CN110713977A - A method for culture expansion of CD8 T cells and K3EC 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 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; 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

A method for culture expansion of CD8 T cells and K3EC cells

technical field

The invention belongs to the technical field of biomedicine, and particularly relates to a method for culturing and expanding T cells.

Background technique

CD8 T cells, also known as cytotoxic T lymphocytes (CTL), are an important line of defense for the adaptive immune system to construct antigen-specific immune protection mechanisms. CD8 T cells specifically recognize antigens through T cell receptors (TCRs), which are expressed in the form of immunogenic peptide epitopes embedded in MHC-I molecules (pMHCI for short) on the cell surface.

Using modern cell engineering techniques to enrich and expand antigen-specific CD8 T cells from bodily fluids (such as blood) or tissues (such as tumor lesions) on a large scale, and then infuse them into patients in need of treatment, this The treatment option is called adoptive T cell therapy or Tcell therapy. Clinical data reveal that this T cell therapy can control refractory viral infections (such as CMV and EB virus) and malignant tumors (such as melanoma, etc.), and achieve significant clinical efficacy. The use of genetic engineering technology to construct TCR expression vectors to prepare genetically modified epitope-specific TCR-T cells is currently under clinical trials. This novel T cell therapy is known as a frontier field leading the development of tumor therapy. attracted the attention of the oncology medical community.

Whether it is primary TCR ⁺ T cells in the enrichment or the construction of genetically engineered TCR-T cells, an efficient expansion platform is required to obtain sufficient T cells for clinical treatment. This platform mainly provides two technical supports, one is to realize large-scale culture and expansion of T cells in accordance with GMP standards; the other is to prevent T cells from undifferentiated and become effector T cells (T _EFF ) while rapidly proliferating. ). Among these two technical supports, the control of cell differentiation is more important, because T cells are implanted into the body for long-term survival after infusion and efficiently expand and differentiate to form _TEFF with specific killing function when they encounter corresponding antigens. 2 prerequisites for clinical efficacy.

T_N)，记忆细胞(memory，T_M)和效应细胞(T_EFF) 等亚群，其中T_M又分为干细胞型(stem cell，T_SCM)、中央型(central，T_CM)和效应型(effector， T_EM)等组份。这些CD8 T细胞亚群及组份具有不同的表型标志，其中T_SCM和T_CM同时表达趋化因子受体CCR7、黏附分子CD62L、共刺激受体(costimulatory receptor)CD27和CD28、 IL7受体α链CD127以及凋亡受体CD95，区别在于T_SCM表达CD45RA而T_CM表达CD45RO (图1)。CD8 T cells are usually divided into naive cells (

T _N ), memory cells (memory, T _M ) and effector cells (T _EFF ) and other subgroups, among which T _M is further divided into stem cell (stem cell, T _SCM ), central (central, T _CM ) and effector cells (effector, _TEM ) and other components. These CD8 T cell subsets and components have different phenotypic markers, wherein T _SCM and T _CM simultaneously express chemokine receptor CCR7, adhesion molecule CD62L, costimulatory receptors CD27 and CD28, IL7 receptors Alpha chain CD127 and apoptosis receptor CD95, differing in that T _SCM expresses CD45RA and T _CM expresses CD45RO (Figure 1).

_TNs are T cells that are released from the thymus and have not encountered antigen. When an antigen stimulates an immune response, _TN differentiates into T _SCM , T _CM , T _EM and T _EFF in turn after proliferation (T _EFF expressing CD45RA is also called T _EMRA ). When the antigen is cleared, _TEFF rapidly disappears from the body by apoptosis, while _TM is preserved, in which _TSCM and _TCM settle in lymphoid hematopoietic organs such as bone marrow, and _TEM distributes in peripheral tissues such as blood circulation. Usually T _SCM and T _CM can survive in the body for decades or even life. These _TM subpopulations have stem cell-like properties (such as self-renewal, etc.), and can be rapidly activated when they encounter the corresponding antigen again and cause a stronger degree of protection. Sexual immune response. Therefore, antigen-specific TCR ⁺ CD8 T cells can be competent for therapeutic function only if they maintain the phenotype and biological characteristics of T _SCM and T _CM after culture and expansion. Indeed, the proportion of the above-mentioned _TM components is closely related to the clinical efficacy after T cell infusion, and at the level of gene expression, CD27 and CD127 expression are important markers for long-term survival of T cells after infusion.

Three signals are required for antigen-induced activation and proliferation of CD8 _TN , namely the first signal of TCR, the second signal of costimulatory receptor and the third signal of cytokine receptor. These signals require antigen-presenting cells (antigen-presenting cells, APC) such as dendritic cells (dendritic cells, DC) to provide, of which CD28 and IL2 receptors are the most functional second and third signals, but the The main role is to induce _TN to proliferate and differentiate to form _TEFF . After the antigen is cleared, the survival of antigen-specific _TM in vivo is a dynamic balance between proliferation and apoptosis, and its proliferation is finely regulated by the mechanism of homeostatic proliferation. This homeostatic proliferation does not require antigen-specific TCR signaling, and cytokine receptor 3 signaling plays a key role.

A major technical hurdle in the large-scale production of antigen-specific CD8 T cells from body fluids or tissues is obtaining sufficient numbers of APCs, especially DCs, as typically obtained from 1 x 10 ⁸ peripheral blood mononuclear cells (PBMCs) (equivalent to 100 ml of blood). Only less than 10 ⁷ DCs can be obtained, so to prepare a T cell therapy preparation (number of cells ≥ 10 ⁸ ), three or more blood collections (or 300–600 ml of blood at one time) are required to meet the needs of DC preparation. Obviously, such a large amount of blood collection is clinically difficult and difficult for patients to accept. Therefore, finding an appropriate alternative to APC (DC) to efficiently expand antigen-specific T cells is a research topic that is generally concerned internationally.

