Compositions and methods for selective depletion and replacement of hematopoietic stem cells

文档序号：1590363 发布日期：2020-01-03 浏览：8次中文

阅读说明：本技术 用于选择性消除和替换造血干细胞的组合物和方法 (Compositions and methods for selective depletion and replacement of hematopoietic stem cells ) 是由 E.奥斯特塔 D.舍德洛克 J.D.道恩于 2018-03-13 设计创作，主要内容包括：公开了消除受试者中的至少一种靶细胞的方法,其包括向受试者施用有效量的包含多种免疫细胞的组合物,其中所述多种中的各免疫细胞表达一种或多种嵌合配体受体(CLR),所述嵌合配体受体各自特异性结合至少一种靶细胞上的靶配体,其中一种或多种CLR与靶标的特异性结合活化免疫细胞,且其中活化的免疫细胞诱导靶细胞的死亡。示例性靶细胞包括,但不限于,造血干细胞(HSC)。(Disclosed are methods of eliminating at least one target cell in a subject comprising administering to the subject an effective amount of a composition comprising a plurality of immune cells, wherein each immune cell in the plurality expresses one or more Chimeric Ligand Receptors (CLRs) that each specifically binds a target ligand on at least one target cell, wherein the specific binding of one or more CLRs to a target activates the immune cell, and wherein the activated immune cell induces death of the target cell. Exemplary target cells include, but are not limited to, Hematopoietic Stem Cells (HSCs).)

1. A method of eliminating at least one target cell in a subject, comprising administering to the subject an effective amount of a composition comprising a plurality of immune cells, wherein each immune cell in the plurality of immune cells expresses one or more Chimeric Ligand Receptors (CLRs) that each specifically binds to a target ligand on at least one target cell, wherein the specific binding of one or more CLRs to a target ligand activates the immune cell, and wherein the activated immune cell induces death of the target cell.

2. The method of claim 1, further comprising the step of eliminating a plurality of immune cells.

3. A method of transplanting the immune system of a subject, comprising:

(a) administering to a subject an effective amount of a composition comprising a plurality of immune cells, wherein each immune cell in the plurality of immune cells expresses one or more Chimeric Ligand Receptors (CLRs) that each specifically binds to a target ligand on at least one target cell, wherein the specific binding of one or more CLRs to a target ligand activates the immune cell, and wherein the activated immune cell induces death of the target cell;

(b) elimination of various immune cells; and

(c) administering to the subject an effective amount of a composition comprising a plurality of therapeutic Hematopoietic Stem Cells (HSCs).

4. The method of any one of claims 1-3, wherein said inducing death of the target cell comprises inducing cell lysis of the target cell.

5. The method of any one of claims 1-4, wherein the at least one target cell is a plurality of target cells.

6. The method of any one of claims 1-5, wherein the at least one target cell or plurality of target cells comprises Hematopoietic Stem Cells (HSCs).

7. The method of claim 5 or 6, wherein the at least one target cell or plurality of target cells comprises immune cells.

8. The method of claim 7, wherein the immune cell is a T lymphocyte (T cell).

9. The method of claim 8, wherein the T cells express CD4 or CD 8.

10. The method of claim 8 or 9, wherein the T cell is helper T (T)_H) A cell.

11. The method of claim 10, wherein the helper T cell (T)_H) Is a type I helper T (T)_H1) A cell.

12. The method of claim 10, wherein the helper T cell (T)_H) Is type 2 helper T (T)_H2) A cell.

13. The method of claim 10, wherein the helper T cell (T)_H) Is T-helper 17 (T)_H17) A cell.

14. The method of claim 8 or 9, wherein the T cell is a regulatory T (T)_REG) A cell.

15. The method of claim 14, wherein the T cell is induced regulatory T (iT)_REG) Cellular or natural regulationSex T (nT)_REG) A cell.

16. The method of claim 14, wherein the T cell is induced regulatory T (iT)_REG) A cell.

17. The method of claim 14, wherein the T cell is a natural regulatory T (nT)_REG) A cell.

18. The method of claim 8, wherein the immune cell is a Natural Killer (NK) cell.

19. The method of any one of claims 8-18, wherein the at least one target cell or plurality of target cells comprises HSCs, wherein the at least one target cell or plurality of target cells further comprises immune cells, and wherein the subject is at risk of rejecting a composition comprising a plurality of immune cells each expressing one or more CLRs.

20. The method of any one of claims 1-19, wherein the composition comprising a plurality of immune cells comprises T cells or NK cells.

21. The method of any one of claims 1-19, wherein the composition comprising a plurality of immune cells comprises T cells and NK cells.

22. The method of any one of claims 1-21, wherein the composition comprising a plurality of immune cells is allogeneic.

23. The method of claim 22, wherein the allogeneic composition is derived from a healthy donor.

24. The method of any one of claims 1-21, wherein the composition comprising a plurality of immune cells is autologous.

25. The method of claim 24, wherein the subject has a disease or disorder, and wherein the autologous composition is derived from a biological sample obtained from the subject prior to development of the disease or disorder, during a period of remission from the disease or disorder, or after treatment of the disease or disorder.

26. The method of any one of claims 1-25, wherein at least one immune cell of the plurality of immune cells comprises a genetic modification, and wherein the genetic modification reduces or inhibits expression of a T-cell receptor or Major Histocompatibility Complex (MHC).

27. The method of any one of claims 1-25, wherein a portion of the plurality of immune cells comprise a genetic modification, and wherein the genetic modification reduces or inhibits expression of a T-cell receptor or Major Histocompatibility Complex (MHC).

28. The method of claim 27, wherein the portion comprises at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of immune cells.

29. The method of any one of claims 1-25, wherein each immune cell of the plurality of immune cells comprises a genetic modification, and wherein the genetic modification reduces or inhibits expression of a T-cell receptor (TCR) or Major Histocompatibility Complex (MHC).

30. The method of any one of claims 26-29, wherein the MHC consists of or comprises MHC I, MHC II, or a combination thereof.

31. The method of any one of claims 26-29, wherein the MHC consists of or comprises MHC I.

32. The method of any one of claims 26-29, wherein the MHC consists of or comprises MHC II.

33. The method of any one of claims 26-32, wherein the genetic modification is a single strand break, a double strand break, a sequence deletion, a sequence insertion, a sequence substitution, or any combination thereof.

34. The method of claim 33, wherein the sequence deletion, sequence insertion, sequence substitution, or a combination thereof comprises a sequence encoding an intron, an exon, a promoter, an enhancer, a transcription repressor, a CpG site, or any combination thereof.

35. The method of any one of claims 26-34, wherein the genetic modification comprises a sequence encoding beta-2 microglobulin (β 2M), and wherein the genetic modification reduces or inhibits expression of MHC I.

36. The method of any one of claims 26-34, wherein the genetic modification comprises a sequence encoding HLA-DR α, CIITA or a combination thereof, and wherein the genetic modification reduces or inhibits expression of MHC II.

37. The method of any one of claims 26-36, wherein the genetic modification comprises a sequence encoding an alpha chain (TCR alpha), a beta chain (TCR beta), or a combination thereof, and wherein the genetic modification reduces or inhibits expression of a TCR.

38. The method of any one of claims 26-37, wherein the genetic modification is introduced by a composition comprising a DNA binding domain and an endonuclease domain.

39. The method of claim 38, wherein the DNA binding domain comprises a guide RNA.

40. The method of claim 38, wherein the DNA-binding domain comprises a sequence isolated or derived from Cas9, a transcription activator-like effector nuclease (TALEN), a centromere, and a promoter factor 1 (Cpf1), or a Zinc Finger Nuclease (ZFN).

41. The method of claim 40, wherein the Cas9 is a catalytically inactive Cas9 (dCas9) or a short and catalytically inactive Cas9 (dsCas 9).

42. The method of any one of claims 38-41, wherein the endonuclease domain comprises a sequence isolated or derived from Cas9, a transcription activator-like effector nuclease (TALEN), or a type IIS endonuclease.

43. The method of claim 42, wherein the type IIS endonuclease is AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BspMI, BsrI, BspMI, BspII, BsgI, BsmI, BspII, BtgII, BtgZI, BbsI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, BspI, BapI, BabXI, CspCI, BspCI, BspIII, BsokMb 36I, FokI, or Clo3836.

44. The method of any one of claims 38-43, wherein the DNA binding domain and the endonuclease domain are linked covalently or non-covalently.

45. The method of claim 44, wherein the DNA binding domain and the endonuclease domain are covalently linked as a fusion protein.

46. The method of any one of claims 38-43, wherein the transposon comprises a composition comprising a DNA binding domain and an endonuclease domain.

47. The method of any one of claims 26-46, wherein the plurality of immune cells comprises resting cells, activated cells, or a combination thereof.

48. The method of any one of claims 26-46, wherein the plurality of immune cells comprises activated cells.

49. The method of any one of claims 26-46, wherein the plurality of immune cells comprises resting cells.

50. The method of any of claims 26-46, wherein the plurality of immune cells comprises resting CAR-T cells, activated CAR-T cells, or a combination thereof.

51. The method of any one of claims 26-46, wherein the plurality of immune cells comprises activated CAR-T cells.

52. The method of any one of claims 26-46, wherein the plurality of immune cells comprises resting CAR-T cells.

53. The method of any one of claims 1-52, wherein at least one immune cell of the plurality of immune cells expresses two or more Chimeric Ligand Receptors (CLRs) that each specifically bind a target ligand on at least one target cell.

54. The method of any one of claims 1-52, wherein a subset of the plurality of immune cells express two or more Chimeric Ligand Receptors (CLRs) that each specifically bind a target ligand on at least one target cell.

55. The method of claim 54, wherein the portion comprises at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of immune cells.

56. The method of any one of claims 1-52, wherein each immune cell of the plurality of immune cells expresses two or more Chimeric Ligand Receptors (CLRs) that each specifically bind a target ligand on at least one target cell.

57. The method of any of claims 53-56, wherein a first CAR specifically binds a first target ligand, wherein a second CAR specifically binds a second target ligand, and wherein said first target ligand and said second target ligand are different.

58. The method of claim 57, wherein the first target ligand and the second target ligand are not homologous.

59. The method of any one of claims 1-58, wherein the at least one target cell or plurality of target cells comprises HSCs, and wherein the target ligand on the at least one target HSC comprises one or more of c-KIT/CD117, CD45, CD34, Thy1/CD90, c-mpl/CD110, CD133, CD49f, ABCG2/CD338, carbonic anhydrase IX/CA9, CD123, and CD 150.

60. The method of any one of claims 1-59, wherein the at least one target cell or plurality of target cells comprises HSCs, wherein the at least one target cell or plurality of target cells further comprises immune cells, and wherein the subject is at risk of rejecting a composition comprising a plurality of immune cells, each of which expresses one or more CLRs.

61. The method of any one of claims 1-60, wherein the at least one target cell or plurality of target cells comprises HSCs, wherein the at least one target cell or plurality of target cells further comprises immune cells, and wherein the subject is at risk of rejecting a composition comprising a plurality of therapeutic HSCs.

62. The method of claim 60 or 61, wherein the target ligand on the target immune cell comprises one or more of CD3, CD4, CD8, CD25, FoxP3, TCR α, TCR β, TCR α β, TCR γ λ, CD52, NK1.1, CD16, CD30, CD31, CD38, CD56, CD94, NKG2A, NKG2C, NKp30, NKp44, NKp46, CD9, CD103, and KIR.

63. The method of any one of claims 1-62, wherein the one or more CLRs each comprise

(a) An extracellular domain comprising a ligand recognition region,

(b) a transmembrane domain, and

The method of claim 63, wherein the ligand recognition region comprises one or more of a protein scaffold, a Centyrin, a single chain variable fragment (scFv), a VHH, an immunoglobulin and an antibody mimetic.

64. The method of claim 63, wherein the immunoglobulin is an antibody or fragment thereof.

65. The method of claim 64, wherein the antibody is an IgA, IgD, IgE, IgG, or IgM isotype.

66. The method of claim 64, wherein the antibody fragment is a Complementarity Determining Region (CDR), a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, a light chain CDR3, an antigen binding fragment (Fab), a variable domain (Fv), a heavy chain variable region, a light chain variable region, a complete heavy chain, a complete light chain, one or more constant domains, an Fc (crystallizable fragment), or any combination thereof.

67. The method of claim 63, wherein said antibody mimetic comprises one or more of affibody, affilin, affimer, affitin, alphabody, anticalin, and avimer, designed ankyrin repeat protein (DARPin), Fynomer, Kunitz domain peptide, and monomer.

68. The method of any one of claims 1-67 wherein at least one of the CLRs is bispecific.

69. The method of any one of claims 1-67, wherein at least one of the CLRs is trispecific.

70. The method of any one of claims 63-69, wherein the extracellular domain of (a) further comprises a signal peptide.

71. The method of claim 70, wherein said signal peptide comprises a sequence encoding a human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide.

72. The method of any one of claims 63-71, wherein the extracellular domain of (a) further comprises a hinge between the ligand recognition region and the transmembrane domain.

73. The method of claim 72, wherein the hinge comprises sequences derived from human CD 8a, IgG4, and/or CD4 sequences.

74. The method of any one of claims 63-73, wherein the transmembrane domain comprises a sequence encoding a human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain.

75. The method of any one of claims 63-74, wherein the endodomain comprises a human CD3 ζ endodomain.

76. The method of any one of claims 63-75, wherein the at least one co-stimulatory domain comprises a human 4-1BB, human CD28, human CD40, human ICOS, human MyD88, human OX-40 intracellular segment, or any combination thereof.

77. The method of any one of claims 63-76, wherein the at least one co-stimulatory domain comprises a human CD28 and/or a human 4-1BB co-stimulatory domain.

78. The method of claim 77, wherein the 4-1BB costimulatory domain is located between the transmembrane domain and the CD28 costimulatory domain.

79. The method of any one of claims 63-78, wherein at least one immune cell in the composition comprising a plurality of immune cells comprises a dividing CLR.

80. The method of claim 79 wherein the dividing CLR comprises two or more CLRs having different intracellular domains that, when expressed simultaneously in at least one immune cell, increase or decrease the activity of the immune cell as compared to an immune cell that does not express a dividing CLR or an immune cell that does not express a CLR.

81. The method of claim 80, wherein the simultaneous expression increases the activity of an immune cell, and wherein the dividing CLR comprises:

(a) a first CLR comprising: an extracellular domain comprising a ligand recognition region, a transmembrane domain and an intracellular domain consisting of a primary intracellular signaling domain, and

(b) a second CLR comprising: an extracellular domain comprising a ligand recognition region, a transmembrane domain, and an intracellular domain consisting of a secondary intracellular signaling domain.

82. The method of claim 80, wherein said primary intracellular signaling domain comprises the intracellular domain of human CD3 ζ.

83. The method of claim 81 or 82, wherein the secondary intracellular signaling domain comprises a human 4-1BB, human CD28, human CD40, human ICOS, human MyD88, or human OX-40 intracellular segment.

84. The method of claim 81 or 82, wherein said secondary intracellular signaling domain comprises human 4-1BB and human CD 28.

85. The method of claim 80 wherein the simultaneous expression reduces the activity of an immune cell, and wherein the dividing CLR comprises:

(a) a first CLR comprising: an extracellular domain comprising a ligand recognition region, a transmembrane domain, and an intracellular domain comprising a primary intracellular signaling domain, a secondary intracellular signaling domain, and

(b) a second CLR comprising: an extracellular domain comprising a ligand recognition region, a transmembrane domain, and an intracellular domain consisting of an inhibitory intracellular signaling domain.

86. The method of claim 85, wherein the primary intracellular signaling domain comprises a human CD3 ζ endodomain and the secondary intracellular signaling domain comprises a human 4-1BB, human CD28, human CD40, human ICOS, human MyD88, or human OX-40 intracellular segment.

87. The method of claim 86, wherein said primary intracellular signaling domain comprises a human CD3 ζ endodomain and said secondary intracellular signaling domain comprises human 4-1BB and human CD 28.

88. The method of any one of claims 85-87, wherein the inhibitory intracellular signaling domain comprises signaling domains derived from PD1, CTLA4, LAG3, B7-H1, B7-1, CD160, BTLA, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, TIGIT, ITIM, ITSM, YVKM, PP2A, SHP2, kiele, and Y265.

89. The method of any of claims 80-88, wherein the second CAR selectively binds to a target ligand on a non-target cell.

90. The method of any one of claims 1-89, wherein the one or more CLRs bind ligand with at least one affinity selected from the group consisting of: less than or equal to 10⁻⁹M, less than or equal to 10⁻¹⁰M, less than or equal to 10⁻¹¹M, less than or equal to 10⁻¹²M, less than or equal to 10⁻¹³M, less than or equal to 10⁻¹⁴M and less than or equal to 10⁻¹⁵K of M_D。

91. The method of claim 90Method in which K_DMeasured by surface plasmon resonance.

92. The method of any one of claims 1-91, wherein the composition comprising a plurality of immune cells further comprises at least one pharmaceutically acceptable carrier.

93. The method of any one of claims 1-92, further comprising administering to the subject an mobilizing composition.

94. The method of claim 93, wherein the mobilizing composition is administered prior to a composition comprising a plurality of immune cells each comprising one or more CLRs.

95. The method of claim 94, wherein the mobilizing composition is administered between 1 day and 7 days, including the endpoints, before the composition comprising a plurality of immune cells each comprising one or more CLRs.

96. The method of any one of claims 93-95, wherein the mobilizing composition comprises granulocyte colony stimulating factor (G-CSF), plerixafor, or a combination thereof.

97. The method of any one of claims 1-96, further comprising administering to the subject an effective amount of a preconditioning composition to enhance the efficacy of the engraftment of the composition comprising a plurality of immune cells each comprising one or more CLRs and the elimination of at least one target cell by the composition comprising a plurality of immune cells each comprising one or more CLRs.

98. The method of claim 97, wherein said preconditioning composition suppresses the immune system.

99. The method of claim 97 or 98, wherein the preconditioning composition comprises an autoimmune therapy, an antirejection agent, a lymphodepleting agent, a myeloablative agent, a chemotherapeutic agent, or a combination thereof.

100. The method of claim 99, wherein the lymphodepleting agent comprises cyclophosphamide or fludarabine.

101. The method of claim 99, wherein the myeloablative agent comprises low dose radiation or partial radiation therapy.

102. The method of claim 99, wherein the chemotherapeutic agent comprises busulfan, troosulfan, melphalan, tiatipa, or a combination thereof.

103. The method of any one of claims 1-102, wherein each immune cell of the plurality of immune cells is pre-irradiated prior to administration to the subject.

104. The method of claim 103, wherein the step of eliminating the plurality of immune cells comprises administering an effective amount of the plurality of pre-irradiated immune cells to the subject, thereby preventing proliferation or shortening survival of the plurality of pre-irradiated immune cells.

105. The method of any one of claims 1-104, wherein each immune cell of the plurality of immune cells comprises an inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide.

106. The method of claim 105, wherein said inducible caspase polypeptide comprises

(a) A ligand-binding domain having a ligand-binding domain,

(b) a joint, and

107. The method of claim 106, wherein the inducible caspase polypeptide does not comprise a non-human sequence.

108. The method of any one of claims 105-107, wherein the step of eliminating the plurality of immune cells comprises administering to the subject an effective amount of an inducing agent to induce the caspase polypeptide, thereby initiating death of the immune cells.

109. The method of any one of claims 105-108, wherein the composition comprising a plurality of immune cells each comprising one or more CLRs further comprises an inducing agent.

110. The method of any of claims 3-109, wherein each HSC in the plurality of therapeutic HSCs comprises an inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide.

111. The method of claim 110, wherein said inducible caspase polypeptide comprises

(a) A ligand-binding domain having a ligand-binding domain,

(b) a joint, and

112. The method of claim 111, wherein the inducible caspase polypeptide does not comprise a non-human sequence.

113. The method of claim 112, further comprising administering to the subject a composition comprising an inducing agent, thereby initiating death of the plurality of therapeutic HSCs.

114. The method of any one of claims 1-113, wherein the subject is a human.

115. The method of any one of claims 1-114, wherein the subject has or is at risk of developing an immune system disease or disorder.

116. The method of any one of claims 1-114, wherein the subject has an autoimmune disease or disorder.

117. The method of claim 116, wherein the autoimmune disease or disorder is Acute Disseminated Encephalomyelitis (ADEM), acute necrotizing leukoencephalitis, addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic nerve abnormality, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immune deficiency, Autoimmune Inner Ear Disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, Autoimmune Thrombocytopenic Purpura (ATP), autoimmune thyroid disease, autoimmunity, axonal and neuronal neuropathy, urticaria, neuro, or neuro, Barlow's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas' disease, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), chronic relapsing multifocal myelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogen syndrome, cold agglutinin disease, congenital heart conduction block, coxsackie myocarditis, CREST disease, basic mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, German Leeb syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evens syndrome, fibrotic paulitis, giant cell arteritis (temporal arteritis), myocarditis, and cardiomyopathy, Glomerulonephritis, Goodpasture's syndrome, granuloma with polyangiitis (GPA), graves' disease, guillain-barre syndrome, hashimoto's encephalitis, hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypoproteinemia, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephropathy, IgG 4-associated sclerosing disease, immunoregulatory lipoprotein, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, kawasaki syndrome, lambert-eaton syndrome, fragmented leukocyte vasculitis, lichen planus, lichen sclerosus, xylem conjunctivitis, linear IgA disease (LAD), lupus (SLE, lyme disease, chronic meniere's disease, microscopic vasculitis, Mixed Connective Tissue Disease (MCTD), muckle ulcer, muller's disease (MCTD), multiple sclerosis, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, lethargy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, recurrent rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorder associated with streptococci), paraneoplastic cerebellar degeneration, Paroxysmal Nocturnal Hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars plana (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous myelitis, pernicious anemia, POEMS, polyarteritis nodosa, autoimmune polyglandular syndrome type I, autoimmune polyglandular syndrome type II, autoimmune polyglandular syndrome type III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progestational dermatitis, and the like, Primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell dysplasia, raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, reiter's syndrome, recurrent polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, Subacute Bacterial Endocarditis (SBE), susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, Undifferentiated Connective Tissue Disease (UCTD), uveitis, vasculitis, vesicular skin disease, or vitiligo.

118. The method of any one of claims 1-117, wherein the subject is immunocompromised.

119. The method of any one of claims 1-117, wherein the subject has an inflammatory disorder.

120. The method of any one of claims 1-119, wherein the subject has a genetic or epigenetic marker of the disease or disorder.

121. The method of any one of claims 1-114, wherein the subject has a genetic or epigenetic marker of a disease or disorder manifested in blood cells, immune cells circulating in the blood, bone marrow cells, or precursor cells thereof.

122. The method of claim 121, wherein said precursor cells are Hematopoietic Stem Cells (HSCs).

123. The method of claim 120, 121, or 122, wherein the disease or disorder is cancer.

124. The method of claim 123, wherein the cancer is lymphoma, leukemia, myeloma, or a malignant immunoproliferative disease.

125. The method of claim 124, wherein said lymphoma is hodgkin's lymphoma, non-hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma (aitt), hepatosplenic T-cell lymphoma, B-cell lymphoma, reticuloendothelial disease, reticulocytosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, Mantle Cell Lymphoma (MCL), burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstr ö m macroglobulinemia, nodular marginal zone B-cell lymphoma, Splenic Marginal Zone Lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphoma-like granuloma, or nodular lymphocyte-dominated hodgkin's lymphoma.

126. The method of claim 124, wherein said leukemia is Plasma Cell Leukemia (PCL), acute erythrocytic and erythrocytic leukemia, acute erythroblastic myelopathy, acute erythroleukemia, Heilmeyer-Sch ö ner disease, acute megakaryoblastic leukemia (AMKL), mast cell leukemia, pan-myelogenous leukemia, acute osteomyelitis with myelofibrosis (APMF), lymphosarcoma cell leukemia, osteogenic chronic myelogenous leukemia, stem cell leukemia, accelerated phase chronic myelogenous leukemia, Acute Myelogenous Leukemia (AML), polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid leukemia, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, B-lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, chronic myelogenous leukemia, or chronic myelogenous leukemia.

127. The method of claim 124, wherein said myeloma is multiple myeloma, Carle's disease, myelomatosis, solitary myeloma, plasma cell leukemia, extramedullary plasmacytoma, a malignant plasma cell tumor, or a plasmacytoma.

128. The method of claim 124, wherein the malignant immunoproliferative disease is an alpha heavy chain disease or a gamma heavy chain disease.

129. The method of claim 120, 121, or 122, wherein the disease or disorder is anemia.

130. The method of claim 129, wherein said anemia is hemolytic anemia, autoimmune hemolytic anemia, congenital hemolytic anemia, aplastic anemia, β -thalassemia, congenital dyserythroid dysplasia, congenital erythropoietic anemia, glucose 6-phosphate dehydrogenase deficiency, fanconi's anemia, hereditary spherocytosis, hereditary elliptocytosis, hereditary hemidysmorphic polycythemia, hereditary persistence of fetal hemoglobin, hereditary stomatocytosis, hexokinase deficiency, excessive anemia, hypopigmentary anemia, erythropoietic inefficiency, megaloblastic anemia, myelogenous anemia, neuroacanthocytosis, chorea-echinocytosis (chorea-acanthosis), paroxysmal nocturnal hemoglobinuria, anemia, megaloblastic anemia, myelogenous anemia, neuroacanthocytosis, chorea-acanthocytosis, acute hemoglobinuria, and other diseases, Pyruvate kinase deficiency, Rh deficiency syndrome, sickle cell disease, iron granulocytopenia, stomatogenic ovoerythrocytosis (stomatocytic ovatososis), thalassemia, triosephosphate isomerase (TPI) deficiency or warm autoimmune hemolytic anemia.

131. The method of claim 120, 121, or 122, wherein the disease or disorder is a coagulation disorder or a bleeding condition.

132. The method of claim 131, wherein the disease or disorder is a blood coagulation disorder.

133. The method of claim 132, wherein the coagulation disorder is defibrination syndrome, protein C deficiency, protein S deficiency, factor V Leiden, thrombocytosis, thrombosis, recurrent thrombosis, antiphospholipid syndrome, primary antiphospholipid syndrome, or Thrombotic Thrombocytopenic Purpura (TTP).

134. The method of claim 131, wherein the disease or disorder is a bleeding condition.

135. The method of claim 134, wherein the bleeding condition is thrombocytopenia, hemophilia a, hemophilia B, hemophilia C, von willebrand disease (vWD), hereditary von willebrand disease (vWD), vWD type 1, vWD type 2, vWD type 3, Glanzmann's thrombocytopenia, or Wiskott-Aldrich syndrome (WAS).

136. The method of any of claims 1-114, wherein the subject has a genetic or epigenetic marker of a disease or disorder manifested in secondary target cells that can be contacted by a composition comprising a plurality of therapeutic HSCs.

137. The method of claim 136, wherein said secondary target cells are stem cells or progenitor cells.

138. The method of claim 137, wherein said stem cell is a somatic stem cell.

139. The method of claim 138, wherein the stem cell is a target HSC, mesenchymal stem cell, epidermal stem cell, epithelial stem cell, neural stem cell.

140. The method of claim 137, wherein said progenitor cells are osteoblasts.

141. The method of claim 136, wherein said secondary target cell is a differentiated cell.

142. The method of claim 141, wherein said differentiated cells are red blood cells, white blood cells, monocytes, granulocytes, platelets, or dendritic cells.

143. The method of claim 136, 137, or 140, wherein at least one HSC in the composition comprising a plurality of therapeutic HSCs is modified to secrete a ligand, peptide, or protein that enhances the activity of osteoblasts.

144. The method of claim 143, wherein said composition comprising a plurality of therapeutic HSCs treats or prevents a disease or disorder associated with abnormal osteoblast function.

145. The method of claim 144, wherein the subject has one or more genetic or epigenetic signatures of a disease or disorder associated with abnormal osteoblast function.

146. The method of claim 144 or 145, wherein the disease or disorder associated with aberrant osteoblastic function is paget's disease, hypophosphatemia or osteoporosis.

147. The method of claim 136, 137, or 142, wherein at least one HSC in the composition comprising a plurality of therapeutic HSCs is modified to secrete a ligand, peptide, or protein that enhances the activity of granulocytes.

