Il-12 activates NK cells transfected with car rna-lnp

Abstract

The present disclosure provides a method for preparing natural killer (NK) cells with increased secretion of cytokines and increased cytotoxicity. The present invention uses IL-12 during transfection to generate CAR-NK cells with increased functional activities (cytotoxicity and secretion of cytokines) against tumor cells.

Description

[0001] IL-12 ACTIVATES NK CELLS TRANSFECTED WITH CAR RNA-LNP

[0002] REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0003] This application contains an ST.26 compliant Sequence Listing, which was submitted in xml format via Patent Center and is hereby incorporated by reference in its entirety. The .xml copy, created on March 3, 2025 is named “Sequence Listing 119995-8061W001.xml” and is 9,200 bytes in size.

[0004] FIELD OF THE INVENTION

[0005] The present invention related to the addition of IL- 12 during transfection of NK cells to boost functional activities of CAR mRNA-LNP (lipid nanoparticle)-transfected NK cells. CAR-NK cells produced with added low dose of IL- 12 during transfection maintain high viability and increase production of INF-gamma to attack tumor cells compared to CAR-NK cells generated without IL- 12 during transfection.

[0006] BACKGROUND OF THE INVENTION

[0007] Natural Killer (NK) Cells are lymphocytes in the same family as T and B cells, coming from a common progenitor. However, as cells of the innate immune system, NK cells are classified as group I Innate Lymphocytes and respond quickly to a wide variety of pathological challenges. NK cells are best known for killing virally infected cells and detecting and controlling early signs of cancer.

[0008] NK cells were first noticed for their ability to kill tumor cells without any priming or prior activation. They are named for this natural killing. Additionally, NK cells secrete cytokines such as IFN-y and TNF-a, which act on other immune cells like macrophage and dendritic cells to enhance the immune response.

[0009] Immunotherapy is emerging as a highly promising approach for the treatment of cancer. NK cells as the armed forces of our immune system, constantly look for foreign antigens and discriminate abnormal (cancer or infected cells) from normal cells.

[0010] Chimeric antigen receptor (CAR)-T cells recently were approved by FDA to treat hematological cancers (leukemia, lymphoma, and multiple myeloma) and demonstrated highly promising results (1-4). CAR-T cell therapy made impressive advancement in the field of cancer therapy but has several limitations such as cytokine release storm (CRS), neurotoxicity and challenges to target solid tumors (5, 6). Another type of promising cell therapy against cancer is CAR-NK cells (5), (7). One of the advantages of NK cells is the low risk of graft- versus-host disease (GVHD) and low toxicity (8, 9). NK cells are also good candidates for allogeneic cell therapy as they are independent of HLA-TCR recognition signaling of T cells (10).

[0011] CAR-NK cells were used in preclinical studies against B-cell malignancies (7, 11, 12), multiple myeloma (13, 14), and against solid tumors such as glioblastoma (15, 16), breast (17, 18) and ovarian cancers (19). There are several clinical trials ongoing with CAR- NK cells against hematological and solid tumors (5) which support use of CAR-NK cells against different cancers.

[0012] The use of viral vectors is associated with high cost, regulatory requirements, and some safety concern (20). There are several non-viral methods such as RNA electroporation and DNA transfection, but these methods have limitations due to low efficiency of transfection (20). Development of non-viral delivery of CAR to NK cells has advantages for manufacturing due to lower cost, easier than viral CAR preparation and convenience for development of allogeneic off-the-shelf CAR-NK cells. However, there are several challenges such as the lower efficacy of the CAR-NK due to transient delivery.

[0013] IL- 12 is a cytokine that stimulates T cells and NK cells to secrete IFN-gamma and increases cytotoxic activity of the cells. Induction of IFN-gamma by IL-12 has been shown to block tumor growth. IL- 12 is a heterodimeric cytokine which is formed of two subunits p40 and p35 that are linked by 3 disulfide bridges to form a p70 protein. IL- 12 was evaluated in the treatment of many tumors in the clinic.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows a DNA template used for in vitro transcription (panel A), and the generation of CAR mRNA with 5-cap, 5’UTR, 3’UTR and poly A tail (panel B).

[0016] FIG. 2 shows high viability (>80%) of CD19-CAR-NK 24 hours after transfection of expanded NK cells in NK medium (AIM-V, 10% FBS with IL-2 (10 ng / mL) and IL-15 (5 ng / mL). IL- 12 (25 pg / ml-10 ng / ml) was added to NK cells during transfection with CAR- mRNA-LNP for 22-24 hours. The data shown are combined from two independent experiments, (i) lxlOA6 NK cells were transfected with 5 pg / mL of CAR-mRNA, with 25- 100 pg / ml IL-12 added, (ii) lxlOA6 NK cells were transfected with 10 pg / mL of CAR- mRNA with 400 pg / mL to 2 ng / mL IL-12. The viability of CAR-NK cells with 25-100 pg / mL of IL- 12 added during transfection was high. FIG. 3 shows increased secretion of IFN-gamma by CD19 CAR-NK cells with IL-12 added during transfection, against Nalm-6 leukemia cells. NK cells were transfected with CD19 CAR mRNA-LNP either without IL-12 or with IL-12 (25-100 pg / ml). Effector (NK / CAR-NK) to target (Nalm-6) cells (E:T) ratio was 5: 1. *p<0.05, CAR-NK vs NK cells, and cells with IL- 12 vs cells with no IL- 12 by Student’s t-test.

