CN113564116B

CN113564116B - Preparation method of specific antiviral adoptive immune cell CE

Info

Publication number: CN113564116B
Application number: CN202110827796.8A
Authority: CN
Inventors: 黄晓军; 董强刚; 涂静溦; 潘程娇
Original assignee: Shanghai Icell Biotechnology Co ltd; Beijing Victory Biotechnology Co ltd
Current assignee: Shanghai Icell Biotechnology Co ltd; Beijing Victory Biotechnology Co ltd
Priority date: 2021-07-21
Filing date: 2021-07-21
Publication date: 2023-08-01
Anticipated expiration: 2041-07-21
Also published as: CN113564116A

Abstract

The invention provides a preparation method of a specific antiviral adoptive immune cell, which comprises the following steps: (1) Collecting peripheral blood, and centrifugally separating to obtain peripheral blood mononuclear cells; (2) Activation of CD3 in peripheral blood mononuclear cells with cytomegalovirus and EB virus antigenic peptides ⁺ T cells; (3) Amplifying the activated T cells of step (2) to the desired number using G-REX10 flask culture. According to the preparation method, the G-REX10 bottle is adopted for cell culture and amplification, pollution and passage in operation are avoided, the blood collection amount is small, the preparation time is short, VST can be infused once through 2-week and 4-week culture respectively, and the obtained immune cells are virus antigen specific T cells, so that the purity is high, the activity is strong, and the treatment effect is better.

Description

Preparation method of specific antiviral adoptive immune cell CE

Technical Field

The invention relates to the field of cellular immunotherapy, in particular to a preparation method of antiviral adoptive immune cells.

Background

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are enveloped double-stranded DNA viruses belonging to the subfamily Herpesvirus (beta-hepesvirus) and the subfamily gamma-hepesvirus. CMV has a broad spectrum of cell tropism (celtprism) such that epithelial cells, endothelial cells, fibroblasts, nerve cells, monocytes, neutrophils and smooth muscle cells are all susceptible to CMV infection. Typically, CMV can be permanently suspended (life-long) within an infected cell, and when the microenvironment of the host cell changes, the suspended CMV can wake up (activation) and cause viral replication, and the CMV can wake up. The specific cellular immunity supervision mechanism of the organism can quickly remove the replicated viruses so as to effectively prevent the viruses from spreading, so that when the immune function of the organism is seriously destroyed (such as SOT and HSCT patients), the risks of CMV awakening and spreading are obviously increased, and the incidence rate reaches 50-80%. ( Paul Griffithset al pathogenesis of human cytomegalovirus J Pathol 2015Jan;235 (2): 288-97. )

In contrast to CMV, the cell tropism of EBV is limited to B cells and a few epithelial cells, EBV can also be permanently lodged within infected cells, which also involves long-lived cells such as memory B cells, and EBV can also wake up within infected cells under appropriate conditions. One major complication of EBV infection is Post-transplant lymphoproliferative disorder (Post-transplant lymphoproliferative disease, PTLD), a potentially malignant lesion with mortality in SOT and HSCT patients as high as 40-60%. ( Jeffrey I Cohen. Primary Immunodeficiencies Associated with EBV disease. Curr Top Microbiol Immunol.2015;390 (Pt 1): 241-65. )

Antiviral drugs are the first option for clinical control of CMV infection, and since the 1980 s 5 drugs have been approved for the treatment of CMV, respectively against DNA polymerase encoded by the viral UL54 gene (Ganciclovir, valganciclovir, foscarnet and Cidofovir) and the terminal enzyme encoded by the UL51 gene (Letermovir). The overall efficacy of these drugs is good, but a significant proportion of patients fail treatment due to intolerance to drug toxicity or viral resistance due to genetic variation. In contrast, the other 3 virus infections have limited alternatives, with many clinical uses of Cidofovir, but no drug has been approved to treat these viruses to date. ( Hantz S et al French CMV Resistance Survey Study group. Drug-resistant cytomegalovirusin transplant recipients: a French core student (2010). J Antimicrob Chemother: 2628-2640. )

Allogeneic hematopoietic stem cell transplantation (Hematopoietic stem cell transplantation, HSCT) has become one of the best therapeutic modalities for hematopoietic-related malignant or genetic diseases. However, in addition to the need to HLA-type hematopoietic stem cell donors, in order to avoid graft versus host disease (Graft versus host disease, GVHD), recipients need to undergo an immune window during which their autoimmunity is greatly destroyed, so that patients are susceptible to viral infection and to develop a range of conditions, serious ones of which may be life threatening. Cytomegalovirus (CMV), epstein Barr Virus (EBV), BK polyomavirus (BKV) and Adenovirus (ADV) infections are reported to be particularly common and are often described as key risk factors affecting the prognosis after HSCT surgery. ( Francesco Saglio et al the time is now: moving towardvirus-specific T cells after allogeneic hematopoietic stem cell transplantation as the standard ofcare. Cytotherapy.2014Feb;16 (2): 149-159. )

Adoptive cell therapy (Adoptive cell therapy) is an emerging therapeutic platform for inducing tumor regression or clearance of certain viral infections following organ or hematopoietic stem cell transplantation. Wherein virus-specific T cells (VST) refer to memory T cells (T) having specific immune response capacity to virus-encoded antigen after amplification culture _M ) VST is another effective option for clinical control of CMV and EBV infection, especially for infected patients who are intolerant to antiviral drugs or who are otherwise refractory to treatment failure, and clinical benefit rate (clinical benefit) after VST infusion is typically 70% or more. (Theresa Kaeuferle et al. Strateies of adaptive T-cell transfer to treat refractory viral infections post allogeneic stem cell transformation. Journal of therapeutics)&Oncology.2019Feb；12：13.)

