CN117701578A

CN117701578A - RNA composition for amplifying NK cells and gene modified NK cell pharmaceutical preparation based on same

Info

Publication number: CN117701578A
Application number: CN202211114933.4A
Authority: CN
Inventors: 聂文冰; 周晨; 杨停停; 阚士凤; 陈婷; 孙振华
Original assignee: Jiangsu Purecell Bio Medicine Technology Co ltd
Current assignee: Jiangsu Purecell Bio Medicine Technology Co ltd
Priority date: 2022-09-14
Filing date: 2022-09-14
Publication date: 2024-03-15

Abstract

The invention relates to the technical field of genetic engineering and cell culture, in particular to an RNA composition for amplifying NK cells and a genetically modified NK cell pharmaceutical preparation based on the RNA composition. The present invention provides an RNA composition for amplifying NK cells, which encodes a target polypeptide comprising one or more of the group consisting of IL-2 polypeptide, IL-7 polypeptide, IL-12 polypeptide, IL-15 polypeptide, IL-18 polypeptide, IL-21 polypeptide, 4-1BBL polypeptide, CD16 alpha polypeptide and IFN-gamma polypeptide. The RNA composition is delivered to PBMC cells, non-NK cells and/or NK cells in PBMC cells by various means, is used for amplifying NK cells in vitro efficiently and stably, and can be used for further carrying out gene modification on the harvested NK cells, so that NK cell medicinal preparations capable of being used for clinical treatment are developed.

Description

RNA composition for amplifying NK cells and gene modified NK cell pharmaceutical preparation based on same

Technical Field

The invention relates to the technical field of genetic engineering and cell culture, in particular to an RNA composition for amplifying NK cells and a genetically modified NK cell pharmaceutical preparation based on the RNA composition.

Background

Natural killer cells (natural killer cell, NK cells) are an important component of the innate immune system of the body, playing a vital role in the body's resistance to viral infection, pathogen invasion and tumor immunity. Unlike T cells, NK cells function independently of their major histocompatibility complex (major histocompatibility, MHC), and function by their interaction between surface inhibitory receptors KIRs, NKG2A, etc. and activating receptors such as NKp30, NKp44, NKp46, and NKG 2D. In addition, since CD16 alpha (FcgammaRIIIalpha) is expressed on the surface of NK cells, the antibody Fc terminal can be specifically combined, and after the NK cells are combined with a monoclonal antibody, an antibody-dependent cell-mediated cytotoxicity (ADCC) effect-mediated killing function can be exerted. Once activated, NK cells rapidly lyse tumor cells or virus-infected cells without prior sensitization.

In recent years, with the rise and development of CAR-T immunotherapy, NK cell-based CAR-NK immunotherapy has also been developed. Compared with CAR-T immunotherapy, the safety of CAR-NK is obviously superior to that of CAR-T cells on the basis of exerting the same anti-tumor curative effect. The CAR-NK cells can obviously reduce toxic and side effects caused by the reinfusion of immune cells, such as cytokine storm, neurotoxicity and the like. Currently, CAR-NK therapies are distributed by several companies such as Fate therapeutics, cytovia Therapeutics.

At present, NK cell sources mainly comprise engineering cell lines NK-92, peripheral blood NK (PB-NK), umbilical cord blood NK (UCB-NK) and NK derived from pluripotent induced stem cells (iPSCs). NK-92 cell strain is model cell, can expand in vitro in a large amount, but because its surface does not express or expresses CD16 molecule lowly, ADCC effect is limited, in addition NK-92 cell strain can apply to the human body after needing to radiate in the clinical application process, has greatly reduced the in vivo pharmacodynamic effect, the clinical application is limited. Cord blood NK cells have a low degree of maturation compared to peripheral blood-derived NK cells, and thus have relatively weak cytotoxicity and ADCC effects. While the potential immunogenicity and the risk of malignant transformation of the NK cells derived from the iPSC need to be considered in clinical application, the in-vitro amplification process is more difficult. In the existing research process, NK cells are mainly derived from peripheral blood. Whether applied in autologous or allogeneic mode, the NK cell preparation from peripheral blood is safer. But the NK cells in the peripheral blood account for only 5% -10%, and the clinical application can be carried out only after the NK cells are amplified to a certain quantity in vitro.

There are two main methods for NK cell in vitro amplification that are commonly used at present: feeder cells stimulated expansion and multifactorial stimulated expansion. The usual feeder cells used in the stimulation of expansion with feeder cells are K562 cells expressing IL-15 or IL-21 and 4-1 BBL. The residual and safety problems of K562 cells in the final preparation need to be considered in clinical application. In addition, when multi-factors such as IL-2, IL-7, IL-12, IL-15, IL-18 and IL-21 are utilized to stimulate NK amplification, NK cells have limited proliferation capacity, lower purity, larger individual variability and high preparation cost. And various cytokines to be introduced in the final product safety assessment are additionally considered.

How to realize the in vitro expansion of NK cells with high efficiency, repeatability (reducing individual variability of expansion efficiency), safety (no exogenous factors and no additional introduction of K562 feeder cells) and economy (controllable cost) is a great difficulty which puzzles the clinical application of NK cells.

NK preparation is short in half-life after being infused into a body, and the killing capacity and survival time of NK cells which are not genetically modified are inferior to those of T cells which are genetically modified, so that in order to improve the survival time of NK cells in the body, the killing capacity of NK cells to tumors in the body and activate systemic immune response, the amplified NK cells are required to be genetically modified. Since the conventional Lentivirus (Lentivirus) gene modification method has low transfection efficiency on NK cell genes, the amplified NK cell genes are modified by using adeno-associated virus (AAV) or electrotransformation. However, due to AAV antibodies prestored in human body, AAV modification can cause certain immune response, and in addition, the AAV preparation process is complex and high in cost, so that clinical popularization is not facilitated. Electrotransfection is achieved by using transient current stimulus physical means, but the conventional electrotransfection of DNA has safety problems of gene integration.

Disclosure of Invention

Problems to be solved by the invention

At present, the common NK cell in-vitro amplification method has the problems of residual feeder cells and stimulus factors, limited proliferation capacity of NK cells, low purity, large individual variability and high preparation cost, and the amplified NK cell preparation also has the problems of short in-vivo survival time, limited killing capacity and potential safety hazard in a gene modification mode. In order to solve the above-mentioned drawbacks, the present invention provides an RNA composition for amplifying NK cells and a genetically modified NK cell pharmaceutical preparation based thereon, which deliver the RNA composition to peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC), non-NK cells and/or NK cells among PBMC cells by various means for efficient in vitro amplification of NK cells, and which can be used to further genetically modify harvested NK cells, thereby developing an NK cell pharmaceutical preparation that can be used for clinical treatment.

Solution for solving the problem

[1] An RNA composition for expanding NK cells encoding a polypeptide of interest comprising one or more of the group consisting of an IL-2 polypeptide, an IL-7 polypeptide, an IL-12 polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, an IL-21 polypeptide, a 4-1BBL polypeptide, a CD16 a polypeptide and an IFN- γ polypeptide.

[2] The RNA composition according to [1], wherein the RNA composition comprises one or more of the mRNAs as shown in the following (a) to (c), or the RNA composition comprises the mRNAs as shown in the following (d):

(a) A first mRNA comprising a first coding sequence encoding an IL-15 polypeptide;

(b) A second mRNA comprising a second coding sequence encoding an IL-21 polypeptide;

(c) A third mRNA comprising a third coding sequence encoding a 4-1BBL polypeptide;

(d) A fourth mRNA comprising a fourth coding sequence encoding a recombinant polypeptide, wherein the fourth coding sequence comprises any two or more of: a first coding sequence encoding an IL-15 polypeptide, a second coding sequence encoding an IL-21 polypeptide, and a third coding sequence encoding a 4-1BBL polypeptide.

Further, the method comprises the steps of,

the first coding sequence comprises a sequence as set forth in SEQ ID NO.2, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO.2 and encoding a polypeptide having or partially having IL-15 polypeptide activity;

the second coding sequence comprises a sequence as set forth in SEQ ID NO.6, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO.6 and encoding a polypeptide having or partially having IL-21 polypeptide activity;

The third coding sequence comprises a sequence as set forth in SEQ ID NO.10, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO.10 and encoding a polypeptide having or partially having 4-1BBL polypeptide activity.

Further, the method comprises the steps of,

the first mRNA further comprises a fifth coding sequence which codes for an IL-15Rα polypeptide;

the second mRNA further comprises a sixth coding sequence encoding a CD8 hinge region polypeptide, a seventh coding sequence encoding a CD8 transmembrane region polypeptide, and an eighth coding sequence encoding a CD8 intracellular region polypeptide.

[3] The RNA composition according to [1] or [2], wherein each mRNA in the RNA composition is independently selected from linear mRNA or circular mRNA.

Further, each mRNA in the RNA composition is a circular mRNA.

[4] The use of the RNA composition according to any one of [1] to [3] in at least one of the following (e) to (f):

(e) Amplifying NK cells;

(f) NK cells were genetically modified.

[5] A method for expanding NK cells, comprising the steps of (i) to (ii) as follows:

(i) The step of preparing feeder cells: delivering the RNA composition of any one of [1] to [3] to a cell to be treated, obtaining a feeder cell containing the RNA composition; wherein the cells to be treated are PBMC cells, non-NK cells and/or NK cells in PBMC cells;

(ii) Amplifying and culturing NK cells: adding the feeder cells to be expanded which do not contain the RNA composition in one time or in batches for co-culture; wherein the cells to be expanded are PBMC cells and/or NK cells.

Further, the source of NK cells includes one or more of NK-92 cell lines, peripheral blood NK cells, umbilical cord blood NK cells and pluripotent stem cell-derived NK cells.

Further, the means of delivery includes one or more of electrotransfection, LNP delivery, and exosome delivery.