[0017] FIG. 4 shows cytotoxicity by RTCA assay of CD19-CAR-NK, GFP-NK cells with Nalm-6 target cells. To generate CD19-CAR-NK and GFP-NK, NK cells were transfected with CD19-CAR mRNA-LNP or GFP mRNA-LNP, respectively, in the presence of IL- 12 at 100 pg / ml for 22 h. Thirty thousand target cells were used for the RTCA assay with different effector to target cell ratio 0.5:1, 1:1 and 2: 1. NK cells from two donors 009 and 710 were used for the assay.

[0018] FIG. 5A-5C show cytokine secretion assay by ELISA using NK cells, CD19-CAR- NK, and GFP-NK, after transfection with mRNA-LNP in the presence of IL- 12 (100 pg / ml) with Nalm-6 leukemia target cells. The supernatant after RTCA assay (FIG.4) was used for ELISA. 5A: IFN-gamma; 5B: Granzyme B; 5C: TNF-alpha secretion. P<0.05 CD19-CAR- NK cells versus control NK cells, Student’s t-test.

[0019] FIG. 6 A and 6B show increased secretion of IFN-gamma by BCMA-CAR-NK cells with multiple myeloma target cells in the presence of IL-12. 6A: MM1S multiple myeloma target cells. 6B: RPMI8226 multiple myeloma target cells. 11-12 was added to the transfection medium at 100 pg / mL. Ratios shown are NK cells to target cells.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Definitions

[0022] As used herein, “about” refers to ± 10% of the recited value.

[0023] As used herein, “activated NK cells” means NK cells activated for proliferation (expansion) and cell killing.

[0024] As used herein, a "chimeric antigen receptor (CAR)" is a receptor protein that has been engineered to give T cells the new ability to target a specific protein. The receptor is chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. CAR is a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. The "extracellular domain capable of binding to an antigen" means any oligopeptide or polypeptide that can bind to a certain antigen. The "intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.

[0025] The "intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.

[0026] As used herein, a "domain" means one region in a polypeptide which is folded into a particular structure independently of other regions.

[0027] As used herein, “feeder cells” consist in a layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate.

[0028] As used herein, “humanized antibodies” are antibodies from non-human species whose non-CDR sequences have been modified to increase their similarity to antibody variants produced naturally in humans.

[0029] As used herein, a "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the ScFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for engineering ScFv are known to a person skilled in the art.

[0030] As used herein, a "tumor antigen" means a biological molecule having expression of which causes cancer.

[0031] Natural Killer (NK) cells are type of cytotoxic lymphocytes which are critical for innate immune system. Engineering NK cells with chimeric antigen receptor (CAR) allows CAR-NK cells to target tumor antigens. The present application uses CAR mRNA-LNP (lipid nanoparticle) technology to effectively transfect NK cells expanded from primary peripheral blood mononuclear cells (PBMC) and to generate functional CAR-NK cells. The nanoparticle -based mRNA delivery provides many advantages, such as high stability, bioavailability, solubility, and low toxicity.

[0032] Systemic administration of IL- 12 has been shown to be toxic for patients. The delivery of IL- 12 would be more effective and less toxic if delivered in the tumor or tumor microenvironment. The present invention uses a low dose of IL- 12 in the transfection medium to generate CAR-NK cells and boost the functional activities (cytotoxicity and secretion of cytokines) of CAR-NK cells against tumor cells, without sacrificing the viability of the NK cells. The present disclosure provides a method for preparing natural killer (NK) cells with increased cytotoxicity and increased secretion of cytokines. The method comprises the steps of: (a) obtaining a mRNA and lipid nanoparticles (LNPs) complex, wherein the mRNA comprises (i) 5'-UTR (untranslated region) coding sequence, (ii) a chimeric antigen receptor fusion protein (CAR) coding sequence that target a tumor antigen, (iii) a 3'-UTR coding sequence, and (iv) a poly A tail sequence; (b) transfecting the mRNA-encapsulated LNPs into NK cells in a medium comprising IL-2, IL15, and 25-100 pg / ml of IL-12, and (c) translating the mRNA in the NK cells to produce the CAR, whereby the NK cells have increased cytotodicity and / or increased secretion of cytokine such as y-interferon.

[0033] The CAR comprises from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) against the tumor antigen, (ii) a transmembrane domain, (iii) at least one costimulatory domain, and (iv) an activating domain.

[0034] In one embodiment, the tumor antigen is BCMA, Her-2, HER-2-t2A-GM-CSF, CD47, CD19, CS1, or Claudin 18.2,

[0035] The mRNAs are embedded in LNPs with an average size in the range of 30-250 nm, or 50-150 nm, or 70-120 nm.

[0036] The present method generates CAR mRNA and deliverers CAR mRNA to activated NK cells to target tumor cells or mating tumor cells. There are several advantages of this delivery: one advantage is to lower costs in manufacturing CAR. mRNAs are generated and delivered inside LNPs and transfected to immune cells, and CAR RNAs are then translated inside immune cells. A second advantage is that there is no viral delivery of CAR that can potentially cause insertion of sequences in different genomic sites to generate unfavorable effects. A third advantage is that the CAR delivery by mRNA is transient compared to viral which persists for several weeks. All these advantages can generate safer CAR-NK or CAR- other types of immune cells to be used against cancer.

[0037] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl] amino] -octanoic acid, 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), Cholesterol, and l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000). [LNP-102 (ii)]

[0038] In another embodiment, the lipid nanoparticles comprise SM-102, DSPC, Cholesterol, or l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)- 2000 (DSPE-PEG2000-MAL). In another embodiment, the lipid nanoparticles comprise 2-hexyLdecanoic acid, 1,1'- [[(4-hydroxybutyl)imino]di-6,l-hexanediyl] ester (ALC-0315), DSPC, Cholesterol, and a-[2- (ditetradecylamino)-2-oxoethyl]-<n-methoxy-poly(oxy-l,2-ethanediyl) (ALC-0159).

