US-12622932-B2 - Immunomodulatory cells and uses thereof

Abstract

Featured are cells and methods of use thereof for modulating an antigen-specific immune response in a subject. The cells comprise a set of transgenes comprising two or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASG or FasL, Ccl21 or Ccl21b, MfgeS and Serpin B9 or Spi6, that shield the cells from immune surveillance (ie. “cloaking genes”). The cells can be used to induce immune tolerance to an antigen (e.g., a donor alloantigen or a self-antigen), or to induce an immune response to (e.g., induce the production of antibodies directed against) a non-self antigen.

Inventors

• Andras Nagy
• Jeffrey Harding
• Kristina NAGY

Assignees

• SINAI HEALTH SYSTEM

Dates

Publication Date

20260512

Application Date

20191213

Claims (17)

1. 1 . A eukaryotic cell genetically modified to comprise i) a set of transgenes comprising the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6; and ii) a transgene encoding a polypeptide comprising a self-antigen associated with an autoimmune disease.
2. 2 . The eukaryotic cell of claim 1 , further comprising one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39 or a gene encoding a biologic that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.
3. 3 . The eukaryotic cell of claim 2 , wherein: (a) the TGF-β or biologic is local acting in the graft environment; or (b) the IFNγR1 d39 transgene encodes a protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 17.
4. 4 . The eukaryotic cell of claim 1 , wherein: (a) the transgene encoding the polypeptide comprising the self-antigen is incorporated into the genome of the cell at a non-endogenous locus; (b) one or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 is expressed at a level that is greater than the expression level of the corresponding endogenous gene in the cell or that is equal to or greater than the expression level of the corresponding endogenous gene in an activated leukocyte; (c) one or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 is expressed at a level that is in the top 5% of gene expression for all genes in the genome of the eukaryotic cell; (d) the transgenes are operably linked to a constitutive promoter; (e) the transgene encoding the polypeptide comprising the self-antigen is operably linked to a constitutive promoter; or (f) the polypeptide comprising the self-antigen is expressed using an inducible expression system selected from the group consisting of a tetracycline response element, a light inducible system, a radiogenetic system, a cumate switch inducible system, an ecdysone inducible system, a destabilization domain system, or a ligand-reversible dimerization system.
5. 5 . The eukaryotic cell of claim 4 , wherein: (a) all eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 are expressed at a level that is greater than the expression level of the corresponding endogenous gene in the cell or that is equal to or greater than the expression level of the corresponding endogenous gene in an activated leukocyte; (b) all eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 are expressed at a level that is in the top 5% of gene expression for all genes in the genome of the cell; or (c) the constitutive promoter is selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1a promoter, the PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of moloney virus, Epstein barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter.
6. 6 . The eukaryotic cell of claim 1 , wherein: (a) the PD-L1 transgene encodes a protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; (b) the HLA-G or H2-M3 transgene encodes a protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 15; (c) the Cd47 transgene encodes a protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; (d) the CD200 transgene encodes a protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6; (e) the FASLG or FasL transgene encodes a protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 9; (f) the Ccl21 or Ccl21b transgene encodes a protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1; (g) the Mfge8 transgene encodes a protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14; and (h) the Serpin B9 or Spi6 transgene encodes a protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 7.
