TRANSPLANTED CELL PROTECTION VIA Fc SEQUESTRATION

Abstract

To provide cells that evade antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).SOLUTION: The invention provides cells that comprise enhanced CD16, CD32, or CD64 expression to evade antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The cells may be pluripotent cells, including hypoimmune pluripotent cells (HIP) or ABO blood type O Rhesus Factor negative HIP cells (HIPO-), that further comprise the enhanced CD16, CD32, or CD64 expression. The invention encompasses cells derived from the pluripotent cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on October 15, 2019, which is incorporated by reference in its entirety. The disclosure of the U.S. Provisional Patent Application No. 62 / 915,601, pursuant to Section 119(e) of the U.S. Patent Act, ) priority is claimed.

[0002] The present invention relates to regenerative cell therapy. In some embodiments, regenerative cell therapy comprises a cell line. In some embodiments, the cell line is transplanted into a patient in need thereof. , including pluripotent cells with elevated CD16, CD32, or CD64 expression. In some forms, they are poorly immunogenic, have O blood type, or are Rh factor negative. In another embodiment, regenerative cell therapy is directed to a method for preventing the immune system of a cell transplant recipient from rejecting the allogeneic material. In some embodiments, regenerative cell therapy reduces the tendency for damaged organs and tissues to become damaged. In some embodiments, the regenerative cell therapy of the present invention is used in the treatment of chimeric Carcinoma antigen receptor (CAR) cells, endothelial cells, dopaminergic neurons, pancreatic islet cells, cardiac myocytes The use of thyroid cells, retinal pigmented endothelial cells, or thyroid cells, which are used to treat disease or repair damaged tissue, Used in retrospect. [Background technology]

[0003] Regenerative cell therapy is an important and promising treatment for regenerating damaged organs and tissues. Due to the low availability of organs and the associated long waiting times, it is necessary to develop a readily available cell line. The possibility of regenerating tissue by transplanting it into a patient is naturally attractive. Promising initial results on the recovery of damaged tissue after transplantation in animal models (e.g., after myocardial infarction) However, the immune system of the transplant recipient tends to reject the allogeneic material. The orientation significantly reduces the potential efficacy of therapeutic agents and reduces the possible favorable effects of such therapeutic agents. This reduces the negative effects.

[0004] Autologous induced pluripotent stem cells (iPSCs) are theoretically a promising method for patient-specific cell-based organ repair. These constitute an unlimited source of cells for needles. However, their production is technically and manufacturing It is a protracted process that poses challenges and conceptually precludes any acute treatment modality. iPSC-based or embryonic stem cell-based therapies are easier from a manufacturing standpoint and are sufficiently It allows for the production of a screened, standardized, high-quality cell product. Due to their allogeneic origin, such cell products are subject to rejection. is eliminated, resulting in the production of a universally acceptable cell product. can be differentiated into any cell type of the three germ layers, making them a potential candidate for stem cell therapy. Differentiation can be achieved ex vivo or in vivo in the organ environment at the transplant site. Ex vivo differentiation can be performed by transplanting progenitor cells that continue to mature. , allowing the researcher or clinician to closely monitor the procedure and ensure the appropriate cell population is generated before porting.

[0005] However, in most cases, undifferentiated pluripotent stem cells are not differentiated due to their tendency to form teratomas. Due to this, transplantation therapy is avoided in clinical practice. Rather, such therapy involves the transfer of differentiated cells (e.g. For example, the trend is to use stem cell-derived cardiomyocytes that are transplanted into the myocardium of patients suffering from heart failure. The clinical application of such pluripotent cells or tissues requires the growth and survival of the cells after transplantation. The system benefits from "safety features" that control

[0006] The art is rich in techniques for producing cells that can be used to regenerate or replace diseased or defective cells. Pluripotent stem cells (PSCs) are a type of stem cell that can be rapidly proliferated and used in many ways. As a family of PSCs, they can be used to differentiate into the cell types that can be obtained. has several members that have been generated via different techniques and have distinct immunogenic characteristics. Patient compatibility with engineered cells or tissues derived from PSCs is important, as it reduces the risk of immune rejection. Determine risk and immunosuppression requirements.

[0007] Embryonic stem cells (ESCs) isolated from the inner cell mass of blastocysts mismatch with recipients. This immunological barrier is mediated by the human leukocyte antigen (HLA) receptors on ESCs. This cannot be resolved by HLA-matched PSC grafts. are also rejected due to mismatches in non-HLA molecules that function as minor antigens. This also applies to allogeneic induced pluripotent stem cells (iPSCs).

[0008] hypoimmunogenic pluripotent (H IP) cells and cell products include, for example, T cells, NK cells, and macrophages. Gene knockouts or transgenes to protect them from cellular components of the immune system They may also be ABO blood group O and Rh negative (HIPO-).

[0009] The two most relevant killing mechanisms involving antibodies are NK cells, macrophages, and B cells or The mechanism of action of granulocytes in the regulation of complement cascade activation and antibody-dependent cellular cytotoxicity (ADCC) is All of these killing mechanisms involve binding to target cells and then killing the target cells through enzyme-dependent cytotoxicity (CDC). Antibodies that activate effector immune cells or complement are used (Figure 1A). IgG antibodies are IgG antibodies can be potent mediators of both ADCC and CDC. The Fab region is highly crystalline and has two variable regions that bind to NK cells, B cells, and It functions for binding of macrophages, granulocytes, or complement.

[0010] There are four different classes of receptors that recognize the Fc portion of IgG: FcγRI (CD64) FcγRII (CD32), FcγRIII (CD16), and FcγRIV. FcγRI has high affinity and restricted isotype specificity for antibody constant regions. FcγRII and FcγRIII show low affinity for the Fc region of IgG. FcγRIV has intermediate affinity but a broader isotype binding pattern. It is a recently identified receptor with gender and restricted subclass specificity. FcγRI functions during early immune responses, whereas FcγRII and RIII function in later immune responses. During the response, IgG is recognized as aggregates that surround multivalent antigens.

[0011] When an antibody binds to an unprotected cell through its Fab region, the FC is generated by NK cells (mostly via its CD16 receptor), macrophages (mostly CD16, CD32, or CD 64), B cells (mostly via CD32), or granulocytes (mostly via CD16, C Complement can also be bound by Fc (via FcD32, or CD64) and mediate ADCC. and activates the cascade that induces the membrane attack complex (MAC) for CDC killing. It can be formed.

[0012] In humans, CD16 occurs as CD16A and CD16B, which are involved in the extracellular immune response. It shares 96% sequence similarity in the globulin-binding domain (also known as FCGR3A and FC GR3B). There are several isoforms of FCGR3A, with a clear major There are no isoforms.

[0013] Pluripotent cells currently have no protection against ADCC or against CDC. Summary of the Invention [Means for solving the problem]

[0014] The present invention avoids antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The present invention provides for the first time cells with enhanced CD16, CD32, or CD64 expression for the treatment of cancer Lack of downstream activation or inhibitory motifs in target cells that do not normally express Fcγ receptors Considering this, the high Fc affinity of CD64 is due to protective Fc sequestration. Overexpression of any one or a combination of γ receptors I to IV may result in some or all of the following: should also show improved efficacy.

[0015] The present invention relates to a method for treating inflammatory bowel disease in mice in which CD16, CD32, or CD64 overexpression blocks the Fc portion of localized antibodies. , thus inhibiting ADCC and CDC (FIG. 1B). Cells, e.g., hypoimmune pluripotent cells (HIP), ABO blood group O rhesus factor negative H IP cells (HIPO-) induced pluripotent stem cells (iPSCs), O- iPSCs, embryonic stem cells The ESCs may be either ESCs that are O- or ESCs that are ES cells, both of which have enhanced CD64 expression. Further includes:

[0016] CD64 is found constitutively only on macrophages and monocytes and not normally on tissue cells. It is not expressed by FcγRI, more commonly known as Fc-gamma receptor 1 (FcγRI). CD64 binds with high affinity to the IgG Fc region. Overexpression of CD64 on target cells inhibits IgG Blocking Fc and binding it to target cells that do not have intracellular motifs for cell activation Even if the free Fab region binds to an adjacent target cell, Fc occupancy remains constant for any ADC. It also inhibits C or CDC.

[0017] Thus, the present invention provides an engineered pluripotent cell, which has a specific pluripotent function compared to a parental form of said engineered pluripotent cell. and having elevated levels of CD16, CD32, or CD64 protein expression, The expression of the protein enhances antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CCL) In some embodiments, modified pluripotent cells are provided that are less susceptible to CD40. 64 protein has at least 90% sequence identity to SEQ ID NO:7. In some embodiments, the CD64 protein has the sequence of SEQ ID NO: 7. 6 proteins have at least 90% sequence identity with SEQ ID NO: 9 or 10. In some embodiments, the CD16 protein has the sequence of SEQ ID NO: 9 or 10. In the present invention, the CD32 protein has at least 90% sequence identity with SEQ ID NO: 11, 12, or 13. In other embodiments, the CD32 protein has sequence identity to SEQ ID NO: 11, 12, or has 13 sequences.

[0018] In some embodiments of the present invention, the modified cells are human hypoimmunogenic pluripotent (HIP) cells, human Low immunogenicity pluripotent ABO blood group O rhesus factor negative (HIPO-) cells, human induced pluripotent They are derived from human inducible stem cells (iPSCs) or human embryonic stem cells (ESCs).

[0019] In other aspects of the invention, the modified cells are human, monkey, bovine, porcine, chicken, hamster, or rat. geese, horses, sheep, goats, donkeys, mules, ducks, geese, buffaloes, camels, From yaks, llamas, alpacas, mice, rats, dogs, cats, hamsters, and guinea pigs The species is selected from the group consisting of:

[0020] The present invention further provides a suicide gene that is activated by a trigger that causes the death of the modified cells. In some embodiments, the modified pluripotent cell further comprises a suicide gene. is the herpes simplex virus thymidine kinase gene (HSV-tk), and the trigger is In another embodiment, the HSV-tk gene is at least one of SEQ ID NO:4. In a preferred embodiment, the HS The V-tk gene encodes a protein comprising the sequence of SEQ ID NO: 4. Other aspects of the invention In Escherichia coli, the suicide gene is the cytosine deaminase The enzyme gene (EC-CD) is triggered by 5-fluorocytosine (5-FC). In a preferred embodiment, the EC-CD gene has at least 90% sequence identity with SEQ ID NO:5. In a more preferred embodiment, the EC-CD gene encodes a protein that contains sequence identity. encodes a protein comprising the sequence of SEQ ID NO:5.

[0021] In some embodiments of the present invention, the suicide gene encodes an inducible caspase protein. In a preferred embodiment, the trigger is a dimer-inducing compound (CID). The present invention relates to a method for producing an inducible caspase protein comprising at least 90% sequence identity with SEQ ID NO:6. In a more preferred embodiment, the gene is an inducible gene comprising the sequence of SEQ ID NO:6. In another preferred embodiment, the CID encodes an AP19 spase protein. The number is 03.

[0022] The present invention relates to chimeric antigen receptor (CAR) cells, endothelial cells, dopaminergic neurons , pancreatic islet cells, cardiomyocytes, retinal pigmented endothelial cells, and thyroid cells; In some embodiments, the present invention provides a cell derived from the modified pluripotent cell described herein. The R cells are CAR-T cells.

[0023] The present invention relates to a method for the treatment of psoriasis by transplanting cells derived from the modified pluripotent cells disclosed herein into a subject. and wherein the subject is a human, a monkey, a cow, a pig, a chicken, a turkey, Horses, sheep, goats, donkeys, mules, ducks, geese, buffaloes, camels, yaks, llamas , alpaca, mouse, rat, dog, cat, hamster, guinea pig. In some embodiments, the cells derived from the modified pluripotent cells express a chimeric antigen receptor (CAR). ) cells, endothelial cells, dopaminergic neurons, pancreatic islet cells, cardiac myocytes, retinal pigment endothelial cells and thyroid cells.

[0024] The invention includes administering cells derived from the modified pluripotent cells disclosed herein. In some embodiments, the derived cells are capable of expressing a chimeric antigen receptor (CACR) in a subject. (CAR) cells, endothelial cells, dopaminergic neurons, pancreatic islet cells, cardiomyocytes, retinal pigment epithelium In another embodiment, the disease is selected from the group consisting of endothelial cells, thyroid cells, and thyroid cells. from the group consisting of type 2 diabetes, heart disease, neurological disease, cancer, eye disease, vascular disease, and thyroid disease Be selected.

[0025] The present invention relates to the expression of CD16, CD32, or CD64 in the parent unmodified form of the pluripotent cells. The present invention provides a method for generating the modified pluripotent cells disclosed herein, comprising increasing In some embodiments, the modified cells are human, monkey, bovine, porcine, chicken, or porcine. Sheep, horses, sheep, goats, donkeys, mules, ducks, geese, buffaloes, camels, yaks , llama, alpaca, mouse, rat, dog, cat, hamster, or guinea pig origin. In some embodiments, the modified pluripotent cells are HIP cells, HIPO-cells, iPSCs, cells, or derived from ESC cells.

[0026] In some embodiments of the invention, increased CD16, CD32, or CD64 expression is At least one of the human CD16, CD32, or CD64 genes under the control of the In a preferred embodiment, the gene is a pluripotent cell, which is a parental type of the modified pluripotent cell. The promoter is a constitutive promoter.

[0027] The present invention relates to a cell derived from an engineered pluripotent cell according to any one of claims 1 to 19, and A pharmaceutical composition for treating a disease is provided, comprising a pharma- ceutical acceptable carrier. In the study, the derived cells include chimeric antigen receptor (CAR) cells, endothelial cells, dopaminergic cells, and from the group consisting of neuronal cells, pancreatic islet cells, cardiac myocytes, retinal pigmented endothelial cells, and thyroid cells. In other embodiments, the disease is selected from the group consisting of type I diabetes, cardiovascular disease, neurological disease, cancer, and ophthalmological disease. , vascular disease, and thyroid disease.

[0028] The present invention relates to a method for treating a disease comprising administering to the patient a therapeutically effective amount of a pluripotent cell .... In some embodiments, the derived cells express a chimeric antigen receptor (CA) R) Cells, endothelial cells, dopaminergic neurons, pancreatic islet cells, cardiac myocytes, retinal pigment endothelium In another embodiment, the disease is selected from the group consisting of type I diabetes mellitus. a disease selected from the group consisting of a heart disease, a neurological disease, a cancer, an ophthalmological disease, a vascular disease, and a thyroid disease. can be.