Therefore, there is an urgent need in the art to develop new and more effective methods for expanding and culturing antigen-specific CD8 T cells.

SUMMARY OF THE INVENTION

The present invention provides a method for expanding and culturing antigen-specific CD8 T cells, as well as a K3EC cell.

The first invention of the present invention provides a method for expanding and culturing antigen-specific CD8 T cells, that is, in the presence of the following substances, inducing, expanding and culturing antigen-specific CD8 T cells:

(1) K3EC cells; the K3EC cells are K562 engineered cells expressing CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L, which are used to provide the second signal of the costimulatory receptor for activating CD8 T cells;

(2) The peptide overlapping library of target antigens is used to provide the first signal of TCR to activate CD8 T cells.

The above target antigen is cytomegalovirus-pp65 protein.

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

Specifically, the co-culture ratio of the PBMC and K3EC cells is between 20:1 and 5:1.

Preferably, when the antigen-specific CD8 T cells are expanded and cultured, cytokines are supplemented as the third signal for the proliferation of CD8 T cells.

The aforementioned cytokines are IL15 and IL2 or IL15 and IL7. The aforementioned cytokines are IL15 and IL2.

The above method further includes the following steps: using paraformaldehyde to fix the K3EC cells; then sorting and enriching the homogeneous CD8 T cells, and then culturing and expanding the CD8 T cells to the required number.

The second invention of the present invention provides a method for expanding and culturing antigen-specific CD8 T cells, which comprises the steps of:

(1) Collect peripheral blood and centrifuge to obtain peripheral blood mononuclear cells;

(2) Co-culturing K3EC cells with peripheral blood mononuclear cells; the K3EC cells are K562 engineered cells expressing CD2 ligand CD58, NKG2D ligand MICA/B, and CD137 ligand CD137L, which are used to provide the ability to activate CD8 T cells. Costimulatory receptor 2 signaling;

(3) step (2) at the same time of co-cultivation, using the whole protein peptide overlapping library of cytomegalovirus-pp65 protein to activate the first signal of TCR of CD8 T cells;

(4) After the T cells are expanded and cultured through steps (2) and (3), cytokines are supplemented as the third signal for CD8 T cell proliferation;

(5) Using paraformaldehyde to fix K3EC cells, and then tissue sorting to enrich homogeneous CD8 T cells;

(6) Culture and expand homogeneous CD8 T cells to the required number.

The aforementioned cytokines are IL15 and IL2 or IL15 and IL7.

The ratio of PBMC and K3EC cells co-cultured above was between 20:1 and 5:1.

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

The above-mentioned cultivation method also comprises the following steps:

(7) Perform quality inspection on the homogeneous CD8 T cells obtained in step (6).

The above quality inspections are:

The characteristics of the CD8 T cells were detected, including the detection of cellular components (TSCM/CM), antigen-specific responsiveness (TSR) and reactive peptides recognized by TCR.

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

The third aspect of the present invention provides antigen-specific CD8 T cells obtained by the above-mentioned method for expanding and culturing antigen-specific CD8 T cells.

The fourth aspect of the present invention provides a K3EC cell, characterized in that the K3EC cell is a K562 engineered cell, which can homogeneously express CD58, MICA/B and CD137L.

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

The patent of the present invention introduces a rapid amplification platform based on MLPC-plus, which has the following characteristics:

1. Bypassing the link of APC processing and presenting peptide epitopes and directly activating antigen-specific CD8 T cells in PBMC. Mixed lymphocyte peptide cultures (MLPC) is a commonly used alternative to APC. During the MLPC process, the peptide epitopes are embedded in the corresponding MHCI on the surface of PBMC to form pMHC, which can be recognized by the specific TCR on the surface of CD8 T and transduce activation signals. This method of antigen presentation is called cross-presentation, which is characterized by direct activation of the corresponding TCR without the need for intracellular antigen processing by APC. However, due to bypassing the process of APC's processing of presenting peptide epitopes, MLPC usually lacks sufficient co-stimulatory signals to activate CD8 T cells, and such T cells have weak proliferation and are prone to activation-induced cell death (activation- induced celldeath, AICD). In order to supplement the costimulatory signal required for CD8 T cell activation in MLPC, this patent uses genetic engineering technology to make K562 cells express CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L, as artificial APC (artificial APC, aAPCs) were co-cultured with PBMCs. This modified MLPC is called MLPC-plus.

2. Has the ability to rapidly form a homogeneous CD8 T cell population. After T cells are activated by MLPC-plus technology, the target cell population is further enriched from proliferating cells, and 108-order, homogeneous CD8 T cells can be obtained from a 20ml peripheral blood sample within 3 weeks. feature.

3. The addition of IL15 and low-dose IL2 provides the third signal to promote the formation of TSCM/CM. After antigen activation of CD8 T cells, IL15 and IL2 3rd signals are required for efficient expansion. The above cytokines play their roles by activating the JAK-STAT5 signaling pathway through specific receptors, but the immunological effects of IL15 receptor and IL2 receptor signaling are different. The binding of IL2 to its receptor promotes the proliferation and differentiation of CD8 T cells into TEFF. The binding of IL15 to its receptor is mainly to maintain the self-stable proliferation of TM. The difference between IL2 and IL15 is that the stimulatory activity of IL2 on the PI3K-AKT signaling pathway is significantly higher than that of IL15, of which AKT (protein kinase B) is a negative regulator of CCR7 and CD62L. The expression of Ccr7 and Cd62l encoding 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 FOXO3a). In TN and TSCM/CM, FOXO1 and FOXO3a localize in the nucleus and induce KLF2 expression. When AKT is activated, FOXO1 and FOXO3a are phosphorylated and then translocated from the nucleus to the cytoplasm and degraded by the proteasome, so the expression of KLF2 decreases, so T cells with high AKT activity usually do not express CCR7 and CD62L. Accordingly, this patent selects a combination of high-concentration IL15 (10ng/ml) and low-concentration IL2 (50U/ml) for culture, so that after CD8 T cell expansion, TSCM and TCM account for 23.5% and 45.3%, respectively. The above stem cell components (stem cell compartment) accounted for 68.8%. Similarly, IL15 and IL7 can also be added as the third signal.