T can be determined based on differences in phenotypic characteristics and biological functions _M Dividing into four sub-populations, namely memory stem cells (memory stem cells, T _SCM ) Central memory cells (central memory cells, T) _CM ) Effector memory cells (effector memory cells, T) _EM ) And tissue-resident memory cells (resident memory cells, T) _RM )。T _SCM And T _CM The phenotypic characteristics of (2) are:

(1) Expression of T _M Core markers such as CD95 and the like;

(2) Expression of lymph node "homing receptors" (home receptors) such as CCR7 and CD62L, etc.;

(3) Expression of co-stimulatory receptors (co-stimulatory receptors) such as CD28 and the like;

(4) Clip variants expressing the leukocyte common antigen CD45 such as CD45RA (T) _SCM ) And CD45RO (T) _CM )。

The two TM subsets have the properties of adult stem cells (somatic stem cells), such as being able to survive in secondary lymphoid organs such as lymph nodes with long life (longevity) without significant differentiation by self-renewal (self-renewing); when encountering corresponding antigen again, the antigen proliferates and differentiates into T with high efficiency _EM And effector cells (T) _EFF ) The latter is also known as terminal differentiation T re-expressing CD45RA _EM (terminally differentiated effector memory T cells re-expressing CD45RA，T _EMRA ). Currently consider T _SCM And T _CM The whole immunity supervision function is born, and plays a key role in immune memory. And T is _sCM/CM Different, T _EM Is unable to express CD28, CD45RA and the homing receptor, and is therefore distributed mainly in peripheral non-lymphoid organs and has a short survival time, but part of T _EM Can differentiate into T in the specific microenvironment (niches) of peripheral organs _RM The latter has long life and takes on regional immunity monitoring functions. ( Charlotte M.Mousset et al computer Phenotyping of T Cells Using Flow cytomet. 2019Jun;95A (6): 647-654. )

Thus, there is an urgent need in the art for methods for the large-scale preparation of antiviral adoptive immune cells.

Disclosure of Invention

The invention uses a medium without animal origin components, i.e. a medium comprising serum replacement, preferably AIM-V medium.

In a first aspect, the present invention provides a method for preparing a specific antiviral adoptive immune cell, the method comprising the steps of:

(1) Collecting peripheral blood, and centrifugally separating to obtain peripheral blood mononuclear cells;

(2) Activation of CD3 in peripheral blood mononuclear cells with two or more viral antigen peptides ⁺ T cells;

(3) Amplifying the activated T cells of step (2) to the desired number using G-REX10 flask culture.

Each of the above virus antigen peptides was 15 peptides in length.

In the step (2), each virus antigen peptide adopts two different sites of virus antigen peptides, and the degradable microsphere loaded with the CD3 and CD28 antibodies is activated.

After the step (2), further detecting CD3 ⁺ T cells and CD137 ⁺ Duty cycle, e.g. CD3 ⁺ T cell ratio is greater than or equal to 50%, CD137 ⁺ The ratio is more than or equal to 2 percent, and continuing the step (3); if an index is not met, restarting the step (1).

The culture medium used in the step (3) for culturing and amplifying T cells is AIM-V culture medium.

The virus antigen peptide in the step (2) is cytomegalovirus and EB virus. The concentration of each virus antigen peptide is 1-3 microgram/10 ⁷ A cell; in the step (3), IL210-30ng/ml, IL4, IL7 and IL15 are added and the concentrations of IL4, IL7 and IL15 are respectively kept at 10-30ng/ml,10-30ng/ml and 10-50ng/ml when the expanded T cells are cultured.

In a second aspect of the present invention, there is provided a method for preparing a specific antiviral adoptive immune cell CE, the method comprising the steps of:

(2) Activation of CD3 in peripheral blood mononuclear cells with cytovirus and EB virus antigenic peptides ⁺ T cells;

(3) Culturing and amplifying the activated T cells in the step (2) to a required number by adopting a G-REX10 bottle;

(4) Taking part of the cells amplified in the step (3), and reactivating the cells by using cytomegalovirus and EB virus antigen peptides;

(5) The activated T cells of step (4) were expanded to the desired number by optimized culture using G-REX10 flasks.

The degradable microsphere in the step (2) is TransACT, and the concentration of the degradable microsphere is kept between 10 and 30 microlitres/10 ⁷ A cell; the concentration of each virus antigen peptide is 1-3 microgram/10 ⁷ And (3) cells.

The peripheral blood of the patient or the healthy person is adopted in the step (1).

When the expanded T cells are cultured in the step (3), 10-30ng/ml of IL2, IL4, IL7 and IL15 are added, and the concentrations of IL4, IL7 and IL15 are maintained at 10-30ng/ml,10-30ng/ml and 10-50ng/ml, respectively.

In the step (4), IL21 was added within six days of reactivation of the viral antigen peptide.

The step (5) is to expand the cultured T cells, and supplement IL7 and IL15 for a plurality of times, and maintain the concentration of the IL7 and IL15 at 10-30ng/ml and 10-50ng/ml respectively.

The culture medium used in the step (3) and the step (5) for culturing and amplifying T cells is AIM-V culture medium.

Each of the above virus antigen peptides was 15 peptides in length.

The concentration of IL21 is 10-30ng/ml.

The patent forms a unique VST large-scale preparation new process through innovation and development on the basis of advanced experience of VST preparation in digestion and absorption internationally. The preparation process is implemented in a clean environment conforming to GMP standards, and various cell therapy products can be formed: lymaVir-CE, which is lymphocytes against virus, and LymaVir-AB, which is ADV and BKV, which are CMV and EBV, respectively. The cultured immune cells have the following main characteristics:

1)CD3 ⁺ t cells, in particular T _SCM/CM Highly enriched subpopulations;

2) The specificity T cell of the virus antigen has high purity and strong activity;

3) The blood sampling amount is small, the preparation time is short, and VST can be infused by 2 weeks and 4 weeks of culture respectively. The new treatment mode of 'one-time blood sampling and two-time infusion' is beneficial to improving or consolidating clinical curative effect.

4) Various specific antiviral adoptive immune cells can be prepared and obtained by the preparation method of the invention.

Drawings

FIG. 1 is a flow chart of a process for preparing an adoptive immune cell;

FIG. 2 is a schematic diagram of the results of an ELISA spot method for detecting the ability of cells to secrete gamma-interferon;

FIG. 3 flow cytometry analysis of immune T cells (CD 3) in blood samples ⁺ ) Schematic of the percentage;

FIG. 4 flow cytometry analysis of VST purity and activation status (CD 137) in immune cell populations ⁺ /CD28 ⁺ ) A schematic diagram;

FIG. 5 flow cytometry analysis of immunocyte composition (CD 3 ⁺ /CD4 ⁺ /CD8 ⁺ ) A schematic diagram;

FIG. 6 flow cytometry analysis of immunocyte subtypes (T _SCM /T _CM /T _EM /T _EMRA ) Schematic of the duty cycle.