[0029] The present invention relates to a method for the detection of inflammatory bowel disorders in which the modified cells exhibit elevated levels of CD16, CD32, or CD 64. A modified cell comprising a protein expression, the elevated protein expression resulting in antibody-dependent Modifications that render the cells less susceptible to ADCC or complement-dependent cytotoxicity (CDC) In some embodiments, the cells are selected from the group consisting of chimeric antigen receptor (CAR) cells, endothelial cells, and the like. cells, dopaminergic neurons, pancreatic islet cells, cardiomyocytes, retinal pigmented endothelial cells, and thyroid glandular cells. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 is a schematic diagram of ADCC and CDC. [Diagram 2] Schematic diagram of the present invention: Cells expressing CD64 block antibody Fc domains and prevent ADCC or CDC. [Diagram 3]We show that CD52+ human macrophages were protected from anti-CD52 antibody (alemtuzumab)-mediated ADCC by their constitutive expression of CD64. Anti-CD64 antibody abolished the protection, which was most evident at lower anti-CD52 antibody concentrations. [Figure 4] Figure 1 shows that CD52+ human HIP iPSC-derived endothelial cells were protected by CD64 from NK cell ADCC induced by anti-CD52 antibody (alemtuzumab). Anti-CD64 antibody abolished the protection. The presence of the CD64 transgene significantly rescued endothelial cells at all but the highest levels of anti-CD52 antibody. [Diagram 5] We show that CD52+ macrophages are protected from anti-CD52 antibody (alemtuzumab)-mediated CDC due to their constitutive expression of CD64. Anti-CD64 antibody abolished the protection, which was most evident at lower anti-CD52 antibody concentrations. [Figure 6] 6A-C show mouse HIP iECs engineered to express CD52 (FIG. 6A), CD52 and CD64 (FIG. 6B), and CD64 (FIG. 6C). [Figure 7] 7A-B show that mouse HIP iECs were unable to bind alemtuzumab Fc (FIG. 7A), whereas mouse HIP iECs (CD64) were able to bind alemtuzumab Fc in a concentration-dependent manner (FIG. 7B). [Figure 8] Figure 1 shows that CD52+ mouse HIP iECs were concentration-dependently sensitive to anti-CD52 antibody (alemtuzumab)-mediated syngeneic NK cell ADCC (top row), but they were protected from NK cell killing when co-expressed CD64 (bottom row). [Figure 9] Figure 1 shows that CD52+ murine HIP iECs were concentration-dependently sensitive to anti-CD52 antibody (alemtuzumab)-mediated syngeneic macrophage ADCC (top row), but they were protected from macrophage killing when co-expressed CD64 (bottom row). [Figure 10]Figure 1 shows that CD52+ mouse HIP iECs were concentration-dependently sensitive to anti-CD52 antibody (alemtuzumab)-mediated syngeneic PMN ADCC (top row), but they were protected from PMN killing when co-expressed CD64 (bottom row). [Figure 11] Figure 1 shows that CD52+ murine B6 HIP iECs were sensitive to anti-CD52 antibody (alemtuzumab)-mediated ADCC by allogeneic BALB / c NK cells in a concentration-dependent manner (top row), but they were protected from BALB / c NK cell killing when co-expressed CD64 (bottom row). [Figure 12] Figure 1 shows that CD52+ murine B6 HIP iECs were concentration-dependently sensitive to anti-CD52 antibody (alemtuzumab)-mediated ADCC by allogeneic BALB / c macrophages (top row), but were protected from BALB / c macrophage killing when they co-expressed CD64 (bottom row). [Figure 13] Figure 1 shows that CD52+ murine B6 HIP iECs were concentration-dependently sensitive to anti-CD52 antibody (alemtuzumab)-mediated ADCC by allogeneic PMNs (top row), but they were protected from BALB / c PMN killing when co-expressed CD64 (bottom row). [Figure 14] Figure 1 shows that CD52+ murine B6 HIP iECs were sensitive to anti-CD52 antibody (alemtuzumab)-mediated CDC in a concentration-dependent manner (top row), whereas anti-CD64 antibody had no effect on this killing because they did not express CD64 (bottom row). [Figure 15] CD52+ CD64+ murine B6 HIP iECs were protected from anti-CD52 antibody (alemtuzumab)-mediated CDC across all concentrations tested, whereas anti-CD64 antibody abolished this protection (bottom row). [Figure 16] Figures 16A-C show human HIP iECs engineered to express CD52 (Figure 16A), CD52 and CD64 (Figure 16B), and CD64 (Figure 16C). [Figure 17]Figures 17A-B show that human HIP iECs were unable to bind alemtuzumab Fc (Figure 17A), but human HIP iECs(CD64) bound to alemtuzumab Fc in a concentration-dependent manner (Figure 17B). [Figure 18] 1 shows that human macrophages constitutively expressing CD64 bound to alemtuzumab Fc in a concentration-dependent manner. [Figure 19] Figure 1 shows that CD52+ human HIP iECs were sensitive to anti-CD52 antibody (alemtuzumab)-mediated NK cell ADCC in a concentration-dependent manner (top row), but were largely protected from allogeneic NK cell killing when they co-expressed CD64 (bottom row). Only the highest anti-CD52 antibody concentrations caused some cytotoxicity. [Figure 20] Figure 1 shows that CD52+ human HIP iECs were sensitive to anti-CD52 antibody (alemtuzumab)-mediated allogeneic macrophage ADCC in a concentration-dependent manner (top row), but they were largely protected from macrophage killing when they co-expressed CD64 (bottom row). Only the highest anti-CD52 antibody concentrations caused some cytotoxicity. [Figure 21] Figure 1 shows that CD52+ human HIP iECs were sensitive to anti-CD52 antibody (alemtuzumab)-mediated CDC in a concentration-dependent manner (top row), whereas anti-CD64 antibody did not affect this killing because they did not express CD64 (bottom row). [Figure 22] Figure 1 shows that CD52+ CD64+ human HIP iECs were largely protected from anti-CD52 antibody (alemtuzumab)-mediated CDC. Only the highest anti-CD52 antibody concentrations caused some cytotoxicity. Anti-CD64 antibody abolished this protection (bottom row). [Diagram 23]Figures 23A-B show human (Figure 23A) and mouse (Figure 23B) thyroid epithelial cells (epiC) engineered to express CD64. Human epiC was unable to bind to alemtuzumab Fc, whereas human epiC(CD64) bound to alemtuzumab Fc in a concentration-dependent manner (Figure 23C). Mouse epiC was unable to bind to alemtuzumab Fc, whereas mouse epiC(CD64) bound to alemtuzumab Fc in a concentration-dependent manner (Figure 23D). [Figure 24] Figure 24A shows that human epiC was unable to bind to anti-TPO Fc, whereas human epiC(CD64) bound to anti-TPO Fc in a concentration-dependent manner (Figure 24A). Mouse epiC was unable to bind to anti-TPO Fc, whereas mouse epiC(CD64) bound to anti-TPO Fc in a concentration-dependent manner (Figure 24B). [Diagram 25] 1 shows that C57BL / 6 thyroid epiC were susceptible to anti-TPO-mediated killing when incubated with syngeneic macrophages as effector cells for ADCC. [Figure 26] We show that CD64-expressing C57BL / 6 thyroid epiCs were not susceptible to anti-TPO-mediated killing when incubated with syngeneic macrophages as effector cells for ADCC. Over a wide range of anti-TPO concentrations, target cells were protected from anti-TPO ADCC. [Figure 27] Figure 27A-B shows a clinically relevant in vivo model for antibody-mediated rejection. Mouse HIP iECs bearing CD52 were killed when syngeneic recipients were treated with alemtuzumab (Figure 27A). However, mouse HIP iECs bearing CD52 and expressing CD64 were completely protected from antibody-mediated rejection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] A. Introduction The present invention avoids antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The present invention provides for the first time cells with enhanced CD16, CD32, or CD64 expression for the treatment of cancer The cells may be pluripotent cells, e.g., hypoimmune pluripotent cells, further comprising enhanced CD64 expression. These cells may be either ABO blood group O rhesus factor negative HIP cells (HIPO-) or ABO blood group O rhesus factor negative HIP cells (HIPO-). do.

[0032] One specific application for this technology may be the cell therapy of autoimmune diseases. Antibodies against epitopes mediate beta cell death and autoimmune thyroiditis in type 1 diabetes The antibodies can bind to HLA class II molecules. Furthermore, HLA-independent antibodies have been described. If the CD6 of the present invention has a CD4+ receptor, it will also be bound and killed by those antibodies. 4 Blockade prevents ADCC or CDC.

[0033] In some embodiments of the invention, hypoimmunogenic pluripotent ("HIP") cells are , CD32, or CD64 expression (HIP / CD16, HI P / CD32, or HIP / CD64 cells). HIP cells can be Due to some genetic manipulation, the cells evade the host immune response. They are engineered to lack key immune antigens and to avoid phagocytosis and NK cell killing. In an embodiment, the HIP cells are characterized by the expression of both the B2M gene and the B2M gene in induced pluripotent stem cells (iPSCs). Eliminate the activity of one allele; activity of both alleles of the CIITA gene in iPSCs and by increasing the expression of CD47 in iPSCs. HIP cells are described in WO 2018132, which is incorporated herein by reference in its entirety. Further details are provided in pamphlet No. 783.

[0034] In another embodiment of the invention, the hypoimmunogenic pluripotent blood type O Rh- ("HIPO-") ) Cells are modified to enhance CD16, CD32, or CD64 expression (HI HIPO- / CD16 cells, HIPO- / CD32, or HIPO- / CD64 cells). PO-cells are a type of cell that exhibits enhanced host immunity due to several genetic or enzymatic manipulations outlined herein. The cells lack the major blood type and immune antigens that trigger an immune response, preventing rejection. These are engineered to avoid starvation, phagocytosis, or killing. This allows the growth of specific tissues and organs. This allows the derivation of a "ready-made" cell product for the production of human allogeneic HIPO-cells and their The advantage of being able to use derivatives in human patients is that they provide significant benefits, e.g., allogeneic transplantation. This provides the ability to avoid the long-term adjuvant immunosuppressive therapy and drug use commonly seen in transplants. These allow cell therapy to be used without the requirement of individual treatment for each patient. This also provides significant cost savings. Alternatively, HIPO- / CD64 cells may be used as a universal cell line for the generation of universally acceptable derivatives. HIPO-cells can serve as a cell source. Nos. 62 / 846,399 and 62 / 855, incorporated herein by reference. This is described in detail in the 499 specification.

[0035] B. Definition The term "pluripotent cell" refers to a cell that is capable of self-renewal and proliferation while maintaining an undifferentiated state; It refers to a cell that can be induced to differentiate into a specialized cell type under appropriate conditions. The term "pluripotent cells" as used herein refers to embryonic stem cells and other types of stem cells, e.g. Exemplary human stem cell systems include fetal, amniotic, or somatic stem cells. 9 human embryonic stem cell line. Additional exemplary stem cell lines include the National Institutes of Health Human Embryonic St em Cell Registry and the Howard Hughes Medicare available through the cal Institute HUES collection. (Cowan, CA, incorporated herein by reference in its entirety). .et.al, New England J.Med.350:13.(2004) ).

[0036] As used herein, "pluripotent stem cells" refer to stem cells that can express one or more of three germ layers: endoderm (e.g., gastric the esophagus (e.g., the gastrointestinal tract, lungs, etc.), the mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.) or the ectoderm The cells have the potential to differentiate into either the epidermis or the nervous system. The term "pluripotent stem cell" as used herein refers to a pluripotent stem cell derived from a non-pluripotent cell. The term also includes the cell type "induced pluripotent stem cells," or "iPSCs." Examples of parent cells include Somatic cells have been reprogrammed by various means to induce a pluripotent undifferentiated phenotype. Such "iPS" or "iPSC" cells induce the expression of certain regulatory genes. These can be produced by induction of a gene or by exogenous application of a protein. Methods for deriving S cells are known in the art and are described further below (e.g., Zhou et al., Stem Cells 27(11):2667-74(20 09);Huangfu et al.,Nature Biotechnol.26( 7):795(2008);Woltjen et al.,Nature 458(7 239):766-770(2009); and Zhou et al., Cell St See em Cell 8:381-384 (2009); each of which is incorporated by reference. The generation of induced pluripotent stem cells (iPSCs) can be carried out as follows: As used herein, "hiPSC" refers to human induced pluripotent stem cells. "miPSC" is a mouse induced pluripotent stem cell.

[0037] "Pluripotent stem cell characteristics" refer to characteristics of a cell that distinguish a pluripotent stem cell from other cells. Under appropriate conditions, cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm) can be associated. The ability of a cell to give rise to progeny capable of undergoing differentiation into cell types that collectively demonstrate the characteristics The expression or non-expression of certain combinations of molecular markers are also characteristic of pluripotent stem cells. For example, human pluripotent stem cells may be characterized by at least one marker from the following non-limiting list: The polypeptide expresses at least some, and in some embodiments all, of: SSEA-3, SSEA-4, SSEA-5, SSEA-6, SSEA-7, SSEA-8, SSEA-9, SSEA-10, SSEA-11, SSEA-12, SSEA-13, SSEA-14, SSEA-15, SSEA-16, SSEA-17, SSEA-18, SSEA-19 EA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, S ox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Pluripotent stem The cell morphology associated with the cells is also characteristic of pluripotent stem cells. Requires that cells undergo pluripotency to be reprogrammed into endometrial progenitor cells and / or hepatocytes. do not have.

[0038] As used herein, "multipotent" or "multipotent cells" refers to a limited number of other specific For example, induced multipotent cells can give rise to endodermal cells. In addition, multipotent blood stem cells can themselves give rise to several types of blood cells. They can differentiate into cells such as lymphocytes, monocytes, and neutrophils.

[0039] As used herein, the term "oligopotent" refers to the differentiation of several different cell types. For example, lymphoid or myeloid stem cells can differentiate into only one of the following lineages: They can give rise to cells of either lymphoid or myeloid lineage.

[0040] As used herein, the term "unipotent" refers to a cell that is capable of forming a single cell type. For example, spermatogonial stem cells can only form sperm cells.

[0041] As used herein, "totipotency" refers to the ability of a cell to form an entire organism. For example, in mammals, only the zygote and the blastomeres in the first cleavage state are totipotent. .

[0042] As used herein, a "non-pluripotent cell" refers to a mammalian cell that is not a pluripotent cell. Examples of such cells include differentiated cells and progenitor cells. Examples include, but are not limited to, from bone marrow, skin, skeletal muscle, adipose tissue and peripheral blood. Exemplary cell types include, but are not limited to, cells from selected tissues. However, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T Cells include those used to generate induced multipotent cells, endodermal progenitor cells, and hepatocytes. The starting cell may be a non-pluripotent cell.

[0043] Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, and unipotent cells. , progenitor cells, and terminally differentiated cells. In certain embodiments, low potency cells are , are considered to be "differentiated" relative to highly potent cells.

[0044] "Somatic cells" are cells that form the body of an organism. Somatic cells include organs, skin, and other parts of an organism. These include cells that make up the skin, blood, bone and connective tissue, but exclude germ cells.

[0045] The cells can be, for example, from a human or a non-human mammal. Exemplary Non-Human Mammals Examples of such animals include, but are not limited to, mice, rats, cats, dogs, rabbits, and guinea pigs. These include mice, hamsters, sheep, pigs, horses, cattle, and non-human primates. In some embodiments, the cells are from an adult human or non-human mammal. In the present invention, the cells are from a neonatal human, an adult human, or a non-human mammal.

[0046] As used herein, the term "subject" or "patient" refers to any animal, For example, a domestic animal, a zoo animal, or a human. For example, the subject may be a dog, a cat, a bird, a livestock animal, or a human. Examples include, but are not limited to, liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood The present invention also includes individuals (particularly humans) who have a disease or disorder associated with the blood, bones, bone marrow, etc.

[0047] The mammalian cells can be from a human or non-human mammal. Exemplary non-human mammals include: Examples include, but are not limited to, mice, rats, cats, dogs, rabbits, and guinea pigs. , hamsters, sheep, pigs, horses, cows, and non-human primates (e.g., chimpanzees, macaques, and apes.

[0048] "Hypoimmunogenic pluripotent" cells or "HIP" cells as used herein are cells that retain their pluripotent characteristics. and still produce a reduced immunological rejection response when transplanted into an allogeneic host. In a preferred embodiment, HIP cells are cells that do not generate an immune response. Thus, "low immunogenicity" refers to the relative immunogenicity of the parent (i.e., refers to an immune response that is significantly reduced or eliminated compared to the immune response of "wt" (i.e., wild-type) cells. In many cases, HIP cells are immunologically silent and still retain pluripotent potential. Assays for HIP characteristics are outlined below.

[0049] As used herein, "HIP / CD16," "HIP / CD32," or "HIP / CD "64" cells are HIP cells with enhanced CD16, CD32, or CD64 expression, respectively. It means cells.

[0050] As used herein, the term "low immunogenic pluripotent cells O-", "low immunogenic pluripotent ORh-" cells or "HIPO-" cells are HIP cells that are ABO blood type O and Rhesus factor Rh-. HIPO- cells are those that have been enzymatically modified to become O- or that have been modified to become O-. The present invention can be produced from genetically engineered O-cells such as

[0051] As used herein, "HIPO- / CD16", "HIPO- / CD32", or "HI PO- / CD64 cells have enhanced CD16, CD32, or CD64 expression IPO-cells are meant.

[0052] The "HLA" or "human leukocyte antigen" complex is the major histocompatibility complex (MHC) in humans. These cells that make up the HLA complex are the gene complexes that code for the HLA proteins. Cell surface proteins are responsible for regulating the immune response to antigens. In humans, there are two MHC There are two types of HLA: class I and class II, "HLA-I" and "HLA-II." I includes three proteins, HLA-A, HLA-B and HLA-C, They present peptides from inside cells and recognize antigens presented by HLA-I complexes. HLA attracts HLA T cells (also known as CD8+ T cells or cytotoxic T cells). The HLA-I protein associates with beta-2 microglobulin (B2M). The five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and These include HLA-DR and HLA-P, which present antigens from the outside of cells to T lymphocytes. "MHC" or "HLA" stimulates CD4+ cells (also known as T helper cells). The use of either "HLA" or "MHC" is based on whether the genes are from human (HLA) or mouse (MHC). It should be understood that this is not meant to be limiting, as it depends on the and when it comes to mammalian cells, the terms are used interchangeably herein. It is possible.

[0053] As used herein, a "gene knockout" refers to the deletion of a particular gene from a host cell in which it resides. a process that renders the protein of interest either non-productive or in an inactive form. As will be appreciated by those of skill in the art, and as described further below, this This can be achieved by a number of different techniques, such as the removal of nucleic acid sequences from genes or the addition of other sequences to the sequences. This can be achieved by disrupting the sequence, altering the reading frame, or altering the regulatory components of the nucleic acid. For example, all or part of the coding region of a gene of interest can be removed, or a "nonsense" sequences, and removing all or part of a regulatory sequence, e.g., a promoter. The translation initiation sequence can be removed or replaced, and the translation initiation sequence can be removed or replaced, etc. Such is the case.

[0054] "Gene knock-in," as used herein, refers to the process of adding gene function to a host cell. This results in increased levels of the encoded protein. As will be appreciated by those skilled in the art, this can be accomplished by a number of techniques, e.g., by introducing one or more genes into a host cell. Addition of additional copies of or regulatory components on endogenous genes to increase protein expression This can be achieved by modifying the promoter and using a different promoter. This can be achieved by adding enhancers or modifying other gene expression sequences. It is possible.

[0055] "Beta-2 microglobulin" or "Beta2M" or "B2M" protein is shown below. This refers to the human β2M protein, which has the amino acid and nucleic acid sequence identified as Session number NC_000015.10:44711487~44718159 do.

[0056] The "CD47 protein" protein has the amino acid and nucleic acid sequences shown below Refers to the human CD47 protein; the human gene is identified under the accession number NC_00001 6.10:10866208-10941562.

[0057] The "CIITA protein" has the amino acid and nucleic acid sequences shown below. This refers to the human CIITA protein; the human gene is available under the accession number NC_000 003.12:108043094-108094200.

[0058] "Wild-type" in the context of a cell means a cell as found in nature. As used herein in the context of pluripotent stem cells, this refers to cells that have pluripotency. The nucleic acid alterations may be indicative of a gene that has undergone the gene editing procedures of the present invention to achieve low immunogenicity. This also refers to iPSCs that could not be produced.

[0059] As used herein, "syngeneic" refers to the genetic similarity or identity of the host organism and the cell transplant. and there is immunological compatibility; e.g., no immune response is generated.

[0060] "Allogeneic" as used herein refers to the genetic dissimilarity of the host organism and the cell transplant, and A response is generated.

[0061] As used herein, "B2M- / -" refers to a diploid cell in which B2 is inactivated in both chromosomes. This means that the individual has the M gene. As described herein, this can be accomplished in a variety of ways. This can be done.

[0062] As used herein, "CIITA- / -" refers to a diploid cell in which the CIITA gene is inactivated in both chromosomes. As described herein, this can be achieved by a variety of techniques. This can be done.

[0063] "CD47 tg" (for "transgene") or "CD47+" herein in some cases by having at least one additional copy of the CD47 gene, It is meant that the host cell expresses CD47.

[0064] "Oct Polypeptide" refers to a naturally occurring member of the octamer family of transcription factors. , or similar (at least 50%, Those variants that maintain transcription factor activity within 80% or 90% of the activity, or A polypeptide that contains at least the DNA binding domain of a naturally occurring family member. Exemplary Oct polypeptides include those that are Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8 Oct3 / 4 (referred to herein as "Oct3 / 4"). t4) is composed of the POU domain, Pit-1, Oct-1, Oct-2 and ur It contains a 150 amino acid sequence conserved among ic-86 (herein incorporated by reference in its entirety). Incorporated into the book, Ryan, AK & Rosenfeld, MG, Genes Dev. 11:1207-1225 (1997). In some embodiments, Variants include naturally occurring Oct polypeptide family members, such as those listed above. or, for example, Genbank Accession No. NP-002692.2 (human Oct4) or NP-038661.1 (mouse Oct4) and at least 85%, 90%, or 95% amino acid sequence identity across their entire sequences. Oct polypeptides (e.g., Oct3 / 4 or Oct4) are human, They may be from mice, rats, cows, pigs, or other animals. The protein is of the same species as the cell species. Oct polypeptides are expressed in non-pluripotent cells. The present invention may be a pluripotency factor that can help induce pluripotency in the cells.

[0065] "Klf polypeptide" refers to a naturally occurring member of the Krüppel-like factor (Klf) family. Member, an amino acid sequence similar to that of the Drosophila embryonic pattern regulator Krüppel or the closest naturally occurring family member Similar (within at least 50%, 80%, or 90% activity) transcription factor activity compared to A naturally occurring variant of a member that maintains the It refers to any polypeptide that contains at least a DNA binding domain, and a transcription activation domain. (Dang, DT, incorporated herein by reference in its entirety. .,Pevsner,J.& Yang,VW,Cell Biol.32:110 3-1121 (2000). Exemplary Klf family members include Klf 1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Kl f9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, K Klf2 and Klf-4 are involved in the regulation of the endothelial cell cycle in mice. It was found to be a factor capable of generating PS cells, and the related genes Klf1 and Klf5 were also The results were similar but less efficient (Nakamura, 2002, incorporated herein by reference in its entirety). gawa,et al.,Nature Biotechnology 26:101- 106 (2007). In some embodiments, the variant is a naturally occurring Kl f polypeptide family members, such as those listed above or, for example, those listed in Genba nk accession number CAX16088 (mouse Klf4) or CAX14962 At least 85% across their entire sequences compared to those listed in (human Klf4) , 90%, or 95% amino acid sequence identity. lf1, Klf4 and Klf5) in humans, mice, rats, cows, pigs or other animals. Generally, proteins of the same species as the cell being engineered are used. The Klf polypeptide may be a pluripotency factor. Expression of the Klf4 gene or polypeptide The expression may aid in the induction of multipotency in a starting cell or population of starting cells.

[0066] "Myc polypeptide" refers to any naturally occurring member of the Myc family. (See, e.g., Adhikary, S. & Eilers, M., Nat. Rev. Mol. Cell Biol. 6:635-64. 5 (2005)), compared to the closest naturally occurring family member. Similar (i.e., within at least 50%, 80%, or 90% activity) transcription factor activity This includes variants that maintain at least the DNA sequence of naturally occurring family members. A-binding domain and may further include a transcription activation domain. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc, and L-Myc. In some embodiments, the variant is a naturally occurring Myc polypeptide. tid family members, such as those listed above or, for example, those listed in the Genbank Accession No. Their full sequences are compared with those listed in session number CAA25015 (human Myc). have at least 85%, 90%, or 95% amino acid sequence identity across the string. c-Myc polypeptides (e.g., c-Myc) can be derived from human, mouse, rat, bovine, porcine, or other Generally, proteins from the same species as the cells being engineered are used. The Myc polypeptide may be a pluripotency factor.

[0067] "Sox polypeptide" refers to the naturally occurring members of the SRY-related HMG box (Sox) transcription factors. It refers to any member of the family that is related to the most closely related naturally occurring family member. Similar (i.e., within at least 50%, 80%, or 90% activity) transcription factor activity These proteins are characterized by the presence of a high mobility group (HMG) domain that maintains a specific affinity for the protein, or a variant thereof. This is a polypeptide that contains at least the DNA binding domain of a naturally occurring family member. and may further comprise a transcription activation domain (see, e.g., the entire disclosure of which is incorporated herein by reference). Dang, DT et al., Int. J. Biochem., incorporated herein by reference. See Cell Biol. 32:1103-1121 (2000). Examples of the peptides include Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, So x13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox 30 Sox1 can generate iPS cells with similar efficiency to Sox2. It has been shown that the genes Sox3, Sox15 and Sox18 also generate iPS cells. has been shown to be somewhat less efficient than Sox2 (herein incorporated by reference in its entirety). Nakagawa, et al., Nature Biotechnology gy 26:101-106 (2007). In some embodiments, the variant are naturally occurring Sox polypeptide family members, such as those listed above. or, for example, those listed in Genbank accession number CAA83435 (human Sox2). at least 85%, 90%, or 95% across their entire sequences compared to those Sox polypeptides (e.g., Sox1, Sox2, Sox 3. Sox15 or Sox18) can be expressed in humans, mice, rats, cows, pigs, or other animals. Generally, proteins of the same species as the cell being engineered are used. The Sox polypeptide may be a pluripotency factor. As discussed herein, SOX2 The proteins find particular use in the generation of iPSCs.

[0068] As used herein, "differentiated hypoimmunogenic pluripotent cells" or "differentiated HIP cells" or "dHI "P cells" are those that have been isolated from mice (e.g., those with knockout of B2M and CIITA and knock-in of CD47). and then ultimately implanted into the subject. Thus, for example, HIP cells are iPS cells that are differentiated into a cell type for The cells may be derived from hepatocytes ("dHIP hepatocytes"), beta-like pancreatic cells or pancreatic islet organoids ("dHIP cells"). beta cells (“HIP beta cells”), endothelial cells (“dHIP endothelial cells”), etc. A similar definition applies to "differentiated HIP / CD64" and differentiated HIPO- / CD64 cells. It fits.

[0069] The term percent "identity" in the context of two or more nucleic acid or polypeptide sequences The sequence comparison algorithms described below (e.g., BLASTP and BLASTN or the like) can be used. Measure and compare using one of the algorithms available to the manufacturer, or by visual inspection. When aligned for maximum matching, they have a defined percentage of the same nucleotides or amino acids. It refers to two or more sequences or subsequences that have the same or similar acid residues. The "identity" may exist across a region of the compared sequences, e.g. across a functional domain, or For sequence comparison, typically 1 One sequence acts as a reference sequence, to which test sequences are compared. If desired, enter the test and reference sequences into a computer and, if necessary, specify subsequence coordinates. and specifying sequence algorithm program parameters. The comparison of the test sequences relative to the reference sequence is based on the designated program parameters. Calculate the percent sequence identity.

[0070] Optimal alignment of sequences for comparison can be performed using, for example, the method of Smith & Waterman n, Adv. Appl. Math. 2:482 (1981) From Needleman & Wunsch, J. Mol. Biol. 48:443 ( The homology alignment algorithm of Pearson & Lipm (1970) an,Proc.Nat'l.Acad.Sci.USA 85:2444(1988) The similarity search method of the Wisconsin Institute for Medical Sciences (WIMS) n Genetics Software Package,Genetics Com puter Group,575 Science Dr.,Madison,Wis. GAP, BESTFIT, FASTA, and TFASTA) or by visual inspection ( This can generally be performed according to Ausubel et al., see below.

[0071] One example of an algorithm suitable for determining percent sequence identity and sequence similarity is BLAST. The algorithm is that of Altschul et al., J. Mol. Biol. 215:403-410 (1990). The software is provided by the National Center for Biotechnology Publicly available via the Information (www.ncbi.nlm.nih.gov / ) is available.

[0072] "Inhibitors," "activators," and "modulators" are used to modify or inhibit the function or activity of biologically relevant molecules. The term "modulator" includes both inhibitors and activators. These include in vitro and in vivo assays for the expression or activity of target molecules. The antibodies can be identified using an assay.

[0073] An "inhibitor" is, for example, an inhibitor that inhibits the expression or binds to a target molecule or protein. These are substances that can partially or totally block stimulation or act as protease inhibitors. These may have inhibitory activity, such as inactivating the activity of the target protein described, desensitizing, The activation of the module may be decreased, reduced, prevented, or delayed, including down-regulated. The inhibitor may be an antagonist of the target molecule or protein.

[0074] An "activator" is, for example, a molecule that induces or activates the function or expression of a target molecule or protein. These are agents that bind to target molecules and stimulate or increase target molecule activity. An activator may activate, release, activate, or promote the activity of a target molecule or protein. It may be an agonist.

[0075] A "homolog" is a molecule that is different from a reference molecule at the nucleotide sequence, peptide sequence, function, or structure level. Homologs are biologically active molecules that are similar in structure and function to a given sequence. Thus, in one embodiment, homologous or Derivative sequences share at least 70 percent sequence identity. In the present specification, a homologous or derivative sequence is one that shares at least 80 or 85 percent sequence identity. In a specific embodiment, the homologous or derivative sequence has at least 90 percent In a specific embodiment, a homologous or derivative sequence shares at least In a more specific embodiment, homologous or derivatives share at least 95 percent sequence identity. The conductor arrangement is 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 Homologous or derivative nucleic acid sequences share sequence identity with each other in a high stringency hybridization assay. and by their ability to remain bound to a reference nucleic acid sequence under annealing conditions. Homologs that have structural or functional similarity to a reference molecule are known to be chemoattractants of the reference molecule. Detecting, generating and screening structural and functional homologs and derivatives Methods for doing so are known in the art.

[0076] "Hybridization" generally occurs when complementary strands are present in an environment below their melting temperature. Hybridization depends on the ability of denatured DNA to reanneal when denatured. The higher the desired degree of homology between the replicable sequences, the greater the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent. whereas lower temperatures tend to make the reaction conditions less stringent. Additional details on the stringency of the hybridization reaction and For a detailed description, see Ausubel et al. al,Current Protocols in Molecular Biolo gy, Wiley Interscience Publishers (1995) .

[0077] The "stringency" of a hybridization reaction can be readily determined by one of skill in the art. and is generally an empirical calculation dependent on probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing; The shorter the probe, the lower the temperature required.

[0078] The terms "stringent conditions" or "high stringency conditions" are used herein. As defined, (1) low ionic strength and high temperature for washing, e.g., 0 at 50°C; .015M Sodium Chloride / 0.0015M Sodium Citrate / 0.1% Dodecyl (2) those that use denaturing agents during hybridization, e.g., sodium persulfate; Formamide, e.g., 50% (v / v) formamide with 0.1% water at 42°C. Serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / Ph6 .5 50Mm Sodium Phosphate Buffer, 750Mm Sodium Chloride, 75Mm with sodium citrate; or (3) 50% formamide, 5 at 42°C. × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM Sodium pyrophosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution , sonicated salmon sperm DNA (50 μl / ml), 0.1% SDS, and 10% sulfuric acid Hybridization overnight in solution with dextran and 0.2×SSC (salt followed by a 10 min wash in 100 mM sodium chloride / sodium citrate at 42°C. A high stringency wash consisting of 0.1x SSC containing EDTA at 55°C for 10 min It can be identified by cleaning.

[0079] Every maximum numerical limitation given throughout this specification is inclusive of every lower numerical limitation. All lower numerical limitations are included as if each was expressly expressly written herein. Every minimum numerical limitation given throughout this specification shall be limited to the fullest extent of every higher numerical limitation. All higher numerical limitations are included, as if all higher numerical limitations were expressly written herein. Every numerical range given throughout the specification includes all narrower numerical ranges. All narrower numbers falling within such broader numerical ranges are treated as if they were explicitly stated in The value range is inclusive.

[0080] As used herein, the term "modification" refers to the modification of a molecule from a parent molecule. In one embodiment, the method of the present invention is prepared according to the method described herein. In the CD47, HSVtk, EC-CD, or iCasp9 variant polypeptides The amino acid changes are the same as those of the corresponding parent that has not been modified according to the methods described herein, e.g. Wild-type proteins, naturally occurring mutant proteins or such variant polypeptides. This distinguishes it from other engineered proteins that do not involve modification of the peptides. The variant polypeptide is obtained by converting an unmodified polypeptide into a variant polypeptide. For example, the amino acid sequence of the variant polypeptide may be modified by one or more modifications that distinguish the function of the variant polypeptide. The alteration affects its receptor binding profile. The polypeptides may include substitution, deletion, or insertion modifications, or any combination thereof. In another embodiment, the variant polypeptide has an affinity comparable to that of the unmodified polypeptide. The polypeptide contains one or more modifications that increase its affinity for the receptor.

[0081] In one embodiment, the variant polypeptide has a 1:1 amino acid sequence similar to the corresponding native or parent sequence. In one embodiment, the variant polypeptide comprises one or more substitutions, insertions, or deletions. is 1, 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, Contains 30, 31 to 40, 41 to 50, or 51 or more modifications.

[0082] As used herein, an "episomal vector" refers to a vector that can exist in the cytoplasm of a cell and replicate autonomously. It refers to a genetic vector that can be produced by, for example, integrating into the genomic DNA of a host cell. A number of episomal vectors are known in the art and are described below. .

[0083] A "knockout" in the context of a gene means that the host cell carrying the knockout has As outlined herein, this means that the Knockout does not produce a functional protein product. Quenching involves removing all or part of the coding sequence, producing a functional protein, by a variety of techniques. Frameshift insertions (either truncated or nonsense sequences) are used to avoid the production of by introducing a natural mutation, or by modifying a regulatory component (e.g., a promoter) so that the gene is not transcribed. These arise from the removal or modification of the nucleotide sequence (e.g., nucleotide sequence) or by binding to mRNA, thereby disrupting translation. Generally, the knockout is carried out in a manner such that the progeny of the cell also permanently carry the knockout. This is effected at the genomic DNA level.

[0084] A "knock-in" in the context of a gene refers to a gene that is capable of expressing itself in a host cell. As outlined herein, A check-in is a process in which at least one transgene (tg), usually encoding a protein, is introduced into the host in a variety of ways. This can be accomplished by introducing into the cell a single copy of both the regulatory components. Replacement can also be used, for example by adding a constitutive promoter to an endogenous gene. Generally, knock-in technology involves the addition of a transgene into a host cell. This results in incorporation of the copy.

[0085] VII. Cells of the Invention The present invention provides a method for generating pluripotent cells with enhanced CD16, CD32, or CD64 expression. In some aspects of the invention, the cells are induced pluripotent. Sexual stem cells (IPSC), O-induced pluripotent stem cells (iPSCO-), embryonic stem cells (ESC) , O-embryonic stem cells (ESCO-), hypoimmunogenic pluripotent (HIP) cells, hypoimmunogenic pluripotent O- (HIPO-) cells, or cells derived therefrom or differentiated therefrom do.

[0086] A. Methods for Genetic Modification The present invention relates to improved CD16 and CD32 expression levels in cells or in a cell-free environment by modifying the nucleic acid sequences. Exemplary techniques include methods to generate both cells with CD64 expression, or both cells with CD64 expression. Recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcriptional activation Clustered regularly interspaced receptor-like effector nuclease (CRISPR) Clustered regularly intermittent short palindromic repeats spaced short palindromic repeat)) / Cas9, and and other site-specific nuclease techniques. These controlled double-stranded breaks are then able to produce double-stranded DNA breaks at specific sites. This process promotes homologous recombination at the site of a gene locus. It focuses on targeting a specific sequence in the chromosome, recognizing and binding to that sequence, and locating a target in the nucleic acid molecule. The double-strand break is an endonuclease that induces a double-strand break in the error-prone non- Repair is accomplished by either homologous end joining (NHEJ) or homologous recombination (HR).

[0087] As will be appreciated by those of skill in the art, a number of different techniques can be used to engineer the pluripotent cells of the invention. iPSCs can be engineered to be less immunogenic as outlined herein. Engineering operations.

[0088] In general, these techniques can be used individually or in combination. In generating IP cells, viral techniques to knock-in CD47 functionality (e.g. , lentivirus) to express active B2M and The expression of the CIITA protein and / or the CIITA protein can be reduced. As will be appreciated, one embodiment includes a CRISPR step to knock out B2M. and the subsequent CRISPR step to knock out CIITA and CD47 function These are sequentially used in the final lentiviral steps to knock-in The genes can be manipulated in different orders using different techniques.

[0089] As discussed in more detail below, transient expression of reprogramming genes is typically performed. to generate / induce pluripotent stem cells.

[0090] a.CRISPR technology In one embodiment, clustered ordered Cells using Critical Interspaced Palindromic Repeats / Cas ("CRISPR") technology Use CRISPR to generate starting iPSCs or to generate HIP cells from iPSCs. There are a number of CRISPR-based techniques available, e.g. Doudna and Charpentier, incorporated herein by reference. See Science doi:10.1126 / science.1258096. CR ISPR technology and kits are commercially available.

[0091] b.TALEN technology In some embodiments, transcription activator-like effector nucleases (TALENs) The HIP cells of the present invention are generated using the methods described herein. TALENs can be used to express virtually any desired DNA. Combined with a nuclease that can be engineered to bind and cleave the sequence. TALEN is a restriction enzyme that has been isolated from other genomic DNA. TALEN kits are commercially available.

[0092] c. Zinc finger technology In one embodiment, cells are engineered using zinc finger nuclease technology. N-finger nucleases convert the zinc finger DNA binding domain into a DNA cleavage domain. The zinc finger domain is an artificial restriction enzyme that is created by fusing it to a They can be engineered to target specific desired DNA sequences, thereby Link-finger nucleases enable targeting of unique sequences within complex genomes By utilizing endogenous DNA repair mechanisms, similar to CRISPR and TALEN, In conclusion, these reagents can be used to precisely modify the genomes of higher organisms.

[0093] d. Virus-based technology Used to generate HIP cells of the present invention (as well as for the initial generation of iPCS) A wide range of viral techniques can be used, including but not limited to retroviruses. vectors, lentivirus vectors, adenovirus vectors and Sendai virus vectors There is a wide range of episomal vectors used in the generation of iPSCs. It is described in.

[0094] e. Downregulation of genes using interfering RNA In another embodiment, genes encoding proteins used in HLA molecules down-regulation by RNAi technology. RNA interference (RNAi) is often used to down-regulate specific m The process by which RNA molecules inhibit gene expression by causing their degradation There are two types of RNA molecules: microRNA (miRNA) and small interfering RNA (SMRNA). A(siRNA)) are at the heart of RNA interference. They bind to target mRNA molecules and RNAi is a method for the inhibition of the expression of parasitic nucleic acids, such as viruses and virions. RNAi also affects development.

[0095] sdRNA molecules are asymmetric siRNA molecules that contain a guide (antisense) strand of 19–21 bases. A class. These are 5' phosphate, 2'Ome or 2'F modified pyrimidines, and Contains six phosphothioates at the 3' position. These are the 3' conjugated sterol moiety, two phosphothioates at the 3' position ( phospotioate), and the sense strand also contains 2'Ome-modified pyrimidines. Both strands contain 2' omega-3 nucleotides with consecutive stretches of unmodified purines not exceeding 3 in length. sdRNA is a nucleic acid that contains purines. As disclosed in US Pat. No. 8,796,443.

[0096] All of these techniques can be used using well known recombinant techniques as outlined herein. In one embodiment, the recombinant nucleic acid (a desired polypeptide, e.g., For example, one or more sequences encoding either CD47 or a disruption sequence may be included in the expression construct. It can be operably linked to a regulatory nucleotide sequence. The regulatory nucleotide sequence can be Generally, the host cell and the subject to be treated are appropriate. For various host cells, many types of Suitable expression vectors and suitable regulatory sequences for the gene are known in the art. The one or more regulatory nucleotide sequences include, but are not limited to, a promoter. -sequences, leader or signal sequences, ribosome binding sites, transcription start and stop sequences, translation Initiation and termination sequences, as well as enhancer or activator sequences, may be mentioned. Constitutive or inducible promoters known in the art are also contemplated. naturally occurring promoters or hybrids that combine elements of two or more promoters The expression construct may be an episome, e.g., a plasmid. The expression construct may be present in the cell on a chromosome or the expression construct may be inserted into a chromosome. In a typical embodiment, the expression vector is In some embodiments, the gene is operably linked to at least one regulatory sequence. an expression vector comprising a nucleotide sequence encoding a variant polypeptide that is linked to Regulatory sequences for use herein include promoters, enhancers, and In one embodiment, the expression vector includes Selection of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the vector The copy number of the vector, the ability to control that copy number, or the amount of the vector-encoded It may be designed for the expression of any other protein, for example an antibiotic marker.

[0097] Examples of suitable mammalian promoters include, for example, promoters from the following genes: Hamster ubiquitin / S27a promoter (International Publication No. WO 97 / 15664) Rett), simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus (R SV) long terminal repeat region, mouse mammary tumor virus promoter (MMTV) , Moloney murine leukemia virus long terminal repeat region, and human cytomegalovirus Examples of heterologous mammalian promoters include the CMV early promoter. is an actin, immunoglobulin or heat shock promoter.

[0098] In additional embodiments, the promoter for use in a mammalian host cell is a viral , e.g., polyomavirus, fowlpox virus (British Patent No. 2,211,504 , released on July 5, 1989), bovine papillomavirus, avian sarcoma virus, cytomegalovirus Roviruses, retroviruses, Hepatitis B virus and Simian virus 40 (SV40) In a further embodiment, the heterologous mammalian promoter can be obtained from the genome of Examples include the actin promoter, the immunoglobulin promoter, and the heat The early and late promoters of SV40 are conveniently It is obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. t al., Nature 273:113-120(1978). Human cytomegalovirus The immediate early promoter of the virus is conveniently obtained as a HindIII E restriction fragment. Greenaway, PJet al., Gene 18:355-360(198 2). The above references are incorporated by reference in their entireties.

[0099] B. Generation of Pluripotent Cells The present invention provides a method for producing non-immunogenic pluripotent cells from pluripotent cells. To this end, the first step is to provide pluripotent stem cells.

[0100] Mouse and human pluripotent stem cells (commonly referred to as iPSCs; miPs for mouse cells) The generation of human pluripotent stem cells (SCs or hiPSCs for human cells) is generally known in the art. As will be appreciated by those of skill in the art, there are a variety of different methods for the generation of iPCS. Viral transduction of four transcription factors, Oct3 / 4, Sox2, c-Myc, and Klf4 Initial derivation was performed from mouse embryonic or adult fibroblasts using The techniques are generally outlined in the literature, and are incorporated herein by reference. See Hi and Yamanaka Cell 126:663-676(2006) Since then, numerous methods have been developed; both in general and to generate hiPSCs specifically. For a review of the method, see Seki et al., which is expressly incorporated herein by reference. t al.,World J.Stem Cells 7(1):116-125(20 15), and Lakshmipathy and Vermuri, editors, M ethods in Molecular Biology: Pluripotent Stem Cells,Methods and Protocols,Springe r 2013 (see, for example, Chapter 3 of the latter reference).

[0101] Generally, iPSCs are expressed in host cells, usually by introducing them using episomal vectors. Under these conditions, the reprogramming factor is generated by transient expression of one or more of the following "reprogramming factors": A small number of cells are induced to become iPSCs (typically because no selection marker is used). (The efficiency of this step is low in humans.) Once a cell is "reprogrammed" and becomes pluripotent, it They lose the episomal vector and use endogenous genes to produce the factor. Loss of the genome vector occurs in cells called "zero footprint" cells. This is because the fewer genetic modifications (especially in the host cell genome) the better. Therefore, it is preferred that the resulting hiPSCs have no permanent genetic modifications. It is nice.

[0102] Also, as will be appreciated by those skilled in the art, reprogramming techniques may be used or may be employed. The number of reprogramming factors can vary. Generally, when fewer reprogramming factors are used, The efficiency of transformation of cells into a pluripotent state is reduced, and so is "pluripotency", e.g., less Reprogramming factors that are not fully pluripotent but can differentiate into only a few cell types This may result in cells that may be capable of

[0103] In some embodiments, a single reprogramming factor, OCT4, is used. In an embodiment, two reprogramming factors are used: OCT4 and KLF4. In an embodiment, three reprogramming factors are used: OCT4, KLF4 and SOX2. In another embodiment, the four reprogramming factors OCT4, KLF4, SO X2 and c-Myc are used. In another embodiment, SOKMNLT; SOX2, OCT4(POU5F1), KLF4, MYC, NANOG, LIN28, and SV40 Five, six or seven reprogramming factors selected from the LT antigens can be used. .

[0104] Generally, on an episomal vector, such as those known in the art and commercially available. These reprogramming factor genes are provided, for example, at ThermoFisher / Invitrogen develops Sendai virus reprogramming for traceless generation of hiPSCs ThermoFisher sells a chromatographic kit, see catalog number A34546. also sells an EBNA-based system, see catalog number A14703.

[0105] In addition, there are numerous commercially available hiPSC lines available; e.g., trace-free and virus-free. Gibco® episomal hiPSC line, an integration-free human iPSC cell line , see K18945 (Burridge et al., 2011, supra).

[0106] Generally, as known in the art, iPSCs can be reproduced as described herein. By transiently expressing programming factors, non-pluripotent cells, e.g., CD34+ umbilical cord blood cells, can be generated. It is made from cord blood cells, fibroblasts, etc.

[0107] For example, good iPSCs also express c-Myc, although this reduces reprogramming efficiency. It was generated by omitting only Oct3 / 4, Sox2 and Klf4.

[0108] In general, iPSCs express genes that express genes that express KLF4, Nanog, OCT4, SOX2, ESRR, etc. The present invention is characterized by the expression of certain factors, including B, TBX3, c-Myc, and TCL1. De novo or increased expression of these factors for identification purposes may be achieved by induction or expression of endogenous loci. or via modulation or expression from a transgene. obtain.

[0109] For example, mouse iPSCs are described in general and in particular in methods and methods for the generation of miPSCs. and reagents are incorporated herein by reference. Rep.2015,Jan.28;5:8081(doi:10.1038 / srep0 For example, the present invention may be produced using the methods of the present application, generally and specifically. Burridge, which is incorporated herein by reference for the methods outlined therein. See also et al., PLoS One, 2011 6(4):18293.

[0110] In some instances, pluripotency of cells can be achieved by, for example, by assaying for reprogramming factors or by performing a differentiation reaction, Measured or confirmed as outlined herein.

[0111] C. Generation of hypoimmunogenic pluripotent (HIP) cells The generation of HIP cells from pluripotent cells is achieved by altering just three genes. It results in minimal disruption of activity but confers immune silencing to the cells.

[0112] As discussed herein, one embodiment includes MHC I and II (where the cells are human) In this case, the reduction or elimination of the protein activity of HLA I and II is utilized. This can be achieved by modifying the genes encoding these components. In this method, the coding region or regulatory sequence of a gene is disrupted using CRISPR. In an embodiment, interfering RNA technology is used to reduce gene translation. and alterations in genes that regulate susceptibility to macrophage phagocytosis, such as CD47. This is generally a "knock-in" of a gene using viral technology.

[0113] In some cases, CRISPR is used to modify genes, allowing for highly efficient editing of cell lines. For example, hiPSC cells containing a Cas9 construct that encodes Lif can be used. Human Episomal Cas9 iPS from e Technologies C cell line, see A33124.

[0114] 1. HLA-I reduction The HIP cells of the invention have MHCI function (HLA I if the cells are derived from human cells). This includes reducing

[0115] As will be appreciated by those of skill in the art, reduction of function can be achieved by a number of techniques, including, for example, deletion of a gene from This can be achieved by removing the nucleic acid sequence, interrupting the sequence with another sequence, or altering the regulatory components of the nucleic acid. For example, all or part of the coding region of the gene of interest can be removed, or can be replaced with a "nonsense" sequence, creating a frameshift mutation and removing or replacing all or part of a regulatory sequence, e.g., a promoter. , the translation initiation sequence can be removed or replaced, etc.

[0116] As will be appreciated by those of skill in the art, MHC I function in pluripotent cells (if the cells are human cells) In the case of HLA I, the successful reduction of HLA I can be achieved using techniques known in the art. and as described below; for example, FA using labeled antibodies that bind to HLA complexes. CS technology; for example, commercially available CS antibodies that bind to the alpha chain of human major histocompatibility HLA class I antigens. It can be measured using HLA-A, B, C antibodies.

[0117] a.B2M change In one embodiment, the reduction in HLA-I activity is determined by the reduction of the beta-2 microglobulin in pluripotent stem cells. This is accomplished by disrupting expression of the globulin gene, the human sequence of which is disclosed herein. This alteration is generally referred to herein as a gene "knockout" and is the HI In P cells, this occurs on both alleles in the host cell. The technique for doing this is the same.

[0118] A particularly useful embodiment uses CRISPR technology to disrupt genes. In some cases, CRISPR technology is used to modify genes so that they do not produce a functional protein. Small deletions / insertions are introduced into the coding region, often resulting in truncated, non-functional transcripts. A frameshift mutation results in the creation of a stop codon so that the protein can be made. will be done.

[0119] Thus, a useful technique is to clone the coding region of the B2M gene in mouse or the coding region of the B2M gene in human. The first step is to use CRISPR sequences designed to target gene sequences. After concentrating, the transfected iPSC cultures are dissociated into single cells. The single cells are then dissociated into full-sized iPSCs. Expand colonies and screen for the presence of aberrant sequences from the CRISPR cleavage sites. Test CRISPR editing by selecting clones with biallelic deletions. Such clones did not express B2M as demonstrated by PCR and did not express B2M by FACS analysis. As demonstrated, they did not express HLA-I (see, eg, Examples 1 and 6).

[0120] Assays for testing whether the B2M gene has been inactivated are known and are described herein. In one embodiment, the assay is performed by probing with an antibody against a B2M protein. 1 is a Western blot of a cell lysate probed with reverse transcriptase. Polymerase chain reaction (rt-PCR) confirms the presence of the inactivating alteration.

[0121] In addition, cells were tested to confirm that HLA I complexes were not expressed on the cell surface. This can be tested against one or more HLA cell surface components as discussed above. The antibodies can be used to assay for the expression of the markers by FACS analysis.

[0122] Notably, when we attempted to silence the B2M gene in both alleles, Others had poor results. For example, Gornalusse et al., ture Biotech.Doi / 10.1038 / nbt.3860).

[0123] 2. HLA-II reduction In addition to reducing HLA I, the HIP cells of the present invention also have MHC II function (the cells are human cells). If it is derived from HLA, it also lacks HLA II).

[0124] As will be appreciated by those of skill in the art, reduction of function can be achieved by a number of techniques, including, for example, deletion of a gene from Removal of nucleic acid sequences, addition of nucleic acid sequences to genes, disruption of reading frames, modification of sequences This can be achieved by an interruption in the sequence or by alteration of the regulatory component of the nucleic acid. It is possible to remove all or part of the coding region of a gene of interest, or to create a "nonsense" In another embodiment, the regulatory sequence, e.g., a promoter, The translation initiation sequence can be removed or replaced. It is possible, etc.

[0125] MHC II function in pluripotent cells or their derivatives (if the cells are derived from human cells) Successful reduction of HLA II can be achieved by techniques known in the art, e.g., protein synthesis. Western blotting, FACS technology, rt-PCR technology, etc. using antibodies against It can be measured using the following:

[0126] a.CIITA Change In one embodiment, the reduction in HLA-II activity is due to the inhibition of the CIITA gene in pluripotent stem cells. This is done by disrupting expression of a gene, the human sequence of which is presented herein. is generally referred to herein as a gene "knockout" and in the HIP cells of the present invention Thus, this occurs on both alleles in the host cell.

[0127] Assays for testing whether the CIITA gene has been inactivated are known. In one embodiment, the assay is for CIITA protein. Western blots of cell lysates probed with antibodies. Transcriptase polymerase chain reaction (rt-PCR) confirms the presence of the inactivating alteration.

[0128] Furthermore, to confirm that HLA II complexes are not expressed on the cell surface, cells were Again, assays are performed as known in the art. Representative assays generally include human HLA class II HLA-DR, as outlined below: Western blot or FA using commercially available antibodies that bind to DP and most DQ antigens CS analysis is one example.

[0129] A particularly useful embodiment uses CRISPR technology to disrupt the CIITA gene. The Ciita gene in mice, which is an essential transcription factor for all MHC II molecules, CRISPR was designed to target the coding sequence of the CIITA gene in humans. After gene editing, the transfected iPSC cultures were dissociated into single cells. The clones were expanded to colonies of different strains and screened for the presence of aberrant sequences from the CRISPR cleavage sites. Clones with deletions were identified by PCR. Does not express CIITA as determined and does not express MHC II / HLA-II was not expressed.

[0130] 3. Reduced Phagocytosis In general, the use of B2M and CIITA knockouts results in the identification of HLA I and II (or In addition to reducing MHC I and II, the HIP cells of the present invention also exhibit reduced macrophage phagocytosis. The resulting HIP cells are sensitive to one or more CD4+ receptors and NK cell killing. 47 Due to the transgene, it “escapes” immune macrophages and the innate pathway.

[0131] A. Increased CD47 In some embodiments, the reduced macrophage phagocytosis and NK cell killing susceptibility is This results from an increase in CD47 on the surface of HIP cells. This can be done in a number of ways, as will be appreciated by those of skill in the art, using genetic techniques. In some embodiments, increased CD47 expression results from one or more CD47 transgenes.

