SIRPα-silenced natural killer (NK) cells

Abstract

To provide natural killer (NK) cells having reduced or ablated Signal Regulatory Protein Alpha (SIRPα-) function that effectively kill a population of cancer cells expressing CD47.SOLUTION: A population of modified natural killer (NK) cells having reduced Signal Regulatory Protein Alpha (SIRPα-) function is provided when compared to a population of NK cells having unmodified SIRPα- function, wherein the modified NK cells effectively kill a population of cancer cells expressing CD47 in an in vitro NK assay.SELECTED DRAWING: None

Description

Technical Field

[0001] I. Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 866,683, filed on Jun. 26, 2019, which is hereby incorporated by reference in its entirety herein under 35 U.S.C. § 119(e).

[0002] II. Field of the Invention The present invention provides natural killer (NK) cells with reduced or lost signal regulatory protein alpha (SIRPα -) function, as compared to non - modified SIRPα - functional NK cells that effectively kill populations of cancer cells expressing CD47.

Background Art

[0003] III. Background of the Invention Natural killer cells or NK cells are important cytotoxic lymphocytes in the innate immune system. The role played by NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response against virus - infected cells and cancer cells. Typically, NK cells are activated by target cells that down - regulate the major histocompatibility complex (MHC), which is one of the main inhibitory NK cell signals. Activation of NK cells causes cytokine release leading to lysis or apoptosis. NK cells are unique in that they can recognize stressed cells by up - regulating other stimulatory NK cell signals without the need for prior exposure to specific cell epitopes. As a result, NK cells respond very rapidly. NK cells are also activated by the strong binding of free antibody Fc ​​​​​Since it is a non-stimulatory NK cell signal, it can also respond rapidly to cells containing antibodies. NK cells, except for exposure to some cytokines such as IL-2 or IL-15, require strong activation to kill cells lacking the MHC class I "self" marker. This role is particularly important because harmful cells with downregulated or absent MHC I markers cannot be detected or destroyed by other immune cells such as T lymphocyte cells.

[0004] NK cells are large granular lymphocytes differentiated from B and T lymphocytes that produce common lymphocyte progenitors. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus and enter the circulation there.

[0005] Current approaches for treating solid tumors use chimeric antigen receptor T cells (CAR-T ). CAR-T cells are produced by removing a patient's own T cells and genetically modifying the T cells to attack cancer cells with specific antigens. Currently, CAR-T treatment targets CD19 and various other antigens in B cell cancer. CAR-T therapy can be effective but does not function well against solid tumors. Tumors can repel invading T cells, inhibit immune cells from invading internally, and suppress the production of antigens targeted by CART cells.

[0006] NK cells are a promising alternative because they can attack solid tumors. NK cells are characterized in the art as lacking the cell surface receptor SIRPα that transmits the CD47 "don't eat me" signal from target cells. SIRPα has been previously Discovered in macrophages.

Summary of the Invention

Means for Solving the Problems

[0007] IV. Summary of the Invention The present invention provides natural killer (NK) cells with reduced or absent signal regulatory protein alpha (SIRPα) function, as compared to NK cells having non-modified SIRPα function that effectively kill populations of cancer cells expressing CD47. In contrast to NK cell lines studied in the literature, the present invention actually recognizes that SIRPα is found in primary NK cells. Reducing or knocking out SIRPα prevents the CD47 protective "don't eat me" signal of target cells from being transmitted from the target cells to NK cells.

[0008] Thus, the present invention provides a population of modified natural killer (NK) cells having reduced signal regulatory protein alpha (SIRPα-) function, as compared to a population of NK cells having non-modified SIRPα function, and these modified NK cells effectively kill populations of cancer cells expressing CD47 in an in vitro NK assay.

[0009] In one aspect of the present invention, in the assay, the killing of cancer cells occurs more rapidly than the killing of cancer cells by a population of non-modified NK cells. In another aspect of the present invention, the modified NK cells are primary NK cells.

[0010] In some aspects of the present invention, the reduction in SIRPα function is due to genetic modification of the population of modified NK cells. In another aspect, the genetic modification is due to SIRPα-knockout, alteration of regulatory sequences, or frameshift mutations. In another aspect of the present invention, the genetic modification is ​​​​​​​​​​​​ , transcription activator-like effector nuclease (TALEN), clustered regularly spaced short palindromic repeats / Cas9 (CRISPR-Cas9), or zinc finger nuclease technology.

[0011] In some aspects of the invention, the reduction in SIRPα function is due to interfering nucleic acid molecules. In other aspects, the interfering RNA is selected from the group consisting of small interfering RNA (siRNA), antisense oligonucleotide nucleotides (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide nucleotides (SSO), and sno-derived RNA (sdRNA). In some aspects of the invention, the reduction in SIRPα function is due to a molecule that binds to SIRP α on the surface of modified NK cells. In other aspects, this molecule is an anti-SIRPα antibody.

[0012] In some aspects of the invention, the cancer is acute myeloid leukemia, non-small cell lung cancer, bladder neoplasm, hepatocellular carcinoma, melanoma, Merkel cell carcinoma, triple-negative breast cancer, ovarian cancer, renal cell carcinoma, colorectal cancer, and sarcoma.

[0013] The present invention provides a population of modified natural killer (NK) cells having a reduced signal regulatory protein alpha (SIRPα-) function compared to NK cells having an unmodified SIRPα function, wherein the modified NK cells effectively kill a population of CD47-expressing low immune cells in an in vitro NK assay. In one aspect, the NK cells are derived from induced pluripotent stem cells (I PSCs). In another aspect, the NK cells are derived from embryonic stem cells (ESCs).

[0014] In another embodiment of the invention, the NK cells comprise a chimeric antigen receptor (CAR-NK).

[0015] The present invention relates to a method for treating cancer comprising administering to a subject a population of modified NK cells of the present invention. In some embodiments, the subject is a human, a mouse, a rat, a cat, a dog, a rabbit, The group consisting of: rats, guinea pigs, hamsters, sheep, pigs, horses, cows, and non-human primates. In other aspects, the cancer is selected from acute myeloid leukemia, non-small cell lung cancer, bladder neoplasm, Hepatocellular carcinoma, melanoma, Merkel cell carcinoma, triple-negative breast cancer, ovarian cancer, renal cell carcinoma, colon cancer The cancer is selected from the group consisting of carcinoma and sarcoma.

[0016] The present invention relates to a method for the detection of transcription activator-like effector nucleases (TALENs), clustered CRISPR-C as9), or by using zinc finger nuclease technology to become SIRPα- The method of the present invention further comprises the steps of: In one embodiment, the SIRPα protein has at least one sequence similar to SEQ ID NO:1. In a preferred embodiment, the SIRPα protein has the sequence It has sequence number 1.

[0017] The present invention relates to small interfering RNA (siRNA), antisense oligonucleotides (AS O), locked nucleic acid (LNA), splice switching oligonucleotide (SSO) or sno-derived RNA (sdRNA) to determine SIR in a population of engineered NK cells The method of the present invention further comprises the steps of: downregulating Pα expression in the expression of modified NK cells; A method for producing a group is provided. V. BRIEF DESCRIPTION OF THE DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

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Mode for Carrying Out the Invention

[0019] VI. DETAILED DESCRIPTION OF THE INVENTION A. First Signal regulatory protein alpha (SIRPα) is a member of the signal regulatory protein (SIRP) family and also belongs to the immunoglobulin superfamily. Members of the SIR P family are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of the receptor tyrosine kinase-binding signal transduction process. SIRPα can be phosphorylated by tyrosine kinases. Phosphotyrosine residues recruit tyrosine phosphatases (PTPs) containing SH2 domains and function as their substrates. SIR Pα is involved in signal transduction mediated by various growth factor receptors. CD47 is a ligand of SIRPα. CD47 is a "self marker" protein that can be widely overexpressed across tumor types. CD47 is a new target for cancer immunotherapy

[0020] ​​​​ has emerged as a potent macrophage immune checkpoint of the rules. CD4 of tumor cells 7 transmits a "don't eat me" signal that inhibits the phagocytosis of macrophages. CD4 7 presents the opportunities and challenges of CD47 inhibitors as both monotherapy and combination therapy for hematological cancers and solid tumors. Some of these agents are currently in clinical trials.

[0021] Previously, the art did not recognize that NK cells express SIRPα. This is presumably because established NK cell lines do not express SIRPα and primary NK cells express SIRPα only after stimulation with cytokines such as IL2 or IL 15. NK cells do not efficiently kill specific solid tumors or hematological tumors. The present invention for the first time recognizes that primary human NK cells express SIRPα upon stimulation and bind to CD47. This results in a reduced killing effect on all types of CD47-expressing tumors. Therefore, the present invention provides for the first time NK cells that are more effective at killing solid and hematological tumors that were previously less sensitive due to reduced or absent expression of SIRPα.

[0022] B. Definitions As used herein, the terms "subject" or "patient" refer to any animal, such as a domestic animal, a zoo animal, or a human. A "subject" or "patient" can be a mammal such as a dog, cat, bird, domestic animal, or a human. Specific examples of "subject" and "patient" include, but are not limited to, an individual (particularly a human) having a disease or disorder associated with the liver, heart, lung, kidney, pancreas, brain, nerve tissue, blood, bone, and bone marrow, etc.

[0023] ​​​Mammalian cells can be derived from human or non-human mammals. Exemplary non-human mammals include , but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, ham sters, sheep, pigs, horses, cows, and non-human primates (e.g., chimpanzees, macaques , and apes).

[0024] As used herein, "low immunogenicity" cells or "HI" cells mean cells that result in a reduced immunological rejection response when transplanted into an allogeneic host. In a preferred embodiment, H I cells do not elicit an immune response. Thus, "low immunogenicity" means that the immune response is significantly reduced or absent compared to the immune response of the pre-immunological engineering parent (i.e., "wt") cells.

[0025] As used herein, "low immunogenic cell O-", "low immunogenic ORh-", or "HI O-" cells mean HI cells that are also ABO blood type O and Rh factor Rh-. HIO - cells can be generated from O- cells, enzymatically modified to become O-, or genetically engineered to become O-.

[0026] The "HLA" or "human leukocyte antigen" complex refers to the gene complex that encodes the human major histocompatibility complex (M HC) proteins. These cell surface proteins that make up the HLA complex are involved in regulating the immune response to antigens. In humans, there are two MHCs, class I and class II, "HLA-I" and "HLA-II" . HLA-I includes three proteins, HLA-A, HLA-B, and HLA-C, which present peptides from within the cell and the antigens presented by the HLA-I complex . ​ attracts killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins are associated with β-2 microglobulin (B2M). HLA-II includes five proteins: HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, which present antigens to T lymphocytes from outside the cell. HLA-II stimulates CD4+ cells (also known as T helper cells). The use of either "MHC" or "HLA" is not meant to be limiting as it depends on whether the gene is of human (HLA) or murine (MHC) origin. Therefore, "MHC" or "HLA" can be used interchangeably herein as they relate to mammalian cells.

[0027] As used herein, "gene knockout" refers to the process of rendering a specific gene inactive in the host cell in which it is present, such that the protein of interest is not produced or is in an inactive form. As understood by those skilled in the art and further described below, gene knockout can be achieved in several ways, including removing a nucleic acid sequence from a gene or inhibiting the sequence with another sequence, changing the reading frame, or altering regulatory components of the nucleic acid. For example, all or part of the coding region of the target gene can be removed or replaced with a "nonsense" sequence, all or part of a regulatory sequence such as a promoter can be removed or replaced, or the translation initiation sequence can be removed or replaced, etc.

[0028] ​​​​​​​​​​​​​​As used herein, "gene knock-in" refers to the process of adding genetic function to a host cell which results in an increase in the level of the encoded protein. As will be understood by those skilled in the art, gene knock-in can be achieved in several ways, including adding one or more additional copies of a gene to a host cell or altering regulatory components of an endogenous gene that increase protein expression. Gene knock-in can be achieved by modifying a promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences. The "β-2 microglobulin" or "β2M" or "B2M" protein refers to the human β2M protein having the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000015.1 0:44711487-44718159.

[0029] The "CD47 protein" refers to the human CD47 protein having the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000016 .10:10866208-10941562.

[0030] The "CIITA protein" refers to the human CIITA protein having the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_0000 03.12:108043094-108094200.

[0031] In the context of cells, "wild-type" means cells found in nature. However, in the context of natural killer (NK) cells as used herein, wild-type means cells Cells may contain nucleic acid changes that result in death, but also means that they have not undergone the gene editing procedures of the present invention to achieve low immunogenicity. This also means that they have not undergone the editing procedure.

[0032] As used herein, "syngeneic" means genetic similarity or identity between a host organism and a cell transplant in which immunological compatibility exists, e.g., no immune response occurs. This also means that they have not undergone the editing procedure.

[0033] As used herein, "allogeneic" refers to genetic dissimilarity between a host organism and a cell transplant in which an immune response occurs. This also means that they have not undergone the editing procedure.

[0034] As used herein, "B2M- / -" means that diploid cells have inactivated the B2M gene on both chromosomes. As described herein, this can be done in various ways. This also means that they have not undergone the editing procedure. This can be done in various ways.

[0035] As used herein, "CIITA- / -" means that diploid cells have inactivated the CIITA gene on both chromosomes. As described herein, this can be done in various ways. This also means that they have not undergone the editing procedure. This can be done in various ways.

[0036] As used herein, "CD47tg" (representing "transgene" or "CD47+") means that the host cell expresses CD47 by having, optionally, at least one additional copy of the CD47 gene. This also means that they have not undergone the editing procedure. This can be done in various ways.

[0037] The term percent "identity" in the context of two or more nucleic acid or polypeptide sequences is measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. This also means that they have not undergone the editing procedure. This can be done in various ways. nucleotides or amino acid residues that are the same when aligned and compared to maximize correspondence refers to two or more sequences or subsequences having a specified percentage of specific depending on the application, percent "identity" can exist over regions of the sequences being compared, e.g., over functional domains or can exist over the full length of the two sequences being compared. In sequence comparisons, typically one sequence serves as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer and, if necessary, subsequence coordinates are designated and program parameters of the sequence algorithm are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the designated program parameters For optimal alignment of sequences for comparison, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981) by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970) by the similarity search method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988)

[0038] by these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970) by the similarity search method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988) 88), or by these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetic s Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) s Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) s Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) s Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) by the modified implementation or by visual inspection (generally, see Ausubel et al. below should be referred to).

[0039] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the A ltschul et al., J. Mol. Biol. 215:403-410 (19 90) described BLAST algorithm. Software for performing BLAST analysis is available from the National Center for Biotechnology Information (www.ncbi.nlm.ni h.gov / ).

[0040] "Inhibitors", "activators", and "modulators" affect the function or expression of biologically relevant molecules. The term "modulator" includes both inhibitors and activators. Modulators can be identified using in vitro and in vivo assays for the expression or activity of the target molecule. "Inhibitor" is, for example, a substance that inhibits expression or binds to a target molecule or protein. Inhibitors can partially or completely block a stimulus or have protease inhibitory activity. Inhibitors can reduce, decrease, prevent, or delay activation, including inactivation, desensitization, or down-regulation of the described target protein activity. A modulator can be an antagonist of the target molecule or protein.

[0041] "Activator" is, for example, an agent that induces or activates the function or expression of a target molecule or protein. Activators bind to stimulate, enhance,

[0042] increase the activity of the target molecule. ​ It can be expanded, activated, or promoted. The activating factor can be an agonist of a target molecule or protein.

[0043] A "homolog" is a bioactive molecule that is similar to a reference molecule at the nucleotide sequence, peptide sequence, functional, or structural level. A homolog can include a sequence derivative that shares a specific percent identity with the reference sequence. Thus, in one embodiment, the homologous sequence or derivative sequence shares at least 70 percent sequence identity. In certain embodiments, the homologous sequence or derivative sequence shares at least 80 or 85 percent sequence identity. In certain embodiments, the homologous sequence or derivative sequence shares at least 90 percent sequence identity. In certain embodiments, the homologous or derived sequence shares at least 95 percent sequence identity. In more specific embodiments, the homologous sequence or derivative sequence shares at least 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity. The homologous nucleic acid sequence or derivative nucleic acid sequence can also be defined by their ability to maintain binding to the reference nucleic acid sequence under high stringency hybridization conditions. A homolog having structural or functional similarity to the reference molecule can be a chemical derivative of the reference molecule. Methods for detecting, generating, and screening for structural and functional homologs and derivatives are known in the art. "Hybridization" generally refers to the situation where complementary strands are present in an environment below their melting temperature.

[0044] ​​​​​​​​when performing, it depends on the ability of the denatured DNA to reanneal. The higher the degree of desired homology between the probe and the repliable sequence, the higher the relative temperature that can be used. As a result, a higher relative temperature tends to make the reaction conditions more stringent, while a lower temperature will be less stringent. For further details and explanations of the stringency of the hybridization reaction, see Ausubel et al, Current Pr otocols in Molecular Biology, Wiley Inter science Publishers (1995), which is hereby incorporated by reference in its entirety. See also.

[0045] The "stringency" of a hybridization reaction can be readily determined by those skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. Generally, the longer the probe, the higher the temperature required for proper annealing, but the shorter the probe, the lower the required temperature.

[0046] "Stringent conditions" or "high stringency conditions" as defined herein can be confirmed by the following: (1) using low ionic strength and high temperature, e.g., 50°C 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate for washing; (2) during hybridization, any denaturing agent such as 50% (v / v) formamide at 42°C and 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate Use buffer (Ph6.5), 750 mM sodium chloride, and 75 mM sodium citrate ; or (3) use a solution of 50% formamide at 42 °C, 5× SSC (0.75 M NaCl, 0 .075 M sodium citrate), 50 mM sodium phosphate (Ph6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μl / ml), 0.1% SDS, and 10% dextran sulfate for overnight hybridization, washing with 0.2× SSC (sodium chloride / sodium citrate) at 42 °C for 10 minutes, followed by high-stringency washing with 0.1× SSC containing EDTA at 55 °C for 10 minutes.

[0047] All maximum numerical limitations given throughout this specification are intended to include all lower numerical limitations as if such lower numerical limitations were expressly recited herein. All minimum numerical limitations given throughout this specification are intended to include all higher numerical limitations as if such higher numerical limitations were expressly recited herein. All numerical ranges given throughout this specification are intended to include all narrower numerical ranges within such broader numerical ranges as if such narrower numerical ranges were expressly recited herein.

[0048] As used herein, the term "modification" refers to a change that physically distinguishes the modified molecule from the parent molecule. In one embodiment, SIR Pα, CD47, HSVtk, EC-CD, or iCasp9 variant polypeptides prepared according to the methods described herein amine ​​​​​​​The amino acid change distinguishes it from the corresponding parent that has not been modified according to the methods described herein, e.g., a wild-type protein, a naturally occurring mutant protein, or another engineered protein that does not include modifications of such mutant polypeptides. In another embodiment, the mutant polypeptide has one or more modifications that distinguish its function from that of the unmodified polypeptide. For example, the amino acid change of the mutant polypeptide affects its receptor binding profile. In other embodiments, the mutant polypeptide includes substitutions, deletions, or insertion modifications, or combinations thereof. In another embodiment, the mutant polypeptide has one or more modifications that enhance its affinity for a receptor as compared to the affinity of the unmodified polypeptide for the receptor.

[0049] In one embodiment, the mutant polypeptide includes one or more substitutions, insertions, or deletions as compared to the corresponding native or parental sequence. In certain embodiments, the mutant polypeptide has 1 one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 - 40, 41 - 50, or 51 or more modifications.

[0050] As used herein, an "episomal vector" means a gene vector that autonomously exists in the cytoplasm of a cell and can be replicated; e.g., an episomal vector is not integrated into the genomic DNA of a host cell. Some episomal vectors are known in the art and are described below.

[0051] "Knockout" in the context of a gene means that the knocked-out host cell does not produce a functional protein product. As outlined herein, knockout can be effected in a variety of ways, such as by removing all or part of the coding sequence, introducing a frameshift mutation (either a cleavage or a nonsense sequence) such that a functional protein is not produced, removing or altering regulatory elements (e.g., a promoter) so that the gene is not transcribed, preventing translation by binding to the mRNA, etc. Generally, knockout is achieved at the genomic DNA level such that the progeny of the cell also permanently have the knockout. "Knockin" in the context of a gene means that the knocked-in host cell has more functional protein active intracellularly. As outlined herein, knockin can be done in a variety of ways, usually by introducing at least one copy of a transgene (tg) encoding a protein into the cell, but this can also be done, for example, by replacing a regulatory element by adding a constitutive promoter to an endogenous gene. Generally, knockin technology results in the integration of an additional copy of the transgene into the host cell. VII. Cells of the Invention The present invention provides compositions and methods for generating SIRPα-NK cells. In some embodiments, the cells are HIO-SIRPα-NK cells. A. Methods of Genetic Modification

[0052]

[0053]

[0054] The present invention includes methods of modifying nucleic acid sequences intracellularly or in cell-free conditions to generate SIRPα-NK cells. Exemplary techniques include homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9, and other site-specific nuclease technologies. These techniques enable the cleavage of double-stranded DNA at a target locus site. These controlled double-strand breaks promote homologous recombination at specific locus sites. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequence and induce double-strand breaks in the nucleic acid molecule. The double-strand breaks are repaired either by error-prone non-homologous end joining (NHEJ) or homologous recombination (HR). As will be understood by those skilled in the art, several different techniques can be used to engineer the NK cells of the present invention and to engineer the NK cells to be less immunogenic as outlined herein. Generally, these techniques can be used individually or in combination. For example, in the generation of SIRPα-NK cells, CRISPR can be used to reduce the expression of active SIRPα protein in engineered cells. In another example, viral technologies (e.g., lentivirus) are used to knock-in genes such as CD47. a. CRISPR technology

[0055]

[0056]

[0057] ​​​​​​​​​​​​​In one embodiment, the cells are clustered regularly as is known in the art spaced short palindromic repeat / Cas ("CRISPR") technology is used to be manipulated. SIRPα-NK cells can be produced using CRISPR . There are a number of CRISPR-based technologies. For example, incorporated herein by reference are Doudna and Charpentier, Science doi:1 0.1126 / science.1258096. See CRISPR technology and kits are commercially available.

[0058] b. TALEN technology In some embodiments, the cells of the present invention are produced using the transcription activator-like effector nuclease (TALEN) method. TALENs are restriction enzymes combined with nucleases that can be engineered to bind and cleave substantially any desired DNA A sequence. TALEN kits are commercially available.

[0059] c. Zinc finger technology In one embodiment, the cells are engineered using Zn finger nuclease technology. Zn finger nucleases are artificial restriction enzymes generated by fusing the DNA binding domain of zinc fingers to a DNA cleavage domain . The zinc finger domain can be engineered to target specific desired DNA sequences, thereby allowing zinc finger nucleases to target unique sequences within complex genomes . By utilizing the endogenous DNA repair machinery, these reagents can be used to accurately modify the genomes of higher organisms, similar to CRISPR and TALENS . to cut.

[0060] d. Virus-based technologies Although not limited to, there are a variety of viral technologies that can be used to form some embodiments of the SIRPα -NK cells of the present invention, including the use of retroviral vectors, lentiviral vectors, adeno viral vectors, and Sendai viral vectors. Episomal vectors used in cell production are described below.

[0061] e. Gene down-regulation using interfering RNAs In other embodiments, the gene encoding the protein used in HLA molecules is down-regulated by RNA i technology. RNA interference (RNAi) is a process by which RNA molecules inhibit gene expression often by degrading specific mRNA molecules. Two types of RNA molecules, microRNA (miRNA) and small interfering RNA (siRNA), are central to RNA interference. These RNA molecules bind to target mRNA molecules and increase or decrease their activity. RNAi helps cells defend against parasitic nucleic acids such as those derived from viruses and transposons. RNAi also affects development.

[0062] siRNA. Small interfering RNA is a double-stranded RNA fragment that can be 21 - 22 nucleotides in length. The first step in using siRNA for therapeutic applications is to design siRNA sequences specific to the target mRNA using a plurality of algorithms known in the art. siRNA can be produced by chemical synthesis or gene expression. siRN When A enters the cell, the process of gene silencing is initiated and carried out via the endogenous RNAi pathway. The antisense strand is loaded into a protein complex called the RNA-induced silencing complex (RISC). The antisense strand functions as a guide to recognize complementary mRNA. Once the target sequence is recognized, the mRNA is cleaved between 10 and 11 nucleotides downstream of the 5' end of the antisense strand by Argonaute 2, which is a component of RISC. This results in a decrease in protein expression from the silenced gene. The advantages of siRNA for drug therapy include high specificity and low toxicity. See Ahmadzada et al, Biophys Rev. 10(1):69-86(2018), which is incorporated herein by reference in its entirety. and is carried out via the endogenous RNAi pathway. The antisense strand is loaded into a protein complex called the RNA-induced silencing complex (RISC). The antisense strand functions as a guide to recognize complementary mRNA. Once the target sequence is recognized, the mRNA is cleaved between 10 and 11 nucleotides downstream of the 5' end of the antisense strand by Argonaute 2, which is a component of RISC. This results in a decrease in protein expression from the silenced gene. The advantages of siRNA for drug therapy include high specificity and low toxicity. See Ahmadzada et al, Biophys Rev. 10(1):69-86(2018), which is incorporated herein by reference in its entirety. and is carried out via the endogenous RNAi pathway. The antisense strand is loaded into a protein complex called the RNA-induced silencing complex (RISC). The antisense strand functions as a guide to recognize complementary mRNA. Once the target sequence is recognized, the mRNA is cleaved between 10 and 11 nucleotides downstream of the 5' end of the antisense strand by Argonaute 2, which is a component of RISC. This results in a decrease in protein expression from the silenced gene. The advantages of siRNA for drug therapy include high specificity and low

[0063] ASO. Antisense oligonucleotides are short synthetic single-stranded oligodeoxynucleotides that can alter RNA and reduce, restore, or alter protein expression through several different mechanisms. First-generation ASOs were typically short synthetic single-stranded oligodeoxynucleotides 8-50 nucleotides in length. ASOs bind to target mRNA by complementary base pairing. This causes knockdown of the RNA transcript via endonuclease, and thus a decrease in the level of the encoded target protein. Second-generation and third-generation ASOs with modified backbones improve pharmacological properties. These improved ASOs can function via alternative mechanisms. For example, these ASOs can sterically block splicing factors to pre-mRNA and is carried out via the endogenous RNAi pathway. The antisense strand is loaded into a protein complex called the RNA-induced silencing complex (RISC). The antisense strand functions as a guide to recognize complementary mRNA. Once the target sequence is recognized, the mRNA is cleaved between 10 and 11 nucleotides downstream of the 5' end of the antisense strand by Argonaute 2, which is a component of RISC. This results in a decrease in protein expression from the silenced gene. The advantages of siRNA for drug therapy include high specificity and low toxicity. See Ahmadzada et al, Biophys Rev. 10(1):69-86(2018), which is incorporated herein by reference in its entirety. and is carried out via the endogenous RNAi pathway. The antisense strand is loaded into a protein complex called the RNA-induced silencing complex (RISC). The antisense strand functions as a guide to recognize complementary mRNA. Once the target sequence is recognized, the mRNA is cleaved between 10 and 11 nucleotides downstream of the 5' end of the antisense strand by Argonaute 2, which is a component of RISC. These ASOs can also alter splicing or inhibit ribosome recruitment. By preventing translation of the mRNA, translation of the mRNA can be blocked. Rinaldi and Wood, Nat. Rev. Ne., incorporated herein by reference. Please see urol.14(1):9-21 2018.

[0064] Some ASOs use phosphorothioate backbones. One of the molecules is replaced by sulfur, which greatly increases resistance to nuclease activity. These changes result in a 50% reduction in serum ASO activity. Although the phase has been extended, this molecule is still being used in applications involving downregulation of target RNA. In addition, the ribose sugar can be modified at the 2' position to provide 2'O-methyl 2'OMe, 2'O-methoxyethyl (2'MOE) oligonucleotides, and lock Another class of drugs with improved safety and efficacy profiles includes LNAs. ASO was obtained.

[0065] To enhance the in vivo activity of LNA:ASO, many artificial nucleic acids have been synthesized and The enzyme resistance, binding properties, RNase H activity, and serum stability have been improved. 2'-O,4'-C-Bridged Nucleic Acid (also known as 2',4'-BNA) The artificial amides contain a methylene bridge connecting the 2'-O and 4'-C positions in the fused ring. It is a nucleic acid derivative. This allows locked nucleic acids to have high binding affinity to complementary RNA. LNA forms a strictly N-type conformation that provides LNAs are also enzyme-resistant. It is used in various gene silencing technologies such as kissing and triplex-forming oligonucleotides. LNA can function in vivo in, for example, mouse models as a splicing switching oligonucleotide (SSO) and can be used in LNA-based SSO (LNA SSO ). See Shimo et.Al. Nucleic Acids Research, 42(12):817 4-8187(2014), which is incorporated herein by reference in its entirety.

[0066] Gapmer ASO is a short single-stranded ASO that contains a central DNA sequence generally flanked by locked nucleic acid (LNA) sequences and interferes with mRNA expression by inducing RNase H activation. Gapmer ASO can exhibit cell penetration that does not require a transfection agent by a process called gymnosis.

[0067] Regulation of splicing induced by SSO oligonucleotides has led to several results in cell culture and in vivo with potential therapeutic value. Splice-switching oligonucleotides (SSO) are oligonucleotides that can regulate pre-mRNA splicing, repair defective RNA, and restore the production of essential proteins. Splice-switching oligonucleotides can also produce novel proteins with desirable properties and regulate the presence of disease-related splice variant proteins. The latter result is achieved by regulating the alternative splicing of pre-mRNA.

[0068] ​​​​​​​​​​​To regulate pre-mRNA splicing, the SSO blocks RNA sequences essential for splicing and prevents the interaction of pre-mRNA with splicing factors (such as RNA-binding proteins, small nuclear RNAs, and other components of the spliceosome). RNA sequences essential for splicing and prevents the interaction of pre-mRNA with splicing factors (such as RNA-binding proteins, small nuclear RNAs, and other components of the spliceosome). RNA sequences essential for splicing and prevents the interaction of pre-mRNA with splicing factors (such as RNA-binding proteins, small nuclear RNAs, and other components of the spliceosome). Chemicals shown to function in animal models include peptide nucleic acids (PNAs), locked nucleic acids (LNAs), deoxynucleotide oligonucleotides, fully modified (non-gapmer) 2'-substituted oligonucleotides, and PMO-based oligomers. Chemicals shown to function in animal models include peptide nucleic acids (PNAs), locked nucleic acids (LNAs), deoxynucleotide oligonucleotides, fully modified (non-gapmer) 2'-substituted oligonucleotides, and PMO-based oligomers. Chemicals shown to function in animal models include peptide nucleic acids (PNAs), locked nucleic acids (LNAs), deoxynucleotide oligonucleotides, fully modified (non-gapmer) 2'-substituted oligonucleotides, and PMO-based oligomers.

[0069] sdRNA.sno-derived RNA molecules are a class of asymmetric siRNAs containing a 19-21 base guide (antisense) strand. sno-derived RNA molecules contain a 5' phosphate, 2'Ome, or 2'F-modified pyrimidine, and six phosphorothioates at the 3' position. sno-derived RNA molecules also contain a sense strand containing a 3' conjugated sterol moiety, two phosphorothioates at the 3' position, and a 2'Ome-modified pyrimidine. Both strands contain 2'Ome purines with an unmodified phosphate stretch not exceeding 3 in length. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety. sdRNA.sno-derived RNA molecules are a class of asymmetric siRNAs containing a 19-21 base guide (antisense) strand. sno-derived RNA molecules contain a 5' phosphate, 2'Ome, or 2'F-modified pyrimidine, and six phosphorothioates at the 3' position. sno-derived RNA molecules also contain a sense strand containing a 3' conjugated sterol moiety, two phosphorothioates at the 3' position, and a 2'Ome-modified pyrimidine. Both strands contain 2'Ome purines with an unmodified phosphate stretch not exceeding 3 in length. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety. sdRNA.sno-derived RNA molecules are a class of asymmetric siRNAs containing a 19-21 base guide (antisense) strand. sno-derived RNA molecules contain a 5' phosphate, 2'Ome, or 2'F-modified pyrimidine, and six phosphorothioates at the 3' position. sno-derived RNA molecules also contain a sense strand containing a 3' conjugated sterol moiety, two phosphorothioates at the 3' position, and a 2'Ome-modified pyrimidine. Both strands contain 2'Ome purines with an unmodified phosphate stretch not exceeding 3 in length. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety. sdRNA.sno-derived RNA molecules are a class of asymmetric siRNAs containing a 19-21 base guide (antisense) strand. sno-derived RNA molecules contain a 5' phosphate, 2'Ome, or 2'F-modified pyrimidine, and six phosphorothioates at the 3' position. sno-derived RNA molecules also contain a sense strand containing a 3' conjugated sterol moiety, two phosphorothioates at the 3' position, and a 2'Ome-modified pyrimidine. Both strands contain 2'Ome purines with an unmodified phosphate stretch not exceeding 3 in length. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety. sdRNA.sno-derived RNA molecules are a class of asymmetric siRNAs containing a 19-21 base guide (antisense) strand. sno-derived RNA molecules contain a 5' phosphate, 2'Ome, or 2'F-modified pyrimidine, and six phosphorothioates at the 3' position. sno-derived RNA molecules also contain a sense strand containing a 3' conjugated sterol moiety, two phosphorothioates at the 3' position, and a 2'Ome-modified pyrimidine. Both strands contain 2'Ome purines with an unmodified phosphate stretch not exceeding 3 in length. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety. sdRNA.sno-derived RNA molecules are a class of asymmetric siRNAs containing a 19-21 base guide (antisense) strand. sno-derived RNA molecules contain a 5' phosphate, 2'Ome, or 2'F-modified pyrimidine, and six phosphorothioates at the 3' position. sno-derived RNA molecules also contain a sense strand containing a 3' conjugated sterol moiety, two phosphorothioates at the 3' position, and a 2'Ome-modified pyrimidine. Both strands contain 2'Ome purines with an unmodified phosphate stretch not exceeding 3 in length. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety. sdRNA.sno-derived RNA molecules are a class of asymmetric siRNAs containing a 19-21 base guide (antisense) strand. sno-derived RNA molecules contain a 5' phosphate, 2'Ome, or 2'F-modified pyrimidine, and six phosphorothioates at the 3' position. sno-derived RNA molecules also contain a sense strand containing a 3' conjugated sterol moiety, two phosphorothioates at the 3' position, and a 2'Ome-modified pyrimidine. Both strands contain 2'Ome purines with an unmodified phosphate stretch not exceeding 3 in length. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety. .

[0070] In all of these techniques, well-known recombinant techniques are used to produce the recombinant nucleic acids outlined herein. In certain embodiments, a recombinant nucleic acid (either encoding a desired polypeptide, such as CD47, or encoding a disrupting sequence) can be operably linked to one or more regulatory nucleotide sequences in an expression construct. In all of these techniques, well-known recombinant techniques are used to produce the recombinant nucleic acids outlined herein. In certain embodiments, a recombinant nucleic acid (either encoding a desired polypeptide, such as CD47, or encoding a disrupting sequence) can be operably linked to one or more regulatory nucleotide sequences in an expression construct. In all of these techniques, well-known recombinant techniques are used to produce the recombinant nucleic acids outlined herein. In certain embodiments, a recombinant nucleic acid (either encoding a desired polypeptide, such as CD47, or encoding a disrupting sequence) can be operably linked to one or more regulatory nucleotide sequences in an expression construct. In all of these techniques, well-known recombinant techniques are used to produce the recombinant nucleic acids outlined herein. In certain embodiments, a recombinant nucleic acid (either encoding a desired polypeptide, such as CD47, or encoding a disrupting sequence) can be operably linked to one or more regulatory nucleotide sequences in an expression construct. The nucleotide sequences are generally appropriate for the host cells and subjects to be treated. A variety of host cells are known in the art with respect to many types of suitable expression vectors and suitable regulatory sequences that are known in the art. Typically, one or more regulatory nucleotide sequences include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, transcription start and termination sequences , translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are also contemplated. The promoter can be either a naturally occurring promoter or a hybrid promoter that combines elements of multiple promoters. The expression construct can be present in the cell on an episome such as a plasmid , or the expression construct can be inserted into the chromosome. In certain embodiments, the expression vector includes a selectable marker gene to enable selection of the transformed host cells . Certain embodiments include an expression vector that includes a nucleotide sequence encoding a variant polypeptide operatively linked to at least one regulatory sequence. Regulatory sequences for use herein include promoters, enhancers , and other expression control elements. In certain embodiments, the expression vector is designed for selection of the host cell to be transformed, the particular variant polypeptide to be expressed, the copy number of the vector, the ability to control that copy number, or the expression of other proteins encoded by the vector such as antibiotic markers .

[0071] Examples of suitable mammalian promoters include, for example, the hamster ubiquitin / S27a promoter (WO 97 / 15664) , simian vacuolar virus 40 (S​​ V40) initial promoter, adenovirus major late promoter, mouse metallothione in-I promoter, long terminal repeat region of Rous sarcoma virus (RSV) , mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus ro ng terminal repeat region, and promoter from the gene of human cytomegalovirus (CMV) is included. Examples of other heterologous mammalian promoters are a ctin, immunoglobulin, or heat shock promoter.

[0072] In additional embodiments, the promoter for use in mammalian host cells is polyoma virus, fowlpox virus (British Patent No. 2,211,50 4 published July 5, 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retro virus, hepatitis B virus, and simian virus 40 (SV40) and the like can be obtained from the genome of the virus. In further embodiments, a heterologous mammalian promoter is used . Examples include an actin promoter, an immunoglobulin promoter, and a heat shock promoter. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment that also includes the origin of replication of the SV40 virus. Fiers et al., Nature 273:113-120 (1978). The major immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIIIE restriction fragment. Greenaw ay, P.J. et al., Gene 18:355-360 (1982). The foregoing references are incorporated by reference in their entirety.

[0073] In some embodiments, the SIRPα-NK cells are derived from stem cells.

[0074] The term "pluripotent cell" refers to a cell that can self-renew and proliferate while remaining in an undifferentiated state and can be induced to differentiate into a specific cell type under appropriate conditions. As used in this specification, the term "pluripotent cell" encompasses embryonic stem cells (ESCs), and other types of stem cells including fetal, amniotic , or somatic stem cells. Exemplary human stem cell lines include the H 9 human embryonic stem cell lines. Additional exemplary stem cell lines include those available through the National Institutes of Health Hum an Embryonic Stem Cell Registry and the Howard Hughes Medica l Institute HUES collection (incorporated herein by reference in its entirety, Cowan, C.A. et. al, New England J. Med. 350:13. (2004)). (described in Cowan, C.A. et al., New England J. Med. 350:13. (2004), which is incorporated herein by reference in its entirety).