FIG. 7 flow cytometry analysis compares the purity and activation status (CD 137) of VST with and without TransACT microspheres ⁺ /CD28 ⁺ ) Schematic diagram of the present invention

FIG. 8 is a bar chart showing the ratio of subpopulations of cells obtained by culturing for 2 weeks in the conventional method to cells obtained by culturing for 2 weeks and 4 weeks in the novel method of the present invention;

FIG. 9 is a histogram of the ability of cells cultured for 2 weeks by the conventional method and cells cultured for 2 weeks and 4 weeks by the novel method of the present invention to secrete gamma-interferon by ELISA spot method.

Detailed Description

Hereinafter, the present invention will be described in more detail and in detail with reference to examples, but the following examples are not intended to limit the present invention.

EXAMPLE 1 preparation of LymaVir-CE adoptive immune cells

LymaVir-CE adoptive immune cells can be prepared by the culture mode, wherein LymaVir is lymphocytes against virus, and CE refers to CMV and EBV respectively.

The 1 st feature of this patent is the related process of establishing a rapid preparation regimen that can be carried out after 2 weeks of culture. The antiviral mechanism of action following adoptive input of VST is that such T cells are able to reconstitute specific immune regulatory mechanisms such as persisting in patients by self-renewal (i.e. long life), proliferate efficiently upon encountering the corresponding viral antigen and exert significant immune effector functions (i.e. immune memory, immunological memory).

The prior data reveals that VST obtained by classical preparation scheme is represented by T _EM Predominance (80% by weight) whereas VST obtained using the rapid preparation protocol is expressed as T _CM Mainly (with a ratio of about 40-60%), but enriched to T after scale-up of VST _SCM There have been no studies reported. The second feature of this project is to obtain T _CM In particular T _SCM Enriched VST. The LymaVir-CE preparation process is shown in FIG. 1.

1. Blood sample discrimination

T cells in peripheral blood of healthy adult account for 2-2.5% of total T cells in human body, and the number is about 5-10×10 ⁹ . After PBMC were isolated by Ficoll gradient centrifugation, the cell yield was roughly estimated to be 1X10 ⁶ Blood sample/ml, wherein CD3 ⁺ T cells 50-80% and CD3 ⁺ CD4 in T cells ⁺ T cells and CD8 ⁺ The ratio of T cells is 2-3:1. VST in peripheral blood T cells can be divided into two cell populations, CD8 ⁺ CD28 ⁺ Cells, the main component of which is T _CM Because of the initial cells (T) _N ) And T _SCM Usually in very small amounts; another type is CD8 ⁺ CD28 ^- Cells, including T _EM And T _EMRA . A common feature of the above T cell subsets is that once subjected to corresponding antigenic stimulation, their reactivation is largely dependent on TCR signaling while the effect of CD28 co-stimulatory signaling is relatively slight. ( Jae-Ouk Kim et al NF-kB and AP-1 regulate activation-dependent CD137 (4-1 BB) expression in T cells FEBS letters 2003:163-170 )

The addition of overlapping short peptides of viral antigen in PBMC was sufficient to activate VST in a manner known as MLPC (mixed lymphocyte peptide culture). ( Jae-Ung Lee et al, review the Concept of Targeting NFAT to Control T Cell Immunity and Autoimmune diseases, front immunol.2018nov;27 (9): 2747. )

GermanyGroup C Sukdolak et al have tested peripheral blood VST in healthy adults using IFN-gamma enzyme-linked spot (ELISPOT) and considered high responders by 204 blood sample analysis authors (high responders, spot forming cells SFC > 50/2.5X10) ⁵ Cells to be examined) are more suitable as donors for the expansion culture of VST. We introduced this detection technique, followed by IFN-. Gamma.ELISPOT detection after MLPC activation of cells and CD137 after activation with the same viral antigen ⁺ The cell ratios were synchronously aligned and statistical analysis showed that the ELISPOT values were approximately 1% CD137 ⁺ Cells (data not shown). Based on this, the screening standard of qualified blood sample, namely CD3, is set ⁺ T cell ratio is greater than or equal to 50% and CD137 ⁺ The cell ratio is more than or equal to 2 percent (each VST is stimulated by 2 virus antigen peptide fragments). ( Cinja Sukdolaket al.CMV-, EBV-and ADV-Specific T Cell Immunity: screening and Monitoring of Potential Third-PartyDonors to Improve Post-transformation Outcome. Biol Blood Marrowtransformation.2013 Oct; )

In the new method, flow cytometry is adopted to analyze and identify the cell phenotype and the subgroup ratio, and CD3 is obtained ⁺ The T cell fraction analysis method is as follows, PBMC (5×105 cells/100. Mu.g) is taken, wherein PerCP-CD3 antibody (5. Mu.l, bioleged Co., U.S.A.) is added to the detection group, no antibody is added to the control group, after incubation at 4 ℃ in the dark for 30min, PBS is added to centrifuge to remove free antibody, and then the analysis is carried out, namely Forward Scattering (FSC) is plotted against Side Scattering (SSC), lymphocyte component P1 gate is set, and the experimental schematic result is shown in FIG. 3A; detection of CD3 after selection of lymphocyte fraction P1 ⁺ Cells, positive reference lines were set according to a control group to which no CD3 antibody was added, and CD3 was detected ⁺ The percentage of cells and the experimental schematic results are shown in 3B.

CD3 ⁺ The cell fraction analysis method is as follows: PBMC (5X 10) ⁵ 50. Mu.l) and CMV antigen (pp 65 and IE1 each 100ng, germany Mei Tianni) or EBV antigen (LMP 2a and EBNA1 each 100ng, germany Mei Tianni) were added, after incubation for 24 hours the test groups were added with APC-CD137 antibody (5. Mu.l, bioleged. Co., USA) and PE-CD28 antibody (20. Mu.l, BD. Co., USA), the control group was not added with antibody, incubated at 4℃for 30min in the absence of lightAfter centrifugation in PBS to remove free antibody, the sample was analyzed on a machine, FSC was plotted against SSC, lymphocyte fraction P1 gate was set, and the experimental results are shown in FIG. 4C.

Post-selection lymphocyte component P1 gate detection of CD137 ⁺ And CD28 ⁺ Cells, positive reference lines were set according to control group without CD137 or CD28 antibody, and CD137 was detected ⁺ And CD28 ⁺ The experimental schematic results are shown in figure 4D.

The healthy volunteer blood samples were tested, showing that all tested blood samples had a CD3T cell fraction > 50% and CD137+ cells fraction > 2%.

Table 1. Ratio screening of peripheral blood immune cells of volunteers and percent detection of specific T cells against a single viral antigen.

Note that: CMVST is a cytomegalovirus specific T cell; EBVST is an epstein barr virus specific T cell.