148. The method of claim 147, wherein the composition comprising a plurality of therapeutic HSCs treats or prevents a disease or disorder associated with abnormal granulocyte function.

149. The method of claim 148, wherein the subject has one or more genetic or epigenetic signatures of a disease or disorder associated with aberrant granulocyte function.

150. The method of claim 148 or 149, wherein the disease or disorder associated with abnormal granulocyte function is chronic granulomatous disease.

151. The method of any one of claims 1-119, wherein the immune system disease or disorder is induced by a medical intervention.

152. The method of any one of claims 1-119, wherein the subject is at risk of developing an immune system disease or disorder due to past, present, or future medical intervention.

153. The method of any one of claims 1-119, wherein the immune system disease or disorder is induced by infection.

154. The method of any one of claims 1-119, wherein the subject is at risk of developing an immune system disease or disorder due to past, present, or potential infection.

155. The method of any one of claims 1-154, wherein administration of the composition comprising a plurality of immune cells is systemic.

156. The method of claim 155, wherein the composition is administered via an intravenous route.

157. The method of any one of claims 1-154, wherein administration of the composition comprising a plurality of immune cells is topical.

158. The method of claim 157, wherein the composition is administered via intraosseous, intraspinal, or intracerebral infusion.

159. The method of any of claims 3-158, wherein said composition comprising a plurality of therapeutic HSCs further comprises at least one pharmaceutically acceptable carrier.

160. The method of any one of claims 111-159, wherein said composition comprising a plurality of therapeutic HSCs further comprises an inducing agent.

161. The method of any of claims 3-160, wherein at least one HSC in the plurality of therapeutic HSCs comprises a genetic modification.

162. The method of any of claims 3-160, wherein a portion of the HSCs in the plurality of therapeutic HSCs comprise a genetic modification.

163. The method of claim 162, wherein the fraction comprises at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of therapeutic HSCs.

164. The method of any of claims 3-160, wherein each HSC in the plurality of therapeutic HSCs comprises a genetic modification.

165. The method of any one of claims 161-164 wherein the genetic modification is a single strand break, a double strand break, a sequence deletion, a sequence insertion, a sequence substitution, or any combination thereof.

166. The method of claim 167, wherein the sequence deletion, sequence insertion, sequence substitution, or a combination thereof comprises a sequence encoding an intron, an exon, a promoter, an enhancer, a transcription repressor, a CpG site, or any combination thereof.

167. The method of any one of claims 161-166 wherein the genetic modification is introduced by a composition comprising a DNA binding domain and an endonuclease domain.

168. The method of claim 167, wherein the DNA binding domain comprises a guide RNA.

169. The method of claim 167, wherein the DNA-binding domain comprises a sequence isolated or derived from Cas9, a transcription activator-like effector nuclease (TALEN), a centromere, and a promoter factor 1 (Cpf1), or a Zinc Finger Nuclease (ZFN).

170. The method of claim 169, wherein the Cas9 is a catalytically inactive Cas9 (dCas9) or a short and catalytically inactive Cas9 (dsCas 9).

171. The method of any one of claims 167-170, wherein the endonuclease domain comprises a sequence isolated or derived from Cas9, a transcription activator-like effector nuclease (TALEN), or a type IIS endonuclease.

172. The method of claim 171, wherein the type IIS endonuclease is AciI, Mn1I, aiwi, BbvI, BccI, bcei, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, hpyiv, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, bmubi, bmgri, BmrI, BpmI, bpmei, BsaI, BseRI, BsgI, BspMI, bsbi, BsrDI, BtgZI, btsis i, EarI, eci, MmeI, nmeiii, NmeAIII, bbvcvcvcci, Bpu10I, bspei, bpei, baxi, csxi, csfiii, bpiii, bmcii, sakii, fokl 2, or Clo.

173. The method of any one of claims 167-172, wherein the DNA binding domain and the endonuclease domain are covalently or non-covalently linked.

174. The method of claim 173, wherein the DNA-binding domain and the endonuclease domain are covalently linked as a fusion protein.

175. The method of any one of claims 161-174, wherein the genetic modification is introduced by inducing homologous recombination, inserting a single stranded oligodeoxynucleotide (ssODN), or a transposition event.

176. The method of claim 175, wherein the genetic modification results in insertion of a sequence.

177. The method of claim 175 or 176, wherein the transposition event results in insertion of a functional and/or therapeutic transgene.

178. The method of any one of claims 175-177, wherein the transposon comprises a functional and/or therapeutic transgene and wherein the transposon is a piggyBac transposon.

179. The method of claim 178, wherein the transposon-containing HSCs further comprise piggybac (pb) transposase.

180. The method of claim 179, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID No. 1.

181. The method of claim 179 or 180, wherein the piggyBac transposase is an hyperactive variant, and wherein the hyperactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282, and 538 of SEQ ID No. 1.

182. The method of claim 181, wherein the amino acid substitution at position 30 of SEQ ID NO 1 is a valine (V) substitution for isoleucine (I) (I30V).

183. The method of claim 181, wherein the amino acid substitution at position 165 of SEQ ID NO. 1 is a serine (S) substituted for glycine (G) (G165S).

184. The method of claim 181, wherein the amino acid substitution at position 282 of SEQ ID NO. 1 is a valine (V) for methionine (M) (M282V).

185. The method of claim 181, wherein the amino acid substitution at position 538 of SEQ ID NO. 1 is a lysine (K) for an asparagine (N) (N538K).

186. The method of any one of claims 180-185, wherein the transposase is a super piggybac (spb) transposase.

187. The composition of claim 186, wherein the superpaggybac (spb) transposase comprises an amino acid sequence comprising SEQ ID No. 2.

188. The method of any one of claims 161-187, wherein the subject has an immune disease or disorder, and wherein the plurality of therapeutic HSCs ameliorate a sign or symptom of the immune disease or disorder.

189. The method of any one of claims 161-187 wherein the subject has a genetic or epigenetic marker of a disease or disorder manifested in blood cells, immune cells circulating in the blood, bone marrow cells, or precursors thereof, and wherein the plurality of therapeutic HSCs ameliorate a sign or symptom of the disease or disorder.

190. The method of claim 189, wherein the disease or disorder is a blood coagulation disorder.

191. The method of claim 190, wherein the plurality of therapeutic HSCs have been modified to secrete proteins that ameliorate the signs or symptoms of a coagulation disorder.

192. The method of claim 191, wherein the plurality of therapeutic HSCs have been modified to secrete one or more clotting factors.

193. The method of any of claims 161-187, wherein the subject has a genetic or epigenetic marker of a glycogen storage disease or disorder, and wherein the plurality of therapeutic HSCs ameliorate a sign or symptom of the glycogen storage disease or disorder.

194. The method of claim 193, wherein the glycogen storage disease or disorder is glycogen storage disease type 0 (GSD), GSD type I, GSD type II, GSD type III, GSD type IV, GSD type V, GSD type VI, GSD type VII, GSD type IX, GSD type X, GSD type XI, GSD type XII, or GSD type XIII.

195. The method of claim 193 or 194, wherein the plurality of therapeutic HSCs have been modified to secrete one or more of glycogen synthase, glucose-6-phosphatase, acid alpha-glucosidase, glycogen debranching enzyme, glycogen branching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphate fructokinase, phosphorylase kinase, glucose transporter GLUT2, aldolase a, or β -enolase, and wherein the plurality of therapeutic HSCs ameliorate a sign or symptom of a type 0 GSD, a type I GSD, a type II GSD, a type III GSD, a type IV GSD, a type V GSD, a type VI GSD, a type VII GSD, a type IX GSD, a type X GSD, a type XI GSD, a type XII GSD, or a type XIII GSD, respectively.

196. The method of any one of claims 161-187, wherein the subject has a genetic or epigenetic marker of an immune system disease or disorder, wherein at least one HSC, a portion of HSCs, or each HSC in the plurality of therapeutic HSCs comprises a genetic modification, and wherein at least one HSC, a portion of HSCs, or each HSC in the plurality of therapeutic HSCs does not comprise a genetic or epigenetic marker.

197. The method of claim 196, wherein the genetic modification removes a genetic or epigenetic marker.

198. The method of claim 196 or 197, wherein said modified HSCs are autologous.

199. The method of claim 196 or 197, wherein the modified HSCs are allogeneic.

200. The method of any one of claims 3-199, wherein the method treats or prevents the onset or progression of graft versus host disease (GvHD).

201. The method of claim 200, wherein the treatment comprises reducing signs or symptoms of GvHD.

202. The method of claim 200 or 201, wherein the GvHD is acute GvHD.

203. The method of claim 200 or 201, wherein said GvHD is chronic GvHD.

204. The method of any one of claims 200-203, wherein the signs or symptoms of GvHD comprise skin rash, blistering skin, nausea, vomiting, abdominal cramps, diarrhea, loss of appetite, jaundice, dry mouth, dry laryngo pharynx, excessive dry mouth, excessive dry laryngo pharynx, ulcers of the mouth or throat, dry bronchial tissue, dry endothelial tissue, dry superficial tissue, skin plaque detachment, skin discoloration, skin scarring, decreased joint mobility associated with skin scarring, hair loss associated with skin injury, loss of tear formation leading to dry eye, or any combination thereof.

205. The method of any one of claims 200-204, wherein the subject is a transplant recipient.

206. The method of claim 205, wherein the composition comprising a plurality of therapeutic HSCs is administered to the subject prior to administration of the transplant, and wherein the plurality of therapeutic HSCs and the transplant are isolated or derived from the same donor.

207. The method of claim 206, further comprising a period of time after administering a composition comprising a plurality of HSCs sufficient to tolerate tolerance of the subject's immune system to the transplant.

208. The method of any one of claims 200-207, wherein the transplant comprises a cell, a tissue transplant, an organ transplant, or any combination thereof.

209. The method of claim 208, wherein said organ is a solid organ.

Technical Field

The present invention relates to molecular biology, and more particularly to cells expressing chimeric ligand receptors that selectively target Hematopoietic Stem Cells (HSCs), and methods of making and using them.

Background

There is a long felt, but unmet need in the art for a method that: methods of selectively ablating endogenous Hematopoietic Stem Cells (HSCs) in a subject prior to replacing these HSCs with a therapeutic HSC composition (e.g., in the context of a bone marrow transplant). The present invention provides compositions and methods for selectively depleting endogenous Hematopoietic Stem Cells (HSCs) in a subject.

SUMMARY

The present invention provides a method of eliminating at least one target cell in a subject comprising administering to the subject an effective amount of a composition comprising a plurality of immune cells, wherein each immune cell in the plurality expresses one or more Chimeric Ligand Receptors (CLRs) each specifically binding to a target ligand on at least one target cell, wherein the specific binding of one or more CLRs to a target ligand activates the immune cell, and wherein the activated immune cell induces death of the target cell. In certain embodiments, the method further comprises the step of eliminating the plurality of immune cells.

The present invention provides a method of transplanting the immune system of a subject, comprising: (a) administering to a subject an effective amount of a composition comprising a plurality of immune cells, wherein each immune cell in the plurality expresses one or more Chimeric Ligand Receptors (CLRs) each specifically binding to a target ligand on at least one target cell, wherein the specific binding of one or more CLRs to a target ligand activates the immune cell, and wherein the activated immune cell induces death of the target cell; (b) elimination of various immune cells; and (c) administering to the subject an effective amount of a composition comprising a plurality of therapeutic Hematopoietic Stem Cells (HSCs).

As used herein, the term "therapeutic HSC" is intended to describe a plurality of HSCs or populations of HSCs administered to a subject following selective elimination of the target cells of the invention. Therapeutic HSCs may include healthy or disease-free autologous or allogeneic HSCs that replace the depleted target HSCs. Alternatively, the therapeutic HSCs may comprise HSCs that differ from the target HSCs in a clinically relevant manner to improve HSC function, regulate an niche or microenvironment, regulate another cell or cell type, or tolerate the immune system of the subject for subsequent transplantation with cells, tissues or organs from the same source as the therapeutic HSCs. Therapeutic HSCs can be isolated or derived from any human source, including but not limited to a subject of the methods of the invention, a twin (e.g., a human not carrying one or more sporadic mutations of the subject), a genetically related individual or combination of genetically related individuals, and individuals having compatible MHCI/MHCII profiles, or a combination of individuals having compatible MHCI/MHCII profiles. Therapeutic HSCs may comprise autologous or allogeneic HSCs that do not include one or more genetic or epigenetic markers of the disease or disorder. In certain embodiments, the therapeutic HSC are not genetically modified. In certain embodiments, the therapeutic HSC are genetically modified. The therapeutic HSCs can be genetically modified to eliminate one or more genetic or epigenetic markers of the disease or disorder. Alternatively or additionally, therapeutic HSCs can be genetically modified to express or secrete one or more ions, small molecules, peptides or proteins at the cell surface to affect the activity of another cell or cell type (e.g., cancer cells, stem or progenitor cells (osteoblasts, mesenchymal stem cells, neural progenitor cells or glial cells) or immune cells) or to modulate a particular biological niche or microenvironment (extracellular matrix, injury site, stem cell niche) to create a more favorable condition for implantation of the therapeutic HSC. Furthermore, in cases where, for example, one or more of the therapeutic HSCs are incompatible with the subject's immune system or undergo malignant transformation, the therapeutic HSCs can be genetically modified to contain an inducible pro-apoptotic polypeptide of the invention (i.e., a safety switch). In certain embodiments, the therapeutic HSCs are administered to the subject to tolerate the subject's immune system for subsequent transplantation with cells, tissues, grafts, or organs from the same donor as the therapeutic HSCs. Once the therapeutic HSCs are tolerant to the subject's immune system, the immune system will respond poorly to subsequent transplants and should not reject subsequent transplants.

In certain embodiments of the methods of the invention, inducing death of the target cell comprises inducing cell lysis of the target cell.

In certain embodiments of the methods of the invention, the at least one target cell is a plurality of target cells.

In certain embodiments of the methods of the invention, the at least one target cell is a plurality of target cells. In certain embodiments, the at least one target cell or the plurality of target cells comprises immune cells. In certain embodiments, the immune cell is a T lymphocyte (T cell). In certain embodiments, the T cell expresses CD4 or CD 8.

In certain embodiments of the methods of the invention, the at least one target cell is a plurality of target cells. In certain embodiments, the at least one target cell or the plurality of target cells comprises immune cells. In certain embodiments, the immune cell is a T lymphocyte (T cell). In certain embodiments, the T cell is a helper T (T)_H) A cell. In certain embodiments, helper T cells (T)_H) Is a type I helper T (T)_H1) A cell. In certain embodiments, helper T cells (T)_H) Is type 2 helper T (T)_H2) A cell. In certain embodiments, helper T cells (T)_H) Is T-helper 17 (T)_H17) A cell.

In certain embodiments of the methods of the invention, the at least one target cell is a plurality of target cells. In certain embodiments, the at least one target cell or the plurality of target cells comprises immune cells. In certain embodiments, the immune cell is a T lymphocyte (T cell). In certain embodiments, the T cell is a regulatory T (T)_REG) A cell. In certain embodiments, the T cell is an induced regulatory T (iT)_REG) Cellular or natural regulatory T (nT)_REG) A cell. In certain embodiments, the T cell is an induced regulatory T (iT)_REG) A cell. In certain embodiments, the T cell is a natural regulatory T (nT)_REG) A cell.

In certain embodiments of the methods of the invention, the at least one target cell is a plurality of target cells. In certain embodiments, the at least one target cell or the plurality of target cells comprises HSCs and immune cells. In certain embodiments, including those in which the at least one target cell or plurality of target cells comprises HSCs and immune cells, the at least one target cell or plurality of target cells comprises HSC cells and T cells or NK cells. In certain embodiments, including those in which the at least one target cell or plurality of target cells comprises HSCs and immune cells, the at least one target cell or plurality of target cells comprises HSC cells and T cells and NK cells. In certain embodiments, wherein the at least one target cell or plurality of target cells comprises HSCs, wherein the at least one target cell or plurality of target cells further comprises immune cells, and wherein the subject is at risk of rejecting a composition comprising a plurality of immune cells, each of which expresses one or more CLRs. In certain embodiments, wherein the at least one target cell or plurality of target cells comprises HSCs, wherein the at least one target cell or plurality of target cells further comprises immune cells, and wherein the subject is at risk of rejection of a composition comprising a plurality of therapeutic HSCs.

In certain embodiments of the methods of the present invention, the composition comprising a plurality of immune cells is allogeneic. In certain embodiments, the allogeneic composition is derived from a healthy donor.

In certain embodiments of the methods of the invention, the composition comprising a plurality of immune cells is autologous. In certain embodiments, including those in which the composition comprising a plurality of immune cells is autologous, the subject has a disease or disorder, and the autologous composition is derived from a biological sample obtained from the subject prior to development of the disease or disorder, during a period of remission from the disease or disorder, or after treatment of the disease or disorder.

In certain embodiments of the methods of the invention, at least one immune cell of the plurality of immune cells comprises a genetic modification, and wherein the genetic modification reduces or inhibits expression of a T-cell receptor or Major Histocompatibility Complex (MHC). In certain embodiments, a portion of the plurality of immune cells comprise a genetic modification, and wherein the genetic modification reduces or inhibits expression of a T-cell receptor or Major Histocompatibility Complex (MHC). In certain embodiments, the portion comprises at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of immune cells. In certain embodiments, each immune cell of the plurality of immune cells comprises a genetic modification, and wherein the genetic modification reduces or inhibits expression of a T-cell receptor (TCR) or Major Histocompatibility Complex (MHC). In certain embodiments, the MHC consists of or comprises MHC I, MHC II, or a combination thereof. In certain embodiments, the MHC consists of or comprises MHC I. In certain embodiments, the MHC consists of or comprises MHC II. In certain embodiments, the genetic modification is a single strand break, a double strand break, a sequence deletion, a sequence insertion, a sequence substitution, or any combination thereof. In certain embodiments, the sequence deletion, sequence insertion, sequence substitution, or a combination thereof comprises a sequence encoding an intron, an exon, a promoter, an enhancer, a transcriptional repressor, a CpG site, or any combination thereof. In certain embodiments, the genetic modification comprises a sequence encoding beta-2 microglobulin (beta 2M), and wherein the genetic modification reduces or inhibits expression of MHC I. In certain embodiments, the genetic modification comprises a sequence encoding HLA-DR α, CIITA, or a combination thereof, and wherein the genetic modification reduces or inhibits expression of MHC II. In certain embodiments, the genetic modification comprises a sequence encoding an alpha chain (TCR alpha), a beta chain (TCR beta), or a combination thereof, and wherein the genetic modification reduces or inhibits expression of a TCR.

In certain embodiments of the methods of the invention, including those in which at least one immune cell of the plurality of immune cells comprises a genetic modification and wherein the genetic modification reduces or inhibits expression of a T-cell receptor or Major Histocompatibility Complex (MHC), the genetic modification is introduced by a composition comprising a DNA-binding domain and an endonuclease domain. In certain embodiments, the DNA binding domain comprises a guide RNA. In certain embodiments, the DNA-binding domain comprises a sequence isolated or derived from Cas9, a transcription activator-like effector nuclease (TALEN), a centromere, and a promoter factor 1 (Cpf1) or a Zinc Finger Nuclease (ZFN). In certain embodiments, Cas9 is catalytically inactive Cas9 (dCas9) or short and catalytically inactive Cas9 (dsCas 9).

In certain embodiments, the dCas9 of the present invention comprises dCas9 isolated or derived from staphylococcus pyogenes. In certain embodiments, dCas9 comprises dCas9 with substitutions at positions 10 and 840 of the amino acid sequence of dCas9 that inactivate catalytic sites. In certain embodiments, these substitutions are D10A and H840A. In certain embodiments, the "X" residue at position 1 of the dCas9 sequence is methionine (M). In certain embodiments, the amino acid sequence of dCas9 comprises the following sequence:

in certain embodiments, the dCas9 of the present invention comprises dCas9 isolated or derived from staphylococcus aureus. In certain embodiments, dCas9 comprises dCas9 with substitutions at positions 10 and 580 of the amino acid sequence of dCas9 that inactivate the catalytic site. In certain embodiments, these substitutions are D10A and N580A. In certain embodiments, dCas9 is a small and inactivated Cas9 (dSaCas 9). In certain embodiments, the amino acid sequence of dSaCas9 comprises the following sequence:

in certain embodiments, the endonuclease domain comprises a sequence isolated or derived from Cas9, a transcription activator-like effector nuclease (TALEN), or a type IIS endonuclease. In certain embodiments, the type IIS endonuclease is AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, bmubi, bmgri, BmrI, BpmI, bpei, bppiei, bsari, BsgI, BspMI, bsbi, BsrDI, btgsi, EarI, eci, MmeI, nmeii, nmeiii, bbvcci, Bpu10I, bspeqi, bapi, baxi, csbsi, pcci, bbifii, bboii, bboqi, fo 36I, or clokii. In certain embodiments, the type IIS endonuclease is Clo 051. In certain embodiments, the DNA binding domain and endonuclease domain are covalently or non-covalently linked. In certain embodiments, the DNA binding domain and the endonuclease domain are covalently linked as a fusion protein.

In certain embodiments of the methods of the invention, including those in which at least one immune cell of the plurality of immune cells comprises a genetic modification and wherein the genetic modification reduces or inhibits expression of a T-cell receptor or Major Histocompatibility Complex (MHC), the plurality of immune cells comprises resting cells, activated cells, or a combination thereof. In certain embodiments, the plurality of immune cells comprises activated cells. In certain embodiments, the plurality of immune cells comprises resting cells. In certain embodiments, the plurality of immune cells comprises resting CAR-T cells, activated CAR-T cells, or a combination thereof. In certain embodiments, the plurality of immune cells comprises activated CAR-T cells. In certain embodiments, the plurality of immune cells comprises resting CAR-T cells.

In certain embodiments of the methods of the invention, at least one immune cell of the plurality of immune cells expresses two or more Chimeric Ligand Receptors (CLRs) that each specifically bind a target ligand on at least one target cell. In certain embodiments, a subset of the plurality of immune cells express two or more Chimeric Ligand Receptors (CLRs) that each specifically bind a target ligand on at least one target cell. In certain embodiments, the portion comprises at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of immune cells. In certain embodiments, each immune cell of the plurality of immune cells expresses two or more Chimeric Ligand Receptors (CLRs) each specifically binding a target ligand on at least one target cell. In certain embodiments, for example, the first CAR specifically binds to a first target ligand, the second CAR specifically binds to a second target ligand, and the first target ligand and the second target ligand are different. In certain embodiments, the first target ligand and the second target ligand are not homologous. In certain embodiments, the third or subsequent CAR specifically binds to a third or subsequent target ligand. In certain embodiments, the first target ligand, the second target ligand, and the third or subsequent target ligand are different. In certain embodiments, the first target ligand, the second target ligand, and the third or subsequent target ligand are not homologous.

In certain embodiments of the methods of the invention, the at least one target cell or plurality of target cells comprises a HSC, and the target ligand on the target HSC comprises one or more of c-KIT/CD117, CD45, CD34, Thy1/CD90, c-mpl/CD110, CD133, CD49f, ABCG2/CD338, carbonic anhydrase IX/CA9, CD123, and CD 150. In certain embodiments, at least one of the plurality of immune cells that deplete the target HSC comprises a CAR that specifically binds c-KIT, and, optionally, the CAR comprises the amino acid sequence:

in certain embodiments, at least one of the plurality of immune cells that deplete the target HSC comprises a CAR that specifically binds c-KIT, and, optionally, the CAR comprises the amino acid sequence:

wherein the sequence comprises an scFv that specifically binds c-KIT. In certain embodiments, at least one of the plurality of immune cells that deplete the target HSC comprises a CAR that specifically binds c-KIT, and, optionally, the CAR comprises the amino acid sequence:

wherein the sequence comprises an scFv that specifically binds c-KIT. In certain embodiments, at least one of the plurality of immune cells that deplete the target HSC comprises a CAR that specifically binds CD133, and, optionally, the CAR comprises the amino acid sequence:

wherein the sequence comprises an scFv that specifically binds CD 133. In certain embodiments, at least one of the plurality of immune cells that deplete the target HSC comprises a CAR that specifically binds CD133, and, optionally, the CAR comprises the amino acid sequence:

wherein the sequence comprises an scFv that specifically binds CD 133.

In certain embodiments of the methods of the invention, the at least one target cell or plurality of target cells comprises an immune cell, and the target ligand on the target immune cell comprises one or more of CD3, CD4, CD8, CD25, FoxP3, TCR α, TCR β, TCR α β, TCR γ λ, CD52, NK1.1, CD16, CD30, CD31, CD38, CD56, CD94, NKG2A, NKG2C, NKp30, NKp44, NKp46, CD9, CD103, and KIR.

In certain embodiments of the methods of the invention, the at least one target cell or plurality of target cells comprises HSCs and immune cells, the target ligand on the target HSCs comprises one or more of c-KIT/CD117, CD45, CD34, Thy1/CD90, c-mpl/CD110, CD133, CD49f, ABCG2/CD338, carbonic anhydrase IX/CA9, CD123, and CD150, and the target ligand on the target immune cells comprises one or more of CD3, CD4, CD8, CD25, FoxP3, TCR α TCR β, TCR γ λ, CD52, NK1.1, CD16, CD30, CD31, CD38, CD56, CD94, NKG2A, NKG2C, NKp30, NKp44, NKp46, CD9, CD103, and KIR.

In certain embodiments of the methods of the present invention, each of the one or more CLRs comprises (a) an extracellular domain comprising a ligand recognition region, (b) a transmembrane domain, and (c) an extracellular domain comprising at least one co-stimulatory domain. In certain embodiments, the ligand recognition region comprises one or more of a protein scaffold, a centryrin, a single chain variable fragment (scFv), a VHH, an immunoglobulin, and an antibody mimetic. In certain embodiments, the immunoglobulin is an antibody or fragment thereof of the IgA, IgD, IgE, IgG, or IgM isotype. In certain embodiments, the antibody fragment is a Complementarity Determining Region (CDR), a heavy chain CDR (including CDR1, CDR2, and/or CDR3), a light chain CDR (including CDR1, CDR2, and/or CDR3), an antigen binding fragment (Fab), a variable domain (Fv), a heavy chain variable region, a light chain variable region, an intact heavy chain, an intact light chain, one or more constant domains, an Fc (crystallizable fragment), or any combination thereof. In certain embodiments, the antibody mimetic comprises one or more of affibody, affilin, affimer, affitin, alphabody, anticalin, and avimer, designed ankyrin repeat protein (DARPin), Fynomer, Kunitz domain peptide, and monomer. In certain embodiments, at least one of the CLRs is bispecific. In certain embodiments, the CLRs are each bispecific. In certain embodiments, at least one of the CLRs is trispecific. In certain embodiments, the CLRs are each trispecific.

In certain embodiments of the methods of the invention, at least one immune cell in the composition comprising a plurality of immune cells comprises a dividing CLR. In certain embodiments, a dividing CLR comprises two or more CLRs having different intracellular domains that, when simultaneously expressed in at least one immune cell, increase or decrease the activity of the immune cell compared to an immune cell that does not express a dividing CLR or an immune cell that does not express a CLR.

In certain embodiments of the methods of the invention, at least one immune cell in the composition comprising a plurality of immune cells comprises a dividing CLR. In certain embodiments, including those in which concurrent expression reduces the activity of an immune cell, dividing CLRs comprises: (a) a first CLR comprising: an extracellular domain comprising a ligand recognition region, a transmembrane domain, and an intracellular domain comprising a primary intracellular signaling domain, a secondary intracellular signaling domain, and (b) a second CLR comprising: an extracellular domain comprising a ligand recognition region, a transmembrane domain, and an intracellular domain consisting of an inhibitory intracellular signaling domain. In certain embodiments, the primary intracellular signaling domain comprises a human CD3 ζ endodomain and the secondary intracellular signaling domain comprises a human 4-1BB, human CD28, human CD40, human ICOS, human MyD88, or human OX-40 intracellular segment. In certain embodiments, the primary intracellular signaling domain comprises a human CD3 ζ endodomain and the secondary intracellular signaling domain comprises human 4-1BB and human CD 28. In certain embodiments, the inhibitory intracellular signaling domain comprises signaling domains derived from PD1, CTLA4, LAG3, B7-H1, B7-1, CD160, BTLA, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, and TIGIT. Additional intracellular signaling components from these inhibitory intracellular signaling domains and other molecules that may be used in whole or in part include, but are not limited to, ITIM, ITSM, YVKM, PP2A, SHP2, KIEELE, and Y265. In certain embodiments, the second CLR selectively binds to a target on a non-target cell, thereby inducing the second CLR to inhibit the activity of the first CLR. In certain embodiments, the second CLR inhibits the ability of the first CLR to induce death of the target cell or a non-target cell.

