NK cells transfected with CAR RNA-LNP

Abstract

The present invention provides a CAR mRNA embedded with lipid nanoparticles to transfect immune NK cells and produce functional CAR-NK cells. The CAR-NK cells target tumor antigens to kill tumors. The present invention provides several advantages: transient expression of CAR-NK cells, lower toxicity, and lower production costs.

Description

Technical Field

[0001] The present application uses CAR mRNA-LNP (lipid nanoparticle) technology to effectively transfect amplified NK cells and produce functional CAR-NK cells. Functional CAR-NK cells effectively attack tumor cells that overexpress tumor extracellular antigens. Background Art

[0002] Natural Killer (NK) cells are lymphocytes that belong to the same family as T cells and B cells, and are derived from a common progenitor cell. However, as cells of the innate immune system, NK cells are classified as class I innate lymphocytes and respond rapidly to a wide variety of pathological challenges. NK cells are best known for killing virus-infected cells, as well as detecting and controlling early signs of cancer.

[0003] NK cells first gained attention for their ability to kill tumor cells without any priming or prior activation. They are named for this natural killing. In addition, NK cells secrete cytokines such as TFN-γ and TNF-α, which act on other immune cells (such as macrophages and dendritic cells) to enhance the immune response.

[0004] Immunotherapy is emerging as a very promising cancer treatment. NK cells, as the armed force of our immune system, constantly search for foreign antigens and distinguish abnormal cells (cancer or infected cells) from normal cells.

[0005] Recently, chimeric antigen receptor (CAR)-T cells have been approved by the FDA for the treatment of hematological cancers (leukemia, lymphoma, and multiple myeloma) and have shown very promising results (1-4). CAR-T cell therapy has made remarkable progress in the field of cancer therapy, but it also has some limitations, such as cytokine release storm (CRS), neurotoxicity, and challenges in targeting solid tumors (5,6).

[0006] Another promising cell therapy for 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 because they do not rely on HLA-TCR recognition signaling of T cells (10).

[0007] CAR-NK cells are used in preclinical studies against B-cell malignancies (7, 11, 12), multiple myeloma (13, 14), and solid tumors such as glioblastoma (15, 16), breast cancer (17, 18), and ovarian cancer (19). There are several ongoing clinical trials using CAR-NK cells against hematologic malignancies and solid tumors (5), which supports the use of CAR-NK cells to target different cancers.

[0008] The use of viral vectors is associated with high costs, regulatory requirements, and some safety concerns (20). Several non-viral methods exist, such as RNA electroporation and DNA transfection, but these methods have limitations due to low transfection efficiency (20). Developing non-viral delivery of CAR to NK cells has production advantages due to lower costs, easier preparation than viral CARs, and ease of developing allogeneic off-the-shelf CAR-NK cells.

[0009] Better delivery systems for CARs are needed. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 : Cap 0 (Cap 0) and cap 1 (Cap 1) structures.

[0011] Figure 2 : Scheme of DNA vector template (A) for in vitro transcription of CAR RNA (B). 5'UTR, 5' untranslated region; 3'UTR, 3' untranslated region; poly A tail for improving stability.

[0012] Figure 3 : Expansion of NK cells using K562-4-BB, IL-21K562 cell lines. Figure 3 Shown are the average fold expansion of NK cells from two different donors.

[0013] Figure 4 : BCMA-CAR in NK cells transfected with BCMA-CAR-LNP was detected by fluorescence by FACS using anti-mouse FAB antibody. RNA was transcribed in vitro with pseudo-UTP and cap 1. The primary antibody was from Jackson Immunoresearch (Cat#115-066-072) biotin-SP-conjugated AffiniPure F(ab')2 fragmented goat anti-mouse IgG, F(ab')2 fragment specific (1:100); the secondary antibody was from Biolegend, PE-streptavidin (1:100); live / dead cells were detected using 7-AAD viability staining solution (1:50) from Biolegend.

[0014] FIG. 5A to FIG. 5D: BCMA-CAR-NK cells kill multiple myeloma cell lines in a dose-dependent manner and secrete higher levels of IFN-γ than NK cells. A: RTCA killing assay using hBCMA-CAR-NK and multiple myeloma RPMI-8226 cells. B: RTCA killing assay using hBCMA-CAR-NK and MM1S multiple myeloma cells. C: hBCMA-CAR-NK cells secrete IFN-γ in a dose-dependent manner as determined by ELISA using multiple myeloma RPM-I8226 target cells (C) and MM1S target cells (D). The bar graph shows the mean + / - standard deviation of 3 independent measurements. P < 0.05, Student's t-test BCMA-CAR-NK cells vs NK cells, IFN-γ secretion vs NK cells.

[0015] Figure 6 :CD19-CAR-NK cells kill leukemia cell lines in a dose-dependent manner and secrete higher levels of IFN-γ than NK cells. A: RTCA killing assay using CD19-CAR-NK effector cells and Daudi target cells. The bar graph shows the average cytotoxicity of effector cells from 3 independent measurements in the RTeA assay (Materials and Methods). *p<0.05CAR-NK vs NK, Student's t test. B: Compared with NK cells, CD19-CAR-NK secretes higher levels of IFN-γ in a dose-dependent manner, p<0.05, and the secretion of IFN-γ in CD19-CAR-NK vs NK cells is detected by ELISA. C: Cytotoxicity assay performed by Nallm-6-luciferase-positive cells with different E:T ratios by luciferase assay (Materials and Methods). The cytotoxicity of CD19-CAR-NK cells is significantly higher than that of NK cells. *p<0.05, CAR-NK vs NK cells, Student's t test. D: ELISA assay showed that the secretion of IFN-γ by CD19-CAR-NK, NK cells and Nalm-6 target cells was dose-dependent. For Nalm-6 target cells, the level of IFN-γ secreted by CD19-CAR-NK cells was significantly higher than that of NK cells. *p<0.05, CD19-CAR-NK vs NK cells, IFN-γ detected by ELISA.

[0016] FIG. 7A to FIG. 7B :CD19-CAR-NK cells can significantly reduce Nalm-6-luciferase tumor growth more than NK cells. 7A:1×10 5 Nalm-6-luciferase positive cells were injected intravenously into NSG mice. Then, 5×10 6Frozen / thawed CD19-CAR-NK cells. Imaging was performed using the Xenogen Ivis system. 7B: Quantification of imaging is shown. *p<0.00002, CD19-CAR-NK vs NK cells, Student's t-test. DETAILED DESCRIPTION

[0017] definition

[0018] As used herein, "about" means ± 10% of the recited value.

[0019] As used herein, "activated NK cells" refer to NK cells that are activated for proliferation and cell killing.

[0020] As used herein, "chimeric antigen receptor (CAR)" is a receptor protein engineered to give T cells a new ability to target specific proteins. The receptors are chimeric because they combine antigen binding function and T cell activation function into a single receptor. CAR is a fusion protein that includes an extracellular domain, a transmembrane domain, and at least one intracellular domain that can bind to an antigen. "Chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor", "T body" or "chimeric immune receptor (CIR)". "Extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide that can bind to a certain antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to act as a domain that transmits signals to activate or inhibit biological processes in cells.

[0021] "Intracellular domain" refers to any oligo- or polypeptide known to function as a domain that transmits signals to activate or inhibit biological processes within a cell.

[0022] As used herein, a "domain" refers to a region of a polypeptide that folds into a specific structure independently of other regions.

[0023] As used herein, "feeder cells" consist of a layer of cells that cannot divide and provide extracellular secretions to help another cell proliferate.

[0024] As used herein, a "humanized antibody" is an antibody from a non-human species whose non-CDR sequences have been modified to increase their similarity to naturally occurring antibody variants in humans.

[0025] As used herein, "single chain variable fragment (scFv)" refers to a single chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA technology, and wherein the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are connected by spacer sequences. Various methods for preparing engineered ScFv are known to those skilled in the art.

[0026] As used herein, "tumor antigen" refers to a biological molecule whose expression causes cancer.

[0027] Natural killer (NK) cells are a class of cytotoxic lymphocytes, which are crucial for the innate immune system. Using chimeric antigen receptor (CAR) engineered NK cells, CAR-NK cells can target tumor antigens. The application uses CAR mRNA-LNP (lipid nanoparticle) technology to effectively transfect cells amplified from primary PBMC NK cells and produce functional CAR-NK cells. Nanoparticle-based mRNA delivery provides many advantages, such as high stability, bioavailability, solubility and low toxicity.

[0028] In the first aspect, the present invention provides a method for amplifying NK (natural killer) cells. The method includes: obtaining K562 feeder cells treated with mitomycin or γ-ray irradiated by expression of IL21, transmembrane domains (such as CD8 or CD28) and 4-1BB ligands (41BBL); NK cells and treated K562 feeder cells are combined in an appropriate ratio; and the mixture is incubated in an expansion medium containing IL-2 and IL-15 and NK cells are amplified.

[0029] In the present method, a transmembrane domain, such as the CD8 transmembrane domain, is important for maintaining IL15 on the cell surface.

[0030] The ratio of the number of NK cells to the number of feeder cells is about 1:2 to 1:1, or about 1:1.

[0031] In the present method, wherein the NK cells are expanded by about at least 500 times or at least 1000 times. For example, the NK cells are expanded by 500 to 1000 times, 500 to 1500 times, 500 to 2000 times, or 1000 to 3000 times, or more than 5000 times, such as 5000 to 10000 times.

[0032] In one embodiment, the expanded NK cells are frozen for storage. In one embodiment, the expanded NK cells are frozen in CS5 or D10 freezing medium.

[0033] NK cells can be obtained from PBMCs, umbilical cord blood, or induced pluripotent stem (iPS) cells.

[0034] In one embodiment, for example, in a research laboratory, 12-well plates, 6-well plates, T25 flasks, T75 flasks are used to expand NK cells.

[0035] In another embodiment, for example, for larger scale and closed system production, NK cells are expanded in a G-rex (Gas-permeable Rapid Expansion) system, which can be scaled from 40 ml to 1 liter.

[0036] In one embodiment, NK cells are expanded using K562 feeder cells that overexpress 41BBL and IL21.

[0037] In one embodiment, NK cells are expanded using a solid phase (such as a plate) coated with IL-21, IL15, 41BBL, or a combination thereof.

[0038] NK cells can also be expanded using P21 particles, which are the membrane fraction of K562-IL21, 41BBL cells, to reduce possible safety concerns when using leukemia cells clinically.

[0039] K562 cells were growth arrested using mitomycin or irradiation.

[0040] In a second aspect, the present invention relates to NK cells transfected with a complex of mRNA and lipid nanoparticles (LNP), wherein the mRNA comprises: (i) a 5'-UTR (untranslated region) coding sequence, (ii) a chimeric antigen receptor fusion protein (CAR) coding sequence targeting a tumor antigen, (iii) a 3'-UTR coding sequence, and (iv) a poly A tail sequence.

[0041] CAR contains, from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) targeting a tumor antigen, (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activation domain.