Primary activation of vst and phenotypic shift

After passing the screening of the blood samples, the VST in the PBMC was activated by MLPC, and the PBMC (1.5X10 ⁷ ) Adjusted to 1X10 ⁷ Per ml, CMV antigen and EBV antigen (CMV pp65, CMV IE1, EBV LMP2a and EBV EBNA1, germany Mei Tianni) were added at a concentration of 2. Mu.g/10 ⁷ Cells), 12-well plates were incubated for 24 hours. However, the VST is activated and amplified for 2 weeks to culture T _CM The subpopulation is dominant (data not listed) in order to promote T _SCM Subgroup formation, the addition of TransACT (CD 3 and CD28 antibody-loaded degradable microspheres, germany Mei Tianni Co.) in MLPC, and titration experiments showed that the amount of TransACT was controlled to be 10-30 microliter/10 ⁷ Cells were cultured for 24 hours and then CD137 ⁺ The cell ratio was not significantly different from that of the virus antigen-activated group without TransACT, and the optimal dosage was 10. Mu.L/10 ⁷ And (3) cells. Amplification culture under this condition for 2 weeks, T _SCM/CM The subgroup ratio was higher than the group without TransACT, as described in detail below.

The cells collected after VST activation were placed in a G-REX10 permeable flask (U.S. Wilson wolf manufacturing Co., ltd., floor area 10 cm) ² ) In this case, 40ml of medium (AIM-V, GICO, incorporated herein by reference, 3%UltraGRO Cell Culture Supplement, cliniScineces, incorporated herein) and Interleukins (IL), including IL2 (50-500 IU/ml, shuanglu pharmaceutical company), IL4 (10-30 ng/ml), IL7 (10-30 ng/ml) and IL15 (10-50 ng/m 1), were supplemented, and IL4/7/15 was a homologous Hairi company. Whereas proliferation differentiation after T cell activation is closely related to cell metabolism, such as IL2 preferentially activates glycolytic metabolism, driving T cell proliferation to form T _EM/EMRA Subpopulations, whereas IL7 and IL15 favor oxidative metabolism of amino acids, promoting T cell proliferation to form T _SCM/CM A sub-population. To amplify and form T _SCM/CM The VST with the dominant subgroup is finally determined by a large number of experiments, 4 cytokines are added, and then different concentrations of the cytokines are optimally arranged and combined, and the optimal combination is formed by compatibility for the experiments.

The method introduces the G-REX culture bottle and has the advantages that:

(1) Seeded cells (cell density controlled at 1X 10) ⁶ /cm ² Or below) without passage in 2 weeks after culture, the risk of pathogen pollution caused by personnel operation can be obviously reduced;

(2) The G-REX bottle is supplied with oxygen by the microporous membrane at the bottom, and excessive culture medium is added, so that cell hypoxia can not be caused, but cell metabolite diffusion dilution can be promoted, and cell proliferation efficiency is obviously improved.

After VST activation, the cells were first cultured in G-REX flasks for 6 days, and then IL4/7/15 was added 3-4 days apart to meet cell growth requirements. In order to ensure that VST is always in active proliferation state, sampling and counting can be performed at 6 days of culture, if cell proliferation is found to be less than 4 times, appropriate amount of TransACT (the dosage is controlled at 20-60 microliter/10) ⁷ Between cells).

3. Quality control inspection

VST can be filled for clinical treatment by amplification culture in G-REX bottle for 15 days, so that part of cells are collected 2 days before filling (i.e. 13 days of culture) for quality control test, and test items comprise:

1) Cell number

The cell number was based on trypan blue exclusion of viable cells, 20 μl of cells were mixed with an equal amount of trypan blue solution, and the ratio and number of viable cells were measured by a cell counter (Shanghai Rui Yu Biotechnology Co., ltd.). The detection results of 2 VST cases show that the ratio of the living cells after 2 weeks of amplification culture is more than 97%, wherein CD3 ⁺ T cell purity averages 99.3%. T cell number was amplified approximately 13.9-fold on average, wherein the culture density of PBMC was controlled at 1.5X10 ⁶ /cm ² In this case, the T cell expansion efficiency is higher.

Table 2: comparison of total immune and T cells expanded for 2 weeks before and after activation;

^* units: 10 ⁶

2) T cell composition and subpopulations

T cell composition including CD3 ⁺ T cell duty cycle and CD4 therein ⁺ T cells and CD8 ⁺ T cell fraction was analyzed as follows, PBMC (5X 10) ⁵ Cells/100. Mu.g), wherein FITC-CD3 antibody, perCP-CD8 antibody and PE-CD4 antibody (5. Mu.l, biolegend, U.S.A.), control group, no antibody, were added, incubated at 4℃in the dark for 30min, centrifuged in PBS to remove free antibody, and then analyzed on a machine, i.e., by Forward Scatter (FSC) versus Side Scatter (SSC), lymphocyte fraction P1 was set, and the experimental schematic results are shown in FIG. 5E;

detection of CD3 after selection of lymphocyte fraction P1 ⁺ Cells, positive reference lines were set according to a control group to which no CD3 antibody was added, and CD3 was detected ⁺ The percentage of cells, the experimental schematic results are shown in FIG. 5F;

selecting a CD3 positive cell population, setting a positive reference line according to a control group without adding CD4 antibody and CD8 antibody, and detecting CD4 ⁺ And CD8 ⁺ The percentage of cells, the experimental schematic results are shown in FIG. 5G. VST is enriched with CD3 after amplification culture ⁺ T cells, averageUp to 99.4% of the CD ⁺ 4 and CD8 ⁺ T cells (including CD 4) ⁺ CD8 ⁺ Cells) were 14.5% and 84.3%, respectively. Generally, expanded T cells can be divided into two types, CD8 ⁺ T cells predominate; second is CD4 ⁺ T cell duty cycle slightly exceeds CD8 ⁺ T cells, and the like, have also been reported abroad. ( Marta Grau-Vorster et al Characation of a Cytomegalovirus-Specific T Lymphocyte Product Obtained Through a Rapid and Scalable Production Process for Use in Adoptive immunology. Front in immunology.2020Feb;11 (271) )