[0075] As used herein, "pluripotent stem cells" can differentiate into any of the three germ layers: endoderm (e.g., stomach junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term "pluripotent stem cells" as used herein also encompasses "induced pluripotent stem cells" or "iPSCs", a type of pluripotent stem cell derived from non-pluripotent cells. Examples of parental cells include somatic cells reprogrammed to induce a pluripotent undifferentiated phenotype by various means. ​ Such "iPS" or "iPSC" cells can be produced by inducing the expression of specific regulatory genes or by adding specific proteins externally. Methods for inducing iPS cells are known in the art and are further described below. (See, for example, Zhou et al., Stem Cells 27(11):2667-74(2009); Huan gfu et al., Nature Biotechnol.26(7):795(2 008); Woltjen et al., Nature 458(7239):766 -770(2009); and Zhou et al., Cell Stem Cell 8:381-384(2009), each of which is incorporated herein by reference in its entirety). The production of induced pluripotent stem cells (iPSCs) is outlined below. As used herein, "hiPSC" refers to human induced pluripotent stem cells, and "miPSC" refers to mouse induced pluripotent stem cells.

[0076] "Characteristics of pluripotent stem cells" refers to the characteristics of cells that distinguish pluripotent stem cells from other cells. Under appropriate conditions, the ability to produce progeny that can differentiate into cell types that collectively exhibit characteristics related to cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm) is a characteristic of pluripotent stem cells. The expression or non-expression of specific combinations of molecular markers is also a characteristic of pluripotent stem cells. For example, human pluripotent stem cells have the following non-limiting list: SSEA-3, SSEA-4 TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, In some cases, all are expressed in some embodiments. Cells related to pluripotent stem cells The morphology is also a characteristic of pluripotent stem cells. As described herein, cells do not need to pass through pluripotency to be reprogrammed into endoderm progenitor cells and / or hepatocytes.

[0077] B. Generation of Low Immunogenicity (HI) SIRPα-NK Cells The generation of HI cells is achieved with just three genetic changes, which immunosuppress the cells while minimally inhibiting cell activity. This technique is disclosed, for example, in WO 2018 / 132783, the entire contents of which are incorporated herein by reference and is briefly described below. As discussed herein, one embodiment utilizes the reduction or elimination of the protein activity of MHC I and II (HLA I and II in the case where the cells are human). This can be done by altering the genes encoding those components. In one embodiment the coding region or regulatory sequences of the gene are disrupted using CRISPR. In another embodiment

[0078] gene translation is reduced using RNA interference technology. Another embodiment is a change in a gene that modulates susceptibility to macrophage phagocytosis, such as CD4 7, which is generally a gene "knock-in" using viral technology.

[0079]

[0080] 1. Reduction of HLA-I The HI SIRPα-NK cells of the present invention include a reduction in MHC I (HLA I when the cells are of human origin) function.

[0080] As will be understood by those skilled in the art, a reduction in function can be the removal of a nucleic acid sequence from the gene, other sequences can be achieved in several ways, including inhibition of the sequence by or alteration of regulatory components of the nucleic acid. For example, all or part of the coding region of the gene of interest can be removed or replaced with a "nonsense" sequence, a frameshift mutation can be introduced, all or part of a regulatory sequence such as a promoter can be removed or replaced,

[0081] the translation start sequence can be removed or replaced, etc. As will be understood by those skilled in the art, success in reducing MHC I function in SIRPα-NK cells (HLA I if the cells are derived from human cells) can be measured using techniques known in the art and the techniques described below; for example, using a labeled antibody that binds to the HLA

[0082] complex; for example, using FACS techniques with commercially available HLA-A, B, C antibodies that bind to the alpha chain of the human major histocompatibility gene HLA class I antigen. a. Alteration of B2M In one embodiment, the reduction of HLA-I activity is achieved by inhibiting the expression of the beta-2 microglobulin gene in HISIRPα-NK

[0083] cells, the human sequence of which is disclosed herein. This alteration is generally referred to in this specification as gene "knockout" and is performed on both alleles of the host cell in the cells of the present As a result, a stop codon is generated and a truncated non-functional protein is produced. This occurs.

[0084] Therefore, a useful technique is to use a CRISPR array designed to target the coding sequence of the mouse B2M gene or the human B2M gene. After gene editing, the transfected SIRPα-NK culture dissociates into single cells. The single cells are expanded into full-sized colonies and tested for CRISPR editing by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones lacking both alleles are selected. Such clones do not express B2M as demonstrated by PCR and do not express HLA-I as demonstrated by FACS analysis (see, for example, Examples 1 and 6).

[0085] Assays for testing whether the B2M gene is inactivated are known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with an antibody against the B2M protein. In another embodiment, reverse transcription polymerase chain reaction (rt-PCR) is used to confirm the presence of the inactivating alteration.

[0086] In addition, cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This can be assayed by FACS analysis using an antibody against one or more HLA cell surface components as described above.

[0087] 2. Reduction of HLA-II In some embodiments, in addition to reducing HLA I, the HI SIRPα-N K cells may also lack MHC II function (HLA II from human-derived cells).

[0088] As will be appreciated by those skilled in the art, a reduction in function can be achieved by the removal of a nucleic acid sequence from a gene, the addition of a nucleic acid sequence to a gene, Addition of a nucleic acid sequence, disruption of the reading frame, interruption of a sequence by another sequence, or This can be accomplished in a number of ways, including altering the regulatory components of the All or part of the coding region of the target gene is removed or replaced with a "nonsense" sequence. In another embodiment, regulatory sequences such as promoters can be removed or replaced. For example, the translation initiation sequence may be removed or replaced.

[0089] Positive MHC II (HLA II) function in SIRPα-NK cells or their derivatives Regular reduction can be achieved, for example, by Western blotting using antibodies against the protein, F It can be measured using techniques known in the art, such as ACS and rt-PCR techniques. can.

[0090] a. Changes in CIITA In one embodiment, the reduction in HLA-II activity is due to CIIT in SIRPα-NK cells. This is accomplished by inhibiting expression of the A gene, the human sequence of which is presented herein. This alteration is generally referred to herein as a gene "knockout" and is used in the manufacture of SIRP genes of the present invention. In α-NK cells, both alleles of the host cell are targeted.

[0091] Assays for testing whether the CIITA gene has been inactivated are known. In one embodiment, the assay is for CIITA protein. A Western blot of cell lysates probed with an antibody against In another embodiment, the presence of modifications to inactivate is confirmed by reverse transcriptase polymerase chain reaction (rt-PCR).

[0092] In addition, cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay is performed as is known in the art. Exemplary assays include Western blot or FACS analysis using commercially available antibodies that bind to human HLA class II HLA-DR, DP, and most DQ antigens, as outlined below. As is known in the art. Exemplary assays include Western blot or FACS analysis using commercially available antibodies that bind to human HLA class II HLA-DR, DP, and most DQ antigens, as outlined below. DR, DP, and most DQ antigens, as outlined below. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II.

[0093] A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II. A particularly useful embodiment uses CRISPR technology to cleave the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, a transcription factor essential for all MHC II molecules. After gene editing, the transfected cell culture is dissociated into single cells. The single cells are expanded into full-sized colonies, and the success of CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions that do not express CIITA are determined by PCR and can be shown by FACS analysis not to express MHC II / HLA-II.

[0094] 3. Reduced phagocytosis In addition to the reduction of HLA I and II (or MHC I and II) generally using B2M and CIITA knockouts, the HIS SIRPα-NK cells of the present invention are macrophages In addition to the reduction of HLA I and II (or MHC I and II) generally using B2M and CIITA knockouts, the HIS SIRPα-NK cells of the present invention are macrophages Phagocytosis by macrophages and susceptibility to killing by NK cells were reduced. The resulting cells "escape" from immune macrophages and innate immune pathways by one or more CD47 transgenes. .

[0095] a. Increase in CD47 In some embodiments, the reduction in macrophage phagocytosis and NK cell killing susceptibility is due to an increase in CD47 on the surface of HI SIRPα-NK cells. This can be done in several ways, as will be understood by those skilled in the art using "knock-in" or transgenic techniques. In some cases, the increase in CD47 expression is due to one or more CD47 transgenes. As will be understood by those skilled in the art using "knock-in" or transgenic techniques, this can be done in several ways. In some cases, the increase in CD47 expression is due to one or more CD47 transgenes. offspring.

[0096] Thus, in some embodiments, one or more copies of the CD47 gene are added to SIRPα-NK cells under the control of an inducible or constitutive promoter (the latter being preferred). In some embodiments, lentiviral constructs are used as described herein or as known in the art. The CD47 gene can be integrated into the genome of the host cell under the control of an appropriate promoter as known in the art. In some embodiments, lentiviral constructs are used as described herein or as known in the art. The CD47 gene can be integrated into the genome of the host cell under the control of an appropriate promoter as known in the art. or as known in the art. The CD47 gene can be integrated into the genome of the host cell under the control of an appropriate promoter as known in the art. As is known in the art, the CD47 gene can be integrated into the genome of the host cell under the control of an appropriate promoter.

[0097] In some embodiments, the expression of the CD47 gene can be increased by altering the regulatory sequences of the endogenous CD47 gene, for example, by replacing the endogenous promoter with a constitutive promoter or a different inducible promoter. This can generally be done using known techniques such as CRISPR. inducible promoter. This can generally be done using known techniques such as CRISPR. RISPR.

[0098] Once altered, the presence of sufficient CD47 expression can be detected by Western blotting using anti-CD47 antibodies. Techniques described in examples such as blotting, ELISA assays, or FACS assays can be used for the assay with any known technique. Generally, "sufficient ness" in this context means an increase in the expression of CD4 7 on the surface of HISIRPα-NK cells that suppresses killing by NK cells. The natural expression level in cells is too low to protect the cells from lysis by NK cells when MHC I

[0099] 4. Suicide gene In some embodiments, the present invention provides HISIRPα-NK cells comprising a "suicide gene" or "suicide switch". These are incorporated to function as a "safety switch" that can cause cell death when the cells grow and divide in an undesirable manner. In the "suicide gene" ablation approach, a suicide gene is included in a gene delivery vector encoding a protein that causes cell death only when activated by a specific compound. The suicide gene can encode an enzyme that selectively converts a non-toxic compound into a toxic metabolite. As a result, cells expressing the enzyme are specifically eliminated. In some embodiments, the suicide gene is the herpes simplex virus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In other embodiments, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5 -fluorocytosine (5-FC) (both of which are incorporated herein by reference in their entireties, Barese et al., Mol. Therap. 20(10):193 2-1943(2012), Xu et al., Cell Res. 8:73-8(1 998)).