7. 7 . A population of genetically modified cells, wherein each of the cells is a eukaryotic cell of claim 1 .
8. 8 . A composition comprising the population of genetically modified cells of claim 7 .
9. 9 . A composition comprising the eukaryotic cell of claim 1 .
10. 10 . The composition of claim 9 , further comprising a pharmaceutically acceptable excipient.
11. 11 . A kit comprising the composition of claim 9 .
12. 12 . The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a stem cell.
13. 13 . The eukaryotic cell of claim 12 , wherein the stem cell is an embryonic stem cell.
14. 14 . The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a kidney cell, an oligodendrocyte, or a pancreatic beta cell.
15. 15 . The eukaryotic cell of claim 1 , wherein the self-antigen comprises a myelin oligodendrocyte glycoprotein (MOG), insulin, or glutamic acid decarboxylase 65 (GAD65).
16. 16 . The eukaryotic cell of claim 1 , wherein the self-antigen comprises 14-3-3ε, 14-3-3ζ, 14-3-3ζ, 14-3-3η, 17 alpha-Hydroxylase, 21-hydroxylase, 21β-4-hydroxylase, 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), trimethylaminobutyraldehyde dehydrogenase, 5′-nucleotidase cytosolic I, 68-kD inner ear protein, actin, ADAM Metallopeptidase with Thrombospondin Type 1 Motif 13 (ADAMTS13), adenine nucleotide translocator, Alpha 3 chain of type IV collagen, Alpha (2)-HSG, alpha-3 chain of type IV collagen, alpha-4 chain of type IV collagen, alpha-5 chain of type IV collagen, Aminoacyl-tRNA synthetase, Aminoacylase 1 (ACY1), AMPA receptors, amphiphysin, amylase-2α, amylase-alpha-2α lactoferrin, amyloid-β, ANNA-1, annexin 5, Annexin A8 Like 1 (ANXA8L1), aquaporin-4, arrestin, Asialoglycoprotein, asialoglycoprotein receptor, AT-Rich Interaction Domain 4B (ARID4B), ATP synthase β chain, ATPase Secretory Pathway Ca2+ Transporting 1 (ATP2C1), B-Cell activating factor, bactericidal permeability increasing (BPI) protein, Basic Leucine Zipper Nuclear Factor 1 (BLZF1), beta actin, beta-2-glycoprotein-1, Bip, BP180, Bruton's tyrosine kinase, bullous pemphigoid antigen 1, Bullous pemphigoid antigen 180 (BP180), bullous pemphigoid antigen 2, bullous pemphigoid antigen 230 (BP230), C1 inhibitor (C1-INH), C1g, C2orf34, C3 convertase, Ca2+/calmodulin-dependent protein kinase II (CamKII), Cadherin 8 (CDH8), Cadherin 9 (CDH9), Calcium/Calmodulin Dependent Protein Kinase IV (CAMK4), calpastatin, calreticulin, carbonic anhydrase, carbonic anhydrase I, Carbonic anhydrase II, carbonic anhydrase IV, carbonic anhydrase-II, carbonic anhydrase-IV, cardiac myosin, cardiolipin, caspase-3, CASPR1, Caspr2, cathepsin G, Cbir1, CD1b Molecule (CD1B), CD33 molecule (CD33), cell-density enhanced protein tyrosine phosphatase-1, CENP-A, CENP-B, CENP-C, CENP-O, Cholinergic Receptor Nicotinic Delta Subunit (CHRND), Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE), chondromodulin 1, CK10, claudin-1, cluster of differentiation 88 (CD88), cochlin, cold agglutinins, collagen, collagen I, collagen II, collagen III, collagen IV, collagen IX, Collagen Type XXI Alpha 1 Chain (COL21A1), collagen V, collagen VII, collagen XI, complement factor B, complement factor H, connective tissue growth factor, connexion, contactin, Contactin 1 (CNTN1), contactin-1, cornea-associated antigen, CTL2, Cyclin Dependent Kinase Inhibitor 1B (CDKN1B), Cyclin Dependent Kinase Like 1 (CDKL1), Cytochrome B5 Type B (CYB5B), Cytochrome P450 Family 1 Subfamily A Member 2 (CYP1A2), Cytochrome P450 Family 2 Subfamily D Member 6 (CYP2D6), Cytochrome P450 Family 2 Subfamily E Member 1 (CYP2E1), Cytochrome P450 Family 3 Subfamily A Member 4 (CYP3A4), cytokeratin 10, cytokeratin 16, Cytokine Induced Apoptosis Inhibitor 1 (CIAPIN1), Cytosolic 5′-nucleotidase 