[0039] Encapsulating mRNA into LNP nanoparticles provides protection of mRNA from degradation and increases the stability mRNA; mRNA is then released from LNPs into cells in vivo to generate protein. mRNA-lipid nanoparticle preparation is described in Schoenmaker (International J. Pharmaceutics, 601: 120856, 2021), the article is incorporated herein by reference in its entirety, regarding the LNPs.

[0040] In a preferred embodiment, the mRNA comprises 5 ’-cap, 5 -end to 5’-UTR, which stabilizes mRNA (FIGs. 1A and IB). All eukaryotic mRNA contains a cap structure - an N7- methylated guanosine (Cap 0) linked to the first nucleotide of the RNA via a reverse 5' to 5' triphosphate linkage. In addition to its essential role of cap-dependent initiation of protein synthesis, the mRNA cap also functions as a protective group from 5' to 3' exonuclease cleavage and a unique identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation and nuclear export. It also acts as the anchor for the recruitment of initiation factors that initiate protein synthesis and the 5' to 3' looping of mRNA during translation. 2'0 methylation of +1 nucleotide (Cap 1) may be central to the non-self-discrimination of innate immune response against foreign RNA.

[0041] The mRNA is transcribed with RNA polymerase in vitro from a DNA sequence comprising (a) a promoter coding sequence, (b) the 5’-UTR coding sequence, (c) the CAR coding sequence, (d) the 3’-UTR coding sequence, and (e) the poly A tail sequence. The poly A tail sequence improves stability and protein translation.

[0042] FIGs. 1A-1B show a linearized DNA template to be used for in vitro transcription with RNA polymerase and nucleotide triphosphates (NTPs) to generate CAR mRNA. The DNA template contains T7 / T7AG or SP6 promoter, followed by 5’UTR (untranslated region), the coding region of CAR, 3’UTR, and >100 poly A tail for RNA stability. The generated mRNA contains a 5 ’-cap such as Cap 0, Capl, or ARCA for increased stability. mRNAs are encapsulated in LNPs to provide protection from degradation and to increase stability. The mRNA-LNPs are transfected and released into NK cells in vivo to generate protein.

[0043] In the DNA sequence, the promoter may be T7, T7AG promoter. Poly A tail sequence is from 20-170 nucleotides. Poly A tail sequence optionally comprises one or more linkers in between the poly A segments. If poly A tail is longer than 60 nucleotides, then it typically contains a linker which includes non-adenosine nucleotides. A linker is 5-30 or 5-25 nucleotides, e.g., 10 nucleotides or 20 nucleotides. In yet another example, poly A tails are 150-160 nucleotides in length, consisting of two linker sequences.

[0044] DNA expression is finely regulated at the post-transcriptional level. Untranslated regions are not translated into amino acids. However, UTRs of mRNAs may control the translation, degradation and localization of stem-loop structures, upstream initiation codons and open reading frames, internal ribosome entry sites and various cis-acting elements that are bound by RNA-binding proteins. UTRs are important in the post-transcriptional regulation of DNA expression, including modulation of the transport of mRNAs out of the nucleus and of translation efficiency, subcellular localization, and stability.

[0045] 5’-UTR typically has 10-1000 nucleotides, or 20-500 nucleotides, or 30-200 nucleotides, or 30-100 nucleotides. For example, 5’-UTR is 40-60 nucleotides (e.g., 50 nucleotides). 3’-UTR typically has 10-3000 nucleotides, for example, 50-500 nucleotides, or 100-300 nucleotides. Preferred 5’-UTRs and 3’-UTRs are UTRs of P-globin, or UTRs of Pfizer CO VID vaccine.

[0046] P-Globin gene is shown in: www.ncbi.nlm.nih.gov / nucleotide / V00497. l?report=genbank&log$=nuclalign&blast rank=5 &RID=TDDZ1K98O16

[0047] In one embodiment, the 5 '-untranslated region is derived from human alpha-globin RNA with an optimized Kozak sequence. The 3 '-untranslated region comprises two sequence elements derived from the amino-terminal enhancer of split (AES) mRNA and the mitochondrial encoded 12S ribosomal RNA to confer RNA stability and high total protein expression.

[0048] Any suitable vector, such as Vector pSP64 Poly(A) (Promega) or pGEM3Z-Vektor (Promega) can be used as a cloning vector for the DNA sequence described above.

[0049] For example, to engineer the pEM3Z-P-globin UTR-UTR-poly A tail, the 3’-UTR of the P-globin molecule flanked by restriction enzyme site can be amplified from human bone marrow. For example, a single (pEM3Z-l P-globin- UTR- A

[0120] ) or 2 serial fragments (pEM3Z-2p-globin-UTR-A

[0120] ) can be inserted in front of the poly(A) tail.

[0050] In general, a chimeric antigen receptor fusion protein (CAR) comprises from N- terminus to C-terminus: (i) a single-chain variable fragment (scFv) against a tumor antigen, (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activating domain.

[0051] In CAR, the co-stimulatory domain is selected from the group consisting of CD28, 4- 1BB, GITR, ICOS-1, CD27, OX-40 and DAP10 domains. A preferred co-stimulatory domain is CD28 or 4- IBB.