4. Antigen-specific immune response ability. CD8 TSCM/CM is characterized by the ability to secrete cytokines such as IFN after the activation of the corresponding antigenic epitope, and to exert effector function through rapid proliferation and differentiation to form TEFF. Detected by IFN capture assay, it was proved that the amount of IFN secretion stimulated by CMV-pp65 antigen was equal to that stimulated by polyclonal CD3 antibody (average 101% calculated by ASR). Further identification by epitope screening (epitope screening) technology, it was found that there were one or more cell clones in the expanded CD8 T cells, and these T cell clones could recognize different HLA-A and/or HLA-B sites. presented antigenic epitopes. Identifying the antigenic epitopes recognized by these specific T cells and their HLA presentation sites will help to select patients with diseases (such as leukemia) with the above sites for targeted infusion therapy, so as to implement precision immunotherapy (precision immunotherapy). immunotherapy) to lay the scientific foundation.

Description of drawings

Figure 1. Phenotypic characterization of CD8 T cell subsets

T cells are divided into naive ( _TN ), memory ( _TM ), and effector ( _TEFF ) subgroups, of which _TM is further subdivided into stem cell ( _TSCM ), central ( _TCM ), tissue Sedentary (T _RM ) and effector ( _TEM ) components. The above cell subsets or components can be identified using 10 phenotypic markers.

Figure 2. Preparation process of antigen-specific CD8 T cells

The T cell preparation process is divided into three steps: induction and expansion, sorting and enrichment, and quality control inspection. The induction and expansion are carried out through MLPC-plus (MLPC+K3EC) and MLPC (there are still K3EC cells at this time, I don’t see any removal step) is implemented. Magnetic cell sorting (MACS) was used for sorting and enrichment to obtain CD8 T cells. When the latter expanded to the order of 10 ⁸ , the antigen-specific response (ASR) and the proportion of stem cell components ( _TSCM and _TCM ) were detected as the main According to the release criteria, some HLA genotypes can also use the corresponding tetramer to detect the proportion of TCR ⁺ cells.

Figure 3. K3EC phenotypic analysis

CD64 (top row, middle) and CD137L (top row, right) introduced by lentiviral transfection on the surface of K3EC cells were detected by FCM, and stained with isotype control antibody (top row left) as a control. MICA/B (bottom row middle) and CD58 (bottom row right) were also detected on the cell surface, while HLA-ABC was slightly expressed (bottom row left). The abscissa is the introduced GFP tracer protein, the ordinate is the fluorescence signal of the antibody to be tested, and the proportion of positive cells is shown in the upper right corner of the figure.

Figure 4. Detection of human IgG antibody binding ability of CD64

The effect of humanized CD3 antibody (IgG1) on the staining signals of mouse CD154 fluorescent antibody (IgG2a), CD58 fluorescent antibody (IgG2a) and CD137L fluorescent antibody (IgG1) was detected by competitive binding assay. K3EC expressed CD64 (upper row right) compared to control group (upper row left). In the absence of CD3 antibodies, CD154 (middle row left), CD58 (middle row middle) and CD137L (middle row right) all tested positive. When a 10-fold excess of CD3 antibody was added, the positive rate of CD154 (bottom row left) detection was significantly reduced, but the detection results of CD58 (bottom row middle) and CD137L (bottom row right) were not affected. The abscissa is the introduced GFP tracer protein, the ordinate is the fluorescence signal of the antibody to be tested, and the proportion of positive cells is shown in the upper right corner of the figure.

Figure 5. CFSE dilution assay to detect T cell proliferation

PBMCs were stained with CFSE and then added with CD3 antibody as the first signal of TCR, and then added with different ratios of K3EC to provide the second signal of co-stimulation (PBMC:K3EC ratios were 5:1, 10:1 and 20:1), cultured for 2 days and Cells were collected at 5 days, and CD3 fluorescent antibody was added to stain T cells in the test group, and no fluorescent antibody was added in the control group. The abscissa is the CFSE signal, and the ordinate is the CD3 antibody fluorescence signal. After T cell proliferation, CD3 is positive but the CFSE signal is weakened. The proportion of such proliferating cells is shown in the upper left corner.

Figure 6. Schematic diagram of CMV structure

CMV has an icosahedral protein capsid structure and contains a double-stranded DNA viral genome. The capsid is surrounded by a spherical lipid membrane, and the body is expressed on the surface of the membrane.

Figure 7. CMV-pp65 whole protein overlapping peptide library

pp65 is the most important structural protein of CMV, and it is also the most abundant integument protein in virus particles. The peptide overlapping library of pp65 whole protein contains 138 peptides, each peptide is 15 amino acids in length, and 11 amino acid sequences overlap between adjacent peptides.

Figure 8. ASR detection results

After adding 20% K3EC to the PBMC (providing the second signal of costimulation), the groups were divided into groups. The positive control group (P) was added with CD3 antibody, the experimental group (E) was added with CMV-pp65 peptide, and the negative control group (N) was not added with TCR No. 1 Signal. After culturing for 24 hrs, the IFNγ secreted in the supernatant was anchored to the cell surface with IFNγ capture antibody, and positive cells (including GFP ⁺ K3EC and GFP ^- PBMC) were detected with IFNγ fluorescent antibody after washing with PBS. The abscissa is the GFP fluorescence signal, the ordinate is the IFNγ fluorescence signal, and the proportion of positive cells is shown in the left and upper right corners, respectively. Antigen-specific response (ASR) was calculated according to the formula listed.