Table 3: the immune cell composition ratio after 2 weeks is prepared by the novel method;

the T cell subset was stained with four fluorescent antibodies (PerCP-CD 3 antibody, APC-CD95 antibody, PE-CD62L and FITC-CD45RA antibodies, all from Biolegend, USA) and FCM analysis was performed by first mapping FSC to SSC, selecting lymphocyte fractions and detecting CD3 ⁺ CD95 ⁺ A cell; the population of cells is then selected for detection of CD62L ⁺ Cell and CD45RA ⁺ And (3) cells. Wherein each subgroup phenotype is T _N (CD3 ⁺ CD95 ^- )、T _SCM (CD3 ⁺ CD95 ⁺ CD45RA ⁺ CD62L ⁺ )、T _CM (CD3 ⁺ CD95 ⁺ CD45RA ^- CD62L ⁺ )、T _EM (CD3 ⁺ CD95 ⁺ CD45RA ^- CD62L ^- ) And T _EMRA (CD3 ⁺ CD95 ⁺ CD45RA ⁺ CD62L ^- ). Mapping FSC to SSC, setting lymphocyte component P1 gate, and experimental schematic result is shown in FIG. 6H;

selecting lymphocyte component P1 gate, setting positive reference line according to control group without CD3 or CD95 antibody, and detecting CD3 ⁺ CD95 ⁺ The schematic results of the cell are shown in FIG. 6I;

selection of CD3 ⁺ CD95 ⁺ Cell fraction according to non-CD addedA control group of 45RA or CD62L antibodies set a positive reference line and detect CD3 ⁺ CD95 ⁺ CD45RA in cells ⁺ And CD62L ⁺ The percentage of cells, the experimental schematic results are shown in FIG. 6J;

t cells in the peripheral blood of healthy adults mainly comprise four sub-populations (i.e., T _N 、T _CM 、T _EM And T _EMRA ) With a ratio of substantially 40%, 30%, 20% and 10%, T _SCM The subpopulations are fewer, typically 2-6% in ratio. The main subgroup formed by VST after amplification culture is T _SCM And T _CM (average ratio of 17.4% and 63.7%) T _N Substantially undetectable and T _EM And T _EMRA The average duty cycle of the subpopulations decreased to 13.9% and 5.07%, respectively, and the possible cause of this T cell phenotype switch was activation of antigen specific TCR signaling leading to CD28 ⁺ T cells proliferate preferentially.

Table 4. Percentage distribution of subpopulations of immune cells obtained after 2 weeks of preparation according to the novel method of the present invention;

3) VST duty cycle and vitality

The ability of cells to secrete gamma-interferon (ifnγ -ELISPOT assay) was examined using enzyme-linked immunospot and regarded as VST viability.

VST (1X 10) ⁶ Tube) is adjusted to 1X10 ⁷ Per ml, ADV antigen and BKV antigen (10. Mu.g/10) ⁷ Cells) or without antigen (negative control), 2-5. Mu.l/tube (i.e., 2-5X 10) were taken after 3-4 hours of incubation ⁴ Cells) were used for ifnγ -ELISPOT assay (Mabtech), and the remaining cells were cultured for a further 20 hours for FCM to detect the percentage of CD137/CD28 expression. ELISPOT assays showed that the negative control had only a small number of spot-forming cells (SFC) while the antigen-activated group had significantly increased SFC, the latter subtracted from the control groupAnd converted to SFC/2X 105 cells to be examined. The experimental schematic results are shown in fig. 2.

The detection results of 5 VST show that CD137 after 2 weeks of amplification culture ⁺ The VST (including CMVST and EBVST) ratio is typically over 60%, indicating significant enrichment of VST after amplification. Such CD137 ⁺ In VST, the average CD28 positive rate reached 86.8%, which is significantly higher than before culture, suggesting such CD137 ⁺ CD28 ⁺ VST belongs to T _SCM/CM The subpopulations were matched with the phenotyping results. IFNgamma-ELISPOT assays showed SFC averages of 2265 (CMVST) and 1795 (EBVST), respectively, which are higher than similar reports abroad. (Ifigeneia Tzannou et al. Off-the-Shell viruses-Specific T Gells to Treat BK Virus, human Herpesvirus 6, cytomegalovirus, epstein-Barr Virus, and Adenovirus Infections After Allogeneic Hematopoietic Stem-Cell transformation. Journal of Clinical oncology.2017nov;35 (31): 3547-3557.) CD137 ⁺ CD28 ⁺ The VST stream detection method comprises the following steps: cells activated with viral antigen peptide for 24 hours were selected, APC-CD137 antibody (5. Mu.l, biolegend, U.S.A.) and PE-CD28 antibody (20. Mu.l, BD, U.S.A.), control group was not added with antibody, incubated at 4℃for 30min in the dark, centrifuged in PBS to remove free antibody, and then analyzed on-machine, with FSC plotted against SSC, lymphocyte component P1 gate set (FIG. 7-K); post-selection lymphocyte component P1 gate detection of CD137 ⁺ And CD28 ⁺ Cells, positive reference lines were set according to control group without CD137 or CD28 antibody, and CD137 was detected ⁺ And CD28 ⁺ Cell percentage of (FIG. 7-L);

TABLE 5 preparation of CD137 of immunocytes obtained after 2 weeks by the novel method of the present invention ⁺ /CD28 ⁺ VST duty cycle and enzyme linked immunosorbent assay spot data table;

^* expanding CD137 in cells ⁺ VST ratio (%); ^** CD137 ⁺ CD28 in VST ⁺ Cell ratio (%); ^*** SFC/2×10 ⁵ cell to be examined

4) Endotoxin, mycoplasma and bacterial culture

Bacterial endotoxin test method: the product is detected according to the fourth section <1143 bacterial endotoxin detection method > of the pharmacopoeia of the people's republic of China, 2020 edition, and should be < 0.5EU/ml. The bacteria detection adopts a BacTALERT3D microorganism detection system, a sample is added into a BACT/ALERT culture flask for 5 days to detect anaerobic bacteria and aerobic bacteria, and the test result of the sample should be negative. The mycoplasma detection adopts a fluorescent staining method, staining is carried out by using sigma company host 33258 staining solution, and the cell nuclei of cells only appear blue fluorescence as negative when observed under a fluorescence microscope.

Reactivation and optimized amplification of VST

Recent foreign studies report that about 20-40% of patients require 2 or more infusions of VST to gain clinical benefit. (Ifigeneia Tzannou et al, "Mini" bank of only 8donors supplies CMV-directed T cells to diverse recipients.blood additives.2019Sep; 3 (17): 2571-2580.) it is thus envisaged that this new mode of treatment of "one-time preparation-two-infusion" would potentially improve or consolidate clinical efficacy by taking a portion of the cells after 2 weeks of expansion culture to obtain VST, continuing expansion culture for 2 weeks, and then re-infusing.