[6]According to [5 ]]The method is characterized in that in the step of preparing feeder cells, the amount of each RNA in the RNA composition is each independently in proportion to the number of cells to be treated (10. Mu.g to 50. Mu.g): (1X 10) ⁷ Individual). Preferably, the total amount of RNA composition is 1 μg to 1mg;

in the step of amplifying and culturing NK cells, the number ratio of the cells to be amplified without containing the RNA composition to the feeder cells is 10:1 to 1:10, and the initial cell density of the co-culture is (3X 10 ⁶ )～(5×10 ⁶ ) And each mL.

[7] A method of genetically modifying an NK cell comprising the step of delivering the RNA composition of any one of [1] to [3] into an NK cell to be modified.

Further, the source of the NK cells includes one or more of NK-92 cell lines, peripheral blood NK cells, umbilical cord blood NK cells and pluripotent stem cell-derived NK cells; preferably, the NK cells are NK cells amplified by the method described in [5] or [6 ].

Further, the means of delivery includes one or more of electrotransfection, LNP delivery, exosome delivery.

Further, the ratio of the amount of each RNA in the RNA composition to the number of NK cells to be modified is (10. Mu.g to 50. Mu.g) independently of each other: (1X 10) ⁷ Individual). Preferably, the total amount of RNA composition is 1 μg to 100mg.

[8] A genetically modified NK cell prepared by the method of [7 ].

[9] A pharmaceutical composition comprising the genetically modified NK cell of [8] and a pharmaceutically acceptable carrier.

[10] Use of the genetically modified NK cell of [8] and/or the pharmaceutical composition of [9] for the preparation of a medicament for the prophylaxis and/or treatment of cancer.

ADVANTAGEOUS EFFECTS OF INVENTION

Compared with the prior art, the method has the beneficial effects that NK cells are amplified in vitro by utilizing different RNA compositions, the safety is higher, exogenous cytokines and additional feeder cells such as K562 cells are not introduced in the process of amplifying the NK cells, and the culture cost of the NK cells is reduced.

Meanwhile, the method for amplifying and culturing NK cells provided by the invention can stably amplify the NK cells, and has good repeatability.

In addition, the RNA composition provided by the invention is used for carrying out gene modification on amplified NK cells, because the gene modification of mRNA belongs to a non-viral system and non-integration expression gene modification method, the mRNA can be quickly degraded after in vivo expression, compared with lentivirus or adenovirus gene modification, the gene modification of mRNA does not introduce virus particles, no gene integration risk exists, and the safety is higher.

Compared with unmodified NK cells, the NK cells subjected to genetic modification provided by the invention can prolong the in-vivo survival time, can play the anti-tumor function of the NK cells, activate the systemic immune response of a subject, enhance the in-vivo and in-vitro curative effect, and have better clinical application prospects.

Drawings

FIG. 1 is a plasmid map of pUC 57-mbiL-15;

FIG. 2 is a plasmid map of pUC 57-mbiL-21;

FIG. 3 is a plasmid map of pUC57-4-1 BBL;

FIG. 4 is a graph showing the mbIL-15 and 4-1BBL expression measurements after transfection of PBMC with mbIL-15 and 4-1BBL mRNA;

FIG. 5 is a graph showing NK cell proliferation fold in vitro;

FIG. 6 shows the purity profile of NK cells cultured in vitro, wherein Panel A is a representative flow chart and Panel B is a statistical chart.

FIG. 7 is a graph showing the in vitro expression of mbIL-15, mbIL-21 and 4-1BBL from NK cells modified with mbIL-15, mbIL-21 and 4-1BBL mRNA;

FIG. 8 is a graph showing the effect of NK cells on killing HEPG2 in vitro after modification with mbiL-15 mRNA.

Detailed Description

The following describes embodiments of the present invention, but the present invention is not limited thereto.

Definition of the definition

In the present invention, the terms "a" or "an" or "the" may mean "one" or "one or more", "at least one", and "one or more".

In the present disclosure, the terms "comprising," "having," "including," or "containing" may be used to specify the presence of stated features, integers, steps, or groups thereof, but do not preclude the presence or addition of other features, integers, steps, or groups thereof. In the meantime, "comprising," "having," "including," or "containing" may also mean enclosed, excluding additional, unrecited elements or method steps.

In the present invention, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In the present invention, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

In the present invention, the term "about" may mean: one value includes the standard deviation of the error of the device or method used to determine the value. Unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used herein are modified by "about".

In the present invention, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

In the present invention, the term "circular RNA" refers to a nucleic acid molecule in the form of a closed loop. In some embodiments, the circular RNA is circular mRNA.

In the present invention, the term "linear RNA" is an RNA molecule that is linear. In some embodiments, the linear RNA is linear mRNA, e.g., a circularized precursor RNA that is capable of forming circular mRNA by a circularization reaction, which is typically transcribed from a linear DNA molecule.

In the present invention, the term "expression" includes any step involving the production of a polypeptide, including, but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

In the present invention, the "RNA composition" is not limited to a combination system that necessarily includes at least two kinds of mRNA, but also covers a combination system that includes only one kind of mRNA. Specifically, the mRNA composition may be one capable of translationally expressing the target polypeptide. In some embodiments, the RNA composition comprises at least two mrnas; in other embodiments, the RNA composition consists of one mRNA.

In the present invention, the term "vector" refers to a DNA construct comprising a DNA sequence operably linked to a suitable control sequence to express a gene of interest in a suitable host.

In the present invention, the term "individual", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

In the present invention, "treatment" means: after suffering from a disease, the subject is contacted (e.g., administered) with an mRNA molecule described in the present invention, thereby alleviating the symptoms of the disease compared to when not contacted, and does not mean that the symptoms of the disease must be completely inhibited. The suffering from the disease is: the body develops symptoms of the disease.

In the present invention, "prevention" means: by contacting a subject with an mRNA molecule described in the present invention prior to the onset of a disease, the probability of the onset of the disease is reduced and/or the symptoms after the onset of the disease are reduced as compared to when not contacted, which does not mean that complete inhibition of the disease is necessary.

In the present invention, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, malignant lentigo melanoma, acromelanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as those associated with scar diseases (komatos), brain metastases and brain tumor associated with brain metastases, brain tumor and brain tumor associated with brain tumor (head and neck and brain tumor syndrome, brain tumor syndrome (brain tumor).

In the present invention, the term "signal peptide" refers to a short (5-30 amino acids in length) peptide chain that directs the transfer of a newly synthesized protein to the secretory pathway. Typically, the signal peptide is located at the N-terminus of the protein (sometimes not necessarily at the N-terminus). The signal peptide is generally relatively hydrophobic and serves primarily to facilitate secretion of the protein out of the cell.

In the present invention, "feeder cells" means that in an in vitro cell culture, single or small number of cells are not easy to survive and reproduce, and other living cells must be added to make them grow and reproduce, and the added cells are feeder cells.

Unless defined otherwise, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

RNA composition

< polypeptide encoded by RNA composition >

The present invention provides an RNA composition encoding a polypeptide of interest comprising one or more of the group consisting of an IL-2 polypeptide, an IL-7 polypeptide, an IL-12 polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, an IL-21 polypeptide, a 4-1BBL polypeptide, a CD16 alpha polypeptide, and an IFN-gamma polypeptide.

In some preferred embodiments, the RNA compositions of the invention encode polypeptides of interest comprising one or more of the group consisting of IL-15 polypeptides, IL-21 polypeptides, and 4-1BBL polypeptides.

The IL-15 polypeptide belongs to gamma receptor cytokine family, NK cells have a certain proliferation response to IL-15 stimulation, and the killing activity is obviously improved, and the survival is maintained. The species origin and sequence of the IL-15 polypeptide are not particularly limited as long as it has IL-15 polypeptide activity.

Further, in some specific embodiments, the IL-15 polypeptide is encoded by a first coding sequence. In some preferred embodiments, the first coding sequence comprises a sequence as set forth in SEQ ID NO.2, or has at least 80% sequence identity to a sequence set forth in SEQ ID NO.2, and encodes a polypeptide having or partially having IL-15 polypeptide activity.

The IL-21 polypeptide is an immunomodulator, can enhance antigen specific reaction of immune cells and promote anti-tumor activity of T cells and NK cells. IL-21 induces maturation of NK cells and enhances cytotoxicity. IL-21 in combination with IL-2 or IL-15 induces proliferation of NK cells, which exert a modulating effect on NK cells. The species origin and sequence of the IL-21 polypeptide are not particularly limited as long as it has IL-21 polypeptide activity.

Further, in some specific embodiments, the IL-21 polypeptide is encoded by a second coding sequence. In some preferred embodiments, the second coding sequence comprises a sequence as set forth in SEQ ID NO.6, or has at least 80% sequence identity to a sequence set forth in SEQ ID NO.6, and encodes a polypeptide having or partially having IL-21 polypeptide activity.

The 4-1BBL polypeptide disclosed by the invention refers to a ligand of 4-1BB, belongs to a member of a tumor necrosis factor receptor family, is encoded by a tumor necrosis factor receptor superfamily member 9 gene, and can activate 4-1BB by the ligand 4-1BBL, so that NK cell activation and proliferation can be stimulated, and NK cell cytotoxicity can be improved. The species origin and sequence of the 4-1BBL polypeptide are not particularly limited as long as it has 4-1BBL polypeptide activity.

Further, in some specific embodiments, the 4-1BBL polypeptide is encoded by the third coding sequence. In some preferred embodiments, the third coding sequence comprises the sequence set forth in SEQ ID NO.10, or has at least 80% sequence identity to the sequence set forth in SEQ ID NO.10, and encodes a polypeptide having or partially having 4-1BBL polypeptide activity.

In order to play a better role in stimulating NK cell proliferation, the target polypeptide coded by the RNA composition can further comprise one or more of IL-15 Ralpha polypeptide, CD8 hinge region polypeptide, CD8 transmembrane region polypeptide and CD8 intracellular region polypeptide.

Further, in some specific embodiments, the IL-15 ra polypeptide is encoded by the fifth coding sequence, the CD8 hinge region polypeptide is encoded by the sixth coding sequence, the CD8 transmembrane region polypeptide is encoded by the seventh coding sequence, and the CD8 intracellular region polypeptide is encoded by the eighth coding sequence.

