CN116286660B

CN116286660B - IPSC- (CAR) natural killer cells, preparation method and application thereof in tumor treatment

Info

Publication number: CN116286660B
Application number: CN202211569386.9A
Authority: CN
Inventors: 原征; 李久续; 山口桥一郎
Original assignee: Global Cell Holdings Guangzhou Co ltd
Current assignee: Global Cell Holdings Guangzhou Co ltd
Priority date: 2022-12-08
Filing date: 2022-12-08
Publication date: 2023-10-03
Anticipated expiration: 2042-12-08
Also published as: CN116286660A

Abstract

The application provides an IPSC- (CAR) natural killer cell, a preparation method and application thereof in tumor treatment, wherein the IPSC-derived natural killer cell can ensure stable and reliable cell source, overcome the immunodeficiency of autologous cells and ensure the anti-tumor effect; the single domain variable region with medium affinity targeting CD19 is taken as an antigen binding domain in the CAR structure, the NKG2D transmembrane region and DAP10 costimulatory factor which are suitable for NK cells are introduced, and meanwhile, the CD28 costimulatory factor is added, so that the activation degree of cells is improved, cancer cells can be effectively killed in vitro and in vivo, the survival period of animals is prolonged, and the secretion of immune factors is regulated.

Description

IPSC- (CAR) natural killer cells, preparation method and application thereof in tumor treatment

Technical field:

the application belongs to the field of anti-tumor drug research, and particularly provides an IPSC- (CAR) natural killer cell, a preparation method and application thereof in tumor treatment.

The background technology is as follows:

tumor immunotherapy has completely changed the knowledge of cancer treatment, and has become the fourth largest means of cancer treatment following surgery, radiotherapy and chemotherapy, and some metastatic cancer patients who have been generally considered incurable in the past can be relieved or even cured for a long period of time by means of immunotherapy. Among the numerous immunotherapies, chimeric antigen receptor (chimeric antigen receptor, CAR) immune cells are of greatest interest, in which the CAR structure is first designed and constructed in vivo, then the gene encoding the CAR is introduced into the immune cells by genetic engineering means, and the genetically modified immune cells are considered to be "live agents" because of their ability to self-replicate and self-renew when they are returned to the patient.

CART cells were the earliest studied and most widely used CAR immune cells, and five CAR T cells have been FDA approved to date, namely Kymriah, yescarta, tecartus, breyanzi and abegma, all of which select for target B cell surface markers, four of which target CD19 and the other of which targets B cell maturation antigen BCMA. Currently, the most successful CAR immune cells are targeted to CD19 CAR-T cells, and cure up to 90% of patients with advanced refractory Acute Lymphoblastic Leukemia (ALL) (see Fleischer LC et al targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions. J Hematol Oncol.2019;12 (1): 141).

Natural killer cells (natural killer cell, NK cells) are innate immune effector cells that are able to rapidly recognize and kill abnormal cells, virus-infected cells, and tumor cells. T cells recognize MHC antigens on antigen presenting cells, and after receiving a proper danger signal, the T cells can kill tumor cells expressing MHC class I, however, the tumor cells usually show reduced or no expression of MHC class I molecules, and the killing effect of the T cells is avoided; unlike T cells, NK cells are lymphocytes lacking antigen-specific receptors, and express neural cell adhesion molecules in large amounts, without relying on MHC-related pathways, and can prevent tumor immune escape. Furthermore, in sharp contrast to T lymphocytes, NK cells do not induce graft versus host disease, but rather exert a regulatory effect in most cases, researchers have found in preclinical studies that CAR-NK cells have similar in vivo activity as CAR-T cells in xenograft mouse models, interestingly, the CAR-NK group shows less cytokine release and better overall survival, showing lower toxic side effects. Therefore, NK cells are considered to be more suitable as ideal materials for CAR immune cells than T cells.

Currently, nearly hundred clinical and preclinical studies on CAR natural killer cells (CAR-NK) have been carried out worldwide, and their antitumor targets include a number of antigens such as CD19, CD20, CD33, epCAM, GPC3, HER2, mesothelin, epidermal growth factor receptor vIII, and the like, and indications also relate to a number of hematological and solid tumors such as leukemia, lymphoma, myeloma, liver cancer, lung cancer, melanoma, colorectal cancer, breast cancer, and the like.

However, research and application of CAR-NK cells also face many challenges, on one hand, the efficacy of CAR-NK cells remains to be enhanced, and some patients have symptoms relieved after receiving CAR-NK cell therapy, but relapse occurs to different degrees after a period of time, and the efficacy is still not stable; on the other hand, the source of NK cells is limited, if the autologous NK cells of a patient are used, the autologous NK cells not only need to undergo complex processes such as extraction, culture, modification, reinfusion and the like, so that the infection risk is increased, the preparation cost is increased, but also the antitumor capability of the autologous NK cells is weakened due to the limitation of tumor microenvironment and the destruction of an immune system in the patient, and the therapeutic effect is poor.

In order to overcome the above difficulties, the present application provides a CAR-natural killer cell and a method for preparing the same, which takes induced pluripotent stem cells (induced pluripotent stem cells, IPSC) as a source to induce and prepare NK cells, thereby solving the drawbacks of autologous NK cells of patients; meanwhile, a novel CAR structure is constructed, so that the anti-tumor activity of the modified natural killer cells is improved, and a novel approach is provided for later clinical application.

Disclosure of Invention

To solve the above technical problem, a first aspect of the present application provides an IPSC-CAR natural killer cell, the CAR structure comprising a signal peptide, a targeting CD19 antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory factor and an intracellular signal domain connected in sequence; the amino acid sequence of the targeted CD19 antigen binding domain is shown as SEQ ID NO. 1, the transmembrane region is an NKG2D transmembrane region, and the amino acid sequence of the targeted CD19 antigen binding domain is shown as SEQ ID NO. 2; the costimulatory factors are DAP10 with the amino acid sequence shown in SEQ ID NO. 3 and CD28 with the amino acid sequence shown in SEQ ID NO. 4.

Further, the amino acid sequence of the CAR structure is shown as SEQ ID NO. 5.

CAR consists essentially of three domains: extracellular domain, transmembrane region and cytoplasmAn activation region. The extracellular domain comprises a single-chain variable fragment (scFv), which is usually derived from an antibody and can provide the capability of specifically recognizing tumor antigens expressed on cancer cells, CD19 which is the most mature in clinical application is taken as a target antigen, a single-domain antibody domain which is specifically combined with the CD19 is screened to obtain the scFv, the domain only comprises a heavy chain structure, the structure is simple and easy to prepare, the scFv has medium affinity with the target antigen, and the KD value of the scFv is 1.38E-8mol.L ^-1 Can prevent adverse reactions such as immune factor storm caused by excessive affinity.

The transmembrane domain anchors the CAR structure to the effector cell membrane, and once the CAR recognizes and is triggered by its specific antigen, the intracellular activation domain of the CAR signals, resulting in a downstream process that promotes killing of the target cell. The common transmembrane region comprises CD8 alpha, CD28, CD3 zeta transmembrane region and the like, but the transmembrane region is more used in CART cells, and in order to obtain a CAR structure suitable for natural killer cells, the application selects the transmembrane region in natural killer cell natural molecule NKG2D to construct a chimeric antigen receptor structure so as to approximate the physiological state of the natural killer cells and strengthen biological signal transmission.

The cytoplasmic activation domain activates NK cells after target antigen recognition, and currently, the chimeric antigen receptor is usually activated by combining CD3 zeta and costimulatory factors. Of the many co-stimulators, 4-1BB and CD28 are most commonly used, and CD28 exhibits a faster effector profile compared to that of CD28 and causes a greater change in lymphocyte-specific protein tyrosine kinase phosphorylation in the signaling pathway, CD28 also induces higher levels of interferon-gamma (IFN-gamma), granzyme B, tumor necrosis factor alpha (tumor necrosis factor alpha, TNF-alpha); in addition, studies have shown that activation of NK cells requires the involvement of the DAP10 complex to effectively initiate NK cell mediated cytotoxicity, and thus DAP10 and CD28 were selected as dual stimulators in the present application (see Lanier LL.Up on the lightning: natural killer cell activation and inhibitor. Nat immunol.2008;9 (5): 495-502.) in order to increase the degree of activation of CAR-NK cells.

Further, the natural killer cells are induced by IPSC cells.

IPSC cells are used as a source of natural killer cells, so that sufficient and reliable immune cells can be ensured to be obtained, the treatment needs are supplied, the influence of individual differences is reduced as much as possible, the controllability and accessibility of the cell preparation process are improved, and the treatment cost is reduced; meanwhile, when the autologous natural killer cells of tumor patients are used, the damage and limitation of the body tumor microenvironment to the immune system can be avoided, and the tumor treatment effect can be ensured and improved.

In a second aspect, the application provides a preparation method of the IPSC-CAR natural killer cell, which is characterized by comprising the steps of preparing the IPSC natural killer cell; preparing a viral vector carrying a nucleotide sequence encoding said CAR structure; transfecting the IPSC natural killer cells with the viral vector to obtain the IPSC-CAR natural killer cells.

Further, the step of preparing the IPSC natural killer cells comprises culturing the IPSC cells until the cell fusion degree reaches more than 80%; final concentrations of 50ng/mL SCF (stem cell factor), 20ng/mL VEGF (vascular endothelial growth factor) and 20ng/mL BMP4 (bone morphogenic protein 4) were added; after induction for about 2 weeks, cells were harvested by centrifugation, and cultured for 2-3 weeks with fresh medium, and final concentrations of 10ng/mL IL-2, 10ng/mL IL-6, 15ng/mL IL-15, 20ng/mL IL-7, 20ng/mL SCF, and 20ng/mL flt3 ligand, to obtain IPSC-natural killer cells.

Further, the viral vector is an adenovirus vector, and the preparation steps include: introducing the nucleotide encoding the CAR into an adenovirus skeleton vector Ad5F35 through enzyme digestion and ligation, and then transfecting the skeleton vector into HEK 293 cells; 37 ℃ and 5% CO ₂ Culturing for 14 days under saturated humidity, centrifuging to collect cells, repeatedly freezing and thawing at-80deg.C and 37deg.C, centrifuging to collect supernatant; the cell supernatant was purified by density gradient centrifugation and then filtered using a 0.22 μm filter to obtain the adenovirus vector.

In a third aspect, the application provides an application of the IPSC-CAR natural killer cells in preparing antitumor drugs.

The CAR of the present application, targeting CD19, has demonstrated expression of this target on a variety of tumors, and researchers have used CD 19-targeted immune cells in clinical studies to treat a variety of liquid and solid tumors. Thus, further, the tumor is at least one selected from leukemia, non-hodgkin lymphoma, multiple myeloma, lung cancer, liver cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, glioma, esophageal cancer, cholangiocarcinoma, endometrial cancer, ovarian cancer, mesothelioma.

Further, the tumor is selected from the group consisting of leukemia selected from acute myeloid leukemia

Advantageous effects

The application provides an IPSC-CAR natural killer cell, a preparation method and application thereof in tumor treatment, and the preparation method has the following advantages:

(1) The single domain antigen binding domain of the target CD19 has a brand new amino acid structure, can be combined with a target antigen with moderate affinity, prevents severe immune factor storm, can effectively identify tumor cells and plays a specific anti-tumor role;

(2) Elements suitable for natural killer cells are introduced into the CAR structure, and comprise an NKG2D transmembrane region and a DAP10 costimulatory factor;

(3) The CAR structure uses DAP10 and CD28 dual-stimulation factors, so that the activation degree of cells can be improved, and the anti-tumor effect can be improved;

(4) The natural killer cells from IPSC can ensure stable and reliable cell sources, overcome the immunodeficiency of autologous cells and ensure the anti-tumor effect.

Drawings

Fig. 1: schematic representation of chimeric antigen receptor structure;

fig. 2: chimeric antigen receptor nucleotide sequence structure;

fig. 3: inhibiting tumor cell growth in vitro;

fig. 4: animal survival curves;

fig. 5: serum IFN-gamma expression levels;

fig. 6: serum TNF- α expression levels;

fig. 7: serum IL-6 expression levels.

Detailed Description

Example 1 design and preparation of chimeric antigen receptor

As shown in fig. 1, the CAR structure of the present application comprises a signal peptide, a targeting CD19 antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory factor and an intracellular signaling domain, connected in sequence; the targeted CD19 antigen binding domain is of a single domain antibody structure, is obtained by immunizing a alpaca with a human CD19 antigen and then carrying out antibody screening, and has a KD value of 1.38E-8 mol.L with a target antigen ^-1 The antigen binding domain has an amino acid sequence shown in a single-domain variable region of SEQ ID NO. 1, and is in a medium affinity range, can be effectively combined with a target antigen and can prevent immune factor storm caused by over immunity. The transmembrane region is an NKG2D transmembrane region, and the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 2. The co-stimulatory factors are DAP10 with an amino acid sequence shown as SEQ ID NO. 3 and CD28 with an amino acid sequence shown as SEQ ID NO. 4, the CAR structure of the application simultaneously comprises the CD28 stimulatory factors with strong immune activation capability and the DAP10 stimulatory factors with NK cell specificity, can strengthen the activation degree of NK cells from different aspects and play a strong anti-tumor role.

The remaining CAR elements select for known domains, specifically as follows:

the amino acid sequence of the signal peptide is: MALPVTALLLPLALLLHAARP;

the amino acid sequence of the hinge region is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACD;

the amino acid sequence of the intracellular signaling domain is: RVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR.

The amino acid sequence of the CAR structure is shown as SEQ ID NO. 5, and the corresponding nucleotide sequence is obtained by a gene synthesis mode, as shown in figure 2.

Example 2 CAR-immune cell preparation

2.1 preparation of immune cells

The IPSC cells used in the present application were obtained earlier by the present inventors by transducing Oct-4, sox2, nanog and Lin28 genes into somatic cells using lentiviral vectors, and then deposited in the laboratory.

The preparation steps of the IPSC-natural killer cell comprise: IPSC cells were inoculated in a 10% FBS-containing alpha-MEM medium at 37℃with 5% CO ₂ Culturing under saturated humidity until cell fusion degree reaches above 80%, adding SCF (stem cell factor) with final concentration of 50ng/mL, VEGF (vascular endothelial growth factor) with final concentration of 20ng/mL, and BMP4 (bone morphogenetic protein 4) with final concentration of 20 ng/mL; after induction culture for about 2 weeks, cells were digested with 0.1% pancreatin, collected by centrifugation, and cell concentration was adjusted with fresh medium to obtain 1X 10 cells ⁵ The individual cells were seeded in 6-well plates, added with IL-2, IL-6, IL-15, IL-7, SCF, and flt3 ligand at final concentrations of 10ng/mL, 15ng/mL, 20ng/mL, 20ng/mL, and flt3 ligand, and cultured for 2-3 weeks, followed by detection by flow cytometry to obtain IPSC-NK cells.

The T cell preparation steps include: peripheral blood of healthy adult was taken, mononuclear cells were isolated using Ficoll lymphocyte separation liquid, and then T cells were isolated using EasySepTM human T Cell screening kit (purchased from Stem Cell company, usa).

2.2. Construction of recombinant adenovirus vectors

Amplifying the chimeric antigen receptor nucleic acid sequence by PCR, and introducing enzyme cutting sites at two ends of the sequence; the nucleotide encoding the CAR was ligated into pcdna3.1 vector by double cleavage and T4 ligase ligation, introduced into DH5 a competent cells by electrotransformation, added LB medium, cultured overnight at 37 ℃ at 250rpm, and plasmids were extracted using plasmid extraction kit (purchased from tencel).