Figure 9. Detection of HLA-A2 ⁺ CD86 ⁺ cells in MLPC-plus culture

PBMCs were sampled after 7 days of MLPC-plus culture, and stained with HLA-A2 and CD86 fluorescent antibodies respectively. FCM detected double-positive cells with the above markers (see the upper right corner of the figure), and these cells have APC function after loading peptide epitopes.

Figure 10. Detection of stem cell components in CD8 T cells

PBMC were sampled before and after culture, and stained with CD8, CD95, CD45RA and CD62L fluorescent antibodies. For FCM detection, the lymphocyte fraction (the left of the figure) was firstly selected to detect CD8 ⁺ CD95 ⁺ cells (in the figure), and then CD45RA and CD62L were detected after selecting this group of cells Expression (right of the figure), where _TSCM is CD45RA ⁺ CD62L ⁺ , and T _CM is CD45RA ^- CD62L ⁺ . The upper row in the figure is the detection result of PBMC before culture, the middle row is the detection result after MLPC-plus culture, and the lower row is the detection result after MACS enrichment of CD8 T cells.

Figure 11. Peptide epitope screening of CD8 T cells

MACS-enriched CD8 T cells were added with 20% K3EC (providing the second signal of co-stimulation) and grouped into groups. CD3 antibody was added to the positive control group (P), and the CMV-pp65 peptide library and 10 HLA were added to the experimental group (E), respectively. Site-specific peptide epitopes, the negative control group (N) does not add the first signal of TCR. IFNγ ⁺ cells were detected by FCM after 24hr of culture (same as ASR detection method).

Detailed ways

The technical solutions of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. The technical solutions of the present invention will be described more clearly. The biochemical reagents used in the examples are all commercially available.

Example 1 The production process of the antigen-specific CD8 T cells of the present invention (Fig. 2)

1. MLPC-plus activates and expands antigen (CMV-pp65) specific CD8 T cells

1) Collect 20ml of peripheral blood, heparin anticoagulation

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

3) Resting overnight in 10% FCS-RPMI

4) After adding 20% K3EC, centrifuge and discard the supernatant, adjust PBMC to 1x 10 ⁷ /ml with 3% SG-AIM-V

5) Add 1 μg/ml CMV-pp65 antigenic peptide (Miltenyi, Miltenyi, Cat. No. 130-093-435), incubate at 37°C for 2hr

6) Supplement 3% SG-AIM-V to 4ml, add 4ml 2x CM, culture and passage in 6-well plate

7) Collect cells after 7 days of culture, adjust cells to 1x 10 ⁷ /ml

8) Add 1 μg/ml CMV-pp65 antigenic peptide and incubate at 37°C for 2hr

9) Fix K3EC cells with paraformaldehyde

10) Adjust cells to 1x 10 ⁶ /ml with 3% SG-AIM-V, dilute to 5x 10 ⁵ /ml with 2x CM, expand the number of cells to ≥ 1x 10 ⁸ (about 3 days)

Note: 3% SG-AIM-V line AIM-V medium (GIBCO company) is supplemented with 3% serum replacement (without xenogeneic protein) 2x CM is 20ng/ml recombinant in 3% SG-AIM-V Human IL15 and 100U/ml recombinant human IL2

2. Enrichment of CD8 T cells and culture expansion

1) Collect cells and enrich CD8 T cells by magnetic bead sorting (MACS) (Invitrogen kit in the United States)

2) 3% SG-AIM-V to adjust CD8 T cells to 1x 10 ⁶ /ml

3) Dilute 2x CM to 5x 10 ⁵ /ml, expand the number of cells to ≥1x 10 ⁸ (about 3-5 days)

3. Quality control inspection

1) FCM detects the characteristics of CD8 T cells, including T _SCM/CM ratio, antigen-specific responsiveness (ASR) and reactive peptides recognized by TCR.

2) Biosafety indicators, including cell viability, pyrogens, pathogens (such as mycoplasma, etc.) and K3EC cell residues.

Example 2 Construction of K562-aAPC (K3EC)

1. Basis for the selection of costimulatory molecules

The main purpose of constructing K3EC is to provide a costimulatory signal for MLPC to activate antigen-specific CD8 T cells. It is currently known that the costimulatory signals required to activate such T cells can be provided by multiple receptors, of which CD28 is the most functional costimulatory receptor. CD28 belongs to the Ig superfamily (IgSF), and its cytoplasmic tail contains three motifs: YMNM, TPRRP and PYAP. When CD28 binds to B7 ligand (CD80/86), YMNM activates AP1 (activating protein 1), NF-kB (κB nuclear factor) and NFAT (activating T cell nuclear factor) through PI3K-AKT pathway and Grb2-Vav-Sos pathway ) resulted in Bcl-xL anti-apoptotic protein and IL2 cytokine expression. The PYAP motif can also activate AP1, NF-kB and NFAT transcription factors through the Grb2-Vav-Sos pathway, while the TPRRP motif mainly activates the tyrosine kinase Itk, whose function is to phosphorylate PLCγ lipase to hydrolyze PIP ₂ form IP3 and DAG second messenger. However, only half of the CD8 T cells in PBMC express CD28, and such CD28 ⁺ cells include _TN , _TSCM , and _TCM , among others.

CD2 is another potent costimulatory signal for CD28 ⁺ cells. CD2 also belongs to IgSF, but different from CD28, the main function of CD2 is to recruit Lck kinase after binding with CD58 ligand, causing CD3ζ and ZAP70 phosphorylation to transduce the proximal signal of TCR. In addition, CD2 activation signal can also cause STAT5 phosphorylation to promote T cell proliferation. Therefore, the CD2 receptor can function as the first signal of TCR and the third signal of γC cytokine receptor. In the absence of B7-CD28 signaling, CD2 cooperates with NKG2D to provide a co-stimulatory signal equivalent to CD28. The specific ligands of NKG2D include MICA/B, etc., and its signal transduction is to activate the PI3K-AKT pathway, but the degree is lower than that of CD28.