[0100] In other embodiments, the suicide gene is an inducible caspase protein. The inducible caspase protein comprises at least a portion of a caspase protein capable of inducing apoptosis. In a preferred embodiment, the inducible caspase protein is iCasp9. iCasp9 is a human FK506 binding protein 12 (FKBP12) with an F36V mutation, which is connected to the gene encoding human caspase 9 via a series of amino acids. FKBP12-F36V contains the sequence of FKBP12. FKBP12-F36V binds with high affinity to the small molecule dimerizer AP1903. Thus, the suicide function of iCasp9 in the present invention is triggered by the administration of a chemical inducer of dimerization (CID). In some embodiments, the CID is the small molecule drug AP1903. Dimerization triggers rapid induction of apoptosis. (See International Publication No. WO 2011 / 146862, which is hereby incorporated by reference in its entirety; Stasi et al., N. Engl. J. Med. 365:18 (2011); Tey et al., Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which is hereby incorporated by reference in its entirety).

[0101] 5. Assay for HI phenotype Once HI cells are generated, the HI cells can be assayed for their low immunogenicity as generally described herein.

[0102] For example, low immunogenicity is assayed using several techniques. One exemplary technique is transplantation into allogeneic hosts and monitoring the survival of HI It contains a ring. Cells can be transfected to express luciferase, and then the organism can be tracked using bioluminescence imaging. Similarly, the response of T cells or B cells of the host animal to HISIRPα-NK cells is tested to confirm that no immune response is induced in the host animal. The function of T cells is evaluated by Elispot, ELISA, FACS, PCR, or mass cytometry (CYTOF). The B cell response or antibody response is evaluated using FACS or luminex. In addition, or alternatively, cells can be assayed for their ability to avoid innate immune responses, such as killing by NK cells. The lytic activity of NK cells is evaluated in vitro or in vivo using techniques known in the art.

[0103] C. Preparation of HISIRPα-NK O-cells In some embodiments of the present invention, the HISIRPα-NK cells prepared as described above are already ABO blood type O and Rh factor negative (-) cells because the process starts with NK cells having blood type O.

[0104] Other embodiments of the present invention include the enzymatic conversion of A and B antigens. In a preferred embodiment, the B antigen is converted to O using an enzyme. In a preferred embodiment, the enzyme is α-galactosidase. This enzyme removes the terminal galactose residue of the B antigen. Other embodiments of the present invention include the enzymatic conversion of the A antigen to O. In a preferred embodiment, the A antigen is converted to O using α-N-acetylgalactosaminidase. For enzymatic conversion, see, for example, each reference Olsson et al., Trans. Usion Clinique et Biologique 11:33-39(20 04); U.S. Patent Nos. 4,427,777, 5,606,042, Specification No. 5,633,130, Specification No. 5,731,426, Specification No. 6,184,0 Specification No. 17, Specification No. 4,609,627, and Specification No. 5,606,042 ; and discussed in WO 9923210.

[0105] Another embodiment of the invention is to knock out exon 7 of the ABO gene or to use SLC Genetically engineer cells by silencing the 14A1(JK) gene. Another embodiment of the present invention involves the use of Rh blood group (RH) C and E antigens. , KEL, Duffy (FY) Fya and Fy3, Kid These include knockouts of Jkb in the JK blood group, or U and S in the MNS blood group. Any method known in the art or described herein, such as SPR, talen, or homologous recombination. Any of the knockout methods described can be used.

[0106] Techniques for generating hypoimmune ABO blood group O Rh factor (-) cells are described in detail in the following references. No. 62 / 846,399, the entirety of which is incorporated herein by reference. It has been done.

[0107] D.SIRPα-CAR-NK cells Chimeric antigen receptors (chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) The CAR-based immunotherapy gives NK cells the new ability to target specific proteins. It is a receptor protein engineered to be as such. The receptor is chimeric because the antigen-binding function and the T cell activation function are combined into a single receptor.

[0108] CAR-NK cell therapy uses NK cells engineered with CAR for cancer treatment. The premise of CAR-NK immunotherapy is to more effectively target and destroy cancer cells by modifying NK cells to recognize cancer cells. Human NK cells expressing chimeric antigen receptors are transplanted into patients to attack tumors. CAR-NK cells can be derived from NK cells (autologous) in the patient's own blood or from NK cells (allogeneic) of another healthy donor. Once isolated from an individual, these NK cells are genetically engineered to express a specific CAR that programs the NK cells to target antigens present on the surface of the tumor. The CAR-NK cells of the present invention can be made SIRPα- by any of the techniques disclosed herein.

[0109] E. Transplantation of HI SIRPα-NK cells As will be understood by those skilled in the art, HI SIRPα-NK cells are transplanted using techniques known in the art to reduce tumors and treat cancer. Exemplary cancers treated using the cells disclosed herein include acute myeloid leukemia, non-small cell lung cancer, bladder neoplasm, hepatocellular carcinoma, melanoma, Merkel cell carcinoma, triple-negative breast cancer, ovarian cancer, renal cell carcinoma, colorectal cancer, and sarcoma.

[0110] Generally, the HI SIRPα-NK cells of the present invention are transplanted intravenously or into the patient's ​​​It is transplanted by injection at a specific location. When transplanting at a specific location, the cells can be suspended in a gel matrix to prevent dispersion while the cells are retained.

[0111] To more fully understand the invention described herein, the following examples are described. These examples are for illustrative purposes only and should not be construed as limiting the invention in any way.

Examples

[0112] VIII. Examples Example 1: Expression of CD47 protects cancer cells from killing mediated by NK cells Cancer cells expressing CD47 are protected from killing mediated by NK cells. K562 cells are a highly malignant immortalized human myeloid leukemia cell line. K562 cells were transfected to overexpress CD47 under a constitutive promoter at an approximately 6-fold increased expression level (Figure 1A).

[0113] Human K562 cells were a gift from Dr Lewis Lanier (UCSF, San Francisco, CA). The cells were cultured as suspension cells in RPMI 1640, 10% FCS hi and 1% Pen / Strep (all from Gibco, Waltham, MA). For transfection, 8 × 10 3 cells per well were plated in a 4 8-well plate. Firefly - luciferase virus particles (Gen target, San Diego, CA) or human CD47 virus particles (Therm o Fisher, Waltham, CA) and 8 μg / mL of polybrene (Sigma ​​​​​​​​​-Aldrich, St. Louis, MO) was added to the cell suspension and centrifuged at 1200 rpm for 2 minutes. The plates were incubated at 37 °C in a cell incubator and the transfection was stopped after 48 hours by changing the medium. The success of the transfection was confirmed by BLI of firefly - luciferase. The cells were incubated with 5 mg / mL D - luciferin (Biosynth AG, Staad, Switzerland) for 10 minutes and the BLI signal was measured on an amiHT bioimaging platform (Spectral Instruments Imaging, Tucson , AZ). For overexpression of CD47, cells were stained with an antibody against CD47 (clone MEM122, Thermo Fisher) or an isotype - matched control Ig (mouse IgM, clone PFR - 03, Thermo Fisher) and analyzed on a FACS Calibur (BD BioSciences). The results were shown as the fold - change relative to untransduced K562.

[0114] Figure 1B shows K562 overexpressing CD47 cultured with NK cells. The survival rate of K562 was measured by luciferase bioluminescence imaging (BLI), and cell death was quantified by the decrease in the BLI signal. K562 overexpressing CD47 was significantly protected from NK - cell - mediated killing compared to K562 cells not overexpressing CD47.

[0115] For the killing of K562, RPMI 1640, 10% FCS hi, 1% Pen / Strep, 1% MEM NEAA (all Gibco), 1% Sodium Pyruv ​​​ with effector cells in 0.2% 2-mercaptoethanol (both from Millipore) co-cultured with 5×10 4 primary NK cells (at a 1:1 ratio, StemCell T Technologies, Vancouver, BC, Canada), 5×10 4 cells of K562 or 5×10 4 K562 CD47 cells were used as target cells and the assay was performed in 24-well plates. NK cells were pre-stimulated overnight and during the assay with human IL-2 (100 ng / mL, Peprotech). The plates were centrifuged at 1200 rpm for 2 minutes. After incubation for 2 hours at 37 °C, 5 mg / mL D-luciferin (Biosynth AG) was added to the wells and the BLI signal was quantified 2 at up to photons / sec·cm / steradian using Ami HT (Spectral Ins truments Imaging) per 24 well. The data were normalized to wells containing only target cells. Some wells were pretreated with SIRPα blocking antibody (2 μg / mL, catalog number MBS822365, MyBioSource

[0116] Example 2: When target cells express CD47, SIRPα inhibits killing by NK cells The present invention is the first to recognize that SIRPα is expressed on NK cells and that target cells expressing CD47 are more protected from killing by NK cells. In particular, primary NK cells were shown to express SIRPα. Expression of SIRPα was detected by flow cytometry (examined in macrophages and primary NK cells using LSR II, BD Biosciences). Figure 2A shows SIRPα on macrophages, a known expression factor ( mean ± s.d., 4 independent experiments per group). Figure 2B shows that SIRPα is inducible by IL-2 on primary human NK cells (70036, Stemcell Technologies, Vancouver , Canada), indicating that SIRPα gradually increased over 5 days. After 5 days, SIRPα on primary human NK cells was similar to that on macrophages. , showing that SIRPα is inducible by IL-2 on primary human NK cells (70036, Stemcell Technologies, Vancouver cells (70036, Stemcell Technologies, Vancouver , Canada) and that SIRPα gradually increased over 5 days. After 5 days, SIRPα on primary human NK cells was similar to that on macrophages.