In some preferred embodiments, the fifth coding sequence comprises the sequence set forth in SEQ ID NO.4, or has at least 80% sequence identity to the sequence set forth in SEQ ID NO.4, and encodes a polypeptide having or partially having IL-15Rα polypeptide activity.

In some preferred embodiments, the sixth coding sequence comprises the sequence set forth in SEQ ID No.7, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID No.7 and encoding a polypeptide having or partially having the activity of a CD8 hinge region polypeptide.

In some preferred embodiments, the seventh coding sequence comprises the sequence set forth in SEQ ID No.8, or has at least 80% sequence identity to the sequence set forth in SEQ ID No.8, and encodes a polypeptide having or partially having the activity of a CD8 transmembrane region polypeptide.

In some preferred embodiments, the eighth coding sequence comprises the sequence set forth in SEQ ID No.9, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID No.9 and encoding a polypeptide having or partially having the activity of a CD8 intracellular domain polypeptide.

In some more preferred embodiments, the invention links the first coding sequence encoding an IL-15 polypeptide to a fifth coding sequence encoding an IL-15Rα polypeptide via a linker (linker) sequence to form a coding sequence encoding an mbiL-15 polypeptide, to better stimulate NK cell proliferation upon expression.

In some more preferred embodiments, the invention links the second coding sequence encoding an IL-21 polypeptide to a sixth coding sequence encoding a CD8 hinge region polypeptide, a seventh coding sequence encoding a CD8 transmembrane region polypeptide, and an eighth coding sequence encoding a CD8 intracellular region polypeptide to form a coding sequence encoding an mbiL-21 polypeptide, to better stimulate NK cell proliferation upon expression.

< mRNA contained in RNA composition >

The target polypeptide coded by the RNA composition is coded by mRNA.

In some embodiments, the RNA composition of the invention comprises one or more of the following mrnas as shown in (a) - (c):

(c) A third mRNA comprising a third coding sequence encoding a 4-1BBL polypeptide.

In some specific embodiments, the first mRNA of the present invention further comprises a fifth coding sequence that encodes an IL-15Rα polypeptide.

In some specific embodiments, the second mRNA of the present invention further comprises a sixth coding sequence encoding a CD8 hinge region polypeptide, a seventh coding sequence encoding a CD8 transmembrane region polypeptide, and an eighth coding sequence encoding a CD8 intracellular region polypeptide.

In some preferred embodiments, the RNA compositions of the present invention comprise one or more of the mRNA as shown in (h) to (c) below:

(h) A first mRNA comprising a coding sequence encoding an mbIL-15 polypeptide;

(g) A second mRNA comprising a coding sequence encoding an mbIL-21 polypeptide;

In other embodiments, the RNA composition of the invention comprises an mRNA as shown in (d): (d) A fourth mRNA comprising a fourth coding sequence encoding a recombinant polypeptide, wherein the fourth coding sequence comprises any two or more of: a first coding sequence encoding an IL-15 polypeptide, a second coding sequence encoding an IL-21 polypeptide, and a third coding sequence encoding a 4-1BBL polypeptide.

In other preferred embodiments, the RNA composition of the present invention comprises an mRNA as shown in (d): (d) A fourth mRNA comprising a fourth coding sequence encoding a recombinant polypeptide, wherein the fourth coding sequence comprises any two or more of: coding sequence for mbIL-15 polypeptide, coding sequence for mbIL-21 polypeptide, coding sequence for 4-1BBL polypeptide.

In the present invention, the mRNA in the RNA composition is used to express the polypeptide of interest in the cell, and the mRNA may be selected for any type of structure that is capable of achieving expression of the protein in the cell. In some embodiments, the mRNA is used to express the polypeptide of interest in eukaryotic cells, e.g., eukaryotic cells are mammalian cells. The mammal may be a primate (monkey, non-human primate, or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse, or other rodent (including any animal of the order rodents), cat, dog, pig, sheep, goat, cow (including cows, e.g., cows, and animals of any genus of cattle), horse (including any equine), donkey, and non-human primate.

In the present invention, each mRNA contained in the RNA composition is independently selected from linear mRNA or circular mRNA. Further, the first mRNA, the second mRNA, the third mRNA and the fourth mRNA are linear mRNA or circular mRNA independently of each other.

In some embodiments, each mRNA comprised by the RNA compositions of the present invention is a circular mRNA. The stable, efficient and durable expression of the target polypeptide can be realized by utilizing the annular mRNA.

In some specific embodiments, the RNA compositions of the invention comprise (a) ₁ )～(c ₁ ) One or more of the circular mRNAs shown:

(a ₁ ) A first circular mRNA comprising a first coding sequence encoding an IL-15 polypeptide;

(b ₁ ) A second circular mRNA comprising a second coding sequence encoding an IL-21 polypeptide;

(c ₁ ) A third circular mRNA comprising a third coding sequence encoding a 4-1BBL polypeptide.

In some preferred embodiments, the RNA composition of the present invention comprises (h ₁ )～(c ₁ ) One or more of the circular mRNAs shown:

(h ₁ ) A first circular mRNA comprising a coding sequence encoding an mbIL-15 polypeptide;

(g ₁ ) A second circular mRNA comprising a coding sequence encoding an mbIL-21 polypeptide;

In other specific embodiments, the RNA compositions of the invention comprise a polypeptide such as (d) ₁ ) The circular mRNA shown: (d) ₁ ) A fourth circular mRNA comprising a fourth coding sequence encoding a recombinant polypeptide, wherein the fourth coding sequence comprises any two or more of: a first coding sequence encoding an IL-15 polypeptide, a second coding sequence encoding an IL-21 polypeptide, and a third coding sequence encoding a 4-1BBL polypeptide.

In other preferred embodiments, the RNA compositions of the invention comprise a polypeptide such as (d) ₂ ) The circular mRNA shown: (d) ₂ ) Comprising braidingA fourth circular mRNA encoding a fourth coding sequence of a recombinant polypeptide, wherein the fourth coding sequence comprises any two or more of: coding sequence for mbIL-15 polypeptide, coding sequence for mbIL-21 polypeptide, coding sequence for 4-1BBL polypeptide.

In some embodiments, the circular mRNA of the present invention has a sequence in the order shown in any one of (j) to (m) as follows:

(j) A first exon sequence, a second exon sequence, a 5 'spacer sequence, a translation initiation sequence, a coding sequence, and a 3' spacer sequence;

(k) A first exon sequence, a second exon sequence, a translation initiation sequence, and a coding sequence;

(l) Translation initiation sequences and coding sequences;

(m) a translation initiation sequence, a coding sequence, and an insertion element sequence.

For the first exon sequence, the second exon sequence, the 5 'spacer sequence, the translation initiation sequence, the 3' spacer sequence and the insert sequence therein, reference may be made to patent applications CN202011408937.4, CN202210200112.6 and CN202210200186.X, the entire contents of which are incorporated herein by reference.

In some preferred embodiments, the first circular mRNA comprises the sequence set forth in SEQ ID No.14, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID No.14 and encoding a polypeptide having or partially having mbIL-15 polypeptide activity.

In some preferred embodiments, the second circular mRNA comprises the sequence set forth in SEQ ID No.15, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID No.15 and encoding a polypeptide having or partially having the activity of an mbIL-21 polypeptide.

In some preferred embodiments, the third circular mRNA comprises the sequence shown as SEQ ID NO.16 or a sequence having at least 80% sequence identity to the sequence shown as SEQ ID NO.16 and encoding a polypeptide having or partially having 4-1BBL polypeptide activity.

< use of RNA composition >

In some embodiments, the RNA compositions of the invention are used to amplify NK cells. In this scheme, the RNA composition can be called mRNA NK amplification composition. The mRNA composition can express NK cell activation amplification factors on the surfaces of non-NK cells and/or NK cell membranes in PBMC cells and PBMC cells, and can also express secreted proteins, so that the cells play a role of feeder cells, and can avoid introducing exogenous cells such as K562 and the like, thereby realizing safe and efficient expansion of NK cells.

In some embodiments, the RNA compositions of the invention are used to genetically modify NK cells. In this scheme, the RNA composition can be called mRNA NK preparation gene modification composition. mRNA has the safety advantage of non-integrated expression of a non-viral system as a genetic modification method. On the basis of adopting the mRNA composition to efficiently amplify NK cells, the invention can also utilize the mRNA composition to carry out gene modification on amplified NK cells, thereby improving the in-vivo survival time and killing capacity of NK cells, preparing NK cell pharmaceutical preparations and providing assistance for clinical treatment of tumors.

In some embodiments, the invention first synthesizes an mRNA molecule, delivers the mRNA molecule composition to PBMC cells, non-NK cells and/or NK cells in PBMC cells by one or more gene delivery, and then performs in vitro expansion by co-culture with PBMC cells and/or NK cells not containing the RNA composition until the number and purity of NK cells meet clinical use requirements; the NK cells which are well grown in vitro are further subjected to gene modification by the mRNA composition, and finally the NK cells with the gene modification are prepared and used for clinical tumor treatment and the like.

Method for amplifying NK cells

The invention provides a method for amplifying NK cells, which comprises the following steps of (i) to (ii):

(i) The step of preparing feeder cells: delivering the RNA composition to a cell to be treated to obtain a feeder cell containing the RNA composition; wherein the cells to be treated are PBMC cells, non-NK cells and/or NK cells in PBMC cells;

In some preferred embodiments, the RNA composition comprises (h ₁ )～(c ₁ ) One, two or three of the cyclic mRNAs shown:

In some embodiments, the source of NK cells comprises one or more of NK-92 cell lines, peripheral blood NK cells, umbilical cord blood NK cells, and NK cells from pluripotent induced stem cells; preferably from peripheral blood NK cells.

In some preferred embodiments, in the step of preparing feeder cells, the cells to be treated are PBMC cells or non-NK cells in PBMC cells. Accordingly, in some preferred embodiments, the method of expanding NK cells further comprises the step of sorting NK cells and non-NK cells from PBMC cells prior to the step of preparing feeder cells.