CD8 T cells also expressed CD137(4-1BB) within 48 hours after activation by TCR signaling, and the combination of the latter and CD137L(4-1BBL) could affect the proliferation ability and differentiation fate. CD137 belongs to the TNFR (tumor necrosis factor receptor) family and mainly exerts its anti-apoptotic function through the TAF2 signaling pathway. In CAR T-cell studies, it was found that insertion of the CD28 signaling domain in a chimeric receptor (CAR) preferentially induced the formation of _TEM , whereas insertion of the CD137 signaling domain mainly formed _TCM . Therefore, providing CD137 signal when CD8 T cells are cultured helps to enrich _TCM .

Based on the above analysis, costimulatory molecules such as CD2 ligand CD58, NKG2D ligand MICA/B and CD137 ligand CD137L were selected to construct aAPC. K562 erythroleukemia cells are a widely used cell line for the construction of aAPC, which are characterized by not expressing HLA-ABC molecules, but positively expressing CD58 and MICA/B. Therefore, only the transgene needs to be introduced into CD137L to meet the demand. In addition, in order to facilitate the study of the function of K562 engineered cells, some transgenic experiments also introduced FcγRI (ie CD64) of human IgG, which can provide the first signal of TCR in combination with CD3 antibody.

2. Gene Transfection and Screening of Stably Transfected Cells

1. Construct human CD137L and CD64 lentiviral expression plasmids, which express green fluorescent protein (GFP) as a tracer to facilitate the identification of transfected cells [entrusted by Shanghai Jikai Company]

2. Transfect K562 cells according to the transfection kit and operating instructions provided by the supplier (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences)

3. After the successful gene transfection was confirmed by FCM detection, the CD137L ⁺ and CD64 ⁺ cells were sorted by MACS step by step.

4. The CD137L ⁺ CD64 ⁺ cells were diluted to 100 cells/ml by limiting dilution method, cultured in a 96-well plate (100 μl/well, 10 cells/well) and passaged for expansion, and 10 μg/ml puromycin was added to screen for resistant cells .

5. FCM detection of CD137L ⁺ CD64 ⁺ stable cells

实验结果

Experimental results

4 days after gene transfection, the positive rates of CD137L and CD64 were 69.4% and 35.8%, respectively. After MACS sorting enriched CD137L ⁺ cells and CD64 ⁺ cells, stably transfected cells were screened in 96-well plates.

Cell proliferation was observed in about 1/3 of the 96 wells, and FCM detected 12 CD64 ⁺ and CD137L ⁺ wells, of which well 6 had the highest positive rate. The cells in this well were passaged and expanded and tested again (Fig. 3), showing that the positive rates of CD64 and CD137L were 74.9% and 99.4%, respectively. The experiments also detected HLA-ABC, CD58 and MICA/B, showing that HLA-ABC was weakly positive, while the positive rates of MICA/B and CD58 were 99.1% and 100%, respectively. After cryopreservation in liquid nitrogen, this group of cells was named K3EC, namely K562 engineering cells with 3 co-stimulatory molecules.

3. Human IgG antibody binding ability test for CD64

As FcγRI, CD64 can bind to human IgG1, IgG3 and IgG4 antibodies, but human CD64 can also bind to murine IgG2 (including IgG2a and IgG2b) antibodies with high affinity. According to this principle, PE fluorescein-labeled mouse IgG2a (CD154) was selected as the tracer, and a competitive binding assay was used to observe whether excess human CD3 antibody (IgG1) could competitively inhibit the binding of the tracer to CD64.

The experiment was divided into the control group without the addition of CD3 antibody and the experimental group with the addition of CD3 antibody (10 μg/ml). For staining with IgG2a) and CD58 (IgG2a) antibodies, the amount of fluorescent antibody was 20 μl/100 μl (containing 5×10 ⁵ K3EC), and the non-binding antibodies were removed by centrifugation and then detected by FCM.

实验结果

Experimental results

1. The detection of the control group 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%. The difference between the two may be the fluorophore labeling. Binding of IgG2a antibody to FcγRI was blocked (Figure 4).

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

Example 3 Polyclonal T cell proliferation assay

aAPC usually needs to be inactivated (ie, the ability to proliferate) before being used for T cell co-culture. This inactivation method includes irradiation, mitomycin treatment, and paraformaldehyde fixation. In order to evaluate whether K3EC still has costimulatory activity after fixation, CD3 antibody was used to provide polyclonal TCR signal, and 4% paraformaldehyde-fixed K3EC provided costimulatory signal. The proliferation activity of T cells was detected by the CFSE dilution method, that is, PBMCs were labeled with CFSE (20 μM). At the same time, a CD3 antibody control group was set up in the plate, and 1 μg/ml CD3 antibody was added to each group. CD3 ⁺ cells and CD3 ⁺ CFSE ^Low proliferating cells were detected after 2 days and 5 days of culture. % CD3 ⁺ proliferating cells = (CD3 ⁺ CFSE ^Low cells/CD3 ⁺ cells) x 100%

实验结果

Experimental results

After 2 days of culture, T cell proliferation could not be detected in each group, but the proportion of CD3 ⁺ cells in the K3EC co-culture group (positive rate 54.0%-55.3%) was significantly higher than that in the antibody group (positive rate 7.8%), suggesting that CD3 antibody Binding may lead to cell surface CD3 protein endocytosis resulting in decreased CD3 expression.