In certain embodiments of the methods of the invention, the one or more CLRs bind the ligand with at least one affinity selected from the group consisting of: less than or equal to 10⁻⁹M, less than or equal to 10⁻¹⁰M, less than or equal to 10⁻¹¹M, less than or equal to 10⁻¹²M, less than or equal to 10⁻¹³M, less than or equal to 10⁻¹⁴M and less than or equal to 10⁻¹⁵K of M_D. In certain embodiments, K_DMeasured by surface plasmon resonance.

In certain embodiments of the methods of the present invention, the composition comprising a plurality of immune cells further comprises at least one pharmaceutically acceptable carrier.

In certain embodiments of the methods of the invention, the method further comprises administering to the subject an mobilizing composition. In certain embodiments, a composition comprising a plurality of immune cells (each comprising one or more CLRs) and an mobilizing composition are administered sequentially. In certain embodiments, wherein the mobilizing composition is administered prior to administration of the composition comprising a plurality of immune cells, each of which comprises one or more CLRs. In certain embodiments, the mobilizing composition is administered a period of time prior to administration of the composition comprising the plurality of immune cells (each of which comprises one or more CLRs), wherein the period of time is sufficient to allow HSCs to migrate from the bone marrow, e.g., to circulating blood, to increase entry of the composition comprising the plurality of immune cells into the target HSCs. In certain embodiments, the mobilizing composition is administered between 1 day and 7 days (inclusive) prior to administration of the composition comprising a plurality of immune cells, each of which comprises one or more CLRs. In certain embodiments, the mobilizing composition comprises granulocyte colony stimulating factor (G-CSF), plerixafor, or a combination thereof.

In certain embodiments of the methods of the present invention, the method further comprises administering to the subject an effective amount of a preconditioning composition to enhance the efficiency of the engraftment of the composition comprising the plurality of immune cells (each comprising one or more CLRs) and the elimination of the at least one target cell by the composition comprising the plurality of immune cells (each comprising one or more CLRs). In certain embodiments, the preconditioning composition suppresses the immune system. In certain embodiments, the preconditioning composition comprises chemotherapy, radiation therapy (including, but not limited to, local radiation and systemic radiation), autoimmune therapy, or an anti-rejection drug. In certain embodiments, the preconditioning composition does not comprise radiation therapy, localized radiation, or systemic radiation. In certain embodiments, the preconditioning composition comprises one or more lymphodepleting agents, myeloablative agents, chemotherapeutic agents, or a combination thereof. In certain embodiments, the preconditioning composition comprises a lymphodepleting agent. Exemplary lymphodepleting agents include, but are not limited to, cyclophosphamide and fludarabine. In certain embodiments, the preconditioning composition comprises a myeloablative agent. Exemplary myeloablative agents include, but are not limited to, low dose and/or partial radiotherapy. In certain embodiments, the preconditioning composition comprises a chemotherapeutic agent selected from the group consisting of busulfan, troosulfan, melphalan, and tiatepa.

In certain embodiments of the methods of the present invention, the method further comprises administering to the subject an effective amount of a preconditioning composition to enhance the efficiency of the engraftment of the composition comprising the plurality of immune cells (each comprising one or more CLRs) and the elimination of the at least one target cell by the composition comprising the plurality of immune cells (each comprising one or more CLRs). In certain embodiments, the preconditioning composition is administered to the subject prior to administering to the subject a composition comprising a plurality of immune cells (each of which comprises one or more CLRs). In certain embodiments, the preconditioning composition is administered to the subject 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or any number of minutes therebetween prior to administering the composition comprising the plurality of immune cells (each of which comprises one or more CLRs) to the subject. In certain embodiments, the preconditioning composition is administered to the subject 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, or any number of hours therebetween prior to administering the composition comprising a plurality of immune cells (each of which comprises one or more CLRs) to the subject.

In certain embodiments of the methods of the invention, at least one immune cell of the plurality of immune cells is pre-irradiated prior to administration to the subject. In certain embodiments of the methods of the invention, a portion of the plurality of immune cells are pre-irradiated prior to administration to the subject. In certain embodiments, the portion comprises at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of immune cells. In certain embodiments, each immune cell of the plurality of immune cells is pre-irradiated prior to administration to the subject.

In certain embodiments of the methods of the present invention, including those in which at least one or each immune cell of the plurality of immune cells is pre-irradiated prior to administration to the subject, the step of eliminating the plurality of immune cells comprises administering an effective amount of the plurality of pre-irradiated immune cells to the subject, thereby preventing proliferation and/or shortening survival of the plurality of pre-irradiated immune cells.

In certain embodiments of the methods of the invention, each immune cell of the plurality of immune cells comprises an inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide. In certain embodiments, the inducible caspase polypeptide comprises (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide. In certain embodiments, the inducible caspase polypeptide does not comprise a non-human sequence.

In certain embodiments of the methods of the invention, each HSC in the plurality of therapeutic HSCs comprises an inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide. In certain embodiments, the inducible caspase polypeptide comprises (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide. In certain embodiments, the inducible caspase polypeptide does not comprise a non-human sequence. In certain embodiments, the method further comprises administering to the subject a composition comprising an inducing agent, thereby initiating death of the plurality of therapeutic HSCs.

In certain embodiments of the methods of the invention, including those in which each immune cell of the plurality of immune cells comprises an inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide, the composition comprising the plurality of immune cells (each comprising one or more CLRs) further comprises an inducing agent. In certain embodiments of the methods of the invention, including those in which each immune cell of the plurality of immune cells comprises an inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide, the composition comprising the plurality of therapeutic HSCs further comprises an inducing agent.

In certain embodiments of the methods of the invention, at least one HSC in the plurality of therapeutic HSCs comprises a genetic modification. In certain embodiments, a portion of HSCs in the plurality of therapeutic HSCs comprise a genetic modification. In certain embodiments, the portion comprises at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween of the plurality of therapeutic HSCs. In certain embodiments, each HSC in the plurality of therapeutic HSCs comprises a genetic modification.

In certain embodiments of the methods of the invention, including those in which at least one HSC of the plurality of therapeutic HSCs comprises a genetic modification, the genetic modification is a single-chain break, a double-chain break, a sequence deletion, a sequence insertion, a sequence substitution, or any combination thereof. In certain embodiments, the sequence deletion, sequence insertion, sequence substitution, or a combination thereof comprises a sequence encoding an intron, an exon, a promoter, an enhancer, a transcriptional repressor, a CpG site, or any combination thereof.

In certain embodiments of the methods of the invention, including those in which at least one HSC of the plurality of therapeutic HSCs comprises a genetic modification, the genetic modification is introduced by a composition comprising a DNA binding domain and an endonuclease domain. In certain embodiments, the DNA binding domain comprises a guide RNA. In certain embodiments, the DNA-binding domain comprises a sequence isolated or derived from Cas9, a transcription activator-like effector nuclease (TALEN), a centromere, and a promoter factor 1 (Cpf1) or a Zinc Finger Nuclease (ZFN).

an exemplary dCas9-Clo051 nuclease domain may comprise, consist essentially of, or consist of the following amino acid sequence (Clo051 sequence underlined (SEQ ID NO:34), linker in bold italics, dCas9 sequence in italics):

in certain embodiments of the methods of the invention, including those in which at least one HSC of the plurality of therapeutic HSCs comprises a genetic modification, the genetic modification is introduced by induction of homologous recombination, insertion of a single stranded oligodeoxynucleotide (ssODN), or a transposition event. In certain embodiments, the genetic modification results in insertion of a sequence. In certain embodiments, the transposition event results in the insertion of a functional transgene. In certain embodiments, the transposon comprises a functional transgene, and wherein the transposon is a piggyBac transposon. In certain embodiments, the transposon-containing HSC further comprises a super piggyBac transposase.

In certain embodiments of the methods of the invention, the at least one target HSC comprises a genetic modification introduced by induction of homologous recombination, insertion of a single stranded oligodeoxynucleotide (ssODN), or transposition event. In certain embodiments, the genetic modification results in insertion of a sequence. In certain embodiments, the transposition event results in the insertion of a functional and/or therapeutic transgene. In certain embodiments, the transposon comprises a functional and/or therapeutic transgene, and wherein the transposon is a piggyBac transposon. In certain embodiments, the at least one target HSC comprising a transposon further comprises a super piggyBac transposase. In certain embodiments, the at least one target HSC is an endogenous HSC of the subject.

The present invention provides compositions comprising the transposons of the invention. In certain embodiments, the composition may further comprise a plasmid comprising a sequence encoding a transposase. The sequence encoding the transposase can be an mRNA sequence.

The transposon of the invention can comprise a piggyBac transposon. The transposons of the invention can include piggyBac transposases or compatible enzymes. In certain embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac or super-piggyBac System (SPB) transposase. In certain embodiments, and particularly those in which the transposase is a super piggyBac Size (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

In certain embodiments of the methods of the invention, the transposase is a piggyBac chamber (PB) transposase. piggybac (pb) transposases can comprise or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percent identity therebetween, with:

in certain embodiments of the methods of the invention, the transposase is a PiggyBac (PB) transposase comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of:

in certain embodiments, the transposase is a piggyBac b (PB) transposase comprising or consisting of an amino acid sequence having amino acid substitutions at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 1. In certain embodiments, the transposase is a piggyBac chamber (PB) transposase comprising or consisting of an amino acid sequence having amino acid substitutions at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 1. In certain embodiments, the transposase is a piggyBac chamber (PB) transposase comprising or consisting of an amino acid sequence having amino acid substitutions at each of the following positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 1. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO:1 is a valine (V) for isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO:1 is a serine (S) to glycine (G) substitution. In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO:1 is a valine (V) for methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO:1 is a lysine (K) for an asparagine (N).

In certain embodiments of the methods of the invention, the transposase is a super piggyBac chamber (SPB) transposase. In certain embodiments, a Super PiggyBac (SPB) transposase of the present invention can comprise or consist of the amino acid sequence of the sequence of SEQ ID NO:1, wherein the amino acid substitution at position 30 is a valine (V) for isoleucine (I), the amino acid substitution at position 165 is a serine (S) for glycine (G), the amino acid substitution at position 282 is a valine (V) for methionine (M), and the amino acid substitution at position 538 is a lysine (K) for asparagine (N). In certain embodiments, a super piggyBac-chamber (SPB) transposase can comprise or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percent identity therebetween, with:

in certain embodiments of the methods of the present invention, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, the piggyBac or super-piggyBac system transposase can further comprise an amino acid substitution at one or more of the following positions of the sequence of SEQ ID No. 1 or SEQ ID No. 2: 3. 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591. In certain embodiments, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, the piggyBac or super piggyBac-system transposase can further comprise amino acid substitutions at one or more of the following positions: 46. 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552, and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO:1 or SEQ ID NO:2 is an asparagine (N) substituted serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO:1 or SEQ ID NO:2 is a serine (S) substitution for alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO:1 or SEQ ID NO:2 is a threonine (T) substitution for alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO:1 or SEQ ID NO:2 is a tryptophan (W) to isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO:1 or SEQ ID NO:2 is a proline (P) substituted for serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO:1 or SEQ ID NO:2 is a proline (P) substituted for arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO:1 or SEQ ID NO:2 is an alanine (A) for a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO:1 or SEQ ID NO:2 is a leucine (L) substituted cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO:1 or SEQ ID NO:2 is a lysine (K) substituted for tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO:1 or SEQ ID NO:2 is a histidine (H) substitution for tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO:1 or SEQ ID NO:2 is a leucine (L) substitution for phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO:1 or SEQ ID NO:2 is an isoleucine (I) substitution for phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO:1 or SEQ ID NO:2 is a valine (V) substitution for phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO:1 or SEQ ID NO:2 is a leucine (L) substituted methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO:1 or SEQ ID NO:2 is a glycine (G) substituted alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO:1 or SEQ ID NO:2 is a tryptophan (W) to phenylalanine (F) substitution. In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO:1 or SEQ ID NO:2 is a proline (P) substituted for valine (V). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO:1 or SEQ ID NO:2 is a phenylalanine (F) substitution for valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO:1 or SEQ ID NO:2 is a phenylalanine (F) substituted methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO:1 or SEQ ID NO:2 is an arginine (R) to leucine (L). In certain embodiments, SEQ ID NO:1 or the amino acid substitution at position 240 of SEQ ID NO:1 is a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO:1 or SEQ ID NO:2 is a leucine (L) substitution for phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO:1 or SEQ ID NO:2 is a lysine (K) substituted for a proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO:1 or SEQ ID NO:2 is a serine (S) substitution for asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO:1 or SEQ ID NO:2 is a tryptophan (W) to leucine (L) substitution. In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO:1 or SEQ ID NO:2 is a tyrosine (Y) substituted leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO:1 or SEQ ID NO:2 is a phenylalanine (F) substituted leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO:1 or SEQ ID NO:2 is a leucine (L) to methionine (M) substitution. In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO:1 or SEQ ID NO:2 is an alanine (A) to methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO:1 or SEQ ID NO:2 is a valine (V) for methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO:1 or SEQ ID NO:2 is an isoleucine (I) substituted proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO:1 or SEQ ID NO:2 is a valine substituted proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO:1 or SEQ ID NO:2 is a lysine (K) substituted for arginine (R). In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO:1 or SEQ ID NO:2 is a glycine (G) substitution for threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO:1 or SEQ ID NO:2 is an arginine (R) substituted for tyrosine (Y). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO:1 or SEQ ID NO:2 is a valine (V) substitution for tyrosine (Y). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO:1 or SEQ ID NO:2 is a glycine (G) substituted cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO:1 or SEQ ID NO:2 is a leucine (L) substituted cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO:1 or SEQ ID NO:2 is a histidine (H) substitution for aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO:1 or SEQ ID NO:2 is an isoleucine (I) substitution for valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO:1 or SEQ ID NO:2 is a tyrosine (Y) substituted methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO:1 or SEQ ID NO:2 is a phenylalanine (F) to leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO:1 or SEQ ID NO:2 is a lysine (K) substituted for a serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO:1 or SEQ ID NO:2 is a leucine (L) to methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO:1 or SEQ ID NO:2 is an isoleucine (I) for methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO:2 or SEQ ID NO:1 is a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO:1 or SEQ ID NO:2 is a threonine (T) substitution for alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO:1 or SEQ ID NO:2 is a proline (P) substituted for glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO:1 or SEQ ID NO:2 is an arginine (R) for glutamine (Q). In certain embodiments of the methods of the present invention, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac ™ transposase can comprise or the super-piggyBac ™ transposase can further comprise an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID No. 1 or SEQ ID No. 2. In certain embodiments of the methods of the present invention, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac ™ transposase can comprise or the super-piggyBac ™ transposase can further comprise amino acid substitutions at 2, 3,4, 5,6 or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID No. 1 or SEQ ID No. 2. In certain embodiments, including those in which the transposase is comprised of the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac-backup transposase can comprise or the super-piggyBac-backup transposase can further comprise amino acid substitutions at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO:1 or SEQ ID NO: 2. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO:1 or SEQ ID NO:2 is a proline (P) substituted for serine (S). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO:1 or SEQ ID NO:2 is a valine (V) for methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO:1 or SEQ ID NO:2 is an alanine (A) to arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID NO:1 or SEQ ID NO:2 is an alanine (A) to lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO:1 or SEQ ID NO:2 is an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO:1 or SEQ ID NO:2 is a glycine (G) substituted serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO:1 or SEQ ID NO:2 is a serine (S) substitution for asparagine (N). In certain embodiments, the piggyBac-box transposase can comprise a substitution of valine (V) for methionine (M) at position 194 of SEQ ID NO: 1. In certain embodiments, including those in which the piggyBac-backup transposase may comprise a substitution of valine (V) for methionine (M) at position 194 of SEQ ID NO:1, the piggyBac-backup transposase may further comprise amino acid substitutions at positions 372, 375, and 450 of the sequence of SEQ ID NO:1 or SEQ ID NO: 2. In certain embodiments, the piggyBac-transposase can comprise a substitution of valine (V) for methionine (M) at position 194 of SEQ ID NO:1, a substitution of alanine (A) for arginine (R) at position 372 of SEQ ID NO:1, and a substitution of alanine (A) for lysine (K) at position 375 of SEQ ID NO: 1. In certain embodiments, the piggyBac-transposase can comprise a substitution of valine (V) for methionine (M) at position 194 of SEQ ID NO:1, a substitution of alanine (A) for arginine (R) at position 372 of SEQ ID NO:1, a substitution of alanine (A) for lysine (K) at position 375 of SEQ ID NO:1, and a substitution of asparagine (N) for aspartic acid (D) at position 450 of SEQ ID NO: 1.

In certain embodiments of the methods of the invention, the subject is a human.

In certain embodiments of the methods of the invention, the subject has, or is at risk for developing, an immune system disease or disorder.

In certain embodiments of the methods of the invention, the subject has an autoimmune disease or disorder. In certain embodiments, the autoimmune disease or disorder is Acute Disseminated Encephalomyelitis (ADEM), acute necrotizing leukoencephalitis, addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic abnormalities, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immune deficiency, Autoimmune Inner Ear Disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, Autoimmune Thrombocytopenic Purpura (ATP), autoimmune thyroid disease, urticaria, axonal and neuronal neuropathies, Barlow's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas' disease, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), chronic relapsing multifocal myelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogen syndrome, cold agglutinin disease, congenital heart conduction block, coxsackie myocarditis, CREST disease, basic mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, German Leeb syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evens syndrome, fibrotic paulitis, giant cell arteritis (temporal arteritis), myocarditis, and cardiomyopathy, Glomerulonephritis, Goodpasture's syndrome, granuloma with polyangiitis (GPA), graves' disease, guillain-barre syndrome, hashimoto's encephalitis, hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypoproteinemia, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephropathy, IgG 4-associated sclerosing disease, immunoregulatory lipoprotein, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, kawasaki syndrome, lambert-eaton syndrome, fragmented leukocyte vasculitis, lichen planus, lichen sclerosus, xylem conjunctivitis, linear IgA disease (LAD), lupus (SLE, lyme disease, chronic meniere's disease, microscopic vasculitis, Mixed Connective Tissue Disease (MCTD), muckle ulcer, muller's disease (MCTD), multiple sclerosis, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, lethargy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, recurrent rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorder associated with streptococci), paraneoplastic cerebellar degeneration, Paroxysmal Nocturnal Hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars plana (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous myelitis, pernicious anemia, POEMS, polyarteritis nodosa, autoimmune polyglandular syndrome type I, autoimmune polyglandular syndrome type II, autoimmune polyglandular syndrome type III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progestational dermatitis, and the like, Primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell dysplasia, raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, reiter's syndrome, recurrent polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, Subacute Bacterial Endocarditis (SBE), susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, Undifferentiated Connective Tissue Disease (UCTD), uveitis, vasculitis, vesicular skin disease, or vitiligo.

In certain embodiments of the methods of the invention, the subject is immunocompromised.

In certain embodiments of the methods of the invention, the subject has an inflammatory disease.

In certain embodiments of the methods of the invention, the subject has or is at risk of developing an immune system disease or disorder. In certain embodiments, the subject has a genetic or epigenetic marker of the immune system disease or disorder. In certain embodiments, the immune system disease or disorder is induced by a medical intervention.

In certain embodiments of the methods of the invention, the subject has a genetic or epigenetic marker of the immune system disease or disorder.

In certain embodiments of the methods of the invention, the subject has a genetic or epigenetic marker of a disease or disorder that is manifested in blood cells, immune cells circulating in the blood, bone marrow cells, or precursor cells thereof, hi certain embodiments, the precursor cells are Hematopoietic Stem Cells (HSCs), hi certain embodiments, the disease or disorder is cancer, hi certain embodiments, the cancer is lymphoma, leukemia, myeloma, or a malignant immunoproliferative disease, hi certain embodiments, the lymphoma is hodgkin's lymphoma, non-hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma (aitt), hepatosplenic T-cell lymphoma, B-cell lymphoma, reticuloendothelial disease, reticulocytoma, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, Mantle Cell Lymphoma (MCL), burkitt's lymphoma, wakstrom's lymphoma, watsell large B-cell lymphoma, lymphoblastic large B-cell lymphoma, lymphomatoid lymphomatosis, lymphomatoid marginal zone lymphoma, lymphomatosis of primary lymphomas, lymphomatosis of the vessels, lymphomatosis of the type of the splenocytes, or lymphomatosis of the type, lymphomatoid granulomatosis, or lymphomatosis of the type, and lymphomatosis of the type of the splen.

In certain embodiments, the leukemia is Plasma Cell Leukemia (PCL), acute erythrocytosis and erythroleukemia, acute erythroblastic myelopathy, acute erythroid leukemia, Heilmeyer-Sch ö ner disease, acute megakaryoblastic leukemia (AMKL), mast cell leukemia, pan-myelogenous leukemia, acute myelofibrosis accompanied by myelofibrosis (APMF), lymphosarcoma cell leukemia, osteogenic chronic myelogenous leukemia, chronic stem cell leukemia, chronic stage leukemia, accelerated myeloid leukemia (acute myeloblastic leukemia), acute myeloblastic leukemia, acute myeloblastic leukemia, acute myeloblastic.

In certain embodiments of the methods of the invention, the subject has a genetic or epigenetic marker of a disease or disorder manifested in blood cells, immune cells circulating in the blood, bone marrow cells, or precursor cells thereof. In certain embodiments, the precursor cell is a Hematopoietic Stem Cell (HSC). In certain embodiments, the disease or disorder is anemia. In certain embodiments, the anemia is hemolytic anemia, autoimmune hemolytic anemia, congenital hemolytic anemia, aplastic anemia, β -thalassemia, congenital erythroid dysplasia, congenital erythropoietic anemia, glucose 6-phosphate dehydrogenase deficiency, fanconi anemia, hereditary spherocytosis, hereditary elliptocytosis, hereditary hemidysmorphic polycythemia, hereditary persistence of fetal hemoglobin, hereditary stomatocytosis, hexokinase deficiency, anemia, hypopigmentary anemia, erythropoietic inefficiency, megaloblastic anemia, myelogenous anemia, neuroacanthocytosis, chorea-acanthocytosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, Rh deficiency syndrome, sickle cell disease, iron granulocytic anemia, stomatogenic ovoerythrocytosis (stomatocytocytic ovatosis), thalassemia, triosephosphate isomerase (TPI) deficiency or warm autoimmune hemolytic anemia.

In certain embodiments of the methods of the invention, the subject has a genetic or epigenetic marker of a disease or disorder manifested in a secondary target cell that can be contacted by a composition comprising a plurality of therapeutic HSCs. In certain embodiments, the secondary target cell is a stem cell or a progenitor cell. In certain embodiments, the progenitor cell is an osteoblast cell. In certain embodiments, at least one HSC in a composition comprising a plurality of therapeutic HSCs is modified to secrete a ligand, peptide, or protein that enhances the activity of osteoblasts. In certain embodiments, a composition comprising a plurality of therapeutic HSCs treats or prevents a disease or disorder associated with abnormal osteoblast function. In certain embodiments, the subject has one or more genetic or epigenetic signatures of a disease or disorder associated with abnormal osteoblast function. In certain embodiments, the disease or disorder associated with aberrant osteoblast function is paget's disease, hypophosphatemia or osteoporosis.

In certain embodiments of the methods of the invention, the subject has a genetic or epigenetic marker of a disease or disorder manifested in a secondary target cell that can be contacted by a composition comprising a plurality of therapeutic HSCs. In certain embodiments, the secondary target cell is a differentiated cell. In certain embodiments, the differentiated cell is a red blood cell, a white blood cell, a monocyte, a granulocyte, a platelet, or a dendritic cell. In certain embodiments, at least one HSC in a composition comprising a plurality of therapeutic HSCs is modified to secrete a ligand, peptide, or protein that enhances the activity of granulocytes. In certain embodiments, a composition comprising a plurality of therapeutic HSCs treats or prevents a disease or disorder associated with abnormal granulocyte function. In certain embodiments, the subject has one or more genetic or epigenetic signatures of a disease or disorder associated with aberrant granulocyte function. In certain embodiments, the disease or disorder associated with abnormal granulocyte function is chronic granulomatous disease.

In certain embodiments of the methods of the invention, the subject has or is at risk of developing an immune system disease or disorder. In certain embodiments, the immune system disease or disorder is induced by infection. In certain embodiments, the subject is at risk of developing an immune system disease or disorder due to past, present, or potential infection. In certain embodiments, the infection is a viral, bacterial, and/or microbial infection. In certain embodiments, the infection is a viral infection. In certain embodiments, the infection is a viral infection, and the subject becomes immunocompromised due to the infection. In certain embodiments, the subject is exposed to or infected with HIV. In certain embodiments, the subject has developed AIDS. In certain embodiments, the infection is a viral infection. In certain embodiments, the infection is a viral infection and the subject develops cancer.

In certain embodiments of the methods of the present invention, administration of the composition comprising a plurality of immune cells is systemic. In certain embodiments, the composition is administered via an intravenous route.

In certain embodiments of the methods of the present invention, administration of the composition comprising a plurality of immune cells is topical. In certain embodiments, the composition is administered via intraosseous, intraspinal, or intracerebral infusion.

In certain embodiments of the methods of the invention, the administration of the composition comprising a plurality of therapeutic HSCs is systemic. In certain embodiments, the composition is administered via an intravenous route.

In certain embodiments of the methods of the invention, administration of the composition comprising a plurality of therapeutic HSCs is topical. In certain embodiments, the composition is administered via intraosseous infusion.

In certain embodiments of the methods of the present invention, the composition comprising a plurality of therapeutic HSCs further comprises at least one pharmaceutically acceptable carrier. In certain embodiments, the composition comprising a plurality of therapeutic HSCs further comprises an inducing agent.

In certain embodiments of the methods of the invention, at least one HSC in the plurality of therapeutic HSCs is genetically modified. In certain embodiments, each HSC in the plurality of therapeutic HSCs is genetically modified. In certain embodiments of the methods of the invention, the subject has an immune disease or disorder, and wherein the plurality of therapeutic HSCs ameliorate a sign or symptom of the immune disease or disorder. In certain embodiments, at least one HSC of the plurality of therapeutic HSCs is genetically modified to improve signs or symptoms of an immune disease or disorder in the subject. In certain embodiments, each HSC in the plurality of therapeutic HSCs is genetically modified to improve signs or symptoms of an immune disease or disorder in the subject.

In certain embodiments of the methods of the invention, the subject has a genetic or epigenetic marker of a disease or disorder that is manifested in blood cells, immune cells circulating in the blood, bone marrow cells, or their precursors, and the plurality of therapeutic HSCs ameliorate a sign or symptom of the disease or disorder. In certain embodiments, the disease or disorder is a blood coagulation disorder. In certain embodiments, at least one HSC of the plurality of therapeutic HSCs has been modified to secrete a protein that ameliorates a sign or symptom of the coagulation disorder. In certain embodiments, a majority of HSCs in the plurality of therapeutic HSCs have been modified to secrete proteins that improve signs or symptoms of a coagulation disorder. In certain embodiments, each HSC in the plurality of therapeutic HSCs has been modified to secrete proteins that improve signs or symptoms of a coagulation disorder. In certain embodiments, at least one HSC, a majority of HSCs, or each HSC in the plurality of therapeutic HSCs is modified to secrete a protein that ameliorates a sign or symptom of the coagulation disorder. In certain embodiments, at least one HSC, a majority of HSCs, or each HSC of the plurality of therapeutic HSCs is modified to secrete one or more coagulation factors.