[0042] In one embodiment, the tumor antigen is BCMA, Her-2, HER-2-t2A-GM-CSF, CD47, CD19, CS1, or Claudin 18.2.

[0043] The mRNA is embedded in LNPs with an average size of 30 nm to 250 nm, or 50 nm to 150 nm, or 70 nm to 120 nm.

[0044] CAR expressed in NK cells can be detected against scFv antibodies, any scFv can be detected using anti-mouse or anti-human Fab, or scFv with fusion tags (Flag, c-myc, HA, His, TF or any other tag) can be detected using different tag antibodies. Tags are useful when there is no known antibody to detect scFv.

[0045] We have produced different CAR RNAs and delivered them to activated NK cells to target tumor cells or mating tumor cells (mating tumor cell). This delivery has several advantages: one advantage is that it reduces the cost of producing CAR. Produce mRNA, the mRNA in the LNP nanoparticles is delivered to immune cells by transfection, and then the CAR RNA is translated in the immune cells. Another advantage is that there is no viral delivery of CAR that may potentially cause insertion of sequences at different genomic sites and have adverse effects. The third advantage is that CAR delivery by mRNA is short-lived compared to viral delivery that lasts for weeks. All of these advantages can produce safer CAR-NK or will be used for other types of immune cells against cancer.

[0046] The present invention also provides a method for producing CAR in NK cells. The method comprises the following steps: obtaining an mRNA-LNP complex; obtaining NK cells that have been amplified at least 500 times; transfecting LNP-encapsulated mRNA into amplified NK cells; and translating mRNA in NK cells to produce CAR. The method can be used for clinical production of allogeneic CAR-NK cells.

[0047] In one embodiment, the lipid nanoparticles include 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). [LNP-102(ii)]

[0048] In one embodiment, the lipid nanoparticles include 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG2000-MAL). [LNP-102(i)]

[0049] In one embodiment, the lipid nanoparticles include 1,1'-[[(4-hydroxybutyl)azadiyl]bis-6,1-hexanediyl]2-hexyldecanoate (ALC-0315), DSPC, cholesterol, and α-[2-(ditetradecylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethanediyl) (ALC-0159). [LNP-315]

[0050] Inserting mRNA into LNP nanoparticles can protect mRNA from degradation and improve the stability of mRNA; mRNA is then released from LNP into cells in vivo to produce protein. The preparation of mRNA-lipid nanoparticles is described in Schoenmaker (International J. Pharmaceutics, 601: 120856, 2021), which is incorporated herein by reference in its entirety, especially with respect to LNP.

[0051] In a preferred embodiment, the mRNA further comprises a 5'-cap, 5-terminally to (i) the 5'-UTR. The 5'-cap stabilizes the mRNA. All eukaryotic mRNAs include a cap structure, i.e., an N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5'-5' triphosphate bond ( Figure 1 , cap 0). In addition to its essential role in cap-dependent initiation of protein synthesis, the mRNA cap also serves as a protective group for 5' to 3' exonuclease cleavage and a unique identifier for the recruitment of protein factors for pre-mRNA splicing, polyadenylation, and nuclear export. It also serves as an anchor for the recruitment of initiation factors that initiate protein synthesis and the 5' to 3' looping of the mRNA during translation. 2'O methylation of the +1 nucleotide (cap 1) may be central to the non-self discrimination of innate immune responses against foreign RNA. The cap 0 structure and cap 1 structure are shown in Figure 2. Figure 1 shown.

[0052] mRNA is transcribed in vitro from a DNA sequence using RNA polymerase, wherein the DNA sequence comprises: (a) a promoter coding sequence, (b) a 5'-UTR coding sequence, (c) a CAR coding sequence, (d) a 3'-UTR coding sequence, and (e) a poly A tail sequence. The poly A tail sequence improves stability and protein translation.

[0053] Figure 2Linearized DNA templates are shown for in vitro transcription with RNA polymerase and NTP to produce CAR mRNA. The DNA template includes a T7 or SP6 promoter, followed by a 5'UTR (untranslated region), a coding region for CAR, and then a 3'UTR and a poly A tail of >100 for RNA stability. The mRNA produced includes a 5'-cap, such as cap 0, cap 1, or ARCA, for improving stability. mRNA can be transfected into NK cells. For in vivo use, mRNA is added to LNPs to provide protection from degradation and improve stability, and then they are released into NK cells in vivo to produce protein.

[0054] In the DNA sequence, the promoter can be a T7, T7AG promoter. The poly A tail sequence is 20 to 170 nucleotides. The poly A tail sequence optionally includes one or more joints between the poly A segments. If the poly A tail is longer than 60 nucleotides, it typically includes a joint containing non-adenosine nucleotides. The joint is 5 to 30 nucleotides, or 5 to 25 nucleotides, for example 10 nucleotides or 20 nucleotides. In another embodiment, the poly A tail has a length of 150 to 160 nucleotides and is composed of two joint sequences.

[0055] DNA expression is finely regulated at the post-transcriptional level. The untranslated region is not translated into amino acids. However, the UTR of mRNA can control the translation, degradation and positioning of various cis-acting elements of stem-loop structure, upstream start codon and open reading frame, internal ribosome entry site and RNA binding protein binding. UTR is important in the post-transcriptional regulation of DNA expression (including regulation of mRNA nuclear transport and translation efficiency, subcellular localization and stability).

[0056] The 5'-UTR typically has 10 to 1000 nucleotides, or 20 to 500 nucleotides, or 30 to 200 nucleotides, or 30 to 100 nucleotides. For example, the 5'-UTR is 40 to 60 nucleotides (e.g., 50 nucleotides). The 3'-UTR typically has 10 to 3000 nucleotides, such as 50 to 500 nucleotides or 100 to 300 nucleotides. Preferred 5'-UTR and 3'-UTR are the UTRs of β-globin, or the UTRs of the Pfizer COVID vaccine.