According to this concept, VST (3X 10) was taken after 2 weeks of amplification culture ⁷ Adjusted to 1X10 ⁷ Per ml) was re-activated by MLPC (TransACT addition) (antigen concentration was reduced to 1. Mu.g/10 as in the initial activation) ⁷ ). However, it was found that these VSTs were subjected to 2-week amplification culture to form T _SCM/CM Dominant T _M The addition of IL4/7/15 after reactivation of the same antigen resulted in a significant cell differentiation, manifested by a decrease in the proportion of the above-mentioned subgroups. To avoid this differentiation, experiments were run with T _M The self-stabilizing proliferation (homeostatic proliferation) factors IL7 and IL15 are used as the basis for optimization combination, and the fact that IL21 (10-30 ng/ml) is adopted to replace IL4 within 6 days after virus antigen activation has obvious differentiation inhibition effect is found. IL4/7/15 amplification culture VST was still used at week 2 of culture, and G-REX10 flasks were also used for the 2-week amplification culture.

Samples were taken for four quality control tests at 13 days of continued culture after antigen reactivation of VST, with the following results:

1) Cell number

The detection results of 2 cases of VST (Table 6) show that the ratio of viable cells after 2 weeks of expansion culture is > 97%, wherein CD3 ⁺ T cell purity averages 99.3%.

T cell number was amplified approximately 13.9-fold on average, wherein the culture density of PBMC was controlled at 1.5X10 ⁶ /cm ² In this case, the T cell expansion efficiency is higher.

* Units: 10 ⁶

Table 6: the method of the invention comprises the steps of comparing the total number of immune cells and the total number of T cells which are amplified for 2 weeks before secondary cell activation and after reactivation;

2) T cell composition and subpopulations

VST is enriched with CD3 after amplification culture ⁺ T cells with an average duty cycle of 99.9% and CD4 ⁺ And CD8 ⁺ T cells (including cd4+cd8+ cells) were 7.55% and 90.6%, respectively (table 7). Due to CD8 ⁺ T cells are the primary antiviral effector cells, and such T cells are highly enriched to facilitate improvement of clinical efficacy.

Table 7: the method of the invention obtains the constitution of cells after secondary activation and expansion for 2 weeks;

the main subgroup formed by VST after amplification culture is T _SCM And T _CM (average ratio of 17.4% and 63.7%) T _N Substantially undetectable and T _EM And T _EMRA The average subgroup duty cycle was reduced to 13.9% and 5.07%, respectively (table 8).

Table 8: the method of the invention obtains the subgroup ratio of the cells after the secondary activation and expansion for 2 weeks;

3) VST duty cycle and vitality

In addition to T _SCM Outside the highly enriched subpopulations, VST was re-activated and cultured for 2 weeks using an optimized amplification system, and VST was also increased in duty cycle and purity (including CD137 ⁺ CMVST and CD137 ⁺ EBVST) and SFC values exceeded the first 2 weeks of culture.

Table 9: the method of the invention obtains VST purity and SFC data table of cells after 2 weeks of secondary activation and amplification;

VST filling and cryopreservation

VST filling can be performed by quality control inspection to reach release standard (release criterion). With reference to the relevant reports of international clinical studies, the release standard should meet the following requirements at the same time:

table 10: VST adoptive immune cells pass through a standard table;

^* units: EU/ml; ^** when VST was cultured for 2 weeks, the CD 8T cell ratio was not used as a release standard due to the individual difference.

The number of VST fills varies from person to person, and the common criteria are:

(1) according to the body surface area, usually 2X 10 ⁷ Cell/m ² The method comprises the steps of carrying out a first treatment on the surface of the (2) According to the weight of the patient, 1X10 is required ⁶ Cells/kg.

VST filling is carried out by centrifuging, washing, removing protein components including cytokine from culture medium, suspending cells in sodium chloride injection (physiological saline) or compound sodium chloride injection (ringer's solution), packaging in polypropylene infusion bag (volume of 50 ml), and transporting by cold chain. If the patient does not have an allergic reaction to Human Serum Albumin (HSA), the appropriate amount (e.g., 1-2%) can improve cell viability. The containers, liquids and proteins adopted in the VST filling process all meet the Chinese pharmacopoeia standards and have batch numbers approved by the medicine monitoring department.

The large number of cells remaining after VST filling is particularly evident in low weight patients (e.g., pediatric patients), and thus the cells need to be cryopreserved using appropriate methods, which can save preparation time and expense for subsequent treatment that may be needed. Cryopreservation (cryopreservation) refers to a technique for preserving living tissue or cells under cryogenic (e.g. liquid nitrogen) conditions involving 2 basic elements, namely cryoprotectants and cooling rates.

When the ambient temperature of human cells is reduced from 37 ℃ to below freezing point, intracellular water aggregates into ice, and the intracellular ice crystals (intracellular ice crystals, ICIC) have significant damage to cell survival and function. The main effect of cryoprotectants (cryoprotective agents, CPA) is to suppress ICIC formation, and the CPA requirements for different types of T cells are different, so that the CPA formula of VST is optimized, and the optimal formula is selected as follows: ringer's solution is mixed with 20% HAS in a 1:1 ratio to form a 10% HSA solution which is then mixed with CryoStor CS10 in a 1:1 ratio (abbreviated as HSA-CS 10). The cell density of VST was adjusted to 5-20X 10 using CPA as described above ⁶ Split charging with 1.25 ml/tube (freezing tube) or 20 ml/bag (freezing bag), slowly cooling (cooling rate controlled at-1 deg.C/min) to 80deg.C with a program cooling instrument (Siemens technology Co.), and storing with liquid nitrogen.

The VST stored at low temperature has higher living cell ratio (usually more than 75%) after being thawed and recovered by water bath at 37 ℃, and at the moment, physiological saline and 1-2% HSA are added for direct filling; or centrifuging the cells to remove protein components, culturing for 2-3 days, and packaging. Compared to direct filling after resuscitation, the living cell fraction of VST after culture is higher (typically > 85%), and thus is more suitable for patients requiring longer cold chain transport after filling. According to our LymaVir-CE series of test results, the optimal culture conditions were selected to be IL7 (20 ng/m 1) and IL15 (10 ng/ml). Culturing under these conditions for 1-3 days, the proportion of living cells being > 85% and the number of living cells remaining relatively constant.