In some embodiments, in the step of preparing feeder cells, each RNA in the RNA composition is used in an amount that is independent of the ratio of the number of cells to be treated (10 μg to 50 μg): (1X 10) ⁷ Individual). Preferably (10. Mu.g to 40. Mu.g): (1X 10) ⁷ Individual). More preferably (10. Mu.g to 30. Mu.g): (1X 10) ⁷ Individual). Even more preferably (15. Mu.g to 25. Mu.g): (1X 10) ⁷ And (c) a). The present invention is not particularly limited in the ratio of the amount of each RNA in the RNA composition.

In some embodiments, in the step of preparing feeder cells, the total amount of RNA composition is 1 μg to 1mg; the preferred amount is 20 μg to 500 μg.

In some embodiments, in the step of preparing feeder cells, the means of delivery comprises one or more of electrotransfection, LNP delivery, and exosome delivery; electrotransfection is preferred.

In some embodiments, in the step of expanding cultured NK cells, the ratio of the number of cells to be expanded that do not contain an RNA composition to the number of feeder cells is from 10:1 to 1:10; preferably 1:1 to 1:5, more preferably 1:1 to 1:3. The proportion is obtained according to the practical NK cell expansion experiment result, and the feeder cells can effectively play a role in stimulating NK cell expansion under the proportion.

In some embodiments, in the step of amplifying the cultured NK cells, the initial cell density of the co-culture is (3X 10) ⁶ )～(5×10 ⁶ ) individual/mL; preferably 4X 10 ⁶ And each mL. The cell density is obtained according to the practical NK cell expansion experimental result, and under the cell density, the NK cell growth state is good and the proliferation speed is high.

In some embodiments, in the step of expanding cultured NK cells, the batch comprises one, two, three, four, and so forth.

In some embodiments, in the step of expanding the cultured NK cells, the co-culturing is accomplished in NK cell expansion medium comprising KBM502 medium, KBM581 medium, X-VIVO 15 medium, GT-T551H 3 medium; preferably KBM502 medium; more preferably, the NK cell expansion medium contains 500U/mL of IL-2 which can further help to stimulate NK cell expansion.

The method for amplifying NK cells provided by the invention can effectively and stably amplify NK cells to obtain high-purity NK cells, and has good repeatability.

Method for genetically modifying NK cells

The present invention provides a method of genetically modifying an NK cell comprising the step of delivering the above RNA composition into the NK cell to be modified.

In some embodiments, the source of NK cells comprises one or more of NK-92 cell lines, peripheral blood NK cells, umbilical cord blood NK cells, and NK cells from pluripotent induced stem cells; preferably, the NK cells are NK cells amplified by the above-mentioned NK cell amplification method.

In some embodiments, the amount of each RNA in the RNA composition is each independently proportional to the number of NK cells to be modified (10 μg to 50 μg): (1X 10) ⁷ Individual). Preferably (10. Mu.g to 40. Mu.g): (1X 10) ⁷ Individual). More preferably (20. Mu.g to 40. Mu.g): (1X 10) ⁷ And (c) a). The present invention is not particularly limited in the ratio of the amount of each RNA in the RNA composition.

In some embodiments, the total amount of RNA composition is 1 μg to 100mg, preferably 200 μg to 50mg.

In some embodiments, the means of delivery comprises one or more of electrotransfection, LNP delivery, and exosome delivery; electrotransfection is preferred. In this way, mRNA compositions can be efficiently delivered into NK cells.

The method for genetically modifying NK cells provided by the invention is a non-viral system and a non-integrally expressed genetic modification method, related polypeptide genes can be quickly degraded after in vivo expression, compared with lentivirus or adenovirus genetic modification, the genetic modification method based on an mRNA composition can not introduce virus particles, has no risk of gene integration, and has higher safety.

Through genetic modificationNK cells of (E) and pharmaceutical compositions thereof

Compared with unmodified NK cells, the genetically modified NK cells obtained by the method can prolong the survival time in vivo, can play the anti-tumor function of the NK cells, activate the systemic immune response of a subject, enhance the in-vivo and in-vitro curative effect, and have better clinical application prospect.

The invention also provides a pharmaceutical composition comprising the genetically modified NK cells and a pharmaceutically acceptable carrier.

In the present invention, the composition is used for promoting the administration to a living body, facilitating the absorption of an active ingredient, and further exhibiting a biological activity. The compositions of the present invention may be administered by any form including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid, oral liquid), rectal, inhalation, implantation, topical (e.g., ocular) administration, and the like. Non-limiting examples of oral solid formulations include, but are not limited to, powders, capsules, lozenges, granules, tablets, and the like. Non-limiting examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, and the like. Non-limiting examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Non-limiting examples of parenteral formulations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection, and the like. The compositions of the invention may also be formulated in controlled or delayed release dosage forms (e.g., liposomes or microspheres).

In the present disclosure, the route of administration can be varied or adjusted in any suitable manner to meet the needs of the nature of the drug, the convenience of the patient and medical personnel, and other relevant factors.

The NK cells subjected to genetic modification and/or the pharmaceutical composition provided by the invention can be used for preparing medicines for preventing and/or treating cancers.

The genetically modified NK cells and/or the pharmaceutical compositions provided by the invention can be used for preventing and/or treating cancers.

The present invention provides a method for preventing and/or treating cancer comprising administering to a subject a prophylactically and/or therapeutically effective amount of the above genetically modified NK cells and/or the above pharmaceutical composition.

Examples

The invention is further illustrated by the following examples, but any examples or combinations thereof should not be construed as limiting the scope or embodiments of the invention. Any modifications or variations of the technical solution of the present invention may be carried out by those skilled in the art without departing from the spirit and scope of the present invention, and such modifications and variations are also included in the scope of the present invention.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other embodiments, methods, means, apparatus and steps well known to those skilled in the art have not been described in detail in order to not obscure the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, all units used in this specification are units of international standard, and the numerical values and numerical ranges appearing in the present invention are understood to include unavoidable systematic errors.

Example 1: construction of pUC57-mbiL-15, pUC57-mbiL-21 and pUC57-4-1BBL plasmids

Human IL-15 (Genbank Accession No: NM-000585.5), IL-15Rα (Genbank Accession No: NM-001243539.2), IL-21 (Genbank Accession No: NM-001207006.3), CD8a (Genbank Accession No: NM-001145873.1) and 41BBL (Genbank Accession No: NM-003811.4) gene sequences were obtained by NCBI query. The IL-15 and IL-15Rα gene sequences are linked by a Linker to form an mbIL-15 gene sequence. The IL-21 sequence was joined to the CD8 hinge region, CD8 transmembrane region and CD8 intracellular region sequences in the CD8a sequence to form the mbiL-21 gene sequence. The above mbIL-15, mbIL-21 and 4-1BBL sequences were subjected to human codon optimization by bioinformatics tools, and the optimized sequences were submitted to company synthesis and cloning. The obtained gene fragments were ligated to pUC57 plasmid vectors, respectively, to obtain pUC57-mbiL-15, pUC57-mbiL-21 and pUC57-4-1BBL plasmids, respectively, and specific plasmid maps were shown in FIGS. 1 to 3, respectively. Wherein the nucleic acid and amino acid sequences involved are as follows:

Nucleic acid sequence:

signal peptide coding sequence in IL-15 (SEQ ID NO. 1): ATGAGAATCAGCAAGCCCCACCTGAGAAGCATCTCCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCC.

IL-15 coding sequence (SEQ ID NO. 2): ATGAGAATCAGCAAGCCCCACCTGAGAAGCATCTCCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGCCTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTCCTGCTGGAGCTGCAGGTGATCAGCCTGGAGAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAATGGCAACGTGACCGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC.

Linker coding sequence (SEQ ID NO. 3): AGCGGCGGCGGCAGCGGAGGCGGCGGCAGCGGCGGCGGGGGCAGCGGCGGAGGCGGCAGCGGCGGCGGCAGCCTGCAG.

IL-15Rα coding sequence (SEQ ID NO. 4): ATCACCTGCCCTCCCCCTATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTACAGCCTGTACAGCAGGGAGAGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTGACCGAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCCAGCCTGAAGTGCATCAGAGATCCCGCCCTGGTGCACCAGAGGCCCGCCCCTCCCAGCACCGTGACCACCGCCGGCGTGACCCCTCAGCCCGAGAGCCTGAGCCCCAGCGGCAAGGAGCCCGCCGCCAGCAGCCCCAGCAGCAACAACACCGCCGCCACCACCGCCGCCATCGTGCCCGGCAGCCAGCTGATGCCCAGCAAGAGCCCCAGCACCGGAACCACAGAGATTAGCTCTCATGAGAGCAGCCACGGCACCCCCAGCCAGACCACCGCCAAGAACTGGGAGCTGACCGCTAGCGCCAGCCACCAGCCCCCTGGCGTGTACCCCCAGGGCCACAGCGACACCACCGTGGCCATCAGCACCTCCACCGTGCTGCTGTGCGGCCTGAGCGCCGTGAGCCTGCTGGCCTGCTACCTGAAGAGCAGGCAGACACCCCCTCTGGCCAGCGTGGAGATGGAGGCCATGGAGGCTCTGCCCGTGACCTGGGGCACCAGCAGCAGAGACGAGGACCTGGAGAACTGCAGCCACCACCTGTGA.

Signal peptide coding sequence in IL-21 (SEQ ID NO. 5): ATGAGAAGCAGCCCCGGCAACATGGAGAGGATCGTGATCTGCCTGATGGTGATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCAGC.

IL-21 coding sequence (SEQ ID NO. 6): ATGAGAAGCAGCCCCGGCAACATGGAGAGGATCGTGATCTGCCTGATGGTGATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCAGCCAGGGCCAAGACAGGCACATGATCAGAATGAGGCAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCCGCCCCCGAGGACGTGGAGACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAGAGCGCCAACACCGGCAACAACGAGAGGATCATCAACGTGAGCATCAAGAAGCTGAAGAGGAAGCCCCCTAGCACCAACGCCGGCAGAAGACAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCTCCCAAGGAGTTCCTGGAGAGATTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCAGAACCCACGGCAGCGAGGACAGC.