In certain embodiments of the methods of the invention, the subject has a genetic or epigenetic marker of a glycogen storage disease or disorder, and the plurality of therapeutic HSCs ameliorate a sign or symptom of the glycogen storage disease or disorder. In certain embodiments, the glycogen storage disease or disorder is glycogen storage disease type 0 (GSD), GSD type I, GSD type II, GSD type III, GSD type IV, GSD type V, GSD type VI, GSD type VII, GSD type IX, GSD type X, GSD type XI, GSD type XII, or GSD type XIII. In certain embodiments, at least one, a majority, or each HSC of the plurality of therapeutic HSCs is modified to secrete one or more of glycogen synthase, glucose-6-phosphatase, acid alpha-glucosidase, glycogen debranching enzyme, glycogen branching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphate fructokinase, phosphorylase kinase, glucose transporter GLUT2, aldolase a, or beta-enolase, and wherein the plurality of therapeutic HSCs ameliorate a sign or symptom of GSD type 0, GSD type I, GSD type II, GSD type III, GSD type IV, GSD type V, GSD type VI, GSD type VII, GSD type IX, GSD type X, GSD type XI, GSD type XII, or GSD type XIII, respectively.

In certain embodiments of the methods of the invention, the subject has a genetic or epigenetic marker of an immune system disease or disorder, at least one HSC, a portion of HSCs, or each HSC of the plurality of therapeutic HSCs comprises a genetic modification, and at least one HSC, a portion of HSCs, or each HSC of the plurality of therapeutic HSCs does not comprise a genetic or epigenetic marker. In certain embodiments, the genetic modification removes a genetic or epigenetic marker.

In certain embodiments of the methods of the invention, at least one HSC in the composition comprising a plurality of therapeutic HSCs is autologous. In certain embodiments, each HSC in the composition comprising a plurality of therapeutic HSCs is autologous. In certain embodiments, at least one genetically modified HSC in the composition comprising a plurality of therapeutic HSCs is autologous. In certain embodiments, each genetically modified HSC in the composition comprising a plurality of therapeutic HSCs is autologous.

In certain embodiments of the methods of the invention, at least one HSC in the composition comprising a plurality of therapeutic HSCs is allogeneic. In certain embodiments, each HSC in the composition comprising a plurality of therapeutic HSCs is allogeneic. In certain embodiments, at least one genetically modified HSC in the composition comprising a plurality of therapeutic HSCs is allogeneic. In certain embodiments, each genetically modified HSC in the composition comprising a plurality of therapeutic HSCs is allogeneic.

In certain embodiments of the methods of the invention, the methods treat or prevent the onset or progression of graft versus host disease (GvHD). In certain embodiments, treating GvHD comprises reducing signs or symptoms of GvHD. In certain embodiments, the GvHD is acute GvHD. In certain embodiments, the GvHD is chronic GvHD. In certain embodiments, signs or symptoms of GvHD include skin rash, blistering skin, nausea, vomiting, abdominal cramps, diarrhea, loss of appetite, jaundice, dry mouth, dry laryngo pharynx, excessive dry mouth, excessive dry laryngo pharynx, ulcers of the mouth or throat, dry bronchial tissue, dry endothelial tissue, dry superficial tissue, shedding of skin plaques, discoloration of skin, scarring of skin, decreased joint mobility with scarring of skin, hair loss with skin injury, loss of tear formation leading to dry eye, or any combination thereof.

In certain embodiments of the methods of the invention, including those wherein the methods treat or prevent the onset or progression of graft versus host disease (GvHD), the subject is a transplant recipient. In certain embodiments, the composition comprising a plurality of therapeutic HSCs is administered to the subject prior to administration of the transplant, and wherein the plurality of therapeutic HSCs and the transplant are isolated or derived from the same donor. In certain embodiments, the method further comprises a period of time after administering a composition comprising a plurality of HSCs sufficient to tolerate tolerance of the subject's immune system to the transplant. In certain embodiments, the graft comprises a cell, a tissue graft, an organ graft, or any combination thereof. In certain embodiments, the organ is a solid organ.

Brief Description of Drawings

Figure 1 is a schematic depicting exemplary inducible truncated caspase 9 polypeptides of the invention.

Fig. 2A-B are a series of graphs depicting results of assessing the in vitro efficacy of an induced pro-apoptotic polypeptide (iC9 safety switch) of the present invention using the exemplary inducer AP 1903. The CARTyrin expressing cells of the invention A) were thawed and left to stand overnight, or B) activated for 5 days with ImmunoCult-s human CD3/CD28/CD 2T cell activator and treated with AP1903 at the indicated concentration for the indicated length of time. All data points were collected in triplicate and relative viability was determined by dividing the number of viable cells in the treated group by the average number of viable cells in the untreated group per 1500 bead events collected. Between all dose levels tested, greater than 80% of the non-activated carbonin-expressing cells were eliminated from culture at 24 hours. In unactivated cells, there was no observable difference between the 24 hour and 48 hour time points. However, in activated CARTyrin-expressing cells, a dose response and a time response were observed. At 12 hours after AP1903 administration, >65% of the cells were killed by concentrations as low as 1 nM. The data indicate that the iC9 safety switch is both functionally expressed and effective in CARTyrin expressing cells. The AP1903/iC9 system is more effective when used against activated cells when compared to unactivated cells. Upon activation of cells, expression of CARTyrin is increased and vector design is provided; expression of iC9 may also be increased. Thus, activated cells can express higher levels of iC9, making them more sensitive to AP 1903. In many embodiments, the activated cell will be the target if and when a safety switch is used. These data confirm that activated cells are indeed more sensitive to AP1903, with >95% of cells being killed at 48 hours.

FIG. 3 is a schematic diagram comparing a conventional method of eliminating HSCs prior to transplantation using a genotoxic agent such as systemic irradiation or busulfan (top sequence) with the method of the present invention (bottom sequence). As shown in this figure, the compositions and methods of the present invention result in a non-genotoxic method that achieves excellent transplantation of HSCs following transplantation that is functional and maintains healthy levels of blood cell production.

Figure 4 is a schematic depicting possible combinations of surface HSC markers for autologous or heterologous CAR tandem targeting to minimize depletion of non-HSCs, including Hematopoietic Progenitor Cells (HPCs).

Figure 5A is a schematic drawing depicting a CAR construct with ScFv sequences against cells expressing c-kit or CD 133. The CAR construct depicts an exemplary CAR sequence coupled to an exemplary signal domain encoded by the mRNA used to generate the CAR-T cells.

FIG. 5B is a series of sequences of exemplary c-kit ScFv (1) (SEQ ID NO:69), c-kit ScFv (2) (SEQ ID NO:70), c-kit ScFv (3) (SEQ ID NO:71), c-kit ScFv (4) (SEQ ID NO:72), c-kit ScFv (5) (SEQ ID NO:73), c-kit ScFv (6) (SEQ ID NO:74), and c-kit ScFv (7) (SEQ ID NO:75) that can be used in the exemplary CAR depicted in FIG. 5A.

FIG. 5C is an exemplary C-kit ScFv (8) (SEQ ID NO: 76); exemplary c-kit ligand (1) (SEQ ID NO:77), c-kit ligand (2) (SEQ ID NO:78) and mouse c-kit ligand (SEQ ID NO: 79); and a series of sequences of exemplary CD133scFv (1) (SEQ ID NO:80), CD133scFv (2) (SEQ ID NO:81), and CD133scFv (3) (SEQ ID NO:82), which can be used in the exemplary CAR depicted in FIG. 5A.

FIG. 5D is a series of sequences of exemplary CD133scFv (4) (SEQ ID NO:83), CD133scFv (5) (SEQ ID NO:84), CD133scFv (6) (SEQ ID NO:85), CD133scFv (7) (SEQ ID NO:86), and CD133scFv (8) (SEQ ID NO:87) that can be used in the exemplary CAR depicted in FIG. 5A. The sequence of the exemplary CAR CAR depicted in FIG. 5A is also provided (SEQ ID NO: 88).

FIGS. 6A-E are a series of graphs depicting results of assessing the in vitro potency of CAR-T cells in specifically targeting c-kit (CD117) or prominin-1(CD133) -expressing human hematopoietic cells. CD3/CD28 stimulated pan T cells isolated from human peripheral blood were electroporated with mRNA encoding each CAR candidate for c-kit or CD133 (fig. 5). The next day after mRNA introduction, CAR expression of antibody-directed ScFv sequences was determined by anti-mouse IgG staining and flow cytometry (fig. 6A). Activation of effector CAR-T cells in the presence of target cells was demonstrated by degranulation based on CD107a expression at 5 hours (effector: target cell ratio 3: 1). TF-1 cells that endogenously and homogeneously expressed c-kit elicited the highest activation of CAR-T cells against c-kit, but less activation when mixed with Raji cells that did not express c-kit at a ratio of 5% (FIG. 6B). CAR-T cell activation was observed similarly to co-culture with human bone marrow cells, but no significant activation was observed beyond the mock CAR-T control cells after co-culture with the C-kit expressing mouse EML-C1 cell line (figure 6B). After electroporation of CD 133-encoding mRNA, TF-1 cells were allowed to express CD133 as determined by anti-CD 133 antibody staining and flow cytometry (data not shown). These transfected cells enabled activation of CAR-T cells carrying four of the eight anti-CD 133ScFv sequences. Less anti-CD 133 CAR-T stimulation was shown for CD133 expressing TF-1 cells mixed with Raji cells not expressing CD133 at a ratio of 5% or for human bone marrow cells (fig. 6C). After 2 days of CAR-T cell co-culture with human bone marrow (effector: target ratio 3:1), cells were stained with anti-human CD34, CD117 and CD133 antibodies and analyzed by flow cytometry or plated in methylcellulose cultures supplemented with human growth factors (methods cult, H4434) for the generation of hematopoietic Colonies (CFU) over 12 days. Flow cytometry analysis within the CD34 positive population showed a reduced proportion of c-kit positive cells for 3 of 6 anti-c-kit CAR-T cell candidates and a reduced proportion of CD133 positive cells for 3 of 7 anti-CD 133 CAR-T candidates (fig. 6D). CFU survival assay showed up to 85% depletion of functional hematopoietic progenitors for 7 of 8 anti-c-kit CAR-T cell candidates (fig. 6E).

FIG. 7 is a depiction of a method for targeting HSCspiggyBac(PB) schematic representation of transposon vector. Elongation factor-1 α (EF1 α) was used as a constitutive promoter to drive the tricistronic cassette consisting of inducible truncated caspase 9 (iCasp9), Chimeric Antigen Receptor (CAR) and dihydrofolate reductase resistance (DHFR) genes. The CAR region comprises the variable regions (VL and VH ScFv sequences) from anti-human C-kit and CD133IgG, which are linked to the VH and VL domainsThe D8a leader peptide, CD8a hinge, CD8a Transmembrane (TM) domain, 41BB costimulatory domain, and the signaling domain consisting of the CD3 zeta chain are coupled. Indicating the SV40 polyA signal and 250 bpcHS4 chromatin insulator. During transposition, the co-delivered PB transposase recognizes transposon-specific Inverted Terminal Repeats (ITRs) located at both ends of the transposon vector and effectively removes the contents from the original site in the delivered DNA plasmid and integrates it into the TTAA chromosomal site efficiently.

FIG. 8A is a series of diagrams depictingpiggyBac(PB) flow cytometric analysis of transposed anti-CD 117 or anti-CD 133 CAR-T cells. Human peripheral blood T-cells were previously treated with PB transposon pDNA (FIG. 7) together with coding super transposon pDNApiggyBac(SPB) electroporation of transposase mRNA. Phenotypic analysis was performed using antibodies against CD3, CD4, CD8, CD56, CD45RA, CD62L, CCR7, CD45RO, PD1, Tim3, Lag3, CD184/CXCR4, CD25, CD127 and CD 28.

Fig. 8B is a series of graphs depicting the proportion of CD4 and CD8 positive T cells present under each of the conditions shown in fig. 8A.

FIG. 8C is a series of graphs depictingpiggyBac(PB) flow cytometric analysis of transposed anti-CD 117 or anti-CD 133 CAR-T cells. Human peripheral blood T-cells were previously treated with PB transposon pDNA (FIG. 7) together with coding super transposon pDNApiggyBac(SPB) electroporation of transposase mRNA. Phenotypic analysis was performed using antibodies against CD3, CD4, CD8, CD56, CD45RA, CD62L, CCR7, CD45RO, PD1, Tim3, Lag3, CD184/CXCR4, CD25, CD127 and CD 28.

Fig. 8D is a series of graphs depicting the proportion of CD4 and CD8 positive T cells present under each of the conditions shown in fig. 8C.

FIGS. 9A-B are a pair of graphs, which are depicted bypiggyBac(PB) percent survival of transposed CAR-T cells targeted to the posterior bone marrow hematopoietic progenitor cells. After 2 days of CAR-T cell co-culture with human or monkey (cynomolgus monkey) bone marrow cells (effector: target ratio of 3:1), cells were plated in methylcellulose cultures supplemented with human growth factors (methods cult., H4434) for the generation of hematopoietic Colonies (CFU) over 12 days. CFU survival assay showed for 8 anti-c-kit CAR-T cell candidatesOf the 3, human functional hematopoietic progenitor cells were depleted by more than 70% (fig. 9A). CAR-T cells encoding these same anti-c-kit ScFv sequences also depleted hematopoietic progenitors from monkey bone marrow to indicate cross-reactivity with this species.

FIGS. 10A-D are a pair of graphs showing that anti-c-kit and anti-CD 133 CAR-T cells deplete cobblestone region-forming cells (CAFC). Human mPB CD34+ cells were co-cultured with anti-c-kit CAR-T cells encoding c-kit ScFv (2) (effector: target ratio 3:1) or anti-CD 133 CAR-T cells encoding CD133ScFv (3) for 24 hours (figure 5). The co-culture was then treated with 10 nM AP1903 for 24 hours to remove the effects due topiggyBacCo-expressed CAR-T cells of iC9 in transposon (FIG. 1) and cells supplemented with 10^-6M hydrocortisone in MyeloCult medium (Stem Cell Technologies) in serial dilutions was plated on a pre-established and irradiated (30 Gy) MS-5 bone marrow stromal Cell layer in 96-well plates. At weeks 2 and 5 in LTC, wells forming positive or negative were counted for CAFCs and CAFC frequency and number determined by limiting dilution analysis using L-Calc software (Stem Cell Technologies).

FIGS. 11A-B are a series of diagrams depicting exemplary PB vector constructs and methods of manufacture: (A) a constitutive promoter is used to drive a tricistronic cassette consisting of a safety switch, a Chimeric Antigen Receptor (CAR) and a selection gene with flanking chromatin insulators; (B) isolation of pan T cells from apheresis products, and then use of anti-CD 117 or anti-CD 133 CARpiggyBacTransposon plasmid DNA and in vitro transcriptionpiggyBacA transposase mRNA is electroporated. The electroporated cells are then activated, expanded, and selected, and then frozen. The process produces>1 x 10⁹A cell in which CAR is expressed>95%。

FIGS. 12A-B are a series of graphs depicting exemplary PB CAR-T phenotypes: following the manufacturing process, PB CAR-T cells for CD117 and CD133 antigens were assessed for typical T cell markers by flow cytometry. (A) Express CD4, CD8, and memory markers that indicate the stem cell memory phenotype of PB CAR-T cells; (B) PB CAR-T cells express CXCR4, a marker commonly associated with bone marrow homing.

FIGS. 13A-B are a series of graphs depicting anti-CD 117 or CD133 CAR-T cells against CD34⁺CD38^-Exemplary Activity of progenitor cell populations and peripheral blood CD34 automated to mobilize⁺CFU of cells: CD34 isolated from mobilized human peripheral blood⁺Cells were incubated with anti-c-kit and CD133 CAR-T cells for 48 hours, followed by FACS phenotypic analysis of the remaining cells (A) and CFU survival assay (B). anti-CD 117 CAR-T depletion from primary CD34⁺CD38^-Group of ckit⁺And CD133⁺Of cells>95% while anti-CD 133 CAR-T depletes CD133+ cells from the population>90% (A). anti-CD 117 and CD133 CAR-T cells also reduced colony formation at all E: T ratios tested.

Fig. 14 is a pair of graphs and corresponding photographs depicting exemplary activity of anti-CD 117 or CD133 CAR-T cells against long-term cobblestone region forming cells (CAFCs): after 2 days of CAR-T cells co-culture with mobilized human peripheral blood CD34+ cells (effector to target ratio of 3:1), cells were plated on MS-5 stromal cells in serial dilutions for CAFC generation over 2 months. 5 weeks after plating, both CAR-T cells significantly reduced the frequency of CAFCs, indicating that these CAR-T cells successfully target very primitive cells.

FIGS. 15A-C are a series of charts depicting bone marrow homing of PB CAR-T cells: PB CAR-T cells were cultured with (+) or without (-) factor to increase CXCR4 expression. Cells from each treatment group were individually labeled, mixed, and IV injected into 4 week old, irradiated NSG mice. (A) Increased expression of CXCR4 following 24 hours of incubation with added factors; (B) inputting a cell ratio; (C) 16h after cell injection, CAR-T cells were found in equal ratios in blood or bone marrow regardless of treatment.

Detailed description of the invention

The compositions and methods of the invention utilize genetically modified immune cells expressing a chimeric ligand/antigen receptor (CLR/CAR) to selectively eliminate target cells in a subject. Furthermore, once the compositions and methods of the invention have target cells that are selectively eliminated, they enable the selective elimination of these CLR/CAR-expressing immune cells. Of particular interest, the compositions and methods of the invention enable the subsequent transplantation of therapeutic cells that may also have been genetically modified to correct genetic defects present in the native cells of the selectively disrupted subject, replace the selectively disrupted cell population or supplement the subject's native cell population to treat genetic, immune and blood based disorders, including cancer.

The compositions and methods of the invention provide "pharmaceutically reversible" CAR-T cells or a variety of cells directed to recipient hematopoietic cells as a selective regulatory strategy for stem cell transplantation. Transplantation of autologous or allogeneic Hematopoietic Stem Cells (HSCs) has demonstrated the ability to treat a wide variety of malignant and non-malignant hematological diseases. However, the preparation protocols generally require invasive and genotoxic treatment with systemic irradiation and/or chemotherapy, which brings serious and even life-threatening complications, limiting their wider application. Previous experimental studies have determined that depletion of recipient HSCs is an essential requirement of these regulatory regimes in allowing successful engraftment of composite donor HSCs. Animal and clinical studies have also shown that allogeneic reactive anti-HSC donor T cells additionally promote stem cell transplantation, but this is often accompanied by a risk of GvHD. This has prompted consideration of alternative regulatory approaches for depleting HSCs with less toxic side effects, such as anti-c-kit and anti-CD 45 antibody directed therapy. In this way, more precise HSC targeting can also be achieved by applying short-lived genetically engineered Chimeric Antigen Receptor (CAR) -T cells for stem cell transplantation regulation.

We developed a novel and controllable CAR-T approach for targeting via genetically modified recipient HSCs using a non-viral PiggyBac (PB) transposon system. In contrast to viral vector delivery systems, the relatively large carrying capacity of PB allows for stable introduction of at least three separate genes encoded in the same tricistronic transgene cassette. This includes second generation CARs that target human c-kit (CD117) or prominin-1(CD133), markers known to be expressed by antigens on the surface of HSCs. In addition, the drug resistance element serves as a selection gene, which in combination with non-genotoxic drugs, provides an efficient method of purifying CAR-T cells during manufacture. Importantly, small molecule drug-inducible safety switch genes are also included to facilitate rapid in vivo clearance of CAR-T cells after recipient HSC depletion and before donor HSC transplantation. Finally, as a result of the manufacturing process, most CAR-T cells express chemokine receptors (such as CXCR4), which can allow more selective trafficking to the Bone Marrow (BM) for eradication of resident HSCs.

To select leading candidates from a panel of anti-HSC CAR constructs, CD3/CD 28-stimulated T cells from human peripheral blood were first electroporated with mRNA encoding each CAR candidate for c-kit or CD 133. CAR surface expression was confirmed in transfected T cells by flow cytometry. In vitro functional assays were performed by co-culturing mRNA-transfected CAR-T cells with mouse or human cell lines expressing C-kit or CD133 (EML-C1, TF-1 and K562) and mouse and human primary BM cells. Leading CAR candidates were identified from the specific activation of CAR-T cells by degranulation based on CD107a expression and secretion of IFN γ. In addition, those CARs are also capable of selectively depleting c-kit or CD133 positive cells. Interestingly, some mRNA transfected CAR-T cells retained effector activity against the target c-kit + TF-1 cells even in the presence of their soluble ligand, stem cell factor. Next, in the same tricistronic transgene, the leading CAR candidates were co-expressed with selection and drug-inducible safety switch genes and then stably delivered to T cells using PB. The manufacturing process produced CAR-T cells that were predominantly of the T memory stem cell (Tscm) phenotype, as determined by positive expression of CD62L and CD45RA, and also expressed high levels of CXCR4 chemokine receptor. Similar to mRNA transfected CAR-T cells, these stably transposed cells can have a broad range of effector functions, including specific depletion of target cells expressing c-kit or CD 133.

Future studies will evaluate PB-producing leading anti-HSC CAR-T cells in immunodeficient NSG mice with pre-established xenogenic human hematopoietic chimeras, along with standard busulfan or irradiation-regulated controls. This approach constitutes a novel targeted biotherapy, envisaging a way to lead to a minimally toxic transplantation regimen of depleting endogenous HSCs in the BM and replacing them with transplanted allogeneic or genetically corrected stem cells.

The need for alternative regulatory therapies prior to HSC transplantation: in the united states, more than 5,000 patients are treated with a myeloablative conditioning regimen prior to HSC transplantation each year. Most of these regulatory regimens consist of high doses of genotoxic irradiation or busulfan, which are mainly applied as HSC depleting agents, but are limited by significant life-threatening complications. Monoclonal antibodies directed against antigens expressed on HSCs (e.g., c-kit and CD45) have been considered as alternatives. CAR-T cells can provide more efficient, selective, and safer depletion of HSCs residing in bone marrow. PiggyBac-generated CAR-T cells are a non-viral system with large cargo capacity that allows the introduction of a variety of genes, including those used for selection and safety switches, that can eliminate CAR-T cells prior to donor HSC transplantation. PB CAR-T cells also exhibit a Stem Cell Memory (SCM) phenotype for enhanced in vivo efficacy and can better home to the bone marrow.

PB CAR-T cells targeted against CD117 or CD133 deplete hematopoietic progenitors from human and monkey bone marrow, and from human CD34⁺Original CAFC of cells. PB CAR-T cells exhibit a stem cell memory phenotype and naturally express CXCR4, although expression can be increased by culturing for 24 hours with added factors. PB CAR-T cells successfully home to bone marrow within 16 hours after injection. This data supports the use of PB CAR-T cells to target endogenous HSCs in the BM as a minimal non-genotoxic HSC transplantation protocol.

The hematopoietic system is maintained by a rare population of primitive Hematopoietic Stem Cells (HSCs) defined by key features of self-renewal and the ability to generate multi-lineage progenitor cell populations that ultimately give rise to functional cells of the blood and immune systems. The normal mammalian hematopoietic system is distributed primarily in the bone marrow around the adult and consists of quiescent stem cells and lineage committed progenitors. The progenitor cells in turn give rise to differentiated cells with defined functions, such as erythrocytes, monocytes, granulocytes, platelets, dendritic cells, B-cells and T-cells. Thus, the proliferative potential of HSCs is enormous because of their unique ability to perpetuate themselves through self-renewal. Methods for differentiating stem cell lineages and developmental potential have used phenotypic and functional characteristics. A defining feature of Hematopoietic Stem Cells (HSCs) that has been found to be useful is the ability of HSCs to re-engraft into the recipient's hematopoietic system following transplantation, particularly following systemic radiotherapy. Thus, it is important to effectively deplete or inactivate host HSCs in the treatment of diseases in which HSCs are involved, such as cancer, immune disorders and transplant rejection. However, this has proven difficult, especially because the frequency of HSCs is very low (in competitive re-propagation experiments, the frequency of HSCs is only 1 to 2 per 100,000 bone marrow cells, making these cells more difficult to target and eradicate). Current treatments typically involve the administration of high doses of cytotoxic agents that eliminate not only HSCs, but also many cells in the hematopoietic system. These therapies have significant drawbacks and serious toxic side effects. Thus, an improved treatment for depleting HSCs (e.g., prior to transplantation of donor HSCs to establish complete or mixed hematopoietic cell chimeras) would be beneficial.

Clinically, bone marrow and hematopoietic stem cell transplants are widely used as a means of providing patients with the ability to generate blood cells, typically where the patient has already depleted endogenous stem cells by high-dose chemotherapy or radiation. Bone marrow and peripheral blood are currently used as a source of autologous and allogeneic stem cells. In the future, cultured stem cells, including those derived from embryonic stem cells and induced pluripotent stem cells (ipscs), may provide an alternative to HSCs for transplants.

Administration of myelosuppressive drugs, graft versus host disease, and early infection after transplantation can cause graft failure or poor graft function. Poor transplantation may also result from microenvironment or marrow stromal dysfunction associated with the patient's underlying disease or prior therapy.

When the recipient is appropriately adjusted to receive the donor transplant, an unresponsive active state is seen with respect to lymphocyte response to one or more specific ligands, such as MHC markers or ligand patterns, due to interaction of lymphocytes with the ligand. Specific tolerance is achieved. A host receiving a bone marrow transplant from an intact allogeneic donor receives a renal allograft from the same donor without immunosuppression. However, fully allogeneic bone marrow transplantation, as currently practiced with extensive myeloablative conditioning, is limited in its suitability for patients of a particular age range and medical history. Myeloablative conditioning regimens, including high dose systemic irradiation, are often employed in HSC transplantation in conjunction with treatments designed to prevent immune rejection (e.g., cyclophosphamide). This modulation is useful for obtaining transplantation of allogeneic donor HSC in recipients. However, these treatments may have undesirable side effects on the recipient, such as toxicity (e.g., enteritis, pneumonia, nephrotoxicity, hyperlipidemia, myelosuppression) and complications of exacerbated GVHD and immunodeficiency (e.g., infection and malignancy). These side effects are believed to be due in part to cytokine-induced adverse reactions and may result in damage to the recipient's organ system. Therefore, less toxic pre-and post-transplant conditioning regimens are highly desirable. The present invention provides compositions and methods for selectively eliminating and replacing HSCs that do not induce any negative side effects resulting from existing treatment options.

Compositions and methods for transplantation of HSCs are provided, wherein endogenous stem cells are selectively eliminated by adoptive transfer of specific CAR-T effector cells, thereby opening the niches for transplantation of donor stem cells. The selective elimination substantially eliminates endogenous stem cells in the targeted tissue without a general elimination of cells in the tissue. The efficiency of transplantation is significantly enhanced by selective elimination compared to transplantation obtained without pretreatment. This selective elimination allows for improved function of the targeted tissue during implantation as compared to methods involving non-selective elimination. Thus, the methods of the present invention provide for efficient HSC transplantation without the use of existing non-selective ablation methods (e.g., radiation or chemotherapy). Irradiation and chemotherapy eliminates differentiated cells involved in targeting tissue function (e.g., progenitor cells to maintain peripheral blood cell numbers), induces undesirable side effects on other tissues (e.g., cells of the gastrointestinal epithelium, lung, liver, and kidney), and increases the risk of secondary malignancies.

In certain embodiments of the methods of the invention, selective depletion is accomplished by administering to the patient, prior to transplantation of the donor stem cells, CAR-T cells capable of specifically depleting endogenous HSCs. After depletion, and after a period of time sufficient to substantially eliminate HSC depleted CAR-T cells from the patient, an effective dose of donor stem cells is introduced into the patient.

For the following non-limiting exemplary uses, the compositions and methods of the invention provide a non-toxic or relatively less toxic regulatory regimen when compared to established non-selective elimination methods (e.g., radiation and chemotherapy) for establishing mixed hematopoietic cell chimeras: (a) treatment of malignant and non-malignant diseases, especially hematological diseases; (b) promote immunological acceptance of cell, tissue and/or solid organ transplantation; (c) preventing or reducing graft versus host disease (GvHD); (d) providing a platform for administering Donor Leukocyte Infusion (DLI); (e) treating enzyme deficient diseases; (f) treatment of autoimmune diseases; (g) congenital diseases affecting HSC derivatives.