[0052] In CAR, a preferred activating domain is CD3-zeta (CD3 Z or CD3Q.

[0053] In CAR, the transmembrane domain may be derived from a natural polypeptide or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a transmembrane domain of a T cell receptor a or P chain, a CD3 zeta chain, CD28, CD3s., CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, 1COS, CD154, or a G1TR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. It is preferable that a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or a polypeptide linker, for example, a linker having a length of 2 to 10 amino acids can be arranged between the transmembrane domain and the intracellular domain. In one embodiment, a linker sequence having a glycine-serine continuous sequence can be used.

[0054] Different CARs against different tumor antigens are inserted into DNA template vector with T7 promoter for RNA polymerase to generate CAR mRNA by in vitro transcription. Tumor antigens include BCMA, Her-2, HER-2-t2A-GM-CSF, CD47, CD19, CS1, or Claudin 18.2. Then the mRNA is transfected into expanded immune cells such as primary NK cells, which is then used to kill tumor cells. In one embodiment, the NK cells are expanded with either P21 particles of K562 cells expressing 41BBL and IL21, or NK-92 cells to translate inside these cells CAR. In one embodiment, the expanded NK cells are frozen and thawed before transfected with mRNA.

[0055] The CAR expressed in NK cells can be detected against ScFv antibodies, using antimouse or anti-human FAB detecting any ScFv, or using different tag antibody to detect ScFv with fused tag (Flag, c-myc, HA, His, TF, or any other tag). The tag is useful when no antibody is known to detect scFv.

[0056] In the present method, a low dose of IL- 12 is added to the medium during transfection of NK cells with CAR mRNA-LNP. The inventors have discovered that low doses (25-100 pg / ml) of IL- 12 added to NK transfection medium containing IL-2 and IL- 15 result in high viability (>80% viable cells) of NK cells. However, high doses (>400 pg / ml) of IL- 12 added to NK medium during transfection result in CAR-NK with a lower viability. The NK transfection medium typically contains serum (fetal bovine serum or human serum), IL-2 and IL- 15. For example, a typical transfection medium may contain AIM-V, 10% FBS, IL-2 (lOng / mL) and IL-15 (5ng / mL). The present method adds low dose IL-12 to a NK transfection medium for transfecting NK cells with LNP-CAR.

[0057] By adding a low dose of IL- 12 (25- 100 pg / ml) to the transfection medium of NK cells, the NK cells boost IFN-gamma, TNF-alpha and Granzyme B secretion, while maintaining viability of CAR-NK cells.

[0058] By adding a low dose of IL- 12 (25-100 pg / ml) to the transfection medium of NK cells, the CAR-NK cells increase cytotoxicity against target cells.

[0059] In one preferred embodiment, the NK cells are expanded before transfection. The NK cells are expanded by (i) obtaining mitomycin-treated or gamma ray-irradiated K562 feeder cells that express IL21 and 4- IBB Ligand (41BBL), (ii) combining NK cells and the treated K562 feeder cells in a proper ratio, and (iii) incubating the mixture in an expansion medium comprising IL-2, and IL- 15 and expanding the NK cells.

[0060] In one embodiment, the NK cells are expanded at least 500-2000-fold before RNA- LNP transfection to have high killing activity and INF-gamma secretion.

[0061] The present application demonstrates non-viral delivery of CAR mRNA to expanded NK cells from primary PBMC cells using mRNA-LNP technology. NK cells are expanded from primary PBMC using K562 feeder cells expressing 4- IBB ligand and membrane-bound IL-21 which activate NK cell activity. This application demonstrates high expansion of NK cells (more than 5000-fold) and high efficiency of CAR mRNA-LNP delivery resulting in >75% CAR-positive NK cells. The present application demonstrates that CAR-NK generated with mRNA-LNP are highly functional in vitro and in vivo and are useful for future preclinical and clinical applications.

[0062] In the present method, CAR mRNA-LNP-transfected NK cells are generated with IL- 12 and tested for functional activity by cytotoxicity and ELISA for IFN-gamma, TNF-alpha and Granzyme B secretion. The invention shows higher activity CD19-CAR-NK cells generated by CAR mRNA-LNP transfection with a low dose of IL- 12 versus CAR-NK cells generated without IL- 12. Low dose of IL- 12 (25-100 ng / ml) does not significantly decrease viability of CAR-NK cells, while at 400 pg / ml, IL- 12 decreases viability to about 50%. The invention demonstrates the manufacturing and production of more functional CAR-NK cells by using mRNA-LNP technology and low dose IL- 12 cytokine addition in the transfection medium. The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as limiting.

[0063] EXAMPLES

[0064] Cells

[0065] HEK-293 cells, K562, Daudi, Nalm-6, MM1S, RPMI-8226 cell lines were purchased from ATCC, and were cultured either in RPMI-1640 or in Dulbecco's Modified Eagle's Medium (DMEM) medium with 10% FBS and penicillin / streptomycin. Nalm-6-luciferase, EGFP-positive positive cell line was obtained after transducing with luciferase-positive, EGFP positive lentivirus. K562-41BBL-IL21 (transmembrane, TM) + feeder cells were obtained after transduction of K562 cells with lentivirus containing 4-1BBL and IL21 TM coding region sequences. Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood obtained in the Stanford Hospital Blood Center, Stanford according to IRB-approved protocol (13942). PBMC cells were isolated by standard density sedimentation over Ficoll-Paque (GE Healthcare) and cryopreserved for later use. All cell lines were cultured in a 5% CO2 incubator.