Introducing the nucleotide encoding the CAR into an adenovirus skeleton vector Ad5F35 through enzyme digestion and ligation, and then transfecting the skeleton vector into HEK 293 cells; 37 ℃ and 5% CO ₂ Culturing under saturated humidity for 14 days, collecting cells at 3000rpm, adding adenovirus frozen stock solution, suspending, precipitating, repeatedly freezing and thawing the suspension four times at-80deg.C and 37deg.C, and centrifuging at 12000g20min, and collecting supernatant. The cell supernatant was purified by density gradient centrifugation, then filtered using a 0.22 μm filter, and the impurities were removed and stored in a-20℃refrigerator with addition of a low-temperature preservative solution.

2.3 CAR-immune cell preparation

Immune cells (IPSC-NK cells and T cells) were inoculated into a petri dish, followed by adding an adenovirus vector carrying the CAR gene at a ratio of 1:10, 37℃and 5% CO ₂ Culturing for 48h under saturated humidity, digesting the cells with 0.1% pancreatin, centrifuging to collect the cells, and obtaining CAR-immune cells, which are respectively marked as IPSC-CARNK cells and CART cells.

Example 3 CAR-immune cells inhibit tumor cell growth in vitro

It was reported that CD 19-targeted chimeric antigen receptor immune cells have killing effect on various hematological tumors including leukemia, lymphoma, myeloma, etc., and human Acute Myeloid Leukemia (AML) cell line HL-60 was selected as a target cell in this example, and the in vitro antitumor activity of CAR-immune cells was studied.

Culturing HL-60 to logarithmic phase, centrifuging at 3000rpm to collect cells, regulating cell concentration with fresh culture medium, inoculating into 96-well plate with 1×10 per well ⁴ Respectively adding the IPSC-CARNK cells and CART cells provided by the application according to the ratio of immune cells to tumor cells of 5:1, uniformly mixing, taking an equal volume of fresh culture medium as a blank control, and adding 5% CO at 37 DEG C ₂ Culturing for 48h under saturated humidity condition. After the co-culture is finished, 10 mu L of CCK8 reagent is added into each hole, after 4 hours of incubation, OD450 value of each hole is measured at the wavelength of 450nm of an enzyme-linked immunosorbent assay, average value of each hole is taken, and the cell inhibition activity corresponding to the absorbance value of the normal control group is 0%, wherein the cell inhibition activity (%) = (OD normal control group-OD dosing culture group)/OD normal control group is multiplied by 100%.

As shown in fig. 3, the CAR immune cells provided in the present application can significantly inhibit HL-60 tumor cell growth, wherein IPSC-calnk exhibits strong tumor inhibition, the cell inhibition activity is more than 90%, and CART is only 60% acting, which indicates that the chimeric antigen receptor designed in the present application is more suitable for NK cells.

Example 4 therapeutic Effect of CAR-immune cells in animal tumor models

4.1 animal model preparation

Selecting 6-8 week old NOD scid mice, and normally feeding for 1 week under SPF level conditions to adapt to the environment; culturing HL-60 cells to logarithmic phase, centrifuging to collect cells, and regulating cell concentration to 1×10 with sterile physiological saline ⁷ Intravenous injection of 0.5mL cell suspension into the tail of the mouse; after the model is constructed for 2-3 weeks, the infiltration condition of the tumor cells of the viscera, the proportion of the tumor cells of the external Zhou Xiezhong and the change condition of the hemogram are detected, and the result shows that the model accords with the pathological characteristics of leukemia, thus indicating that the construction of the leukemia animal model is successful.

4.2 animal grouping and administration treatments

The constructed animal model mice were randomly divided into 3 groups of 10 mice each, each injected with 1×10 injections ⁷ IPSC-CARNK cells, 1X 10 ⁷ The CART cells and an equal volume of physiological saline were injected 1 time per week for 2 total injections.

4.3 observations of animal survival

Dosing when astronomical was day 0, after which the physiological status of each group of experimental animals was observed daily and animal survival was recorded and animal survival curves were plotted using GraphPad Prism 9 software after the end of the experiment. As shown in FIG. 4, the IPSC-CARNK cells and CART cells can obviously prolong the survival time of animals, wherein the effects of the IPSC-CARNK cells are more obvious, and compared with a control group, the maximum survival time is prolonged by nearly one time, which indicates that the IPSC-CARNK cells can play a remarkable role in inhibiting tumors in animals.