[0117] PBMCs were isolated from fresh blood by Ficoll separation and resuspended in RPMI -1640 containing 10% heat-inactivated fetal bovine serum (FCS hi), 1% pen / strep (all from Gibco), and 10 ng / ml human M-CSF (Peprotech, Rocky Hill, NJ). Cells were plated at a cell concentration of 1 × 10 6 cells per ml in 24-well plates, and the medium was changed every 2 days. From day 6, 1 μg / ml human IL-2 (Peprotech) was added to the medium for 24 hours before the assay was performed. Human primary NK cells were purchased from Stemcell Technologies and assayed after culturing in RPMI- 1640 with 10% FCS hi, 1% pen / strep, 1% MEM NEAA, 1% glutamine (all from Gibco). Cell culture was performed in uncoated flasks (Coming) treated with T175 TC, and the medium was changed every 2 days. Before the assay, anti-human CD3 MACS beads (Milteny were used. TC and the medium was changed every 2 days. Before the assay, anti-human CD3 MACS beads (Milteny Biotec) were used. Cells that are CD3- were sorted using (Auburn, CA). Human primary NK cells were stimulated with 100 ng / mL of IL2 (Peprotech) for various periods up to 5 days Macrophages or NK cells were incubated with anti-SIRPα (clone: 15-414, Bio legend) and mouse IgG2a isotype control (clone X39, BD Bio science) at 4°C for 45 minutes. The results were shown as the fold change in mean fluorescence intensity relative to isotype-matched control Ig staining Figure 2C shows that macrophages bind to CD47. Chimeric proteins were used

[0118] to establish CD47 binding. Figure 2D shows an increase in CD47 binding to primary human NK cells in parallel with the increase in SIRPα expression over 5 days shown in Figure 2B. Binding of chimeric proteins was quantified using flow cytometry Macrophages were generated as previously described. Human primary NK cells were purchased from StemC ell Technologies and cell culture was performed as described

[0119] NK cells were stimulated with human IL2 (100 ng / mL, Peprot ech) for various periods up to 5 days. Macrophages or NK cells were incubated with CD47 chimeric protein (46 70-CD, R&D systems) at 4°C for 4 hours. Human I gG1 antibody was used as secondary staining (polyclonal, Life Technologies) at 4°C for 45 minutes. The results were shown as the fold change in mean fluorescence intensity relative to Ig staining with secondary antibody only to control for non-specific binding

[0120] ​​​NK cell lines require IL2 medium for continuous culture. Therefore, Figure 3A shows primary human NK cells expressing SIRPα and four established NK cell lines in IL2 medium. In contrast to primary human NK cells showing SIRPα expression, the four established NK cell lines do not express SIRPα upon IL2 stimulation. Figure 3B shows that none of the four NK cell lines bind to CD47. Primary NK cells interact with CD47, while NK cell lines do not.

[0121] The NK cell lines NKL, NK-RL12, and NK-CT604 were cultured in RPMI-1640 containing 10% FCS-hi, 1% Pen / Strep, 1% L-glutamine, 1% HEPES, 1% sodium pyruvate, 1 % MEM-NEAA (all from Gibco), 0.1% 2-mercaptoethanol (Millipore), and 100 ng / mL human IL-2 (Peprotech). The NK cell line NK-92 was cultured in alpha-MEM containing 10% FCS-hi, 10% horse serum hi, 1% Pen / Strep, 1% L-glutamine (all from Gibco), 0.2 mM Myo-inositol, 0.02 mM folic acid (both from Sigma), 0.1% 2- mercaptoethanol (Millipore), and 100 ng / mL human IL-2 (P eprotech). Primary human NK cells were cultured as above and further stimulated with 100 ng / mL human IL2. Cell cultures were performed in uncoated flasks treated with T175 TC, and the medium was changed every two days. Flow cytometry was performed as described above for SIRPα expression and CD47 binding. Since expression and binding were shown as multiples of isotype controls, a value of 1 indicates no expression. And corresponds without conjugation.

[0122] Many tumors downregulate HLA class I and class II and upregulate CD47. This makes these tumors "low-immune" and helps them evade immune surveillance. and helps them evade immune surveillance. Engineered B2M- / -CIITA- / -CD47 tg human induced endothelial cells (hiEC) that do not express HLA class 1 and class 2 and overexpress CD47 were used as a low-immune tumor model. endothelial cells (hiEC) that do not express HLA class 1 and class 2 and overexpress CD47 were used as a low-immune tumor model.

[0123] 3 plasmid, 7 factor (SOKMNLT; SOX2, OCT4 (POU5F1), KL F4, MYC, NANOG, LIN28, and SV40L T antigen) EBNA-based episomal system was used to generate CD34 + human episomal iPSC lines derived from umbilical cord blood were used (Thermo Fisher Scientific, Waltham, MA). CR ISPR-Cas9 technology was used to generate B2M hiPSCs by cleaving the B2M and CIITA genes as described in Deuse T, et al. Nat Biotechnol. 2019 Mar;37:252-258. and CIITA genes as described in Deuse T, et al. Nat Biotechnol. 2019 Mar;37:252-258. - / - CIITA - / - hiPSCs were generated . To achieve overexpression of CD47, CD47 cDNA was synthesized, cloned into a lentiviral plasmid, and cells were transfected to obtain a pool of B2M CIITA - / - CIITA - / - CD47 tg hiPSCs. Human iPSCs were cultured in Essential 8 Flex medium (Thermo Fisher Scientific) at low Cultured in a 10 cm dish coated with Matrigel feeder-free (hESC certified, BD Biosciences, San Jose, CA). The medium was changed every 24 hours, and Versene (Gibco) was used for cell passage at a ratio of 1:6. Differentiation into hiEC began at 60% confluence, and the medium was replaced with RPMI-1640 containing 2% B-27 minus insulin (both from Gibco) and 5 μM CHIR-99021 (Selleckchem). On day 2, the medium was replaced with RPMI-1640 containing 2% B-27 minus insulin (Gibco) and 2 μM CHIR-99021 (Selleckchem). From day 4 to day 7, the cells were exposed to RPMI-1640 EC medium, i.e., RPMI-1640 containing 2% B-27 minus insulin, 50 ng / ml human vascular endothelial growth factor (VEGF; R&D Systems), 10 ng / ml human fibroblast growth factor basic (FGFb; R&D Systems), 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB431542 (Sigma-Aldrich). Endothelial cell clusters became visible on day 7, and the cells were maintained in endothelial cell basal medium 2 (PromoCell, Heidelberg, Germany) with supplements, 10% FCS hi (Gibco), 1% pen / strep, 25 ng / ml VEGF, 2 ng / ml FGFb, 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB431542 (Sigma-Aldrich). The differentiation process was completed after 14 days, and undifferentiated cells were separated during the differentiation process. TrypLE Express (Gibco) was used for 1:3 cell passage every 3 - 4 days. ​​​​​​​​​​​​​ It was used for cell passage.

[0124] NK cell lines lacking SIRPα were more aggressive in killing B2M- / - -CIITA- / -CD47 tg hiEC. Figure 4A shows that hiEC lacking HLA class I and HLA class II (B2M- / -CIITA- / -) were rapidly and efficiently killed by primary NK cells. However, target cells containing additional overexpression of CD47 were not killed. Figures 4B - 4E show the same pattern when the target cells lack CD47. However, when the target cells overexpress CD47, the target cells were killed more rapidly than primary cells. Figure 4B shows NK L cells. Figure 4C shows NK-CT604 cells. Figure 4D shows NK-RL12 cells and Figure 4E shows NK-92 cells. The graphs show the mean ± s.d. of three independent replicates for each group and time point, with three different effector cell:target cell (E:T) ratios.

[0125] The killing assay by NK cells was performed on the xCelligence SP platform and MP platform (ACEA BioSciences, San Diego, CA .). A special 96-well E-plate (ACEA BioSciences) was coated with collagen (Sigma-Aldrich), and 4×10 5 wt, B2M - / - CIITA - / - or B2M - / - CIITA - / - CD47 tg (pool or single clone) hiEC were plated in 100 μl of cell-specific medium. The cells When the index value reached 0.7, human NK cells were added at an E:T ratio of 0.5:1, 0.8:1, or 1:1 together with 1 ng / ml human IL-2 (Peprot ech).

[0126] Example 3: Blocking SIRPα on primary NK cells improves killing efficiency Blocking SIRPα on primary NK cells with an anti-SIRPα antibody led to a significant increase in the killing of CD47+ cells against target cells overexpressing CD47 compared to killing by NK cell lines. Figures 5A - 5E show the killing curves of B2M- / -CIITA- / -CD47 tg hiEC from killing by primary NK cells and NK cell lines in the presence of IL2. In some experiments, the binding of CD47 - SIRPα was prevented using specific antibodies against CD47 or SIRPα. Figure 5A shows that primary NK cells killed target cells very rapidly when either CD47 or SIRPα was blocked. Figure 5B shows the NKL cell line. Figure 5C shows the NK-CT604 cell line. Figure 5D shows the NK-RL12 cell line. Figure 5E shows the NK-92 cell line. Overall, neither the block of CD47 nor the block of SIRPα significantly altered the killing properties of the four NK cell lines. There was a mild tendency towards more rapid killing by anti-CD47 in the NK cell lines. This could be a non-specific effect of either the block of anti-CD47 or the block of FcR. This could promote antibody-mediated cytotoxicity of cells. The graph shows the mean ± s.d. of three independent replicates for each group and time point, and three different E:T ratios. The killing assay by NK cells was performed as described above. Some wells were treated with anti-CD47 block

[0127] ​Blocking antibody (10 μg / ml, clone B6.H12, BioXCell, West Lebanon, NH) was used for pretreatment for 2 hours and during the assay, or anti-SIRPα blo cking antibody (2 μg / ml, catalog number MBS822365, MyBi oSource) was used for pretreatment for 2 hours and during the assay. In some cases, NK cells were pretreated with human Fc receptor (FcR) block (catalog number 130-059-901, Milte nyi) for 4 hours before adding target cells.

[0128] The antibody blocked the interaction between target cell CD47 and primary NK cell SIRPα. The antibody did not show a clear effect on any of the four NK cell lines.