[0057] The β-globin gene is shown in:

[0058] www.ncbi.nlm.nih.gov / nucleotide / V00497.1?report=genbank&log$= nuclalign&blast_rank=5&RID=TDDZ1K98016

[0059] In one embodiment, the 5'-untranslated region is derived from human α-globin RNA with an optimized Kozak sequence. The 3'-untranslated region contains two sequence elements derived from the amino-terminal enhancer of split (AES) mRNA and the mitochondrially encoded 12S ribosomal RNA to confer RNA stability and high total protein expression.

[0060] Any suitable vector, such as vector pSP64Poly(A) (Promega) or pGEM3Z-Vektor (Promega) can be used as a cloning vector for the above DNA sequence.

[0061] For example, the 3'-UTR of the β-globin molecule flanked by restriction enzyme sites can be amplified from human bone marrow to engineer pEM3Z-β-globin UTR-UTR-poly A tail. For example, a single (pEM3Z-1p-globin-UTR-A

[120] ) or two tandem fragments (pEM3Z-2p-globin-UTR-A

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

[0062] Typically, a chimeric antigen receptor fusion protein (CAR) comprises, from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) directed against a tumor antigen, (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activation domain.

[0063] In CAR, the costimulatory domain is selected from the group consisting of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAP10 domains. The preferred costimulatory domain is CD28 or 4-1BB.

[0064] In CAR, the preferred activation domain is CD3-ζ (CD3 ζ or CD3ζ).

[0065] In CAR, the transmembrane domain can be derived from a natural polypeptide, or it can be artificially designed. The transmembrane domain derived from a natural polypeptide can be derived from any membrane-bound protein or transmembrane protein. For example, the transmembrane domain of T cell receptor α chain or β chain, CD3ζ chain, CD28, CD3ε., CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 or GITR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues (such as leucine and valine). Preferably, the triplets of phenylalanine, tryptophan and valine are located at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or a polypeptide linker, such as a linker with 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 with a continuous sequence of glycine-serine can be used.

[0066] Different CARs for different tumor antigens are inserted into a DNA template vector with a T7 promoter for RNA polymerase to produce 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, mRNA is transfected into amplified immune cells, such as primary NK cells, and then used to kill tumor cells. In one embodiment, NK cells are amplified with P21 particles or NK-92 cells of K562 cells expressing 41BBL and IL21 to translate CAR in these cells. In one embodiment, the amplified NK cells are frozen and thawed before transfection with mRNA.

[0067] After the T2A self-cleaving peptide (EGRGSLLTCGDVEENPGP), different cytokines (secreted or bound to the membrane) or ligands can be added to the CAR, wherein the T2A self-cleaving peptide is added after the CAR sequence before the stop codon and allows the translated protein to be cut before the last P of its sequence to produce two proteins CAR and the added cytokines or other proteins. The cytokines or ligands can be IL-12, IL15, GM-CSF, IL-2, IL-18 or FLT-3 to reduce the exhaustion of NK cells and stimulate their activity.

[0068] The present invention shows that the freezing medium of CAR-NK cells is important for maintaining high activity and expression of CAR, which has functional killing activity and secretes IFN-γ against antigen-positive target cells. Donor selection is important for high activity of CAR-NK cells.

[0069] The present invention suggests that NK cells should be expanded at least 500 to 2000 times before RNA-LNP transfection to have high killing activity and INF-γ secretion.

[0070] The application confirms that CAR mRNA is delivered non-virally to amplified NK cells from primary PBMC cells using mRNA-LNP technology. Using K562 feeder cells expressing 4-1BB ligands and membrane-bound IL-21 that activates NK cell activity, NK cells are amplified from primary PBMC. The application confirms the high amplification (more than 5000 times) of NK cells and the high efficiency of CAR mRNA-LNP delivery, thereby producing> 75% of CAR positive NK cells. In addition, BCMA and CD19-CAR-NK cells effectively kill multiple myeloma and lymphoma cancer cells, respectively, and secrete high levels of IFN-γ. In addition, CD19-CAR-NK cells significantly block the growth of Nalm-6 leukemia tumors in vivo. The application confirms that CAR-NK produced with mRNA-LNP is highly functional both in vitro and in vivo, and is useful for future preclinical and clinical applications.

[0071] The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting the present invention.

[0072] Example

[0073] cell

[0074] HEK-293 cells, K562, Daudi, Nalm-6, MM1S, RPMI-8226 cell lines were purchased from ATCC and cultured in RPMI-1640 or Dulbecco's modified Eagle medium (DMEM) containing 10% FBS and penicillin / streptomycin. After transduction with luciferase-positive, EGFP-positive lentivirus, Nalm-6-luciferase, EGFP-positive positive cell lines were obtained. After transduction of K562 cells with lentivirus containing 4-1BBL and IL21 TM coding region sequences, K562-41BBL-IL21 (transmembrane, TM) + feeder cells were obtained. According to the IRB-approved protocol (#13942), human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood obtained from the Stanford Hospital Blood Center (Stanford, Stanford) of Stanford. PBMC cells were separated by standard density sedimentation on Ficoll-Paque (GE Healthcare) and frozen for later use. All cell lines were cultured in a 5% CO2 incubator.