[0066] Antibodies

[0067] Goat Anti-Mouse IgG, F(ab’)2 fragment antibodies were obtained from (Jackson Immunoresearch. Anti-Flag tag and Secondary PE-Streptavidin antibodies and 7-AAD Viability Staining Solution were obtained from Biolegend. 4- IBB ligand, IL-21 antibodies were from Biolegend. Isotype, CD3, and CD56 antibodies were from Biolegend. Lentivirus generation

[0068] FACS

[0069] FACS was performed as described in (21). In brief, 0.25 million cells were suspended in 100 pL of buffer (PBS containing 2 mM EDTA pH 8 and 0.5% BSA) and incubated on ice with 1 |iL of human serum for 10 min. The diluted primary antibody was used with cells for 30 min at 4 °C, and then after washing secondary antibody was added for 30 min at 4C. The cells were rinsed with 3 mL of washing buffer, then stained for 10 min with 7-AAD, and FACS analysis was performed on FACS Calibur (BD Biosciences). IFN-gamma, TNF-alpha and Granzyme B secretion assay by ELISA

[0070] Nonadherent target cells were cultured with the effector cells at different effector to target (E:T) ratio in U-bottom 96-well plates with 200 pL of AIM V-AlbuMAX medium containing 10% FBS, in triplicate. After 16 h, the top 150 pL of medium was transferred to V-bottom 96-well plates and centrifuged at 300x g for 5 min. The top 120 pL of supernatant was transferred to a new 96-well plate and analyzed by ELISA for human IFN-y, TNF-alpha or Granzyme B levels using the R&D Systems Human IFN-gamma Quantikine Kit (Minneapolis, MN, USA) according to the manufacturer’s protocol. The supernatant after RTCA cytotoxicity assay, described in (21) with adherent tethered target cells was collected and analyzed as above.

[0071] Statistical analyses

[0072] Comparisons between two groups were performed by Student’s t-test. Differences with p < 0.05 were considered significant. GraphPad software 9.5 version was used to prepare graphs.

[0073] Example 1. Preparation of linearized DNA template for in vitro transcription.

[0074] DNA was digested with appropriate restriction Bgl II (AGATCT) or Asc I (GGCGCGCC) enzyme, which cut DNA at 3 ’-end after poly A tail, at 37 °C overnight following manufacturer’s protocol. The digested DNA was treated with 50-100 pg / mL Proteinase K and 0.5% SDS for 30 minutes at 50°C. Then phenol / chloroform extraction and ethanol precipitation of DNA were performed. The DNA was used for in vitro RNA transcription reaction.

[0075] Example 2. Preparing mRNA by in vitro transcription reaction.

[0076] For DNA templates with T7AG promoter, we used the below protocol.

[0077] 2.1. The in vitro transcription reaction:

[0078] The DNA template for generating RNA had T7AG promoter in front of coding sequence of protein. The reaction was the following:

[0079] Standard RNA synthesis protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080) was used as described below:

[0080] 1. Thaw the necessary components, keep the T7 RNA Polymerase Mix on ice.

[0081] 2. Mix and pulse-spin in a microfuge to collect the solutions to the bottom of the tubes. Set up the reaction at room temperature in the following order: Final

[0082] Volume concentration mM

[0083] Nuclease-free Water

[0084] 10X Reaction Buffer 2 0.5x

[0085] 100 mM ATP 2 5 mM final

[0086] 100 mM GTP 2 5 mM final

[0087] 100 mM methyl-Pseudo-UTP

[0088] (N-1081) 2 5 mM final

[0089] 100 mM CTP 2 5 mM final

[0090] 100 mM CleanCapAG (3’ OMe

[0091] N-7413) 1.6 4 mM final

[0092] Linear Template DNA 1 pg total

[0093] T7 RNA Polymerase Mix 4

[0094] Total 40 pl

[0095] 3. Gently mix the reaction by pipetting up and down and microfuge briefly. Incubate at 37 °C for 2 hours.

[0096] 4. Optional: The reaction volume can be up to 50 pl with nuclease-free water. Add 2 pl of DNase I, mix well and incubate at 37 °C for 15 minutes.

[0097] 2.2. Cleaning in vitro transcribed RNA

[0098] For cleaning RNA, MONACH® RNA Cleanup Kits (NEB, T2050) was used according to manufacturer’s protocol.

[0099] 2.3. Assessing RNA yield

[0100] The concentration of RNA was determined using a spectrophotometer at 260 nm. The concentration (pg / mL) of RNA was calculated as follows: A260 x dilution factor x 40 pg / mL.

[0101] 2.4. mRNA in vitro transcription mRNA was in vitro transcribed from a DNA template with T7AG promoter using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080). In vitro transcription detail reaction conditions are shown in Examples 2.1. GFP coding sequence was inserted into DNA template vector for in vitro transcription with T7 AG promoter in front and 5’UTR, 3’UTR flanking open reading frame of the codon sequence and 152 poly A tail after the stop codon. For CD19 CAR, CD19 scFv (FMC63) Flag tag-CD28-CD3 sequence was used for inserting into the DNA template vector. For BCMA-CAR, humanized BCMA scFv-41BB- CD3 CAR was used in the DNA template vector. In brief, a DNA template, 0.5 x T7 CleanCap Reagent AG Reaction Buffer, 5 mM of ATP, CTP, pseudo-UTP, and GTP were added to 4 mM of CleanCapAG and T7 polymerase mix for 2 h at 37 °C. After DNAse I treatment for 15 min at 37 °C, the mRNA was purified with the Monarch RNA Cleanup Kit (T2050) according to the manufacturer’s protocol. After each reaction, mRNA was checked on agarose gel with molecular weight ladder, and concentration of mRNA was detected with Nanodrop.

[0102] Example 3. NK cell isolation and expansion

[0103] NK cells were isolated from PBMC using NK Cell Isolation Kit, Human (Miltenyi Biotec) according to the manufacturer’s protocol. The NK cells were expanded using K562- 41-BBL, IL-21 feeder cells pre-treated with Mitomycin C (Sigma) using regular cell culture flasks or gas-permeable static cell culture flasks (G-Rex) (Wilson-Wolf). The medium for expansion was AIM-V, 10% FBS with IL-2 [10 ng / mL] and IL-15 [5 ng / mL]. NK cells can be frozen using NutriFreez DIO Cryopreservation Medium, without phenol red (Satorius). IL- 12 was added to the medium for expanded NK cells overnight (22-24 h) at different concentrations to increase activity of NK cells.

[0104] Example 4. CAR sequences

[0105] A. CD19-CAR

[0106] The DNA template for in vitro transcription of CD19-Flagtag-CD28-CD3-CAR (PMC1637) is shown below. T7AG promoter is underlined, bold, followed by 5’UTR. The CAR sequence in shown in capital letters bold, followed by 3’UTR, and 152 nucleotide poly A tail in italics.

[0107] TAATACGACTCACTATAAGGAGAAAGCTTacatttgcttctgacacaactgtgttcactagcaacctcaaa cagacacc

[0108] ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCA GCATTCCTCCTGATCCCAGACATCCAGATGACACAGACTACATCCTCCCTGT CTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACA TTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACT CCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGT GGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAA GAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGT TCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCCGGCA AGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGT CAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTG TCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCC ACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATA CTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAG AGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTT ACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTG GGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAGACTACAAAGA CGATGACGACAAGATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAG

[0109] AAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTC CCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGG AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACT CCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCA CGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAC GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTA

[0110] GGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCT GAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAAT GAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA GGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAG TACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC

[0111] TCGCTAAGctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaag ggccttgagcatctggattctgcctaataaaaaacatttattttcattgcagctcgctttcttgctgtccaatttctattaaaggttcctttgttcc ctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaGTC GACTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGATCCCCGG

[0112] GCGAGCTCCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 1)

[0113] Translated CD19-CAR amino acid sequence (SEQ ID NO: 2):

[0114] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYL NWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG NTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTV SGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLK MNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAADYKDDDDKIEVM YPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFII

[0115] FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA

[0116] YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK

[0117] MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0118] B. Humanized BCMA-CAR

[0119] The DNA template for in vitro transcription of hBCMA-41BB-CD3 CAR (PMC 1767) is shown below. T7AG promoter is underlined, bold, followed by 5’UTR. The CAR sequence in shown in capital letters bold, then followed by 3’UTR and 152 nucleotide poly A tail in italics.

[0120] TAATACGACTCACTATAAGGAGAAAGCTTacatttgcttctgacacaactgtgttcactagcaacctcaaa cagacacc

[0121] ATG GCG CTC CCA GTG ACT GCC CTT CTG TTG CCA TTG GCC TTG CTT TT G CAC GCC GCG AGG CCC GCC TCC CAA GTG CAA CTC GTT CAG TCC GGG

[0122] GCG GAG GTC AAA AAG CCA GGC TCC TCC GTC AAG GTA TCC TGT AAG G CG TCC GGA TAC ACA TTC ACC TCC TAC GTC ATG CAC TGG GTC AGA CA A GCA CCG GGC CAA GGG CTC GAG TGG ATG GGC TAC ATT ATA CCG TAC AAC GAC GCG ACG AAG TAC AAC GAA AAG TTC AAA GGG AGA GTA ACC A TA ACG GCC GAC AAA AGC ACA AGC ACT GCG TAC ATG GAA CTC TCC TC C CTC CGA TCC GAA GAT ACC GCA GTA TAC TAC TGT GCC AGA TAC AAC TAC GAT GGT TAC TTC GAC GTC TGG GGA CAG GGC ACC CTG GTA ACA G TA TCA TCA GGA GGA GGG GGC AGC GGA GGC GGC GGA TCA GGG GGC G GC GGC AGC GAC GTC GTG ATG ACG CAG AGC CCG GCA TTC CTC TCT GT C ACA CCC GGA GAG AAG GTC ACA ATC ACT TGC CGC GCA TCC CAA AGC ATA TCA GAC TAC CTG CAC TGG TAC CAG CAG AAA CCC GAC CAA GCC C CC AAA CTC TTG ATA AAG TAC GCC AGT CAA AGC ATA TCA GGA GTC CC C TCC CGG TTC AGT GGC AGT GGC TCC GGA ACG GAC TTC ACG TTC ACC ATC TCA TCA TTG GAA GCG GAA GAC GCG GCA ACA TAC TAC TGC CAA A AT GGC CAC AGC TTC CCG CCC ACG TTC GGG GGC GGA ACA AAA GTC GA A ATA AAG TTG GAG AAA CCC ACC ACT ACA CCA GCC CCC AGA CCA CCC ACT CCC GCA CCG ACC ATC GCG AGC CAG CCA CTC TCT CTG AGA CCC G AG GCC TCA CGC CCG GCC GCA GGG GGC GCG GTC CAC ACG CGC GGG C TC GAT TTT GCC TCC GAC AAA CCC TTC TGG GTC CTG GTC GTA GTA GGA

[0123] GGA GTC CTG GCC TGC TAC TCC TTG TTG GTA ACC GTT GCG TTC ATC A TC TTC TGG GTC AAG AGA GGC CGA AAG AAA CTG CTC TAC ATC TTC AA G CAA CCC TTC ATG CGC CCG GTC CAA ACA ACA CAA GAA GAG GAC GGC TGC TCA TGC CGC TTT CCG GAG GAG GAG GAA GGG GGC TGT GAA TTG A GG GTG AAA TTC AGC CGG TCT GCG GAC GCC CCC GCC TAC CAA CAG GG C CAG AAT CAA CTC TAC AAC GAA CTC AAC TTG GGG AGA CGC GAG GAA TAC GAT GTA CTG GAT AAG CGA CGC GGG CGC GAC CCT GAG ATG GGG GGC AAG CCC CAG AGG AGG AAG AAC CCC CAA GAG GGC CTG TAC AAC GAG CTG CAG AAG GAC AAA ATG GCG GAG GCC TAC TCA GAG ATC GGG ATG AAG GGC GAA CGG AGA CGC GGA AAA GGG CAC GAC GGG CTC TAC CAA GGC TTG TCA ACA GCT ACC AAG GAC ACC TAT GAC GCG CTC CAC A TG CAA GCG TTG CCA CCC AGA TAA

[0124] Gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggcc ttgagcatctggattctgcctaataaaaaacatttattttcattgcagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaa gtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaGTCGAC TCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGATCCCCGGGC GAGCTCCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCGAATTCCTGCAGCTCGAGA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 3)

[0125] Translated amino acid sequence of BCMA-CAR: (SEQ ID NO: 4)

[0126] MALPVTALLLPLALLLHAARPASQVQLVQSGAEVKKPGSSVKVSCKASGYTF TSYVMHWVRQAPGQGLEWMGYIIPYNDATKYNEKFKGRVTITADKSTSTAYMELSS LRSEDTAVYYCARYNYDGYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQ SPAFLSVTPGEKVTITCRASQSISDYLHWYQQKPDQAPKLLIKYASQSISGVPSRFSGS GSGTDFTFTISSLEAEDAATYYCQNGHSFPPTFGGGTKVEIKLEKPTTTPAPRPPTPAP TIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP

[0127] Example 5. Transfection of NK cells with CAR-mRNA-LNP

[0128] K562-4-1BB-IL21 K562 cell line was used for expansion of NK cells (see Example 3). The NK cells were expanded >500 and showed high CAR expression after transfection with CAR mRNA-LNP. CAR expression was verified by FACS with anti-mouse FAB or with Flag-tag for Flag-tagged CD19 Scfv-CAR (not shown). CD19-CAR-NK cells generated with CAR mRNA transfection demonstrated high functional activity. IL- 12 was added to the NK or CAR-NK cells in the NK medium overnight. IL- 12 was added during transfection with CAR-mRNA-LNP to the medium with NK cells. IxlO6NK cells were transfected with 5 or 10 microliters of mRNA-LNP (50 microgram / ml) in NK medium for 22-24 hours.

[0129] Example 6. mRNA-LNP generation and transfection of NK cells

[0130] Materials

[0131] SM-102: 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1- octylnonyl ester; CAS number: 2089251-47-6

[0132] DMG-PEG2000: 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DSPC: Distearoylphosphatidylcholine Cholesterol (Sigma)

[0133] To generate an mRNA-LNP complex, an aqueous solution of mRNA in 100 mM sodium acetate (pH 4.0) was combined with a lipid mix containing the ethanol phase of SM- 102 (Cayman), DSPC (Avanti), cholesterol (Sigma), and DMG-PEG2000 (Cayman) (at a molar % ratio of 50:10:38.5: 1.5, respectively).

[0134] To generate mRNA-LNP the above mix was processed with PreciGenome Flex S System (San Jose, CA, USA) at a flow rate ratio of 3:1 (aqueous: organic phase). The mRNA-LNPs were purified and concentrated using Amicon® Ultra- 15 centrifugal filter units (30-100 kDa). The polydispersity index (PDI), size, and zeta-potential of mRNA-LNPs were detected using an Anton Paar Litesizer 500 System.

[0135] The size of nanoparticles is confirmed usingDynamic Light Scattering (DLS) system. The size of RNA-LNP nanoparticles is usually in the range of 90-105 nM or 75-100 nM.

[0136] Example 7. CD19-CAR-NK cells secreted higher level of IFN-gamma after treatment with IL-12 than CAR-NK without treatment with IL-12

[0137] CD19-CAR-NK cells were generated by transfection of NK cells after being expanded for 12 days, with CD19-CAR-mRNA-LNP, in the presence of different concentrations ( of IL- 12. CAR-NK and NK cells were tested for viability (Figure 2). Transfection of CD19-CAR mRNA LNP in the CAR-NK medium with IL- 12 (25-100 pg / ml) resulted in >80% viability of CAR-NK cells (Figure 2). When IL- 12 was increased to 400 pg / ml and more up to 10 ng / ml, viability decreased to 49% in transfected with CAR mRNA-LNP NK cells (Figure 2). Thus, we consider a therapeutic window of optimal dose starting from 25 to 400 pg / ml of IL- 12 when transfection with CAR mRNA-LNP NK cells with IL- 12 is not toxic to NK cells.

