TRANSPLANTED CELL PROTECTION VIA MODIFIED Fc RECEPTORS
20240325536 ยท 2024-10-03
Assignee
Inventors
Cpc classification
A61K2239/26
HUMAN NECESSITIES
C07K14/70535
CHEMISTRY; METALLURGY
A61K39/4611
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K35/36
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
Abstract
The invention provides, for the first time, cells that express truncated or modified Fc Receptor proteins (e.g. CD16t, CD32t, or CD64t) 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 express a truncated or modified Fc Receptor. The invention encompasses cells derived from the pluripotent cells as well as primary cells. The cells may also be differentiated cells, including chimeric antigen receptor (CAR) cells, T cells, natural killer (NK) cells, endothelial cells, dopaminergic neurons, neuroglial cells, pancreatic islet cells, pancreatic beta cells, thyroid cells, fibroblasts, hepatocytes, cardiomyocytes, or retinal pigment endothelium cells.
Claims
1. A modified cell, wherein said modified cell expresses an F.sub.c receptor protein comprising a truncation or modification, wherein said F.sub.c receptor protein expression causes said modified cell to be less susceptible to antibody dependent cellular cytoxicity (ADCC) or complement-dependent cytotoxicity (CDC), wherein said truncation or modification reduces or eliminates said F.sub.c receptor intracellular signaling.
2. The modified cell of claim 1, wherein said F.sub.c receptor protein is selected from the group consisting of a truncated CD16 (CD16t), truncated CD32 (CD32t), and truncated CD64 (CD64t).
3. The modified cell of claim 1, wherein said cell is a pluripotent cell.
4. (canceled)
5. The modified cell of claim 4, wherein said CD64t protein has the sequence of SEQ ID NO:16.
6. The modified cell of claim 1, wherein said modified cell is derived from a human hypo-immunogenic pluripotent (HIP) cell or a human hypo-immunogenic pluripotent ABO blood group O Rhesus Factor negative (HIPO?) cell.
7-10. (canceled)
11. The modified cell of claim 1, further comprising a suicide gene that is activated by a trigger that causes said modified cell to die.
12-21. (canceled)
22. The modified cell of claim 1, wherein said cell is selected from the group consisting of a chimeric antigen receptor (CAR) cell, a T cell, an NK cell, an ILC, an endothelial cell, a dopaminergic neuron, a neuroglial cell, a pancreatic islet cell, a pancreatic beta cell, a thyroid cell, an endocrine cell, a fibroblast, a hepatocyte, a cardiomyocyte, and a retinal pigment endothelium cell.
23-26. (canceled)
27. A method of treating a disease, comprising administering the modified cell of claim 1 to a subject.
28. The method of claim 27, wherein said modified cell is selected from the group consisting of a chimeric antigen receptor (CAR) cell, a T cell, an NK cell, an ILC, an endothelial cell, a dopaminergic neuron, a neuroglial cell, a pancreatic islet cell, a pancreatic beta cell, a thyroid cell, a fibroblast, a hepatocyte, a cardiomyocyte, and a retinal pigment endothelium cell.
29. The method of claim 27, wherein said disease is selected from the group consisting of Type I Diabetes, a cardiac disease, a neurological disease, an endocrine disease, a cancer, an ocular disease, and a vascular disease.
30. A method for generating the modified cell of claim 1, comprising expressing said CD16t, CD32t, or CD64t protein in a parental non-modified version of said cell.
31. (canceled)
32. The method of claim 30, wherein said modified cell is derived from a HIP cell, a HIPO? cell, an iPSC cell, or an ESC cell.
33-37. (canceled)
38. A pharmaceutical composition for treating a disease, comprising a cell derived from the modified cell of claim 1 and a pharmaceutically-acceptable carrier.
39. The pharmaceutical composition of claim 38, wherein said cell is selected from the group consisting of a chimeric antigen receptor (CAR) cell, a T cell, an NK cell, an ILC, an endothelial cell, a dopaminergic neuron, a neuroglial cell, a pancreatic islet cell, a pancreatic beta cell, a thyroid cell, a fibroblast, a hepatocyte, a cardiomyocyte, and a retinal pigment endothelium cell.
40-45. (canceled)
46. The modified cell of claim 1, wherein said cell comprises an Fc receptor chimera comprising a cytoplasmic domain that does not mediate an Fc receptor signaling pathway and wherein said cytoplasmic domain promotes endocytosis of an antibody bound by its Fc to an extracellular domain of said Fc receptor chimera.
47. The modified cell of claim 46, wherein said cytoplasmic domain is from a transferrin receptor.
48. (canceled)
49. The modified cell of any one of claims 46-48, wherein said F.sub.c receptor chimera comprises a CD16, CD32, or CD64 cell surface domain and a TfR1 or TfR2 cytoplasmic domain.
50. The modified cell of claim 1, wherein said cell expresses a recombinant SIRP? engager protein.
51. The modified cell of claim 50, wherein said SIRP? engager protein comprises an immunoglobulin superfamily domain, an antibody Fab domain, or a single chain variable fragment (scFV).
52. (canceled)
53. The modified cell of claim 22, wherein said cell comprises a B2M?/? phenotype, a CIITA?/? phenotype, a CD64t, and a SIRP?-engager molecule.
54. The modified cell of claim 53, wherein said SIRP?-engager protein is CD47.
55. The modified cell of claim 53, wherein said cell is an engineered NK cell.
Description
IV. BRIEF DESCRIPTION OF THE DRAWINGS
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[0036] A truncated or modified CD16, CD32, or CD64 coding sequence (CDS) lacks a functioning intracellular signaling domain. The protein domains according to Uniprot are shown. The cytoplasmic domains for truncation or modification are indicated.
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[0066] Only CAR-T.sup.CD64t were able to bind IgG1 in a concentration-dependent manner (mean?SD, three independent replicates per group and time point).
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V. DETAILED DESCRIPTION OF THE INVENTION
A. Introduction
[0079] The invention provides, for the first time, cells that comprise truncated CD16, CD32, or CD64 expression to evade antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) while eliminating intracellular signaling. The cells may be primary cells or pluripotent cells, including hypoimmune pluripotent cells (HIP) or ABO blood type O Rhesus Factor negative HIP cells (HIPO?), that further comprise the enhanced CD64 expression. The cells may also be pancreatic islet cells, thyroid cells, chimeric antigen receptor (CAR) cells, T cells, NK cells, ILCs, endothelial cells, dopaminergic neurons, cardiomyocytes, or retinal pigment endothelium cells used for treating diseases or rehabilitating damaged tissues.
[0080] Antibody-mediated rejection (AMR) after solid organ transplantation was first discovered as a distinct clinicopathological entity in 1997. Decades after the concept of cellular rejection had been widely accepted, and standardized nomenclature and diagnostic criteria were developed for kidney, heart, lung, liver, and pancreatic transplantation. A hallmark of AMR is the presence of graft-specific antibodies in combination with graft damage.
[0081] When an antibody binds to an HLA or non-HLA antigen on a cell via its Fab regions, the F.sub.c is bound by NK cells (mostly via their CD16 receptor) and mediate antibody-mediated cellular cytotoxicity (ADCC) or AMR in a transplant setting. Complement can also bind to F.sub.c and activate its cascade and form the membrane attack complex (MAC) for CDC killing (
[0082] There are three layers of antigens to which antibodies can be directed. First and most relevant in the transplant setting are human leukocyte antigens (HLA), the most polymorphic histocompatibility complexes crucial for T cell activation. Patients with antibodies to their grafts were consistently shown to have inferior graft survival across organ systems.
[0083] Second are the highly polymorphic non-HLA antigens such as the MHC Class I-Related Sequence A (MICA), a cell surface glycol-protein encoded within the human HLA genes. It does not associate with ?2-microglobulin nor present peptides. Anti-MICA antibodies have been associated with accelerated graft failure in solid organ transplantation.
[0084] MICA polymorphism and expression on pancreatic islet cells is associated with type 1 diabetes mellitus (T1DM).
[0085] Third, antibodies against cell type-specific surface antigens generated after solid organ transplantation mediate AMR.
[0086] These AMR reactions occur in transplant recipients despite the use of effective systemic immunosuppression. In some autoimmune diseases, autoantibodies cause or enhance the destruction of the target cells and persist as part of the disease. Patients with autoimmune thyroiditis or type 1 diabetes mellitus (T1DM) have a high prevalence of anti-thyroid epithelial cell or anti-? cell antibodies, respectively, which may persist for many years.
[0087] Patients with cellular grafts using long-term benign regenerative approaches in immunocompetent patients may eventually experience some form of antibody-mediated immune attack, dependent on the cell source and disease treated. Thyroid cells and islet cells express all three layers of antigens and are most vulnerable against all forms of AMR. For this reason, these cell types exemplify the antibody evasive technology disclosed herein.
[0088] Other cell types are implicated and include allogeneic human embryonic stem cell-derived cardiac progenitors and mesenchymal stroma cell derivatives.
[0089] The invention provides a gene editing strategy that effectively protects transplanted cells such as islet cells and thyroid cells from AMR. It establishes protection from antibodies against HLA-, non-HLA-, and cell type-specific antigens. In some embodiments it is combined with additional hypoimmune edits to silence cellular rejection.
[0090] The invention provides antibody resistance that builds upon the HIP concept and utilizes non-immunogenic components. The systemic use of microbial IgG-degrading enzymes has successfully been used to deplete total IgG and HLA antibodies in highly sensitized patients before their kidney transplantation(Jordan, S. C. et al., N Engl J Med 377, 442-453 (2017)). More recently, this endopeptidase was shown to cleave IgG bound to target cells (Peraro, L. et al., Mol Ther (2021)). Pre-existing antibodies against these bacterial enzymes, however, are widely prevalent in the healthy population (Akesson, P. et al., J Infect Dis 189, 797-804 (2004)). They spike during streptococcal infections (Lei, B. et al., Nat Med 7, 1298-1305 (2001); Okamoto, S., et al. Vaccine 23, 4852-4859 (2005)), and might themselves add unwanted immunogenicity. Human CD64 and its truncated form CD64t are non-immunogenic, have high affinity for IgG(Bruhns, P. et al., Blood 113, 3716-3725 (2009)), and are very effective against antibody killing in several translationally relevant cell types.
[0091] The invention applies this technology to regenerative cell therapeutics, especially for diseases with an underlying autoimmune component in which antibodies are present that would destroy the transplanted cells. Regenerative cell therapeutics would be destroyed similarly to the native cells if autoimmunity is not circumvented (Hollenberg, A. N. et al., Mol Cell Endocrinol 445, 35-41 (2017)).
[0092] The invention shows that engineered epithelial cells (epC) expressing thyroid peroxidase (TPO) and CD64 (epiCs.sup.TPO,CD64t) were protected against clinically relevant anti-TPO killing. Current stem cell-derived pancreatic islet cells in clinical trials for patients with Type-I Diabetes Melitis are currently produced from embryonic stem cells (Melton, D. The promise of stem cell-derived islet replacement therapy. Diabetologia 64, 1030-1036 (2021)). They are transplanted across an HLA mismatch. Despite the use of encapsulation strategies to protect the islet cells from the host immune system, however, antibodies against microencapsulated graft cells have been observed. (Desai, T. & Shea, L. D., Nat Rev Drug Discov 16, 338-350 (2017); Duvivier-Kali et al., Am J Transplant 4, 1991-2000 (2004)). Forced overexpression of CD64t on human beta cells was sufficient to protect them from anti-HLA ADCC and CDC.
[0093] Most success in CAR-T cell therapy has been achieved with B cell neoplasms in which patients are treated with lymphocytotoxic drugs and the CAR-T cells directly target the source for antibody production (Brudno, J. N. & Kochenderfer, J. N., Nat Rev C/in Oncol 15, 31-46 (2018)). Antibody induction, however, is still regularly observed (Till, B. G. et al. Blood 112, 2261-2271 (2008)). An antibody response against the CAR-T cells has been shown to prevent expansion of CAR-T cells upon re-infusion (Peraro, L. et al. Incorporation of bacterial immunoevasins to protect cell therapies from host antibody-mediated immune rejection. Mol Ther (2021)) and hamper persistence. The expression of CD64t on CAR-T cells makes them resistant to anti-CAR-T cell antibodies without affecting their specific killing capacity. Since early immune clearance is even more common in non-B cell cancer, CAR-T.sup.CD64t may be more efficient for these indications. The F.sub.c sequestration mechanism reliably builds protection against antibodies in several cell types and further advances the immune evasion concept for allogeneic regenerative and immune-oncology cell therapeutics. (See WO2021076427, incorporated by reference herein in its entirety.)