[0129] K562 and K562 overexpressing CD47 were transduced to express firefly luciferase. Figure 6A shows that when primary NK cells are stimulated with IL2, K562 is very effectively killed by primary NK cells in the BLI assay. Blocking of NK cell SIR Pα does not promote killing, suggesting that basal CD47 levels do not protect K562 from killing by NK cells. However, Figure 6B shows that K562 overexpressing CD47 is less susceptible to killing by primary NK cells and killing is much more inefficient. pNK cells stimulated with IL2 killed only about half of the K562 cancer cells, suggesting a survival effect associated with high levels of CD47. Blocking S IRPα improved the killing ability of primary NK cells against K562 overexpressing CD47. Blocking of SIRPα rendered cancer cells more susceptible to killing by NK. Made it easy and covered the protection by CD47 (mean ± s.d., 3 independent experiments per group) )

[0130] The BLI killing assay was performed as described above

[0131] The data show that the CD47 - SIRPα immune checkpoint is utilized by cancer cells to reduce NK cell - based immune attack

[0132] Example 4: Improved killing efficiency by blocking CD47 on cancer cell lines Different CD47 levels affect the killing of human cancer cell lines. All cancer cells were transduced to express firefly luciferase and cultured. 1×10 3 cancer cells were grown as targets in 96 - well plates. Human primary NK cells were sorted for the CD3 - CD7 + C D56 + population and then stimulated with human IL - 2 for 3 days. Then, NK cells were used as effector cells at a ratio of 10:1 against target cells. In some experiments, human FcR - blocking Ab and 10 μg / mL CD47 - blocking Ab (BioXcell, Lebanon, NH, catalog number: BE0019 - 1) were incubated and then NK cells were added. After 120 minutes, luciferase expression was detected by adding D - luciferin . Triton X served as a control

[0133] Cancer cell lines Hutu80 (Figure 7A, upper panel) and NCCIT (Figure 7B, upper panel) showed low levels of surface CD47 expression. However, Detroit 562 showed very high CD47 expression (Figure 7C, upper panel). The expression was compared to isotype control Shown as multiples of illumination, a value of 1 corresponds to no expression. Highly selected primary human N The CD3-CD7+CD56+ population of NK cells was stimulated with IL-2 for 3 days. Then, these stimulated NK cells were added to firefly luciferase-expressing cancer cells, and the survival of the cancer cells was monitored by bioluminescence imaging (BLI). The decrease in the BLI signal was correlated with cell death. In FIGS. 7A and 7B (lower panels), NK cells stimulated with IL-2 showed efficient cancer cell death that was not affected by the CD47 blocking antibody. Thus, CD47 had no protective effect on these cell lines at all. However, in FIG. 7C (lower panel), CD47 blocking significantly increased the killing by NK cells. This showed a high protective effect of CD47 on Detroit 56 2. All killing experiments using anti-CD47 blocking antibodies were performed using human FcR blocks to prevent antibody-dependent cellular cytotoxicity from affecting the readout. Thus, high CD47 expression was correlated with an inhibitory signal that reduced killing by NK cells in cancer

[0134] Example 5: Killing effect by human SIRPα- / - iPSC-derived NK cells SIRPα- / - iPSC-derived NK cells efficiently kill CD47+ cancer cells . Human HIP iPSCs that had undergone CRISPR / Cas9 inactivation of both the SIRPα allele and SIRPA- / - were confirmed by Sanger sequencing. The NK cell differentiation protocol was used as described above (bioRxiv preprint; doi:h ttp: / / dx.doi.org / 10.1101 / 614792), HIP iPS ttp: / / dx.doi.org / 10.1101 / 614792), HIP iPS NK cells were generated from C(iNK) and SIRPA- / - HIP iPSC (iNK(iNK SIRP-KO ). K562 cancer cells were generated to express firefly luciferase . Then, some K562 were further transduced with lentiviral particles carrying CD47 cDNA to achieve high CD47 expression (K562-CD47OV). K5 62 or K562-CD47OV was used as a target in the BLI killing assay as described above .

[0135] Additional gene editing was performed on human HIP iPSC (B2M- / -CIITA- / -CD47tg) to knockout the SIRPα gene. These SIRP A- / - iPSC were then differentiated into NK cells (iNK(SIRP-KO)). Human HIP iPSC-derived iNK cells served as a control. When using K562 as a target cell , both iNK and iNK(SIRP-KO) showed similar killing effects (Figure 8 A). When using K562 targets overexpressing CD47, iNK(SIRP-KO) was more aggressive, while iNK showed a certain degree of reduction in killing ability (Figure 8B). Therefore , for target cells expressing CD47 at a protective level, engineered iNK (SIRP-KO) was more potent.

[0136] IX. Exemplary Sequences: SEQ ID NO: 1 - Human SIRPα >NP_001317657.1 Tyrosine-protein phosphatase non-receptor type substrate 1 isoform 2 precursor [Homo sapiens]

Chem.

[0137] All publications and patent documents disclosed or referenced in this specification are incorporated by reference in their entirety. The foregoing description is presented for purposes of illustration and explanation only. This description is not intended to limit the invention to the exact form disclosed. The scope of the invention is intended to be defined by the claims appended hereto. incorporated. The foregoing description is presented for purposes of illustration and explanation only. This description is not intended to limit the invention to the exact form disclosed. The scope of the invention is intended to be defined by the claims appended hereto.

Claims

1. Signal Regulatory Proteins: Lower Relative to NK Cell Populations with Unaltered SIRPα-Function A population of engineered natural killer (NK) cells with alpha (SIRPα-) function. and wherein the modified NK cells are capable of inhibiting the proliferation and proliferation of cancer cells expressing CD47 in an in vitro NK assay. A population of modified NK cells that effectively kills the population.

2. In the assay, killing of the cancer cells occurs more rapidly than in the population of unmodified NK cells. The modified NK cell population of claim 1.

3. 3. The population of modified NK cells of claim 1 or 2, wherein the modified NK cells are primary NK cells. 。

4. the reduced SIRPα-function is due to a genetic modification of the population of modified NK cells. A population of modified NK cells according to any one of claims 1 to 3.

5. The genetic modification is SIRPα-knockout, alteration of the regulatory sequence, or frameshift. The population of modified NK cells according to claim 4, which is due to mutation.

6. The genetic modification may be a transcription activator-like effector nuclease (TALEN), Starred regularly interspaced short palindromic repeats / Cas9 (CRIS PR-Cas9), or zinc finger nuclease technology, 5. A population of modified NK cells according to claim 4.

7. The method according to any one of claims 1 to 3, wherein the reduction in SIRPα-function is due to an interfering nucleic acid molecule. A population of modified NK cells according to paragraph 1.

8. The interfering RNA may be a small interfering RNA (siRNA), an antisense oligonucleotide, ASO, locked nucleic acid (LNA), splice switching oligonucleotide ( SSO), and sno-derived RNA (sdRNA), A population of modified NK cells as described.

9. The reduction in SIRPα-function binds to the SIRPα- on the surface of the modified NK cell. A population of modified NK cells according to any one of claims 1 to 3, resulting from a molecule which inhibits the proliferation and proliferation of the NK cells.

10. 10. The population of modified NK cells of claim 9, wherein the molecule is an anti-SIRPα antibody.

11. The cancer is selected from the group consisting of acute myeloid leukemia, non-small cell lung cancer, bladder neoplasms, hepatocellular carcinoma, melanoma, and mercury. The group consisting of breast cancer, triple-negative breast cancer, ovarian cancer, renal cell carcinoma, colorectal cancer, and sarcoma The population of modified NK cells according to any one of claims 1 to 10, selected from:

12. Lower signal-regulatory protein α expression compared to NK cells with unaltered SIRPα function A population of engineered natural killer (NK) cells having α-function, The modified NK cells are capable of selectively isolating low-level immune cells expressing CD47 in an in vitro NK assay. A population of modified NK cells that effectively kills the population.

13. The method according to any one of claims 1 to 12, wherein the NK cells are derived from induced pluripotent stem cells (IPSCs). A population of modified NK cells according to any one of claims 1 to 4.

14. The method according to any one of claims 1 to 12, wherein the NK cells are derived from embryonic stem cells (ESCs). A population of modified NK cells.

15. 13. The method according to claim 1, wherein the NK cells comprise a chimeric antigen receptor (CAR-NK). A population of modified NK cells according to any one of claims 1 to 4.

16. The method comprises administering to a subject a population of modified NK cells according to any one of claims 1 to 15. , a method of treating cancer.

17. The subject may be a human, a mouse, a rat, a cat, a dog, a rabbit, a guinea pig, a hamster, 17. The method of claim 16, wherein the animal is selected from the group consisting of sheep, pigs, horses, cattle, and non-human primates. The method described.

18. The cancer is selected from the group consisting of acute myeloid leukemia, non-small cell lung cancer, bladder neoplasms, hepatocellular carcinoma, melanoma, and mercury. The group consisting of breast cancer, triple-negative breast cancer, ovarian cancer, renal cell carcinoma, colorectal cancer, and sarcoma The method of claim 16 , wherein the .alpha.-amino acid is selected from the group consisting of .alpha.-amino acid and .beta.-amino acid.

19. A method for producing a population of modified NK cells according to any one of claims 1 to 5, comprising the steps of: Transcription activator-like effector nucleases (TALENs), clustered ordered Interspaced short palindromic repeats / Cas9 (CRISPR-Cas9), or Using zinc finger nuclease technology to convert SIRPα+ NK cells to SIRPα- The method includes modifying the compound to produce a compound having a molecular weight of 100 or more.

20. The SIRPα protein has at least 90% sequence identity with SEQ ID NO:

1.

20. The method of claim 19.

21. 21. The method of claim 20, wherein the SIRPα protein has the sequence of SEQ ID NO:

1.

22. A method for producing a population of modified NK cells according to any one of claims 1 to 5, comprising: Interfering RNA (siRNA), antisense oligonucleotides (ASO), locked Nucleic acid (LNA), splice switching oligonucleotide (SSO), or sno Using sdRNA, the expression of SIRPα in the population of modified NK cells is downregulating

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