After 5 days of culture, it was found that the proportion of CD3 ⁺ cells in the co-culture group (the positive rate was 78.2%-82.9%) was still higher than that in the antibody group (the positive rate was 52.3%), and the percentage of CD3 ⁺ proliferating cells in the co-culture group was higher in K3EC. When the ratio was high (PBMC:K3EC=5:1 and 10:1), it was higher than the antibody group, while the low ratio was lower than the antibody group. Further increasing the K3EC ratio (PBMC:K3EC=2.5:1 and 1:1) did not significantly improve T cell proliferation activity (results not shown). Therefore, the K3EC ratio was chosen to be 20%, ie PBMC:K3EC=5:1. Figure 5 shows the detection results of CD3 ⁺ CFSE ^Low proliferating cells cultured for 5 days.

Table 1 Polyclonal T cell proliferation

●CMV antigen selection

The CMV structure is an icosahedral protein capsid containing a double-stranded DNA viral genome of about 235 kb (Figure 6). The CMV capsid is wrapped by lipid membrane and tegument, and the tegument is encoded by the viral late gene.

CMV integument proteins include pp65, pp71, pp150 and pp28, among which pp65 is the most important structural protein of CMV and the most abundant integument protein in virus particles. The function of pp71 is to initiate viral-immediate earlygene expression and cause viral genome replication. The role of pp150 and pp28 is to promote the assembly and egress of viral particles. The above integument proteins all have strong immunogenicity, among which pp65 is the most commonly used viral antigen in clinic.

This patent uses the peptide overlapping library of pp65 whole protein (pepMix, each peptide is 15 amino acids in length, and 11 amino acid sequences overlap between adjacent peptides) for MLPC. The pepMix contains 138 peptides (Figure 7), any of which can provide the first signal of TCR by binding to MHCI on the surface of PBMC, so it has the advantage of not needing to select the HLA genotype of the donor.

MLPC-plus culture activates antigen-specific CD8 T cells

In MLPC-plus, CD8 T cells were activated by antigenic peptides and firstly secreted cytokines including IL2 and IFNγ, and the cells started to proliferate after about 72 hours. In order to evaluate whether the combination of MLPC and K3EC can specifically activate T cells, the experiment detected the IFNγ secreted by T cells after 24 hours of culture.

PBMC and K3EC were mixed at a ratio of 5:1, and 1 μg/ml CMV-pp65 peptide or Epstein-Barr virus membrane antigen peptide (EBV-LMP2a, Miltenyi, Germany) was added to the experimental group (E), and a negative control group (N ) and positive control group (P), the former did not add TCR activation signal, and the latter added 1 μg/ml functional grade CD3 antibody. After culturing for 24 hours, IFNγ capture antibody (Miltenyi Company) was added, which was formed by coupling IFNγ antibody and CD45 antibody, wherein CD45 antibody was combined with PBMC and K3EC, and IFNγ antibody was combined with IFNγ in the culture supernatant. After centrifugation and washing, the cells were stained with APC fluorescently labeled IFNγ detection antibody, and the percentage of positive cells was detected by FCM. Antigen-specific response (ASR) was calculated according to the following formula: ASR={(E-N)/(P-N)} x 100%.

实验结果

Experimental results

ASR assay was performed on 7 PBMCs (Figure 8), and the results showed that both viral antigens could specifically activate TCR ⁺ T cells to secrete IFNγ under MLPC-plus conditions, and the average ASR of CMV-pp65 antigen was 23.5% (range). 91.6%-1.7%), while the ASR for the EBV-LMP2a antigen averaged 7.3% (range 16.1%-0%).

Table 2 ASR detection of PBMC

*nd not detected

Example 4 Analysis of the technical effect of MLPC-plus culture to promote the expansion of antigen-specific CD8 T cells

According to the preparation process, 4 cases of PBMC were cultured and expanded and the following technical performance indicators were evaluated.

1. The expansion capacity of CD8 T cells

Three time points were selected to evaluate the proliferation status of CD8 T cells stimulated by MLPC-plus. The results showed that the average yield of PBMC before culture was 1×10 ⁶ /ml of whole blood, and CD8 T cells accounted for about 1/4 of the lymphocyte population. One week after antigen stimulation, the proportion of CD8 T cells reached 1/2, and the number increased by 3.8 times.

After 1 week of culture, about 77.2% (range 62.2%-91.8%) of PBMC cells detected the simultaneous expression of HLA-A2 and CD86 (Fig. 9), these double positive cells will have APC function after loading with antigen peptides. According to this, PBMCs were cultured for 1 week after re-antigen stimulation at the ratio of 1 μg antigen peptide/10 ⁷ cells, and continued to culture for 3–5 days when the total number of cells reached 1–2x 10 ⁸ , MACS sorting enriched CD8 T cells , the purity of CD8 T cells after enrichment was 84.4% on average (range 69%-97.1%), and the number reached 30.22 x 10 ⁶ . Such T cells can be expanded to the order of 10 ⁸ if cultured for 3–5 days.

Table 3 MLPC-plus culture promotes the proliferation of antigen-specific TCR ⁺ T cells

2. Subpopulation composition of CD8 T cells

Four-color fluorescent staining was used to detect the phenotype of CD8 T cells, and two control groups were set up at the same time. The blank control group (without antibody) was used to set the positive benchmark for CD8 (PE marker) and CD95 (APC marker), and the CD8/CD95 control The group (without CD45RA and CD62L antibodies) was used to set the positive benchmark for CD45RA (PerCP marker) and CD62L (FITC marker), and then the proportion of 5 subpopulations in the experimental group cells was detected, of which the _TN phenotype was CD8 ⁺ CD95 ^- , T _SCM phenotype is CD8 ⁺ CD95 ⁺ CD45RA ⁺ CD62L ⁺ , T _CM phenotype is CD8 ⁺ CD95 ⁺ CD45RA ^- CD62L ⁺ , T _EM phenotype is CD8 ⁺ CD95 ⁺ CD45RA ^- CD62L ^- , while T _EMRA phenotype is CD8 ⁺ CD95 ⁺ CD45RA ⁺ CD62L ⁻ .

实验结果

Experimental results

The proportion of _TN in CD8 T cells before culture was about twice that of _TEMRA (44.4% and 21.4%, respectively), while the proportions of the three subpopulations of _TM decreased sequentially, 17.4% ( _TSCM ), 9.4% (T _CM ) and 7.2% (T _EM ). After 1 week of culture, the subpopulation composition of CD8 T cells changed significantly, and the proportion of _TN and _TEMRA decreased significantly, while the proportion of T _SCM/CM reached 90.2%, of which T _CM was the main component. After MACS enriched CD8 T cells, it was detected again, showing that the proportion of T _SCM/CM reached an average of 68.8%, and the dominant cell was still T _CM . Figure 10 shows the detection results of a representative experiment.

Table 4 Subpopulation composition of CD8 T cells after MACS enrichment

3. Antigen-specific responsiveness

The ability of CD8 T cells to respond to CMV-pp65 antigen after MACS enrichment was significantly enhanced compared with that before culture, and the detection showed that the ASR reached 101.0%, indicating that the amount of IFNγ secreted by such T cells under antigen stimulation was equal to that of polyclonal CD3 antibody stimulation. Another characteristic of these homogeneous CD8 T cells is their high antigen specificity, when EBV-LMP2a responsiveness was tested concurrently, the ASR was found to be only 3.3%, which was not significantly different from pre-culture.

Table 5 The specific response ability of CD8 T cells to CMV-pp65 antigen after MACS enrichment

4. Screening of reactive peptides

The peptide epitopes in CMV-pp65 antigen are mainly presented by HLA-A and HLA-B. In the Chinese Han population, the dominant alleles in the above loci are HLA-A*11:01 and HLA-B*40:01 respectively, while the internationally favored HLA-A*02:01 allele Gene, in my country's Han population, is relegated to the third place. According to the detection data of 170,000 large samples of this project, we selected the 5 most common HLA-A and HLA-B alleles respectively, and synthesized the corresponding CMV-pp65 peptide epitopes, the purpose is to analyze the antigen Cell subsets that recognize these epitopes in specific CD8 T cells.

Table 6 HLA-A and HLA-B epitopes and their CMV-pp65 peptide epitopes

Peptide epitope screening still uses IFNγ detection method, the difference is that in addition to the whole protein pepMix, the above 10 peptide epitopes are also included in the experimental group. Results are expressed as % of IFNγ ⁺ cells.

实验结果

Experimental results

In the experiment, 1 case was randomly selected among the above 4 cases of HLA-A2 ⁺ CD8 T cells for detection. The results in Figure 11 show that there are 2 subsets of T cells in this case, and 1 subset recognizes HLA-A*02:01. NLV peptides presented by HLA-A*11:01 were recognized by another subset, but ATV-stimulated IFNγ ⁺ cells were only 40% of NLV.

Example 5 Safety assessment of CMV-pp65-specific CD8 T cells

We once cooperated with the Department of Hematology, Peking University Affiliated People's Hospital to carry out a clinical study of CMV-pp65-specific T cell therapy. Through this study, we established a set of safety evaluation indicators for T cell preparations, including (1) the viability of T cells. (2) Pyrogen detection; (3) Mycoplasma detection and (4) Bacterial (aerobic and anaerobic) detection, when it is proved that the above detection indicators meet the release criteria, the infusion of T cells is safe , no obvious adverse reactions were observed.

The above-mentioned safety indicators were also tested for 4 cases of CD8 T cell culture, and the results all met the release criteria.

Table 7 Safety assessment of CMV-pp65-specific CD8 T cells

Different from previous studies, the K562 engineered cell K3EC was added to the CD8 T cell culture prepared by the MLPC-plus platform, which has a potential carcinogenic risk, so it needs to be strictly tested. First, the proliferative capacity of K3ECs after paraformaldehyde fixation was assessed. Experiment The fixed K3ECs were placed in a 24-well culture plate (2×10 ⁶ /well) for 7 consecutive days, and 6-well cells were counted on the 1st, 3rd, 5th and 7th days of culture, respectively. The results showed that K3EC could survive for 7 days after being fixed with 4% paraformaldehyde for 30 minutes, but no increase in the number of cells was detected during the period, indicating that the cells had completely lost their ability to proliferate after being fixed with paraformaldehyde. tumorigenic. Subsequently, experiments assessed whether K3EC remained in CD8 T cells after MACS sorting. The research group of Baylor College of Medicine in the United States also used K562 engineered cells to expand NK cells, and proved that when the residual amount of K562 engineered cells in the NK cell culture is ≤0.2%, the infusion of such NK cells is safe. Accordingly, we also take K3EC residue ≤0.2% as the release standard. In the experiment, the transgenic green fluorescent protein (GFP) tracer was used as the detection marker. The results showed that the proportion of K3EC cells in the CD8 T cell culture was lower than the detection limit (no positive cells were detected in 5× ^{10 4} cells by FCM analysis). It was revealed that K3EC not only lost the proliferation ability after being fixed with paraformaldehyde, but also the residual amount in T cell culture after CD8 T cell sorting and expansion had reached the release standard.

K3EC paraformaldehyde fixation steps:

Add 10ml PBS to K3EC, centrifuge at 400g for 5min, wash and remove supernatant; add 2ml 4% paraformaldehyde, add 6ml PBS, room temperature for 30min; centrifuge at 400g for 5min, wash and remove supernatant; add 10ml PBS to resuspend, centrifuge at 400g for 5min, wash away Supernatant; add 10ml SG-AIM-V medium and store at 4°C.