[0094] CD64 is the high-affinity human IgG receptor Fc?RI capable of capturing free serum IgG. In order to achieve resistance against AMR, the Fc binding capacity of CD64 was utilized. To avoid alterations of the engineered cells from unwanted intracellular signaling, however, the invention provides a truncated form of CD64 (CD64t) on cells that is incapable of inducing intracellular signaling. The intracellular tail was cut off in the CD64 coding sequence (CDS) before it was cloned into a lentivirus for expression in pluripotent cells. The CD64t captures free IgG Fc without inducing downstream signaling (
[0095] One specific indication for this technology is the cellular treatment of autoimmune diseases. Antibodies against autologous epitopes cause beta cell death in type 1 diabetes and thyroid cells in autoimmune thyroiditis. Antibodies may bind to HLA class II molecules. Additionally, HLA-independent antibodies have been described. Should a cell that is used for regenerative therapy have the same epitope, it will also be bound by these antibodies and be killed. The CD64 sequestration of the invention prevents ADCC and CDC.
[0096] In some embodiments of the invention, HypoImmunogenic Pluripotent (HIP) cells are modified to express CD16t, CD32t, or CD64t (HIP/CD16t, HIP/CD32t, or HIP/CD64t cells). HIP cells avoid host immune responses due to several genetic manipulations as outlined herein. The cells lack major immune antigens that trigger immune responses and are engineered to avoid phagocytosis and NK cell killing. In some embodiments, the HIP cells are made by eliminating the activity of both alleles of a B2M gene in an induced pluripotent stem cell (iPSC); eliminating the activity of both alleles of a CIITA gene in the iPSC; and increasing the expression of CD47 in the iPSC. HIP cells are described in detail in WO2018132783, incorporated by reference herein in its entirety. In other embodiments, HIP cells express a SIRP?-engager molecule as disclosed in PCT/US2021/062008, incorporated by reference herein in its entirety.
[0097] In other embodiments of the invention, HypoImmunogenic Pluripotent Blood group O Rh-(HIPO?) cells are modified to express CD16t, CD32t, or CD64t (HIPO?/CD16t cells, HIPO?/CD32t, or HIPO?/CD64t cells). HIPO? cells avoid host immune responses due to several genetic or enzymatic manipulations as outlined herein. The cells lack major blood group and immune antigens that trigger immune responses and are engineered to avoid rejection, phagocytosis, or killing. This allows the derivation of off-the-shelf cell products for generating specific tissues and organs. The benefit of being able to use human allogeneic HIPO? cells and their derivatives in human patients provides significant benefits, including the ability to avoid long-term adjunct immunosuppressive therapy and drug use generally seen in allogeneic transplantations. They also provide significant cost savings as cell therapies can be used without requiring individual treatments for each patient. HIPO?/CD16, HIPO?/CD32, or HIPO?/CD64 cells may serve as a universal cell source for the generation of universally-acceptable derivatives. HIPO? cells are described in detail in U.S. Prov. Appl. No. 62/846,399 and U.S. Pat. No. 62,855,499 each of which are incorporated by reference herein in their entirety.
B. Definitions
[0098] The term pluripotent cells refers to cells that can self-renew and proliferate while remaining in an undifferentiated state and that can, under the proper conditions, be induced to differentiate into specialized cell types. The term pluripotent cells, as used herein, encompass embryonic stem cells and other types of stem cells, including fetal, amnionic, or somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Additional exemplary stem cell lines include those made available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (as described in Cowan, C. A. et. al, New England J Med. 350:13. (2004), incorporated by reference herein in its entirety.)
[0099] Pluripotent stem cells as used herein have the potential to differentiate into any of the three germ layers: endoderm (e.g. the stomach linking, gastrointestinal tract, lungs, etc), mesoderm (e.g. muscle, bone, blood, urogenital tissue, etc) or ectoderm (e.g. epidermal tissues and nervous system tissues). The term pluripotent stem cells, as used herein, also encompasses induced pluripotent stem cells, or iPSCs, a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such iPS or iPSC cells can be created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the induction of iPS cells are known in the art and are further described below. (See, e.g., Zhou et al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al., Nature Biotechnol. 26 (7): 795 (2008); Woltjen et al., Nature 458 (7239): 766-770 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009); each of which is incorporated by reference herein in their entirety.) The generation of induced pluripotent stem cells (iPSCs) is outlined below. As used herein, hiPSCs are human induced pluripotent stem cells, and miPSCs are murine induced pluripotent stem cells.
[0100] Pluripotent stem cell characteristics refer to characteristics of a cell that distinguish pluripotent stem cells from other cells. The ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. Expression or non-expression of certain combinations of molecular markers are also pluripotent stem cell characteristics. For example, human pluripotent stem cells express at least several, and in some embodiments, all of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics. As described herein, cells do not need to pass through pluripotency to be reprogrammed into endodermal progenitor cells and/or hepatocytes.
[0101] As used herein, multipotent or multipotent cell refers to a cell type that can give rise to a limited number of other particular cell types. For example, induced multipotent cells are capable of forming endodermal cells. Additionally, multipotent blood stem cells can differentiate itself into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc.
[0102] As used herein, the term oligopotent refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of either the lymphoid or myeloid lineages, respectively.
[0103] As used herein, the term unipotent means the ability of a cell to form a single cell type. For example, spermatogonial stem cells are only capable of forming sperm cells.
[0104] As used herein, the term totipotent means the ability of a cell to form an entire organism. For example, in mammals, only the zygote and the first cleavage stage blastomeres are totipotent.
[0105] As used herein, non-pluripotent cells refer to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, but are not limited to, cells from a tissue selected from bone marrow, skin, skeletal muscle, fat tissue and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells. The starting cells employed for generating the induced multipotent cells, the endodermal progenitor cells, and the hepatocytes can be non-pluripotent cells.
[0106] Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In particular embodiments, a less potent cell is considered differentiated in reference to a more potent cell.
[0107] A somatic cell is a cell forming the body of an organism. Somatic cells include cells making up organs, skin, blood, bones and connective tissue in an organism, but not germ cells.
[0108] Cells can be from, for example, human or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, and non-human primates. In some embodiments, a cell is from an adult human or non-human mammal. In some embodiments, a cell is from a neonatal human, an adult human, or non-human mammal.
[0109] As used herein, the terms subject or patient refers to any animal, such as a domesticated animal, a zoo animal, or a human. The subject or patient can be a mammal like a dog, cat, bird, livestock, or a human. Specific examples of subjects and patients include, but are not limited to, individuals (particularly human) with a disease or disorder related to the liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bone, bone marrow, and the like.
[0110] Mammalian cells can be from humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, and non-human primates (e.g., chimpanzees, macaques, and apes).
[0111] By hypo-immunogenic pluripotent cell or HIP cell herein is meant a pluripotent cell that retains its pluripotent characteristics and yet gives rise to a reduced immunological rejection response when transferred into an allogeneic host. In preferred embodiments, HIP cells do not give rise to an immune response. Thus, hypo-immunogenic refers to a significantly reduced or eliminated immune response when compared to the immune response of a parental (i.e. wt) cell prior to immunoengineering as outlined herein. In many cases, the HIP cells are immunologically silent and yet retain pluripotent capabilities. Assays for HIP characteristics are outlined below.
[0112] By HIP/CD16t, HIP/CD32t, or HIP/CD64t cell herein is meant a HIP cell that has a truncated CD16, CD32, or CD64 protein, respectively, on the cell surface. The truncation removes the intracellular signaling domain. In an alternative embodiment, the signaling domain is not truncated, but rather, modified to eliminate a signaling function. Such modifications may be amino acid substitutions or internal deletions.
[0113] By hypo-immunogenic pluripotent cell O? hypo-immunogenic pluripotent ORh? cell or HIPO? cell herein is meant a HIPO? cell that is also ABO blood group O and Rhesus Factor Rh?. HIPO? cells may have been generated from O? cells, enzymatically modified to be O?, or genetically engineered to be O?.
[0114] By HIPO?/CD16t, HIPO?/CD32t, or HIPO?/CD64t cell herein is meant a HIPO? cell that has a truncated CD16, CD32, or CD64 protein, respectively, on the cell surface. The truncation removes the intracellular signaling domain. In an alternative embodiment, the signaling domain is not truncated, but rather, mutated to eliminate a signaling function.
[0115] By HLA or human leukocyte antigen complex is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. These cell-surface proteins that make up the HLA complex are responsible for the regulation of the immune response to antigens. In humans, there are two MHCs, class I and class II, HLA-I and HLA-II. HLA-I includes three proteins, HLA-A, HLA-B and HLA-C, which present peptides from the inside of the cell, and antigens presented by the HLA-I complex attract killer T-cells (also known as CD8+ T-cells or cytotoxic T cells). The HLA-I proteins are associated with ?-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outside the cell to T lymphocytes. This stimulates CD4+ cells (also known as T-helper cells). It should be understood that the use of either MHC or HLA is not meant to be limiting, as it depends on whether the genes are from humans (HLA) or murine (MHC). Thus, as it relates to mammalian cells, these terms may be used interchangeably herein.
[0116] By gene knock out herein is meant a process that renders a particular gene inactive in the host cell in which it resides, resulting either in no protein of interest being produced or an inactive form. As will be appreciated by those in the art and further described below, this can be accomplished in a number of different ways, including removing nucleic acid sequences from a gene, or interrupting the sequence with other sequences, altering the reading frame, or altering the regulatory components of the nucleic acid. For example, all or part of a coding region of the gene of interest can be removed or replaced with nonsense sequences, all or part of a regulatory sequence such as a promoter can be removed or replaced, translation initiation sequences can be removed or replaced, etc.
[0117] By gene knock in herein is meant a process that adds a genetic function to a host cell. This causes increased levels of the encoded protein. As will be appreciated by those in the art, this can be accomplished in several ways, including adding one or more additional copies of the gene to the host cell or altering a regulatory component of the endogenous gene increasing expression of the protein is made. This may be accomplished by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.
[0118] ?-2 microglobulin or 02M or B2M protein refers to the human 02M protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000015.10:44711487-44718159.
[0119] CD47 protein protein refers to the human CD47 protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000016.10:10866208-10941562. CD47 is a ligand for SIRP? (a SIRP? engager molecule). CD47 is a marker-of-self protein that can be overexpressed broadly across tumor types. It is emerging as a novel potent macrophage immune checkpoint for cancer immunotherapy. CD47 in tumor cells sends a don't-eat-me signal that inhibits macrophage phagocytosis.
[0120] CIITA protein protein refers to the human CIITA protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000003.12:108043094-108094200.
[0121] By wild type in the context of a cell means a cell found in nature. However, in the context of a pluripotent stem cell, as used herein, it also means an iPSC that may contain nucleic acid changes resulting in pluripotency but did not undergo the gene editing procedures of the invention to achieve hypo-immunogenicity.
[0122] By syngeneic herein refers to the genetic similarity or identity of a host organism and a cellular transplant where there is immunological compatibility; e.g. no immune response is generated.
[0123] By allogeneic herein refers to the genetic dissimilarity of a host organism and a cellular transplant where an immune response is generated.
[0124] By B2M?/? herein is meant that a diploid cell has had the B2M gene inactivated in both chromosomes. As described herein, this can be done in a variety of ways.
[0125] By CIITA ?/? herein is meant that a diploid cell has had the CIITA gene inactivated in both chromosomes. As described herein, this can be done in a variety of ways.
[0126] By CD47 tg (standing for transgene) or CD47+) herein is meant that the host cell expresses CD47, in some cases by having at least one additional copy of the CD47 gene.
[0127] An Oct polypeptide refers to any of the naturally-occurring members of Octamer family of transcription factors, or variants thereof that maintain transcription factor activity, similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. Oct3/4 (referred to herein as Oct4) contains the POU domain, a 150 amino acid sequence conserved among Pit-1, Oct-1, Oct-2, and uric-86. (See, Ryan, A. K. & Rosenfeld, M. G., Genes Dev. 11:1207-1225 (1997), incorporated herein by reference in its entirety.) In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Oct polypeptide family member such as to those listed above or such as listed in Genbank accession number NP-002692.2 (human Oct4) or NP-038661.1 (mouse Oct4). Oct polypeptides (e.g., Oct3/4 or Oct 4) can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. The Oct polypeptide(s) can be a pluripotency factor that can help induce multipotency in non-pluripotent cells.
[0128] A Klf polypeptide refers to any of the naturally-occurring members of the family of Krnppel-like factors (Klfs), zinc-finger proteins that contain amino acid sequences similar to those of the Drosophila embryonic pattern regulator Kruppel, or variants of the naturally-occurring members that maintain transcription factor activity similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain. (See, Dang, D. T., Pevsner, J. & Yang, V. W., Cell Biol. 32:1103-1121 (2000), incorporated by reference herein in its entirety.) Exemplary Klf family members include, Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Kifl0, Klfl1, Klfl2, Klfl3, Klfl4, Klfl5, Klfl6, and Klfl7. Klf2 and Klf-4 were found to be factors capable of generating iPS cells in mice, and related genes Klf1 and Klf5 did as well, although with reduced efficiency. (See, Nakagawa, et al., Nature Biotechnology 26:101-10.sup.6 (2007), incorporated by reference herein in its entirety.) In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Klf polypeptide family member such as to those listed above or such as listed in Genbank accession number CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klf polypeptides (e.g., Klf1, Klf4, and Klf5) can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. The Klf polypeptide(s) can be a pluripotency factor. The expression of the Klf4 gene or polypeptide can help induce multipotency in a starting cell or a population of starting cells.
[0129] A Myc polypeptide refers to any of the naturally-occurring members of the Myc family. (See, e.g., Adhikary, S. & Eilers, M., Nat. Rev. Mol. Cell Biol. 6:635-645 (2005), incorporated by reference herein in its entirety.) It also includes variants that maintain similar transcription factor activity when compared to the closest related naturally occurring family member (i.e. within at least 50%, 80%, or 90% activity). It further includes polypeptides comprising at least the DNA-binding domain of a naturally occurring family member, and can further comprise a transcriptional activation domain. Exemplary Myc polypeptides include, e.g., c-Myc, N-Myc and L-Myc. In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Myc polypeptide family member, such as to those listed above or such as listed in Genbank accession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc) can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. The Myc polypeptide(s) can be a pluripotency factor.
[0130] A Sox polypeptide refers to any of the naturally-occurring members of the SRY-related HMG-box (Sox) transcription factors, characterized by the presence of the high-mobility group (HMG) domain, or variants thereof that maintain similar transcription factor activity when compared to the closest related naturally occurring family member (i.e. within at least 50%, 80%, or 90% activity). It also includes polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain. (See, e.g., Dang, D. T. et al., Int. J. Biochem. Cell Biol. 32:1103-1121 (2000), incorporated by reference herein in its entirety.) Exemplary Sox polypeptides include, e.g., Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30. Sox1 has been shown to yield iPS cells with a similar efficiency as Sox2, and genes Sox3, Sox15, and Sox18 have also been shown to generate iPS cells, although with somewhat less efficiency than Sox2. (See, Nakagawa, et al., Nature Biotechnology 26:101-10.sup.6 (2007), incorporated by reference herein in its entirety.) In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Sox polypeptide family member such as to those listed above or such as listed in Genbank accession number CAA83435 (human Sox2). Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. The Sox polypeptide(s) can be a pluripotency factor. As discussed herein, SOX2 proteins find particular use in the generation of iPSCs.
[0131] By differentiated hypo-immunogenic pluripotent cells or differentiated HIP cells or dHIP cells herein is meant iPS cells that have been engineered to possess hypoimmunogenicity (e.g. by the knock out of B2M and CIITA and the knock in of CD47) and then are differentiated into a cell type for ultimate transplantation into subjects. Thus, for example HIP cells can be differentiated into hepatocytes (dHIP hepatocytes), into beta-like pancreatic cells or islet organoids (dHIP beta cells), into endothelial cells (dHIP endothelial cells), etc. Parallel definitions apply to differentiated HIP/CD64t and differentiated HIPO?/CD64t cells.
[0132] The term percent identity, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent identity can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
[0133] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
[0134] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
[0135] Inhibitors, activators, and modulators affect a function or expression of a biologically-relevant molecule. The term modulator includes both inhibitors and activators. They may be identified using in vitro and in vivo assays for expression or activity of a target molecule.
[0136] Inhibitors are agents that, e.g., inhibit expression or bind to target molecules or proteins. They may partially or totally block stimulation or have protease inhibitor activity. They may reduce, decrease, prevent, or delay activation, including inactivation, desensitizion, or down regulation of the activity of the described target protein. Modulators may be antagonists of the target molecule or protein.
[0137] Activators are agents that, e.g., induce or activate the function or expression of a target molecule or protein. They may bind to, stimulate, increase, open, activate, or facilitate the target molecule activity. Activators may be agonists of the target molecule or protein.
[0138] Homologs are bioactive molecules that are similar to a reference molecule at the nucleotide sequence, peptide sequence, functional, or structural level. Homologs may include sequence derivatives that share a certain percent identity with the reference sequence.
[0139] Thus, in one embodiment, homologous or derivative sequences share at least a 70 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least an 80 or 85 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least a 90 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least a 95 percent sequence identity. In a more specific embodiment, homologous or derivative sequences share at least an 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity.
[0140] Homologous or derivative nucleic acid sequences may also be defined by their ability to remain bound to a reference nucleic acid sequence under high stringency hybridization conditions. Homologs having a structural or functional similarity to a reference molecule may be chemical derivatives of the reference molecule. Methods of detecting, generating, and screening for structural and functional homologs as well as derivatives are known in the art.
[0141] Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), incorporated by reference herein in its entirety.
[0142] Stringency of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.
[0143] Stringent conditions or high stringency conditions, as defined herein, can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50? C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 Mm sodium phosphate buffer at Ph 6.5 with 750 Mm sodium chloride, 75 Mm sodium citrate at 42? C.; or (3) overnight hybridization in a solution that employs 50% formamide, 5?SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 Mm sodium phosphate (Ph 6.8), 0.1% sodium pyrophosphate, 5?Denhardt's solution, sonicated salmon sperm DNA (50 ?l/ml), 0.1% SDS, and 10% dextran sulfate at 42? C., with a 10 minute wash at 42? C. in 0.2?SSC (sodium chloride/sodium citrate) followed by a 10 minute high-stringency wash consisting of 0.1?SSC containing EDTA at 55? C.
[0144] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
[0145] As used herein the term modification refers to an alteration that physically differentiates the modified molecule from the parent molecule. In one embodiment, an amino acid change in a CD16, CD32, CD64, CD47, HSVtk, EC-CD, or iCasp9 variant polypeptide prepared according to the methods described herein differentiates it from the corresponding parent gene or cell that has not been modified according to the methods described herein, such as wild-type proteins, a naturally occurring mutant proteins or another engineered protein that does not include the modifications of such variant polypeptide. In another embodiment, a variant polypeptide includes one or more modifications that differentiates the function of the variant polypeptide from the unmodified polypeptide. For example, an amino acid change in a variant polypeptide affects its receptor binding profile. In other embodiments, a variant polypeptide comprises substitution, deletion, or insertion modifications, or combinations thereof. In another embodiment, a variant polypeptide includes one or more modifications that increases its affinity for a receptor compared to the affinity of the unmodified polypeptide.
[0146] In one embodiment, a variant polypeptide includes one or more substitutions, insertions, or deletions relative to a corresponding native or parent sequence. In certain embodiments, a variant polypeptide includes 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, 30, 31-40, 41 to 50, or 51 or more modifications.
[0147] By episomal vector herein is meant a genetic vector that can exist and replicate autonomously in the cytoplasm of a cell; e.g. it is not integrated into the genomic DNA of the host cell. A number of episomal vectors are known in the art and described below.
[0148] By knock out in the context of a gene means that the host cell harboring the knock out does not produce a functional protein product of the gene. As outlined herein, a knock out can result in a variety of ways, from removing all or part of the coding sequence, introducing frameshift mutations such that a functional protein is not produced (either truncated or nonsense sequence), removing or altering a regulatory component (e.g. a promoter) such that the gene is not transcribed, preventing translation through binding to mRNA, etc. Generally, the knock out is effected at the genomic DNA level, such that the cells' offspring also carry the knock out permanently.
[0149] By knock in in the context of a gene means that the host cell harboring the knock in has more functional protein active in the cell. As outlined herein, a knock in can be done in a variety of ways, usually by the introduction of at least one copy of a transgene (tg) encoding the protein into the cell, although this can also be done by replacing regulatory components as well, for example by adding a constitutive promoter to the endogeneous gene.
[0150] In general, knock in technologies result in the integration of the extra copy of the transgene into the host cell.
VI. Cells of the Invention
[0151] The invention provides compositions and methodologies for generating pluripotent cells that express CD16t, CD32t, or CD64t. In some aspects of the invention, the cells will be derived from induced pluripotent 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 or differentiated therefrom. In other aspects, the cells are primary cells, including gene-edited primary cells. The cells of the invention may also be pancreatic islet cells, thyroid cells, chimeric antigen receptor (CAR) cells, endothelial cells, dopaminergic neurons, cardiomyocytes, or retinal pigment endothelium cells used for treating diseases or rehabilitating damaged tissues.
A. Methodologies for Genetic Alterations
[0152] The invention includes methods of modifying nucleic acid sequences within cells or in cell-free conditions to generate both cells with CD16t, CD32t, or CD64t expression. Exemplary technologies include homologous recombination, knock-in, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9, and other site-specific nuclease technologies. These techniques enable double-strand DNA breaks at desired locus sites. These controlled double-strand breaks promote homologous recombination at the specific locus sites. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequences and induce a double-stranded break in the nucleic acid molecule. The double-strand break is repaired either by an error-prone non-homologous end-joining (NHEJ) or by homologous recombination (HR).
[0153] As will be appreciated by those in the art, a number of different techniques can be used to engineer the pluripotent cells of the invention, as well as the engineering of the iPSCs to become hypo-immunogenic as outlined herein.
[0154] In general, these techniques can be used individually or in combination. For example, these techniques can be used to generate F.sub.c receptor-expressing cells where the receptor is truncated to remove some or all of the cytoplasmic signalling domain.
[0155] Alternatively, this domain may be modified by deletion, substitutions, or nonsense mutations.
[0156] Upon Fc binding, the cytoplasmic tail of CD64 interacts with the src-related family of tyrosine kinases (such as Fyn and Lyn) and Syk family kinases. They phosphorylate a cytoplasmic amino acid motif termed the immunoreceptor tyrosine-based activation motif (ITAM) on the associated FcR gamma chain (coded by its own gene FCER1G). This mediates the activation of immune cells.
[0157] Thus, the invention provides modifying the cytoplasmic tail that interact with the src-related family of tyrosine kinases and the Syk family kinases to prevent the activation of the FcR gamma chain ITAMs and thus prevent downstream signaling.
[0158] The CD64 cytoplasmic tail is the following 60 amino acids from SEQ ID NO:7: [0159] RKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGV HRKEPQGAT
[0160] In some embodiments, the cytoplasmic domain of the Fc receptors are exchanged for another cytoplasic domain with a different physiology. For instance, a cytoplasmic domain of a transferrin receptor (e.g. TfR1 or TfR2) promotes continuous endocytosis leading to a high receptor turnover. Thus, in certain embodiments, the Fc receptors are a chimera between an Fc receptor extracellular domain and a transferrin receptor cytoplasmic domain. This will promote endocytosis and disintegration of an IgG antibody that was initially bound to the Fc receptor domain on the cell surface. Thus, in preferred embodiments, the cells of the invention express a chimera comprising a CD16, CD32, or CD64 cell surface domain and a TfR1 or TfR2 cytoplasmic domain.
[0161] Moreover, these techniques may be used in the generation of the HIP cells. CRISPR may be used to reduce the expression of active B2M and/or CIITA protein in the engineered cells, with viral techniques (e.g. lentivirus) to knock in expression of CD47 or other SIRP?-Engager proteins. Also, as will be appreciated by those in the art, although one embodiment sequentially utilizes a CRISPR step to knock out B2M, followed by a CRISPR step to knock out CIITA with a final step of a lentivirus to knock in expression of CD47 or other SIRP?-Engager proteins, these genes can be manipulated in different orders using different technologies.
[0162] As is discussed more fully below, transient expression of reprogramming genes is generally done to generate/induce pluripotent stem cells.
a. CRISPR Technologies
[0163] In one embodiment, the cells are manipulated using clustered regularly interspaced short palindromic repeats)/Cas (CRISPR) technologies as is known in the art. CRISPR can be used to generate the starting iPSCs or to generate the HIP cells from the iPSCs. There are a large number of techniques based on CRISPR, see for example Doudna and Charpentier, Science doi:10.1126/science.1258096, hereby incorporated by reference. CRISPR techniques and kits are sold commercially.
b. TALEN Technologies
[0164] In some embodiments, the HIP cells of the invention are made using Transcription Activator-Like Effector Nucleases (TALEN) methodologies. TALEN are restriction enzymes combined with a nuclease that can be engineered to bind to and cut practically any desired DNA sequence. TALEN kits are sold commercially.
c. Zinc Finger Technologies
[0165] In one embodiment, the cells are manipulated using Zn finger nuclease technologies. Zn finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms, similar to CRISPR and TALENs.
d. Viral Based Technologies
[0166] There are a wide variety of viral techniques that can be used to generate the HIP cells of the invention (as well as for the original generation of the iPCSs), including, but not limited to, the use of retroviral vectors, lentiviral vectors, adenovirus vectors and Sendai viral vectors. Episomal vectors used in the generation of iPSCs are described below.
e. Down regulation of genes using interfering RNA
[0167] In other embodiments, genes that encode proteins used in HLA molecules are downregulated by RNAi technologies. RNA interference (RNAi) is a process where RNA molecules inhibit gene expression often by causing specific mRNA molecules to degrade. Two types of RNA moleculesmicroRNA (miRNA) and small interfering RNA (siRNA)are central to RNA interference. They bind to the target mRNA molecules and either increase or decrease their activity. RNAi helps cells defend against parasitic nucleic acids such as those from viruses and transposons. RNAi also influences development.