CD8 hinge region coding sequence (SEQ ID NO. 7): ACCACCACCCCCGCCCCTAGGCCCCCTACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGGCCCGAGGCCTGCAGGCCCGCCGCCGGCGGCGCCGTGCACACCAGGGGCCTGGACTTCGCCTGCGAT.

CD8 transmembrane region coding sequence (SEQ ID NO. 8): ATCTACATCTGGGCCCCCTTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACC.

CD8 intracellular region coding sequence (SEQ ID NO. 9): CTGTACTGCAACCACAGGAACAGAAGAAGGGTGTGCAAGTGCCCCAGGCCCGTGGTGAAGAGCGGCGACAAGCCCAGCCTGAGCGCCAGATACGTGTGA.

4-1BBL coding sequence (SEQ ID NO. 10): ATGGAGTACGCCAGCGACGCCAGCCTGGACCCCGAGGCCCCCTGGCCTCCCGCTCCCAGGGCCAGAGCCTGCAGGGTGCTGCCCTGGGCCCTGGTGGCCGGCCTGCTGCTGCTCCTGCTGCTGGCTGCCGCCTGCGCCGTGTTCCTGGCCTGCCCCTGGGCTGTGAGCGGCGCTAGGGCCAGCCCCGGCAGCGCCGCCAGCCCCAGACTGAGGGAGGGCCCCGAGCTGAGCCCCGACGATCCCGCTGGCCTGTTGGATCTGAGGCAGGGCATGTTTGCCCAACTGGTGGCCCAAAATGTTCTGCTTATCGATGGCCCCCTGAGCTGGTACTCTGACCCTGGCCTGGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTATAAAGAGGACACCAAGGAGTTAGTGGTGGCCAAGGCTGGAGTGTATTATGTGTTCTTTCAACTTGAGCTGAGGAGGGTGGTGGCCGGCGAGGGCTCTGGCAGCGTTTCTCTTGCTCTGCACCTGCAGCCCCTGAGGAGCGCTGCTGGCGCCGCCGCCCTGGCCCTCACCGTGGACCTGCCTCCCGCCAGCAGCGAGGCCAGGAACAGCGCCTTCGGCTTCCAGGGCAGGCTGCTGCACCTGAGCGCCGGCCAGAGGCTGGGCGTGCACCTGCACACCGAGGCCAGGGCCCGGCACGCCTGGCAGCTGACCCAGGGCGCCACCGTGCTGGGCCTGTTCAGGGTGACCCCCGAGATCCCCGCCGGCCTGCCCAGCCCCAGGAGCGAGTGA.

Amino acid sequence:

the amino acid sequence corresponding to the mbIL-15 gene (SEQ ID NO. 11):

in SEQ ID No.11, the single underlined part is the amino acid sequence corresponding to IL-15, the wavy underlined part is the amino acid sequence corresponding to Linker, and the bold font is the amino acid sequence corresponding to IL-15Rα.

The amino acid sequence corresponding to the mbIL-21 gene (SEQ ID NO. 12):

in SEQ ID NO.12, the single underlined part is the amino acid sequence corresponding to IL-21, the wavy underlined part is the amino acid sequence corresponding to the CD8 hinge region, the double underlined part is the amino acid sequence corresponding to the CD8 transmembrane region, and the bold font is the amino acid sequence corresponding to the CD8 intracellular region.

4-1BBL gene (SEQ ID NO. 13): MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE.

Example 2: mbIL-15, mbIL-21 and 4-1BBL mRNA Synthesis

pUC57-mbiL-15, pUC57-mbiL-21 and pUC57-4-1BBL plasmids prepared as described in example 1 were extracted according to the instructions of the Tiangen endotoxin-free plasmid big extraction kit. The resulting plasmid was digested with XbaI restriction enzyme at 37℃overnight. The cleavage system is shown in Table 1:

table 1 XbaI restriction enzyme system

Reagent(s)	Volume of
		Plasmid(s)	10μg
Enzyme (1000 units)	5μl
		10x cutsmart buffer	50μl
Nuclease free，H ₂ O	Total 500μl

Then, the digested product was recovered by using a universal DNA gel recovery kit (Tiangen Biochemical Co., ltd.) and the concentration was measured. The digested product was identified by 1% agarose gel electrophoresis and used for in vitro transcription.

mRNA was synthesized using the T7 in vitro transcription kit (APExBIO T7 High Yield RNA Synthesis Kit) and further purified by column purification (Thermo, geneJET RNA Purification Kit) to obtain a purified linear mRNA molecule. Further cyclizing the purified linear mRNA molecules via a cyclizing system to obtain cyclic mbiL-15, mbiL-21 and 4-1BBL mRNA molecules, wherein the cyclizing system is shown in Table 2:

1) Cyclization reagent:

GTP Buffer：50mM Tris-HCl，10mM MgCl ₂ 1mM DTT, pH 7.5 or so;

2) Cyclization system and conditions:

TABLE 2 cyclization system

Solution	Volume of
		mRNA	25μg mRNA
GTP solution(20mM)	50μl
		GTP buffer	Make up to 500. Mu.l

The above solution was heated at 55℃for 15min, then placed on ice, and the above cyclic product was further purified according to GeneJET RNA Purification Kit instructions to obtain a purified cyclic mRNA molecule. The concentration was determined and identified by 1% agarose gel electrophoresis and stored at-80℃for further use.

Nucleic acid sequence:

nucleic acid sequence of mbIL-15 circular mRNA (SEQ ID NO. 14): TTTAAAACAGCTCTAGGGTTGTTCCCACCCTAGAGGCCCAAGTGGCGGCTAGCACTCTGGTATTACGGTACCTTTGTGCGCCTGTTTTATATCCCTTCCCCCATGTAACTTAGAAGATATTAAACAAAGTTCAATAGGAGGGGGTACAAACCAGTGCCACCACGAACAAACACTTCTGTTTCCCCGGTGAAGCTACATAGACTGTTCCCACGGTTGAAAGTGGCAGATCCGTTATCCGCTTTGGTACTTCGAGAAACCTAGTACCACCTTGGAATCTTCGATGCGTTGCGCTCAGCACTCAACCCCAGAGTGTAGCTTAGGTCGATGAGTCTGGACGATCCTCACTGGCGACAGTGGTCCAGGCTGCGTTGGCGGCCTACCTGTGGCGAAAGCCACAGGACGCTAGTTGTGAACAAGGTGTGAAGAGTCTATTGAGCTACCAAAGAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACCACGGAGCAAGTGCCCACAAACCAGTGGGTGGCTTGTCGTAATGCGTAAGTCTGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTTTTATCATGGCTGCTTATGGTGACAATCTAAGATTGTTATCATATAGCTATTGGATTGGCCATCCGGTGACTAACAGAGATCTTGCATACCTGTTTTTGGGTCTGACTAAACTAGATATAGTTACATTTAAAACTCTTCTTTATATCATACAGTTGAATAGTAGAAAGAGAAAATGAGAATCAGCAAGCCCCACCTGAGAAGCATCTCCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGCCTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTCCTGCTGGAGCTGCAGGTGATCAGCCTGGAGAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAATGGCAACGTGACCGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCAGCGGCGGCGGCAGCGGAGGCGGCGGCAGCGGCGGCGGGGGCAGCGGCGGAGGCGGCAGCGGCGGCGGCAGCCTGCAGATCACCTGCCCTCCCCCTATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTACAGCCTGTACAGCAGGGAGAGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTGACCGAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCCAGCCTGAAGTGCATCAGAGATCCCGCCCTGGTGCACCAGAGGCCCGCCCCTCCCAGCACCGTGACCACCGCCGGCGTGACCCCTCAGCCCGAGAGCCTGAGCCCCAGCGGCAAGGAGCCCGCCGCCAGCAGCCCCAGCAGCAACAACACCGCCGCCACCACCGCCGCCATCGTGCCCGGCAGCCAGCTGATGCCCAGCAAGAGCCCCAGCACCGGAACCACAGAGATTAGCTCTCATGAGAGCAGCCACGGCACCCCCAGCCAGACCACCGCCAAGAACTGGGAGCTGACCGCTAGCGCCAGCCACCAGCCCCCTGGCGTGTACCCCCAGGGCCACAGCGACACCACCGTGGCCATCAGCACCTCCACCGTGCTGCTGTGCGGCCTGAGCGCCGTGAGCCTGCTGGCCTGCTACCTGAAGAGCAGGCAGACACCCCCTCTGGCCAGCGTGGAGATGGAGGCCATGGAGGCTCTGCCCGTGACCTGGGGCACCAGCAGCAGAGACGAGGACCTGGAGAACTGCAGCCACCACCTGTGAAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAAAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAAGGGG.