Example 2 preparation of LymaVir-AB adoptive immune cells

The method is not only suitable for preparing LymaVir-CE adoptive immune cells, but also can be used for harvesting corresponding virus specific immune cells by using other virus antigen peptides in the activation stage. For example, ADV antigen and BKV antigen (ADV Hexon, ADV Panten, BKV LargeT and BKV VP1, germany Mei Tianni company) were used at a concentration of 2. Mu.g/10 ⁷ Cells) to obtain the LymaVir-AB adoptive immune cells. The preparation and detection method are the same as in example 1.

1. Blood sample discrimination

Blood samples from 10 healthy volunteers were tested and showed CD3 in all blood samples tested ⁺ T cell ratios were > 50% (average 65.5%, range 54.8% -76.5%) and CD137 ⁺ The cell fractions were all > 2% (average 5.6% and 6.1%, respectively, ranging from 2.0% to 10.2%) and CD137 ⁺ CD28 in cells ⁺ About half of the cells (average values of 63.2% and 62.9%, respectively, in the range of 44.4% -84.2%) were used, and experimental data are shown in Table 11.

Table 11. Percentage of peripheral blood immune cells of volunteers were screened for specific T-cell percentage against a single viral antigen.

^* CD137 in cell ⁺ VST ratio (%); ^** CD137 ⁺ CD28 in VST ⁺ Cell ratio (%);

note that: ADVST is an adenovirus-specific T cell; BKVST is RK polyomavirus specific T cells.

Primary activation of vst and phenotypic shift

VST in PBMC was activated using MLPC, PBMC (1.5×10 ⁷ ) Adjusted to 1X10 ⁷ Per ml, ADV antigen and BKV antigen (ADV Hexon, ADV Panten, BKV LargeT and BKV VP1, germany Mei Tianni company) were added at a concentration of 2. Mu.g/10 ⁷ Cells) and TransACT,12 well plates were incubated for 24 hours. Other details are the same as in example 1.

3. Quality control inspection

1) Cell number

The detection results of 5 VST cases show that the ratio of the living cells after 2 weeks of amplification culture is more than 97%, wherein CD3 ⁺ T cell purity averages 96.5%. T cell number was amplified approximately 7.8-fold on average, wherein the culture density of PBMC was controlled at 1.5X10 ⁶ /cm ² When the T cells are amplified more efficiently, the data are shown in Table 12;

table 12: comparison of total immune and T cells expanded for 2 weeks before and after activation;

^* units: 10 ⁶

2) T cell composition and subpopulations

VST is enriched with CD3 after amplification culture ⁺ T cells with an average duty cycle of 96.5%, wherein CD ⁺ 4 and CD8 ⁺ T cells (including CD 4) ⁺ CD8 ⁺ Cells) were 34.2% and 62.0%, respectively, as shown in table 13.

Table 13: the novel method prepares the immune cell composition ratio after 2 weeks

The main subgroup formed by VST after amplification culture is T _SCM And T _CM (average ratio of 32.0% and 57.4%, respectively), TN was substantially undetectable and T was _EM And T _EMRA The average subgroup duty cycle was reduced to 6.3% and 2.4%, respectively. The data are shown in Table 14;

table 14. Percentage distribution of subpopulations of immune cells obtained after 2 weeks of preparation according to the novel method of the present invention;

the detection results of 5 VST show that CD137 after 2 weeks of amplification culture ⁺ The VST (including ADVST and BKVST) ratio is typically over 60%, 2 of which are CD137 ⁺ The purity of VST is even more than 90%, which indicates that the amplified VST is obviously enriched. Such CD137 ⁺ In VST, the average CD28 positive rate reached 95.1%, which is significantly higher than before culture, suggesting such CD137 ⁺ CD28 ⁺ VST belongs to T _SCM/CM The subpopulations were matched with the phenotyping results. IFNgamma-ELISPOT detection shows that SFC average values are 2607 (ADVST) and 1761 (BKVST) respectively, the values are higher than those reported in the same class abroad, and the data are shown in Table 15; ( Ifigeneia Tzannou et al off-the-shell viruses-Specific T Cells to Treat BK Virus, human Herpesvirus 6, cytomegalovirus, epstein-Barr Virus, and Adenovirus Infections After Allogeneic Hematopoietic Stem-Cell transformation. Journal of Clinical oncology.2017Nov:35 (31): 3547-3557. )

TABLE 15 preparation of CD137 of immunocytes obtained after 2 weeks by the novel method of the present invention ⁺ /CD28 ⁺ VST duty cycle and enzyme linked immunosorbent assay spot data table;

Reactivation and optimized amplification of VST

1) Cell number

After 2 weeks of reactivation and expansion culture of VST, the cell number increased approximately 3-fold on average (table 16).

^* Units: 10 ⁶

Table 16: the method of the invention comprises the steps of comparing the total number of immune cells and the total number of T cells which are amplified for 2 weeks before secondary cell activation and after reactivation;

2) T cell composition and subpopulations

The composition of T cells after VST reactivation and expansion culture is significantly changed, and is expressed as CD8 ⁺ T cells predominate (average 86.8%) while CD4 ⁺ The T cell ratio is reduced to above and below 10%. The data are shown in Table 17;

table 17: the method of the invention obtains the constitution of cells after 2 weeks of secondary activation and expansion

Another significant change in cells after VST has been reactivated and expanded through the optimization protocol for 2 weeks is T _SCM Overrunning T _CM Becomes a dominant cell subset (average up to 73.7%) (table 18).

Table 18: the method of the invention obtains the subgroup ratio of the cells after the secondary activation and expansion for 2 weeks

3) VST duty cycle and vitality

After VST was re-activated and cultured for 2 weeks using the optimized amplification system, the VST duty cycle was also increased, and the purity (including CD137 ⁺ ADVST and CD137 ⁺ BKVST) and SFC values exceeded the first 2 weeks of culture. (Table 19)

Table 19: the method of the invention obtains VST purity and SFC data table of cells after 2 weeks of secondary activation and amplification;

VST filling and cryopreservation were performed as described in example 1.