Stem cell microenvironment

The interaction of stem cells with their microenvironment provides important cues for maintenance, proliferation and differentiation. This physical environment in which the stem cells are located may be referred to as the stem cell microenvironment or niche. Stromal and other cells involved in this niche provide soluble and bound factors that have multiple roles in HSC regulation.

Various models for the interaction between stem cells and niches have been proposed. In its simplest form, a model has been proposed in which when a stem cell divides, only one daughter cell remains in the niche and the other daughter cell leaves the niche to differentiate.

A particular advantage of the compositions and methods of the invention is the ability to activate CLR/CAR-expressing T cells only within close proximity or only within a specific microenvironment. This physical selectivity minimizes the effect of the compositions of the invention on cells, niches and microenvironments that are not targets of a given therapy.

Furthermore, since the microenvironment may be defined by the secretory set of one or more target cells, the CLR/CAR-expressing immune cells of the invention may be modified such that the CLR/CAR is activated only when the CLR/CAR-expressing immune cells of the invention are contacted with a secreted protein or with a given concentration of a secreted protein. Furthermore, CLR/CAR-expressing immune cells of the invention can be modified such that upon contact with an apoptosis-inducing agent of the invention or a component of the endogenous secretory component of a non-target cell, the CLR/CAR is inactivated or eliminated.

The microenvironment of the present invention may be defined by the expression of proteins on the surface of one or more target cells. Thus, a CLR/CAR-expressing immune cell of the invention can be modified such that the CLR/CAR is activated only when the CLR/CAR-expressing immune cell of the invention contacts one or more cell-surface bound proteins on a target cell. Furthermore, CLR/CAR-expressing immune cells of the invention can be modified such that CLR/CAR is inactivated or eliminated upon contact with an apoptosis-inducing agent of the invention or a cell surface-bound protein of a non-target cell.

For example, a CLR/CAR-expressing immune cell of the invention can be modified to express a CLR/CAR that specifically binds to one or more ligands on a target cancer cell, but may also need to bind to one or more secreted proteins (e.g., one or more cytokines, one or more factors that induce angiogenesis, one or more factors that break down the extracellular matrix, etc.) present in the microenvironment of the target cancer cell to be activated. As in the digital world, this two-factor authentication system ensures that the CLR/CAR-expressing immune cells of the invention eliminate only target cells and do not negatively affect non-target cells or non-target environments. As described above, if one or more of the desired signals do not match the target cell and target microenvironment, the CLR/CAR-expressing immune cells of the invention can be modified to induce apoptosis rather than eliminate the risk of non-target cells. Rather than the digital world, CLR/CAR-expressing immune cells of the invention can be modified to require multi-factor authentication from, for example, at least 2, 3,4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different ligands (which can include cell surface bound ligands, secreted ligands, or combinations thereof). As used herein, the term ligand may be used to describe any sequence, nucleic acid, or amino acid to which a CAR of the invention specifically binds.

Chimeric ligand/antigen receptor (CLR/CAR)

The terms "Chimeric Ligand Receptor (CLR)" and "Chimeric Antigen Receptor (CAR)" are used interchangeably throughout the disclosure. The chimeric receptors of the invention can specifically bind to the target antigens and/or target ligands of the invention.

Exemplary CLR/CARs of the invention comprise (a) an extracellular domain comprising a ligand recognition region, (b) a transmembrane domain, and (c) an extracellular domain comprising at least one costimulatory domain. In certain embodiments, the ligand recognition region comprises one or more of a protein scaffold, a centryrin, a single chain variable fragment (scFv), a VHH, an immunoglobulin, and an antibody mimetic. In certain embodiments, the immunoglobulin is an antibody or fragment thereof of the IgA, IgD, IgE, IgG, or IgM isotype. In certain embodiments, the antibody fragment is a Complementarity Determining Region (CDR), a heavy chain CDR (including CDR1, CDR2, and/or CDR3), a light chain CDR (including CDR1, CDR2, and/or CDR3), an antigen binding fragment (Fab), a variable domain (Fv), a heavy chain variable region, a light chain variable region, an intact heavy chain, an intact light chain, one or more constant domains, an Fc (crystallizable fragment), or any combination thereof. In certain embodiments, the antibody mimetic comprises one or more of affibody, affilin, affimer, affitin, alphabody, anticalin, and avimer, designed ankyrin repeat protein (DARPin), Fynomer, Kunitz domain peptide, and monomer. In certain embodiments, at least one of the CLRs is bispecific. In certain embodiments, the CLRs are each bispecific. In certain embodiments, at least one of the CLRs is trispecific. In certain embodiments, the CLRs are each trispecific.

In certain embodiments of the methods of the invention, at least one immune cell in the composition comprising a plurality of immune cells comprises a dividing CLR/CAR. In certain embodiments, a dividing CLR/CAR comprises two or more CLR/CARs having different intracellular domains that, when simultaneously expressed in at least one immune cell, increase or decrease the activity of the immune cell compared to an immune cell that does not express a dividing CLR/CAR or an immune cell that does not express a CLR/CAR.

In certain embodiments of the methods of the invention, at least one immune cell in the composition comprising a plurality of immune cells comprises a dividing CLR/CAR. In certain embodiments, including those in which concurrent expression reduces the activity of an immune cell, dividing CLR/CAR comprises: (a) a first CLR/CAR comprising: an extracellular domain comprising a ligand recognition region, a transmembrane domain, and an intracellular domain comprising a primary intracellular signaling domain, a secondary intracellular signaling domain, and (b) a second CLR/CAR comprising: an extracellular domain comprising a ligand recognition region, a transmembrane domain, and an intracellular domain consisting of an inhibitory intracellular signaling domain. In certain embodiments, the primary intracellular signaling domain comprises a human CD3 ζ endodomain and the secondary intracellular signaling domain comprises a human 4-1BB, human CD28, human CD40, human ICOS, human MyD88, or human OX-40 intracellular segment. In certain embodiments, the primary intracellular signaling domain comprises a human CD3 ζ endodomain and the secondary intracellular signaling domain comprises human 4-1BB and human CD 28. In certain embodiments, the inhibitory intracellular signaling domain comprises signaling domains derived from PD1, CTLA4, LAG3, B7-H1, B7-1, CD160, BTLA, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, and TIGIT. Additional intracellular signaling components from these inhibitory intracellular signaling domains and other molecules that may be used in whole or in part include, but are not limited to, ITIM, ITSM, YVKM, PP2A, SHP2, KIEELE, and Y265. In certain embodiments, the second CLR/CAR selectively binds to a target on a non-target cell, thereby inducing the second CLR/CAR to inhibit the activity of the first CLR/CAR. In certain embodiments, the second CLR/CAR inhibits the ability of the first CLR/CAR to induce death of the target cell or a non-target cell.

In certain embodiments of the methods of the invention, the one or more CLR/CARs bind the ligand with at least one affinity selected from the group consisting of: less than or equal to 10⁻⁹M, less than or equal to 10⁻¹⁰M, less than or equal to 10⁻¹¹M, less than or equal to 10⁻ ¹²M, less than or equal to 10⁻¹³M, less than or equal to 10⁻¹⁴M and less than or equal to 10⁻¹⁵K of M_D. In certain embodiments, K_DMeasured by surface plasmon resonance.

Scaffold proteins

The protein scaffolds of the invention may be derived from fibronectin type III (FN3) repeat proteins, encoding or complementary nucleic acids, vectors, host cells, constructs, combinations, formulations, devices, and methods of making and using the same. In a preferred embodiment, the protein scaffold comprises a consensus sequence from multiple FN3 domains of human tenascin-C (hereinafter "tenascin"). In a more preferred embodiment, the protein scaffold of the invention is a consensus sequence of the 15 FN3 domain. The protein scaffolds of the invention can be designed to bind a variety of molecules, for example, cellular target proteins. In a preferred embodiment, the protein scaffold of the invention may be designed to bind epitopes of the ligand in wild-type and/or variant form.

The protein scaffold of the invention may include additional molecules or moieties, for example, the Fc region of an antibody, albumin binding domain, or other half-life affecting moieties. In a further embodiment, the protein scaffold of the invention may be bound to a nucleic acid molecule which may encode a protein scaffold.

The present invention provides at least one method of expressing at least one protein scaffold based on a consensus sequence of multiple FN3 domains in a host cell, comprising culturing a host cell as described herein under conditions wherein the at least one protein scaffold is expressed in detectable and/or recoverable amounts.

The present invention provides at least one composition comprising (a) a protein scaffold based on a consensus sequence of multiple FN3 domains and/or encoding nucleic acids as described herein; and (b) a suitable and/or pharmaceutically acceptable carrier or diluent.

The present invention provides methods of generating protein scaffold libraries based on a fibronectin type III (FN3) repeat protein, preferably a consensus sequence of multiple FN3 domains, and more preferably a consensus sequence of multiple FN3 domains from human tenascin. Successive generations of scaffolds are prepared to form a scaffold library by altering (by mutation) the number of amino acids or amino acids at a particular position in the molecule in a portion of the scaffold, such as a loop region. A scaffold library can be generated by altering the amino acid composition of a single loop or simultaneously altering additional positions of multiple loops or scaffold molecules. The modified ring may be lengthened or shortened accordingly. Such a library may be generated to contain all possible amino acids, or a subset of designed amino acids, at each position. Library members can be used for screening by display, such as in vitro or CIS display (DNA, RNA, ribosome display, etc.), yeast, bacterial and phage display.

The protein scaffolds of the present invention provide enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations; they can be expressed and folded in prokaryotic systems, such as E.coli, in eukaryotic systems, such as yeast, and in vitro transcription/translation systems, such as the rabbit reticulocyte lysis system.

The invention provides methods of generating scaffold molecules that bind to a particular target by panning a scaffold library of the invention with the target and detecting the binding agent. In other related aspects, the invention includes screening methods that can be used to generate or bind mature protein scaffolds with desired activities, e.g., capable of binding to a target protein with a certain affinity. Affinity maturation can be accomplished by iterative mutagenesis and selection using systems such as phage display or in vitro display. Mutagenesis in this process may be the result of site-directed mutagenesis of specific scaffold residues, random mutations due to error-prone PCR, DNA shuffling (shuffling), and/or a combination of these techniques.

The present invention provides isolated, recombinant, and/or synthetic protein scaffolds, including but not limited to mammalian-derived scaffolds, based on the consensus sequence of fibronectin type III (FN3) repeat proteins, as well as nucleic acid molecules that constitute and encode protein scaffolds comprising at least one polynucleotide encoding a protein scaffold based on the consensus FN3 sequence. The invention also includes, but is not limited to, methods of making and using such nucleic acid and protein scaffolds, including diagnostic and therapeutic compositions, methods and devices.

The protein scaffolds of the present invention provide advantages over traditional therapies, such as the ability to be administered topically, orally, or across the blood-brain barrier, the ability to be expressed in e.coli, the resource increase that allows expression of the protein as a function of comparative mammalian cell expression ability (bispecific or tandem molecules that would be engineered to bind multiple targets or multiple epitopes of the same target), the ability to be conjugated to drugs, polymers, and probes, the ability to be formulated in high concentrations, and the ability of such molecules to effectively penetrate diseased tissues and tumors.

Furthermore, protein scaffolds possess a number of antibody properties related to their ability to mimic the folding of the variable regions of antibodies. This orientation enables the FN3 loop to be exposed to antibody Complementarity Determining Regions (CDRs) similar to antibodies. They should be capable of binding to cellular targets and the loops can be altered, e.g., affinity matured, to improve certain binding or related properties.

3 of the 6 loops of the protein scaffold of the invention correspond topologically to the complementarity determining regions (CDR 1-3) of the antibody, i.e., the ligand-binding region, while the remaining three loops are exposed on the surface in a manner similar to the CDR of the antibody. These loops span or are near residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO. 1. Preferably, the loop regions at or near residues 22-28, 51-54 and 75-81 are altered for binding specificity and affinity. One or more of these loop regions are randomized with other loop regions and/or other chains of sequences that retain them as scaffold moieties to populate the library, while effective binders can be selected from libraries that have high affinity for a particular protein target. One or more of the loop regions can interact with a target protein, similar to the interaction of antibody CDRs with proteins.

The scaffolds of the invention may comprise an antibody mimetic.

The term "antibody mimetic" is intended to describe an organic compound that specifically binds to a target sequence and has a different structure from a naturally occurring antibody. The antibody mimetic can comprise a protein, a nucleic acid, or a small molecule. The target sequence to which the antibody mimetic of the present invention specifically binds may be a ligand. Antibody mimetics can provide superior properties over antibodies, including, but not limited to, superior solubility, tissue permeability, stability to heat and enzymes (e.g., resistance to enzymatic degradation), and lower production costs. Exemplary antibody mimetics include, but are not limited to, affibodies, affilins, affimers, affitins, alphabodies, anticalins and avimers (also known as affinity polymers), darpins (designed ankyrin repeat proteins), fynomers, Kunitz domain peptides and monomers.

The affibody molecules of the invention comprise a protein scaffold comprising or consisting of one or more alpha helices without any disulfide bonds. Preferably, the affibody molecule of the invention comprises or consists of 3 alpha helices. For example, the affibody molecules of the invention may comprise an immunoglobulin binding domain. The affibody molecules of the invention may comprise the Z domain of protein a.

The Affilin molecules of the invention comprise a protein scaffold created by modifying exposed amino acids of, for example, either gamma-B crystallin or ubiquitin (ubiquitin). The Affilin molecule functionally mimics the affinity of an antibody for a ligand, but does not structurally mimic an antibody. In any protein scaffold used to prepare affilin, those amino acids accessible to solvents or potential binding partners in the correctly-folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids may be modified to specifically bind a target ligand sequence or ligand.

The Affimer molecules of the present invention comprise a protein scaffold comprising a highly stable protein engineered to display peptide loops that provide high affinity binding sites for a particular target sequence. Exemplary Affimer molecules of the invention comprise a protein scaffold based on a cystatin protein or its tertiary structure. Exemplary Affimer molecules of the invention may share a common tertiary structure comprising an alpha-helix lying on an antiparallel beta-sheet.

The Affitin molecules of the invention comprise an artificial protein scaffold, e.g., the structure of which may be derived from a DNA binding protein (e.g., DNA binding protein Sac7 d). Affitinns of the invention selectively bind to a target sequence, which may be all or part of a ligand. Exemplary affitinns of the invention are prepared by randomizing one or more amino acid sequences on the binding surface of a DNA binding protein and subjecting the resulting protein to ribosome display and selection. The target sequences of affitinns of the invention may for example be found in the genome or on the surface of peptides, proteins, viruses or bacteria. In certain embodiments of the invention, affitin molecules may be used as specific inhibitors of enzymes. The Affitin molecule of the invention may comprise a thermostable protein or a derivative thereof.

The Alphabody molecules of the present invention may also be referred to as cell-penetrating alphabodies (cpab). The Alphabody molecules of the present invention comprise small proteins (typically less than 10 kDa) that bind various target sequences, including ligands. Alphabody molecules are capable of reaching and binding intracellular target sequences. Structurally, the Alphabody molecules of the present invention comprise an artificial sequence (similar to a naturally occurring coiled-coil structure) that forms a single-chain alpha-helix. The Alphabody molecules of the present invention may comprise a protein scaffold comprising one or more amino acids modified to specifically bind to a target protein. The Alphabody molecules of the present invention maintain proper folding and thermal stability regardless of the binding specificity of the molecule.

The Anticalin molecules of the invention comprise an artificial protein that binds to a target sequence or site in a protein or small molecule. The Anticalin molecule of the present invention may comprise an artificial protein derived from human lipocalin (lipocalin). Instead of, for example, monoclonal antibodies or fragments thereof, the Anticalin molecules of the invention may be used. The Anticalin molecules may exhibit superior tissue penetration and thermostability to monoclonal antibodies or fragments thereof. Exemplary Anticalin molecules of the invention may comprise about 180 amino acids, having a mass of about 20 kDa. Structurally, the Anticalin molecules of the invention comprise a barrel structure containing antiparallel beta-strands connected in pairs by loops and attached alpha helices. In a preferred embodiment, the inventive Anticalin molecule comprises a barrel structure comprising 8 antiparallel β -strands connected in pairs by loops and an attached α helix.

The Avimer molecules of the present invention comprise an artificial protein that specifically binds to a target sequence (which may also be a ligand). Avimer of the invention can recognize multiple binding sites within the same target or within different targets. Avimer of the invention mimics the function of a bispecific antibody when it recognizes more than one target. The artificial protein avimer may comprise two or more peptide sequences of about 30-35 amino acids each. These peptides may be linked via one or more linker peptides. The amino acid sequence of one or more peptides of Avimer may be derived from the a domain of the membrane receptor. Avimer has a rigid structure that may optionally contain disulfide bonds and/or calcium. The Avimer of the present invention may exhibit greater thermal stability compared to antibodies.

Darpins (designed ankyrin repeat proteins) of the invention comprise genetically engineered, recombinant or chimeric proteins with high specificity and high affinity for the target sequence. In certain embodiments, the darpins of the invention are derived from ankyrin, and optionally comprise at least 3 repeat motifs (also referred to as repeat building blocks) of the ankyrin. Ankyrin mediates high affinity protein-protein interactions. Darpins of the invention comprise large target interaction surfaces.

The fynomers of the present invention comprise small binding proteins (about 7 kDa) derived from the human Fyn SH3 domain and engineered to bind target sequences and molecules with equivalent affinity and equivalent specificity to antibodies.

The Kunitz domain peptides of the present invention comprise a protein scaffold comprising a Kunitz domain. The Kunitz domain contains an active site that inhibits protease activity. Structurally, the Kunitz domain of the present invention comprises a disulfide-rich α + β sheet. This structure is exemplified by bovine pancreatic trypsin inhibitor. The Kunitz domain peptide recognizes specific protein structures and acts as a competitive protease inhibitor. The Kunitz domain of the invention may comprise ecalapide (derived from human lipoprotein-associated coagulation inhibitor (LACI)).

The monomers of the invention are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) comparable in size to single chain antibodies. These genetically engineered proteins specifically bind target sequences including ligands. The monomers of the invention can be specifically targeted to one or more different proteins or target sequences. In a preferred embodiment, the monomers of the invention comprise a protein scaffold mimicking the structure of human fibronectin, and more preferably, mimicking the structure of the tenth extracellular type III domain of fibronectin. The tenth extracellular type III domain of fibronectin, and its monomeric mimic, contains 7 barrel-forming β sheets and 3 exposed loops on each side corresponding to the 3 Complementarity Determining Regions (CDRs) of the antibody. In contrast to the structure of the variable domains of antibodies, monomers lack any binding sites for metal ions as well as a central disulfide bond. Multispecific monomers can be optimized by modifying the loops BC and FG. The monomers of the invention may comprise an adnectin.

Such methods may comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one scaffold protein to a cell, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention or reduction of symptoms, effects or mechanisms. An effective amount may comprise an amount of about 0.001-500 mg/kg per single (e.g., bolus), multiple, or consecutive administration, or to achieve a serum concentration of 0.01-5000 μ g/ml per single, multiple, or consecutive administration, or any effective range or value therein, as determined and measured using known methods (e.g., as described herein or known in the relevant art).

Production and Generation of scaffold proteins

At least one scaffold protein of the invention may optionally be produced by a cell line, mixed cell line, immortalized cell or clonal population of immortalized cells as is well known in the art. See, e.g., Ausubel, et al, eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al, eds., Current Protocols in immunology, John Wiley & Sons, NY (1994-2001); Colligan et al, Current Protocols in Protein sciences, John Wiley & Sons, N.1997).

Amino acids from the scaffold protein may be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic known in the art.

Optionally, the scaffold protein may be engineered to retain high affinity for the ligand and other favorable biological properties. To achieve this goal, scaffold proteins can optionally be prepared by an analytical process using three-dimensional models of the parental and engineered sequences for the parental sequences and various conceptually engineered products. Three-dimensional models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structure of a selected candidate sequence and can measure the likely immunogenicity (e.g., the immunolilter program by Xencor, inc. Examination of these displays allows analysis of the likely role of the residues in candidate sequence function, i.e., analysis of residues that affect the ability of a candidate scaffold protein to bind its ligand. In this way, residues can be selected and combined from the parent and reference sequences such that the desired characteristics, such as affinity for the target ligand, are achieved. Alternatively, or in addition to the above procedures, other suitable engineering methods may be used.

Screening for scaffold proteins

Screening for protein scaffolds that specifically bind to similar proteins or fragments can be conveniently accomplished using a nucleotide (DNA or RNA display) or peptide display library, e.g., in vitro display. This method involves screening large collections of peptides for individual members with desired functions or structures. The displayed nucleotide or peptide sequence may be 3-5000 or more nucleotides or amino acids in length, typically 5-100 amino acids in length, and often about 8-25 amino acids in length. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of peptide sequences on the surface of a phage or cell. Each bacteriophage or cell contains a nucleotide sequence encoding a particular displayed peptide sequence. Such methods are described in PCT patent publication nos. 91/17271, 91/18980, 91/19818 and 93/08278.

Other systems for generating peptide libraries have aspects of in vitro chemical synthesis and recombinant methods. See, PCT patent publication nos. 92/05258, 92/14843, and 96/19256. See also, U.S. patent nos. 5,658,754; and 5,643,768. Peptide display libraries, vectors and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, Calif.), and Cambridge Antibody Technologies (Cambridge shire, UK). See, e.g., U.S. Pat. nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260, 5856456 to Enzon; 5,223,409, 5,403,484, 5,571,698, 5,837,500 to Dyax, 5,427,908, 5,580,717 to Affymax; 5,885,793 to Cambridge antibody Technologies; 5,750,373 to Genentech, 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417 to Xoma, Colligan, supra; ausubel, supra; or Sambrook, supra.

The protein scaffolds of the invention can bind human or other mammalian proteins with a wide range of affinities (KD). In a preferred embodiment, at least one protein scaffold of the invention can optionally bind a target ligand with high affinity, e.g., with a KD of equal to or less than about 10-7M, such as but not limited to 0.1-9.9 (or any range or value therein) X10-8, 10-9, 10-10, 10-11, 10-12, 10-13, 10-14, 10-15, or any range or value therein, as determined by one of skill in the art by performing surface plasmon resonance or the Kinexa method.

The affinity (affinity) or avidity (avidity) of a protein scaffold for a Ligand may be determined experimentally using any suitable method (see, e.g., Berzofsky, et al, "Antibody-Ligand Interactions," InFundamental Immunology, Paul, W.E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H. Freeman and Company: New York, N.Y. (1992); and the methods described herein). The measured affinities of a particular protein scaffold-ligand interaction may differ if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other ligand-binding parameters (e.g., KD, Kon, Koff) are preferably performed with a standardized solution of protein scaffold and ligand and a standardized buffer (such as the buffers described herein).

Competitive assays can be performed with the protein scaffolds of the invention to determine which proteins, antibodies, and other antagonists compete with the protein scaffold of the invention for binding to a target protein and/or share epitope regions. These assays, which are readily known to one of ordinary skill in the art, can assess competition between antagonists or ligands for binding sites on a limited number of proteins. The protein and/or antibody are immobilized or insoluble before and after the competition, and the sample bound to the target protein is separated from the unbound sample, for example, by pouring (where the protein/antibody has been pre-solubilized) or by centrifugation (where the protein/antibody precipitates after the competition reaction). Likewise, competitive binding can be determined by: whether binding or lack of binding of the protein scaffold to the target protein alters the function, e.g., whether the protein scaffold molecule inhibits or enhances enzymatic activity of, e.g., a label. ELISA and other functional assays can be used, as is well known in the art.

Centrins and CARTyrins

The present invention provides a chimeric ligand/antigen receptor (CLR/CAR) comprising: (a) an extracellular domain comprising a ligand recognition region, wherein the ligand recognition region comprises at least one centrin; (b) a transmembrane domain, and (c) an extracellular domain comprising at least one costimulatory domain. As used throughout this disclosure, CLR/CAR comprising centrin is referred to as CARTyrin. In certain embodiments, the ligand recognition region may comprise two centrins to produce a dual specific or tandem CLR/CAR. In certain embodiments, the ligand recognition region may comprise three centrins to produce a trispecific CLR/CAR. In certain embodiments, the extracellular domain may further comprise a signal peptide. Alternatively or additionally, in certain embodiments, the extracellular domain may further comprise a hinge between the ligand recognition region and the transmembrane domain.

The present invention provides a chimeric ligand/antigen receptor (CLR/CAR) comprising: (a) an extracellular domain comprising a ligand recognition region, wherein the ligand recognition region comprises at least one protein scaffold or antibody mimetic; (b) a transmembrane domain, and (c) an extracellular domain comprising at least one costimulatory domain. In certain embodiments, the ligand recognition region may comprise two scaffold proteins or antibody mimics to produce a dual specific or tandem CLR/CAR. In certain embodiments, the ligand recognition region may comprise three protein scaffolds to generate a trispecific CLR/CAR. In certain embodiments, the extracellular domain may further comprise a signal peptide. Alternatively or additionally, in certain embodiments, the extracellular domain may further comprise a hinge between the ligand recognition region and the transmembrane domain.

In certain embodiments of the CLRs/CARs of the invention, the signal peptide may comprise a signal peptide encoding human CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD8 alpha, CD19, CD28, 4-1BB, or GM-CSFRThe sequence of (a). In certain embodiments of the CLRs/CARs of the invention, the signal peptide may comprise a sequence encoding a human CD8a signal peptide. The human CD8 alpha signal peptide may comprise

The amino acid sequence of (a). The human CD8 alpha signal peptide may comprise

Or with an amino acid sequence containing

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The human CD8 alpha signal peptide can be prepared by

The nucleic acid sequence of (a).

In certain embodiments of the CLRs/CARs of the invention, the transmembrane domain may comprise a sequence encoding a human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain. In certain embodiments of the CLRs/CARs of the invention, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain. The CD8 alpha transmembrane domain may compriseOr with an amino acid sequence containing

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The CD8 alpha transmembrane domain may be composed of

The nucleic acid sequence of (a).

In certain embodiments of the CLRs/CARs of the invention, the endodomain may comprise the human CD3 ζ endodomain.

In certain embodiments of the CLRs/CARs of the invention, the at least one co-stimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the CLRs/CARs of the invention, the at least one co-stimulatory domain may comprise a CD28 and/or a 4-1BB co-stimulatory domain. The CD28 co-stimulatory domain may comprise a domain comprising

Or with an amino acid sequence containing

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The CD28 co-stimulatory domain may be comprised of

The nucleic acid sequence of (a). The 4-1BB co-stimulatory domain may comprise a polypeptide comprising

Or with an amino acid sequence containing

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The 4-1BB co-stimulatory domain may be comprised of

The nucleic acid sequence of (a). The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

In certain embodiments of the CLRs/CARs of the invention, the hinge may comprise sequences derived from human CD8 α, IgG4, and/or CD4 sequences. In certain embodiments of the CLRs/CARs of the invention, the hinge may comprise a sequence derived from the human CD8a sequence. The hinge may comprise

Or with a polypeptide comprising the human CD8 alpha amino acid sequence

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The human CD8 alpha hinge amino acid sequence may be composed of

The nucleic acid sequence of (a).

The centryrin of the invention may comprise a protein scaffold, wherein the scaffold is capable of specifically binding a ligand. The centryrin of the invention may comprise a protein scaffold comprising at least one consensus sequence of fibronectin type III (FN3) domain, wherein the scaffold is capable of specifically binding a ligand. At least one fibronectin type III (FN3) domain may be derived from a human protein. The human protein may be tenascin-C. The consensus sequence may comprise

The consensus sequence may be formed from

The nucleic acid sequence of (a). The consensus sequence may be modified at one or more positions within: (a) an A-B loop comprising or consisting of amino acid residues TEDS (SEQ ID NO: 63) at positions 13-16 of the consensus sequence; (b) a B-C loop comprising or consisting of amino acid residues TAPDAAF (SEQ ID NO:64) at positions 22-28 of the consensus sequence; (c) a C-D loop comprising or consisting of amino acid residues SEKVGE (SEQ ID NO:65) at positions 38-43 of the consensus sequence; (D) a D-E loop comprising or consisting of the amino acid residues GSER at positions 51-54 of the consensus sequence (SEQ ID NO: 66); (e) an E-F loop comprising or consisting of amino acid residues GLKPG at positions 60-64 of the consensus sequence (SEQ ID NO: 67); (f) a F-G loop comprising or consisting of amino acid residues KGGHRSN at positions 75-81 of the consensus sequence (SEQ ID NO: 68); or (g) any combination of (a) - (f). The centryrin of the invention may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains or at least 15 fibronectin type III (FN3) domains. The scaffold may be selected from at least one affinity binding ligand of: less than or equal to 10⁻⁹M, less than or equal to 10⁻¹⁰M, less than or equal to 10⁻¹¹M, less than or equal to 10⁻¹²M, less than or equal to 10⁻¹³M, less than or equal to 10⁻¹⁴M and less than or equal to 10⁻¹⁵K of M_D。K_DCan be determined by surface plasmon resonance.