1A, D2 receptors, DEAD-Box Helicase 17 (DDX17), Delta/Notch Like EGF Repeat Containing (DNER), desmocollin, Desmocollin 1 (DSC1), Desmocollin 3 (DSC3), desmoglein 1, desmoglein 3, DNA topoisomerase II, DNER, Dopamine D2 receptor, DPPX, E (HLA-E), E2 subunit of pyruvate dehydrogenase, ECGF, elastase, Endophilin B1 (SH3GLB1), Enolase, enolase a, Ephrin A3 (EFNA3), epidermal transglutaminase, ER60, erythropoietin, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2), extracellular matrix protein 1, factor VIII, Fc Fragment Of IgG Receptor IIa (CD32), FcγRIIIb, FcεRIa of FCER1, ferritin, FH, fibrillin, fibrin, fibrinogen, filaggrin, FLII actin remodeling protein (FLII), formiminotransferase cyclodeaminase, FSH receptor, GA1, GABAA receptor associated protein, GABAA receptors, GABAB receptors, GAD65, ganglionic acetylcholine receptor (α3-nicotinic AChR), ganglioside GD1a, ganglioside GM1, ganglioside GM1b, Ganglioside GQ1b, ganglioside GT1a, GD1b, GDP Dissociation Inhibitor 2 (GDI2), gliadin, gliomedin, glucose-6-phosphate isomerase, glutamic acid decarboxylase, Glutamic acid decarboxylase (GAD), glutamic acid decarboxylase 65 (GAD65), Glutaminase 2 (GLS2), Gly receptors, glycine receptor a1, glycogen, glycophorin A, Glycoprotein IIb/IIIa (GPIIb/IIIa), glypican 3, glypican 4, GM1, GOR, GP120, GP1BA, gp50-64, gp70-95, GPIb/IX, GSG1 Like (GSG1L), H+/K (+)-ATPase, Heat Shock Protein Family A (Hsp70) Member 8 (HSPA8), helicase/histone deacetylase protein complex, high-mobility group (HMG)-1, histone H1, histone proteins, HLA, HLA-B27, HMG-2, HMGCR, hnRNP H1, Hsp10, Hsp60, Hsp65, Hsp70, Hsp70-2, Hu, Human leukocyte antigen B27 (HLA B27), I antigens of red blood cells, I2, ICQK, IF-R7, IgE, Immunoglobulin Heavy Constant Gamma 1 (IGHG1), insulin, insulinoma antigen 2 protein, Integrin Subunit Alpha L (CD11a), Integrin Subunit Alpha M (CD11b), interleukin 6 (IL-6), interphotoreceptor retinoid binding protein (IRBP), interphotoreceptor retinoid-binding protein, Jo-1, keratin, kinectin, Kpb antigen of Kell blood group system, Ku, Ky1.4 voltage-gated potassium channel, La, La/SSB, LABD97 (97 kDa fragment of bullous pemphigoid antigen 180), lactoferrin, LAD-1 (120 kDa fragment of bullous pemphigoid antigen 180), lamin A, lamin C, laminin, laminin 5, laminin 5 a3 subunit, laminin B receptor, laminin-5, laminin-6, LC1, LDL Receptor Related Protein 4 (LRP4), Leucine Rich Glioma Inactivated 1 (LGI1), LFA-1, LH receptor, LIM And Calponin Homology Domains 1 (LIMCH1), LKM-1, LKM-3, Lyn tyrosine kinase, M1 muscarinic AChR, M2, M2 muscarinic acetylcholine receptor, M2 muscarinic AChR, M3 muscarinic AChR, M4, M8, M9, MAGE Family Member A4 (MAGEA4), Major Histocompatibility Complex Class I, major peripheral myelin protein P0, major zymogen granule membrane glycoprotein 2, MART-1/Melan A, maternal antigen that embryos require (Mater/Nalp5), Matrilin 2 (MATN2), Matrix GIa protein, Matrix metalloproteinase 10 (MMP10), MDA5, Melanin associated antigen (MAA), melanocortin 4 receptor, melanoma differentiation antigen 5, Methyltransferase-like 22, Mg2+/Mn2+ Dependent 1A (PPM1A), mGluR1, Mi-2, Mi2, MJ, MLLT6, muscarinic acetylcholine receptor, Muscarinic Acetylcholine Receptor M3 (CHRM3), muscarinic M2 acetylcholine receptor, muscarinic M3 receptor, Muscle Associated Receptor Tyrosine Kinase (MuSK), mutant transthyretin (V30M), Myelin associated glycoprotein (MAG), Myelin oligodendrocyte glycoprotein, Myelin oligodendrocyte glycoprotein (MOG), myelin peroxidase zero, Myelin Protein Zero (MPZ), myeloperoxidase, Myocyte Enhancer Factor 2D (MEF2D), myofibrillar protein, myosin, NADH: Ubiquinone Oxidoreductase Core Subunit S1 (NDUFS1), NC1 domain of Alpha-3 chain of type IV collagen, NDP52, nebulin-related anchoring protein, Neural Cell Adhesion Molecule 1 (NCAM), Neural Cell Adhesion Molecule 2 (NCAM2), Neurofascin-186, Neurofascin-186 (NF186), neuronal cell adhesion molecule (NrCAM), NF140, NF155, NF186, nicotinic acetylcholine receptor, NIMA Related Kinase 7 (NEK7), NMDA receptors, Nuclear