[0138] IFN-gamma secretion ELISA assay was used to test functional activity of CAR-NK after transfection with CD19 CAR mRNA-LNP in the presence of IL-12 (25-100 pg / ml) (Figure 3). CD19-CAR-NK cells secreted IFN-gamma in a dose-dependent manner at significantly at higher level than NK cells against target Nalm-6 cells (Figure 2). Nalm-6 is a B cell precursor leukemia cell line initiated from an adolescent male with acute lymphoblastic leukemia. In addition, treatment with IL- 12 increased dose-dependent secretion of INF-gamma by CAR-NK cells against target Nalm-6 cells compared to the level of INF-gamma secreted by NK, CAR-NK cells without IL- 12 (Figure 3). Thus, the addition of IL- 12 to the medium for 22-24 h with NK cells or CAR-NK cells generated by transfection with CD19-CAR mRNA activates CD19- CAR-NK and NK cells which secrete more IFN-gamma against leukemia tumor cells.

[0139] Example 8. CD19-CAR-NK generated by transfection with CAR mRNA-LNP with IL- 12 expressed higher cytotoxicity and secreted higher levels of IFN-gamma, TNF-alpha and Granzyme B than NK cells generated by transfection with GFP mRNA-LNP

[0140] IL-12 was added at 100 pg / ml to the medium during transfection with CAR mRNA- LNPs, and NK cells from two donors (donor 009 and 710) were used as effector cells with Nalm-6 target cells (Figure 4). The cytotoxicity (RTCA) assay was performed as described in (21) using 30,000 target cells and NK cells transfected with green fluorescent protein GFP mRNA, CD 19-CAR-NK effector cells at different ratio to target cells from 0.5 : 1 to 2: 1 (FIG. 4). CD19-CAR-NK cells secreted higher level of cytotoxicity than NK cells (Figure 4).

[0141] The supernatant after cytotoxicity assay was collected and used for ELISA assay to detect IFN-gamma (FIG. 5A), Granzyme B (FIG. 5B) and TNF-alpha (FIG. 5C). ELISA showed that CD 19-CAR-NK cells from two donors had significantly higher secretion of cytokines (IFN-gamma, Granzyme B, and TNF-alpha) versus control NK and NK cells transfected with GFP mRNA against Nalm-6 target cells (FIGs. 5A-5C).

[0142] Example 9. BCMA-CAR-NK has increased secretion of IFN-gamma in the presence of IL-12.

[0143] Similar to Example 7, the same effect of increased activity of CAR was observed with BCMA-CAR (FIG. 6). In the presence of IL-12 in the transfection medium during transfection, BCMA-CAR-NK cells secreted more IFN-gamma than CAR-NK cells without IL- 12 in the transfection medium, against two different multiple myeloma cell lines (FIGs. 6A and 6B). References

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[0151] L. Li, D. Marin, W. Wierda, M. Keating, R. Champlin, E. Shpall and K. Rezvani: Cord blood NK cells engineered to express IL- 15 and a CD19-targeted CAR show long-term persistence and potent anti-tumor activity. Leukemia (2017) doi:10.1038 / leu.2017.226

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Claims

What is claimed is:

1. A method for preparing natural killer (NK) cells, comprising the steps of: obtaining a mRNA and lipid nanoparticles (LNPs) complex, wherein the mRNA comprises (i) 5'-UTR (untranslated region) coding sequence, (ii) a chimeric antigen receptor fusion protein (CAR) coding sequence that target a tumor antigen, (iii) a 3'-UTR coding sequence, and (iv) a poly A tail sequence. transfecting the mRNA-encapsulated LNPs into K cells in a medium comprising 1L-2, IL15, and 25-100 pg / ml of IL-12, and translating the mRNA in the NK cells to produce the CAR.

2. The method of claim 1, wherein the NK cells were expanded at least 500-fold before the transfection.

3. The method of claim 2, wherein the expanded NK cells were frozen and thawed before expansion.

4. The method of claim 1, wherein the tumor antigen is CD 19, BCM A, Her-2, HER-2- t2A-GM-CSF, CD47, CS1, or Claudin 18.2.

5. The method of claim 1, wherein the tumor antigen is CD 19, and the nucleic acid sequence of a DNA template for transcribing the mRNA is SEQ ID NO: 1.

6. The method of claim 5, wherein the translated CAR has the amino acid sequence of SEQ ID NO: 2.

7. The method of claim 1, wherein the tumor antigen is BCMA, and the nucleic acid sequence of a DNA template for transcribing the mRNA is SEQ ID NO: 3.

8. The method of claim 7, wherein the translated CAR has the amino acid sequence of SEQ ID NO: 4.

9. The method of claim 1, wherein the LNPs have an average size of 30-250 nm.

10. The method of claim 1, wherein the LNP comprises (a) 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl] amino] -octanoic acid, 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), cholesterol, and l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000), or (b) SM-102, DSPC, Cholesterol, orl,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)- 2000 (DSPE-PEG2000-MAL), or (c) 2-hexyl-decanoic acid, l,l'-LL(4-hydroxybutyl)imino]di- 6,1 -hexanediyl] ester, DSPC, Cholesterol, and a-[2-(ditetradecylamino)-2-oxoethyl]-(D- methoxy-poly(oxy- 1 ,2-ethanediyl).

11. The method of any one of claims 1-10, wherein the CAR comprises from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) against the tumor antigen, (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activating domain.

12. The method of any one of claims 1-11, wherein the mRNA further comprises 5 ’-cap.

13. The method of claim 1 or 2, wherein the mRNA-encapsulated LNPs are transfected into the expanded NK cells in a G-rex (Gas Permeable Rapid expansion) system.

14. A method for treating cancer in a patient, comprising the step of: administering the NK cells of any one of claims 1-13 to a patient, whereby the NK cells target the tumor antigen and kill tumors.

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