The invention provides compositions comprising the CLR/CAR of the invention and at least one pharmaceutically acceptable carrier.

The invention provides transposons comprising the CLR/CAR of the invention.

The transposons of the invention can comprise a selection gene for identifying, enriching and/or isolating cells that express the transposons. Exemplary selection genes encode any gene product (e.g., transcripts, proteins, and enzymes) necessary for cell viability and survival. Exemplary selection genes encode any gene product (e.g., transcripts, proteins, and enzymes) that is critical for conferring resistance to drug attack, and cells are susceptible to drug attack (or may be lethal to the cell) in the absence of the gene product encoded by the selection gene. Exemplary selection genes encode any gene product (e.g., transcript, protein, enzyme) necessary for viability and/or survival in a cell culture medium lacking one or more nutrients necessary for cell viability and/or survival in the absence of the selection gene. Exemplary selection genes include, but are not limited to,neo(conferring resistance to neomycin), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), TYMS (encoding thymidylate synthase), MGMT (encoding O (6) -methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding aldehyde dehydrogenase family 1, member A1), FRANCF, RAD51C (encoding RAD51 paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 homeobox 2).

The transposons of the invention are maintained as episomes or integrated into the genome of the recombinant/modified cell. The transposon may be part of a two-component piggyBac system that utilizes a transposon and a transposase for enhanced non-viral gene transfer. In certain embodiments of the method, the transposon is a plasmid DNA transposon having a sequence encoding a chimeric ligand/antigen receptor, flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac or super-piggyBac System (SPB) transposase.

In certain embodiments of the methods of the invention, the transposon is a plasmid DNA transposon having a sequence encoding a ligand/antigen receptor, flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac or super-piggyBac System (SPB) transposase. In certain embodiments, and particularly those in which the transposase is a super piggyBac Size (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

Inducible pro-apoptotic polypeptides

The inducible pro-apoptotic polypeptides of the invention are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides of the invention are much less immunogenic. Although the inducible pro-apoptotic polypeptides of the invention are recombinant polypeptides, therefore, the non-naturally occurring sequences that are recombined to produce the inducible pro-apoptotic polypeptides of the invention do not comprise non-human sequences that the host human immune system can recognize as "non-self" and thus induce an immune response in a subject receiving the inducible pro-apoptotic polypeptides of the invention, cells comprising the inducible pro-apoptotic polypeptides or compositions comprising the inducible pro-apoptotic polypeptides or cells comprising the inducible pro-apoptotic polypeptides.

The transposons of the invention can comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction enzyme site. In certain embodiments, the ligand binding region can be a multimeric ligand binding region. The induced pro-apoptotic polypeptides of the invention may also be referred to as "iC 9 safety switches". In certain embodiments, a transposon of the invention can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, a transposon of the invention can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, a transposon of the invention can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the induced pro-apoptotic polypeptides, induced caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the ligand binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence comprising the ligand binding region of the FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide consists of

The amino acid sequence of (1) encodes. In some embodiments of the present invention, the substrate is,FKBP12 polypeptide consisting of

The nucleic acid sequence of (a). In certain embodiments, the inducer specific for the ligand binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide having a valine (V) substituted for a phenylalanine (F) at position 36 (F36V), including both AP20187 and/or AP1903 synthetic drugs.

In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the linker region is encoded by an amino acid comprising GGGGS (SEQ ID NO:47) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 48). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction enzyme site.

In certain embodiments of the truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not include arginine (R) at position 87 of the sequence. Alternatively or additionally, in certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise alanine (a) at position 282 of the sequence. In certain embodiments of the induced pro-apoptotic polypeptides, induced caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is comprised of

Or comprise

The nucleic acid sequence of (a).

In certain embodiments of the induced pro-apoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the induced pro-apoptotic polypeptide is comprised of

Or comprises an amino acid sequence of

The nucleic acid sequence of (a).

Transposons and transposases

The transposons of the invention can comprise at least one self-cleaving peptide located, for example, between one or more of the protein scaffold, centrin, or CARTyrin of the invention and the selection gene of the invention. The transposons of the invention can comprise at least one self-cleaving peptide located, for example, between one or more of the protein scaffold, centrin, or CARTyrin of the invention and the inducible pro-apoptotic polypeptide of the invention. The transposon of the invention can comprise at least two self-cleaving peptides, a first self-cleaving peptide located, e.g., upstream or immediately upstream of the inducible pro-apoptotic polypeptide of the invention, and a second self-cleaving peptide located, e.g., downstream or immediately upstream of the inducible pro-apoptotic polypeptide of the invention.

The at least one self-cleaving peptide may comprise, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. The T2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-T2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprisingA sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-T2A peptide may comprise a peptide comprisingThe nucleic acid sequence of (1). The E2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprisingA sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-E2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The F2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprisingA sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-F2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprisingA sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The P2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprisingA sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-P2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a).

The transposons of the invention can comprise a first and a second self-cleaving peptide, the first self-cleaving peptide being located, for example, upstream of one or more of the protein scaffold, centryrin, or CARTyrin of the invention, and the second self-cleaving peptide being located, for example, downstream of one or more of the protein scaffold, centryrin, or CARTyrin of the invention. The first and/or second self-cleaving peptide may comprise, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. The T2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-T2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-T2A peptide may comprise a peptide comprising

The nucleic acid sequence of (1). The E2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-E2A peptide may comprise a peptide comprisingOr with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The F2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-F2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The P2A peptide may comprise a peptide comprisingOr with an amino acid sequence comprisingA sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-P2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a).

The transposon of the invention can comprise a piggyBac transposon. The transposons of the invention can include piggyBac transposases or compatible enzymes. In certain embodiments of the method, the transposon is a plasmid DNA transposon having a sequence encoding a chimeric ligand/antigen receptor, flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. The transposons of the invention can include piggyBac transposases or compatible enzymes. In certain embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac or super-piggyBac System (SPB) transposase. In certain embodiments, and particularly those in which the transposase is a super piggyBac Size (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

Carrier

The invention provides a vector comprising a CAR of the invention. In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.

The viral vectors of the invention may comprise sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. The viral vector may comprise sequences isolated or derived from an adeno-associated virus (AAV). The viral vector may comprise a recombinant aav (raav). Exemplary gonadal-associated viruses and recombinant adeno-associated viruses of the invention comprise two or more Inverted Terminal Repeat (ITR) sequences immediately in cis (cis) to sequences encoding the protein scaffold, centrin or CARTyrin of the invention. Exemplary adeno-associated and recombinant viruses of the invention include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV 9). Exemplary adeno-associated and recombinant viruses of the invention include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids comprising a genome of one serotype and a capsid of another serotype (e.g., AAV2/5, AAV-DJ and AAV-DJ 8). Exemplary gonadal-associated viruses and recombinant adeno-associated viruses of the invention include, but are not limited to, rAAV-LK 03.

The viral vectors of the invention may comprise a selection gene. The selection gene may encode a gene product necessary for cell viability and survival. When challenged by selective cell culture conditions, the selection gene may encode a gene product that is essential for cell viability and survival. The selective cell culture conditions may comprise a compound detrimental to cell viability or survival, and wherein the gene product confers resistance to said compound. Exemplary selection genes of the invention can include, but are not limited toneo(conferring resistance to neomycin), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), TYMS (encoding thymidylate synthase), MGMT (encoding O (6) -methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding the aldehyde dehydrogenase family 1, member a1), FRANCF, RAD51C (encoding RAD51 paralog C), GCS (encoding glucosyl ceramide synthase), NKX2.2 (encoding NK2 homeobox 2), or any combination thereof.

The viral vector of the invention may comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction enzyme site. In certain embodiments, the ligand binding region can be a multimeric ligand binding region. The induced pro-apoptotic polypeptides of the invention may also be referred to as "iC 9 safety switches". In certain embodiments, the viral vectors of the invention may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the viral vectors of the invention may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the viral vectors of the invention may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the induced pro-apoptotic polypeptides, the inducible caspase polypeptide or truncated caspase 9 polypeptide of the invention, the ligand binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence comprising the ligand binding region of the FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide consists of

The amino acid sequence of (1) encodes. In certain embodiments, the FKBP12 polypeptide consists of

The nucleic acid sequence of (a). In certain embodiments, the inducer specific for the ligand binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide having a substitution of phenylalanine (F) at position 36 with valine (V) (F36V), comprising both AP20187 and/or AP1903 synthetic drugs.

In certain embodiments of the induced pro-apoptotic polypeptides, induced caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the linker region is comprised of

Amino group of (2)Acid or comprises

The nucleic acid sequence of (a). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction enzyme site.

In certain embodiments of the truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is encoded by an amino acid sequence of arginine (R) at position 87 that does not include the sequence. Alternatively, or in addition, in certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not include alanine (a) at position 282 of the sequence. In certain embodiments of the induced pro-apoptotic polypeptides, induced caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is comprised of

Or comprise

The nucleic acid sequence of (a).

In certain embodiments of the induced pro-apoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the induced pro-apoptotic polypeptide is comprised of

Or comprises an amino acid sequence of

The nucleic acid sequence of (a).

The viral vector of the present invention may comprise at least one self-cleaving peptide. In some embodiments, the vector can comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located upstream of the CAR and the second self-cleaving peptide is located downstream of the CAR. The viral vector of the invention may comprise at least one self-cleaving peptide located, for example, between one or more of the protein scaffold, centrin or CARTyrin of the invention and the inducible pro-apoptotic polypeptide of the invention. The viral vector of the invention may comprise at least two self-cleaving peptides, a first self-cleaving peptide located, e.g., upstream or immediately upstream of the inducible pro-apoptotic polypeptide of the invention, and a second self-cleaving peptide located, e.g., downstream or immediately upstream of the inducible pro-apoptotic polypeptide of the invention. Self-cleaving peptides can comprise, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. The T2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-T2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-T2A peptide may comprise a peptide comprising

The nucleic acid sequence of (1). The E2A peptide may comprise a peptide comprisingOr with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-E2A peptide may comprise a peptide comprisingOr with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The F2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-F2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The P2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-P2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a).

The invention provides a vector comprising a CAR of the invention. In certain embodiments, the carrier is a nanoparticle. Exemplary nanoparticle carriers of the invention include, but are not limited to, nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified synthetic nucleotides, or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymers), micelles, lipids (e.g., liposomes), organic molecules (e.g., carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g., calcium phosphate or gold), or any combination thereof. The nanoparticle carrier can pass through the cell membrane passively or actively.

The nanoparticle vectors of the present invention may comprise a selection gene. The selection gene may encode a gene product necessary for cell viability and survival. When challenged by selective cell culture conditions, the selection gene may encode a gene product that is essential for cell viability and survival. The selective cell culture conditions may comprise a compound detrimental to cell viability or survival and wherein the gene product confers resistance to said compound. Exemplary selection genes of the invention can include, but are not limited toneo (conferring resistance to neomycin), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), TYMS (encoding thymidylate synthase), MGMT (encoding O (6) -methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding aldehyde dehydrogenase family 1, member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding RAD51 Paralog C)Glucosylceramide synthase), NKX2.2 (encoding NK2 homeobox 2), or any combination thereof.

The nanoparticle vectors of the invention can comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction enzyme site. In certain embodiments, the ligand binding region can be a multimeric ligand binding region. The induced pro-apoptotic polypeptides of the invention may also be referred to as "iC 9 safety switches". In certain embodiments, the nanoparticle vectors of the invention can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the nanoparticle vectors of the invention can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the nanoparticle vectors of the invention can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the induced pro-apoptotic polypeptides, the inducible caspase polypeptide or truncated caspase 9 polypeptide of the invention, the ligand binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence comprising the ligand binding region of the FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide consists of

The amino acid sequence of (1) encodes. In some implementationsIn one embodiment, the FKBP12 polypeptide comprises

The nucleic acid sequence of (a). In certain embodiments, the inducer specific for the ligand binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide (valine (V) with phenylalanine (F) at substitution position 36 (F36V)), comprising two synthetic drugs, AP20187 and/or AP 1903.

In certain embodiments of the induced pro-apoptotic polypeptides, induced caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the linker region is comprised ofOr comprise

The nucleic acid sequence of (a). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction enzyme site.

In certain embodiments of the truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is encoded by an amino acid sequence of arginine (R) at position 87 that does not include the sequence. Alternatively, or in addition, in certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not include the alanine (a) at position 282 of the sequence. In certain embodiments of the induced pro-apoptotic polypeptides, induced caspase polypeptides, or truncated caspase 9 polypeptides of the invention, the truncated caspase 9 polypeptide is comprised of

Or comprise

The nucleic acid sequence of (a).

In certain embodiments of the induced pro-apoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the induced pro-apoptotic polypeptide is comprised of

Or comprises an amino acid sequence of

The nucleic acid sequence of (a).

The nanoparticle carrier of the present invention may comprise at least one self-cleaving peptide. In some embodiments, the nanoparticle carrier can comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the nanoparticle. In some embodiments, the nanoparticle carrier may comprise at least one self-cleaving peptideWherein the first self-cleaving peptide is located upstream of the CAR and the second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector can comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle and the second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector can comprise at least one self-cleaving peptide, wherein a first self-cleaving peptide is located between the CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR, e.g., between the CAR and the selection gene. The nanoparticle vectors of the invention may comprise at least one self-cleaving peptide located, for example, between one or more of the protein scaffold, centrin or CARTyrin of the invention and the inducible pro-apoptotic polypeptide of the invention. The nanoparticle vectors of the invention may comprise at least two self-cleaving peptides, a first self-cleaving peptide located, for example, upstream or immediately upstream of an inducible pro-apoptotic polypeptide of the invention, and a second self-cleaving peptide located, for example, downstream or immediately upstream of an inducible pro-apoptotic polypeptide of the invention. Self-cleaving peptides can comprise, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. The T2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-T2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-T2A peptide may comprise a peptide comprising

The nucleic acid sequence of (1). The E2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-E2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The F2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-F2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprisingA sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The P2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a). The GSG-P2A peptide may comprise a peptide comprising

Or with an amino acid sequence comprising

A sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence of (a).

The invention provides compositions comprising a vector of the invention.

CAR-expressing cells

The invention provides a cell comprising a CAR of the invention. The invention provides a cell comprising a transposon of the invention. In certain embodiments, a cell comprising a CAR, transposon, or vector of the invention can express the CAR on the cell surface. The cells may be any type of cells.

In certain embodiments of the invention, the cell is an immune cell. The immune cell can be a T-cell, a Natural Killer (NK) -like cell (e.g., a cytokine-induced killer (CIK) cell), a hematopoietic progenitor cell, a Peripheral Blood (PB) -derived T cell, or a Umbilical Cord Blood (UCB) -derived T cell.

In certain embodiments of the invention, the immune cell is a T-cell. The T cell may be a helper T cell, a helper type 1T cell, a helper type 2T cell, a helper 17T cell, a regulatory T cell, a natural regulatory T cell or an induced regulatory T cell. The T cell may be CD4⁺。

In certain embodiments of the invention, the cells may be artificial ligand presenting cells, which may optionally be used to stimulate and expand the modified immune cells or T cells of the invention.

In certain embodiments of the invention, the cell may be a tumor cell, which may optionally be used as an artificial or modified antigen presenting cell.

The modified cells of the invention that are useful in adoptive therapy may be autologous or allogeneic.

Method of making CAR-expressing cells

The present invention provides a method of expressing a chimeric ligand/antigen receptor (CAR) on the surface of a cell, comprising: (a) obtaining a population of cells, (b) contacting the population of cells with a composition comprising a CAR or a sequence encoding a CAR of the invention under conditions sufficient for the CAR to migrate across the cell membrane of at least one cell in the population of cells, thereby producing a modified population of cells; (c) culturing the modified population of cells under conditions suitable for transposon integration; and (d) expanding and/or selecting at least one cell expressing the CAR on the cell surface from the modified population of cells.

In certain embodiments of such methods of expressing a CAR, the cell population can comprise leukocytes and/or CD4+ and CD8+ leukocytes. The cell population may comprise an optimized ratio of CD4+ and CD8+ leukocytes. The optimal ratio of CD4+ to CD8+ leukocytes did not occur naturally in vivo. The cell population may comprise tumor cells.

In certain embodiments of this method of expressing a CAR, the transposon or vector comprises a CAR or a sequence encoding a CAR.

In certain embodiments of such methods of expressing a CAR, the conditions sufficient to transfer the sequence encoding the CAR across the cell membrane of at least one cell in the population of cells comprise nuclear transfection.

In certain embodiments of such methods of expressing a CAR, wherein the conditions sufficient to transfer the sequence encoding the CAR across the cell membrane of at least one cell in the population of cells comprise at least one of: application of one or more current pulses at a specified voltage, a buffer, and one or more complementary factors. In certain embodiments, the buffer may comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nuclear transfection buffer, or any combination thereof. In certain embodiments, the one or more supplemental factors can comprise (a) a recombinant human cytokine, chemokine, interleukin, or any combination thereof; (b) a salt, a mineral, a metabolite, or any combination thereof; (c) a cell culture medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof; and (e) an agent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, chemokine, interleukin, or any combination thereof may comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12 p 13, IL-12/IL-35 p 13, IL-13, IL-17/IL-17A-17F 72, IL-17A/17F 72, IL-17F-17, IL-32, IL-13, IL-3632, IL-13, IL-17F-17, IL-17F-, LAP (TGF-. beta.1), lymphotoxin-. alpha./TNF-. beta., TGF-. beta., TNF-. alpha., TRANCE/TNFSF11/RANK L, or any combination thereof. The salt, mineral, metabolite, or any combination thereof may comprise HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS/FCS, human serum, serum replacement, antibiotics, pH adjuster, erlotin salt, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell culture medium may comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, or any combination thereof. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof, include TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, inhibitors of Wnt3A, inhibitors of glycogen synthase kinase-3 β (GSK-3 β) (e.g., TWS119), Bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-TD-FMK, or any combination thereof. The agent that modifies or stabilizes one or more nucleic acids comprises a pH adjusting agent, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without an NLS sequence, a TREX1 enzyme, or any combination thereof.

In certain embodiments of such methods of expressing a CAR, conditions suitable for integration of a CAR or CAR-encoding sequence of the invention include at least one of: a buffer and one or more supplemental factors. In certain embodiments, a transposon or vector of the invention comprises a CAR of the invention or a sequence encoding a CAR. In certain embodiments, the buffer may comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nuclear transfection buffer, or any combination thereof. In certain embodiments, the one or more supplemental factors can comprise (a) a recombinant human cytokine, chemokine, interleukin, or any combination thereof; (b) a salt, a mineral, a metabolite, or any combination thereof; (c) a cell culture medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof; and (e) an agent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, chemokine, interleukin, or any combination thereof may comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12 p 13, IL-12/IL-35 p 13, IL-13, IL-17/IL-17A-17F 72, IL-17A/17F 72, IL-17F-17, IL-32, IL-13, IL-3632, IL-13, IL-17F-17, IL-17F-, LAP (TGF-. beta.1), lymphotoxin-. alpha./TNF-. beta., TGF-. beta., TNF-. alpha., TRANCE/TNFSF11/RANK L, or any combination thereof. The salt, mineral, metabolite, or any combination thereof may comprise HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS/FCS, human serum, serum replacement, antibiotics, pH adjuster, erlotin salt, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell culture medium may comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, or any combination thereof. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof include TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, inhibitors of Wnt3A, inhibitors of glycogen synthase kinase-3 β (GSK-3 β) (e.g., TWS119), Bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-TD-FMK, or any combination thereof. Agents that modify or stabilize one or more nucleic acids include pH adjusting agents, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, or any combination thereof.

In certain embodiments of this method of expressing a CAR, the amplification and selection steps occur sequentially. Amplification may occur prior to selection. Amplification may occur after selection, and optionally, further (i.e., second) selection may occur after amplification.

In certain embodiments of such methods of expressing a CAR, the amplification and selection steps can occur simultaneously.

In certain embodiments of such methods of expressing a CAR, expanding can comprise contacting at least one cell of the modified population of cells with a ligand to stimulate the at least one cell by the CAR, thereby generating an expanded population of cells. The ligands may be presented on the surface of the matrix. The substrate can have any form including, but not limited to, a surface, a well, a bead, or a plurality thereof and a substrate. The matrix may further comprise a paramagnetic or magnetic component. In certain embodiments of this method of expressing a CAR, the ligand can be presented on the surface of a substrate, wherein the substrate is a magnetic bead, and wherein a magnet can be used to remove or separate the magnetic bead from the modified and expanded cell population. The ligand may be presented on the surface of a cell or an artificial ligand-presenting cell. The artificial ligand presenting cells of the present invention may include, but are not limited to, tumor cells and stem cells.

In certain embodiments of such methods of expressing a CAR, wherein the transposon or vector comprises a selection gene, and wherein the selecting step comprises contacting at least one cell of the modified population of cells with a compound that confers resistance to the selection gene, thereby identifying the cell that expresses the selection gene as viable in selection and identifying the cell that fails to express the selection gene as failing to survive in the selecting step.

In certain embodiments of such methods of expressing a CAR, the amplifying and/or selecting step may last for a period of 10-14 days, inclusive.

The invention provides compositions comprising cell populations modified, expanded, and selected using the methods of the invention.

Hematopoietic stem cells

The compositions of the invention may comprise a plurality of Hematopoietic Stem Cells (HSCs) for transplantation following selective removal of native HSCs from a subject.

Hematopoietic Stem Cells (HSCs) are pluripotent, self-renewing progenitors. All differentiated blood cells from lymphoid and myeloid lineages are from HSCs. HSCs can be found in adult bone marrow, peripheral blood and cord blood.

Typically, HSC transplantation in the form of bone marrow transplantation fails because remnants of the subject's immune system attack the transplanted cells or create conditions that are detrimental to the survival of the transplanted cells. Prior to the development of the compositions and methods of the present invention, HSCs were either eliminated or rendered ineffective prior to bone marrow transplantation, or caused damage to cell populations other than the intended HSCs. The compositions and methods of the present invention provide methods for selectively eliminating HSCs, which are damaged, dysfunctional, or carry genetic defects that cause disease, by targeting these HSCs with immune cells that express chimeric ligand receptors (CARs) that specifically target HSC surface ligands. Once the composition comprising the immune cells expressing the CAR performs its function, they can be eliminated by pre-irradiating the immune cells or by further modifying these cells to contain an inducible pro-apoptotic polypeptide that initiates apoptosis of immune cells expressing only the exogenous CAR containing the inducible pro-apoptotic polypeptide upon administration of the inducer (otherwise referred to as a "safety switch").

The compositions and methods of the invention further provide for transplantation of multiple HSCs. Preferably, the transplanted HSCs of the invention are genetically modified.

The HSCs of the invention may be modified by a composition comprising a DNA localization domain and an effector domain. In certain embodiments, the DNA localization domain may comprise a DNA binding domain of Cas9, inactivated Cas9, short Cas9, short and inactivated Cas9, TALEN, or zinc finger protein. In certain embodiments, the effector comprises an endonuclease. Preferably, the endonuclease is a type IIS endonuclease. In certain embodiments, the IIS-type endonuclease is one or more of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, hpyaav, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, bmubi, bminbi, BmrI, BpmI, bpei, BsaI, BseRI, BsgI, BspMI, bsbi, BsrDI, btgsi, EarI, eci, mmeii, nmeiii, bbvcci, Bpu10I, bspeqi, bapi, baxi, csbsi, bpii, bbciii, bboqi 36I, fokl, or Clo. For more details on genome editing tools, see PCT/US2016/037922, the contents of which are incorporated herein by reference in their entirety). The composition comprising the DNA localization domain and the effector domain may be comprised in a transposon. Compositions comprising a DNA localization domain and an effector domain, including those contained in a vector, may be further contained in a vector for expression and/or delivery to a cell.

HSCs of the invention may be modified to remove genetic or epigenetic markers of a disease or disorder.

HSCs of the invention can be modified to express or overexpress nucleic acids or proteins or secretory molecules, peptides, proteins or compounds to treat the diseases or disorders of the invention.

HSCs of the invention can be modified to express or overexpress nucleic acids or proteins or secretory molecules, peptides, proteins or compounds to modify the immune response of the invention.

HSCs of the invention can be modified to express or overexpress cell surface ligands to modify the activity of CAR-expressing immune cells of the invention. For example, the transplanted HSCs may express a ligand that inactivates immune cells upon binding to CAR-expressing immune cells of the invention to prevent any residual CAR-expressing immune cells from selectively eliminating the transplanted HSC cells.

Nucleic acid molecules

The nucleic acid molecules of the invention encoding a protein scaffold can be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including, but not limited to, CDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triplex, double stranded or single stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

The isolated nucleic acid molecules of the invention may include nucleic acid molecules comprising an Open Reading Frame (ORF), optionally with one or more introns, such as, but not limited to, at least one specific portion of at least one protein scaffold; a nucleic acid molecule comprising a coding sequence that binds to a protein scaffold or loop region of a target protein; and nucleic acid molecules comprising nucleotide sequences substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode a protein scaffold as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it will be routine for a person skilled in the art to generate degenerate nucleic acid variants encoding a particular protein scaffold of the invention. See, e.g., Ausubel, et al, supra, for such nucleic acid variants to be included in the present invention.

As indicated herein, nucleic acid molecules of the invention comprising a nucleic acid encoding a protein scaffold may include, but are not limited to, those encoding the amino acid sequence of the protein scaffold fragment itself; a coding sequence for a complete protein scaffold or a portion thereof; the coding sequence of the protein scaffold, fragment or portion, and additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron along with additional non-coding sequences, including but not limited to non-coding 5 'and 3' sequences, such as transcribed, non-translated sequences that function in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and stability of mRNA); additional coding sequences that encode additional amino acids, such as those that provide additional functions. Thus, the sequence encoding the protein scaffold may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of a fusion protein scaffold comprising a protein scaffold fragment or portion.

Polynucleotides that selectively hybridize to polynucleotides as described herein

The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect and/or quantify nucleic acids comprising such polynucleotides. For example, the polynucleotides of the invention may be used to identify, isolate or amplify partial or full length clones in a deposited library. In some embodiments, the polynucleotide is a genomic or isolated cDNA sequence, or alternatively, is complementary to a cDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% of the full-length sequence, preferably at least 85% or 90% of the full-length sequence, and more preferably at least 95% of the full-length sequence. The cDNA library can be normalized to increase the representation of rare sequences. Hybridization conditions of low or moderate stringency are generally, but not exclusively, used for sequences having reduced sequence identity relative to the complementary sequence. Neutralizing high stringency conditions can optionally be used for sequences with greater identity. Low stringency conditions allow selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

Optionally, the polynucleotides of the invention will encode at least a portion of the protein scaffold encoded by the polynucleotides described herein. The polynucleotides of the invention comprise nucleic acid sequences that are useful for selective hybridization to polynucleotides encoding the protein scaffolds of the invention. See, e.g., Ausubel, supra; colligan, supra, each is incorporated herein by reference in its entirety.

Construction of nucleic acids

The isolated nucleic acids of the invention can be prepared using the following methods: (a) recombinant methods, (b) synthetic techniques; (c) purification techniques, and/or (d) combinations thereof, as are well known in the art.

The nucleic acid may conveniently comprise further sequences in addition to the polynucleotide of the invention. For example, a multiple cloning site comprising one or more endonuclease restriction sites can be inserted into a nucleic acid to aid in the isolation of a polynucleotide. Likewise, translatable sequences may be inserted to aid in the isolation of the translated polynucleotides of the invention. For example, a hexa-histidine tag sequence provides a convenient method of purifying the protein of the invention. The nucleic acids of the invention (excluding coding sequences) may optionally be vectors, aptamers, or linkers for cloning and/or expression of the polynucleotides of the invention.

Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in the isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Cloning vectors, expression vectors, aptamers, and linkers are well known in the art (see, e.g., Ausubel, supra; or Sambrook, supra).

Recombinant method for constructing nucleic acid

An isolated nucleic acid composition of the invention, such as RNA, CDNA, genomic DNA, or any combination thereof, can be obtained from a biological source using any number of cloning methods known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize to polynucleotides of the invention are used to identify a desired sequence in a cDNA or genomic DNA library under stringent conditions. The isolation of RNA, and the construction of cDNA and genomic libraries, are well known to those of ordinary skill in the art (see, e.g., Ausubel, supra; or Sambrook, supra).

Nucleic acid screening and isolation method

cDNA or genomic libraries can be screened using probes based on the sequences of the polynucleotides of the invention. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. One skilled in the art will appreciate that varying degrees of hybridization intensity can be used in the assay; and whether hybridization or wash media may be stringent. As the conditions for hybridization become more stringent, a greater degree of complementarity between the probe and target must be achieved in order for a duplex to form. Stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of partially denaturing solvents such as formamide. For example, by changing the polarity of the reactant solution, the stringency of hybridization can be conveniently changed, for example, by manipulating the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization medium and/or wash medium. The degree of complementarity will preferably be 100%, or 70-100%, or any range or value therein, however, it will be appreciated that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.

Methods for amplifying RNA or DNA are well known in the art and may be employed in accordance with the present invention based on the teachings and guidance presented herein without undue experimentation.

Known methods of DNA or RNA amplification include, but are not limited to, Polymerase Chain Reaction (PCR) and related amplification procedures (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis, et al; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 to Innis; 5,122,464 to Wilson, et al; 5,091,310 to Innis; 5,066,584 to Gylensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, et al; 4,766,067 to Biswas; 4,656,134 to Ringold), and RNA-mediated amplification (using antisense RNA to a target sequence as a template for double-stranded DNA synthesis) (U.S. Pat. No. 5,130,238 to Malek, et al, trade name BA; incorporated herein by Sambro, et al; see, et al; supra; incorporated herein by reference, et al; for example, supra).

For example, Polymerase Chain Reaction (PCR) techniques can be used to amplify the sequences of the polynucleotides of the invention and the associated genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be used, for example, to clone nucleic acid sequences encoding proteins to be expressed, to prepare nucleic acids for use as probes to detect the presence of desired mRNA in a sample, to sequence nucleic acids, or for other purposes. Examples of techniques sufficient to guide a skilled artisan by in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, and Mullis, et al, U.S. Pat. No. 4,683,202 (1987); and Innis, et al, PCR Protocols A guides to Methods and Applications, eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., the Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to increase the yield of long PCR products.

Synthetic method for constructing nucleic acid

Isolated nucleic acids of the invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al, supra). Chemical synthesis generally produces single-stranded oligonucleotides that can be converted to double-stranded DNA by hybridization to a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One skilled in the art will recognize that although chemical synthesis of DNA may be limited to sequences of about 100 bases or more, longer sequences may be obtained by ligating shorter sequences.

Recombinant expression cassette

The invention further provides recombinant expression cassettes comprising a nucleic acid of the invention. The nucleic acid sequences of the invention, e.g., cDNA or genomic sequences encoding the protein scaffold of the invention, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. Recombinant expression cassettes generally comprise a polynucleotide of the invention operably linked to transcription initiation control sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters may be used to direct expression of the nucleic acids of the invention.

In some embodiments, an isolated nucleic acid that acts as a promoter, enhancer, or other element may be introduced at an appropriate location (upstream, downstream, or in an intron) of a non-heterologous form of a polynucleotide of the invention in order to up-or down-regulate expression of the polynucleotide of the invention. For example, endogenous promoters may be altered in vivo or in vitro by mutation, deletion, and/or substitution.

Vectors and host cells

The invention also relates to vectors comprising the isolated nucleic acid molecules of the invention, host cells genetically engineered with recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al, supra; ausubel, et al, supra, each of which is incorporated herein by reference in its entirety.

For example, the PB-EF1a vector may be used. The vector comprises the following nucleotide sequence:

the polynucleotide may optionally be linked to a vector comprising a selectable marker for propagation in a host. Generally, the plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell.

The DNA insert should be operably linked to a suitable promoter. The expression construct will also contain sites for transcription initiation, termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct will preferably include translation from the beginning and a stop codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferably being used for mammalian or eukaryotic cell expression.

The expression vector will preferably (but optionally) comprise at least one selectable marker. Such markers include, for example, but are not limited to, ampicillin, bleomycin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/geneticin (neo gene), mycophenolic acid or glutamine synthetase (GS, U.S. Pat. No. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes to eukaryotic cells and ampicillin, bleomycin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/neomycin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B or tetracycline, resistance genes to cultivation in E.coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entirety). Suitable culture media and conditions for the above-described host cells are known in the art. Suitable vectors will be apparent to the skilled person. Introduction of the vector construct into the host cell can be carried out by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, e.g., Sambrook, supra, sections 1-4 and 16-18, Ausubel, supra, sections 1, 9, 13, 15, 16.

The expression vector will preferably (but optionally) include at least one selectable cell surface marker for use in isolating cells modified by the compositions and methods of the invention. The selectable cell surface marker of the present invention comprises a surface protein, glycoprotein or proteome that distinguishes a cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes those cells modified by the compositions and methods of the invention from those cells not modified by the compositions and methods of the invention. Such cell surface markers include, for example, but are not limited to, "designated cluster" or "determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. The cell surface marker also included the suicide gene marker RQR8 (Philip B et al blood. 2014 Aug 21; 124(8): 1277-87).

The expression vector will preferably (but optionally) include at least one selectable drug resistance marker for use in isolating cells modified by the compositions and methods of the invention. The selectable resistance marker of the invention may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

At least one protein scaffold of the invention may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. For example, additional amino acid regions, particularly charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and persistence in the host cell during purification, or during subsequent handling and storage. Likewise, peptide moieties may be added to the protein scaffold of the invention to facilitate purification. Such regions may be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, sections 17.29-17.42 and 18.1-18.74, Ausubel, supra, sections 16, 17 and 18.

A large number of nucleic acid expression systems which can be used to express nucleic acids encoding the proteins of the present invention are well known to those of ordinary skill in the art. Alternatively, the nucleic acid of the invention may be expressed in a host cell by opening (by manipulation) in a host cell containing endogenous DNA encoding the protein scaffold of the invention. Such methods are well known in the art, for example, as described in U.S. Pat. nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are all incorporated herein by reference.

Illustrative cell cultures that can be used to produce protein scaffolds, specific portions or variants thereof, are bacterial, yeast and mammalian cells known in the art. Mammalian cell systems often exist as monolayers of cells, although mammalian cell suspensions or bioreactors may also be used. Many suitable host cell lines capable of expressing the entire glycosylated protein have been developed in the art and include COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells, and the like, readily available from, for example, the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2/0-Ag14 cells (ATCC accession number CRL-1851). In a particularly preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2/0-Ag14 cell.

Expression vectors for use in these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; promoters (e.g., late or early SV40 promoter, CMV promoter (U.S. Pat. No. 5,168,062; 5,385,839), the HSV tk promoter, the pgk (phosphoglycerate kinase) promoter, the EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer, and/or a processing information site, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., SV40 large T Ag poly A addition sites), see, e.g., Ausubel et al, supra; other cells useful for producing the nucleic acids or proteins of the invention are known and/or may be obtained, for example, from the American type culture Collection Cell line and hybridoma catalog (American type culture Collection of Cell Lines and hybrids) (www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are utilized, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is a polyadenylation sequence from the bovine growth hormone gene. It is also possible to include sequences of the transcript that are spliced exactly. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al, J. Virol. 45:773-781 (1983)). In addition, gene sequences that control replication of the host cell may be incorporated into the vector, as is known in the art.

Purification of protein scaffolds

Protein scaffolds can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein a purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997) 2001, e.g., sections 1, 4,6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

Protein scaffolds of the invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, E.coli, yeast, higher plants, insects, and expression cells. Depending on the host used in the recombinant production procedure, the protein scaffold of the invention may be glycosylated or may be non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42, Ausubel, supra, Sections 10, 12, 13, 16, 18, and 20, Colligan, Protein Science, supra, Sections 12-14, all of which are incorporated herein by reference in their entirety.

Amino acid code

The amino acids that make up the protein scaffold of the invention are generally abbreviated. Amino acid nomenclature may be represented by assigning an amino acid by its single letter code, its three letter code, name, or three nucleotide codons as is commonly understood in The art (see Alberts, b., et al, Molecular Biology of The Cell, 3 rd edition, Garland Publishing, inc., New York, 1994). The protein scaffolds of the invention may comprise one or more amino acid substitutions, deletions or additions, whether from natural mutations or artificial manipulations, as specified herein. Amino acids essential for function in the protein scaffolds of the invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, section 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces a single alanine mutation at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. The key sites for protein scaffold binding can also be determined by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al, J. mol. biol. 224:899-904 (1992) and de Vos, et al, Science 255:306-312 (1992)).

As the skilled artisan will appreciate, the present invention includes at least one biologically active protein scaffold of the present invention. Biologically active protein scaffolds have a specific activity of at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% -99% or more of the specific activity of the native (non-synthetic), endogenous, or related and known protein scaffold. Methods for the determination and quantitative measurement of enzymatic activity and substrate specificity are well known to those skilled in the art.

In another aspect, the invention relates to protein scaffolds and fragments as described herein, which are modified by covalent attachment of organic molecules. Such modifications can result in protein scaffold fragments with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moiety may be a linear or branched hydrophilic polymer group, a fatty acid group or a fatty acid ester group. In particular embodiments, the hydrophilic polymer group may have a molecular weight of about 800 to about 120,000 daltons and may be a polyalkylene glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group may contain from about 8 to about 40 carbon atoms.

The modified protein scaffolds and fragments of the invention may comprise one or more organic moieties covalently bound, directly or indirectly, to an antibody. Each organic moiety bound to a protein scaffold or fragment of the invention may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" includes monocarboxylic acids and dicarboxylic acids. The term "hydrophilic polymer group" as used herein refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, the present invention encompasses protein scaffolds modified by covalent attachment of polylysine. Hydrophilic polymers suitable for modifying the protein scaffold of the present invention may be linear or branched and include, for example, polyalkanediols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkylene oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymer modifying the protein scaffold of the present invention has a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity. For example, PEG5000 and PEG 20,000 can be used, where the subscripts are the average molecular weight (in daltons) of the polymer. The hydrophilic polymer groups may be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by employing suitable methods. For example, a polymer comprising amine groups can be coupled to a carboxylate salt of a fatty acid or fatty acid ester, while an activated carboxylate salt on a fatty acid or fatty acid ester (e.g., activated with carbonyldiimidazole) can be coupled to a hydroxyl group of the polymer.

Fatty acids and fatty acid esters suitable for modifying the protein scaffold of the invention may be saturated or may contain one or more units of unsaturation. Fatty acids suitable for modifying the protein scaffold of the present invention include, for example, n-dodecanoic acid (C12, lauric acid), n-tetradecanoic acid (C14, myristic acid), n-octadecanoic acid (C18, stearic acid), n-eicosanoic acid (C20, arachidic acid), n-docosanoic acid (C22, behenic acid), n-triacontanoic acid (C30), n-tetracontanoic acid (C40), cis- Δ 9-octadecanoic acid (C18, oleic acid), all cis- Δ 5,8,11, 14-eicosatetraenoic acid (C20, arachidonic acid), suberic acid, tetradecanoic acid, octadecanedioic acid, docosanoic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing linear or branched lower alkyl groups. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6, carbon atoms.

Modified protein scaffolds and fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. The term "modifying agent" as used herein, refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid ester) that comprises an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as toluenesulfonic acid, methanesulfonic acid, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinyl ester (NHS), and the like. The activating group reactive with thiol includes, for example, maleimide, iodoacetyl, acryloyl (acrylolyl), pyridine disulfide (pyridine disulfides), 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. The aldehyde functional group can be coupled to a molecule containing an amine or hydrazide, while the azide group can react with a trivalent phosphorus group to form phosphoramidate (phosphoramidate) or phosphoramide linkages (phosphoramide linkages). Suitable methods for introducing activating groups into molecules are known in the art (see, e.g., Hermanson, G.T., Bioconjugate techniques; Academic Press: San Diego, Calif. (1996)). The activating group can be directly attached to an organic group (e.g., a hydrophilic polymer, a fatty acid ester), or through a linker moiety, e.g., a divalent C1-C12 group, wherein one or more carbon atoms can be replaced by a heteroatom, such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol (tetraethylene glycol), - (CH2)3-, -NH- (CH2)6-NH-, - (CH2)2-NH-, and-CH 2-O-CH2-CH2-O-CH2-CH 2-O-CH-NH-. For example, a modifier comprising a linker moiety can be prepared by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by: treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate, as described, or can be reacted with maleic anhydride, and cyclizing the resulting product to an activated derivative of the maleimide fatty acid (see, e.g., Thompson, et al, WO 92/16221, the entire teachings of which are incorporated herein by reference).

The modified protein scaffolds of the invention may be prepared by reacting a protein scaffold or fragment with a modifying agent. For example, organic moieties can be bound to a protein scaffold in a non-site specific manner by using amine-reactive modifying agents, such as NHS esters of PEG. Modified Protein scaffolds and fragments comprising an organic moiety that binds to a specific site of a Protein scaffold of the invention may be prepared using suitable methods, such as reverse proteolysis (Fisch et al, Bioconjugate chem., 3:147-153(1992); Werlen et al, Bioconjugate chem., 5:411-417 (1994); Kumaran et al, Protein Sci.6 (10):2233-2241 (1997); Itoh et al, Bioorg. chem., 24(1): 59-68 (1996); Capella et al, Biotechnol. Bioeng., 56(4):456-463 (1997)), and in Hermanson, G.T., Bioconjugate Techniques; academic Press: San Diego, Calif. (1996).

Isolation of T cells from Leukapheresis (Leukapheresis) products

Leukapheresis products or blood can be collected from subjects at the clinical site using closed systems and standard methods (e.g., the COBE Spectra Apheresis System). Preferably, the product is collected in a standard leukapheresis collection bag according to standard hospital or institutional leukapheresis procedures. For example, in a preferred embodiment of the method of the invention, no additional anticoagulant or blood additive (heparin or the like) is included beyond those typically used during leukopheresis.

Alternatively, White Blood Cells (WBC)/Peripheral Blood Mononuclear Cells (PBMC) (using Biosafe Sepax 2 (closed/automated)) or T cells (using CliniMACS ® Prodigy) can be isolated directly from whole blood. However, in certain subjects (e.g., those diagnosed and/or treated for cancer), the yield of WBC/PBMCs isolated from whole blood may be significantly lower than when isolated by leukapheresis.

Leukopheresis procedures and/or direct cell separation procedures may be used with any subject of the present invention.

The leukapheresis products, blood, WBC/PBMC compositions, and/or T-cell compositions are packaged using insulated containers and should be maintained at controlled room temperatures (+19 ℃ to +25 ℃) in accordance with standard hospital facility blood collection procedures approved for clinical protocols. The leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should not be cryopreserved.

During transport, the cytoconcentration leukopheresis product, blood, WBC/PBMC composition and/or T-cell composition should not exceed 0.2x10⁹cells/mL. Intensive mixing of leukopheresis products, blood, WBC/PBMC compositions and/or T-cell compositions should be avoided.

If the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition must be stored, for example overnight, it should be maintained at a controlled room temperature (as above). The concentration of leukopheresis product, blood, WBC/PBMC composition and/or T-cell composition during storage should never exceed 0.2X10⁹Individual cells/mL.

Preferably, the leukopheresis product, blood, cells of the WBC/PBMC composition and/or the T-cell composition should be stored in autologous plasma. In certain embodiments, if the cell concentration of the leukapheresis product, blood, WBC/PBMC composition, and/or T-cell composition is greater than 0.2x10⁹Individual cells/mL, product diluted with autologous plasma.

Preferably, the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should not exceed 24 hours when the labeling and separation procedure is initiated. Leukapheresis products, blood, WBC/PBMC compositions, and/or T-cell compositions can be processed and/or prepared for cell labeling using a closed and/or automated system (e.g., CliniMACS diagnosis).

Automated systems may perform additional buffy coat separations (e.g., leukopheresis products, blood, WBC/PBMC compositions, and/or T-cell compositions) by percolation (flocculation) and/or washing of cell products.

Closed and/or automated systems can be used to prepare and label cells for T-cell separation (from, e.g., leukapheresis products, blood, WBC/PBMC compositions, and/or T-cell compositions).

Although WBCs/PBMCs can be directly transfected nuclearly (which is easier and preserves other steps), the methods of the invention can include first isolating T cells prior to nuclear transfection. The simpler strategy of direct nuclear transfection of PBMCs requires selective amplification of CAR + cells mediated via CAR signaling, which in itself turns proves to be a poor amplification method that directly reduces the in vivo efficiency of the product by depleting T cell function. The product may be a heterogeneous composition of CAR + cells, including T cells, NK cells, NKT cells, monocytes, or any combination thereof, which increases the variability of the product from patient to patient and makes dosing and CRS management more difficult. Because T cells are thought to be the primary effector of tumor inhibition and killing, the use of T cell isolation for the manufacture of autologous products may result in significant benefits over other, more heterogeneous compositions.

T cells can be isolated directly by enriching for labeled cells or depleting labeled cells in a one-way labeling procedure, or indirectly in a two-step labeling procedure. According to certain enrichment strategies of the present invention, T cells can be collected in a cell collection bag, while unlabeled cells (non-target cells) are collected in a negative fraction bag. In contrast to the enrichment strategy of the present invention, unlabeled cells (target cells) are collected in a cell collection bag, while labeled cells (non-target cells) are collected in a negative fraction bag or a non-target cell bag, respectively. The selection reagent may include, but is not limited to, antibody coated beads. The antibody-coated beads can either be removed prior to the modification and/or amplification step or remain on the cells prior to the modification and/or amplification step. One or more of the following non-limiting examples of cell markers can be used to isolate T-cells: CD3, CD4, CD8, CD25, anti-biotin, CD1c, CD3/CD19, CD3/CD56, CD14, CD19, CD34, CD45RA, CD56, CD62L, CD133, CD137, CD271, CD304, IFN- γ, TCR α/β, and/or any combination thereof. Methods for isolating T-cells may include one or more reagents that specifically bind to and/or detectably label one or more of the following non-limiting examples of cellular markers useful for isolating T-cells: CD3, CD4, CD8, CD25, anti-biotin, CD1c, CD3/CD19, CD3/CD56, CD14, CD19, CD34, CD45RA, CD56, CD62L, CD133, CD137, CD271, CD304, IFN- γ, TCR α/β, and/or any combination thereof. These agents may or may not be of the "good manufacturing practice" ("GMP") grade. Reagents may include, but are not limited to Thermo DynaBeads and Miltenyi CliniMACS products. Methods of isolating T-cells of the invention may comprise multiple iterations of the labeling and/or isolation steps. At any point in the method of isolating the T-cells of the invention, unwanted cells and/or unwanted cell types may be depleted from the T-cell product composition of the invention by positive or negative selection of unwanted cells and/or unwanted cell types. T cell product compositions of the invention may contain additional cell types that may express CD4, CD8, and/or another T cell marker.

The method of the invention for nuclear transfection of T cells may eliminate the step of T cell isolation by methods such as transfection of T cell nuclei in a population or composition of WBCs/PBMCs, which includes an isolation step or a selective amplification step via TCR signaling after nuclear transfection.

Certain cell populations may be eliminated by positive or negative selection before or after T cell enrichment and/or sorting. Examples of cell compositions that can be depleted from the cell product composition can include myeloid cells, CD25+ regulatory T cells (TRegs), dendritic cells, macrophages, erythrocytes, mast cells, γ - δ T cells, Natural Killer (NK) -like cells (e.g., cytokine-induced killer (CIK) cells), Induced Natural Killer (iNK) T cells, NK T cells, B cells, or any combination thereof.

The T cell product compositions of the invention may include CD4+ and CD8+ T-cells. During the isolation or selection procedure, CD4+ and CD8+ T-cells may be isolated into separate collection bags. CD4+ T cells and CD8+ T cells can be further treated separately or at a specific ratio after reconstitution (combined into the same composition).

The particular ratio of reconstitutable CD4+ T cells and CD8+ T cells may depend on the type and potency of the expansion technique used, the cell culture medium, and/or the growth conditions used to expand the T-cell product composition. Examples of possible CD4+: CD8+ ratios include, but are not limited to, 50%:50%, 60%:40%, 40%: 60%: 75%:25% and 25%: 75%.

CD8+ T cells exhibit a strong ability to kill tumor cells, while CD4+ T cells provide many cytokines required to support the proliferative capacity and function of CD8+ T cells. Because T cells isolated from normal donors are predominantly CD4+, the T-cell product composition is artificially modulated in vitro according to the CD4+: CD8+ ratio to increase the ratio of CD4+ T cells to CD8+ T cells, which otherwise occur in vivo. The optimized ratio can also be used for ex vivo expansion of autologous T-cell product compositions. In view of the CD4+: CD8+ ratio of the artificially regulated T-cell product composition, it is important to note that the product composition of the present invention may differ significantly and provide greater advantages compared to any naturally occurring T-cell population.

Preferred methods for T cell isolation may include a negative selection strategy for generating untouched pan T cells, meaning that the resulting T-cell composition includes T-cells that are not manipulated and contain the species/ratio of naturally occurring T-cells.

Reagents that can be used for positive or negative selection include, but are not limited to, magnetic cell separation beads. The magnetic cell separation beads may or may not be removed or depleted from selected CD4+ T cells, CD8+ T cell populations, or mixed populations of both CD4+ and CD8+ T cells prior to performing the next step in the T-cell separation method of the invention.

T cell compositions and T cell product compositions can be prepared for cryopreservation, storage in standard T cell culture media, and/or genetic modification.

The T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof can be cryopreserved using standard cryopreservation methods optimized for storing and recovering human cells with high recovery, viability, phenotype, and/or functional capacity. Commercially available cryopreservation media and/or protocols can be used. The cryopreservation methods of the invention can include a DMSO-free cryopreservative (e.g., a Cryofree ™ DMSO-free cryopreservation medium) to reduce freeze-related toxicity.

The T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof can be maintained in a culture medium. The T cell culture media of the invention can be optimized for cell storage, cell genetic modification, cell phenotype, and/or cell expansion. The T cell culture medium of the invention may comprise one or more antibodies. Because the inclusion of antibiotics in the cell culture medium may reduce transfection efficiency and/or cell yield following genetic modification via nuclear transfection, the specific antibiotics (or combinations thereof) and their respective concentrations may be varied to obtain optimal transfection efficiency and/or cell yield following genetic modification via nuclear transfection.

The T cell culture medium of the present invention may include serum, and, moreover, the serum composition and concentration may be varied to obtain optimal cell results. Human AB serum is more suitable for T cell culture than FBS/FCS because, although considered for use in the T cell culture medium of the invention, FBS/FCS can introduce foreign proteins. Serum may be isolated from blood intended for administration to a T-cell composition cultured by a subject, and thus, the T-cell culture medium of the invention may comprise autologous serum. Serum-free media or serum-substitutes may also be used in the T cell culture media of the invention. In certain embodiments of the T-cell culture media and methods of the invention, serum-free media or serum-substitutes can provide advantages over supplementing the media with xenogenic serum, including, but not limited to, healthier cells with higher viability, performing nuclear transfection with higher efficiency, exhibiting greater viability following nuclear transfection, exhibiting more desirable cell phenotypes, and/or greater/faster expansion following increased expansion techniques.

The T cell culture medium may comprise commercially available cell growth media. Exemplary commercially available cell growth media include, but are not limited to, PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, or any combination thereof.

T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any portion thereof can be prepared for genetic modification. Preparation of T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any portion thereof for genetic modification can include cell washing and/or resuspension in a desired nuclear transfection buffer. Cryopreserved T-cell compositions can be thawed and prepared for genetic modification by nuclear transfection. Cryopreserved cells can be thawed according to standard or known protocols. Thawing and preparation of cryopreserved cells can be optimized to produce cells that have greater viability, are more efficiently transfected, exhibit greater viability following nuclear transfection, exhibit a more desirable cell phenotype, and/or are expanded more/faster following the addition of expansion techniques. For example, Grifols albumin (25% human albumin) can be used in the thawing and/or preparation process.

Genetic modification of T cells

The T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof can be genetically modified using, for example, a nuclear transfection strategy such as electroporation. The total number of cells to be subjected to nuclear transfection, the total volume of the nuclear transfection reaction and the precise time to prepare the sample can be optimized to produce cells that have greater viability, are transfected with higher efficiency, exhibit greater viability following nuclear transfection, exhibit a more desirable cell phenotype, and/or are expanded more/more quickly following increased expansion techniques.

Nuclear transfection and/or electroporation can be accomplished using, for example, Lonza Amaxa, MaxCyte pulseAgile, Harvard Apparatus BTX, and/or Invitrogen Neon. Non-metal electrode systems, including but not limited to plastic polymer electrodes, may be preferred for nuclear transfection.

Prior to genetic modification by nuclear transfection, the T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof can be resuspended in nuclear transfection buffer. The nuclear transfection buffer of the present invention includes commercially available nuclear transfection buffers. The nuclear transfection buffers of the present invention can be optimized to produce cells that have greater viability, nuclear transfection at higher efficiency, exhibit greater viability following nuclear transfection, exhibit a more desirable cell phenotype, and/or greater/faster expansion following increased expansion techniques. The nuclear transfection buffer of the present invention may include, but is not limited to, PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T nuclear transfection buffer, and any combination thereof. The nuclear transfection buffers of the present invention may comprise one or more supplemental factors to produce cells that have greater viability, are transfected with greater efficiency, exhibit greater viability following nuclear transfection, exhibit a more desirable cell phenotype, and/or expand more/more rapidly following increased expansion techniques. Exemplary complementing factors include, but are not limited to, recombinant human cytokines, chemokines, interleukins, and any combination thereof. Exemplary cytokines, chemokines, and interleukins include, but are not limited to, IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, GM-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12 p 13, IL-12/IL-35 p 13, IL-13, IL-17/IL-17A-17F 72, IL-17A/F72, IL-17F-17, IL-32, IL-13, IL-3632, IL-13, IL-17F-32, IL-17F-17, IL-17, LAP (TGF-. beta.1), lymphotoxin-. alpha./TNF-. beta., TGF-. beta., TNF-. alpha., TRANCE/TNFSF11/RANK L, and any combination thereof. Exemplary supplemental factors include, but are not limited to, salts, minerals, metabolites, or any combination thereof. Exemplary salts, minerals, and metabolites include, but are not limited to, HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solutions, ascorbic acid, nucleosides, FBS/FCS, human serum, serum substitutes, antibiotics, pH modifiers, Erer's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, and any combination thereof. Exemplary supplemental factors include, but are not limited to, media such as PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC media, CTS OpTimizer T cell expansion SFM, TexMACS media, PRIME-XV T cell expansion media, ImmunoCult-XF T cell expansion media, and any combination thereof. Exemplary complementing factors include, but are not limited to, inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, apoptotic pathways, and combinations thereof. Exemplary inhibitors include, but are not limited to, TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, inhibitors of Wnt3A, inhibitors of glycogen synthase kinase-3 β (GSK-3 β) (e.g., TWS119), Bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, IEZ-TD-FMK, and any combination thereof. Exemplary complementing factors include, but are not limited to, agents that modify or stabilize one or more nucleic acids in a manner that enhances cellular delivery, enhances nuclear delivery or transport, enhances the transport of nucleic acids into the nucleus, enhances degradation of epichromosomal nucleic acids, and/or reduces DNA-mediated toxicity. Exemplary agents that modify or stabilize one or more nucleic acids include, but are not limited to, pH adjusting agents, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, and any combination thereof.