histone 1 of polymorphonuclear leukocytes, nuclear matrix protein 2, NXP-2, NXP2, Ompc, osteoglycin, osteonectin, Outer Dense Fiber of Sperm Tails 2 (ODF-2), P antigens of red blood cells, p155/140, p200, P2RX7, P450scc, p57, P62, pancreas secretory trypsin inhibitor, pancreatic secretory trypsin inhibitor, pancreatic trypsinogen, PCA-1, Peripheral Myelin Protein 2 (PMP2), Peripheral Myelin Protein 22 (PMP22), peroxiredoxin, PHD Finger Containing (MLLT6), phosphatidylserine, phosphatidylserine-prothrombin complex, phosphodiesterase, phosphoglycerate kinase 1, phospholipid cofactor, Plakophilin 3 (PKP3), plasminogen-binding protein, Pm/ScI, Pmel17/gp100, PML, PMP22, PNMA2 (Ma-2/Ta), Poly (ADPRibose) Polymerase Family Member 3 (PARP3), pro-opiomelanocortin-1 (POMC-1), progesterone, proteasome complex component 2 and zeta chain, Protein Kinase D1 (PRKD1), protein phosphatase, protein tyrosine phosphatase, Protein Tyrosine Phosphatase Non-Receptor Type 22 (PTPN22), proteinase 3, Pyruvate Dehydrogenase E1 Alpha 1 Subunit (PDHA1), RA33/hnRNP A2, Raf-1, rapsyn, recoverin, red blood cell antigens, Regulator Of G Protein Signaling 10 (RGS10), retinal arrestin, retinal soluble antigen, rheumatoid factor, rhodopsin, RNAP I, RNAP II, RNAP III, RNP, Ro, Ro/La, Ro/SSA, Ro52, Ryanodine receptor (RyR), Sa, SAE, SAGA-1, Scl-70, Scl70, Serpin family A member 5 (SERPINA5), serum andolase, SH3 Domain Containing GRB2 Like, side-chain cleavage enzyme, signal recognition particle, Sm, Sm/RNP, small ubiquitin-like modifier activating enzyme, SOD2, Solute Carrier Family 36 Member 4 (SLC36A4), SP100, SP140, SPARC Related Modular Calcium Binding 1 (SMOC1), SSA, SSb, sulfoglucuronosyl glycolipids, Superoxide dismutase 2 (SOD2), synapsin synthetase, Th/To, thyroglobulin, thyroid peroxidase, thyroid stimulating hormone, thyroperoxidase, thyrotropin receptor, TIF1-gamma, TIF1-γ, titin, transcriptional intermediary factor 1, transferrin, transglutaminase, Transglutaminase 1 (TGM1), transglutaminase 2, transglutaminase 3, transglutaminase 6, transthyretin, trichohyalin, Tropomyosin, tropomyosin 5, troponin, trypsin, trypsinogen, tubulin-α-1c, tubulointerstitial nephritis antigen, type II collagen, type IX collagen, Tyrosinase (Tyr), tyrosinase 1, tyrosinase 2, tyrosinase-related protein 1 (TRP1), tyrosinase-related protein 2, tyrosinase-related protein 2 (TRP2), tyrosine hydroxylase isoform B1, tyrosine hydroxylase isoform B2, U1 ribonucleoprotein (U1-RNP), U1-RNP, U1A, Ubiquitin Conjugating Enzyme E2 W (UBE2W), UDP-glucuronosyltransferases, UGA-suppressor-tRNA-associated protein, Vascular cell adhesion molecule 1 (VCAM-1), VGCC, vimentin, vinculin, Yo, zinc transporter 8, zonulin, α enolase, a (1) subunits of voltage gated calcium channels, α-Enolase, α-fodrin, a1-adrenaline receptor, α3 chain of type IV collagen, a6-integrin subunit, β(3) subunits of voltage gated calcium channels, β-glucuronidase, B1 adrenergic receptor, β1-adrendergic receptor, β2 glycoprotein 1 (β2-GPI), β2-glycoprotein I, β2-glycoprotein-I, B4-integrin, or β4-integrin subunit.
17. 17 . The eukaryotic cell of claim 1 , wherein the set of transgenes comprises: PD-L1, HLA-G, Cd47, Cd200, FASLG, Ccl21, Mfge8, and Serpin B9; or PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a U.S. National Stage Application of PCT/CA2019/051808, filed Dec. 13, 2019, which claims the benefit of the earlier filing date of U.S. Provisional Application Ser. No. 62/779,449, filed Dec. 13, 2018, each of which is incorporated herein by reference its entirety as if fully set forth herein. BACKGROUND OF THE DISCLOSURE The immune system is essential for the maintenance of health and protection against disease. The immune system is also capable of causing harm or impeding therapeutic treatment. For example, although the immune system is designed to detect and attack foreign antigens from bacteria, viruses, and other pathogens, an immune response can be mistakenly mounted against endogenous “self” antigens, which may lead to autoimmune disease. In