Nucleic acid sequence of mbIL-21 circular mRNA (SEQ ID NO. 15): GGGGTTTAAAACAGCTCTAGGGTTGTTCCCACCCTAGAGGCCCAAGTGGCGGCTAGCACTCTGGTATTACGGTACCTTTGTGCGCCTGTTTTATATCCCTTCCCCCATGTAACTTAGAAGATATTAAACAAAGTTCAATAGGAGGGGGTACAAACCAGTGCCACCACGAACAAACACTTCTGTTTCCCCGGTGAAGCTACATAGACTGTTCCCACGGTTGAAAGTGGCAGATCCGTTATCCGCTTTGGTACTTCGAGAAACCTAGTACCACCTTGGAATCTTCGATGCGTTGCGCTCAGCACTCAACCCCAGAGTGTAGCTTAGGTCGATGAGTCTGGACGATCCTCACTGGCGACAGTGGTCCAGGCTGCGTTGGCGGCCTACCTGTGGCGAAAGCCACAGGACGCTAGTTGTGAACAAGGTGTGAAGAGTCTATTGAGCTACCAAAGAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACCACGGAGCAAGTGCCCACAAACCAGTGGGTGGCTTGTCGTAATGCGTAAGTCTGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTTTTATCATGGCTGCTTATGGTGACAATCTAAGATTGTTATCATATAGCTATTGGATTGGCCATCCGGTGACTAACAGAGATCTTGCATACCTGTTTGTTGGGTCTACTAAACTAGATATAGTTACATTTAAAACTCTTCTTTATATCATACAGTTGAATAGTAGAAAGAGAAAATGAGAAGCAGCCCCGGCAACATGGAGAGGATCGTGATCTGCCTGATGGTGATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCAGCCAGGGCCAAGACAGGCACATGATCAGAATGAGGCAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCCGCCCCCGAGGACGTGGAGACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAGAGCGCCAACACCGGCAACAACGAGAGGATCATCAACGTGAGCATCAAGAAGCTGAAGAGGAAGCCCCCTAGCACCAACGCCGGCAGAAGACAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCTCCCAAGGAGTTCCTGGAGAGATTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCAGAACCCACGGCAGCGAGGACAGCACCACCACCCCCGCCCCTAGGCCCCCTACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGGCCCGAGGCCTGCAGGCCCGCCGCCGGCGGCGCCGTGCACACCAGGGGCCTGGACTTCGCCTGCGATATCTACATCTGGGCCCCCTTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCACAGGAACAGAAGAAGGGTGTGCAAGTGCCCCAGGCCCGTGGTGAAGAGCGGCGACAAGCCCAGCCTGAGCGCCAGATACGTGTGAAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAAAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAA.

4-1BBL circular mRNA nucleic acid sequence (SEQ ID NO. 16): GGGGTTTAAAACAGCTCTAGGGTTGTTCCCACCCTAGAGGCCCAAGTGGCGGCTAGCACTCTGGTATTACGGTACCTTTGTGCGCCTGTTTTATATCCCTTCCCCCATGTAACTTAGAAGATATTAAACAAAGTTCAATAGGAGGGGGTACAAACCAGTGCCACCACGAACAAACACTTCTGTTTCCCCGGTGAAGCTACATAGACTGTTCCCACGGTTGAAAGTGGCAGATCCGTTATCCGCTTTGGTACTTCGAGAAACCTAGTACCACCTTGGAATCTTCGATGCGTTGCGCTCAGCACTCAACCCCAGAGTGTAGCTTAGGTCGATGAGTCTGGACGATCCTCACTGGCGACAGTGGTCCAGGCTGCGTTGGCGGCCTACCTGTGGCGAAAGCCACAGGACGCTAGTTGTGAACAAGGTGTGAAGAGTCTATTGAGCTACCAAAGAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACCACGGAGCAAGTGCCCACAAACCAGTGGGTGGCTTGTCGTAATGCGTAAGTCTGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTTTTATCATGGCTGCTTATGGTGACAATCTAAGATTGTTATCATATAGCTATTGGATTGGCCATCCGGTGACTAACAGAGATCTTGCATACCTGTTTGTTGGGTCTACTAAACTAGATATAGTTACATTTAAAACTCTTCTTTATATCATACAGTTGAATAGTAGAAAGAGAAAATGGAGTACGCCAGCGACGCCAGCCTGGACCCCGAGGCCCCCTGGCCTCCCGCTCCCAGGGCCAGAGCCTGCAGGGTGCTGCCCTGGGCCCTGGTGGCCGGCCTGCTGCTGCTCCTGCTGCTGGCTGCCGCCTGCGCCGTGTTCCTGGCCTGCCCCTGGGCTGTGAGCGGCGCTAGGGCCAGCCCCGGCAGCGCCGCCAGCCCCAGACTGAGGGAGGGCCCCGAGCTGAGCCCCGACGATCCCGCTGGCCTGTTGGATCTGAGGCAGGGCATGTTTGCCCAACTGGTGGCCCAAAATGTTCTGCTTATCGATGGCCCCCTGAGCTGGTACTCTGACCCTGGCCTGGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTATAAAGAGGACACCAAGGAGTTAGTGGTGGCCAAGGCTGGAGTGTATTATGTGTTCTTTCAACTTGAGCTGAGGAGGGTGGTGGCCGGCGAGGGCTCTGGCAGCGTTTCTCTTGCTCTGCACCTGCAGCCCCTGAGGAGCGCTGCTGGCGCCGCCGCCCTGGCCCTCACCGTGGACCTGCCTCCCGCCAGCAGCGAGGCCAGGAACAGCGCCTTCGGCTTCCAGGGCAGGCTGCTGCACCTGAGCGCCGGCCAGAGGCTGGGCGTGCACCTGCACACCGAGGCCAGGGCCCGGCACGCCTGGCAGCTGACCCAGGGCGCCACCGTGCTGGGCCTGTTCAGGGTGACCCCCGAGATCCCCGCCGGCCTGCCCAGCCCCAGGAGCGAGTGAAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAAAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAA.

Example 3: PBMC separation and extraction

A donor peripheral blood sample was collected with an anticoagulant tube and diluted by equal volumes of Du's phosphate buffer DPBS (Thermo Fisher Scientific). The diluted blood sample was carefully and slowly added to a sample density separation solution (daceae, biotechnology limited) at a volume ratio of 1:2, 700g, and centrifuged for 30min. Centrifugal acceleration was 1 and deceleration was 0. And after centrifugation, collecting the cells in the middle tunica media into a centrifuge tube, adding washing liquid (DPBS containing 5% human serum albumin) for washing, uniformly mixing, centrifuging at 1500rpm at room temperature for 10min, discarding the supernatant, and washing the cell sediment again for 2 times by the washing liquid to obtain the PBMC cells. The obtained PBMC cells can be directly transfected with the mRNAs of mbIL-15, mbIL-21 and 4-1BBL prepared in the example 2 to amplify NK cells, or can be stored in liquid nitrogen for standby after frozen storage.

Example 4: NK cell sorting

PBMC cells were obtained with reference to example 3 and further sorted using human CD3 microblads coupled magnetic beads (Miltenyi, CD3 microblads, human) or human NK cell sorting kit (Miltenyi, NK Cell Isolation Kit, human) with reference to the instructions.

The following describes in detail the sorting of NK cells by taking specific operation in the specification of a human NK cell sorting kit as an example:

Every 1X 10 after counting PBMCs ⁷ mu.L of sorting buffer was added to each cell at 1X 10 ⁷ mu.L of NK Cell Biotin-Antibody Cocktail was added to each Cell, and the cells were incubated at 4℃for 5min after mixing. Every 1X 10 after incubation is completed ⁷ mu.L of sorting buffer was added to each cell at 1X 10 ⁷ The individual cells were added to 20 mu L NK Cell Microbeads Cocktail and incubated at 4℃for 10min after homogenization. Every 1X 10 after incubation is completed ⁷ Adding 1mL of sorting buffer solution into each cell, centrifuging at 300g at room temperature for 10min, discarding supernatant, adding sorting buffer solution into cell precipitate, and adjusting cell density to 2×10 ⁸ And (3) per mL, flowing out the cell suspension through a sorting column, washing 3 times with 3 mL/time sorting buffer solution, obtaining the effluent as NK cells, collecting NK cells, directly culturing or transfecting the mRNAs of mbIL-15, mbIL-21 and 4-1BBL prepared in the example 2 to amplify the NK cells, and storing the NK cells in liquid nitrogen for standby after freezing. The non-NK cells can be directly cultured or transfected to prepare the mRNAs of mbIL-15, mbIL-21 and 4-1BBL obtained in the example 2 to amplify NK cells, or can be frozen and stored in liquid nitrogen for standby.

Example 5: mRNA molecules (mbIL-15 and 4-1BBL mRNA) electrotransfection of PBMC

Fresh or cryopreserved recovered PBMC cells were taken, centrifuged at 1500rpm at room temperature for 5min, the supernatant was discarded, the cell pellet was resuspended in electrotransfer buffer opti-MEM, and after mixing well, counted by trypan blue staining. Cells were divided into two groups according to the count: (1) An untransfected group, (2) a group of transfected mbIL-15 and 4-1BBL mRNA compositions, wherein the cells of the untransfected group are compared to the transfected group The cell ratio was 1:3. Centrifuging the cell solution requiring electrotransformation at 1500rpm at room temperature for 5min, discarding supernatant, resuspending the cell pellet with opti-MEM and adjusting cell density to 1×10 ⁸ Each mL, 100. Mu.L/tube was dispensed into 1.5mL sterile EP tubes. Each tube was charged with 20. Mu.g of mbiL-15 circular mRNA and/or 4-1BBL circular mRNA, and the final volume of opti-MEM was made up to 150. Mu.L/tube, and after air-beating and mixing, the cell suspension was transferred to an electrorotating cup. The electrotransfection conditions were set at 350V,1000 μs/time, 3 shocks, 1000ms intervals, and electrotransfection was performed by an EBXP-H1 one-day electrotransfection apparatus.

Test results: FIG. 4 shows the results of IL-15 and 4-1BBL expression detection after PBMC transfection with mbIL-15 and 4-1BBL circular mRNA, and the expression efficiency after PBMC transfection with IL-15 and 4-1BBL circular mRNA was 80% or more, and NK cells were effectively activated and amplified.

Example 6: mRNA molecules (mbIL-21 and 4-1BBL mRNA) electrotransfection of PBMC

Fresh or cryopreserved recovered PBMC cells were taken, centrifuged at 1500rpm at room temperature for 5min, the supernatant was discarded, the cell pellet was resuspended in electrotransfer buffer opti-MEM, and after mixing well, counted by trypan blue staining. Cells were divided into two groups according to the count: (1) An untransfected group, (2) a group of transfected mbIL-21 and 4-1BBL mRNA compositions, wherein the ratio of cells in the untransfected group to cells in the transfected group is 1:4. Centrifuging the cell solution requiring electrotransformation at 1500rpm at room temperature for 5min, discarding supernatant, resuspending the cell pellet with opti-MEM and adjusting cell density to 1×10 ⁸ Each mL, 100. Mu.L/tube was dispensed into 1.5mL sterile EP tubes. Each tube was charged with 20. Mu.g of mbiL-21 circular mRNA and/or 4-1BBL circular mRNA, and the final volume of opti-MEM was made up to 150. Mu.L/tube, and after air-beating and mixing, the cell suspension was transferred to an electrorotating cup. The electrotransfection conditions were set at 350V,1000 μs/time, 3 shocks, 1000ms intervals, and electrotransfection was performed by an EBXP-H1 one-day electrotransfection apparatus.

