CN110713977B - Culture amplification method of CD8T cells - Google Patents

Abstract

The invention provides a method for expanding and culturing CD8T cells, which is characterized in that CD8T cells are induced to expand and culture in the presence of the following substances: k3EC cells; the K3EC cell is a K562 engineering cell expressing CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L, and is used for providing a2 nd signal of a costimulatory receptor for activating CD8T cells; a peptide overlapping library of target antigens for use in providing TCR 1 st signals that activate CD8T cells. The method realizes the rapid and efficient amplification of antigen (CMV-pp65) specific CD8T cells; the CD8T cells obtained by amplification have high clinical application value in the prevention and treatment of CMV infection and CMV-related cancer.

Description

Culture amplification method of CD8T cells

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a culture and amplification method of T cells.

Background

CD8T cells, also known as Cytotoxic T Lymphocytes (CTL), are an important defense line for adaptive immune system (adaptive immune system) to construct antigen-specific immune protection mechanisms. CD8T cells specifically recognize antigens via T Cell Receptors (TCRs), and such antigens are expressed on the cell surface in such a manner that immunogenic peptide epitopes (pMHCI) are embedded in MHC class I molecules.

Modern cell engineering techniques are used to enrich (enrich) and expand (expand) antigen-specific CD8T cells from body fluids (e.g., blood) or tissues (e.g., tumor lesions) and then transfuse them to patients in need of treatment, a treatment option known as adoptive T cell therapy (adoptive T cell therapy) or T cell therapy (T cell therapy). Clinical data reveal that this T cell therapy allows refractory (refractory) viral infections (such as CMV and EB viruses) and malignancies (such as melanoma) to be controlled and achieve significant clinical efficacy. The genetically modified epitope specific TCR-T cell prepared by constructing a TCR expression vector by adopting a genetic engineering technology is also in clinical test at present, and the novel T cell treatment is known as a leading-edge field for guiding the development of tumor treatment and is highly concerned by the tumor medicine field.

Primary (primary) TCR, whether enriched in vivo⁺T cells or genetically engineered TCR-T cells are constructedSufficient T cells for clinical therapy are obtained through an efficient expansion platform (expansion platform). The platform mainly provides two technical supports, namely, the large-scale culture and amplification of T cells are realized according to GMP standard; secondly, the T cells are prevented from being terminally undifferentiated to become effector T cells (T cells) while being rapidly proliferated_EFF). In the two technical supports, the control of cell differentiation is more important, because the T cells are implanted into the body for long-term survival after being infused and efficiently expand and differentiate to form T with specific killing function when meeting corresponding antigens_EFFIt is 2 preconditions for clinical efficacy.

CD8T cells are generally divided into naive cells: (

T_N) Memory cells (T)_M) And effector cells (T)_EFF) Subgroup of which T_MAnd stem cell type (T)_SCM) Central, T_CM) And effect type (T, effector)_EM) And the like. These CD8T cell subsets and fractions have distinct phenotypic markers, where T_SCMAnd T_CMSimultaneously expresses chemokine receptor CCR7, adhesion molecule CD62L, costimulatory receptor (costimulatory receptor) CD27 and CD28, IL7 receptor alpha chain CD127 and apoptosis receptor CD95, and is distinguished by T_SCMExpression of CD45RA and T_CMExpressing CD45RO (fig. 1).

T_NAre T cells that are released from the thymus and have not encountered antigen. When the antigen elicits an immune response, T_NProliferation followed by sequential differentiation to form T_SCM、T_CM、T_EMAnd T_EFF(T in which CD45RA is expressed_EFFAlso known as T_EMRA). When the antigen is cleared, T_EFFRapid apoptosis disappears from the body, and T_MThen it is saved, wherein T_SCMAnd T_CMColonizing lymphoid hematopoietic organs such as bone marrow, T_EMDistributed in peripheral tissues such as the blood circulation. Usually T_SCMAnd T_CMCan survive in vivo for decades and even for life, these T' s_MSubset toolHaving stem cell-like properties (e.g., self-renewal, etc.), being rapidly activated upon re-encounter with the corresponding antigen and eliciting a more responsive protective immune response. Thus antigen-specific TCR⁺CD8T cell only maintains T after being cultured and expanded_SCMAnd T_CMThe phenotype and biological characteristics of the cells are sufficient for therapeutic function. Indeed, the above-mentioned T_MThe proportion of the components is closely related to the clinical efficacy of the T cells after infusion, and at the gene expression level, the expression of CD27 and CD127 is an important mark for long-term survival of the T cells after infusion.

Antigen elicits CD8T_NActivation and proliferation requires 3 signals, namely the TCR 1 st signal, the costimulatory receptor 2 nd signal and the cytokine receptor 3 rd signal. These signals are required to be provided by antigen-presenting cells (APCs), such as Dendritic Cells (DCs), in which the CD28 and IL2 receptors are the most functional 2 nd and 3 rd signals, but the main role of these signals is to induce T_NProliferate and differentiate to form T_EFF. When the antigen is cleared, the antigen-specific T_MSurvival in vivo is a dynamic balance of proliferation and apoptosis, with proliferation being finely regulated by a homeostatic proliferation (homeostatic proliferation) mechanism. This self-sustained proliferation does not require antigen-specific TCR signaling, and cytokine receptor No. 3 signaling plays a key role.

One major technical obstacle to the large-scale production of antigen-specific CD8T cells from body fluids or tissues is the availability of sufficient numbers of APCs, particularly DCs, e.g., from 1X 10⁸Less than 10 can usually be obtained in Peripheral Blood Mononuclear Cells (PBMC) (equivalent to 100ml of blood)⁷Thus preparing one part of a T cell therapeutic preparation (cell number. gtoreq.10)⁸) Blood sampling is required to be performed 3 times or more (or one-time blood sampling is 300-. Obviously, the blood collection amount in such a scale is difficult clinically and is not easy to be accepted by patients. Therefore, the search for an appropriate alternative to apc (dc) for efficient expansion of antigen-specific T cells is a research topic of current international general interest.