Transposable reagents, including transposons and transposases, can be added to the nuclear transfection reactions of the invention before, simultaneously with, or after the cells are added to the nuclear transfection buffer (optionally contained in the nuclear transfection reaction vial or cuvette). Transposons of the invention can comprise plasmid DNA, linear plasmid DNA, PCR products, DOGGYBONE ™ DNA, mRNA templates, single-or double-stranded DNA, protein-nucleic acid combinations, or any combination thereof. Transposons of the invention can comprise one or more sequences encoding one or more TTAA sites, one or more Inverted Terminal Repeats (ITRs), one or more Long Tail Repeats (LTRs), one or more insulators, one or more promoters, one or more full-length or truncated genes, one or more polya signals, one or more self-cleaving 2A peptide cleavage sites, one or more Internal Ribosome Entry Sites (IRES), one or more enhancers, one or more regulatory genes, one or more origins of replication, and any combination thereof.

The transposons of the invention may comprise one or more sequences encoding one or more full-length or truncated genes. The full-length and/or truncated gene introduced by the transposon of the invention can encode one or more signal peptides, centryrin, single-chain variable fragments (scFv), hinges, transmembrane domains, co-stimulatory domains, chimeric ligand/antigen receptors (CLR/CAR), chimeric T-cell receptors (CAR-T), CARTyrin (CAR-T comprising centryrin), receptors, ligands, cytokines, drug-resistant genes, tumor ligands, alloligands or auto-ligands, enzymes, proteins, peptides, polypeptides, fluorescent proteins, muteins, or any combination thereof.

The transposon of the present invention can be prepared in water, TAE, TBE, PBS, HBSS, culture medium, a complementing factor of the present invention, or any combination thereof.

The transposons of the present invention can be designed to optimize clinical safety and/or improve manufacturability. As a non-limiting example, transposons of the invention can be designed to optimize clinical safety and/or improve manufacturability by eliminating unnecessary sequences or regions and/or including non-antibiotic selection markers. The transposons of the invention may or may not be of GMP grade.

Transposases of the invention can be encoded by one or more sequences of plasmid DNA, mRNA, protein-nucleic acid combinations, or any combination thereof.

Transposases of the invention can be prepared in water, TAE, TBE, PBS, HBSS, culture media, supplements of the invention, or any combination thereof. The transposases of the invention or the sequences/constructs encoding or delivering them may or may not be GMP grade.

The transposons and transposases of the invention can be delivered to cells by any means.

Although the compositions and methods of the invention include delivering the transposons and/or transposases of the invention to cells via plasmid DNA (pdna), the use of plasmids for delivery may allow the transposons and/or transposases to be integrated into the chromosomal DNA of the cells, which may result in continuous transposase expression. Thus, the transposons and/or transposases of the invention can be delivered to cells as mRNA or protein to remove the possibility of any chromosomal integration.

The transposons and transposases of the invention may be preincubated separately or in combination prior to the introduction of the transposons and/or transposases into the nuclear transfection reaction. The absolute amounts, as well as the relative amounts, e.g., the ratio of transposon to transposase, of each transposon and transposase can be optimized.

After preparation of the nuclear transfection reaction, optionally in a vial or cuvette, the reaction may be loaded into a nuclear transfectator device and activated for delivery of a current pulse according to the manufacturer's protocol. Current pulsing conditions for delivering a transposon and/or transposase of the invention (or a sequence encoding a transposon and/or transposase of the invention) to a cell can be optimized to produce cells with enhanced viability, higher nuclear transfection efficiency, greater viability following nuclear transfection, a more desirable cell phenotype, and/or increased amplification greater/faster following amplification techniques. For Amaxa 2B or 4D nuclear transfectants, each different nuclear transfection procedure was considered.

After the nuclear transfection reaction of the present invention, the cells may be gently added to the cell culture medium. For example, when T cells undergo a nuclear transfection reaction, the T cells may be added to the T cell culture medium. The post-nuclear transfection cell culture medium of the present invention may comprise any one or more commercially available media. The post-nuclear transfection cell culture media of the invention (including the post-nuclear transfection T cell culture media of the invention) can be optimized to produce cells that have greater viability, higher nuclear transfection efficiency, exhibit greater viability following nuclear transfection, exhibit more desirable cell phenotypes, and/or are expanded more/faster following the addition of expansion techniques. Post-nuclear transfection cell culture media of the invention (including post-nuclear transfection T cell culture media of the invention) may comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, and any combination thereof. Post-nuclear transfection cell culture media of the invention (including post-nuclear transfection T cell culture media of the invention) may comprise one or more of the supplemental factors of the invention to enhance viability, nuclear transfection efficiency, efficiency of viability after nuclear transfection, cell phenotype, and/or greater/faster expansion following increased expansion techniques. Exemplary complementing factors include, but are not limited to, recombinant human cytokines, chemokines, interleukins, and any combination thereof. Exemplary cytokines, chemokines and interleukins include, but are not limited to, IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12 p 13, IL-12/IL-35 p 13, IL-13, IL-17/IL-17A-17F 72, IL-17A/F72, IL-17F-17, IL-17F-32, IL-3632, IL-13, IL-3632, IL-13, IL-17F-17, IL-17F-17, LAP (TGF-. beta.1), lymphotoxin-. alpha./TNF-. beta., TGF-. beta., TNF-. alpha., TRANCE/TNFSF11/RANK L, and any combination thereof. Exemplary supplemental factors include, but are not limited to, salts, minerals, metabolites, or any combination thereof. Exemplary salts, minerals, and metabolites include, but are not limited to, HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solutions, ascorbic acid, nucleosides, FBS/FCS, human serum, serum substitutes, antibiotics, pH modifiers, Erer's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, and any combination thereof. Exemplary supplemental factors include, but are not limited to, media such as PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC media, CTSOpTimizer T cell expansion SFM, TexMACS media, PRIME-XV T cell expansion media, ImmunoCult-XF T cell expansion media, and any combination thereof. Exemplary complementing factors include, but are not limited to, inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, apoptotic pathways, and combinations thereof. Exemplary inhibitors include, but are not limited to, TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNAfield III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, inhibitors of Wnt3A, inhibitors of glycogen synthase kinase-3 beta (GSK-3 beta) (e.g., TWS119), Bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, IEZ-TD-FMK, and any combination thereof. Exemplary complementing factors include, but are not limited to, agents that modify or stabilize one or more nucleic acids in a manner that enhances cellular delivery, enhances nuclear delivery or transport, enhances the transport of nucleic acids into the nucleus, enhances degradation of epichromosomal nucleic acids, and/or reduces DNA-mediated toxicity. Exemplary agents that modify or stabilize one or more nucleic acids include, but are not limited to, pH adjusting agents, DNA-binding proteins, lipids, phospholipids, CaPO4, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, and any combination thereof.

The post-nuclear transfection cell culture media of the invention (including the post-nuclear transfection T cell culture media of the invention) can be used at room temperature or pre-warmed to, for example, between 32 ℃ and 37 ℃ (inclusive). The post-nuclear transfection cell culture medium of the invention (including the post-nuclear transfection T cell culture medium of the invention) can be pre-warmed to any temperature that maintains or increases cell viability and/or expresses the transposons or portions thereof of the invention.

The post-nuclear transfection cell culture media of the invention (including the post-nuclear transfection T cell culture media of the invention) may be contained in tissue culture flasks or dishes, G-Rex flasks, bioreactors or cell culture bags, or any other standard receptacle. The post-nuclear transfection cell cultures of the invention (including post-nuclear transfection T cell cultures of the invention) may be kept stationary or, alternatively, they may also be agitated (e.g., shaken, rotated or shaken).

The cell culture may comprise genetically modified cells after nuclear transfection. The post-nuclear transfection T cell culture may comprise genetically modified T cells. The genetically modified cells of the invention may be allowed to rest for a defined period of time or stimulated to expand by, for example, the addition of the T cell Expander technology. In certain embodiments, the genetically modified cells of the invention may either rest for a specific period of time or immediately stimulate expansion, for example, by adding the T cell Expander technology. The genetically modified cells of the invention can be quiescent to allow them sufficient time to adapt, sufficient time for transposition to occur, and/or time for positive or negative selection, resulting in cells with enhanced viability, higher nuclear transfection efficiency, greater viability following nuclear transfection, desired cell phenotype, and/or greater/faster expansion following increased expansion techniques. The genetically modified cells of the invention can be quiescent, e.g., for 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours. In certain embodiments, the genetically modified cells of the invention may be quiescent, e.g., overnight. In certain aspects, the overnight is about 12 hours. The genetically modified cells of the invention can be quiescent, e.g., for 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

The genetically modified cells of the invention can be selected after the nuclear reaction and before the expander technique is added. For optimal selection of genetically modified cells, the cells may be allowed to rest in cell culture media for at least 2-14 days following nuclear transfection to facilitate identification of modified cells (e.g., to distinguish modified from non-modified cells).

Expression of the CAR/CARTyrin and selectable marker of the invention can be detected in modified T cells following successful nuclear transfection of the transposon of the invention as early as 24-hours after nuclear transfection. Expression of the selectable marker alone is unable to distinguish modified T cells (those in which the transposon has successfully integrated) from unmodified T cells (those in which the transposon has not successfully integrated) due to epichromosomal expression of the transposon. When the episomal expression of the transposon masks the modified cells detected by the selectable marker, the nuclear transfected cells (both modified and unmodified cells) may be allowed to sit for a period of time (e.g., 2-14 days) to allow the cells to cease expression or lose all of the episomal transposon expression. After this extended rest period, only the modified T cells should remain positive for expression of the selectable marker. The length of this extended resting phase can be optimized for each nuclear transfection reaction and selection procedure. When the episomal expression of the transposon masks the modified cells detected by the selectable marker, selection can be performed without this extended resting phase, however, additional selection steps can be included at a later point in time (e.g., either during or after the amplification stage).

The selection of the genetically modified cells of the invention can be performed in any manner. In certain embodiments of the methods of the invention, selection of the genetically modified cells of the invention can be performed by isolating cells that express a particular selectable marker. The selectable marker of the invention may be encoded by one or more sequences in a transposon. The selectable marker of the invention may be expressed by the modified cell as a result of successful transposition (i.e., one or more sequences in the transposon are not encoded). In certain embodiments, the genetically modified cells of the invention contain a selectable marker that confers resistance to the target compound of the cell culture medium following nuclear transfection. The target compound may comprise, for example, an antibiotic or drug, which may result in cell death in the absence of the selectable marker conferring resistance to the modified cell. Exemplary selectable markers include, but are not limited to, Wild Type (WT) or mutated forms of one or more of the following genes: neo, DHFR, TYMS, ALDH, MDR1, MGMT, FANCF, RAD51C, GCS, and NKX 2.2. Exemplary selectable markers include, but are not limited to, surface-expressed selectable markers or surface-expressed tags that can be targeted by Ab-coated magnetic bead technology or column selection, respectively. Cleavable tags such as those used for protein purification can be added to the selection markers of the present invention for efficient column selection, washing and elution. In certain embodiments, the selectable markers of the invention are not expressed by naturally modified cells (including modified T cells) and, therefore, can be used to modify the physical separation of cells (e.g., by cell sorting techniques). Exemplary selectable markers of the invention are not expressed by naturally modified cells (including modified T cells), including, but not limited to, full-length, mutated, or truncated forms of CD271, CD19, CD52, CD34, RQR8, CD22, CD20, CD33, and any combination thereof.

The genetically modified cells of the invention can be selectively amplified following a nuclear transfection reaction. In certain embodiments, a modified T cell comprising CAR/CARTyrin can be selectively expanded by CAR/CARTyrin stimulation. Modified T cells comprising CAR/CARTyrin can be stimulated by contact with a target-coated agent (e.g., a tumor cell line or a normal cell line expressing the target or expanded beads coated in the target). Alternatively, modified T cells comprising CAR/CARTyrin can be stimulated by contact with irradiated tumor cells, irradiated allogeneic normal cells, irradiated autologous PBMCs. To minimize contamination of the cell product composition of the invention with the target-expressing cells for stimulation, for example, expanded beads coated with the CAR/CARTyrin target protein can be used for stimulation when the cell product composition can be administered directly to a subject. Selective expansion of modified T cells comprising CAR/CARTyrin by CAR/CARTyrin stimulation can be optimized to avoid functionally depleting modified T-cells.

The selected genetically modified cells of the invention may be cryopreserved, rested for a defined period of time, or expanded by the addition of cellular Expander technology to stimulate expansion. The selected genetically modified cells of the invention may be cryopreserved, rested for a defined period of time, or immediately stimulated to expand by the addition of the cell Expander technology. When the selected genetically modified cell is a T cell, the expansion of the T cell can be stimulated by the addition of the T-cell Expander technology. Selected genetically modified cells of the invention may be quiescent, e.g., for 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours. In certain embodiments, the selected genetically modified cells of the invention may be quiescent, e.g., overnight. In certain aspects, overnight is about 12 hours, and selected genetically modified cells of the invention can be quiescent, e.g., for 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. The genetically modified cells of the invention selected may be rested for any period of time, resulting in cells with enhanced viability, higher efficiency of nuclear transfection, greater viability following nuclear transfection, more desirable cell phenotype, and/or greater/faster expansion following increased expansion techniques.

Selected genetically modified cells (including selected genetically modified T cells of the invention) can be cryopreserved using any standard cryopreservation method, which can be optimized for storage and/or recovery of human cells with high recovery, viability, phenotype and/or functional capacity. The cryopreservation methods of the invention can include commercially available cryopreservation media and/or protocols.

The efficiency of transposition of selected genetically modified cells, including selected genetically modified T cells of the invention, can be assessed by any means. For example, transposons expressed by selected genetically modified cells, including selected genetically modified T cells of the invention, can be measured by fluorescence-activated cell sorting (FACS) prior to application of the expander technology. The determination of the transposition efficiency of the selected genetically modified cells, including the selected genetically modified T cells of the invention, can comprise determining the percentage of selected cells that express a transposon (e.g., CAR). Alternatively, or in addition, the purity of the T cell, the Mean Fluorescence Intensity (MFI) of transposon expression (e.g., CAR expression), the ability of the CAR (delivered in the transposon) to mediate degranulation and/or killing of target cells expressing the CAR ligand, and/or the phenotype of selected genetically modified cells (including selected genetically modified T cells of the invention) can be assessed by any method.

The cell product compositions of the invention can be released for administration to a subject after certain release criteria are met. Exemplary release criteria can include, but are not limited to, a specific percentage of T cells that express detectable levels of modification, selection, and/or expansion of the CAR on the cell surface.

Generation of CAR-expressing T cells

Genetically modified cells of the invention (including genetically modified T cells) can be expanded using the Expander technology. The Expander technology of the present invention may comprise commercially available Expander technology. Exemplary Expander technology of the invention includes stimulating the genetically modified T cells of the invention via a TCR. Although all means of stimulating the genetically modified T cells of the invention are contemplated, stimulation of the genetically modified T cells of the invention via a TCR is a preferred method, resulting in a product with excellent levels of lethality.

To stimulate the genetically modified T cells of the invention via TCR, Thermo Expander DynaBeads can be used at a 3:1 bead to T cell ratio. If the expander beads are not biodegradable, the beads can be removed from the expander composition. For example, after about 5 days, the beads can be removed from the expander composition. To stimulate the genetically modified T cells of the invention via the TCR, Miltenyi T Cell Activation/Expansion reagents (Miltenyi T Cell Activation/Expansion Reagent) can be used. To stimulate the genetically modified T cells of the invention via TCR, ImmunoCult Human CD3/CD28 or CD3/CD28/CD 2T cell activator reagents from stemCell technologies can be used. This technique may be preferred because soluble tetrameric antibody complexes degrade over time and do not need to be removed from the process.

Artificial ligand presenting cells (APCs) may be engineered to co-express a target ligand and may be used to stimulate cells or T-cells of the invention via TCRs and/or CARs of the invention. The artificial APCs can comprise or can be derived from a tumor cell line (including, for example, the immortalized myeloid leukemia cell line K562) and can be engineered to co-express a variety of co-stimulatory molecules or technologies (e.g., CD28, 4-1BBL, CD64, mbIL-21, mbIL-15, CAR target molecules, etc.). When the artificial APCs of the present invention are combined with co-stimulatory molecules, conditions can be optimized to prevent the development or emergence of undesirable phenotypic and functional capacity, i.e., terminally differentiated effector T cells.

Irradiated PBMCs (autologous or allogeneic) may express some target ligand, such as CD19, and may be used to stimulate the cells or T-cells of the invention via the TCRs and/or CARs of the invention. Alternatively, or in addition, the irradiated tumor cells may express some target ligands and may be used to stimulate the cells or T-cells of the invention by the TCRs and/or CARs of the invention.

Plate-bound and/or soluble anti-CD 3, anti-CD 2, and/or anti-CD 28 stimulation may be used to stimulate the cells or T-cells of the invention via the TCRs and/or CARs of the invention.

The ligand-coated beads can display a target protein and can be used to stimulate cells or T-cells of the invention via a TCR and/or CAR of the invention. Alternatively, or additionally, expander beads coated with CAR/CARTyrin target proteins can be used to stimulate cells or T-cells of the invention via a TCR and/or CAR of the invention.

The expansion method involves stimulating the cells of the invention or T-cells by TCR or CAR/CARTyrin and other markers on the T-cells via surface-expressed CD2, CD3, CD28, 4-1BB and/or genetic modification.

Amplification techniques can be applied to cells of the invention immediately after nuclear transfection until about 24 hours after nuclear transfection. While various cell culture media can be used during the expansion procedure, the desired T cell expansion media of the present invention can produce cells with, for example, greater viability, cell phenotype, total expansion or greater capacity for in vivo persistence, engraftment, and/or CAR-mediated killing. The cell culture medium of the invention can be optimized to improve/enhance the amplification, phenotype and function of the genetically modified cells of the invention. Preferred phenotypes of expanded T cells may include a mixture of T stem cell memory, T central and T effector memory cells. Expander Dynabeads can produce predominantly central memory T cells, which can lead to superior clinical manifestations.

Exemplary T Cell Expansion media of the invention may include some or all of PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium (CellGro DC Medium), CTS Optimizer T Cell Expansion (T Cell Expansion) SFM, TexMACS Medium (TexMACS Medium), PRIME-XV T Cell Expansion Medium (PRIME-XV T Cell Expansion Medium), ImmunoCult-XF T Cell Expansion Medium (ImmunoCult-XF T Cell Expansion Medium), or any combination thereof. The T cell expansion medium of the present invention may further comprise one or more supplemental factors. Supplemental factors that can be included in the T cell expansion media of the invention enhance viability, cell phenotype, total expansion, or increase in vivo persistence, engraftment, and/or CAR-mediated killing ability. The supplementary factors that can be included in the T cell expansion medium of the present invention include, but are not limited to, recombinant human cytokines, chemokines and/or interleukins such as IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, GM-IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12 p-35, IL-12 p-72, IL-13, IL-17F-17A/F17, IL13, IL-17F-17, IL13, IL-17F-17, IL13, IL-17F-17/17F-17, IL13, IL-32, IL-32 β, IL-32 γ, IL-33, LAP (TGF- β 1), lymphotoxin- α/TNF- β, TGF- β, TNF- α, TRANCE/TNFSF11/RANK L, or any combination thereof. Supplemental factors that can be included in the T cell expansion media of the invention include, but are not limited to, salts, minerals, and/or metabolites, such as HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM nonessential amino acid solution, ascorbic acid, nucleosides, FBS/FCS, human serum, serum replacement, antibiotics, pH regulators, el's salt, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. Supplemental factors that may be included in the T cell expansion media of the invention include, but are not limited to, inhibitors of cellular DNA sensing, metabolic, differentiation, signal transduction, and/or apoptotic pathways, such as TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferons, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3 β (GSK-3 β) (such as TWS119), Bafilomycin, chloroquinacrine, AC-YVAD-FMK, Z-Wnt FMK, Z-TD-IEK, VAD, or any combination thereof.

Supplemental factors that may be included in the T cell amplification medium of the invention include, but are not limited to, agents that modify or stabilize nucleic acids in a manner that enhances cell delivery, enhances nuclear delivery or transport, enhances transport of nucleic acids into the nucleus, enhances degradation of episomal nucleic acids, and/or reduces DNA-mediated toxicity, such as pH regulators, DNA-binding proteins, lipids, phospholipids, CaPO4, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, or any combination thereof.

The genetically modified cells of the invention can be selected during the amplification process by using alternative drugs or compounds. For example, in certain embodiments, when the transposons of the invention can encode a selection that confers resistance to a drug added to the culture medium on genetically modified cells, the selection can occur during the amplification process and may require approximately 1-14 days of culture for selection. Examples of drug resistance genes that can be used as selectable markers encoded by the transposons of the invention include, but are not limited to, the wild-type (WT) or mutant forms of the genes neo, DHFR, TYMS, ALDH, MDR1, MGMT, FANCF, RAD51C, GCS, NKX2.2, or any combination thereof. Examples of corresponding drugs or compounds that can be added to the medium in which the selection marker can confer resistance include, but are not limited to, G418, puromycin, ampicillin, kanamycin, methotrexate, melphalan, temozolomide, vincristine, etoposide, doxorubicin, bendamustine, fludarabine, Aredia (disodium pamidronate), Becenum (carmustine), BiCNU (carmustine), Bortezomib (Bortezomib), Carfilzomib (Carfilzomib), carmubin (carmustine), carmustine, Clafen (cyclophosphamide), cyclophosphamide, Cytoxan (cyclophosphamide), darunavir (Darzalex), doxorubin (Darzalex), Doxil (liposome), doxorubicin hydrochloride liposome, doxorubin-SL (doxorubin hydrochloride liposome), lutumumab, empilirubicin (etotuzumab)), and doxorubin (evtuzumab (hydrochloric acid)) Farydak (Panobinostat), ixazofamid (Ixazomib) citrate, Kyprolis (carfilzomib), lenalidomide, LipoDox (liposomal doxorubicin hydrochloride), Mozobil (Plerixafor), Neosar (cyclophosphamide), nilaro (ixazofamid citrate), disodium pamidronate, panobistat (Panobinostat), Plerixafor (Plerixafor), Pomalidomide (pomidomide)/pomalyt (Pomalidomide), revlimide (lenalidomide), Synovir (thalidomide), thalidomide, Thalomid (thalidomide), velcadede (bortezomib), Zoledronic Acid (Zoledronic Acid), zeta (Zoledronic Acid), or any combination thereof.

The T-cell expansion process of the present invention may take place in a WAVE bioreactor, a G-Rex flask, or in a cell culture bag in any other suitable container and/or reactor.

The cells or T-cell cultures of the invention may be kept stable, shaken, rotated or shaken.

The cell or T-cell expansion process of the invention may optimize certain conditions including, but not limited to, culture duration, cell concentration, schedule of T-cell culture medium addition/removal, cell size, total cell number, cell phenotype, purity of the cell population, percentage of genetically modified cells in the growing cell population, use and composition of supplements, addition/removal of expander techniques, or any combination thereof.

The cell or T-cell expansion process of the invention may be continued up to a predefined endpoint and the resulting expanded cell population then formulated. For example, the cell or T-cell expansion process of the invention may be continued for a predetermined amount of time: at least 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours; at least 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 days; at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 weeks; for at least 1, 2, 3, 4, 5,6 months, or for at least 1 year. The cell or T-cell expansion process of the invention may be continued until the resulting culture reaches a predetermined total cell density: 1. 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010 cells/volume (μ Ι, ml, L) or any density in between. The cell or T-cell expansion process of the invention may be continued until the genetically modified cells of the resulting culture exhibit a predetermined level of expression of the transposon of the invention: 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or any percentage therebetween of a threshold level of expression (a minimum, maximum, or average level of expression indicates that the resulting genetically modified cell is clinically effective). The cell or T-cell expansion process of the invention may be continued until the resulting culture has a ratio of genetically modified cells to unmodified cells reaching a predetermined threshold: at least 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9: 110: 1, or any ratio therebetween.

Quality control analysis of CAR-expressing T cells prior to administration

The percentage of genetically modified cells can be assessed during or after the amplification process of the invention. The cell expression of the transposon of the genetically modified cell of the invention can be measured by fluorescence-activated cell sorting (FACS). For example, FACS can be used to determine the percentage of CAR-expressing cells or T cells of the invention. Alternatively, or additionally, the purity of the genetically modified cell or T cell, the Mean Fluorescence Intensity (MFI) of the CAR expressed by the genetically modified cell or T cell of the invention, the ability of the CAR to mediate degranulation and/or killing of target cells expressing the CAR ligand, and/or the phenotype of the CAR + T cell can be assessed.

Compositions of the invention intended for administration to a subject may be required to meet one or more "release criteria" that indicate that the composition is safe and effective for formulation as a pharmaceutical and/or administration to a subject. The release criteria may include the requirement that a composition of the invention (e.g., a T-cell product of the invention) comprises a specific percentage of T cells that express detectable levels of a CAR of the invention on their cell surface.

The expansion process should continue until certain criteria have been met (e.g., achieving a certain total cell number, achieving a certain memory cell number, achieving a certain size population).

The amplification process should end at that point with a specific standard signal. For example, once cells reach a cell size of 300fL (otherwise, cells reaching a size above this threshold may begin to die), the cells should be formulated, reactivated, or cryopreserved. Cryopreservation immediately once the cell population reaches an average cell size of less than 300fL can result in better cell recovery after thawing and culture because the cells have not reached a fully quiescent state before cryopreservation (fully quiescent size is about 180 fL). Prior to expansion, T cells of the invention may have a cell size of about 180 fL, but 3 days after expansion, their cell size may be more than four times, to about 900 fL. Over the next 6-12 days, the population of T cells will slowly reduce cell size to complete quiescence at 180 fL.

Methods for preparing a cell population for a formulation may include, but are not limited to, the steps of concentrating the cells of the cell population, washing the cells, and/or further selecting the cells via drug resistance or magnetic bead sorting for a particular surface-expressed marker. The method for preparing a cell population for formulation may further comprise a sorting step to ensure safety and purity of the final product. For example, if tumor cells from a patient have been used to stimulate the genetically modified T-cells of the invention or have been engineered to stimulate the genetically modified T-cells of the invention that are being prepared for formulation, it is important that the patient's tumor cells are not included in the final product.

Administration and preservation of CAR-expressing cells

The pharmaceutical preparation of the present invention may be packaged in bags for infusion, cryopreservation and/or storage.

The pharmaceutical formulations of the present invention may be cryopreserved using standard protocols and optionally infusible cryopreservation media. For example, a DMSO-free cryopreservative (e.g., a Cryofree ™ DMSO-free cryopreservation medium) can be used to reduce freeze-related toxicity. The cryopreserved pharmaceutical formulation of the present invention may be stored for infusion into a patient at a later date. Effective treatment may require multiple administrations of the pharmaceutical formulation of the invention, and therefore, the pharmaceutical formulation may be packaged in pre-aliquoted "doses" which may be stored frozen, but may be thawed separately for a single dose.

The pharmaceutical formulations of the present invention may be stored at room temperature. Effective treatment may require multiple administrations of the pharmaceutical formulation of the invention, and thus, the pharmaceutical formulation may be packaged in pre-aliquoted "doses" that may be stored together, but divided for thawing of the individual doses.

The pharmaceutical formulations of the invention may be archived, use subsequently re-expanded and/or selected for generating additional doses in the case of allotherapy for the same patient who may need to be administered at some later date, e.g., after remission and recurrence of the condition.

Infusion of modified cells as adoptive cell therapy

The present invention provides modified immune cells and HSCs for administration to a subject in need thereof. The modified cells of the invention can be formulated for storage at any temperature, including room temperature and body temperature. The modified cells of the invention can be formulated for cryopreservation and subsequent thawing. The modified cells of the invention can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. The modified cells of the invention can be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and/or CAR/CARTyrin expression level to ensure a minimum level of cell function and CAR/CARTyrin expression. The modified cells of the invention can be formulated at a defined density in a pharmaceutically acceptable carrier with one or more agents that inhibit further expansion and/or prevent cell death.

Examples

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