Test results: after the PBMC cells are transfected by the mbIL-21 and 4-1BBL circular mRNA, the PBMC cells can effectively express related polypeptides, and NK cells can be effectively activated and amplified.

Example 7: mRNA molecules (mbIL-15, mbIL-21 and 4-1BBL mRNA) electrotransfection of PBMC

Taking fresh or cryopreserved recovered PBMC cells, centrifuging at 1500rpm at room temperature for 5min, discarding supernatant, and collecting cellsThe pellet was resuspended in electrotransfer buffer opti-MEM and counted by trypan blue staining after homogenization. Cells were divided into two groups according to the count: an untransfected group, a group of mbIL-15, mbIL-21 and 4-1BBL mRNA compositions were transfected, wherein the ratio of untransfected cells to transfected cells was 1:5. Centrifuging the cell solution requiring electrotransformation at 1500rpm at room temperature for 5min, discarding supernatant, resuspending the cell pellet with opti-MEM and adjusting cell density to 1×10 ⁸ Each mL, 100. Mu.L/tube was dispensed into 1.5mL sterile EP tubes. Each tube was filled with 20. Mu.g of mbiL-15 circular mRNA, mbiL-21 circular mRNA and/or 4-1BBL circular mRNA, and the final volume of opti-MEM was made up to 150. Mu.L/tube, and after pipetting, the cell suspension was transferred to an electrorotating cup. The electrotransfection conditions were set at 350V,1000 μs/time, 3 shocks, 1000ms intervals, and electrotransfection was performed by an EBXP-H1 one-day electrotransfection apparatus.

Test results: after the PBMC cells are transfected by the mbIL-15, mbIL-21 and 4-1BBL circular mRNA, the PBMC cells can effectively express related polypeptides, and NK cells can be effectively activated and amplified.

Example 8: mRNA molecules (mbIL-15 and 4-1BBL mRNA) electrotransfected and sorted non-NK cells

Reference example 4 sorted NK cells and non-NK cells were obtained using a human NK cell sorting kit, counted and resuspended in NK cell expansion medium, and transferred into a suitable flask for culture. Wherein the NK cell expansion medium is KBM502 medium (Corning, KBM502, serum-free medium for NK and CTL) containing 500U/mL IL-2 (Beijing Shuanglu pharmaceutical Co., ltd., recombinant human interleukin-2 for injection). Centrifuging all non-NK cells after sorting at 1500rpm at room temperature for 5min, discarding supernatant, resuspending cell pellet with opti-MEM and adjusting cell density to 1×10 ⁸ Each mL, 100. Mu.L/tube was dispensed into 1.5mL sterile EP tubes. Each tube was charged with 20. Mu.g of mbiL-15 circular mRNA and 4-1BBL circular mRNA, and with opti-MEM added to a final volume of 150. Mu.L/tube, and after air-beating and mixing, the cell suspension was transferred to an electrorotating cup. The electrotransfection conditions were set at 350V,1000 μs/time, 3 shocks, 1000ms intervals, and electrotransfection was performed by an EBXP-H1 one-day electrotransfection apparatus. Adding non-NK cells after electrotransformation into non-electrotransformation NK cells, and supplementing NK cell expansion medium to adjust cell density to 4×10 ⁶ Activation and expansion of NK cells per mL。

Test results: after the mbIL-15 and 4-1BBL circular mRNA are transfected into non-NK cells in PBMC cells, the non-NK cells can effectively express related polypeptides, and NK cells can be effectively activated and amplified.

Example 9: mRNA molecule (mbIL-15 and 4-1BBL mRNA) and NK cells were amplified by two electrotransfection

Reference example 4 sorted NK cells and non-NK cells were obtained using a human NK cell sorting kit, counted and resuspended in NK cell expansion medium, and transferred into a suitable flask for culture. Centrifuging a part of non NK cells after sorting at 1500rpm at room temperature for 5min, discarding supernatant, resuspending cell pellet with opti-MEM and adjusting cell density to 1×10 ⁸ Each mL, 100. Mu.L/tube was dispensed into 1.5mL sterile EP tubes. Each tube was charged with 20. Mu.g of mbiL-15 circular mRNA and 4-1BBL circular mRNA, and with opti-MEM added to a final volume of 150. Mu.L/tube, and after air-beating and mixing, the cell suspension was transferred to an electrorotating cup. The electrotransfection conditions were set at 350V,1000 μs/time, 3 shocks, 1000ms intervals, and electrotransfection was performed by an EBXP-H1 one-day electrotransfection apparatus. Adding non-NK cells after electrotransformation into non-electrotransformation NK cells, and supplementing NK cell expansion medium to adjust cell density to 4×10 ⁶ And (3) activating and amplifying NK cells per mL. The remaining non-NK cells were either cultured in vitro continuously or frozen in liquid nitrogen for subsequent secondary transfection. After NK cells were cultured for 7 days, cultured or frozen non-NK cells were centrifuged at 1500rpm at room temperature for 5min, the supernatant was discarded, and the cell pellet was resuspended in opti-MEM and the cell density was adjusted to 1X 10 ⁸ Each mL, 100. Mu.L/tube was dispensed into 1.5mL sterile EP tubes. Each tube was charged with 20. Mu.g of mbiL-15 circular mRNA and 4-1BBL circular mRNA, and with opti-MEM added to a final volume of 150. Mu.L/tube, and after air-beating and mixing, the cell suspension was transferred to an electrorotating cup. The electrotransfection conditions were set at 350V,1000 μs/time, 3 shocks, 1000ms intervals, and electrotransfection was performed by an EBXP-H1 one-day electrotransfection apparatus. Adding the non-NK cells after electrotransformation into NK cells cultured for 7 days, and continuing to amplify the NK cells after uniformly mixing.

Test results: non-NK cells in PBMC cells transfected with mbiL-15 and 4-1BBL circular mRNA can be effectively stimulated to expand by adding to NK cells in batches.

Example 10: NK cell in vitro expansion

The non-electrokinetic PBMC cells were centrifuged at 1500rpm for 5min at room temperature and the supernatant discarded. The cell pellet is resuspended in NK cell expansion medium and transferred into a suitable culture flask for culture. Adding PBMC cells of the electrotransfected mRNA composition to untransfected PBMC cells, and adding NK cell expansion medium to adjust cell density to 4×10 ⁶ Gently mixing the materials at 37 ℃ and 5 percent CO per mL ₂ Culturing in an incubator. Observing cell state every day, supplementing NK cell expansion medium according to cell proliferation condition, sampling every 1 day after cell culture for 7 days to measure NK cell purity and count, and regulating cell density to 1×10 ⁶ And culturing in each mL. NK cells can be further genetically modified by mRNA compositions for functional assays after 12 days of in vitro culture.

Test results: as shown in FIG. 5 and FIG. 6, the NK cell purity between different individuals after 14 days of culture is over 80% and the cell expansion multiple can reach 400 times by using the culture method provided by the embodiment.

Example 11: gene modification of NK cells by delivery of mbIL-15 and 4-1BBL mRNA via Lipid Nanoparticles (LNP)

The mbIL-15 and 4-1BBL circular mRNA molecules were obtained as described in reference example 2. Further, by dissolving ionizable cationic lipid, neutral helper phospholipid, cholesterol and polyethylene glycol modified phospholipid in ethanol at a molar ratio of 50:10:38.5:0.75, mbIL-15 and 4-1BBL cyclic mRNA were dissolved in 50mM citrate buffered saline solution at pH 4.0 to give mRNA concentrations of 30 μg/mL, respectively. The volume ratio of the ethanol solution to the citric acid solution is 1:3, and the ethanol is removed by dialysis or tangential flow continuous dilution after the two phases are rapidly mixed by a microfluidic technology. And due to the reduced solubility of the lipid, gradually precipitating and solidifying in the mixed solution to form lipid nanoparticles, and simultaneously, efficiently encapsulating mRNA, thereby obtaining LNP-mbiL-15 and LNP-4-1BBL.

Collecting NK cells with good growth, centrifuging at 1500rpm at room temperature for 10min, discarding supernatant, and re-suspending cell pellet with NK cell amplification medium to cell density of 1×10 ⁶ LNP-mbiL-15 and LNP-4-1BBL prepared in this example were added to the cell suspension at a final concentration of 15. Mu.g/mL, and mixed wellTransferring the cell suspension into a proper culture container, wherein the temperature is 37 ℃ and the concentration of CO is 5% ₂ After culturing in an incubator for 1h, the culture solution is replaced. Collecting cells, centrifuging at 1500rpm at room temperature for 10min, discarding supernatant, and re-suspending cell pellet with NK cell amplification medium to cell density of 1×10 ⁶ Individual/mL, continued at 37 ℃,5% CO ₂ Culturing in an incubator.

Test results: the mbIL-15 and 4-1BBL annular mRNA delivered by LNP carry out gene modification on NK cells, and both molecules can be effectively expressed, so that the survival and proliferation of NK cells can be promoted, the systemic immune system can be activated after the NK cells are applied to the body, and the anti-tumor effect is improved.