[0075] Antibody

[0076] Goat anti-mouse IgG, F(ab')2 fragment antibody was obtained from Jackson Immunoresearch. Anti-Flag tag and PE-streptavidin secondary antibodies and 7-AAD viability stain were obtained from Biolegend. 4-1BB ligand, IL-21 antibodies were from Biolegend. Isotype, CD3 and CD56 antibodies were from Biolegend.

[0077] Lentivirus production

[0078] HEK-293 cells were used to produce EGFP, luciferase, 4-1BBL, IL21 lentiviruses as described (24). Lentiviruses were used to transduce different cell lines and protein expression was verified by FACS or luciferase assays.

[0079] FACS

[0080] FACS was performed as described in (24, 28, 29). Briefly, 250,000 cells were suspended in 100 μL buffer (PBS containing 2 mM EDTA, pH 8 and 0.5% BSA) and incubated with 1 μL human serum on ice for 10 minutes. The diluted primary antibody was used with the cells at 4°C for 30 minutes, and then after washing, the secondary antibody was added and kept at 4°C for 30 minutes. The cells were rinsed with 3 mL of washing buffer, then stained with 7-AAD for 10 minutes, and FACS analysis was performed on a FACS Calibur (BD Biosciences).

[0081] Cytotoxicity assay

[0082] The impedance-based real-time cytotoxicity assay (RTCA) for Daudi and multiple myeloma cell lines was performed by the CELLigence system (Agilent). 4 Target cells were seeded into 96-well E-plates coated with CD40 for leukemia cells or CD9 for multiple myeloma cells to allow the cells to attach to the plates (Agilent / Acea Biosciences, San Diego, CA, USA). The next day, the culture medium was removed and the plates were replaced with 10% FBS ± 1 × 10 5The AIM V-AlbuMAX medium with effector cells was replaced with different (effector cells to target cells) E: T ratios in triplicate. The cells were monitored for an additional 24 to 48 hours using the RTCA system, and the impedance was plotted over time. The percentage of cytotoxicity was calculated according to ((impedance of target cells without effector cells minus impedance of target cells with effector cells) / impedance of target cells without effector cells × 100). For Nalm-6 cells, luciferase-positive, EGFP-positive cells were treated with NK and CD19-CAR-NK cells at different E: T ratios. Cytotoxicity was quantified by luciferase assay using luciferase assay substrate from the Steady-Glo Luciferase Assay System (Promega). Luciferase-positive live cells were normalized to untreated Nalm-6-luc+ cells in duplicate, and the percentage of cytotoxicity of NK and CAR-NK cells at different E: T ratios was calculated.

[0083] IFN-γ secretion assay by ELISA

[0084] Non-adherent target cells were cultured in triplicate at different effector to target (E:T) ratios in U-bottom 96-well plates containing 200 μL of AIM V-AlbuMAX medium with 10% FBS. After 16 hours, the top 150 μL of medium was transferred to a V-bottom 96-well plate and centrifuged at 300 × g for 5 minutes. The top 120 μL of supernatant was transferred to a new 96-well plate and analyzed for human IFN-γ levels by ELISA using the R&D Systems Human IFN-gamma Quantikine Kit (Minneapolis, MN, USA) according to the manufacturer's protocol. Supernatants with adherent cells after RTCA were collected and analyzed as described above.

[0085] NSG mouse model and imaging

[0086] Six-week-old NSG mice (Jackson Laboratories, Bar Harbor, ME, USA) were housed according to the Institutional Animal Care and Use Committee (IACUC) protocol. Each mouse was injected subcutaneously on day 0 with 100 μL of 1 × 10 5 5×10 Nalm-6-luciferase positive cells were injected intravenously in NK medium on days 1, 3, 6, and 8. 6NK cells or CAR-NK cells. After injection of luciferin, imaging was performed using the Xenogen Ivis system (Perkin Elmer, Waltham, MA, USA). Quantification was accomplished by measuring bioluminescence (BLI) in photons / second signals.

[0087] Statistical analysis

[0088] Comparison between two groups was performed by Student's t-test. Differences of p<0.05 were considered significant. Graphs were prepared using GraphPad software version 9.5.

[0089] Example 1. Preparation of linearized DNA template for in vitro transcription

[0090] Following the manufacturer's protocol, the DNA was digested with the appropriate restriction enzyme Bgl II (AGATCT) or Asc I (GGCGCGCC), which cleaves the DNA at the 3' end after the poly A tail at 37°C overnight. The digested DNA was treated with 50 μg / mL to 100 μg / mL proteinase K and 0.5% SDS at 50°C for 30 minutes. Phenol / chloroform extraction and ethanol precipitation of the DNA were then performed. The DNA was used for in vitro RNA transcription reactions.

[0091] Example 2. Preparation of mRNA by in vitro transcription reaction

[0092] For DNA templates with T7AG promoter, we used the following protocol.

[0093] 2.1. Complete the in vitro transcription reaction by the following protocol:

[0094] The DNA template used to produce RNA had the T7AG promoter preceding the protein coding sequence. The reaction was as follows:

[0095] Use the standard RNA synthesis protocol with the HiScribe T7 mRNA Kit (NEB #E2080) with CleanCap Reagent AG as follows:

[0096] 1. For RNA without pseudo-UTP, set up the reaction at room temperature in the following order:

[0097]

[0098] 2. Gently mix the reaction by pipetting up and down and centrifuge briefly. Incubate at 37°C for 2 hours.

[0099] 3. Increase the reaction volume to 50 μl with nuclease-free water. Add 2 μl of DNase I, mix well and incubate at 37°C for 15 minutes.

[0100] 4. Purify mRNA

[0101] For reactions with pseudo-UTP, we used the following protocol:

[0102] 1. Thaw necessary components and keep T7 RNA polymerase mix on ice.