4.4 peripheral blood cytokine detection

30 days after the first dose of therapy, mice were bled from their tail veins, serum was collected by centrifugation at 3000rpm at 4℃and assayed for IFN-gamma, TNF-alpha and IL-6 levels using ELISA kits (available from BioLegend Inc. of America) following the kit instructions.

The results are shown in figures 5-7, after CAR-immune cell treatment, the expression level of immune factors such as IFN-gamma, TNF-alpha, IL-6 and the like in animals is obviously improved, and the immune response of organisms can be effectively mediated to resist invasion of tumor cells. Compared with CART cells, the IPSC-CARNK cells have obvious advantages in promoting IFN-gamma and TNF-alpha secretion, especially the expression level of TNF-alpha is obviously increased, but the expression level of IL-6 does not show obvious difference, thus indicating that the IPSC-CARNK cells have certain specificity for regulating immune factors.

The results show that the chimeric antigen receptor constructed in the application is suitable for being applied to IPSC-NK cells, and the IPSC-NK cells modified by the CAR gene can obtain strong tumor inhibition capability and immune regulation capability, can effectively inhibit the growth of tumor cells in vivo and in vitro, and provides a direction for the research and development of novel antitumor drugs.

The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.

Claims

1. An IPSC-CAR natural killer cell, wherein the CAR structure comprises a signal peptide, a targeting CD19 antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory factor, and an intracellular signaling domain, connected in sequence; the amino acid sequence of the targeted CD19 antigen binding domain is shown as SEQ ID NO. 1; the transmembrane region is an NKG2D transmembrane region, and the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 2; the costimulatory factors are DAP10 with the amino acid sequence shown in SEQ ID NO. 3 and CD28 with the amino acid sequence shown in SEQ ID NO. 4.

2. The IPSC-CAR natural killer cell according to claim 1, wherein the amino acid sequence of the CAR structure is shown in SEQ ID No. 5.

3. The IPSC-CAR natural killer cell according to claim 1, wherein said natural killer cell is induced by IPSC cells.

4. A method of producing an IPSC-CAR natural killer cell according to any one of claims 1-3, wherein said method of producing comprises producing an IPSC natural killer cell; preparing a viral vector carrying a nucleotide sequence encoding said CAR structure; transfecting an IPSC natural killer cell with the viral vector to obtain the IPSC-CAR natural killer cell.

5. The method for producing an IPSC-CAR natural killer cell according to claim 4, wherein the step of producing an IPSC natural killer cell comprises culturing the IPSC cell until a cell fusion degree reaches 80% or more; adding final concentration of 50ng/mL SCF, 20ng/mL VEGF and 20ng/mL BMP4; after 2 weeks of induction culture, cells were collected by centrifugation, and fresh medium was added, and final concentrations of 10ng/mL IL-2, 10ng/mL IL-6, 15-11 ng/mL IL-15, 20ng/mL IL-7, 20ng/mL SCF and 20ng/mL flt3 ligand were maintained for 2-3 weeks to obtain IPSC-natural killer cells.

6. The method for producing IPSC-CAR natural killer cells according to claim 4, wherein the viral vector is an adenovirus vector, the producing step comprising: introducing the nucleotide encoding the CAR into an adenovirus skeleton vector Ad5F35 through enzyme digestion and ligation, and then transfecting the skeleton vector into HEK 293 cells; 37 ℃ and 5% CO ₂ Culturing for 14 days under saturated humidity, centrifuging to collect cells, repeatedly freezing and thawing at-80deg.C and 37deg.C, centrifuging to collect supernatant; the cell supernatant was purified by density gradient centrifugation and then filtered using a 0.22 μm filter to obtain the adenovirus vector.

7. Use of an IPSC-CAR natural killer cell according to any one of claims 1-3 for the manufacture of an anti-tumor medicament, said tumor being human acute myeloid leukemia.