[0132] Thus, in some embodiments, one or more copies of the CD47 gene are expressed in HIP cells. The gene is added to the cell under the control of an inducible or constitutive promoter, the latter being preferred. In the present invention, lentiviral constructs can be prepared as described herein or as disclosed in the art. The CD47 gene is used in conjunction with a suitable promoter as known in the art. The vector may be integrated into the genome of the host cell under the control of a promoter.

[0133] The HIP cell line was generated from B2M- / - CIITA- / - iPSCs. Isolate cells containing lentiviral vectors expressing CD47 using an immunomarker The CD47 gene sequence was synthesized and a plasmid containing blasticidin resistance was created. Tivirus pLenti6 / V5 (Thermo Fisher Scientific The DNA was cloned into the Escherichia coli K5711B (Waltham, MA).

[0134] In some embodiments, the regulatory sequence of the endogenous CD47 gene is altered, e.g. For example, the endogenous promoter can be replaced with a constitutive promoter or with a different inducible promoter. By transfecting the CD47 gene, the expression of the CD47 gene can be increased. This can be done using techniques such as CRISPR.

[0135] Once modified, the antibody can be prepared by known techniques, e.g., those described in the Examples, e.g., by adding an anti-CD47 antibody. Use Western blot, ELISA assay or FACS assay to confirm sufficient In general, the term "sufficient" in this context can be used to assay for the presence of sufficient CD47 expression. " increased expression of CD47 on the HIP cell surface, silencing NK cell killing. This means that when MHC I is removed from cells, the natural expression levels on the cells are too low to stimulate NK cells. They are unable to protect them from cytolysis.

[0136] 4. The suicide gene In some embodiments, the present invention relates to a method for the treatment of hypoimmune disorders comprising a "suicide gene" or "suicide switch." These provide immunogenic pluripotent cells that, when growing and dividing undesirably, produce low immunogenic pluripotent cells. The IL-1 receptor is internalized to function as a "safety switch" that can trigger the death of a cytotoxic cell. The "suicide gene" deletion approach involves the deletion of a gene that is activated in cells only when activated by a defined compound. Included are suicide genes in gene transfer vectors that code for proteins that cause killing. Suicide genes may code for enzymes that selectively convert non-toxic compounds into highly toxic metabolites. The result is the specific elimination of cells that express the enzyme. The gene is the herpesvirus thymidine kinase (HSV-tk) gene, and the trigger is In another embodiment, the suicide gene is ganciclovir. hia coli cytosine deaminase (EC-CD) gene, and the trigger is 5- and fluorocytosine (5-FC), both of which are incorporated herein by reference in their entireties. Barese et al., Mol. Therap. 20(10):1932 -1943(2012), Xu et al., Cell Res.8:73-8(19 98)).

[0137] In another embodiment, the suicide gene is an inducible caspase protein. The caspase protein is at least a portion of a caspase protein that can induce apoptosis. In one embodiment, a portion of a caspase protein is exemplified in SEQ ID NO:6. In a preferred embodiment, the inducible caspase protein is iCasp9. , F36V linked via a stretch of amino acids to the gene encoding human caspase-9 It contains the sequence of human FK506 binding protein, FKBP12, with the mutation. P12-F36V binds with high affinity to the small molecule dimerizer AP1903. In the present invention, the suicide function of iCasp9 is regulated by administration of a dimer-inducing compound (CID). In some embodiments, the CID is the small molecule drug AP1903. The somatization leads to the rapid induction of apoptosis (each of which is incorporated herein by reference in its entirety). International Publication No. 2011146862, incorporated herein by reference; t al,N.Engl.J.Med 365;18(2011);Tey et al .,Biol.Blood Marrow Transplant.13:913-92 4 (2007).

[0138] 5. CD16, CD32, or CD64 expression The cells of the present invention exhibit ADCC resulting from increased CD16, CD32, or CD64 expression. and reduced susceptibility to CDC. The resulting cells have In one embodiment, the cells are immunoreactive with one or more of CD16, CD32, or CD40. Contains the D64 transgene.

[0139] Increased CD16, CD32, or CD64 In some embodiments, the decreased ADCC or CDC susceptibility is due to increased This arises from CD16, CD32, or CD64, as will be appreciated by those skilled in the art. In some approaches, this is done using "knock-in" or transgenic techniques. In some instances, increased CD16, CD32, or CD64 expression is indicative of one or more of the following: It arises from genes.

[0140] Thus, in some embodiments, one of the CD16, CD32, or CD64 genes One or more copies of the gene are added to the cell under the control of an inducible or constitutive promoter, the latter being preferred. In some embodiments, the lentiviral constructs described herein are Genes are used as described in the literature or as known in the art. The vector may be integrated into the genome of the host cell under the control of a suitable promoter as known in the art.

[0141] Using the blasticidin marker, cells expressing CD16, CD32, or CD64 were identified. Cells containing the antiviral vector are selected. The gene sequence is synthesized and then transfected into, for example, a Brass The lentivirus pLenti6 / V5 (Thermo DNA was cloned into a 100-well plate (Fisher Scientific, Waltham, MA). It can be done.

[0142] In some embodiments, the regulatory sequence of the endogenous CD16, CD32, or CD64 gene For example, the endogenous promoter can be changed to a constitutive promoter or to a different By replacing the promoter with an inducible promoter, expression of the gene can be increased. This can be done using commonly known techniques, for example, CRISPR.

[0143] Once modified, the antibody can be used in a variety of ways, including those described in the Examples, such as anti-CD16, CD 32, or Western blot, ELISA assay, or FAC using CD64 antibody The S assay can be used to assay for the presence of sufficient expression. "Sufficient" in the context of vasopressin is the expression of vasopressin on the cell surface that blocks antibodies and inhibits ADCC or CDC. This means an increase in the present.

[0144] 6. Assay of HIP Phenotype and Retention of Pluripotency Once the HIP cells are generated, they are generally cultured as described herein and in the Examples. These can be assayed for low immunogenicity and / or retention of pluripotency.

[0145] For example, low immunogenicity is assayed using a number of techniques. For example, transplantation into an allogeneic host and HIP cell growth (e.g., teratomas) that evades the host immune system. The HIP-derived product is then made to express luciferase. can be transduced and then tracked using bioluminescence imaging. The T cell and / or B cell responses of the host animal to the HIP cells are examined to determine whether the HIP cells are host Confirm that the antibody does not induce immune responses in animals. T cell function is evaluated by Elispot , Elisa, FACS, PCR, or mass cytometry (CYTOF) B cell or antibody responses are assessed using FACS or luminex. Alternatively, the cells can be modified to improve their ability to evade innate immune responses, e.g., NK cell killing. NK cytolytic activity can be assayed in vitro or in vivo. Assays are performed using techniques known in the art.

[0146] Similarly, retention of pluripotency is tested in a number of ways. As described herein, pluripotency is assayed by expression of certain pluripotency-specific factors. Or alternatively, the HIP cells are differentiated into one or more cell types as an indication of pluripotency.

[0147] D. Generation of HIPO-CD16, CD32, or CD64 overexpressing cells In some embodiments of the present invention, the HIP cells produced as described above are To start with pluripotent cells with O blood type, they are already HIPO- cells.

[0148] Another embodiment of the invention involves the enzymatic conversion of A and B antigens. In a preferred embodiment, the enzyme In a more preferred embodiment, the enzyme is α-galactosyltransferase. This enzyme eliminates the terminal galactose residues of the B antigen. The method involves the enzymatic conversion of the A antigen to O. In a preferred embodiment, α-N-acetylglucosamine is The enzyme catalyzes the conversion of the A antigen to O. The enzymatic conversion is Olsson et al., Trans. Fusion Clinique et Biologique 11:33-39(2 004); U.S. Pat. Nos. 4,427,777 and 5,606,042; Specification No. 5,633,130, Specification No. 5,731,426, No. 6,184, Specification No. 017, Specification No. 4,609,627, and Specification No. 5,606,042 as well as in WO 9923210.

[0149] Other embodiments of the invention include knocking out ABO gene exon 7 or SLC14 This involves genetically engineering cells by silencing the A1(JK) gene. Other embodiments of the present invention include the C and E antigens of the Rh blood group system (RH), the Kell blood group (KEL) K, FYa and FY3 of the Duffy system, Jkb of the Kidd system, or MNS This includes knocking out the U and S blood groups. Any of the knockout methods described herein, e.g., CRISPR, talen, or homologous recombination. can be used.

[0150] E. Preferred Embodiments of the Invention The present invention relates to HIP, HIPO-, iP overexpressing CD16, CD32, or CD64. SC, iPSC-, ESC, or ESCO-cells, or their derivatives, e.g. These include type 1 diabetes, heart disease, neurological diseases, cancer, blindness, vascular diseases, and those that respond to regenerative drug therapy. In particular, the present invention provides a method for the differentiation of any cell type. The use of cells with enhanced CD16, CD32, or CD64 expression is therefore contemplated. and exhibit pluripotency, but the pluripotent cells or their differentiation products are not transferred to an allogeneic host, e.g., a human patient. These cells do not elicit a host ADCC or CDC response when transplanted as either Provided in the specification.

[0151] In one embodiment, the cell of the present invention comprises a nucleic acid encoding a chimeric antigen receptor (CAR). CARs express either CD16, CD32, or CD64 on the extracellular domain. , a transmembrane domain, and an intracellular signaling domain.

[0152] In some embodiments, the extracellular domain is CD19, CD20, CD22, CD3 8. CD123, CS1, CD171, BCMA, MUC16, ROR1, and WT1 In one embodiment, the extracellular domain binds to an antigen selected from the group consisting of: In some embodiments, the transmembrane domain comprises a CD3ζ fragment variable fragment (scFv). , CD4, CD8α, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD In one embodiment, the intracellular signaling domain includes IL-1, IL-2, IL-3, and BTLA. The main ones are CD3ζ, CD28, 4-1BB, OX40, ICOS, CTLA-4, and PD. -1, LAG-3, and BTLA.

[0153] In one embodiment, the CAR comprises an anti-CD19 scFv domain, a CD28 transmembrane domain, and In some embodiments, the IL-16 polypeptide comprises a CD3 nucleotide sequence, a CD4 nucleotide sequence, and a CD3 zeta signaling intracellular domain. CAR is a fusion protein that contains an anti-CD19 scFv domain, a CD28 transmembrane domain, and a 4-1BB signaling domain. It contains the CD3 β signaling intracellular domain, the CD3 β signaling intracellular domain, and the CD3 zeta signaling intracellular domain.

[0154] In another embodiment of the present invention, the in vitro differentiation of any one of the cells described herein The present invention provides isolated CAR-T overexpressing cells CD16, CD32, or CD64 produced by the method of the present invention. In some embodiments, the cells are cytotoxic hypoimmune CAR-T cells.

[0155] In various embodiments, in vitro differentiation is achieved by the use of bFGF, EPO, Flt3L, IGF , IL-3, IL-6, IL-15, GM-CSF, SCF, and VEGF. The CAR construct is supported in a culture medium containing one or more growth factors or cytokines selected from the group consisting of In some embodiments, the culture medium comprises culturing cells that contain a BMP-activating factors, GSK3 inhibitors, ROCK inhibitors, TGFβ receptor / ALK inhibitors, and NOTC H activators.

[0156] In certain embodiments, the isolated CAR-T cells of the present invention are produced by in vitro differentiation. The cells are used as a treatment for cancer.

[0157] In another embodiment of the invention, a therapeutically effective amount of any of the isolated CAR-T cells described herein is administered. Methods of treating a patient with cancer by administering a composition comprising any of the following are provided. In some embodiments, the composition further comprises a therapeutically effective carrier.

[0158] In some embodiments, the administering step comprises administering intravenously, subcutaneously, intralymphatically, In some instances, administration includes intratumoral administration, intrathecal administration, intrathoracic administration, and intraperitoneal administration. Administration by infusion, bolus or continuous perfusion is further included.

[0159] In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, and myeloma. In various embodiments, the cancer is a solid tumor cancer or a liquid tumor cancer.

[0160] In another aspect, the present invention relates to an isolated CAR-T / CD16, CD32 The present invention provides a method for producing any one of the CD64 cells, CD65 cells, or CD64 cells of the present invention. The in vitro differentiation includes any one of bFGF, EPO, Flt3 L, IGF, IL-2, IL-3, IL-6, IL-7, IL-15, GM-CSF, S CF, and VEGF. In some embodiments, the culture medium comprises culturing the cells in a culture medium containing B MP activators, GSK3 inhibitors, ROCK inhibitors, TGFβ receptor / ALK inhibitors, and and NOTCH activators.

[0161] In some embodiments, in vitro differentiation involves culturing HIPO-cells on feeder cells. In various embodiments, in vitro differentiation includes culturing in simulated microgravity. In one example, the incubation in simulated microgravity includes incubation for at least 72 hours. It is nourishment.

[0162] In some embodiments, cells with enhanced CD16, CD32, or CD64 expression are Provided herein are isolated engineered hypoimmunogenic cardiac cells differentiated from the cardiac cells. do.

[0163] In some embodiments, provided herein are methods for treating a patient suffering from a cardiac condition or disease. The method includes administering a therapeutically effective amount of an isolated engineered cell derived from the cells of the invention described herein. The method includes administering a composition comprising any one of a population of immune-suppressing cardiac cells to a patient. In embodiments, the composition further comprises a therapeutically effective carrier.

[0164] In some embodiments, administration is by implantation into the patient's cardiac tissue, intravenous injection, intra-arterial injection, or the like. injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, transepicardial injection , or infusion.

[0165] In some embodiments, the cardiac condition or disease is pediatric cardiomyopathy, age-related cardiomyopathy, dilated heart Myopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, Other cardiomyopathies, myocarditis, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary Arterial disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina, rheumatoid arthritis The present invention is selected from the group consisting of pulmonary heart disease, arteritis, or cardiovascular disease.

[0166] In some embodiments, in vitro differentiation results in HIPO- / CD16, CD32, or C A method for producing a population of hypoimmune cardiac cells from a population of D64 cells, comprising: In the present study, endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II CITRA gene activity is eliminated, and CD47 expression is increased. Provided herein are methods for culturing a GSK-inhibitor-containing HI (b) culturing a population of PO cells in a culture medium containing a WNT antagonist; (c) culturing the population of PO-cells to produce a population of cardiac progenitor cells; and and culturing the population of cardiac progenitor cells in a culture medium comprising: Includes.

[0167] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof or a In some embodiments, the GSK inhibitor is in the range of about 2 μM to about 10 μM. In some embodiments, the WNT antagonist is an inhibitor of IWR1, its induction In some embodiments, the WNT antagonist is at about 2 μM to about 2 μM. At concentrations in the range of approximately 10 μM.

[0168] In some embodiments, isolated engineered CD16, CD32, differentiated from HIPO- cells. Alternatively, CD64-overexpressing endothelial cells are provided herein. Isolated engineered endothelial cells include capillary endothelial cells, vascular endothelial cells, aortic endothelial cells, and brain endothelial cells. and renal endothelial cells.

[0169] In some aspects, methods of treating a patient suffering from a vascular condition or disease are provided herein. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of isolated engineered endothelial cells of the invention. The method includes administering a composition comprising a population of cells.

[0170] The method includes administering a therapeutically effective amount of an isolated engineered CD16, CD32, or CD6 In some embodiments, the method includes administering a composition comprising a population of endothelial cells overexpressing any one of the four. In some embodiments, the composition may further comprise a therapeutically effective carrier or excipient. In some embodiments, the administration includes implantation into the patient's cardiac tissue, intravenous injection, , intra-arterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, This includes epicardial injection or infusion.

[0171] In some embodiments, the vascular condition or disease is vascular injury, cardiovascular disease, vascular disease, ischemic heart disease, Hemolytic disease, myocardial infarction, congestive heart failure, hypertension, ischemic tissue damage, limb ischemia, stroke, and neurological disorders and cerebrovascular disease.

[0172] In some embodiments, in vitro differentiation results in overexpression of CD16, CD32, or CD64. A method for producing a population of hypoimmune endothelial cells from a population of endothelial cells, comprising the steps of: In this study, endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transcriptome were abrogated CIT-activating factor (CIITA) gene activity and increased CD47 expression A method is provided herein. The method includes: (a) culturing H in a first culture medium that contains a GSK inhibitor. (b) culturing the population of IPO-cells; (b) a second culture medium comprising VEGF and bFGF. (c) culturing the population of HIPO- cells in a culture medium to produce a population of endothelial progenitor cells; and Culturing the population of endothelial progenitor cells in a third culture medium containing a ROCK inhibitor and an ALK inhibitor. The method includes generating a population of hypoimmune endothelial cells by inducing endothelial cell proliferation and proliferation.

[0173] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or In some embodiments, the GSK inhibitor has a concentration in the range of about 1 μM to about 10 μM. In some embodiments, the ROCK inhibitor is Y-27632, its derivative In some embodiments, the ROCK inhibitor is from about 1 μM to about 20 μM. In some embodiments, the ALK inhibitor is SB-43154. 2, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is about 0. The concentration range is from 5 μM to approximately 10 μM.