addition, immune system activation against foreign antigens can make treatments, such as organ and tissue transplants, challenging and uncertain due a possibility that the immune system of a recipient will reject the organ or tissue transplant as foreign. Both transplant recipients and subjects with an autoimmune disease may be treated with immunosuppressive medication to dampen the response of the immune system, but this approach is accompanied by undesirable side effects and an increased risk of infection. The adaptive immune response to foreign antigens involves the generation of antibodies, which leads to immunity against such antigens given that the antibodies can be rapidly generated by the immune system upon detection of the same antigen in the future. This feature of the immune response can be harnessed by vaccine technology, and a number of vaccines have been created using antigens from common viruses to produce widespread immunity. There is interest in using vaccines to treat a variety of different diseases and conditions, but the development of vaccines is a long and complex process that often lasts 10-15 years. For this reason, some have turned to other approaches to induce antibody-based immunity in a subject. One approach involves cell-based immunization, in which a cell is used to present an antigen to the immune system. This approach can be unreliable and cumbersome and may lead to the production of multiple non-specific antibodies against various proteins contained in the cell rather than production of a specific therapeutic antibody against the target antigen. There is a need for targeted immunomodulatory therapies that can be used to generate a desired immune response to an antigen without off target effects or the need for widespread suppression of the immune system. SUMMARY OF THE DISCLOSURE Featured are cells and methods of use thereof for modulating an antigen-specific immune response in a subject. The cells contain a set of transgenes that shield the cells from immune surveillance. The cells may further contain a polypeptide containing a donor alloantigen or a polypeptide containing a self-antigen. The cells can be used to induce immune tolerance to an antigen (e.g., a donor alloantigen or a self-antigen). The cells can also be used to induce immune tolerance in a subject in a method of inducing an immune response to (e.g., induce the production of antibodies directed against) a non-self antigen. In a first aspect, the invention features a cell genetically modified to contain at least one mechanism for inducing immune tolerance to an antigen when administered to a subject, the genetically modified cell containing a transgene selected from one or more (e.g., one, two, three, four, five, six, seven, or all eight, e.g., a set of transgenes containing two, three, four, five, six, seven, or all eight) of the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 or a gene encoding a biologic that acts as an agonist of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, or Serpin B9 or Spi6; and a transgene encoding a polypeptide containing an antigen. In some embodiments, each transgene of the set of transgenes encodes a gene product that is cytoplasmic, membrane bound, or local acting, and that has one or more of the following functions: a) to mitigate antigen presenting cell activation and function; b) to mitigate graft attacking leukocyte activity or cytolytic function; c) to mitigate macrophage cytolytic function and phagocytosis of allograft cells; d) to induce apoptosis in graft attacking leukocytes; e) to mitigate local inflammatory proteins; and f) to protect against leukocyte-mediated apoptosis. In some embodiments, the cell contains a transgene selected from two or more (e.g., a set of transgenes containing two, three, four, five, six, seven, or all eight) of the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 or a gene encoding a biologic that acts as an agonist of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl2