14+ cell, adding maturation inducing factor combination (rhIFN-gamma, POLYI: C, rh TNF-alpha and rh IL-1 beta) and loaded tumor antigen polypeptide, inducing in vitro to differentiate into mature DC cell

1. Taking the CD14+ cells prepared in step two, adding 1% HSAThe DC cell culture medium of (1), adding rh GM-CSF and rh IL-4 to final concentrations of 50ng/ml and 100ng/ml, respectively, 37 ℃, 5% ₂ The culture was carried out for 4 days.

2. After the step 1 is finished, rhIFN-gamma, POLYI C, rhTNF-alpha and rhIL-1 beta are added into the system to obtain an induction system; in an induction system, the concentration of rhIFN-gamma is 5ng/ml, the concentration of POLYI is 5 mu g/ml, the concentration of rh TNF-alpha is 10ng/ml, and the concentration of rh IL-1 beta is 10ng/ml.

4. After the step 3 is completed, adding the antigen polypeptide HLA0201-74 into the system to obtain a system 2; in the system 2, the final concentration of the antigen polypeptide HLA0201-74 is 2. Mu.g/ml. Adding antigen polypeptide HLA0201-13 into the system to obtain a system 3; in the system 3, the final concentration of antigen polypeptide HLA0201-13 is 2. Mu.g/ml. Adding antigen polypeptide C005-19 (numbered as C005-19, and the amino acid sequence is VTFHIPFEV) into the system to obtain system 4; in the system 4, the final concentration of the antigenic polypeptide C005-19 was 2. Mu.g/ml. Adding antigen polypeptide HLA0201-2 (number is HLA0201-2, amino acid sequence is AVGSYVYSV) into the system to obtain system 5; in the system 5, the final concentration of the antigen polypeptide HLA0201-2 is 2. Mu.g/ml.

5. After completion of step 4, the DC cells of the above-mentioned system 2, system 3, system 4 or system 5 were taken, and the content of CO was 5% at 37 ℃% ₂ Mature DC cells were obtained after 1 day of culture.

In subsequent T cell stimulation experiments, equal amounts of DC cells induced by the

systems

2, 3, 4 and 5 were mixed and co-cultured with CD8+ cells.

The whole culture process does not change liquid and does not supplement liquid.

4. Freezing and storing

The mature DC cells obtained in step three were subjected to cryopreservation using the cryopreservation agent prepared in example 3.

Example 5 and example 4 detection of mature DC cells prepared by the methods established

1. 3 batches of mature DC cells, designated batch A, batch B and batch C, were prepared using the method established in example 4.

2. The CD14+ yield and CD14+ ratio of the three batches of CD14+ cells obtained were examined according to the method of example 1.

The results are shown in FIG. 5. The result shows that the yield of CD14+ is 14-25%; the proportion of CD14+ is 99.6-99.9%. It follows that CD14+ cells are sufficiently pure and abundant for subsequent preparation of mature DC cells.

3. The three batches of mature DC cells were examined by beckmann flow cytometry for the expression of surface marker molecules (CD 83+, CD86+ or CD80 +).

The results are shown in FIG. 6. The results showed that CD86 expression was above 94.0%, CD80 expression was above 98.6%, and CD83 expression was above 90.3%. It can be seen that the DC cells prepared by the method established in example 4 are phenotypically highly mature.

4. The amount of IL-12 secreted in the cell supernatants obtained from the three batches was determined using an IL-12ELISA (R & D) kit. The amount of IL-23 secreted in the cell supernatants obtained from the three batches was determined using IL-23ELISA (R & D) kit.

The cells were the cells obtained in step 1 of example 4 (i.e., the cells on day 4), the cells obtained in step 3 of example 4 (i.e., the cells on day 5), or the cells obtained in step 5 of example 4 (i.e., the cells on day 6).

The results are shown in FIG. 7. The results show that after the mature induction factor is added, the secretion of IL-12 and IL-23 shows the trend of increasing and then decreasing, and the peak value is reached after the mature induction factor is added for 24 hours. In general, the secretion of IL-12 is lower than that of IL-23.

5. The three batches were tested for mature DC cell induction yield (in CD14+ inoculum size) and viability.

DC cell induction yield = (number of mature DC cells/number of CD14+ seeded cells) × 100%

DC cell viability (%) = (number of viable cells recovered 2 h/total number of cells recovered 2 h) × 100%

The results are shown in FIG. 8. The result shows that the DC cell yield is 62-69%, and the magnitude order is 10 ⁸ The above; the survival rate of DC cells reaches more than 97 percent. It can be seen that the method established in example 4 can be used onceMature DC cells were prepared on a large scale in accordance with GMP grade.

6. The mature DC cells obtained from three batches were used to stimulate the activation of CD8+ T cells into CTL cells, and the IFN-gamma secretion activity of the CTL cells was examined by ELISPOT to evaluate the antigen presenting ability of the DC. Three batches of 2 replicates each.

The results are shown in FIG. 9 (negative group is co-incubation of T2 cells without loaded polypeptide and T cells, positive group is co-incubation of PHA-loaded T2 cells and T cells, and test group is co-incubation of T2 cells with loaded polypeptide and T cells). The result shows that the number of IFN-gamma spots secreted by the CTL cells (test sample group) induced by the mature DC cells obtained by the invention is far more than 3 times of the number of the spots in the negative group, namely the signal-to-noise ratio is more than 3. Therefore, the mature DC obtained by the invention has strong presenting capability, and the specific immunogenicity of the stimulated CTL cell is positive.

7. Three batches of mature DC cells were used to stimulate activation of CD8+ T cells into CTL cells. The melanoma A375 tumor cell line after CFSE staining was a target cell with CTL cells as effector cells, and was killed in vitro for 20-24h according to a 20. Adding 7-AAD before loading on the machine, and detecting by using a Beckmann CytoFLEX flow cytometer.

Percent killing (%) = CFSE and 7-AAD double positive/CFSE positive

The results are shown in FIG. 10. The results show that the mature DC cells obtained by the present invention are activated into CTL cells having a target specific killing rate of 90.61%, 71.08% and 50.33% for a375 cells 20. Therefore, the mature DC cell obtained by the invention can effectively stimulate the activated CD8+ T cell to expand into CTL cell, and has obvious killing effect on tumor cell.

8. The cryopreservation protective agent prepared in example 3 was used to perform ultra-low temperature storage (i.e., liquid nitrogen storage) on the mature DC cells obtained from three batches, with a storage time of 0-12 months; the survival rate of mature DC cells and the expression of surface marker molecules are then examined. The detection method refers to step 3.

Some of the results are shown in FIG. 11. The results show that the survival rate of the mature DC cells is more than 80% and the expressions of CD86+, CD80+ and CD83+ are all more than 80% when the cells are frozen and stored for 12 months. Therefore, the frozen mature DC cells are frozen by the freezing protective agent provided by the invention for less than 12 months, and the quality is stable.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.

Claims

1. A method of making GMP-grade mature DC cells, comprising the steps of, in order:

(1) Sorting CD14+ cells from monocytes;

(2) Adding a DC cell culture medium containing 1-6% HSA to the CD14+ cells, adding rh GM-CSF and rh IL-4 to a concentration of 10-120ng/ml and 10-120ng/ml in the system, respectively, and culturing for 3-4 days;

(3) Adding rhIFN-gamma, POLYI C, rhTNF-alpha and rhIL-1 beta into the system in the step (2) to obtain an induction system, and culturing for 1 day;

2. The method of claim 1, wherein: in the step (2), the DC Cell culture medium is that of AIM-V, X-VIVO or Cell Genix.

3. The method of claim 1, wherein: in the step (3), the concentration of rhIFN-gamma in the induction system is 0-50ng/ml, the concentration of poly I: C is 1-30 mu g/ml, the concentration of rh TNF-alpha is 10-50ng/ml, and the concentration of rh IL-1 beta is 5-40ng/ml.

4. The method of claim 1, wherein: in the step (4), after the tumor antigen polypeptides are added, the final concentration of each tumor antigen polypeptide in the system is 1-3 mu g/ml.

5. The method of claim 4, wherein: the tumor antigen polypeptide is at least one of antigen polypeptide HLA0201-74, antigen polypeptide HLA0201-13, antigen polypeptide C005-19 and antigen polypeptide HLA 0201-2.

6. The method of any of claims 1 to 5, wherein: the method further comprises step (5): after the step (4) is finished, preserving the mature DC cells by adopting a cryopreservation protective agent;

7. The method of claim 6, wherein: the cryopreservation protective agent consists of 15-60% (v/v) of compound electrolyte, 5-40% (v/v) of glucose injection, 0-40% (v/v) of dextran, 10-60% (v/v) of human serum albumin and 0-15% (v/v) of dimethyl sulfoxide.

8. The cryopreservation agent of claim 6 or 7.

9. A kit comprising the cryoprotectant of claim 6 or 7; the kit is used for preserving mature DC cells.

10. Use of the cryopreservation agent of claim 6 or 7 for preserving mature DC cells.