[0168] sdRNA molecules are a class of asymmetric siRNAs comprising a guide (antisense) strand of 19-21 bases. They contain a 5 phosphate, 2Ome or 2F modified pyrimidines, and six phosphotioates at the 3 positions. They also contain a sense strand containing 3 conjugated sterol moieties, 2 phospotioates at the 3 position, and 2Ome modified pyrimidines. Both strands contain 2 Ome purines with continuous stretches of unmodified purines not exceeding a length of 3. sdRNA is disclosed in U.S. Pat. No. 8,796,443, incorporated herein by reference in its entirety.
[0169] For all of these technologies, well known recombinant techniques are used, to generate recombinant nucleic acids as outlined herein. In certain embodiments, the recombinant nucleic acids (either than encode a desired polypeptide, e.g. CD47, or disruption sequences) may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate for the host cell and subject to be treated. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are also contemplated. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow the selection of transformed host cells. Certain embodiments include an expression vector comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequence for use herein include promoters, enhancers, and other expression control elements. In certain embodiments, an expression vector is designed for the choice of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the vector's copy number, the ability to control that copy number, or the expression of any other protein encoded by the vector, such as antibiotic markers.
[0170] Examples of suitable mammalian promoters include, for example, promoters from the following genes: ubiquitin/S27a promoter of the hamster (WO 97/15664), Simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, the long terminal repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus Long Terminal repeat region, and the early promoter of human Cytomegalovirus (CMV). Examples of other heterologous mammalian promoters are the actin, immunoglobulin or heat shock promoter(s).
[0171] In additional embodiments, promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). In further embodiments, heterologous mammalian promoters are used. Examples include the actin promoter, an immunoglobulin promoter, and heat-shock promoters. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers et al., Nature 273: 113-120 (1978). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. Greenaway, P. J. et al., Gene 18: 355-360 (1982). The foregoing references are incorporated by reference in their entirety.
B. Generation of Pluripotent Cells
[0172] The invention provides methods of producing non-immunogenic pluripotent cells from pluripotent cells. Thus, the first step is to provide the pluripotent stem cells.
[0173] The generation of mouse and human pluripotent stem cells (generally referred to as iPSCs; miPSCs for murine cells or hiPSCs for human cells) is generally known in the art. As will be appreciated by those in the art, there are a variety of different methods for the generation of iPCSs. The original induction was done from mouse embryonic or adult fibroblasts using the viral introduction of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4; see Takahashi and Yamanaka Cell 126:663-676 (2006), hereby incorporated by reference in its entirety and specifically for the techniques outlined therein. Since then, a number of methods have been developed; see Seki et al., World J Stem Cells 7(1):116-125 (2015) for a review, and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013, both of which are hereby expressly incorporated by reference in their entirety, and in particular for the methods for generating hiPSCs (see for example Chapter 3 of the latter reference).
[0174] Generally, iPSCs are generated by the transient expression of one or more reprogramming factors in the host cell, usually introduced using episomal vectors. Under these conditions, small amounts of the cells are induced to become iPSCs (in general, the efficiency of this step is low, as no selection markers are used). Once the cells are reprogrammed, and become pluripotent, they lose the episomal vector(s) and produce the factors using the endogeneous genes. This loss of the episomal vector(s) results in cells that are called zero footprint cells. This is desirable as the fewer genetic modifications (particularly in the genome of the host cell), the better. Thus, it is preferred that the resulting hiPSCs have no permanent genetic modifications.
[0175] As is also appreciated by those of skill in the art, the number of reprogramming factors that can be used or are used can vary. Commonly, when fewer reprogramming factors are used, the efficiency of the transformation of the cells to a pluripotent state goes down, as well as the pluripotency, e.g. fewer reprogramming factors may result in cells that are not fully pluripotent but may only be able to differentiate into fewer cell types.
[0176] In some embodiments, a single reprogramming factor, OCT4, is used. In other embodiments, two reprogramming factors, OCT4 and KLF4, are used. In other embodiments, three reprogramming factors, OCT4, KLF4 and SOX2, are used. In other embodiments, four reprogramming factors, OCT4, KLF4, SOX2 and c-Myc, are used. In other embodiments, 5, 6 or 7 reprogramming factors can be used selected from SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen.
[0177] In general, these reprogramming factor genes are provided on episomal vectors such as are known in the art and commercially available. For example, ThermoFisher/Invitrogen sell a sendai virus reprogramming kit for zero footprint generation of hiPSCs, see catalog number A34546. ThermoFisher also sells EBNA-based systems as well, see catalog number A14703.
[0178] In addition, there are a number of commercially available hiPSC lines available; see, e.g., the Gibco? Episomal hiPSC line, K18945, which is a zero footprint, viral-integration-free human iPSC cell line (see also Burridge et al, 2011, supra).
[0179] In general, as is known in the art, iPSCs are made from non-pluripotent cells such as CD34+ cord blood cells, fibroblasts, etc., by transiently expressing the reprogramming factors as described herein.
[0180] For example, successful iPSCs were also generated using only Oct3/4, Sox2 and Klf4, while omitting the C-Myc, although with reduced reprogramming efficiency.
[0181] In general, iPSCs are characterized by the expression of certain factors that include KLF4, Nanog, OCT4, SOX2, ESRRB, TBX3, c-Myc and TCL1. New or increased expression of these factors for purposes of the invention may be via induction or modulation of an endogenous locus or from expression from a transgene.
[0182] For example, murine iPSCs can be generated using the methods of Diecke et al, Sci Rep. 2015, Jan. 28; 5:8081 (doi:10.1038/srep08081), hereby incorporated by reference in its entirety and specifically for the methods and reagents for the generation of the miPSCs.
[0183] See also, e.g., Burridge et al., PLoS One, 2011 6(4):18293, hereby incorporated by reference in its entirety and specifically for the methods outlined therein.
[0184] In some cases, the pluripotency of the cells is measured or confirmed as outlined herein, for example by assaying for reprogramming factors or by conducting differentiation reactions as outlined herein and in the Examples.
C. Generation of Hypo-Immunogenic Pluripotent (HIP) Cells
[0185] Generating HIP cells from pluripotent cells is done with as few as three genetic changes, resulting in minimal disruption of cellular activity but conferring immunosilencing to the cells.
[0186] As discussed herein, one embodiment utilizes a reduction or elimination in the protein activity of MHC I and II (HLA I and II when the cells are human). This can be done by altering genes encoding their component. In one embodiment, the coding region or regulatory sequences of the gene are disrupted using CRISPR. In another embodiment, gene translation is reduced using interfering RNA technologies. The third change is a change in a gene that regulates susceptibility to macrophage phagocytosis, such as CD47 or SIRP?-Engager, and this is generally a knock in of a gene using viral technologies.
[0187] In some cases, where CRISPR is being used for the genetic modifications, hiPSC cells that contain a Cas9 construct that enable high efficiency editing of the cell line can be used; see, e.g., the Human Episomal Cas9 iPSC cell line, A33124, from Life Technologies.
1. HLA-I Reduction
[0188] The HIP cells of the invention include a reduction in MHC I function (HLA I when the cells are derived from human cells).
[0189] As will be appreciated by those in the art, the reduction in function can be accomplished in a number of ways, including removing nucleic acid sequences from a gene, interrupting the sequence with other sequences, or altering the regulatory components of the nucleic acid. For example, all or part of a coding region of the gene of interest can be removed or replaced with nonsense sequences, frameshift mutations can be made, all or part of a regulatory sequence such as a promoter can be removed or replaced, translation initiation sequences can be removed or replaced, etc.
[0190] As will be appreciated by those in the art, the successful reduction of the MHC I function (HLA I when the cells are derived from human cells) in the pluripotent cells can be measured using techniques known in the art and as described below; for example, FACS techniques using labeled antibodies that bind the HLA complex; for example, using commercially available HLA-A,B,C antibodies that bind to the the alpha chain of the human major histocompatibility HLA Class I antigens.
a. B2M Alteration
[0191] In one embodiment, the reduction in HLA-I activity is done by disrupting the expression of the ?-2 microglobulin gene in the pluripotent stem cell, the human sequence of which is disclosed herein. This alteration is generally referred to herein as a gene knock out, and in the HIP cells of the invention it is done on both alleles in the host cell. Generally the techniques to do both disruptions is the same.
[0192] A particularly useful embodiment uses CRISPR technology to disrupt the gene. In some cases, CRISPR technology is used to introduce small deletions/insertions into the coding region of the gene, such that no functional protein is produced, often the result of frameshift mutations that result in the generation of stop codons such that truncated, non-functional proteins are made.
[0193] Accordingly, a useful technique is to use CRISPR sequences designed to target the coding sequence of the B2M gene in mouse or the B2M gene in human. After gene editing, the transfected iPSC cultures are dissociated to single cells. Single cells are expanded to full-size colonies and tested for a CRISPR edit by screening for presence of aberrant sequence from the CRISPR cleavage site. Clones with deletions in both alleles are picked.
[0194] Such clones did not express B2M as demonstrated by PCR and did not express HLA-I as demonstrated by FACS analysis (see examples 1 and 6, for example).
[0195] Assays to test whether the B2M gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with antibodies to the B2M protein. In another embodiment, reverse transcriptase polymerase chain reactions (rt-PCR) confirms the presence of the inactivating alteration.
[0196] In addition, the cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This may be assayed by FACS analysis using antibodies to one or more HLA cell surface components as discussed above.
[0197] It is noteworthy that others have had poor results when trying to silence the B2M genes at both alleles. See, e.g. Gornalusse et al., Nature Biotech.
[0198] Doi/10.1038/nbt.3860). 2. HLA-II Reduction
[0199] In addition to a reduction in HLA I, the HIP cells of the invention also lack MHC II function (HLA II when the cells are derived from human cells).
[0200] As will be appreciated by those in the art, the reduction in function can be accomplished in a number of ways, including removing nucleic acid sequences from a gene, adding nucleic acid sequences to a gene, disrupting the reading frame, interrupting the sequence with other sequences, or altering the regulatory components of the nucleic acid. In one embodiment, all or part of a coding region of the gene of interest can be removed or replaced with nonsense sequences. In another embodiment, regulatory sequences such as a promoter can be removed or replaced, translation initiation sequences can be removed or replaced, etc.
[0201] The successful reduction of the MHC II function (HLA II when the cells are derived from human cells) in the pluripotent cells or their derivatives can be measured using techniques known in the art such as Western blotting using antibodies to the protein, FACS techniques, rt-PCR techniques, etc.
a. CIITA Alteration
[0202] In one embodiment, the reduction in HLA-II activity is done by disrupting the expression of the CIITA gene in the pluripotent stem cell, the human sequence of which is shown herein. This alteration is generally referred to herein as a gene knock out. In the HIP cells of the invention it is done on both alleles in the host cell.
[0203] Assays to test whether the CIITA gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cells lysates probed with antibodies to the CIITA protein. In another embodiment, reverse transcriptase polymerase chain reactions (rt-PCR) confirms the presence of the inactivating alteration.
[0204] In addition, the cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay is done as is known in the art. Exemplary analyses include Western Blots or FACS analysis using commercial antibodies that bind to human HLA Class II HLA-DR, DP and most DQ antigens as outlined below.
[0205] A particularly useful embodiment uses CRISPR technology to disrupt the CIITA gene. CRISPRs were designed to target the coding sequence of the Ciita gene in mouse or the CIITA gene in human, an essential transcription factor for all MHC II molecules. After gene editing, the transfected iPSC cultures were dissociated into single cells.
[0206] They were expanded to full-size colonies and tested for successful CRISPR editing by screening for the presence of an aberrant sequence from the CRISPR cleavage site. Clones with deletions did not express CIITA as determined by PCR and did not express MHC II/HLA-II as determined by FACS analysis.
3. Phagocytosis Reduction
[0207] In addition to the reduction of HLA I and II (or MHC I and II), generally using B2M and CIITA knock-outs, the HIP cells of the invention have a reduced susceptibility to macrophage phagocytosis and NK cell killing. The resulting HIP cells escape the immune macrophage and innate pathways due to one or more CD47 transgenes.
a. CD47 Increase
[0208] In some embodiments, reduced macrophage phagocytosis and NK cell killing susceptibility results from increased CD47 on the HIP cell surface. This is done in several ways as will be appreciated by those of skill in the art using knock in or transgenic technologies. In some cases, increased CD47 expression results from one or more CD47 transgene.
[0209] Accordingly, in some embodiments, one or more copies of a CD47 gene is added to the HIP cells under control of an inducible or constitutive promoter, with the latter being preferred. In some embodiments, a lentiviral construct is employed as described herein or known in the art. CD47 genes may integrate into the genome of the host cell under the control of a suitable promoter as is known in the art.