[0103] 2. Mix and pulse spin in a microcentrifuge to collect the solution to the bottom of the tube. Set up the reaction at room temperature in the following order:

[0104]

[0105] 3. Gently mix the reaction by pipetting up and down and centrifuge briefly. Incubate at 37°C for 2 hours.

[0106]

[0107] 4. Optional: Increase the reaction volume to 50 μl with nuclease-free water. Add 2 μl DNase I, mix well and incubate at 37°C for 15 minutes.

[0108] 2.2. Cleaning of in vitro transcribed RNA

[0109] To clean RNA, we used the NEB The Monarch RNA Cleanup Kit (T2050) according to the manufacturer's protocol.

[0110] 2.3. Assessment of RNA Yield

[0111] RNA concentration was determined by diluting an aliquot of the preparation (usually 1:50 to 1:100 dilution) in 1xTE (10 mM Tris-HCl pH 8, 1 mM EDTA) buffer and reading the absorbance at 260 nm in a spectrophotometer. The concentration of RNA (μg / mL) was therefore calculated as follows: A260 × dilution factor × 40 μg / mL.

[0112] 2.4. mRNA in vitro transcription

[0113] The HiScribe T7 mRNA kit (NEB #E2080) with CleanCap Reagent AG was used to transcribe mRNA in vitro from a DNA template with a T7AG promoter. The detailed reaction conditions for in vitro transcription are shown in Example 2.1. For the GFP coding sequence inserted into the DNA template vector for in vitro transcription, there is a T7AG promoter in front, an open reading frame with codon sequences flanking the 5'UTR and 3'UTR, and a 152 poly A tail after the stop codon. For CD19 CAR, the CD19 scFv (FMC63) Flag tag-CD28-CD3 sequence is used for insertion into the above vector (26). For BCMA-CAR, a humanized BCMA scFv-41BB-CD3 CAR (27) is used in the DNA template vector. Briefly, DNA template, 0.5×T7 CleanCapReagentAG reaction buffer, 5mM ATP, CTP, pseudo-UTP and GTP were added to 4mM CleanCapAG and T7 polymerase mixture at 37°C for 2 hours. After treatment with DNAse I for 15 minutes at 37°C, mRNA was purified using Monarch RNA Cleanup Kit (T2050) according to the manufacturer's protocol. After each reaction, mRNA was checked on an agarose gel with a molecular weight ladder marker, and the concentration of mRNA was detected using Nanodrop.

[0114] Example 3. CAR DNA template sequence

[0115] 3.1A. BCMA-CAR DNA Template

[0116] We used the BCMACAR DNA template (PMC1538) with 5' and 3' UTRs and a poly A tail. The T7 promoter is underlined; the CAR is shown in bold; the 150-nucleotide poly A tail is shown in italics:

[0117]

[0118]

[0119] T7 promoter

[0120]

[0121] 5'UTR

[0122]

[0123] BCMA-CAR Nucleotide Sequence:

[0124]

[0125] Poly A tail (152 nucleotides), (SEQ ID NO: 6)

[0126]

[0127] Translated BCMA-41BB-CD3 CAR amino acid sequence (SEQ ID NO: 7)

[0128]

[0129] 3.1B. BCMA-CAR DNA template (uridine-deficient RNA)

[0130] We also used a DNA template (capital letters below) in which the T in the third (underlined, bold) of the trinucleotide codon was replaced with a C or A nucleotide base without changing the amino acid to generate a uridine-deficient RNA sequence for BCMA (PMC1767) for higher expression of CAR.

[0131] The nucleic acid sequence of PMC 1767 is shown below. The amino acid sequence is the same as shown in 3.1A above.

[0132]

[0133]

[0134] The vector we used carries the kanamycin R gene instead of Amp.

[0135] 3.2A.CD19-CAR DNA Template

[0136] We also used CD19-CAR (PMC1643) to generate CD19-41BB-CD3 CAR-RNA. The CAR DNA template has 5' and 3' UTR and poly A tail. The T7 promoter is shown underlined; CD19-CAR is shown in bold; the 150-nucleotide poly A tail is shown in italics.

[0137]

[0138] Amino acid sequence of CD19-41BB-CD3 CAR (SEQ ID NO: 10)

[0139]

[0140] 3.2B.CD19-CAR DNA template (CD28 signal and tag)

[0141] We also used CD19-CARs with a CD28 signaling domain and either a Flag tag or a TF tag following the CD19 scFv.

[0142] PMC1637 with CD19Flag-CD28-CD3 mRNA template for mRNA is shown below. The T7AG promoter is shown in bold underline, and the CD19-Flag tag-Cd28-CD3 sequence is shown in bold. The Flag tag is shown in bold, italic, and underlined.

[0143]

[0144]

[0145] Amino acid sequence: Flag tag is underlined

[0146]

[0147] The TF tag was used instead of the Flag tag in PMC2039. The T7Ag promoter is underlined and the coding sequence CD19TF-28-CD is in bold.

[0148]

[0149]

[0150] The amino acids are as follows:

[0151]

[0152] GFP DNA Template

[0153] We also used GFP in DNA templates with different 5'UTR and 3'UTR and poly A tail. For preparation of PMC1634 (GFP RNA):

[0154]

[0155] T7 promoter

[0156]

[0157] 5'UTR:

[0158]

[0159]

[0160] GFP

[0161]

[0162] Translation of amino acids to GFP:

[0163]

[0164] 3'UTR

[0165]

[0166] Poly A tail (152 nucleotides)

[0167]

[0168] Example 4. NK cell isolation and expansion

[0169] NK cells were isolated from PBMC using a human NK cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. NK cells were expanded using K562-41-BBL, IL-21 feeder cells pretreated with mitomycin C (Sigma) using vented static cell culture flasks (G-Rex) (Wilson-Wolf) (25). The culture medium used for expansion was AIM-V, 10% FBS containing IL-2 [10 ng / mL] and IL-15 [5 ng / mL]. NK cells were frozen using NutriFreez D10 cryopreservation medium (Satorius) without phenol red.