[0174] In some embodiments, the first culture medium contains between about 2 μM and about 10 μM CHIR-9902. In some embodiments, the second culture medium comprises 50 ng / mL VEGF and In another embodiment, the second culture medium contains Y-276 32 and SB-431542. In various embodiments, the third culture medium further comprises In one embodiment, the composition comprises 10 μM Y-27632 and 1 μM SB-431542. In certain embodiments, the third culture medium further comprises VEGF and bFGF. The first and / or second medium lacks insulin.

[0175] In some embodiments, a single cell differentiated from CD16, CD32, or CD64 overexpressing cells. Provided herein are isolated, engineered, hypoimmune dopaminergic neurons (DNs). In one embodiment, endogenous beta-2 microglobulin (B2M) gene activity and endogenous clones are The rasII transactivator (CIITA) gene activity is eliminated, and CD47 expression is increased and the neurons are blood type O and Rh-.

[0176] In some embodiments, the isolated dopaminergic neurons are derived from neural stem cells, neural progenitors, or cells, immature dopaminergic neurons, and mature dopaminergic neurons. is selected from the group.

[0177] In some embodiments, a method of treating a patient suffering from a neurodegenerative disease or condition is provided herein. In some embodiments, the method comprises administering to a subject a therapeutically effective amount of an isolated hypoimmune dopamine The method includes administering a composition comprising any one of a population of receptor neurons. In some embodiments, the composition further comprises a therapeutically effective carrier. A population of isolated hypoimmune dopaminergic neurons is present on a biodegradable scaffold. In some embodiments, the neurodegenerative disease or condition is Parkinson's disease. The disease is selected from the group consisting of Son's disease, Huntington's disease, and multiple sclerosis.

[0178] In some embodiments, in vitro differentiation results in overexpression of CD16, CD32, or CD64. Methods for producing a population of living dopaminergic neurons are provided herein. In an embodiment, endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity is eliminated, and CD47 expression is increased. In some embodiments, the method includes: (a) administering to the subject a solution of Nick hedgehog (SHH), BDNF, EGF, bFGF, FGF8, WNT1, Selected from the group consisting of thinoic acid, GSK3β inhibitors, ALK inhibitors, and ROCK inhibitors. The population of cells is cultured in a first culture medium containing one or more factors that are associated with the production of immature dopamine receptors. (b) producing a population of mobile neurons; and (b) culturing the cells in a second culture medium different from the first culture medium. Cultivate a population of immature dopaminergic neurons in culture to generate dopaminergic neurons The method includes producing a population of

[0179] In some embodiments, the GSKβ inhibitor is CHIR-99021, a derivative thereof, or is a variant thereof. In some embodiments, the GSKβ inhibitor is at about 2 μM to about 10 μM In some embodiments, the ALK inhibitor is SB-431542, or a derivative or variant thereof. In some embodiments, the ALK inhibitor is at least about 1 μM In some embodiments, the first culture medium and / or The second culture medium lacks animal serum.

[0180] In some embodiments, the method comprises the steps of: In some embodiments, the method also includes isolating a population of minergic neurons. The method further comprises cryopreserving the isolated population of hypoimmune dopaminergic neurons.

[0181] In some embodiments, a single cell differentiated from CD16, CD32, or CD64 overexpressing cells. Provided herein are isolated, engineered hypoimmune pancreatic islet cells. In some embodiments, endogenous β-2 microglobulin (B2M) gene activity and endogenous class II transactivators (CIITA) gene activity is eliminated, CD47 expression is increased, and blood type is O and Rh-.

[0182] In some embodiments, the isolated hypoimmune pancreatic islet cells include islet progenitor cells, immature pancreatic islet cells, and and mature pancreatic islet cells.

[0183] In some aspects, provided herein are methods of treating a patient suffering from diabetes. The method includes administering a therapeutically effective amount of an isolated CD16, CD32, or CD64 overexpression antibody described herein. In some embodiments, the method comprises administering a composition comprising any one of a population of pancreatic islet cells. In some embodiments, the composition further comprises a therapeutically effective carrier. The population of infected pancreatic islet cells is present on a biodegradable scaffold. In some instances, administration is by transplantation or Including injections.

[0184] In some embodiments, in vitro differentiation results in overexpression of CD16, CD32, or CD64. Provided herein are methods for producing a population of hypoimmune pancreatic islet cells from a population of current cells. In one embodiment, endogenous beta-2 microglobulin (B2M) is expressed in HIPO-cells. Gene activity and endogenous class II transactivator (CIITA) gene activity were eliminated. The patient had elevated CD47 expression and was blood type O and Rh-. Insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast Cell Growth Factor (EGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Sonic Transforming hedgehog (SHH) and vascular endothelial growth factor (VEGF) Transforming Growth Factor-β (TGFβ) Superfamily, Bone Morphogenetic Protein-2 (BMP2), Bone Morphogenetic protein-7 (BMP7), GSK3β inhibitor, ALK inhibitor, BMP type 1 receptor A first culture medium containing one or more factors selected from the group consisting of an inhibitor, and retinoic acid. Cultivating populations of CD16, CD32, or CD64-overexpressing cells in culture to identify immature islet cells and (b) producing a population of immature islets in a second culture medium different from the first culture medium. Culturing the population of cells to produce a population of hypoimmune pancreatic islet cells.

[0185] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or In some embodiments, the GSK inhibitor has a concentration in the range of about 2 μM to about 10 μM. In some embodiments, the ALK inhibitor is SB-431542, its derivative. In some embodiments, the ALK inhibitor is from about 1 μM to about 10 In some embodiments, the first culture medium and / or the second culture medium are at a concentration in the range of 1 μM. The nutrient medium lacks animal serum.

[0186] In some embodiments, the method further comprises the step of: detecting CD16, CD32, or CD64 from non-islet cells. In some embodiments, the method further comprises isolating a population of hypoxic IL-1-1 overexpressing pancreatic islet cells. The method further comprises cryopreserving the isolated population of infected islet cells.

[0187] In some embodiments, a single cell differentiated from CD16, CD32, or CD64 overexpressing cells. Isolated, hypoimmune retinal pigment epithelial (RPE) cells expressing endogenous β-2 microglobulin B2M gene activity and the intrinsic class II transactivator (CIITA) gene Cells with abolished activity, increased CD47 expression, and blood type O and Rh- is provided herein.

[0188] In some embodiments, the isolated hypoimmune RPE cells are RPE progenitor cells, immature RPE cells, The RPE cells are selected from the group consisting of: mature RPE cells, and functional RPE cells.

[0189] In some embodiments, provided herein are methods of treating a patient suffering from an ophthalmic condition. The method includes administering a therapeutically effective amount of an isolated CD16, CD32, or CD64 overexpressing antibody described herein. In some embodiments, the method comprises administering a composition comprising any one of the populations of current RPE cells. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of isolated hypoimmune RPE cells is present on a biodegradable scaffold. In some embodiments, the administration comprises implantation or injection into the retina of the patient. , wet macular degeneration, dry macular degeneration, juvenile macular degeneration, Leber's congenital amaurosis, retinal pigment epithelium degeneration, and retinal detachment.

[0190] In some embodiments, the population of cells is differentiated by in vitro differentiation to detect CD16, CD32, or Provided herein are methods for producing a population of CD64-overexpressing retinal pigment epithelial (RPE) cells. In some embodiments, endogenous beta-2 microglobulin is expressed in HIPO- cells. (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity The cytotoxicity was eliminated and CD47 expression was increased. GF, BMP4 / 7, DKK1, IGF1, Noggin, BMP inhibitors, ALK inhibitors, RO The present invention relates to a method for treating a CK disorder and a method for treating a CK disorder. Culturing the population of HIPO- cells in the first culture medium to produce a population of RPE progenitor cells. and (b) culturing the population of RPE progenitor cells in a second culture medium that is different from the first culture medium. The method involves culturing the cells to produce a population of hypoimmune RPE cells.

[0191] In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a In some instances, the ALK inhibitor is a variant of In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof or a variant thereof. In some embodiments, the ROCK inhibitor is from about 1 μM to about 10 Concentrations in the μM range.

[0192] In some embodiments, the first culture medium and / or the second culture medium lacks animal serum. .

[0193] In some embodiments, the method comprises isolating a population of hypoimmune RPE cells from non-RPE cells. In some embodiments, the method further comprises isolating and collecting hypoimmune RPE cells. The method further includes cryopreserving the group.

[0194] In one embodiment, human pluripotent stem cells (PSCs) are characterized by the expression of CD16, CD32, or CD64 Overexpression of these proteins results in resistance to ADCC or CDC. These are: a) B2 in each allele; Disruption of the M gene (e.g., B2M- / -), b) CIITA gene in each allele (e.g., CIITA- / -), and c) overexpression of the CD47 gene (CD47+ For example, via the introduction of one or more additional copies of the CD47 gene or genomic gene activation. This is because the hiPSC population is considered to be less immunogenic by using B2M- / -CIITA- In a preferred embodiment, the cells are non-immunogenic. In an embodiment, the HIP cells are non-immunogenic B2M- / -CIITA- / - as described above. CD47tg is an inducible gene that can be induced to kill cells in vivo on demand. It is further modified by the inclusion of a suicide gene. 2, or CD64-overexpressing HIPO cells, which have ABO gene exon 7 knockout, Or, by silencing the SLC14A1(JK) gene, HIP cells were able to differentiate into blood types. O, C and E antigens of the Rh blood group system (RH), K of the Kell blood group (KEL), and Duffy blood group ( FY (Fyya and FY3), Kidd (JK) (Jkb), or MNS (U and S) blood types It is produced when cells are made Rh- by knocking out

[0195] F. Maintenance of HIPO- / CD16, CD32, or CD64 cells Once generated, iPSCs were maintained using HIPO-CD16, CD32, or HIPO-CD32 as described previously. Alternatively, CD64 cells can be maintained in an undifferentiated state, e.g., differentiation can be prevented and pluripotency can be maintained. Culture HIP cells on Matrigel using maintaining culture medium.

[0196] G. Differentiation of HIPO- / CD16, CD32, or CD64 Cells The present invention relates to HIPO- / HEPATIC cells that are differentiated into different cell types for subsequent transplantation into a subject. As will be appreciated by those of skill in the art, the present invention provides CD16, CD32, or CD64 cells. The method of differentiation depends on the desired cell type using known techniques. and then in the form of a gel matrix, e.g., matrigel, gelatin, or fibrils. Differentiation is achieved by placing the phosphodiesterase (phosphoryl) and thrombin in a phosphodiesterase / thrombin form to promote cell survival. As described above, the assay is generally performed by assessing the presence of cell-specific markers.

[0197] In some embodiments, the HIPO- / CD16, CD32, or CD64 cells are To address the loss of hepatocyte function or cirrhosis of the liver, HIPO-cells are differentiated into hepatocytes. There are a number of techniques that can be used to differentiate between the whole and the Specifically, the methods and reagents for differentiation are expressly incorporated herein by reference. Included, Pettinato et al., doi:10.1038 / spre32 888, Snykers et al., Methods Mol Biol 698: 305-314(2011), Si-Tayeb et al, Hepatology 51:297-305 (2010) and Asgari et al., Stem Cel See Rev(:493-504(2013). Generally, hepatocyte-associated and / or specific myeloma. Markers, including but not limited to albumin, alpha-fetoprotein and by assessing the presence of fibrinogen, as known in the art. Differentiation is assayed. Ammonia metabolism, LDL storage and uptake, ICG uptake and Differentiation can also be measured functionally, such as release and glycogen storage.

[0198] In some embodiments, HIPO is administered for transplantation to treat type 1 diabetes mellitus (T1DM). - / CD16, CD32, or CD64 cells differentiate into beta-like cells or pancreatic islet organoids Cell lines are a promising approach to address T1DM. Ellis et al., doi / 10.1038 / nrgast, incorporated herein by reference. ro.2017.93. Furthermore, Pagliuca et al. Here we report successful differentiation of β cells from human pluripotent stem cells (overall, specifically functional The methods and reagents outlined for large-scale production of targeted human β-cells are incorporated herein by reference. (See doi / 10.106 / j.cell.2014.09.040, which is incorporated herein by reference). In addition, Vegas et al. reported the generation of human β cells from human pluripotent stem cells and their (in general, and in particular in human pluripotent stem cells) to avoid immune rejection by the host after See the references for methods and reagents outlined for the large-scale production of functional human β-cells from stem cells. doi:10.1038 / nm.4030, incorporated herein by reference.

[0199] Generally, β-cell associated or specific markers, such as, but not limited to, insulin, insulin, insulin-like peptides ... Differentiation is assayed by assessing the presence of thrombopoietin, as known in the art. Differentiation can also be measured functionally, such as by measuring glucose metabolism. For a detailed overview of the biomarkers, see Mura et al., incorporated herein by reference. See ro et al, doi:10.1016 / j.cels.2016.09.002 Light.

[0200] Once dHIPO- / CD16, CD32, or CD64 beta cells have been generated, they It can be implanted in the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous pouch (see the present specification). either as a cell suspension or within a gel matrix as discussed herein.

[0201] In some embodiments, HIPO- / CD16, CD32, or CD64 cells are The RPE differentiates the retina to treat vision-threatening eye diseases. The methods and reagents outlined in the publication are incorporated herein by reference. Kamao et al.,Stem Cell Reports 2014:2: Using the techniques outlined in 205-18, human pluripotent stem cells have been differentiated into RPE cells. The techniques for generating RPE cell sheets and transplanting them into patients are generally similar. Incorporated in Mandai et al., doi:10.1056 / NEJMoa1 See also 608368.

[0202] As known in the art, generally, RPE-associated and / or specific markers include Assaying differentiation by assessing the presence of, or functionally measuring, For example, the markup outlined in general and specifically in the first paragraph of the Results section can be used to Kamao et al., doi:10.1017 / 002214, which is also incorporated herein by reference. See also i:10.1016 / j.stemcr.2013.12.007.

[0203] In some embodiments, HIPO- / CD16, CD32, or CD64 cells are administered to myocardium. Addressing cardiovascular disease by differentiating into cells: Techniques for differentiation of hiPSCs into cardiomyocytes As is known in the art, and is discussed in the Examples. and generally by assessing the presence of cardiomyocyte-associated or specific markers or by assessing the function Differentiation can be assayed by measuring differentiation in a systematic way; for example, in the aggregate or specifically The present application is incorporated herein by reference for methods of differentiating stem cells, e.g., cardiomyocytes. Loh et al., doi:10.1016 / j.cell.2016.06 See .001.

[0204] In some embodiments, HIPO- / CD16, CD32, or CD64 cells are To treat peripheral arterial disease by differentiating into colony forming cells (ECFCs) and forming new blood vessels Techniques for differentiating endothelial cells are known. The present invention provides methods and methods for the generation of endothelial cells from human pluripotent stem cells, as well as for transplantation techniques. and reagents are incorporated by reference in Prasain et al., doi:10. 1038 / nbt.3048. As is known in the art, generally, endothelial cell-associated by assessing the presence of related or specific markers, or by functional measurement Differentiation can then be assayed.

[0205] In some embodiments, HIPO- / CD16, CD32, or CD64 cells are The cells were differentiated into thyroid gland progenitor cells and thyroid follicular organoids capable of secreting thyroid hormone. Techniques for differentiating thyroid cells are known in the art. For example, the generation of thyroid cells from human pluripotent stem cells in general and in particular For methods and reagents for the same, as well as for further transplantation techniques, see, for example, US Pat. Kurmann et al., doi:10.106 / j.stem As known in the art, thyroid cell-related by assessing the presence of related or specific markers, or by functional measurement Differentiation can then be assayed.

[0206] H. Transplantation of Differentiated HIPO- / CD16, CD32, or CD64 Cells As will be appreciated by those skilled in the art, differentiation HIPO- / CD16, CD32, or CD64 Derivatives are well known in the art depending on both the cell type and the end use of those cells. The cells are implanted using known techniques. Generally, the cells of the invention are injected intravenously or into a specific location in the patient. For implantation at a specific location, the cells are suspended in a gel matrix. It is possible to prevent dispersion by making them turbid and retaining them.

[0207] In order that the invention described herein may be more fully understood, the following examples are provided herein. Examples are provided. These examples are for illustrative purposes only and are not to be construed as limiting the invention in any way. It is understood that this should not be the case. EXAMPLES

[0208] HIP and HIPO-cells are each incorporated herein by reference in their entirety. International Publication No. 2018 / 132783 and PCT / US19 / 42123 No. 62 / 698,977, and PCT / US19 / 42117. Specification No. 3, Specification No. 62 / 698,978, Specification No. 62 / 698,981, Specification No. 62 / 698,984, Specification No. 62 / 846,399, Specification No. 62 / 8 It is produced as disclosed in US Pat. No. 5,55499.

[0209] A. Example 1: CD64 Protected Macrophages from NK Cell ADCC Killing Constitutive CD64 expression is consistent with naturally expressing CD52 when challenged with anti-CD52 antibodies. It was shown that CD52 constitutively expressing macrophages protects macrophages from NK cell ADCC. Hypoimmune macrophages (B2M) expressed in - / - CIITA - / - CD47 tg HIP iPSC-derived (XCelligen) for in vitro impedance assay ce platform (ACEA BioSciences, San Diego, CA ) on a plastic dish. They attached to the plastic dish and grew to confluence. The FDA-approved humanized IgG antibody alemtuzumab (Bio-Rad, Herceggio, MD) was used to form a layer. ules, CA, Catalog No. MCA6101) to 0.001, 0.01, 0.1, or Alemtuzumab was added at a concentration of 1.0 μg / ml. The NK cells were then added to the assay. Impedance measurements between electrodes were performed on a 96-well E-plate (ACEA BioSciences). Macrophage killing was assessed by measuring the .