The method can also be used to inactivate K3EC cells, and then wash and remove the supernatant for several times to obtain CD8 T cells, so that the residual amount of K3EC cells reaches the specified standard, but the paraformaldehyde fixation effect is better; The cells are sorted and expanded, and the residual amount of K3EC cells naturally meets the release standards.

The CD8 T cells of peripheral blood mononuclear cells cultured and expanded according to the above method have high clinical application value in the prevention and treatment of CMV infection and CMV-related cancer. The CMV infection includes leukemia patients after hematopoietic stem cell transplantation, and CMV-related cancers include glioblastoma multitype.

The K3EC cell provided by the present invention is a K562 engineered cell, which can homogeneously express CD58, MICA/B and CD137L, and can simultaneously provide co-stimulatory receptor (co-stimulatory receptor) signals such as CD2, NKG2D and CD137, etc. Its advantages are:

(1) When antigenic peptides are embedded in MHCI molecules (such as HLA-A and HLA-B) on the surface of PBMCs to form the first signal of TCR, K562 engineered cells can induce activation of CD8 T cells by providing the second signal of the above-mentioned costimulatory receptors . Since the antigenic peptides use a full-protein overlapping peptide library of the target antigen (ie, CMV-pp65), as long as one of the peptides binds to the MCI on the cell surface, the addition of K562 engineered cells can induce activation of specific CD8 T cells.

(2) Due to the lack of CD28 ligands (such as CD80 and CD86) on the surface of K562 engineered cells, CD8 T cells are less likely to be undifferentiated after activation, resulting in a significant increase in the proportion of stem cells after culture expansion.

The present invention is a kind of expansion culture method, using MLPC-plus platform technology can realize the rapid and efficient expansion of antigen (CMV-pp65) specific CD8 T cells without preparing DC as antigen presenting cells; meanwhile, by optimizing the configuration of CD8 Costimulatory receptor 2 signaling required for T cell proliferation can significantly increase the proportion of stem cell components (ie, T _SCM and T _CM ) in CD8 T cells, and cytokine receptor 3 signaling can significantly increase post-expansion The proportion of stem cell components (ie, T _SCM and T _CM ) in CD8 T cells can obtain antigen-specific CD8 T cells, which has clinical application value in the prevention and treatment of CMV infection and CMV-related cancer.

The above embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that, without departing from the spirit and scope of the present invention, all similar substitutions and modifications should be included within the protection scope of the present invention.

SEQUENCE LISTING

<110> Shanghai Saiao Biotechnology Co., Ltd.

Chongqing Guolian Stem Cell Technology Co., Ltd.

Beijing Saiao Biotechnology Co., Ltd.

Guangzhou Saiao Biotechnology Co., Ltd.

Xuzhou Cell Medical Co., Ltd.

<120> A method for culture expansion of CD8 T cells and K3EC cells

<130> 180524

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<170> PatentIn version 3.3

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Claims (19)

1. A method for culturing CD8T cells in an amplification manner, which is characterized in that the CD8T cells are cultured in an induction and amplification manner 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;

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

2. The method for expanding and culturing the CD8T cells of claim 1, wherein the target antigen is cytomegalovirus-pp 65 protein.

3. The method for expanding and culturing the CD8T cells according to claim 2, wherein the method comprises co-culturing the K3EC cells with peripheral blood mononuclear cells.

4. The method for expanding and culturing the CD8T cells according to claim 3, wherein the ratio of the PBMC cultured with the K3EC cells is between 20:1 and 5: 1.

5. The method for expanding and culturing CD8T cells according to any one of claims 1 to 4, wherein the antigen-specific CD8T cells are expanded and cultured by supplementing cytokines as the 3 rd signal for CD8T cell proliferation.

6. The method for expanding and culturing CD8T cells according to claim 5, wherein the cytokines are IL15 and IL2 or IL15 and IL 7.

7. The method for expanding and culturing the CD8T cells according to any one of claims 1 to 4, further comprising the steps of fixing the K3EC cells with paraformaldehyde; the cells were then sorted to enrich for homogenous CD8T cells and cultured to expand CD8T cells to the desired number.

8.A method for expanding and culturing CD8T cells, which is characterized by comprising 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 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 CD8T cells when the T cells are cultured by the amplification in 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.

9. The method for expanding and culturing CD8T cells according to claim 8, wherein the cytokines are IL15 and IL2 or IL15 and IL 7.

10. The method for expanding and culturing the CD8T cells according to claim 8, wherein the ratio of the PBMC to the K3EC cells in the co-culture is between 20:1 and 5: 1.

11. The method for expanding and culturing CD8T cells according to claim 9, wherein the ratio of PBMC to K3EC cells in coculture is between 20:1 and 5: 1.

12. The method for expanding and culturing CD8T cells according to claim 9, wherein the ratio of PBMC to K3EC cells in coculture is 5: 1.

13. The method for expanding and culturing the CD8T cells according to any one of claims 7 to 12, further comprising: (7) performing quality inspection on the homogeneous CD8T cells obtained in the step (6).

14. The method for expanding and culturing the CD8T cells according to claim 13, wherein the quality test is:

detecting said CD8T cell characteristic, including detecting a cellular component (T)_SCM/CM) Reactive peptide fragments for antigen specific response ability and TCR recognition.

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

16. The method for expanding and culturing the CD8T cells according to claim 14, wherein the quality test further comprises detecting biosafety indicators including cell viability, pyrogen, pathogen and K3EC cell residual.

17. CD8T cells obtained by the method for the expanded culture of CD8T cells according to any one of claims 1 to 16.

18. A K3EC cell, wherein the K3EC cell is a K562 engineering cell, and can homogenously express CD58, MICA/B and CD 137L.

19. The K3EC cell of claim 18, wherein the K3EC cell has CD137L and CD64 genes introduced therein.

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