Therefore, there is an urgent need in the art to develop new and more efficient methods for expanding and culturing antigen-specific CD8T cells.

Disclosure of Invention

The invention provides an amplification culture method of antigen-specific CD8T cells and simultaneously provides a K3EC cell.

The first invention of the present invention provides a method for expansion culture of antigen-specific CD8T cells, which comprises inducing expansion culture of antigen-specific CD8T cells in the presence of:

(1) k3EC cells; the K3EC cell is a K562 engineered cell expressing CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L, and is used for providing a2 nd signal of a costimulatory receptor for activating CD8T cells;

(2) a peptide overlapping library of target antigens for use in providing TCR 1 signals that activate CD8T cells.

The target antigen is cytomegalovirus-pp 65 protein.

In the above culture method, K3EC cells were co-cultured with peripheral blood mononuclear cells.

Specifically, the ratio of the PBMC to the K3EC cell coculture is between 20:1 and 5: 1.

Preferably, the cytokine is supplied as a 3 rd signal for CD8T cell proliferation when antigen-specific CD8T cells are expanded in culture.

The above cytokines are IL15 and IL2 or IL15 and IL 7. The above cytokines are IL15 and IL 2.

The method also comprises the following steps of fixing the K3EC cells by using paraformaldehyde; the enriched homogenous CD8T cells were then sorted and expanded in culture to the desired number of CD8T cells.

The second invention of the present invention provides an expansion culture method of antigen-specific CD8T cells, the culture method comprising the steps of:

(1) collecting peripheral blood, and performing centrifugal separation to obtain peripheral blood mononuclear cells;

(2) co-culturing K3EC cells with peripheral blood mononuclear cells; the K3EC cell is a K562 engineered cell expressing CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L, and is used for providing a2 nd signal of a costimulatory receptor for activating CD8T cells;

(3) activating TCR 1 signal of CD8T cells by adopting a complete protein peptide overlapping library of cytomegalovirus-pp 65 protein during co-culture in the step (2);

(4) supplementing cytokines as a 3 rd signal for proliferation of the CD8T cells after the T cells are cultured by the steps (2) and (3);

(5) fixing K3EC cells by using paraformaldehyde, and then carrying out tissue sorting to enrich homogeneous CD8T cells;

(6) culture expanded homogeneous CD8T cells to the required number.

The above cytokines are IL15 and IL2 or IL15 and IL 7.

The ratio of the PBMC to the K3EC cell in the co-culture process is 20:1 to 5: 1.

Preferably, the ratio of the PBMC to the K3EC cells in the co-culture is 5: 1.

The culture method further comprises the following steps:

(7) performing quality inspection on the homogeneous CD8T cells obtained in the step (6).

The quality inspection comprises the following steps:

detecting the characteristics of the CD8T cells, including detecting cell components (TSCM/CM), antigen specific response capability (TSR) and TCR recognition reactivity peptide fragments.

The quality test also includes the detection of biosafety indicators including cell viability, pyrogens, pathogens, and K3EC cell residues.

In a third aspect of the present invention, there is provided an antigen-specific CD8T cell obtained by the above method for expansion culture of an antigen-specific CD8T cell.

In a fourth aspect, the invention provides a K3EC cell, wherein the K3EC cell is a K562 engineered cell that can homogeneously express CD58, MICA/B and CD 137L.

The CD137L and CD64 genes were introduced into the K3EC cells.

The invention discloses a rapid amplification platform based on MLPC-plus, which has the following characteristics:

1. bypassing the process of APC processing to present peptide epitope, antigen-specific CD8T cells in PBMC were directly activated. Mixed Lymphocyte Peptide Cultures (MLPCs) are currently a more common alternative to APC. During MLPC, peptide epitopes are embedded in the corresponding MHCI on the surface of PBMC to form pMHC, which is recognized by the specific TCR on the surface of CD8T and transduces the activation signal. This form of antigen presentation is called cross presentation (cross presentation) and is characterized by direct activation of the corresponding TCR without the need for APC to perform intracellular antigen processing. However, due to bypassing the process of processing presented peptide epitopes by APCs, MLPCs often lack sufficient costimulatory signals to activate CD8T cells, which are less proliferative and are susceptible to activation-induced cell death (AICD). In order to supplement the costimulatory signal required for the activation of CD8T cells in MLPC, the patent uses genetic engineering technology to make K562 cells express CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L, and co-culture the cells with PBMC as artificial APC (aAPC). This modified MLPC is referred to as MLPC-plus.

2. Has the ability to rapidly form a homogeneous CD8T cell population. After T cells are activated by MLPC-plus technology, target cell populations are further enriched from the proliferated cells, 108-magnitude and homogeneous CD8T cells can be obtained from 20ml of peripheral blood samples within 3 weeks, and the method has the characteristic of rapid and efficient preparation.

3. Addition of IL15 and low dose IL2 provided a 3 rd signal to promote TSCM/CM formation. Antigen activation of CD8T cells requires the provision of IL15 and IL2 No. 3 signals for efficient expansion. The cytokines activate the JAK-STAT5 signal pathway through specific receptors, but the immunological effects generated by signals of IL15 receptors and IL2 receptors are different, the combination of IL2 and the receptors promotes the proliferation and differentiation of CD8T cells into TEFF, and the combination of IL15 and the receptors mainly maintains the self-stable proliferation of TM. The difference between the effects of IL2 and IL15 was that IL2 had significantly higher stimulatory activity on the PI3K-AKT signaling pathway than IL15, where AKT (protein kinase B) is a negative regulator of CCR7 and CD 62L. The expression of Ccr7 and Cd62l coding genes of CCR7 and CD62L is regulated by Kruppel-like factor 2(KLF2, Kruppel-like factor 2), which in turn is regulated by Forkhead transcription factors (such as FOXO1 and FOXO3 a). In TN and TSCM/CM, FOXO1 and FOXO3a are located in the nucleus and induce expression of KLF 2. Upon activation of AKT, FOXO1 and FOXO3a, modified by phosphorylation, translocate from the nucleus into the cytoplasm and are degraded by proteasomes (proteosomes), so that KLF2 expression is reduced, and thus T cells with high activity of AKT do not normally express CCR7 and CD 62L. Accordingly, the present patent selects a combination of high concentration IL15(10ng/ml) and low concentration IL2(50U/ml) for culturing, so that the ratio of TSCM and TCM respectively accounts for 23.5% and 45.3% after CD8T cell expansion, and the ratio of stem cell component (stem cell component) reaches 68.8%. Similarly, IL15 and IL7 may also be added as signal No. 3.