[0210] The top row of Figure 3 shows that high concentrations of alemtuzumab are required for NK cell-mediated ADCC, whereas The results show that the phages were killed only at 1.0 μg / ml. At maximum alemtuzumab concentrations, however, all CD64 receptors were occupied. and sufficient alemtuzumab Fc domain is available to block and mitigate ADCC. It is assumed that blocking antibodies against CD64 (Thermo Fisher Scientific ientific, Carlsbad, CA, clone 10.1, catalog number MA1- 10270) was added to the assay at a concentration of 5 μg / ml (bottom row), ADC Target cell killing was improved at alemtuzumab concentrations of 0.001 μg / ml. As the concentration of alemtuzumab increased, the mAb activity in the presence of CD64 blocking antibodies increased. phage killing became more efficient.

[0211] B. Example 2: CD64 Protected Macrophages from CDC Killing CD64 mediates CD52+ macrophages activation when challenged with anti-CD52 antibodies. As in Example 1, macrophages were cultured in Xcellige nce plates and challenged with alemtuzumab. Serum from a blood group compatible with the blood type of the target cells was added to the assay to detect additional blood type antibodies. However, all complement components were present. Macrophages were not involved in the CDC study because high concentrations of alemtuzumab did not result in significant killing. The CD64 protection was 0.001 and 0. This was clearly demonstrated at 0.01 μg / ml of alemtuzumab, which indicates that anti-CD64 antibodies This is because it caused significant CDC death in macrophages (Fig. 5, bottom row). However, the construct against CDC via its CD64 expression can be suppressed at high antibody concentrations. This indicates that the compound has intellectual property protection.

[0212] C. Example 3: CD64 Protected Engineered Endothelial Cells from ADCC Killing by NK Cells CD64 mediates ADCC of CD52+ endothelial cells when challenged with anti-CD52 antibodies. Endothelial cells were differentiated from HIP cells as follows. The protocol was initiated at 60% HIP confluency followed by 2% B-27 m inus insulin(Gibco,Thermo Fisher Scienti Fic brand) and 5 μM CHIR-99021 (Selleckchem, Munich, Germany) The medium was replaced with RPMI-1640 (Gibco) containing riboflavin (Chicago, Germany). Day 2, reduced medium: 2% B-27 minus insulin (Gibco) and RPMI-164 containing 2 μM CHIR-99021 (Selleckchem) The medium was changed to 0. From the 4th to 7th day of culture, the cells were cultured in RPMI-1640 EC medium, 2% B-27 minus insulin and 50ng / ml human vascular endothelial growth factor (V EGF;R&D Systems,Minneapolis,MN), 10ng / ml Basic human fibroblast growth factor (FGFb; R&D Systems), 10 μM Y- 27632 (Sigma-Aldrich, St. Louis, MO), and 1 μM S Exposure to RPMI-1640 containing B 431542 (Sigma-Aldrich) Endothelial cell clusters were visible from day 7 onwards, and the cells were identified as Endothelial Cells. ll Basal Medium 2(PromoCell,Heidelberg,G ermany) and supplements, 10% FCS hi (Gibco), 1% pen / strep, 25ng / ml VEGF, 2ng / ml FGFb, 10μM Y-27 632 (Sigma-Aldrich), and 1 μM SB 431542 (Sigma The differentiation protocol was completed after 14 days; undifferentiated cells were maintained in 10% HO (Aldrich). TrypLE Express (Gibco) was used every 3-4 days. The HIP-derived epithelial cells were then cultured at a 1:3 ratio to express CD52. For transfection experiments, the cells were transfected with lentiviral vectors in 6-well plates. 1.5×10 per well 5 People B2M - / - CIITA - / - CD47 tg EC The cells were incubated overnight at 37°C in a cell incubator. The next day, the cells were incubated with Fugene (Promaga, Fitchburg, WI) and 5 μl g CD52 expressing particles (Origene, Rockville, MD, Catalog no.: R Transfection was performed using a 3:2 ratio of Transfection Reagent Solution Pipette into OptiMEM (Gibco), mix, and incubate at room temperature for 10 minutes. The DNA transfection complex was added to 2 ml of cell culture medium. After 24 hours, the cells were transformed. The transfection was stopped and the cells were grown in endothelial cell medium (Gibco) for an additional 48 hours. Successful transfection was confirmed by flow cytometry analysis and was analyzed by FACS Aria (CD52 Via FACS sorting on APCs (clone HI186, Biolegend) CD52 positive cells were enriched using ELISA.

[0213] CD52+ B2M - / - CIITA - / - A subpopulation of CD47 tg ECs was cultured using C Further transfection was performed using the same protocol with lentivirus to express D64. Fugene (Promaga) and 5 μg of CD64 expression plasmid (Origine ne, Rockville, MD, Catalog Number: RC207487L2V) in a 3:2 ratio. Transfection was performed using a ratio of 1:1. Transfection was stopped as outlined above and cells were cultured for a further 4 The cells were expanded for 8 hours. Successful transfection was confirmed by flow cytometry analysis and FACS via FACS sorting on Aria (CD64PE: clone 10.1, BD) CD64 positive cells were enriched. This CD52 / CD64 double positive population was then culture expanded. did.

[0214] Both EC lines were evaluated in the XCelligence assay. The bottom row shows that CD64-expressing CD52 tg EC was significantly better for ADCC, especially at lower alemtuzumab concentrations Protected.

[0215] Using a different assay, CD64 was again upregulated when challenged with anti-CD52 antibodies. The combination of these antibodies was shown to protect CD52+ mouse endothelial cells from ADCC. Mouse HIP iPSCs were differentiated as described above. Mouse HIP iPSCs were cultured in 6-well plates. The cells were plated on gelatin in vitro and maintained in mouse iPSC medium. After reaching 0% confluency, differentiation was initiated and the cells were cultured in 2% B-27 minus Ins ulin (Gibco) and 5 μM CHIR-99021 (Selleckchem, The medium was RPMI-1640 (Gibco, Munich, Germany) containing On the second day, the reduced medium was replaced with 2% B-27 minus insulin (Gibco ) and 2 μM CHIR-99021 (Selleckchem) The medium was changed to RPMI-1640 (Gibco). From days 4 to 7, the cells were incubated in RPMI-1640. EC medium, 2% B-27 minus insulin and 50ng / mL mouse vascular Endothelial growth factor (mVEGF; R&D Systems, Minneapolis, MN) , 10 ng / mL of basic mouse fibroblast growth factor (mFGFb; R&D System ms), 10 μM Y-27632 (Sigma-Aldrich, Saint Lou is, MO), and 1 μM SB 431542 (Sigma-Aldrich). Endothelial cell clusters were visible from day 7 onwards, and the cells were exposed to RPMI-1640, which is a nutrient EGM-2 SingleQuots medium (Lonza) and 10% FCS hi (Gi bco), 25ng / mL mVEGF, 2ng / mL mFGFb, 10μM Y-2 7632 (Sigma-Aldrich), and 1 μM SB 431542. The differentiation process was completed after 21 days, and undifferentiated cells were detached during the differentiation process. After MACS purification, cells were incubated with anti-CD15m for negative selection according to the manufacturer's protocol. Provided using Ab-coated magnetic microbeads (Miltenyi, Auburn, CA). Highly purified miECs from the flow-through were cultured in EGM-2 SingleQuot The cells were cultured in TrypLE medium with supplements and 10% FCS for 3-4 days. The cells were passaged 1:3 every 2 days. These phenotypes were CD31 (ab28364, Abcam), and VE-cadherin (sc-6458, Santa Cruz Bio by immunofluorescence (IF) at 100 nm chromatin (100 nm chromatin) and 100 nm chromatin (100 nm chromatin) at 100 nm chromatin (100 nm chromatin). Confirmed.

[0216] A portion of HIP-derived mouse iECs were transduced with a lentiviral vector expressing CD52. Transfected: 1.5 × 10 per well in a 6-well plate for transfection experiments. 5 pcs Mouse HIP iECs were plated. The cells were incubated at 37°C in a cell incubator. The next day, the plates were incubated overnight in Fugene (Promaga, Fitchbuch). rg, WI) and 5 μg of CD52-expressing particles (Origene, Rockville, MA) Transfections were performed using a 3:2 ratio of 1:1 ... The transfection reagent solution was pipetted into OptiMEM (Gibco), mixed, and The DNA transfection complex was added to 2 ml of cell culture medium and incubated at room temperature for 10 min. After 24 hours, the transfection was stopped and the cells were further incubated in endothelial cell medium (Gibco). The cells were grown for 48 hours.

[0217] A subpopulation of mouse HIP iECs (CD52) was transfected with lentivirus to express CD64. The virus was further transformed using the same protocol. a) and 5 μg of CD64 expression plasmid (Origene, Rockville, MD). Transfections were performed using 3:2 ratio of 1000 ribosomal RNA (Cat. No. RC207487L2V) The transfection was stopped as outlined above and the cells were expanded for a further 48 hours.

[0218] A portion of HIP-derived mouse iECs were cultured to express only CD64 according to the protocol described above. The mutant was transduced with .

[0219] 6A-6C show successful transfection with robust expression of the transgene. iECS (CD52, Figure 6A), mouse HIP iECs (CD52 CD64, Figure 6B ), and mouse HIP iECs (CD64, Fig. 6C). The following immunohistochemistry programs are shown: CD52 (CD52 APC: clone HI186, Biolegend) and and CD64 (CD64 PE: clone 10.1, BD) were used to perform flow cytometry. Cytometric analysis was performed. All iEC pools were culture expanded for subsequent assays. did.

[0220] Assess the ability of iECs to bind alemtuzumab Fc using flow cytometry The cells were treated with alemtuzumab (catalog) at concentrations of 0.0001 μg / ml to 1.0 μg / ml. MCA6101, BioRad, Hercules, CA) Goat anti-human IgG(H+L)F(ab')2 secondary antibody (Cat. No. Q-11221M P, Qdot655 tag, Invitrogen) to detect alemtuzumab Fc binding The analysis was performed using an LSRFortessa cytometer (BD Bioscience s) above.

[0221] Figure 7A shows that mouse HIP iECs did not bind any alemtuzumab. However, mouse HIP iECs (CD64) were not able to bind alemtuzumab Fc at high concentrations. They were able to bind in a cytotoxic T cell-dependent manner (Fig. 7B).

[0222] Mouse B6 iEC (CD52) and mouse B6 iEC (CD52, CD64) were XCelligenc using different syngeneic B6 effector immune cells capable of ADCC The upper row of Figure 8 shows that CD52+ iECs were concentration-dependently expressed in alemtuzumab. The lower row shows that CD64-expressing CD52 tg i cells underwent NK cell ADCC mediated by CD64. EC protected against NK cell killing across all concentrations tested.

[0223] FIG. 9 shows the Xcellige antibody assay using syngeneic B6 macrophages as effector cells. nce assay. CD52+ iECs induce alemtuzumab-mediated macrophage proliferation in a concentration-dependent manner. CD64-expressing CD52 tg iECs (bottom row) were subjected to phage ADCC. Protected against macrophage killing across a range of concentrations.

[0224] FIG. 10 shows the results of Xcel ELISA using syngeneic B6 polymorphonuclear cells (PMN) as effector cells. The CD52+ iECs showed a concentration-dependent alemtuzumab-mediated ligation assay. PMNs underwent ADCC. CD64 co-expression in the bottom row indicates that PMNs across all concentrations tested Thus, CD64 expression on iECs was significantly increased with 1.0 μg / mL of IgG1. Even at high alemtuzumab concentrations of 100-150 ml, protection against ADCC was observed in all patients.

[0225] CD64-expressing HIP cells continue to be protected from allogeneic NK cells, macrophages, and PMNs To further confirm that the above assays could be performed using allogeneic effector immune cells, In these assays, effector cells were stimulated by target cell antibody binding or direct cellular The mice were stimulated with either an alloantigen that was detected via cell-cell interactions.

[0226] The top row of Figure 11 shows that CD52+ B6 HIP iECs were cultured with allogeneic BALB / c NK cells. The lower row shows that CD64 expression was mediated by alemtuzumab in a concentration-dependent manner. Current CD52 tg HIP iECs were highly resistant to allogeneic NK cell killing across all concentrations tested. There was no additional direct killing by allogeneic NK cells.

[0227] FIG. 12 shows the expression of Xce using allogeneic BALB / c macrophages as effector cells. lligence assay. CD52+ HIP iECs were cultured with allogeneic macrophages. The lower row shows CD64 expression and CD52 expression, and the lower row shows alemtuzumab-mediated ADCC in a concentration-dependent manner. tg HIP iECs were protected against allogeneic macrophage killing across all concentrations tested. There was no additional direct killing by allogeneic macrophages.

[0228] FIG. 13 shows Xcelli using allogeneic BALB / c PMN as effector cells. Figure 1 shows a concentration-dependent expression assay of CD52+ HIP iECs with allogeneic PMNs. Underwent selective alemtuzumab-mediated ADCC. CD64 co-expression in the bottom row again corresponds to all Targets were protected against allogeneic PMN killing across the concentrations tested. Thus, CD64-expressing HIP iECs were able to mediate ADCC and Protected against syngeneic and allogeneic innate immune cell killing via both direct cytotoxicity.

[0229] FIG. 14 shows the results of incubation with matched syngeneic B6 serum and increasing concentrations of alemtuzumab. Mouse B6 HIP iEC (CD52) on the Xcelligence platform The CD52+ mouse B6 HIP iECs were stained with extremely low levels of 0.0001 μg / ml. Even at low alemtuzumab concentrations, the patients were susceptible to alemtuzumab-mediated CDC (upper row) These targets do not express CD64, so blocking antibodies against CD64 do not bind to the target cells. There was no effect on kill (bottom row).

[0230] Figure 15 shows that CD52+ mouse B6 HIP iECs co-expressing CD64 outgrow all apoptotic cells. The lemtuzumab concentration ranged across all tested concentrations and protected against CDC (top row). A corresponding blocking antibody abolished protection, rendering targets vulnerable to CDC (bottom row).

[0231] D. Example 4: CD64 Protected Engineered Human Endothelial Cells from ADCC and CDC Killing Human HIP iPCs were differentiated into endothelial cells (iECs). The differentiation protocol was performed at 60% H IP iPSCs were started at confluency and incubated with 2% B-27 minus ins ulin (Gibco, Thermo Fisher Scientific brand) and 5 μM CHIR-99021 (Selleckchem, Munich, Germany). The medium was replaced with RPMI-1640 (Gibco) containing any of the following: Baseline: 2% B-27 minus insulin (Gibco) and 2 μM CHIR The medium was replaced with RPMI-1640 containing -99021 (Selleckchem). From day 4 to day 7 of culture, cells were cultured in RPMI-1640 EC medium, 2% B-27 min. us insulin and 50ng / ml human vascular endothelial growth factor (VEGF; R&D S systems, Minneapolis, MN), 10 ng / ml basic human fibroblast FGFb (R&D Systems), 10 μM Y-27632 (Sig ma-Aldrich), and 1 μM SB 431542 (Sigma-Aldrich The endothelial cells were exposed to RPMI-1640 containing 100 mM NaCl (St. Louis, MO). The stars were visible from day 7 onwards and the cells were cultured as Endothelial Cell Basal Medium 2 (PromoCell, Heidelberg, Germany) and supplement, 10% FCS hi (Gibco), 1% pen / strep, 25n 1 μg / ml VEGF, 2 ng / ml FGFb, 10 μM Y-27632 (Sigma -Aldrich), and 1 μM SB 431542 (Sigma-Aldrich). The differentiation protocol was completed after 14 days; undifferentiated cells were detached during the differentiation process. TrypLE Express (Gibco) was added every 3–4 days at a dilution of 1:3 to the cells. was used for passage.

[0232] We then transduced some human HIP iECs with lentiviral vectors expressing CD52. Transformed: 1.5 × 10 per well of a 6-well plate for transfection analysis. 5 pieces Human HIP iECs were plated on the plate. The cells were incubated at 37°C in a cell incubator. The next day, the plates were incubated overnight in Fugene (Promaga, Fitchbuch). rg, WI) and 5 μg of CD52-expressing particles (Origene, Rockville, MA) Transfections were performed using a 3:2 ratio of 1:1 ... The transfection reagent solution was pipetted into OptiMEM (Gibco), mixed, and The DNA transfection complex was added to 2 ml of cell culture medium and incubated at room temperature for 10 min. After 24 hours, the transfection was stopped and the cells were further incubated in endothelial cell medium (Gibco). The cells were grown for 48 hours.

[0233] A subpopulation of human HIP iECs (CD52) was transfected with lentil-derived pluripotent stem cells to express CD64. The virus was further transfected using the same protocol with Fugene (Promagazine). ) and 5 μg of CD64 expression plasmid (Origene, Rockville, MD, Transfections were performed using 100% ribosomal RNA (catalog number: RC207487L2V) at a ratio of 3:2. The transfection was stopped as outlined above and the cells were expanded for a further 48 hours.

[0234] A portion of HIP-derived human iECs were cultured to express only CD64 according to the protocol above. was transduced into

[0235] Figures 16A-16C show successful transfection with strong expression of the transgene. P iECs (CD52, Fig. 16A), human HIP iECs (CD52 CD64, Fig. 16 B) and mouse HIP iECs (CD64, Fig. 16C). The figures show the number of CD52 APCs (CD52 APCs: clone HI186, Biolegend). ) and CD64 (CD64 PE: clone 10.1, BD) were used to Flow cytometry analysis was performed. All iEC pools were cultured for subsequent assays. Expanded.