[0210] The HIP cell lines were generated from B2M?/? CIITA?/? iPSCs. Cells containing lentivirus vectors expressing CD47 were selected using a Blasticidin marker. The CD47 gene sequence was synthesized and the DNA was cloned into the plasmid Lentivirus pLenti6/V5 with a blasticidin resistance (Thermo Fisher Scientific, Waltham, MA)
[0211] In some embodiments, the expression of the CD47 gene can be increased by altering the regulatory sequences of the endogenous CD47 gene, for example, by exchanging the endogenous promoter for a constitutive promoter or for a different inducible promoter. This can generally be done using known techniques such as CRISPR.
[0212] Once altered, the presence of sufficient CD47 expression can be assayed using known techniques such as those described in the Examples, such as Western blots, ELISA assays or FACS assays using anti-CD47 antibodies. In general, sufficiency in this context means an increase in the expression of CD47 on the HIP cell surface that silences NK cell killing. The natural expression levels on cells is too low to protect them from NK cell lysis once their MHC I is removed.
[0213] SIRP? engagement maybe be accomplished via engineered or alternative molecules. In some aspects of the invention, the engager molecule is a protein. In other aspects, the protein is a fusion protein. In other aspects, the fusion protein comprises a CD47 extracellular domain (ECD). In other aspects of the invention, the SIRP-? engager cell comprises an immunoglobulin superfamily domain. In other aspects of the invention, the engager molecule comprises an antibody Fab or a single chain variable fragment (scFV) that binds to SIRPa. In other aspects, the Fab or scFV binds to SIRP? with an affinity measured by its dissociation constant (Kd), wherein the Kd is between about 10.sup.?7 and 10.sup.?13 M. See PCT/US2021/062008, incorporated by reference herein in its entirety.
[0214] In some aspects of the invention, the engager molecule comprises one or more antibody complimentarity determining regions (CDRs) that binds to SIRPa.
4. Suicide Genes
[0215] In some embodiments, the invention provides hypoimmunogenic pluripotent cells that comprise a suicide gene or suicide switch. These are incorporated to function as a safety switch that can cause the death of the hypoimmunogenic pluripotent cells should they grow and divide in an undesired manner. The suicide gene ablation approach includes a suicide gene in a gene transfer vector encoding a protein that results in cell killing only when activated by a specific compound. A suicide gene may encode an enzyme that selectively converts a nontoxic compound into highly toxic metabolites. The result is specifically eliminating cells expressing the enzyme. In some embodiments, the suicide gene is the herpesvirus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In other embodiments, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC) (Barese et al., Mol. Therap. 20(10):1932-1943 (2012), Xu et al., Cell Res. 8:73-8 (1998), both incorporated herein by reference in their entirety.)
[0216] In other embodiments, the suicide gene is an inducible Caspase protein. An inducible Caspase protein comprises at least a portion of a Caspase protein capable of inducing apoptosis. In one embodiment, the portion of the Caspase protein is exemplified in SEQ ID NO:6. In preferred embodiments, the inducible Caspase protein is iCasp9. It comprises the sequence of the human FK506-binding protein, FKBP12, with an F36V mutation, connected through a series of amino acids to the gene encoding human caspase 9. FKBP12-F36V binds with high affinity to a small-molecule dimerizing agent, AP1903. Thus, the suicide function of iCasp9 in the instant invention is triggered by the administration of a chemical inducer of dimerization (CID). In some embodiments, the CID is the small molecule drug AP1903. Dimerization causes the rapid induction of apoptosis. (See WO2011146862; Stasi et al, N. Engl. J Med 365;18 (2011); Tey et al., Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which are incorporated by reference herein in their entirety.)
5. CD16t, CD32t, or CD64t Expression
[0217] The cells of the invention have a reduced susceptibility to ADCC and CDC resulting from CD16t, CD32t, or CD64t expression. The resulting cells sequester antibodies due to the increased expression. In one embodiment, the cells comprise one or more CD16t, CD32t, or CD64t transgenes.
[0218] In some embodiments, reduced ADCC or CDC susceptibility results from CD16t, CD32t, or CD64t on the cell surface. This is done in several ways as will be appreciated by those in the art using knock in or transgenic technologies. In some cases, CD16t, CD32t, or CD64t expression results from one or more transgenes.
[0219] Accordingly, in some embodiments, one or more copies of a CD16t, CD32t, or CD64t gene is added to the cells under control of an inducible or constitutive promoter, with the latter being preferred. In some embodiments, a lentiviral construct is employed as described herein or known in the art. The genes may integrate into the genome of the host cell under the control of a suitable promoter as is known in the art.
[0220] Cells containing lentivirus vectors expressing CD16t, CD32t, or CD64t are selected using a Blasticidin marker. The gene sequence is synthesized and the DNA may be cloned, for instance, into the plasmid Lentivirus pLenti6/V5 with a blasticidin resistance (Thermo Fisher Scientific, Waltham, MA)
[0221] In some embodiments, the expression of the gene can be increased by altering the regulatory sequences of the endogenous CD16, CD32, or CD64 gene combined with a truncation or mutation as described herein. This may be accomplished, for example, by exchanging the endogenous promoter for a constitutive promoter or for a different inducible promoter. This can generally be done using known techniques such as CRISPR.
[0222] Once altered, the presence of sufficient expression can be assayed using known techniques such as those described in the Examples, such as Western blots, ELISA assays or FACS assays using anti-CD16, CD32, or CD64 antibodies. In general, sufficiency in this context means an increase in expression the cell surface that sequesters antibodies and inhibis ADCC or CDC.
6. Assays for HIP Phenotypes and Retention of Pluripotency
[0223] Once the HIP cells have been generated, they may be assayed for their hypo-immunogenicity and/or retention of pluripotency as is generally described herein and in the examples.
[0224] For example, hypo-immunogenicity are assayed using a number of techniques One exemplary technique includes transplantation into allogeneic hosts and monitoring for HIP cell growth (e.g. teratomas) that escape the host immune system. HIP derivatives are transduced to express luciferase and can then followed using bioluminescence imaging. Similarly, the T cell and/or B cell response of the host animal to the HIP cells are tested to confirm that the HIP cells do not cause an immune reaction in the host animal. T cell function is assessed by Elispot, Elisa, FACS, PCR, or mass cytometry (CYTOF). B cell response or antibody response is assessed using FACS or luminex. Additionally, or alternatively, the cells may be assayed for their ability to avoid innate immune responses, e.g. NK cell killing. NK cell lytolytic activity is assessed in vitro or in vivo using techniques known in the art.
[0225] Similarly, the retention of pluripotency is tested in a number of ways. In one embodiment, pluripotency is assayed by the expression of certain pluripotency-specific factors as generally described herein. Additionally or alternatively, the HIP cells are differentiated into one or more cell types as an indication of pluripotency.
D. Generation of HIPO? CD16t, CD32t, or CD64t-expressing Cells
[0226] In some aspects of the invention, the HIP cells generated as above will already be HIPO? cells because the process will have started with pluripotent cells having an 0-blood type.
[0227] Other aspects of the invention involve the enzymatic conversion of A and B antigens. In preferred aspects, the B antigen is converted to 0 using an enzyme. In more preferred aspects, the enzyme is an a-galactosidase. This enzyme eliminates the terminal galactose residue of the B antigen. Other aspects of the invention involve the enzymatic conversion of A antigen to 0. In preferred aspects, the A antigen is converted to 0 using an a-N-acetylgalactosaminidase. Enzymatic conversion is discussed, e.g., in Olsson et al., Transfusion Clinique et Biologique 11:33-39 (2004); U.S. Pat. Nos. 4,427,777, 5,606,042, 5,633,130, 5,731,426, 6,184,017, 4,609,627, and 5,606,042; and Int'l Pub. No. WO9923210, each of which are incorporated by reference herein in their entirety.
[0228] Other embodiments of the invention involve genetically engineering the cells by knocking out the ABO gene Exon 7 or silencing the SLC14A1 (JK) gene. Other embodiments of the invention involve knocking out the C and E antigens of the Rh blood group system (RH), K in the Kell system (KEL), Fya and Fy3 in the Duffy system (FY), Jkb in the Kidd system (JK), or U and S in the MNS blood group system. Any knockout methodology known in the art or described herein, such as CRISPR, talens, or homologous recombination, may be employed.
E. Preferred Embodiments of the Invention
[0229] The CD16t, CD32t, or CD64t expressing HIP, HIPO?, iPSC, iPSCO?, ESC, or ESCO? cells, or derivatives thereof, of the invention may be used to treat, for example, Type 1 diabetes, cardiac diseases, neurological diseases, cancer, blindness, vascular diseases, and others that respond to regenerative medicine therapies. In particular, the invention contemplates using the cells for differentiation into any cell type. Thus, provided herein are cells that express CD16t, CD32t, or CD64t and exhibit pluripotency but do not result in a host ADCC or CDC response when transplanted into an allogeneic host such as a human patient, either as pluripotent cells or as the differentiated products of them. Additionally, the CD16, CD32, or CD64 intracellular signaling function is reduced or eliminated by truncation or mutation.
[0230] In one aspect, the cells of the present invention comprise a nucleic acid encoding a chimeric antigen receptor (CAR), with CD16t, CD32t, or CD64t expression. The CAR can comprise an extracellular domain, a transmembrane domain, and an intracellular signaling domain.
[0231] In some embodiments, the extracellular CAR domain binds to an antigen selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CS1, CD171, BCMA, MUC16, ROR1, and WT1. In certain embodiments, the extracellular domain comprises a single chain variable fragment (scFv). In some embodiments, the CAR-Transmembrane domain comprises CD3?, CD4, CD8?, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, and BTLA. In certain embodiments, the CAR intracellular signaling domain comprises CD3?, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, and BTLA.
[0232] In certain embodiments, the CAR comprises an anti-CD19 scFv domain, a CD28 transmembrane domain, and a CD3 zeta signaling intracellular domain. In some embodiments, the CAR comprises anti-CD19 scFv domain, a CD28 transmembrane domain, a 4-1BB signaling intracellular domain, and a CD3 zeta signaling intracellular domain.
[0233] In another aspect of the invention, an isolated CAR-T cell that expresses CD16t, CD32t, or CD64t is produced by in vitro differentiation of any one of the cells described herein. In some embodiments, the cell is a cytotoxic hypoimmune CAR-T cell.
[0234] In various embodiments, the in vitro differentiation comprises culturing the cell carrying a CAR construct in a culture media comprising one or more growth factors or cytokines selected from the group consisting of bFGF, EPO, Flt3L, IGF, IL-3, IL-6, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture media further comprises one or more selected from the group consisting of a BMP activator, a GSK3 inhibitor, a ROCK inhibitor, a TGF? receptor/ALK inhibitor, and a NOTCH activator.
[0235] In particular embodiments, the isolated CAR-T cell of the invention produced by in vitro differentiation is used as a treatment of cancer.
[0236] Another aspect of the invention provides is a method of treating a patient with cancer by administering a composition comprising a therapeutically effective amount of any of the isolated CAR-T cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier.
[0237] In some embodiments, the administration step comprises intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, and intraperitoneal administration. In certain instances, the administration further comprises a bolus or by continuous perfusion.
[0238] In some embodiments, the cancer is a blood cancer 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.
[0239] In another aspect, the present invention provides a method of making any one of the isolated CAR-T/CD16t, CD32t, or CD64t cells described herein. The method includes in vitro differentiating of any one of the cells of the invention wherein in vitro differentiating comprises culturing them in a culture media comprising one or more growth factors or cytokines selected from the group consisting of bFGF, EPO, Flt3L, IGF, IL-2, IL-3, IL-6, IL-7, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture media further comprises one or more selected from the group consisting of a BMP activator, a GSK3 inhibitor, a ROCK inhibitor, a TGF? receptor/ALK inhibitor, and a NOTCH activator.
[0240] In some embodiments, the in vitro differentiation comprises culturing the HIPO-cells on feeder cells. In other embodiments, the in vitro differentiating comprises culturing in simulated microgravity. In certain instances, the culturing in simulated microgravity is for at least 72 hours.
[0241] In some aspects provided herein is an isolated, engineered hypoimmune cardiac cell (hypoimmunogenic cardiac cell) differentiated from a cell that expresses CD16t, CD32t, or CD64t.
[0242] In some aspects, provided herein is a method of treating a patient suffering from a heart condition or disease. The method comprises administering a composition comprising a therapeutically effective amount of a population of any one of the isolated, engineered hypoimmune cardiac cells derived from cells of the invention as described herein. In some embodiments, the composition further comprises a therapeutically effective carrier.
[0243] In some embodiments, the administration comprises implantation into the patient's heart tissue, intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, trans-endocardial injection, trans-epicardial injection, or infusion.
[0244] In some embodiments, the heart condition or disease is selected from the group consisting of pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
[0245] In some aspects, provided herein is a method of producing a population of hypoimmune cardiac cells from a population of HIPO?/CD16t, CD32t, or CD64t cells by in vitro differentiation, wherein endogenous ?-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated and expression of CD47 or another SIRP?-Engager protein has been increased in the HIPO-cells. The method comprises: (a) culturing a population of HIPO-cells in a culture medium comprising a GSK inhibitor; (b) culturing the population of HIPO-cells in a culture medium comprising a WNT antagonist to produce a population of pre-cardiac cells; and (c) culturing the population of pre-cardiac cells in a culture medium comprising insulin to produce a population of hypoimmune cardiac cells.
[0246] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 2 ?M to about 10 ?M. In some embodiments, the WNT antagonist is IWR1, a derivative thereof, or a variant thereof. In some instances, the WNT antagonist is at a concentration ranging from about 2 ?M to about 10 ?M.
[0247] In some aspects, provided herein is an isolated, engineered CD16, CD32, or CD64-overexpressing endothelial cell differentiated from HIPO? cells. In other aspects, the isolated, engineered endothelial cell of the invention is selected from the group consisting of a capillary endothelial cell, vascular endothelial cell, aortic endothelial cell, brain endothelial cell, and renal endothelial cell.
[0248] In some aspects, provided herein is a method of treating a patient suffering from a vascular condition or disease. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of a population of isolated, engineered endothelial cells of the invention.
[0249] The method comprises administering a composition comprising a therapeutically effective amount of a population of any one of the isolated, engineered CD16, CD32, or CD64-overexpressing endothelial cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier or excipient. In some embodiments, the administration comprises implantation into the patient's heart tissue, intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, trans-endocardial injection, trans-epicardial injection, or infusion.
[0250] In some embodiments, the vascular condition or disease is selected from the group consisting of, vascular injury, cardiovascular disease, vascular disease, ischemic disease, myocardial infarction, congestive heart failure, hypertension, ischemic tissue injury, limb ischemia, stroke, neuropathy, and cerebrovascular disease.
[0251] In some aspects, provided herein is a method of producing a population of hypoimmune endothelial cells from a population of CD16t, CD32t, or CD64t-expressing cells by in vitro differentiation, wherein endogenous ?-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated and CD47 expression has been increased in the HIPO-cells. The method comprises: (a) culturing a population of HIPO-cells in a first culture medium comprising a GSK inhibitor; (b) culturing the population of HIPO-cells in a second culture medium comprising VEGF and bFGF to produce a population of pre-endothelial cells; and (c) culturing the population of pre-endothelial cells in a third culture medium comprising a ROCK inhibitor and an ALK inhibitor to produce a population of hypoimmune endothelial cells.
[0252] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 1 ?M to about 10 ?M. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some instances, the ROCK inhibitor is at a concentration ranging from about 1 ?M to about 20 ?M. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 0.5 ?M to about 10 ?M.
[0253] In some embodiments, the first culture medium comprises from 2 ?M to about 10 ?M of CHIR-99021. In some embodiments, the second culture medium comprises 50 ng/ml VEGF and 10 ng/ml bFGF. In other embodiments, the second culture medium further comprises Y-27632 and SB-431542. In various embodiments, the third culture medium comprises 10 ?M Y-27632 and 1 ?M SB-431542. In certain embodiments, the third culture medium further comprises VEGF and bFGF. In particular instances, the first culture medium and/or the second medium is absent of insulin.
[0254] In some aspects, provided herein is an isolated, engineered hypoimmune dopaminergic neuron (DN) differentiated from a CD16t, CD32t, or CD64t-expressing cell.
[0255] In some embodiments, the endogenous ?-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, CD47 or another SIRP?-Engager expression has been increased, the neuron is blood type O and Rh?.
[0256] In some embodiments, the isolated dopaminergic neuron is selected from the group consisting of a neuronal stem cell, neuronal progenitor cell, immature dopaminergic neuron, and mature dopaminergic neuron.
[0257] In some aspects, provided herein is a method of treating a patient suffering from a neurodegenerative disease or condition. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of a population of any one of the isolated hypoimmune dopaminergic neurons. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of the isolated hypoimmune dopaminergic neurons is on a biodegradable scaffold.
[0258] The administration may comprise transplantation or injection. In some embodiments, the neurodegenerative disease or condition is selected from the group consisting of Parkinson's disease, Huntington disease, and multiple sclerosis.
[0259] In some aspects, provided herein is a method of producing a population of CD16t, CD32t, or CD64t-expressing dopaminergic neurons by in vitro differentiation. In some embodiments, the endogenous ?-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, CD47 or another SIRP?-Engager expression has been increased, the blood group is O and Rh?. In some embodiments, the method comprises (a) culturing the population of cells in a first culture medium comprising one or more factors selected from the group consisting of sonic hedgehog (SHH), BDNF, EGF, bFGF, FGF8, WNT1, retinoic acid, a GSK3? inhibitor, an ALK inhibitor, and a ROCK inhibitor to produce a population of immature dopaminergic neurons; and (b) culturing the population of immature dopaminergic neurons in a second culture medium that is different than the first culture medium to produce a population of dopaminergic neurons.
[0260] In some embodiments, the GSKO inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSKO inhibitor is at a concentration ranging from about 2 ?M to about 10 ?M. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 1 ?M to about 10 ?M. In some embodiments, the first culture medium and/or second culture medium are absent of animal serum.
[0261] In some embodiments, the method also comprises isolating the population of hypoimmune dopaminergic neurons from non-dopaminergic neurons. In some embodiments, the method further comprises cryopreserving the isolated population of hypoimmune dopaminergic neurons.
[0262] In some aspects, provided herein is an isolated engineered hypoimmune pancreatic islet cell differentiated from a CD16t, CD32t, or CD64t-expressing cell. In some embodiments, the endogenous ?-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, CD47 or another SIRP?-Engager expression has been increased, the blood type is O and Rh?.
[0263] In some embodiments, the isolated hypoimmune pancreatic islet cell is selected from the group consisting of a pancreatic islet progenitor cell, immature pancreatic islet cell, and mature pancreatic islet cell.
[0264] In some aspects, provided herein is a method of treating a patient suffering from diabetes. The method comprises administering a composition comprising a therapeutically effective amount of a population of any one of the isolated CD16t, CD32t, or CD64t-expressing pancreatic islet cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of the isolated hypoimmune pancreatic islet cells is on a biodegradable scaffold.
[0265] In some instances, the administration comprises transplantation or injection.
[0266] In some aspects, provided herein is a method of producing a population of hypoimmune pancreatic islet cells from a population of CD16t, CD32t, or CD64t-expressing cells by in vitro differentiation. In some embodiments, the endogenous ?-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, CD47 or another SIRP?-Engager expression has been increased, the blood type is O and Rh-in the HIPO? cells. The method comprises: (a) culturing the population of CD16t, CD32t, or CD64t-expressing cells in a first culture medium comprising one or more factors selected from the group consisting insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH), and vascular endothelial growth factor (VEGF), transforming growth factor-?(TGF?) superfamily, bone morphogenic protein-2 (BMP2), bone morphogenic protein-7 (BMP7), a GSK3? inhibitor, an ALK inhibitor, a BMP type 1 receptor inhibitor, and retinoic acid to produce a population of immature pancreatic islet cells; and (b) culturing the population of immature pancreatic islet cells in a second culture medium that is different than the first culture medium to produce a population of hypoimmune pancreatic islet cells.
[0267] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 2 ?M to about 10 ?M. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 1 ?M to about 10 ?M. In some embodiments, the first culture medium and/or second culture medium are absent of animal serum.
[0268] In some embodiments, the method also comprises isolating the population of CD16t, CD32t, or CD64t-expressing pancreatic islet cells from non-pancreatic islet cells. In some embodiments, the method further comprises cryopreserving the isolated population of hypoimmune pancreatic islet cells.
[0269] In some aspects, provided herein is an isolated, engineered hypoimmune retinal pigmented epithelium (RPE) cell differentiated from a CD16t, CD32t, or CD64t-expressing cell, wherein endogenous ?-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, CD47 or another SIRP?-Engager expression has been increased, the blood type is O and Rh?.
[0270] In some embodiments, the isolated hypoimmune RPE cell is selected from the group consisting of a RPE progenitor cell, immature RPE cell, mature RPE cell, and functional RPE cell.
[0271] In some aspects, provided herein is a method of treating a patient suffering from an ocular condition. The method comprises administering a composition comprising a therapeutically effective amount of a population of any one of a population of the isolated CD16t, CD32t, or CD64t-expressing RPE cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of the isolated hypoimmune RPE cells is on a biodegradable scaffold. In some embodiments, the administration comprises transplantation or injection to the patient's retina.
[0272] In some embodiments, the ocular condition is selected from the group consisting of wet macular degeneration, dry macular degeneration, juvenile macular degeneration, Leber's Congenital Ameurosis, retinitis pigmentosa, and retinal detachment.
[0273] In some aspects, provided herein is a method of producing a population of CD16t, CD32t, or CD64t-expressing retinal pigmented epithelium (RPE) cells from a population of cells by in vitro differentiation. In some embodiments, the endogenous ?-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated and CD47 or another SIRP?-Engager expression has been increased in the HIPO-cells. The method comprises: (a) culturing the population of HIPO? cells in a first culture medium comprising any one of the factors selected from the group consisting of activin A, bFGF, BMP4/7, DKK1, IGF1, noggin, a BMP inhibitor, an ALK inhibitor, a ROCK inhibitor, and a VEGFR inhibitor to produce a population of pre-RPE cells; and (b) culturing the population of pre-RPE cells in a second culture medium that is different than the first culture medium to produce a population of hypoimmune RPE cells.
[0274] In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 2 ?M to about 10 ?M. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some instances, the ROCK inhibitor is at a concentration ranging from about 1 ?M to about 10 ?M.
[0275] In some embodiments, the first culture medium and/or second culture medium lack animal serum.
[0276] In some embodiments, the method further comprises isolating the population of hypoimmune RPE cells from non-RPE cells. In some embodiments, the method further comprises cryopreserving the isolated population of hypoimmune RPE cells.
[0277] In one aspect, human pluripotent stem cells (PSCs) resist ADCC or CDC by CD16t, CD32t, or CD64t-expressing. In some embodiments, they are hypoimmune pluripotent stem cells (hiPSC). They are rendered hypo-immunogenic by a) the disruption of the B2M gene at each allele (e.g. B2M ?/?), b) the disruption of the CIITA gene at each allele (e.g. CIITA ?/?), and c) by the overexpression of the CD47 gene (CD47+, e.g. through introducing one or more additional copies of the CD47 gene or activating the genomic gene).
[0278] This renders the hiPSC population B2M?/? CIITA ?/? CD47tg. In a preferred aspect, the cells are non-immunogenic. In another embodiment, the HIP cells are rendered non-immunogenic B2M?/? CIITA ?/? CD47tg as described above but are further modified by including an inducible suicide gene that is induced to kill the cells in vivo when required. In other aspects, CD16, CD32, or CD64-overexpressing HIPO cells are created when HIP cells are rendered blood type O by by knocking out the ABO gene Exon 7 or silencing the SLC14A1 (JK) gene and the cells are rendered Rh-by knocking out the C and E antigens of the Rh blood group system (RH), K in the Kell system (KEL), Fya and Fy3 in the Duffy system (FY), Jkb in the Kidd system (JK), or U and S in the MNS blood group system.
F. Maintenance of HIPO?/CD16t, CD32t, or CD64t Cells
[0279] Once generated, the HIPO-CD16t, CD32t, or CD64t cells can be maintained in an undifferentiated state as is known for maintaining iPSCs. For example, HIP cells are cultured on Matrigel using culture media that prevents differentiation and maintains pluripotency.
G. Differentiation of HIPO?/CD16t, CD32t, or CD64t Cells
[0280] The invention provides HIPO?/CD16t, CD32t, or CD64t cells that are differentiated into different cell types for subsequent transplantation into subjects. As will be appreciated by those in the art, the methods for differentiation depend on the desired cell type using known techniques. The cells are differentiated in suspension and then put into a gel matrix form, such as matrigel, gelatin, or fibrin/thrombin forms to facilitate cell survival. Differentiation is assayed as is known in the art, generally by evaluating the presence of cell-specific markers.
[0281] In some embodiments, the HIPO?/CD16t, CD32t, or CD64t cells are differentiated into hepatocytes to address loss of the hepatocyte functioning or cirrhosis of the liver. There are a number of techniques that can be used to differentiate HIPO? cells into hepatocytes; see for example Pettinato et al., doi:10.1038/spre32888, Snykers et al., Methods Mol Biol 698:305-314 (2011), Si-Tayeb et al, Hepatology 51:297-305 (2010) and Asgari et al., Stem Cell Rev (:493-504 (2013), all of which are hereby expressly incorporated by reference in their entirety and specifically for the methodologies and reagents for differentiation. Differentiation is assayed as is known in the art, generally by evaluating the presence of hepatocyte associated and/or specific markers, including, but not limited to, albumin, alpha fetoprotein, and fibrinogen. Differentiation can also be measured functionally, such as the metabolization of ammonia, LDL storage and uptake, ICG uptake and release and glycogen storage.
[0282] In some embodiments, the HIPO?/CD16t, CD32t, or CD64t cells are differentiated into beta-like cells or islet organoids for transplantation to address type I diabetes mellitus (T1DM). Cell systems are a promising way to address T1DM, see, e.g., Ellis et al., doi/10.1038/nrgastro.2017.93, incorporated herein by reference. Additionally, Pagliuca et al. reports on the successful differentiation of 0-cells from hiPSCs (see doi/10.106/j.cell.2014.09.040, hereby incorporated by reference in its entirety and in particular for the methods and reagents outlined there for the large-scale production of functional human 0 cells from human pluripotent stem cells). Furthermore, Vegas et al. shows the production of human R cells from human pluripotent stem cells followed by encapsulation to avoid immune rejection by the host; (doi:10.1038/nm.4030, hereby incorporated by reference in its entirety and in particular for the methods and reagents outlined there for the large-scale production of functional human R cells from human pluripotent stem cells).
[0283] Differentiation is assayed as is known in the art, generally by evaluating the presence of R cell associated or specific markers, including but not limited to, insulin.
[0284] Differentiation can also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al, doi:10.1016/j.cels.2016.09.002, hereby incorporated by reference in its entirety, and specifically for the biomarkers outlined there.
[0285] Once the dHIPO?/CD16t, CD32t, or CD64t beta cells are generated, they can be transplanted (either as a cell suspension or within a gel matrix as discussed herein) into the portal vein/liver, the omentum, the gastrointestinal mucosa, the bone marrow, a muscle, or subcutaneous pouches.
[0286] In some embodiments, the HIPO?/CD16t, CD32t, or CD64t cells are differentiated into retinal pigment epithelium (RPE) to address sight-threatening diseases of the eye. Human pluripotent stem cells have been differentiated into RPE cells using the techniques outlined in Kamao et al., Stem Cell Reports 2014:2:205-18, hereby incorporated by reference in its entirety and in particular for the methods and reagents outlined there for the differentiation techniques and reagents; see also Mandai et al., doi:10.1056/NEJMoa1608368, also incorporated in its entirety for techniques for generating sheets of RPE cells and transplantation into patients.
[0287] Differentiation can be assayed as is known in the art, generally by evaluating the presence of RPE associated and/or specific markers or by measuring functionally. See for example Kamao et al., doi:10.1016/j.stemcr.2013.12.007, hereby incorporated by reference in its entirety and specifically for the markers outlined in the first paragraph of the results section.
[0288] In some embodiments, the HIPO?/CD16t, CD32t, or CD64t cells are differentiated into cardiomyocytes to address cardiovascular diseases. Techniques are known in the art for the differentiation of hiPSCs to cardiomyoctes and discussed in the Examples. Differentiation can be assayed as is known in the art, generally by evaluating the presence of cardiomyocyte associated or specific markers or by measuring functionally; see for example Loh et al., doi:10.1016/j.cell.2016.06.001, hereby incorporated by reference in its entirety and specifically for the methods of differentiating stem cells including cardiomyocytes.
[0289] In some embodiments, the HIPO?/CD16t, CD32t, or CD64t cells are differentiated into endothelial colony forming cells (ECFCs) to form new blood vessels to address peripheral arterial disease. Techniques to differentiate endothelial cells are known.
[0290] See, e.g., Prasain et al., doi:10.1038/nbt.3048, incorporated by reference in its entirety and specifically for the methods and reagents for the generation of endothelial cells from human pluripotent stem cells, and also for transplantation techniques. Differentiation can be assayed as is known in the art, generally by evaluating the presence of endothelial cell associated or specific markers or by measuring functionally.
[0291] In some embodiments, the HIPO?/CD16t, CD32t, or CD64t cells are differentiated into thyroid progenitor cells and thyroid follicular organoids that can secrete thyroid hormones to address autoimmune thyroiditis. Techniques to differentiate thyroid cells are known the art. See, e.g. Kurmann et al., doi:10.106/j.stem.2015.09.004, hereby expressly incorporated by reference in its entirety and specifically for the methods and reagents for the generation of thyroid cells from human pluripotent stem cells, and also for transplantation techniques. Differentiation can be assayed as is known in the art, generally by evaluating the presence of thyroid cell associated or specific markers or by measuring functionally.
H. Transplantation of Differentiated HIPO?/CD16t, CD32t, or CD64t Cells
[0292] As will be appreciated by those in the art, the differentiated HIPO?/CD16t, CD32t, or CD64t derivatives are transplated using techniques known in the art that depend on both the cell type and the ultimate use of these cells. In general, the cells of the invention are transplanted either intravenously or by injection at particular locations in the patient. When transplanted at particular locations, the cells may be suspended in a gel matrix to prevent dispersion while they take hold.
[0293] In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
VII. EXAMPLES
[0294] HIP and HIPO? cells are generated as disclosed in WO2018/132783, PCT/US19/42123, PCT/US19/42117, and Prov. Appl. Nos. 62/698,973, 62/698,978, 62/698,981, 62/698,984, 62/846,399, and 62/855,499. Protection from NK cell and ADCC killing when CD64 is overexpressed on pluripotent cells is disclosed in PCT/US20/55120.
[0295] Each of the foregoing are incorporated herein by reference in their entirety.
Example 1: CD64t expression sequesters antibodies by the F.SUB.c .Domain
[0296]
Example 2: HIP iECs expressing CD64t are protected from ADCC and CDC
[0297] HIP iECs do not express CD64 (
[0298] HIP iECs were transduced to express CD64 (
[0299] To avoid intracellular signaling, HIP iECs were transduced to express intracellularly truncated CD64 (CD64t) (
[0300] HIP iECs and HIP iECs.sup.CD64t were then transduced to additionally express CD52, the target for the highly cytotoxic IgG1 antibody alemtuzumab. Human primary NK cells were purchased from Stemcell Technologies (70036, Vancouver, Canada) and were cultured in RPMI-1640 plus 10% FCS hi and 1% pen/strep before performing the assays. In CDC assays, target cells were incubated with fresh ABO-compatible human serum.
[0301] Real-time killing assays were performed on the XCelligence SP platform and MP platform (ACEA Biosciences, San Diego, CA.). Special 96-well E-plates (ACEA Biosciences) coated with collagen (Sigma-Aldrich) were used. A total of 4?10.sup.4 human iECs, epiCs, beta cells, or CAR-T cells were plated in 100 ?l medium. After the cell index reached 0.7, 4?10.sup.4 human NK cells was added to ADCC assays or 50 ?l blood-type compatible human serum to CDC assays. Patient serum samples were treated with DTT to eliminate blood type IgM antibodies before use.
[0302] For Nalm6 killing assays, 4?10.sup.4 Nalm6 cells were plated and 4?10.sup.4 CAR-T cells were used as effector cells. The following antibodies were used and added after mixing with the NK cells in 50 ?l medium or in the serum as indicated: humanized anti-CD52 IgG1 (alemtuzumab, ichorbio, catalog no. ICH4002), humanized anti-HLA-A2 IgG1 (clone 3PF12, Absolute Antibody, catalog no. AB00947-10.0), humanized anti-Rh(D) IgG1 (clone F5, Creative Biolabs, catalog no. FAMAB-0089WJ), humanized anti-TPO IgG1 (clone B8, Creative Biolabs, catalog no. FAMAB-0014JF), humanized anti-CD3 IgG1 (Creative Biolabs, custom product), humanized anti-CD19 scFv (FMC63) IgG1 (clone 136.20.1, Creative Biolabs, catalog no. HPAB-0440-YJ-m/h). Different concentrations ranging from 0.0001 ?g/ml to 1 ?g/ml were used. As a negative control, cells were treated with 2% Triton X-100 in medium (data not shown). Data were standardized and analyzed with the RTCA software (ACEA).
[0303] In ADCC and CDC killing assays, HIP iECs.sup.CD52 were killed by NK cells (FIG. 9) or complement (
Example 3: Engineered human thyroid epithelial cells evade antibody-mediated autoimmune killing
[0304] Hashimoto's thyroiditis is a prototypic disease in which cytotoxic autoantibodies lead to the destruction of thyroid tissue. Anti-TPO antibodies, primarily of IgG1 subclass, are present at high concentrations in 90% of patients (Rapoport, B. & McLachlan, S. M., Thyroid autoimmunity. J Clin Invest 108, 1253-1259 (2001); Doullay, F. et al., Autoimmunity 9, 237-244 (1991)). They mediate both ADCC (Bogner, U. et al., J Cin EndocrinolMetab 59, 734-738 (1984); Stathatos, N. & Daniels, G. H., Autoimmune thyroid disease. Curr Opin Rheumatol 24, 70-75 (2012); Stassi, G. & De Maria, R. Autoimmune thyroid disease: new models of cell death in autoimmunity. Nat Rev Immunol 2, 195-204 (2002)) and CDC (Rebuffat, S. et al., J Cin Endocrinol Metab 93, 929-934 (2008); Chiovato, L. et al., J Clin Endocrinol Metab 77, 1700-1705 (1993)). This diminishes the function of the thyroid gland (Pearce, E. N., et al., NEngl J Med 348, 2646-2655 (2003)). The invention provides thyroid epithelial cells (epiCs) resistant to TPO antibodies for surviving innate immune responses and re-establishing organ function when transplanted into hypothyroid patients.
[0305] Immortalized human thyroid epithelial cells (epiCs, Inscreenex, Braunschweig, Germany, Catalog #INS-CI-1017) and primary human islet cells (Takara, Mountain View, CA, Catalog #Y10106) were used. Since the epiCs did not show a physiologic response to thyroid stimulating hormone (TSH) in vitro, their thyroid peroxidase (TPO) levels were well-below physiologic expression levels. Thus, a lentiviral transduction approach was used to artificially increase their TPO expression. One hundred thousand human thyroid epiCs were plated on gelatin-coated six-well plates and incubated overnight at 37? C. at 5% CO.sub.2. The next day, media was changed and 200 ?l of lentiviral particles (at 1?10.sup.7 IFU/ml) carrying the transgene for human TPO reengineered EF1a promotor (GenTarget) was added to 1.5 ml media. After 36 h, 1 ml of cell media was added.
[0306] These epiCs.sup.TPO showed good TPO expression but did not express CD64 (
[0307] Thyroxine production was measured by ELISA. A 96 well plate was coated with gelatin and 3?10.sup.4 human thyroid epiCs.sup.TPO or epiCs.sup.TPO,CD64t per well were seeded in 100 ?l h7H media and incubated for 24 hours at 370 C in 5% CO.sub.2. The next day, the h7H media was changed and supplemented with 1 mU/mL native bovine thyroid stimulating hormone protein (TSH, catalog no. TSH-1315B, Creative Biomart, Shirley, NY). Three wells per epiC group were also supplemented with 1 ?g/mL anti-CD52 IgG1 (alemtuzumab, clone Campath-1H, Biorad). After 72 hours, the supernatant was collected and the level of thyroxine was assessed using the thyroxine (T4) competitive ELISA kit (catalog no. EIAT4C, Invitrogen) according to manufacturer's instructions. Results are presented as change in optical density (OD) between groups with and without alemtuzumab. Both thyroid epiCs.sup.TPO and epiCs.sup.TPO,CD64t produced thyroxine irrespective of the presence of IgG1 antibodies (
[0308] epiCs (TPO) with and without CD64t expression were plated on the XCelligence platform for in vitro impedance assays (
[0309] Although rabbit IgG F.sub.c binds human CD64 and CD64t, we next used a humanized anti-TPO IgG1 antibody. This human IgG1 antibody was effective in killing thyroid epiCs.sup.TPO in NK cell ADCC assays (
[0310] Serum samples from three patients with Hashimoto's thyroiditis and anti-TPO antibody titers 21-fold, 26.3-fold, and 29.6-fold the upper level of normal (0.03 U/ml) rapidly killed thyroid epiCs.sup.TPO in CDC assays (
[0311] NSG (NOD.Cg-Prkdc.sup.scidI12rg.sup.tm1WjI/SzJ, 005557) mice 6-12 weeks old were purchased from the Jackson Laboratories (Sacramento, CA). The number of animals used in the examples is presented in each figure. NSG recipients were subcutaneously injected with 5?10.sup.4 epiCs.sup.TPO or epiCs.sup.TPO,CD64t one million human NK cells, and 1 mg doses of anti-TPO on days 0, 1, and 2 (
Example 4: Engineered human beta cells evade HLA antibody-mediated killing
[0312] Type 1 diabetes mellitus (T1DM) is another autoimmune disease, which is T cell-mediated with an accompanying antibody response. We aimed to test whether CD64t expression can make them resistant against HLA antibody killing. Human iPSC-derived pancreatic beta cells were purchased from TaKaRa (ChiPSC22, catalog no. Y10106) and were cultured in Cellartis hiPS Beta Cell Media Kit (TaKaRa, catalog no. Y10108). Cells were plated in 12-well plates according to the manufacturer protocol. Some cells were transduced with CD64t lentiviral particles (GenTarget). Human iPSC-derived pancreatic islet cells (beta cells) do not express CD64 (
[0313] Insulin production was measured by ELISA. A 24 well plate was coated with gelatin and 5?10.sup.4 iPSC-derived beta cells and beta cells.sup.CD64t cells (catalog no. Y10108, Takara Bio, San Jose, CA) per well were seeded in 500 ?l Cellartis hiPS beta cell media and incubated for 24 hours at 370 C in 5% CO.sub.2. The next day, the Cellartis hiPS beta cell media was changed to RPMI 1640 without glucose (catalog no. 11879-020, Gibco) for 2 hours. After 2 hours the media was changed to RPMI without glucose supplemented with 2 mM glucose (catalog no. G7528, Sigma Aldrich). Three wells per beta cell group were also supplemented with 1 ?g/mL anti-CD52 IgG1 (alemtuzumab, clone Campath-1H, catalog no. MCA6101, Biorad). After 20 minutes, the supernatant was collected and the media was changed to RPMI without glucose supplemented with 20 mM glucose. Again, 1 ?g/mL Alemtuzumab was added to 3 wells per group. After 20 minutes, the supernatant was collected and the level of human insulin was determined using the human insulin ELISA kit (catalog no. KAQ1251, Invitrogen) according to manufacturer's instructions. Results are presented as change in optical density (OD) between groups with and without alemtuzumab. Both beta cells (
[0314] Islet cells (pancreatic beta cells) with and without CD64t expression were then plated on the XCelligence platform (
[0315] HLA-A2 expressing beta cells were killed increasingly quickly with increasing anti-HLA-A2 IgG1 antibody concentrations in CDC assays (
[0316] We then subcutaneously injected NSG mice with 5?10.sup.4 human beta cells or beta cells.sup.CD64t together with one million human NK cells (
Example 5: Engineered human CAR-T cells evade HLA-, non-HLA-, and CAR-directed antibody killing
[0317] Clinical CAR-T cell therapy induces an antibody response, which is even more pronounced in patients with solid tumors. Antibody protection was thus engineered into CAR-T cells. Human T cells were transduced to express a CD19 scFv-4-1BB-CD3? construct with or without additional CD64t expression. Human anti-CD19 CAR-T cells were generated from human PBMCs using lentiviral particles carrying a transgene for the CD19 scFv-4-1BB-CD3? construct (ProMab, catalog no. PM-CAR1002-V). PBMCs were stimulated with IL-2 overnight and seeded in 96-well U-bottom plates at a density of 10.sup.5 cells per well containing protamine sulfate and 1 ?g/ml IL-2 (Peprotech). Lentiviruses were added to the wells at a MOI of 20. Some wells were transduced additionally with CD64t lentiviral particles at a MOI of 20 (GenTarget). Spinfection was carried out at 1800 rpm for 30 min at 25? C. After that, the cells were returned to a humidified 5% CO.sub.2 incubator overnight. The medium was changed after 2 days and cells were seeded at a density of 10.sup.6 per ml in T cell media (OpTmizer, Thermo Fisher Scientific). CD3/CD28 beads (Thermo Fisher Scientific) were used for T-cell expansion. Cells were sorted for CAR.sup.+ and CAR.sup.+/C64t.sup.+ populations on BD Aria Fusion and used for further assays.
[0318] CAR-T cells showed marked expression of the CAR receptor (anti-CD19 scFv) but did not express CD64t (
[0319] While CAR-T cells did not bind any free IgG1 (using an anti-TPO antibody,
[0320] The killing efficacy of CAR-T, CAR-T.sup.CD64t, and regular T cells was assessed against CD19-positive NALM target cells (
[0321] ADCC (
Example 6: Engineered human NK cells evade ADCC and CDC killing
[0322] To show that antibody protection can be engineered into NK cells, human NK cells were transduced to express CD64t. Peripheral human NK cells do not express CD64t (
[0323] While NK cells did not bind any free IgG1 (using an anti-TPO antibody,
[0324] ADCC (
VIII. Exemplary sequences
[0325]
TABLE-US-00001 Human?-2-Microglobulin SEQIDNO:1 MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNELNC YVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEF TPTEKDEYACRVNHVTLSQPKIVKWDRDI HumanCIITAprotein,160aminoacidN-terminus SEQIDNO:2 MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPL CLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEE TREAYANIAELDQYVFQDSQLEGLSKDIFKHIGPDEVIGESMEMP AEVGQKSQKRPFPEELPADLKHWKP HumanCD47 SEQIDNO:3 MWPLVAALLLGSACCGSAQLLENKTKSVEFTFCNDTVVIPCFVTN MEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDESSAKIEVSQ LLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVS WESPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALL VAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHY YVESTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPL LISGLSILALAQLLGLVYMKFVE HerpesSimplexVirusThimidineKinase(HSV-tk) SEQIDNO:4 MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQK MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVT DAVLAPHVGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMG SMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGE RLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQ GAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVL AKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPT ICDLARTFAREMGEAN EscherichiacoliCytosineDeaminase(EC-CD) SEQIDNO:5 MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITEN SLDAEQGLVIPPEVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWA ERKALLTHDDVKQRAWQTLKWQIANGIQHVRTHVDVSDATLTALK AMLEVKQEVAPWIDLQIVAFPQEGILSYPNGEALLEEALRLGADV VGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQSR EVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSG INFVANPLVNIHLQGREDTYPKRRGITRVKEMLESGINVCFGHDD VFDPWYPLGTANMLQVLHMGLHVCQLMGYGQINDGLNLITHHSAR TLNLQDYGIAAGNSANLIILPAENGEDALRRQVPVRYSVRGGKVI ASTQPAQTTVYLEQPEAIDYKR TruncatedhumanCaspase9 SEQIDNO:6 GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRT RTGSNIDCEKLRRRESSLHFMVEVKGDLTAKKMVLALLELAQQDH GALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNG TSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEP DATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKS GSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCF NFLRKKLFFKTS HumanCD64 NM_000566 SEQIDNO:7 MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEV LHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLS GRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNV LYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAG ISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQL YFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVL KRSPELELQVLGLQLPTPVWFHVLFYLAVGIMFLVNTVLWVTIRK ELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQ LQEGVHRKEPQGAT HumanCD52 NM_001803 SEQIDNO:8 MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSSMSGGI FLFFVANAIIHLFCES HumanCD16FCGR3A NP_000560.6 SEQIDNO:9 MAEGTLWQILCVSSDAQPQTFEGVKGADPPTLPPGSFLPGPVLWW GSLARLQTEKSDEVSRKGNWWVTEMGGGAGERLFTSSCLVGLVPL GLRISLVTCPLQCGIMWQLLLPTALLLLVSAGMRTEDLPKAVVFL EPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYF IDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVEKE EDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLK DSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVS FCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK HumanCD16FCGR3B NP_001231682.2 SEQIDNO:10 MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYSVLEKDSVTL KCQGAYSPEDNSTQWFHNENLISSQASSYFIDAATVNDSGEYRCQ TNLSTLSDPVQLEVHIGWLLLQAPRWVEKEEDPIHLRCHSWKNTA LHKVTYLQNGKDRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKN VSSETVNITITQGLAVSTISSFSPPGYQVSFCLVMVLLFAVDTGL YFSVKTNI HumanCD32FCGR2A >NP_001129691.1 SEQIDNO:11 MTMETQMSQNVCPRNLWLLQPLTVLLLLASADSQAAAPPKAVLKL EPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSY RFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQE GETIMLRCHSWKDKPLVKVTFFQNGKSQKFSHLDPTESIPQANHS HSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGIIVAVVIA TAVAAIVAAVVALIYCRKKRISANSTDPVKAAQFEPPGRQMIAIR KRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNS NN HumanCD32FCGR2B >NP_003992.3 SEQIDNO:12 MGILSFLPVLATESDWADCKSPQPWGHMLLWTAVLFLAPVAGTPA APPKAVLKLEPQWINVLQEDSVTLTCRGTHSPESDSIQWFHNGNL IPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVL QTPHLEFQEGETIVLRCHSWKDKPLVKVTFFQNGKSKKFSRSDPN ESIPQANHSHSGDYHCTGNIGYTLYSSKPVTITVQAPSSSPMGII VAVVTGIAVAAIVAAVVALIYCRKKRISALPGYPECREMGETLPE KPANPTNPDEADKVGAENTITYSLLMHPDALEEPDDQNRI HumanCD32FCGR2C >NP_963857.3 SEQIDNO:13 MGILSFLPVLATESDWADCKSPQPWGHMLLWTAVLFLAPVAGTPA APPKAVLKLEPQWINVLQEDSVTLTCRGTHSPESDSIPWFHNGNL IPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVL QTPHLEFQEGETIVLRCHSWKDKPLVKVTFFQNGKSKKFSRSDPN ESIPQANHSHSGDYHCTGNIGYTLYSSKPVTITVQAPSSSPMGII VAVVTGIAVAAIVAAVVALIYCRKKRISANSTDPVKAAQFEPPGR QMIAIRKRQPEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPP NDHVNSNN TruncatedhumanCD16 SEQIDNO:14 MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTL KCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQ TNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTA LHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKN VSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGL YFSV TruncatedhumanCD32 SEQIDNO:15 MTMETQMSQNVCPRNLWLLQPLTVLLLLASADSQAAAPPKAVLKL EPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSY RFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQE GETIMLRCHSWKDKPLVKVTFFQNGKSQKFSHLDPTFSIPQANHS HSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGIIVAVVIA TAVAAIVAAVVALIY TruncatedhumanCD64 SEQIDNO:16 MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEV LHLPGSSSTQWELNGTATQTSTPSYRITSASVNDSGEYRCQRGLS GRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNV LYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAG ISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQL YFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVL KRSPELELQVLGLQLPTPVWFHVLFYLAVGIMFLVNTVLWVTI TfR1cytoplasmicdomain(positions1-67) SEQIDNO:17 MMDQARSAFSNLEGGEPLSYTRESLARQVDGDNSHVEMKLAVDEE ENADNNTKANVTKPKRCSGSIC TfR1transmembranedomain(positions68-88) SEQIDNO:18 YGTIAVIVFFLIGFMIGYLGY TfR1extracellulardomain(positions89-760) SEQIDNO:19 CKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSE KLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKL SKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYS KAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKV ANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGF PSENHTQFPPSRSSGLPNIPVQTISRAAAEKLEGNMEGDCPSDWK TDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYV VVGAQRDAWGPGAAKSGVGTALLLKLAQMESDMVLKDGFQPSRSI IFASWSAGDEGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSN FKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLD NAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPEL NKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQY RADIKEMGLSLQWLYSARGDFFRATSRLTTDEGNAEKTDRFVMKK LNDRVMRVEYHELSPYVSPKESPFRHVFWGSGSHTLPALLENLKL RKQNNGAFNETLERNQLALATWTIQGAANALSGDVWDIDNEF TfR2cytoplasmicdomain(positions1-83) SEQIDNO:20 MERLWGLFQRAQQLSPRSSQTVYQRVEGPRKGHLEEEEEDGEEGA ETLAHFCPMELRGPEPLGSRPRQPNLIPWAAAGRRAAP TfR2transmembranedomain(positions84-104) SEQIDNO:21 YLVLTALLIFTGAFLLGYVAF TfR2extracellulardomain(positions105-801) SEQIDNO:22 RGSCQACGDSVLVVSEDVNYEPDLDFHQGRLYWSDLQAMFLQFLG EGRLEDTIRQTSLRERVAGSAGMAALTQDIRAALSRQKLDHVWTD THYVGLQFPDPAHPNTLHWVDEAGKVGEQLPLEDPDVYCPYSAIG NVTGELVYAHYGRPEDLQDLRARGVDPVGRLLLVRVGVISFAQKV TNAQDFGAQGVLIYPEPADESQDPPKPSLSSQQAVYGHVHLGTGD PYTPGFPSFNQTQFPPVASSGLPSIPAQPISADIASRLLRKLKGP VAPQEWQGSLLGSPYHLGPGPRLRLVVNNHRTSTPINNIFGCIEG RSEPDHYVVIGAQRDAWGPGAAKSAVGTAILLELVRTFSSMVSNG FRPRRSLLFISWDGGDFGSVGSTEWLEGYLSVLHLKAVVYVSLDN AVLGDDKFHAKTSPLLTSLIESVLKQVDSPNHSGQTLYEQVVFTN PSWDAEVIRPLPMDSSAYSFTAFVGVPAVEFSFMEDDQAYPFLHT KEDTYENLHKVLQGRLPAVAQAVAQLAGQLLIRLSHDRLLPLDFG RYGDVVLRHIGNLNEFSGDLKARGLTLQWVYSARGDYIRAAEKLR QEIYSSEERDERLTRMYNVRIMRVEFYFLSQYVSPADSPFRHIFM GRGDHTLGALLDHLRLLRSNSSGTPGATSSTGFQESRFRRQLALL TWTLQGAANALSGDVWNIDNNE
[0326] All publications and patent documents disclosed or referred to herein are incorporated by reference in their entirety. The foregoing description has been presented only for purposes of illustration and description. This description is not intended to limit the invention to the precise form disclosed. It is intended that the scope of the invention be defined by the claims appended hereto.