4. The antigen specificity immune response ability is strong. CD8 TSCM/CM is characterized by being capable of secreting cytokines such as IFN and the like after being activated by corresponding epitope and playing an effector function by forming TEFF through rapid proliferation and differentiation. The IFN capture assay was used to demonstrate that the amount of IFN secretion following CMV-pp65 antigen stimulation was equal to that of polyclonal CD3 antibody stimulation (averaged to 101% as calculated for ASR). Further identified using epitope screening (epitope screening) technology, it was found that 1 or more cell clones existed in the expanded CD8T cells, and these T cell clones could recognize epitopes presented by different HLA-a and/or HLA-B sites. The identification of the antigen epitope recognized by the specific T cells and the HLA presenting site thereof is helpful for selecting the patients with diseases (such as leukemia) with the site to carry out targeted infusion therapy, thereby laying a scientific foundation for implementing precision immunotherapy.

Drawings

FIG. 1 phenotypic characterization of CD8T cell subpopulations

T cells are classified into primitive types (T)_N) Memory type (T)_M) Sum effect type (T)_EFF) Subgroup of which T_MSubdivided into stem cell types (T)_SCM) Central type (T)_CM) Tissue colonisation type (T)_RM) Sum effect type (T)_EM) And the like. The use of 10 phenotypic markers allows the identification of the above cell subsets or components.

FIG. 2 preparation of antigen-specific CD8T cells

The T cell preparation process comprises three links of induced amplification, sorting enrichment and quality control inspection, wherein the induced amplification is performed through MLPC-plus (MLPC + K3EC) and MLPC (again, this time bar with K3EC cells, no removal step was seen). Sorting enrichment Using magnetic cell sorting (MACS) CD8T cells were obtained, which expanded to 10⁸Magnitude-time detection of antigen-specific response (ASR) and stem cell component (T)_SCMAnd T_CM) Certain HLA genotypes can also be tested for TCR's using the corresponding tetramer (tetramer) as a major release criterion⁺Cell proportion.

FIG. 3. phenotypic analysis of K3EC

FCM detects CD64 (upper row) and CD137L (upper right row) introduced by lentiviral transfection on the surface of K3EC cells and controls stained with isotype control antibody (upper left row). MICA/B (lower row) and CD58 (lower right row) were also detected on the cell surface, while HLA-ABC was micro-expressed (lower left row). The transverse coordinate system introduces GFP tracer protein, the longitudinal coordinate is the fluorescence signal of the antibody to be detected, and the positive cell proportion is shown in the upper right corner of the figure.

FIG. 4 detection of human IgG antibody binding ability of CD64

The influence of humanized CD3 antibody (IgG1) on the staining signals of murine CD154 fluorescent antibody (IgG2a), CD58 fluorescent antibody (IgG2a) and CD137L fluorescent antibody (IgG1) was examined by a competitive binding assay. K3EC expressed CD64 (upper right) compared to control (upper left). In the absence of CD3 antibody, CD154 (left middle row), CD58 (middle row) and CD137L (right middle row) were all positive. When a 10-fold excess of CD3 antibody was added, the positive rate of CD154 (left lower panel) detection decreased significantly, but did not affect the CD58 (left lower panel) and CD137L (right lower panel) detection results. The transverse coordinate system introduces GFP tracer protein, the longitudinal coordinate is the fluorescence signal of the antibody to be detected, and the positive cell proportion is shown in the upper right corner of the figure.

FIG. 5 detection of T cell proliferation by CFSE dilution

The PBMC are subjected to CFSE fluorescent staining, then CD3 antibody is added as a TCR 1 st signal, then K3EC with different proportions is added to provide a co-stimulation 2 nd signal (the proportion of PBMC: K3EC is 5:1, 10:1 and 20:1 respectively), cells are collected after 2 days and 5 days of culture, wherein CD3 fluorescent antibody is added to a detection group to stain T cells, and no fluorescent antibody is added to a control group. The horizontal coordinate is CFSE signal, the vertical coordinate is CD3 antibody fluorescence signal, after T cell proliferation, CD3 detects positive but CFSE signal is weakened, and the ratio of the proliferating cells is shown in the upper left corner of the figure.

FIG. 6 is a schematic diagram of CMV structure

CMV is an icosahedral protein capsid structure that contains a double-stranded DNA viral genome. The capsid is enveloped by a spherical lipid membrane, and is expressed on the membrane surface by proteins.

FIG. 7 CMV-pp65 full protein overlapping peptide fragment library

pp65 is the most major structural protein of CMV, and is also the most abundant one of the integument proteins in viral particles. The overlapping library of peptide fragments of pp65 holoprotein contains 138 peptide fragments, each peptide fragment is 15 amino acids in length, and adjacent peptide fragments are overlapped by 11 amino acid sequences.

FIG. 8 ASR detection results

PBMCs were grouped after addition of 20% K3EC (providing co-stimulatory signal No. 2), positive control (P) with addition of CD3 antibody, experimental (E) with addition of CMV-pp65 peptide, and negative control (N) without addition of TCR No. 1 signal. Culturing for 24hr, anchoring IFN γ secreted in the supernatant to the cell surface with IFN γ capture antibody, rinsing with PBS, and detecting positive cells (including GFP) with IFN γ fluorescent antibody⁺K3EC and GFP^-PBMC). The horizontal coordinate is GFP fluorescence signal, the vertical coordinate is IFN gamma fluorescence signal, and the positive cell proportion is respectively shown in the left upper corner and the right upper corner of the figure. The Antigen Specific Response (ASR) was calculated according to the formula set forth.

FIG. 9 MLPC-plus culture of HLA-A2⁺CD86⁺Cell detection

PBMC were cultured in MLPC-plus for 7 days, then sampled, stained with HLA-A2 and CD86 fluorescent antibody, and FCM detected the above marker double positive cells (see upper right corner of the figure), which had APC function after loading peptide epitope.

FIG. 10 detection of Stem cell fraction in CD8T cells

PBMC were sampled before and after culture, stained with fluorescent antibodies CD8, CD95, CD45RA and CD62L, FCM assay first selected lymphocyte fraction (left panel) for detection of CD8⁺CD95⁺Cells (in the figure), the population of cells selected for detection of CD45RA and CD62L expression (right panel), wherein T_SCMIs CD45RA⁺CD62L⁺，T_CMIs CD45RA^-CD62L⁺. In the figure, the results of PBMC detection before culture are shown in the upper row, the results of MLPC-plus detection after culture are shown in the middle row, and the results of MACS CD8T cell enrichment are shown in the lower row.

FIG. 11 peptide fragment epitope screening of CD8T cells

MACS-enriched CD8T cells were grouped after addition of 20% K3EC (providing co-stimulatory signal No. 2), the positive control group (P) was supplemented with CD3 antibody, the experimental group (E) was supplemented with CMV-pp65 peptide library and 10 HLA site-specific peptide epitopes, respectively, and the negative control group (N) was not supplemented with TCR 1 signal. FCM detection of IFN gamma after 24hr incubation⁺Cells (same as ASR detection method).

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. The technical solution of the present invention will be described more clearly. The biochemical reagents used in the examples are all commercially available.

Example 1 Process flow for preparing antigen-specific CD8T cells of the invention (FIG. 2)

MLPC-plus activation and amplification of antigen (CMV-pp65) -specific CD8T cells

1) Collecting 20ml of peripheral blood and heparin anticoagulation

2) PBMC were collected by Ficoll gradient centrifugation and washed 3 times with PBS centrifugation

3) Resting (resting) culture in 10% FCS-RPMI overnight

4) After addition of 20% K3EC, the supernatant was centrifuged off and PBMC was adjusted to 1X 10 with 3% SG-AIM-V⁷/ml

5) Adding 1. mu.g/ml CMV-pp65 antigen peptide fragment (Miltenyi, America gentle, cat # 130-

6) Supplementing 3% SG-AIM-V to 4ml, adding 4ml 2 XCM, culturing in 6-well plate, and subculturing

7) Collecting cells after 7 days of culture, adjusting the cells to 1X 10⁷/ml

8) Adding CMV-pp65 antigen peptide 1 μ g/ml, and incubating at 37 deg.C for 2hr

9) Paraformaldehyde fixed K3EC cells

10) 3% SG-AIM-V adjusted cells to 1X 10⁶Per ml, 2 XCM diluted to 5X 10⁵Per ml, expanding the cell number to be more than or equal to 1x 10⁸(about 3 days)

Note: 3% SG-AIM-V Medium (GIBCO Co.) supplemented with 3% serum replacement (free of foreign proteins) 2 XCM 20ng/ml recombinant human IL15 and 100U/ml recombinant human IL2

2. Enrichment of CD8T cells and expansion in culture

1) Cells were collected, magnetic bead sorting (MACS) enriched for CD8T cells (U.S. Invitrogen kit)

2) 3% SG-AIM-V regulated CD8T cells to 1X 10⁶/ml

3)2 XCM dilution to 5X 10⁵Per ml, expanding the cell number to more than or equal to 1x 10⁸(about 3-5 days)

3. Quality control test

1) FCM measures CD8T cell characteristics, including T_SCM/CMOccupancy, antigen specific response capability (ASR) and TCR recognition of reactive peptides.

2) Biological safety indexes include cell survival rate, pyrogen, pathogen (such as mycoplasma, etc.) and K3EC cell residue.

EXAMPLE 2 construction of K562-aAPC (K3EC)

1. Selection of costimulatory molecules

K3EC was constructed primarily to provide a costimulatory signal for MLPC to activate antigen-specific CD8T cells. It is now known that the costimulatory signals required to activate such T cells can be provided by multiple receptors, with CD28 being the most powerful costimulatory receptor. CD28 belongs to Ig superfamily (IgSF), and its cell tail region (cytoplastic tail) contains three motifs of YMNM, TPRP and PYAP. After CD28 binds to B7 ligand (CD80/86), YMNM activates AP1 (activin 1), NF-kB (kappa B nuclear factor) and NFAT (activated T nuclear factor) via the PI3K-AKT pathway and the Grb2-Vav-Sos pathway, resulting in the expression of Bcl-xL anti-apoptotic proteins and IL2 cytokines. The PYAP motif also activates AP1, NF-kB and NFAT transcription factors through the Grb2-Vav-Sos pathway, while the TPRP motif primarily activates the tyrosine kinase Itk, which functions to phosphorylate PLC gamma lipase, causing it to hydrolyze PIP₂Forming IP3 and DAG second messengers. However, only half of the CD8T cells expressed CD in PBMC28, such CD28⁺The cell comprises T_N、T_SCMAnd T_CMAnd the like.

CD2 is CD28⁺The cell has another effective costimulatory signal. CD2 also belongs to IgSF, but unlike CD28, CD2 has the primary role of recruiting Lck kinase upon binding to CD58 ligand, causing CD3 ζ and ZAP70 to phosphorylate proximal signals transducing TCRs. In addition, CD2 activation signals can also cause STAT5 phosphorylation to promote T cell proliferation. Thus, the CD2 receptor may function as a TCR 1 st signal and a γ C cytokine receptor 3 rd signal. In the absence of B7-CD28 signaling, CD2 in concert with NKG2D provides an equivalent costimulatory signal to CD 28. Specific ligands for NKG2D include MICA/B, among others, whose signaling activates the PI3K-AKT pathway, but to a lesser extent than CD 28.

CD8T cells also express CD137(4-1BB) within 48hr after TCR signaling activation, which in combination with CD137L (4-1BBL) can affect proliferative capacity and differentiation fate. CD137 belongs to the TNFR (tumor necrosis factor receptor) family and exerts anti-apoptotic functions primarily through the TAF2 signaling pathway. In CAR T cell studies, it was found that insertion of the CD28 signaling domain in chimeric receptors (CARs) preferentially induces T formation_EMAnd after inserting CD137 signal domain, it mainly forms T_CM. Thus, providing CD137 signaling in CD8T cell culture helps to enrich for T_CM。

Based on the above analysis, co-stimulatory molecules such as CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L were selected to construct aAPC. K562 erythroleukemia cells, which are a cell line widely used for the construction of aAPC, are characterized by the absence of HLA-ABC molecules, but positive expression of CD58 and MICA/B. Thus, only transgene introduction of CD137L was required to meet the requirements. In addition, to facilitate the study of the function of K562 engineered cells, part of the transgenic experiments also introduced Fc γ RI of human IgG (i.e., CD64), which binds to CD3 antibody and provides TCR 1 st signal.

2. Gene transfection and selection of stably transfected cells

1. Construction of lentiviral expression plasmids of human CD137L and CD64, which express Green Fluorescent Protein (GFP) as a tracer to facilitate identification of transfected cells [ construct by Gkey, Shanghai Co., Ltd ]

2. Transfection of K562 cells (from cell resource center, Shanghai Life sciences institute of Chinese academy of sciences) according to the transfection kit and instructions provided by the supplier

Step-sorting MACS CD137L after successful gene transfection confirmed by FCM assay⁺And CD64⁺Cells

4.CD 137L was diluted by limiting dilution method⁺CD64⁺Cells were diluted to 100 cells/ml, cultured in 96-well plates (100. mu.l/well, 10 cells/well) and subcultured for expansion, while simultaneously selecting resistant cells by adding 10. mu.g/ml puromycin.

FCM detection of CD137L⁺CD64⁺Stable transfer of cells

Results of the experiment

The positive rates of CD137L and CD64 were 69.4% and 35.8%, respectively, as measured 4 days after gene transfection. MACS sorting enriched CD137L⁺Cells and CD64⁺Cells were then screened for stable transfectants in 96-well plates.

Cell proliferation was observed at about 1/3 in 96 wells, with FCM detecting 12 CD64⁺And CD137L⁺Wells, of which well No. 6 had the highest positive rate. The well cells were retested after passage expansion (fig. 3), showing a 74.9% and 99.4% positive rate for CD64 and CD137L, respectively. The experiment also detected HLA-ABC, CD58 and MICA/B, showing that HLA-ABC is weakly positive, while the positive rates of MICA/B and CD58 are 99.1% and 100%, respectively. The population of cells was frozen in liquid nitrogen and named K3EC, K562 engineered cells expressing 3 costimulatory molecules (K562 engineering cells with 3 co-stimulatory molecules).

Human IgG antibody binding Capacity test for CD64

CD64 was able to bind to human IgG1, IgG3 and IgG4 antibodies as Fc γ RI, but human CD64 also binds with high affinity to murine IgG2 (including IgG2a and IgG2b) antibodies. According to this principle, PE fluorescein-labeled murine IgG2a (CD154) was chosen as tracer and a competition binding assay was used to see if excess human CD3 antibody (IgG1) competed for inhibition of tracer binding to CD 64.

The experiment was divided into a control group without addition of CD3 antibody and an experiment group with addition of CD3 antibody (10. mu.g/ml), each group was divided into 4 tubes and stained with PE-labeled mouse anti-CD 64(IgG1), CD137L (IgG1), CD154(IgG2a) and CD58 (IgG2a) antibodies, respectively, in an amount of 20. mu.l/100. mu.l volume (5X 10. mu.l inclusion) of fluorescent antibody⁵K3EC), FCM detection after centrifugation and washing to remove unbound antibody.

Results of the experiment

1. The control group tests showed that the positive rates of CD58 and CD137L were 100% and 99.0%, respectively, the positive rate of CD64 was 72.1%, and the positive rate of tracer (CD154) was 43.2%, which are different from each other in that the fluorophore labeling prevented the IgG2a antibody from binding to Fc γ RI (fig. 4).

2. The results of the experimental group confirmed (fig. 4) that the preincubation with the CD3 antibody had no significant effect on the detection of CD58 and CD137L, and the positive rates were 99.9% and 98.6%, respectively. However, the binding of tracer to Fc γ RI was completely inhibited after pre-incubation with CD3 antibody, and the positive rate decreased to the level of the blank control (1.6%).

EXAMPLE 3 polyclonal T cell proliferation assay

aapcs typically require inactivation (i.e., elimination of proliferative capacity) for T cell co-culture by irradiation, mitomycin treatment, paraformaldehyde fixation, and the like. To assess whether co-stimulatory activity was still present after K3EC fixation, experiments provided polyclonal TCR signals with CD3 antibody and co-stimulatory signals with 4% paraformaldehyde-fixed K3 EC. The proliferation activity of T cells was measured by CFSE dilution, i.e., PBMC was labeled with CFSE (20. mu.M), washed, mixed and cultured with K3EC at different ratios (PBMC: K3EC ═ 5:1, 10:1 and 20:1) in a 96-well plate, and CD3 antibody controls were established, each supplemented with 1. mu.g/ml of CD3 antibody. Detection of CD3 after 2 and 5 days of culture⁺Cells and CD3⁺CFSE^LowAnd (5) proliferating the cells. CD3⁺Proliferation cell% (% CD 3)⁺CFSE^Lowcell/CD 3⁺Cell) x 100%

Results of the experiment

After 2 days of culture, it was revealed that T cell proliferation could not be detected in each group, but CD3 in the K3EC co-cultured group⁺The cell proportion (positive rate of 54.0-55.3%) is significantly higher than that of the antibody group (positive rate of 7.8%), which suggests that the CD3 antibody binding may cause the endocytosis of CD3 protein on the cell surface to reduce the expression of CD 3.

After 5 days of culture, CD3 was found in the co-cultured group⁺The proportion of cells (positive rate of 78.2-82.9%) is still higher than that of the antibody group (positive rate of 52.3%), and the CD3 of the co-culture group⁺The% proliferating cells were higher in the K3EC high proportion (PBMC: K3EC ═ 5:1 and 10:1) than in the antibody group, and lower in the low proportion. Further increases in the K3EC ratio (PBMC: K3EC ═ 2.5: 1 and 1:1) did not significantly improve T cell proliferative activity (results not shown). Therefore, the K3EC ratio was chosen to be 20%, i.e. PBMC: K3 EC-5: 1. FIG. 5 shows CD3 cultured for 5 days⁺CFSE^LowAnd (5) detecting the proliferation cells.

TABLE 1 polyclonal T cell proliferation

● CMV antigen selection

The CMV structure is an icosahedral protein capsid (icosahedral protein capsid) containing approximately 235kb of the double-stranded DNA viral genome (fig. 6). The CMV capsid is enclosed by the lipid membrane and by integrins (proteins) encoded by viral late genes.

The CMV integument proteins include pp65, pp71, pp150, pp28, etc., wherein pp65 is the most major structural protein of CMV and is also the integument protein with the highest content in virus particles. The function of pp71 is to initiate viral-mediated early gene expression leading to viral genome replication. The role of pp150 and pp28 is to promote assembly (assembly) and excretion (egresses) of viral particles. The integument proteins have strong immunogenicity, and pp65 is the most common virus antigen in clinic.

This patent uses overlapping library of pp65 full protein peptide (pepMix, each peptide length is 15 amino acids, 11 amino acid sequence overlap between adjacent peptide) to carry out MLPC. This pepMix contains 138 peptides (FIG. 7), any of which binds to MHCI on the surface of PBMC to provide the TCR 1 signal, thus providing the advantage of not requiring the selection of the HLA genotype of the donor.

● MLPC-plus culture activated antigen-specific CD8T cells

After activation of CD8T cells by antigenic peptides in MLPC-plus, cytokines including IL2 and IFN γ were first secreted, and after about 72hr, the cells began to proliferate. To assess whether MLPC in combination with K3EC specifically activated T cells, the secretion of IFN γ by T cells after 24hr culture was experimentally examined.

PBMC and K3EC were mixed at a ratio of 5:1, and 1. mu.g/ml of CMV-pp65 peptide fragment or EB virus membrane antigen peptide fragment (EBV-LMP2a, Miltenyi, Germany) was added to the experimental group (E), while a negative control group (N) and a positive control group (P) were set, the former containing no TCR activation signal and the latter containing 1. mu.g/ml functional grade CD3 antibody. After 24hr incubation, an IFN γ capture antibody (Miltenyi) was added, formed by coupling an IFN γ antibody to CD45, wherein the CD45 antibody binds to PBMC and K3EC, and the IFN γ antibody binds to IFN γ in the culture supernatant. After centrifugal washing, the mixture is stained by adopting an APC fluorescence labeled IFN gamma detection antibody, and the FCM detects positive cells%. The antigen-specific response (ASR) was calculated according to the following equation: ASR { (E-N)/(P-N) } x 100%.

Results of the experiment

ASR assays were performed on 7 PBMCs (FIG. 8), and the results showed that 2 viral antigens were able to specifically activate TCR under MLPC-plus conditions⁺T cells secrete IFN γ, with the CMV-pp65 antigen averaging 23.5% (range 91.6% -1.7%) ASR, while the EBV-LMP2a antigen averaging 7.3% (range 16.1% -0%).

TABLE 2 ASR detection of PBMC

Nd not detected

Example 4 analysis of the Effect of MLPC-plus culture on the proliferation of antigen-specific CD8T cells

Culture amplification of 4 PBMCs was performed according to the preparation process and the following technical performance index was evaluated.

Expansion Capacity of CD8T cells

The proliferation status of MLPC-plus stimulated CD8T cells was evaluated after 3 time points were selected. The results show that the average yield of PBMC before culture is 1x 10⁶Whole blood/ml with CD8T cells in the lymphocyte population at a ratio of about 1/4. 1 week after antigen stimulation, the ratio of CD8T cells reached 1/2, and the number increased 3.8 times.

Simultaneous expression of HLA-a2 and CD86 was detected in approximately 77.2% (ranging from 62.2% to 91.8%) cells in PBMCs after 1 week of culture (fig. 9), and these double positive cells were loaded with antigenic peptides and then had APC function. Accordingly, 1. mu.g of the antigenic peptide/10 of the PBMC was cultured for 1 week⁷The ratio of cells is again antigen-stimulated, and the total amount of cells reaches 1-2 x 10 after further culture for 3-5 days⁸In time, MACS sorted enriched CD8T cells, which averaged 84.4% pure (range 69% -97.1%) with CD8T cells after enrichment, in amounts up to 30.22X 10⁶. If the culture is continued for 3-5 days, the T cells can be expanded to 10⁸Magnitude.

TABLE 3 MLPC-plus culture driven antigen-specific TCR⁺T cell proliferation

Subgroup constitution of CD8T cells

The CD8T cell phenotype is detected by adopting a four-color fluorescent staining method, 2 control groups are simultaneously established, a blank control group (without adding antibodies) is used for setting positive benchmarks of CD8(PE marks) and CD95(APC marks), a CD8/CD95 control group (without adding CD45RA and CD62L antibodies) is used for setting positive benchmarks of CD45RA (PerCP marks) and CD62L (FITC marks), and then the ratio of 5 subgroups in the cells of the experimental group is detectedExample, where T_NPhenotype is CD8⁺CD95^-，T_SCMPhenotype is CD8⁺CD95⁺CD45RA⁺CD62L⁺，T_CMPhenotype is CD8⁺CD95⁺CD45RA^-CD62L⁺，T_EMPhenotype is CD8⁺CD95⁺CD45RA^-CD62L^-And T is_EMRAPhenotype is CD8⁺CD95⁺CD45RA⁺CD62L^-。

Results of the experiment

T in CD8T cells before culture_NA ratio of about T _EMRA2 times (44.4% and 21.4% respectively) of the total weight of the composition, and T_MThe ratio of the 3 subgroups decreases in turn, and is respectively 17.4 percent (T)_SCM)、9.4％(T_CM) And 7.2% (T)_EM). A major change in the subpopulation composition of CD8T cells after 1 week of culture was noted as T_NAnd T_EMRAThe ratio of the components is remarkably reduced, and T_SCM/CMThe ratio of the component reaches 90.2 percent, wherein T is used_CMMainly comprises the following steps. Detection of MACS after enrichment of CD8T cells again revealed T_SCM/CMThe ratio of the cells is 68.8 percent on average, and the dominant cells are still T_CM. FIG. 10 shows the results of 1 representative experiment.

TABLE 4 subpopulation composition of CD8T cells after MACS enrichment

3. Antigen specific response capacity

After MACS enrichment, the response capacity of CD8T cells to CMV-pp65 antigen is obviously enhanced compared with that before culture, and detection shows that ASR reaches 101.0%, which indicates that the secretion amount of IFN gamma of the T cells is equal to that of polyclonal CD3 antibody stimulation under antigen stimulation. Another feature of these homogeneous CD8T cells was that they were highly antigen specific, and when the response capacity of EBV-LMP2a was tested simultaneously, ASR was found to be only 3.3%, with no significant difference compared to pre-culture.

TABLE 5 specific response Capacity of CD8T cells to CMV-pp65 antigen after MACS enrichment

4. Reactive peptide fragment screening

The peptide epitopes in the CMV-pp65 antigen are mainly presented by HLA-A and HLA-B. In Chinese Han population, the dominant alleles in the above loci are HLA-A11: 01 and HLA-B40: 01, respectively, while the internationally favored allele of HLA-A02: 01 regresses to position 3 in Chinese Han population. Based on the data from the 17 ten thousand human oversize samples, we selected the 5 most common HLA-A and HLA-B alleles, respectively, and synthesized the corresponding CMV-pp65 peptide fragment epitopes in order to analyze the subpopulation of cells in the antigen-specific CD8T cells that can recognize these epitopes.

TABLE 6 HLA-A and HLA-B epitopes and CMV-pp65 peptide fragment epitope thereof

The screening of the peptide fragment epitope still adopts an IFN gamma detection method, and the difference is that the 10 peptide fragment epitopes are included in an experimental group besides the whole protein pepMix. Results were expressed as IFN γ⁺Cell% indicates.

Results of the experiment

The experiment was carried out in the 4 cases mentioned above for HLA-A2 ⁺1 of the CD8T cells was randomly selected and tested, and the results in FIG. 11 show that there were 2 subsets of T cells, of which 1 subset recognized the NLV peptide fragment presented by HLA-A02: 01 and the other 1 subset recognized the ATV peptide fragment presented by HLA-A11: 01, but IFN γ after ATV stimulation⁺The cells were only 40% of NLV.

Example 5 safety assessment of CMV-pp 65-specific CD8T cells

The clinical research of CMV-pp65 specific T cell treatment is developed by matching with the Hematology department of affiliated people hospital of Beijing university, and a set of safety evaluation indexes of T cell preparations is established through the research, including (1) the survival rate detection of T cells; (2) detecting a heating source; (3) mycoplasma detection and (4) bacteria (aerobes and anaerobes) detection, which prove that when the detection indexes meet the release standard (release criterion), the infusion of T cells is safe, and no obvious adverse reaction is observed.

The safety index detection is also carried out on 4 cases of CD8T cell culture, and the results all meet the release standard.

TABLE 7 safety assessment of CMV-pp 65-specific CD8T cells

Unlike previous studies, the culture of CD8T cells prepared using the MLPC-plus platform was supplemented with K562 engineered cells K3EC, which have potential carcinogenic risks and thus need to be rigorously detected. First, the proliferation potency of K3EC after fixation with paraformaldehyde was evaluated. Experiment immobilized K3EC was placed in 24-well culture plates (2X 10)⁶/well) were cultured continuously for 7 days, and cell counts of 6 wells were collected on days 1, 3, 5 and 7 of culture, respectively. The results showed that K3EC survived for 7 days after 30min of fixation with 4% paraformaldehyde, but no increase in cell number was detected during this period, indicating that the cells had completely lost their proliferative capacity after fixation with paraformaldehyde, and that these cells were not tumorigenic even if remaining. Subsequently, experiments evaluated whether K3EC remained in CD8T cells after MACS sorting. The topic group of the Beller medical college of America also adopts K562 engineering cells to culture and amplify NK cells, and proves that when the residual quantity of the K562 engineering cells in the NK cell culture is less than or equal to 0.2 percent, the infusion of the NK cells is safe. Accordingly, the release standard is that the residual K3EC content is less than or equal to 0.2%. Fruit of Chinese wolfberryThe test takes a transgenically introduced Green Fluorescent Protein (GFP) tracer as a detection marker, and the results prove that the K3EC cell ratios in the culture of CD8T cells are all lower than the lower limit of detection (FCM analysis 5x 10)⁴No positive cells could be detected in the cells). It was thus revealed that not only did K3EC lose proliferative capacity after paraformaldehyde fixation, but also the residual amount in T cell cultures reached the release criteria after sorting and expansion of CD8T cells.

K3EC paraformaldehyde fixing step:

adding 10ml PBS into K3EC, centrifuging for 5min at 400g, and washing to remove supernatant; adding 2ml of 4% paraformaldehyde and 6ml of PBS, and keeping the temperature at room temperature for 30 min; centrifuging at 400g for 5min, and washing to remove supernatant; adding 10ml PBS for resuspension, centrifuging for 5min at 400g, and washing supernatant; adding 10ml SG-AIM-V culture medium, and storing at 4 deg.C.

Or the method can be adopted to inactivate the K3EC cells, and then the supernatant is washed away by multiple times to obtain CD8T cells, so that the residual quantity of the K3EC cells reaches the specified standard, but the fixing effect of the paraformaldehyde is better; in addition, after K3EC cells are fixed, the residual K3EC cells naturally reach the release standard after the cells are sorted and amplified.

The CD8T cell of the peripheral blood mononuclear cell cultured and amplified according to the method has higher clinical application value in preventing and treating CMV infection and CMV related cancer. The CMV infection comprises a leukemia patient following hematopoietic stem cell transplantation, and the CMV-associated cancer comprises a glioblastoma multiforme.

The K3EC cell provided by the invention is a K562 engineering cell, can uniformly express CD58, MICA/B and CD137L, can simultaneously provide co-stimulatory receptor (co-stimulatory receptor) signals such as CD2, NKG2D and CD137, and has the advantages that:

(1) when MHC molecules (such as HLA-A and HLA-B) with antigen peptide fragments embedded on the surface of PBMC form TCR 1 signal, K562 engineering cells can induce activation of CD8T cells by providing the 2 nd signal of the costimulatory receptor. Since the antigen peptide fragment is a whole protein overlapping peptide fragment library of a target antigen (namely CMV-pp65), the addition of K562 engineering cells can induce the activation of specific CD8T cells as long as one of the peptide fragments is combined with MHCI on the cell surface.

(2) Because of the lack of CD28 ligand (such as CD80 and CD86) on the surface of K562 engineering cells, CD8T cells are less likely to be terminally undifferentiated after activation, so that the proportion of stem cell components is obviously increased after culture expansion.

The invention relates to an amplification culture method, which adopts MLPC-plus platform technology to realize the rapid and efficient amplification of antigen (CMV-pp65) specific CD8T cells without preparing DC as antigen presenting cells; meanwhile, the stem cell component (namely T) in the CD8T cell can be greatly increased by optimally configuring the 2 nd signal of a costimulatory receptor required by the proliferation of the CD8T cell (namely T cell)_SCMAnd T_CM) The proportion, and cytokine receptor 3 rd signal, significantly increased the stem cell component (i.e., T) in expanded CD8T cells_SCMAnd T_CM) In proportion, the antigen-specific CD8T cell is obtained, and has clinical application value in preventing and treating CMV infection and CMV-related cancer.

The above embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that all such similar changes and modifications can be made without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> Shanghai Aao Biotechnology Ltd

Chongqing United Stem Cell Technology Co.,Ltd.

Beijing victory Biotechnology Co.,Ltd.

Guangzhou victory Biotechnology Co.,Ltd.

Xuzhou cell Medical Co.,Ltd.

<120> culture amplification method of CD8T cells and K3EC cells

<130> 180524

<160> 10

<170> PatentIn version 3.3

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Claims (9)

1. A method for expanding and culturing CD8T cells, which is characterized in that the CD8T cells are induced to be expanded and cultured in the presence of the following substances:

(1) k3EC cells; the K3EC cell is a K562 engineered cell expressing CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L, and is used for providing a2 nd signal of a costimulatory receptor for activating CD8T cells; co-culturing K3EC cells with peripheral blood mononuclear cells; the ratio of the peripheral blood mononuclear cells to the K3EC cells in the co-culture process is between 20:1 and 5: 1;

(2) peptide fragment of cytomegalovirus-pp 65 protein of America whirlpool, cat # 130-093-435, for providing TCR 1 signal for activating CD8T cells;

(3) supplementing cytokines as the 3 rd signal for CD8T cell proliferation when expanding cultured antigen-specific CD8T cells; the cytokines are IL15 and IL2 or IL15 and IL 7.

2. The method for expanding and culturing CD8T cells according to claim 1, further comprising the steps of fixing K3EC cells with paraformaldehyde; the enriched homogenous CD8T cells were then sorted and expanded in culture to the desired number of CD8T cells.

3. An expansion culture method of CD8T cells, which comprises the following steps:

(1) collecting peripheral blood, and performing centrifugal separation to obtain peripheral blood mononuclear cells;

(2) co-culturing K3EC cells with peripheral blood mononuclear cells; the K3EC cell is a K562 engineered cell expressing CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L, and is used for providing a2 nd signal of a costimulatory receptor for activating CD8T cells;

(3) activating the TCR 1 signal of the CD8T cell by adopting the peptide segment of the cytomegalovirus-pp 65 protein which is gentle in America and has the accession number of 130-;

(4) supplementing cytokines as a 3 rd signal for proliferation of CD8T cells when the T cells are cultured by the steps (2) and (3); the cytokines are IL15 and IL2 or IL15 and IL 7;

(5) fixing K3EC cells by using paraformaldehyde, and then carrying out tissue sorting to enrich homogeneous CD8T cells;

(6) culture expanded homogeneous CD8T cells to the required number.

4. The method for expanding and culturing the CD8T cells, according to claim 3, wherein the ratio of the peripheral blood mononuclear cells to the K3EC cells in the co-culture is between 20:1 and 5: 1.

5. The method for expanding and culturing the CD8T cells, according to claim 3, wherein the ratio of the peripheral blood mononuclear cells to the K3EC cells in the co-culture is 5: 1.

6. The method for expanding and culturing the CD8T cells according to any one of claims 3-5, wherein the method further comprises:

(7) performing quality inspection on the homogeneous CD8T cells obtained in the step (6).

7. The method for expanding and culturing the CD8T cells according to claim 6, wherein the quality test is:

detecting said CD8T cell characteristic comprising detecting cell component T_SCM/CMReactive peptide fragments for antigen specific response ability and TCR recognition.

8. The method for expanding and culturing the CD8T cells according to claim 6, wherein the quality test further comprises detecting biosafety indicators comprising cell viability, pyrogen, pathogen and K3EC cell residual.

9. The method for expanding and culturing the CD8T cells according to claim 7, wherein the quality test further comprises detecting biosafety indicators comprising cell viability, pyrogen, pathogen, and K3EC cell residual.

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