[0236] Binding of human HIP iECs to alemtuzumab Fc using flow cytometry The ability of the cells to inhibit the growth of erythrocytes was evaluated by injecting them with alemtuzumab at concentrations ranging from 0.0001 μg / ml to 1.0 μg / ml. Mab (catalog no. MCA6101, BioRad, Hercules, CA) and ink Goat anti-human IgG(H+L)F(ab')2 secondary antibody (catalog no. Q- Alemtizumab was used to identify the 11221MP (Qdot655 tag, Invitrogen). The analysis was performed using an LSRFortessa cytometer (BD Biosciences). This was done on the NIRS.

[0237] Figure 17A shows that human HIP iECs did not bind to either alemtuzumab. However, human HIP iEC (CD64) responded to alemtuzumab Fc in a concentration-dependent manner. The antibody was able to bind selectively to the IgG1 antibody (Figure 17B).

[0238] Macrophages constitutively express CD64, and therefore they are the primary markers of human HIP iECs. Fresh PBMCs were used as a control to compare alemtuzumab Fc binding capacity. The cells were isolated from the blood of the subjects by Ficoll separation and then incubated in 10% FCS hi, 1% pen-st rep (all Gibco) and 10 ng / ml human M-CSF (Peprotech). The cells were resuspended in RPMI 1640 (Gibco) containing 100% ethanol. 1 × 10 per ml in 6 At a cell concentration of 1 ml per 24-well plate The macrophages were plated in a 100% PBS-containing medium and the medium was changed every other day until day 6. Cells were assayed after stimulation with μg / ml human IL2 (Peprotech) for 24 hours. Used for.

[0239] Figure 18 shows the ability of human macrophages to bind alemtuzumab Fc. Zumab Fc binding was concentration dependent.

[0240] The top row of Figure 19 shows that CD52+ human HIP iECs inhibited concentration-dependent activation by allogeneic NK cells. The bottom row shows CD64-expressing CD52 tg cells undergoing lemtuzumab-mediated ADCC. HIP iECs were largely protected against allogeneic NK cell killing. Killing was greater at 1.0 μg This occurred only in patients with anti-CD52 antibody (alemtuzumab) at 0.05 mg / ml. There was no direct killing of any additional individuals.

[0241] The top row of Figure 20 shows that CD52+ human HIP iECs were stimulated by allogeneic macrophages in a concentration-dependent manner. The bottom row shows CD64 expression, CD52 expression, and the bottom row shows CD64 expression. tg HIP iECs were largely protected against allogeneic NK cell killing. Killing was 1. This occurred only in the presence of 0 μg / ml of anti-CD52 antibody (alemtuzumab). There was no additional direct killing by the CD64-expressing human HIP iECs recruit allogeneic NK cells and macrophages via both ADCC and direct cytotoxicity. Protected against page killing.

[0242] FIG. 21 shows a comparison of the 100% IgG4-associated ... Human HIP iECs (CD52) on the XCelligence platform CD52+ human HIP iECs were cultured at extremely low concentrations of alemtuzumab at 0.001 μg / ml. Even at these concentrations, the cells were susceptible to alemtuzumab-mediated CDC (top row). These targets did not express CD64, so blocking antibodies against CD64 were not effective in killing the target cells. It had no effect (bottom row).

[0243] Figure 22 shows that CD52+ human HIP iECs co-expressing CD64 were significantly more potent against CDC. The killing was achieved by the addition of 1.0 μg / ml of anti-CD52 antibody ( Blocking antibodies against CD64 abolished the protection, and the target Made the target vulnerable to CDC (bottom row).

[0244] E. Example 5: CD64 Expression on Thyroid Epithelial Cells Blocked IgG Antibody Fc on target cells to block IgG Fc and protect them from ADCC and CDC CD64 expression has been demonstrated in thyroid epithelial cells. These cells are typically associated with Hashimoto's thyroiditis. is invaded by autoimmune antibodies in mice and is therefore a clinically relevant cell type. B6 thyroid epithelial cells (Cat. No.: mGFP-6040, Cell Biologic s, Chicago, IL) and immortalized human thyroid epithelial cells (catalog number: INS-C I-1017, InSCREENeX, Braunschweig, Germany) CD64 expression was achieved as outlined above.

[0245] FIG. 23 shows the results of human thyroid epithelial cells (epiC, FIG. 23A) and mouse epiC (FIG. 23B). ) showing good expression of CD64 in 100+ / - ... The binding assay was performed as described below. Human epiC binds to alemtuzumab Fc. However, CD64+ human epiC showed concentration-dependent binding of IgG Fc (Figure 2 3C). Similarly, mouse epiC was unable to bind alemtuzumab Fc, but was unable to bind CD64+ Mouse epiC showed concentration-dependent binding of IgG Fc (FIG. 23D).

[0246] Next, the blockade of anti-TPO Fc by CD64-expressing human and mouse epiC was analyzed. Anti-TPO antibodies induced in autoimmune thyroiditis (Hashimoto's disease) are a thyroid Responsible for the destruction of cells and the establishment of hypothyroidism. Rabbit anti-mouse TPO antibody (polyclonal antibody) (Nal, Catalog Number: ab203057, Abcam, Cambridge, MA) We designed a disease model using this antibody. It can bind to human CD64 via the Fc domain.

[0247] Human and mouse thyroid epA binding to anti-TPO Fc using flow cytometry The ability of iC to inhibit the proliferation of cells was evaluated by injecting the cells with anti-T PO (polyclonal, catalog number: ab203057, Abcam, Cambridge ge, MA). Goat anti-rabbit IgG(H+L)F(ab')2 secondary Antibody (catalog no. Q11422MP, Qdot655 tag, Invitrogen) Anti-TPO Fc binding was quantified using an LSR Fortessa cytometer. (BD Biosciences).

[0248] Figure 24A and B show that human epiC was unable to bind anti-TPO Fc, but was able to bind CD64+ Human epiC showed concentration-dependent binding of anti-TPO Fc (FIG. 24A). In contrast, mouse epiC was unable to bind anti-TPO Fc, whereas CD64+ mouse epiC showed concentration-dependent binding of anti-TPO Fc (Figure 23B). Thus, the concept of Fc blockade has been identified in clinically relevant cell types.

[0249] Figure 25 shows a C57BL / 6 thyroid epitope on the XCelligence platform. The in vitro killing of C. target cells was treated with different concentrations of anti-TPO (0.001 μg / ml - 1.0 μg / ml) and syngeneic macrophages as effector cells for ADCC. Target cell killing was observed in mice incubated with 0.01 μg / ml to 1.0 μg / ml of anti-T This was observed at 100% TPO concentration, indicating that mouse epiC inhibits TPO in vitro. It was shown to be sensitive to C.

[0250] FIG. 26 shows the same assay design, but using C57BL / 6 thyroid epiC cells expressing CD64. When incubated with anti-TPO and syngeneic macrophages, the anti-TPO concentration No killing was observed in any of the mice. CD64 expression inhibits mouse thyroid epiC. It was found to be effective in protecting against anti-TPO ADCC.

[0251] F. Example 6: CD64 Expression Protects Mouse Epithelial Cells from Killing In Vivo An in vivo model demonstrated that CD64 protected mouse epithelial cells from antibody-mediated rejection. To avoid allogeneic cell rejection, the recipients of C57BL / 6HIP iEC were Syngeneic C57BL / 6 mice were chosen, thus ensuring a purely antibody-driven model. One million CD52-expressing mouse HIP iECs were subcutaneously implanted. The animals were monitored daily using BLI. Graft cell survival was assessed by imaging.

[0252] Figures 27A and 27B show graft survival in vivo. Untreated (left) and alemtuzumab-treated (right) C57BL / 6HIP iECs (CD52 ) survival. Alemtuzumab administration resulted in 100% graft loss at day 5. FIG. 27B shows C57BL / Survival of 6HIP iEC (CD52, CD64). All grafts showed antibody-mediated rejection. The grafts were protected from injury and all grafts survived the study period without an associated drop in BLI signal. This analysis confirms that CD64 expression renders target cells resistant to antibody-mediated rejection. This in vivo model combines ADCC and CDC killing mechanisms.

[0253] IX. Exemplary Sequences: SEQ ID NO:1 - Human beta-2-microglobulin MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKS NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDI SEQ ID NO:2 - Human CIITA protein, 160 amino acids N-terminus [ka] SEQ ID NO:3 - Human CD47 [ka] SEQ ID NO:4 - Herpes simplex virus thymidine kinase (HSV-tk) [ka] SEQ ID NO:5 - Escherichia coli cytosine deaminase (EC -CD) [ka] SEQ ID NO:6 - Truncated human caspase 9 [ka] SEQ ID NO:7 - Human CD64 NM_000566 [ka] SEQ ID NO:8 - Human CD52 NM_001803 MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSN ISGGIFLFFVANAIIHLFCFS SEQ ID NO:9 - Human CD16 FCGR3A NM_001803 [ka] SEQ ID NO:10 - Human CD16 FCGR3B NM_001803 [ka] SEQ ID NO:11 - Human CD32 FCGR2A NM_001803 [ka] SEQ ID NO:12 - Human CD32 FCGR2B NM_001803 [ka] SEQ ID NO:13 - Human CD32 FCGR2C NM_001803 [ka]

[0254] All publications and patent documents disclosed or referenced herein are incorporated by reference in their entirety. The above description has been presented for purposes of illustration and description only. It is not intended to limit the invention to the precise forms disclosed. The scope of the invention is defined by the appended claims. The scope of the present invention is defined by the claims appended hereto.

Claims

1. A modified pluripotent cell, comprising an altered pluripotent cell that exhibits elevated levels of CD40 as compared to a parental form of the modified pluripotent cell. 16, CD32, or CD64 protein expression, and has low susceptibility to antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Engineered pluripotent cells that become pluripotent.

2. said elevated protein expression being CD64 protein, The modified pluripotent vector of claim 1 , wherein the modified pluripotent vector has at least 90% sequence identity with SEQ ID NO:

7. cell.

3. The modified pluripotent cell of claim 2, wherein the CD64 protein has the sequence of SEQ ID NO:

7. cell.

4. 2. The modified pluripotent cell of claim 1 , which is derived from a human hypoimmunogenic pluripotent (HIP) cell.

5. Derived from human low immunogenic pluripotent ABO blood group O rhesus factor negative (HIPO-) cells The modified pluripotent cell of claim 1 .

6. 2. The modified pluripotent cell of claim 1 , which is derived from a human induced pluripotent stem cell (iPSC).

7. The modified pluripotent cell of claim 1 , which is derived from a human embryonic stem cell (ESC).

8. Humans, monkeys, cows, pigs, chickens, turkeys, horses, sheep, goats, donkeys, rats, etc. Babies, ducks, geese, buffaloes, camels, yaks, llamas, alpacas, mice, rats, from a species selected from the group consisting of dog, cat, hamster, and guinea pig The modified pluripotent cell of claim 1.

9. and a suicide gene that is activated by a trigger that causes the death of the modified cells. The modified pluripotent cell of any one of claims 1 to 8.

10. The suicide gene is the herpes simplex virus thymidine kinase gene (HSV-tk). and the trigger is ganciclovir.

11. The HSV-tk gene is a protein that contains at least 90% sequence identity to SEQ ID NO:

4. The modified pluripotent cell of claim 10 , wherein the modified pluripotent cell encodes a protein.

12. The HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO:

4.

12. The modified pluripotent cell according to 11.

13. The suicide gene is Escherichia coli cytosine deaminase The trigger is 5-fluorocytosine (5-FC). The modified pluripotent cell of claim 9.

14. The EC-CD gene encodes a protein that contains at least 90% sequence identity to SEQ ID NO:

5. The modified pluripotent cell of claim 13 , wherein the modified pluripotent cell encodes a gene encoding a pluripotent protein.

15. The EC-CD gene encodes a protein comprising the sequence of SEQ ID NO:

5.

5. The modified pluripotent cell according to claim 4.

16. The suicide gene encodes an inducible caspase protein and the trigger is a dimer The modified pluripotent cell of claim 9, which is an inducing compound (CID).

17. The gene is an inducible caspase kinase inhibitor that contains at least 90% sequence identity to SEQ ID NO:

6. The modified pluripotent cell of claim 16 , wherein the modified pluripotent cell encodes a protein.

18. The gene encodes an inducible caspase protein comprising the sequence of SEQ ID NO:

6. The modified pluripotent cell of claim 17.

19. The modified polynucleotide according to any one of claims 16 to 18, wherein the CID is AP1903. Competent cells.

20. Chimeric antigen receptor (CAR) cells, endothelial cells, dopaminergic neurons, pancreatic islet cells , cardiomyocytes, retinal pigmented endothelial cells, and thyroid cells.

20. A cell derived from the modified pluripotent cell of any one of claims 19.

21. The modified pluripotent cell of claim 20, wherein the CAR cell is a CAR-T cell.

22. Transplantation of cells derived from the modified pluripotent cells according to any one of claims 1 to 19 into a subject. The subject is a human, a monkey, a cow, a pig, a chicken, a turtle, a hamster, a hamster, a hamster, a reptile, a reptile, a hamster, a turtle, a hamster, a hamster, a reptile, a hamster ...hamster, a turtle, a hamster geese, horses, sheep, goats, donkeys, mules, ducks, geese, buffaloes, camels, Yaks, llamas, alpacas, mice, rats, dogs, cats, hamsters, and guinea pigs Law.

23. The cells derived from the modified pluripotent cells include chimeric antigen receptor (CAR) cells, endothelial cells, It consists of blastocysts, dopaminergic neurons, pancreatic islet cells, cardiomyocytes, and retinal pigmented endothelial cells.

23. The method of claim 22, selected from the group:

24. Administering cells derived from the modified pluripotent cells of any one of claims 1 to 19. A method for treating a disease comprising:

25. The derived cells include chimeric antigen receptor (CAR) cells, endothelial cells, dopaminergic neurons, and the like. a pancreatic islet cell, a cardiac muscle cell, a retinal pigmented endothelial cell, and a thyroid cell. The method of claim 24 .

26. The diseases include type I diabetes, heart disease, neurological diseases, cancer, ophthalmological diseases, vascular diseases, and thyroid diseases.

25. The method of claim 24, wherein the patient is selected from the group consisting of:

27. Increasing expression of CD16, CD32, or CD64 in the parental unmodified form of said pluripotent cells A method for generating the modified pluripotent cell of any one of claims 1 to 19, comprising: Law.

28. The modified cells may be human, monkey, bovine, porcine, chicken, turkey, equine, ovine, , goats, donkeys, mules, ducks, geese, buffalo, camels, yaks, llamas, alpacas, 28. The method of claim 27, which is of mouse, rat, dog, cat, hamster, or guinea pig origin. The method described.

29. 28. The method of claim 27, wherein the modified pluripotent cells are derived from HIP cells.

30. The method of claim 27, wherein the modified pluripotent cells are derived from HIPO- cells.

31. 28. The method of claim 27, wherein the modified pluripotent cells are derived from iPSCs.

32. 28. The method of claim 27, wherein the modified pluripotent cells are derived from ESCs.

33. The increased CD16, CD32, or CD64 expression is achieved by the use of a human At least one copy of the CD16, CD32, or CD64 gene is introduced into the modified pluripotent cells.

28. The method of claim 27, resulting from the introduction into the parent form of a cell.

34. 34. The method of claim 33, wherein the promoter is a constitutive promoter.

35. A cell derived from the modified pluripotent cell according to any one of claims 1 to 19 and a pharma- ceutical acceptable carrier. A pharmaceutical composition for treating a disease comprising an acceptable carrier.

36. The derived cells include chimeric antigen receptor (CAR) cells, endothelial cells, dopaminergic neurons, and the like. a pancreatic islet cell, a cardiac muscle cell, a retinal pigmented endothelial cell, and a thyroid cell. The pharmaceutical composition of claim 35.

37. The diseases include type I diabetes, heart disease, neurological diseases, cancer, ophthalmological diseases, vascular diseases, and thyroid diseases.

36. The pharmaceutical composition of claim 35, wherein the therapeutic agent is selected from the group consisting of:

38. A method for treating a disease comprising administering to the patient a cell derived from the modified pluripotent cell according to any one of claims 1 to 19. Medicines for treatment.

39. The derived cells include chimeric antigen receptor (CAR) cells, endothelial cells, dopaminergic neurons, and the like. a pancreatic islet cell, a cardiac muscle cell, a retinal pigmented endothelial cell, and a thyroid cell. The pharmaceutical product of claim 38.

40. The diseases include type I diabetes, heart disease, neurological diseases, cancer, ophthalmological diseases, vascular diseases, and thyroid diseases. The pharmaceutical agent of claim 38, selected from the group consisting of:

41. Elevated levels of CD16, CD32, or CD64 proteins compared to parental types of modified cells. wherein the elevated protein expression induces antibody-dependent cell proliferation and / or cell death. Modified cells that become less susceptible to complement dependent cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).

42. Chimeric antigen receptor (CAR) cells, endothelial cells, dopaminergic neurons, pancreatic islet cells , cardiomyocytes, retinal pigmented endothelial cells, and thyroid cells. The modified cell described herein.

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