Example 12: genetic modification of NK cells by delivery of mbIL-15mRNA by electrotransfection

Well grown NK cells were taken, centrifuged at 1500rpm at room temperature for 10min, the supernatant was discarded, the cell pellet was resuspended in electrotransfer buffer opti-MEM, and after passing through a 70 μm cell screen, samples were taken and counted by trypan blue staining. Centrifuging the cell solution at 1500rpm at room temperature for 10min, discarding supernatant, resuspending the cell pellet with opti-MEM and adjusting cell density to 1×10 ⁸ The mIL-15 circular mRNA was added at 300. Mu.g/mL and the opti-MEM was added to give a final cell suspension concentration of 6.67X 10 ⁷ And each mL. After being blown and mixed uniformly, the centrifuge tube containing the cell suspension is connected with an electrotransport F1 system consumable (EBXP-F1 consumable package of Yida biotechnology Co., suzhou). The electrotransformation condition is set to 250V,1000 mu s/time, electric shock is carried out 3 times, the flow rate is 4.71mL/min, and the electrotransformation operation is carried out through an EBXP-F1 one electric transfection instrument. Transferring the cells into NK cell expansion medium after electrotransformation, placing the cell suspension at 37 ℃ and 5% CO ₂ Culturing in an incubator.

Test results: the mIL-15 annular mRNA is delivered through electrotransfection to carry out gene modification on NK cells, the mIL-15 annular mRNA can be effectively expressed, the proliferation of the NK cells is promoted, the survival time of the NK cells is prolonged, and the mIL-15 annular mRNA can activate the whole body immune system after being applied to the body, thereby improving the anti-tumor effect.

Example 13: gene modification of NK cells by electrotransfection delivery of mbIL-15mRNA, mbIL-21mRNA and 4-1BBL mRNA

Collecting NK cells with good growth, centrifuging at 1500rpm at room temperature for 10min, and discarding supernatantThe cell pellet was resuspended in electrotransfer buffer opti-MEM, and after passing through a 70 μm cell screen, samples were counted by trypan blue staining. Centrifuging the cell solution at 1500rpm at room temperature for 10min, discarding supernatant, resuspending the cell pellet with opti-MEM and adjusting cell density to 1×10 ⁸ Adding mbIL-15 circular mRNA, mbIL-21 circular mRNA and 4-1BBL circular mRNA to final concentration of 300 μg/mL, adding opti-MEM to final concentration of 6.67×10 cell suspension ⁷ And each mL. After being blown and mixed uniformly, the centrifuge tube containing the cell suspension is connected with an electrotransport F1 system consumable (EBXP-F1 consumable package of Yida biotechnology Co., suzhou). The electrotransformation condition is set to 250V,1000 mu s/time, electric shock is carried out 3 times, the flow rate is 4.71mL/min, and the electrotransformation operation is carried out through an EBXP-F1 one electric transfection instrument. The cells after electrotransformation are transferred into NK cell expansion culture medium which is KBM502 culture medium (Corning, KBM502, serum-free medium for NK and CTL) containing 500U/mL IL-2 (Beijing Shuanglu pharmaceutical Co., ltd., recombinant human interleukin-2 for injection). The cell suspension was placed at 37℃in 5% CO ₂ Culturing in an incubator.

Test results: as shown in FIG. 7, the mIL-15 circular mRNA, the mIL-21 circular mRNA and the 4-1BBL circular mRNA are delivered through electrotransfection to carry out gene modification on NK cells, and three molecules are expressed at 85% -95%, so that the survival and proliferation of NK cells are promoted, and the anti-tumor effect is improved after the anti-tumor agent is applied to the whole body immune system.

Example 14: in vitro killing HEPG2 function detection of mbIL 15mRNA gene modified NK cells

Referring to example 9, NK cells modified mbIL-15 circular mRNA were obtained. HEPG2 cells well grown were subjected to cellrace Violet dye (Thermo, cellrace ^TM Violet Cell Proliferation Kit) post-marking plate 48-well plate, 8×10 ⁴ And/or holes. Collecting NK cells of the transgenic 24h modified mbIL-15 annular mRNA, washing the NK cells without the transgenic NK cells, plating the NK cells in the effective target ratio of 2:1, 1:1, 1:2, 1:4 and 1:8, uniformly mixing the cells, and then placing the cells at 37 ℃ and 5% CO ₂ The incubator cultures for 4 hours. After 4h, cells per well are collected and stained with dead cells such as PI, 7AAD, eBioscience ^TM Fixable Viability Dye eFluor ^TM 780, etc. after marking, the flow type detection body is in vitroKilling efficiency.

Test results: FIG. 8 is a graph showing the effect of NK cell in killing HEPG2 after in vitro modification of mbiL-15 circular mRNA. Because the IL-15 has the main function of activating the whole body immune system after being applied in vivo, the killing effect of the modified mbIL-15 is similar to that of unmodified NK cells in the in vitro killing process without obvious difference.

Example 15: NK cell gene modified mbIL-15mRNA in vivo killing HEPG2 function detection

Referring to example 9, NK cells modified mbIL-15 circular mRNA were obtained. Taking 6-8 week old male Balbc nude mice, subcutaneously injecting 3×10 ⁶ HEPG2-luciferase tumor cells. After 7 days of molding, the mice were divided into three groups: blank, NK, and NK-modified mbIL-15mRNA (NK-IL-15) groups. Tail vein injection of 1X 10 of NK group and NK-IL-15 group mice every 1-2 days ⁷ The NK cells and NK-modified mbIL-15 circular mRNA cells were dosed 6 times in total. Weekly imaging observations, mice were sacrificed 4 weeks after molding, and tumor tissue was isolated to observe tumor size. NK-IL-15 group showed good antitumor effect in HEPG2 tumor-forming nude mice.

In summary, the present invention provides an mRNA composition for highly efficient NK cell expansion and a genetically modified NK cell pharmaceutical preparation based thereon, wherein the composition of one or more mRNAs selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, 4-1BBL, CD 16. Alpha., IFN-. Gamma.is transfected into PBMC cells, non-NK cells and/or NK cells in PBMC cells by one or more electrotransformation or the like, and NK cells satisfying the number of clinical applications can be obtained by co-culturing with PBMC cells and/or NK cells not containing an RNA composition in vitro after continuous culture in NK cell expansion medium. Exogenous feeder cells are not introduced in the whole culture process, so that the use of cytokines is reduced, the in-vitro culture cost of NK cells is reduced, the safety is higher, and the repeatability is good. The NK cells obtained are further subjected to genetic modification by the mRNA composition, so that the survival of the NK cells can be prolonged, the NK cells can be killed by themselves, and the whole immune system can be activated, so that the NK cells have a good application prospect.

The technical means and test schemes related to the present invention have been described above, but the scope of the present invention is not limited thereto by way of example only. Alterations and modifications as would occur to those skilled in the relevant art are intended to be included within the scope of the invention.

Claims

1. An RNA composition for expanding NK cells encoding a polypeptide of interest comprising one or more of the group consisting of an IL-2 polypeptide, an IL-7 polypeptide, an IL-12 polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, an IL-21 polypeptide, a 4-1BBL polypeptide, a CD16 a polypeptide and an IFN- γ polypeptide.

2. The RNA composition of claim 1, wherein the RNA composition comprises one or more of the following mrnas as shown in (a) to (c), or wherein the RNA composition comprises an mRNA as shown in (d):

(d) A fourth mRNA comprising a fourth coding sequence encoding a recombinant polypeptide, wherein the fourth coding sequence comprises any two or more of: a first coding sequence encoding an IL-15 polypeptide, a second coding sequence encoding an IL-21 polypeptide, a third coding sequence encoding a 4-1BBL polypeptide;

Alternatively to this, the method may comprise,

the third coding sequence comprises a sequence as set forth in SEQ ID NO.10, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO.10 and encoding a polypeptide having or partially having 4-1BBL polypeptide activity;

alternatively to this, the method may comprise,

3. The RNA composition of claim 1 or 2, wherein each mRNA in the RNA composition is independently selected from linear mRNA or circular mRNA;

preferably, each mRNA in the RNA composition is a circular mRNA.

4. The use of the RNA composition of any one of claims 1 to 3 in at least one of the following (e) to (f):

(e) Amplifying NK cells;

(f) NK cells were genetically modified.

5. A method of expanding NK cells comprising the steps of (i) to (ii) as follows:

(i) The step of preparing feeder cells: delivering the RNA composition of any one of claims 1-3 to a cell to be treated, obtaining a feeder cell comprising the RNA composition; wherein the cells to be treated are PBMC cells, non-NK cells and/or NK cells in PBMC cells;

(ii) Amplifying and culturing NK cells: adding the feeder cells to be expanded which do not contain the RNA composition in one time or in batches for co-culture; wherein the cells to be amplified are PBMC cells and/or NK cells;

optionally, the source of NK cells comprises one or more of NK-92 cell lines, peripheral blood NK cells, umbilical cord blood NK cells, and pluripotent stem cell-derived NK cells;

optionally, the means of delivery includes one or more of electrotransfection, LNP delivery, and exosome delivery.

6. The method of claim 5, wherein in the step of preparing feeder cells, each RNA in the RNA composition is used in an amount that is independent of the ratio of the number of cells to be treated to (10 μg to 50 μg): (1X 10) ⁷ Individual). Preferably, the total amount of RNA composition is 1 μg to 1mg;

7. A method of genetically modifying NK cells comprising the step of delivering the RNA composition of any one of claims 1-3 into an NK cell to be modified;

optionally, the source of NK cells comprises one or more of NK-92 cell lines, peripheral blood NK cells, umbilical cord blood NK cells, and pluripotent stem cell-derived NK cells; preferably, the NK cells are NK cells amplified by the method according to claim 5 or 6;

optionally, the means of delivery comprises one or more of electrotransfection, LNP delivery, exosome delivery;

alternatively, the amount of each RNA in the RNA composition is each independently in proportion to the number of NK cells to be modified (10. Mu.g to 50. Mu.g): (1X 10) ⁷ Individual). Preferably, the total amount of RNA composition is 1 μg to 100mg.

8. A genetically modified NK cell produced by the method of claim 7.

9. A pharmaceutical composition comprising the genetically modified NK cell of claim 8 and a pharmaceutically acceptable carrier.

10. Use of a genetically modified NK cell according to claim 8 and/or a pharmaceutical composition according to claim 9 for the manufacture of a medicament for the prevention and/or treatment of cancer.