[0170] Example 5. Efficient expansion of NK cells and transfection with GFP-mRNA-LNP

[0171] Expansion of NK cells using K562-4-1BB-IL21 K562 cell line (see Example 5). FACS showed expression of 4-1BBL and IL21 in K562 after transduction with lentivirus encoding 4-1BB and IL21.

[0172] Figure 3 Shown is the expansion of NK cells using K562-4-BB, IL-21K562 cell lines. Figure 3 The average fold expansion of NK cells from two different donors is shown. Using the G-Rex system, NK cells expanded 5754-fold in 18 days with these feeder cells.

[0173] In the characterization of the expanded NK cells, FACS by anti-CD56 and CD3 antibodies showed that 98% of the expanded NK cells were CD56 positive and CD3 negative.

[0174] To examine the efficiency of transfection of NK cells with mRNA-LNPs, we prepared GFP mRNA encapsulated into LNPs using NanoSystem and transfected expanded NK cells. The expanded NK cells were transfected with GFP mRNA-LNPs, frozen in D10 cryopreservation medium, and then thawed and cultured in NK expansion medium at different time points to examine the stability of GFP expression.

[0175] The transfection efficiency was high, with 98% of NK cells being GFP positive 16 hours after transfection, and this efficiency was maintained after freezing and thawing in D10 cryopreservation medium for up to 72 hours. 94% of NK cells were positive 72 hours after freeze / thaw in culture. Thus, NK cells were efficiently expanded with K562-41BB ligand, IL21 positive cells, and efficiently transfected with GFP mRNA-LNP.

[0176] Example 6. mRNA-LNP production and transfection of NK cells

[0177] Material

[0178] SM-102: 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid 1-octylnonyl ester; CAS No.: 2089251-47-6

[0179] DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0180] DSPC: Distearoylphosphatidylcholine

[0181] Cholesterol (Sigma)

[0182] To generate mRNA-LNP complexes, an aqueous solution of mRNA in 100 mM sodium acetate (pH 4.0) was mixed with an ethanolic lipid mixture containing SM-102 (Cayman), DSPC (Avanti), cholesterol (Sigma), and DMG-PEG2000 (Cayman) (molar % ratios of 50:10:38.5:1.5, respectively).

[0183] To generate mRNA-LNPs, the above mixture was processed using the PreciGenome Flex S system (San Jose, CA, USA) at a flow rate ratio of 3:1 (aqueous phase: organic phase). Ultra-15 centrifugal filter units (30-100 kDa) were used to purify and concentrate mRNA-LNPs. The polydispersity index (PDI), size, and zeta potential of mRNA-LNPs were determined using the Anton Paar Litesizer 500 system.

[0184] The size of the nanoparticles was confirmed using a dynamic light scattering (DLS) system. The size of the RNA-LNP nanoparticles was typically in the range of 90 to 105 nM or 75 to 100 nM.

[0185] LNPs can also be prepared with other ionized lipids, such as 50 mM 1 ml (23.15 mM LNP-0315; 4.7 mM DSPC; 21.35 mM cholesterol; 0.8 mM ALC-0159, formulated in a ratio of 46.3:9.4:42.7:1.6 mol %, respectively).

[0186] Example 7. Transfection of BCMA CAR RNA-LNP into NK cells to generate BCMA-CAR-NK cells

[0187] BCMA CAR RNA was produced from a template with (PMC1538) DNA template. The RNA was embedded in LNPs and transfected into amplified NK cells. CAR expression was detected 24, 48, 72 and 96-144 hours after RNA-LNP transfection. Similar results were obtained using PMC1767 (uridine-deficient template). We found that NK cells should be expanded in the range of 500 to 1000 to obtain high expression of CAR. We found that CAR expression varies in different donors, and due to the variability of CAR-positive cells, donor selection is important for the preparation of NK cells. We obtained 648-fold expanded NK cells, of which BCMA+CAR+NK cells were >95% (see Figure 4 ).

[0188] Example 8. BCMA-CAR-NK cells (frozen / thawed) generated with BCMA-CAR-RNA-LNP have cytotoxic activity against BCMA-positive cells

[0189] To test the functional activity of CAR-NK, we first used BCMA-CAR-NK in a real-time cytotoxicity assay against multiple myeloma RPMI8226 cells with different E:T (effector cell to target cell) ratios. Figure 5A BCMA-CAR-NK cells killed RPML8226 cells more effectively than NK cells in a dose-dependent manner ( Figure 5A Similar results were obtained using another multiple myeloma cell line, MM1S ( Figure 5BBCMA-CAR-NK cells are more effective at killing target cells than NK cells ( Figure 5B ).

[0190] Supernatants were collected after the killing assay to test IFN-γ secretion by NK cells and CAR-NK cells ( Figure 5C , Figure 5D ). and NK cells with RPML-8226 target cells ( Figure 5C ) and NK cells with MM1S target cells ( Figure 5D ) Compared with BCMA-CAR-NK cells, BCMA-CAR-NK cells secrete significantly higher levels of IFN-γ in a dose-dependent manner. Therefore, transfection of BCMA CAR mRNA-LNP into NK cells produces functional BCMA CAR-NK cells with high cytotoxic activity and secretion of IFN-γ.

[0191] Example 9. CD19-CAR-NK cells kill leukemia cells and secrete higher levels of IFN-γ than NK cells

[0192] CD19-CAR-NK cells generated by CD19-CAR-mRNA-LNP transfection were functionally tested using killing and IFN-γ secretion ELISA assays. Using a real-time cytotoxicity assay, CD19-CAR-NK cells were significantly more able to kill Daudi target cells than NK cells ( Figure 6 A). CD19-CAR-NK cells secrete significantly higher levels of IFN-γ than NK cells ( Figure 6 B). The same results were obtained in a luciferase killing assay performed with Nalm-6-luciferase positive target cells ( Figure 6 C). CD19-CAR-NK killed significantly more Nalm-6-luciferase-positive cells than NK cells ( Figure 6 C). Compared with NK cells, CD19CAR-NK secreted significantly higher levels of IFN-γ in a dose-dependent manner ( Figure 6 D). Therefore, transfection of CD19-CAR mRNA-LNP into NK cells generated highly functional CD19-CAR-NK cells against leukemia tumor cells.

[0193] Example 10. In vivo activity of CD19-CAR-NK cells

[0194] To test the in vivo activity of CAR-NK, we used Nalm-6-luciferase cells in the NSG mouse model. First, 1×10 5 Then, on days 1, 3, 6, and 8, 5×10 6Frozen NK and CD19 CAR-NK cells were injected into mice. Both NK and CD19-CAR-NK blocked Nalm-6 tumor growth ( Fig. 7A Importantly, at day 15, CD19-CAR-NK cells significantly outperformed NK cells in blocking Nalm-6-luc+ tumor growth (p<0.00002)( Figure 7B ). The CD19-CAR-NK cells generated with mRNA-LNP showed high efficacy against Nalm-6 lymphoma tumors.

[0195] Example 11. Freezing culture medium of CAR-NK to maintain high viability and CAR expression

[0196] For developing allogeneic CAR-NK, it is important to have freezing medium to maintain a high percentage of viable cells and high CAR expression. CS5 is a uniquely formulated, serum-free, animal-free, 5% dimethyl sulfoxide (DMSO) defined cryopreservation medium designed for storage of cells in a cryogenic environment (-80°C to -196°C). D10 medium (NutriFreez D10 Cryopreservation Medium) is a ready-to-use solution for animal-free, xeno-free, serum-free cryopreservation of human embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and mesenchymal stem cells. We tried 4 different culture media to freeze CAR-NK cells, and found that CS5 and D10 were the best culture media to preserve CAR expression and provide high viability CAR-NK cells. In both culture media, CAR-NKs were frozen and thawed, and maintained high viability and high expression 24 to 48 hours after thawing.

[0197] GFP (PMC1634) RNA-LNP was transfected into NK cells, frozen in D10 medium, and stored in NK medium for different time periods after thawing. After thawing, their GFP positive cell percentage and count intensity were tested. 48 hours after thawing, 95.6% of the cells expressed GFP; 72 hours after thawing, 94.5% of the cells expressed GFP.

[0198] BCMA-CAR-NK cells transfected with mRNA-LNP complexes were frozen in D10 medium and detected by FACS using anti-mouse FAB antibody against BCMA at different time periods of 0, 24 and 48 hours after thawing, and 99.8%, 98.6% and 71.2% of positive BCMA-CAR-NK cells were detected, respectively.

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Claims

1. Natural killer (NK) cells transfected with a complex of mRNA and lipid nanoparticles (LNP), wherein: The mRNA comprises: (i) a 5'-UTR (untranslated region) coding sequence, (ii) a chimeric antigen receptor fusion protein (CAR) coding sequence targeting a tumor antigen, (iii) a 3'-UTR coding sequence, and (iv) a poly A tail sequence.

2. The NK cell according to claim 1, wherein The tumor antigen is BCMA, Her-2, HER-2-t2A-GM-CSF, CD47, CD19, CS1 or Claudin 18.

2.

3. The NK cell according to claim 1 or 2, wherein The LNPs have an average size of 30 nm to 250 nm.

4. The NK cell according to claim 1, 2 or 3, wherein The LNPs include: (a) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), or (b) SM-102, DSPC, cholesterol or 1,2-distearoyl-sn-glycerol -3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-MAL), or (c) 1,1'-[[(4-hydroxybutyl)azepinediyl]bis-6,1-hexanediyl]2-hexyldecanoate (ALC-0315), DSPC, cholesterol and α-[2-(ditetradecylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethanediyl) (ALC-0159).

5. The NK cell according to any one of claims 1 to 4, 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 activation domain.

6. The method according to any one of claims 1 to 5, wherein The mRNA further comprises a 5'-cap.

7. A method for preparing the NK cell according to any one of claims 1 to 6, comprising the following steps: obtaining the mRNA-LNP complex; Obtaining NK cells that have been expanded at least 500-fold; The LNP-encapsulated mRNA was transfected into the expanded NK cells; as well as The mRNA is translated in NK cells to produce CAR.

8. The method of claim 7, wherein: The expanded NK cells were frozen and thawed prior to transfection with mRNA.

9. A method for treating cancer in a patient, comprising the steps of: Administering the NK cells of any one of claims 1 to 6 to a patient, The NK cells thereby target the tumor antigens and kill the tumor.

10. The method of claim 7, wherein: The expanded NK cells are prepared according to claim 1.

11. The method of claim 7, wherein: The LNP-encapsulated mRNA was transfected into NK cells expanded in the G-rex (Gaser-Rapid Expansion) system.

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