Example 3 comparison of cell subsets obtained by the preparation method of the invention with data from conventional methods

The conventional method uses MLPC method to obtain 1.5X10 ⁷ The cells were placed in 15ml centrifuge tubes and added with viral antigen-encoded overlapping peptide (CMV/EBV/ADV/BKV) (100 ng/1.5X10) ⁷ Cells) were cultured at 37℃for 30min, transferred to G-REX10, and then cultured with IL4 (400U,/ml) and IL7 (10 ng,/ml). The cells were cultured for about 2 weeks for safety and functional assays, and all cells were collected and prepared as cell preparations for reinfusion. ( Anastasia Papadopoulou et al activity of broadcast-select T-cells as treatment for AdV, EBV, CMV, BKV and HHV6 inputs after HSCT. Sci Transl Med.2014June 25;6 (242): 242ra83. )

The preparation method of the invention is innovated on the basis of the conventional method: taking 1.5X10 ⁷ Cells were placed in 12 well plates, added with viral antigen-encoded overlapping peptide (CMV/EBV) at 37℃overnight, transferred to G-REX10 the next day, cultured for 2 weeks with IL4 (10-30 ng/ml) IL7 (10-30 ng/ml) and IL15 (10-30 ng/ml) for safety and functional assays, part of the cells were prepared as cell preparations for reinfusion, and the remaining cells were subjected to a second round of activation expansion phase and a second reinfusion after 2 weeks.

Unlike conventional method, the new mode of primary blood sampling and secondary reinfusion is favorable to clinical treatment effect, and the cells obtained in week 4 of the preparation method of the invention are sub-dividedThe cloth tends to be more dry and has the characteristics of adult stem cells, namely T _SCM/CM The ratio is higher, the cells maintain the stem cell attribute of the cells without differentiation, and the SFC value obtained by the detection of IFNgamma-ELISPOT is higher than that obtained by the conventional method, so that the cells obtained by the preparation method have obvious advantages in functionality.

Take LymaVir-CE cells of example 1 as an example:

as shown in FIG. 8, T cells obtained using the novel method have higher CD8 ⁺ The ratio suggests that it has higher killing activity. Culturing by conventional method to facilitate T _SCM Subgroup (CD 45 RA) ⁺ CD62L ⁺ ) Differentiation into T _CM (CD45RA ^- CD62L ⁺ ) And T _EM (CD45RA ^- CD62L ^- ) Therefore, the conventional method can only obtain T _CM Is a duty cycle data of (a). Compared with the conventional method, the T of the cells obtained by the novel method _SCM/CM Higher duty cycle, in particular T can be avoided by the novel method _SCM Differentiation of subpopulations of cells, T after 2 weeks of culture _SCM/CM The ratio of the cell to the cell is more than 80 percent, which is far higher than that of the conventional method, and the cell obtained by the novel method has the characteristic of memory stem cells more than that obtained by the conventional method.

As shown in FIG. 9, CMVST secreted IFNγ in the cells obtained by the conventional method in an amount of about 1300 SFCs/2X 10 ⁵ Cells, EBVST secreted IFN gamma amount about 400 SFCs/2X 10 ⁵ A cell; and the same is the cells obtained by culturing for 2 weeks, the CMVST secretes IFN gamma amount in the cells of the novel method of the invention is about 2264SFCs/2×10 ⁵ Cells, EBVST secreted IFNgamma amount about 1795 SFCs/2X 10 ⁵ A cell; whereas cells cultured for 4 weeks had CMVST of 2832 SFCs/2X 10 ⁵ To, and EBVST is 1915SFCs/2×10 ⁵ . Therefore, the cells obtained by the novel method have stronger specific antiviral capability than the cells obtained by the conventional method. Furthermore, it should be understood that the above embodiments are only for illustrating the technical scheme of the present invention, and not for limiting the scope of the present invention, and that although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will understand that various modifications or changes can be made to the technical scheme of the present invention without departing from the scope and spirit of the present inventionEquivalent substitutions.

Claims

1. The preparation method of the specific antiviral adoptive immune cell is characterized by comprising the following steps:

(2) Activation of CD3 in peripheral blood mononuclear cells with cytomegalovirus and EB virus antigenic peptides ⁺ T cells;

(3) Culturing and amplifying the activated T cells in the step (2) to a required number by adopting a G-REX10 bottle; the length of the virus antigen peptide is 15 peptide, and the concentration of each virus antigen peptide is 1-3 microgram/10 ⁷ A cell; when the step (3) is used for culturing and expanding T cells, 10-30ng/ml of IL2, IL4, IL7 and IL15 are added, and the concentration of IL4 and IL7 and the concentration of IL15 are respectively kept at 10-30ng/ml,10-30ng/ml and 10-50ng/ml; AIM-V medium was used for culturing expanded T cells.

2. The method of claim 1, wherein each of the viral antigen peptides in step (2) is activated by two different sites of viral antigen peptide and the addition of a degradable microsphere loaded with CD3 and CD28 antibodies.

3. The method of claim 1 or 2, wherein after step (2), CD3 is further detected ⁺ T cells and CD137 ⁺ Duty cycle, e.g. CD3 ⁺ T cell duty cycle ≡ 50%, CD137 ⁺ The ratio is equal to or greater than 2%, and continuing the step (3); if an index is not met, restarting the step (1).

4. A method for preparing specific antiviral adoptive immune cells CE, comprising the steps of:

(3) Culturing and amplifying the activated T cells in the step (2) to a required number by adopting a G-REX10 bottle; IL210-30ng/ml, IL4, IL7, IL15 are added while culturing and expanding T cells, and the concentrations of IL4, IL7 and IL15 are respectively kept at 10-30ng/ml,10-30ng/ml and 10-50ng/ml;

(5) Optimizing and culturing and amplifying the T cells activated in the step (4) to the required quantity by adopting a G-REX10 bottle; each of the viral antigen peptides in step (2) and step (4) is 15 peptides in length.

5. The method of claim 4, wherein each of the viral antigen peptides in step (2) is activated by two different sites of viral antigen peptide and the addition of a degradable microsphere loaded with CD3 and CD28 antibodies.

6. The method of claim 5, wherein the degradable microspheres in step (2) are TransACT, and the concentration of the degradable microspheres is maintained at 10-30 microliters/10 ⁷ A cell; the concentration of each virus antigen peptide is 1-3 microgram/10 ⁷ And (3) cells.

7. The method according to claim 4, wherein the peripheral blood in the step (1) is peripheral blood of a patient or a healthy person.

8. The method of any one of claims 4-7, wherein IL21 is added within six days of reactivation of the viral antigen peptide in step (4).

9. The method of claim 7, wherein the step (5) comprises expanding the cultured T cells, and supplementing IL7 and IL15 a plurality of times, and maintaining the concentrations at 10-30ng/ml and 10-50ng/ml, respectively; the concentration of IL21 is 10-30ng/ml.