Senescence vaccine

Abstract

Methods, and compositions of matter useful for enhancing activity of endogenous and / or exogenous regenerative cells by selectively eliminating senescent cells through induction of immunity against said senescent cells or components thereof. Fusion of autologous patient cells made senescent, autologous antigen presenting cells and utilized as a vaccine. Autologous fibroblasts can be made senescent by genotoxic stress and fused with dendritic cells. Said dendritic cells may be generated from circulating monocytes, CD34 cells or autologous iPSC cells.

Description

SENESCENCE VACCINECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and benefit from U.S Provisional Application No. 63 / 560,359, titled “Senescence Vaccine” and filed on March 1, 2024, the entire contents of which are hereby expressly incorporated by reference.SEQUENCE LISTING

[0002] UUAUAAUGACUGGAUGUUC (SEQ ID NO: 1)

[0003] GUCUGGUGUAUGAAGGGUU (SEQ ID NO: 2)

[0004] CUCCUAUUUUGGUUUAUGC (SEQ ID NO: 3)

[0005] GCAGCGUCUUUCAGUGCUU (SEQ ID NO: 4)

[0006] EFDVILKAAGANKVAVIKAVRGATGLGLKEAKDLVESAPAALKEGVSKDDAEALKKALEEAGAEVEVK (SEQ ID NO: 5)FIELD OF INVENTION

[0007] The invention relates to the field of vaccines, more specifically the invention pertains to utilization of immune modulation to remove senescent cells in a subject.BACKGROUND

[0008] Historically, the process of aging has been associated with telomere shortening, which results in the cells reaching a critical stage in which they either die by programmed cell death such as apoptosis or become senescent. Senescent cells accumulate in tissues and organs of individuals as they age and are found at sites of age- related pathologies. While senescent cells are believed important to inhibiting proliferation of dysfunctional or damaged cells and particularly to constraining development of malignancy, the presence of senescent cells in an aging individual may contribute to aging and aging-related dysfunction. Given that senescent cells have been causally implicated in certain aspects of age-related decline in health and may contribute to certain diseases, and are also induced as a result of necessary life-preserving chemotherapeutic and radiation treatments, the presence of senescent cells may have deleterious effects to millions of patients worldwide (e.g., fatigue, weakness, loss of physical agility, decrease in cognitive function). Accordingly, treatments aimed atclearing aging-induced and therapy-induced senescent cells and improving age-sensitive traits have the potential to markedly improve the health, lifespan, and quality of life for patients exposed to senescence-inducing stimuli. The present disclosure addresses these needs and offers numerous means of clearing senescent cells through leveraging immunological mechanisms.SUMMARY

[0009] A summary of the invention is provided below with respect to the following numbered aspects.

[0010] 1. A method for reducing content of senescent cells in a mammal comprising the steps of: a) Selecting an immune competent subject or if said subject is not immune competent, enhancing immunity in said subject so as to reach immune competency; b) immunizing said subject with a composition capable of stimulating immunity towards senescent cells and / or antigens; and c) optionally repeating said immunization until sufficient immunity is generated to reduce load of said senescent cells.

[0011] 2 The method of aspect 1, wherein said senescent cells are host cells treated with one or genotoxins.

[0012] 3. The method of aspect 2, wherein said genotoxin is a chemotherapy or a radiotherapy.

[0013] 4. The method of aspect 2, wherein said genotoxin is doxorubicin

[0014] 5. The method of aspect 2, wherein said genotoxin is a DNA damaging agent.

[0015] 6. The method of aspect 1, wherein said senescence is associated with production of enhanced levels of TNF-alpha as compared to a non-senescent cell.

[0016] 7 The method of aspect 6, wherein said senescent cell produces enhancedTGF-beta as compared to a non-senescent cell.

[0017] 8. The method of aspect 1, wherein said senescent cells are autologous expanded fibroblasts treated with a genotoxin and fused with an antigen presenting cell.

[0018] 9. The method of aspect 8, wherein said antigen presenting cell is a dendritic cell.

[0019] 10. The method of aspect 1, wherein said senescence is associated with enhanced angiogenesis and said anti-senescence vaccine concurrently targets angiogenic antigens.

[0020] 11. The method of aspect 10, wherein said angiogenesis associated antigens are selected from a group comprising of: a) CD-248; b) Robo 1-8; c) VEGF-rl; d) VEGF- R2; e) FGF1-R; f) FGF2-R; g) endosialin; h) Integrin avP3; i) PDGF-BB receptor; j) EGF-R; and k) human chorionic gonadotropin receptor.

[0021] 12. The method of aspect 1, wherein said composition capable of stimulating immunity towards senescent cells contains beta galactosidase as well as senescent endothelial cells.

[0022] 13. The method of aspect 12, wherein said endothelial cells are derived from the umbilical vein.

[0023] 14. The method of aspect 13, wherein said umbilical vein cells expressCD133.

[0024] 15. The method of aspect 13, wherein said umbilical vein cells expressCD34.

[0025] 16. The method of aspect 13, wherein said umbilical vein cells express c-kit.

[0026] 17. The method of aspect 13, wherein said umbilical vein cells express c- met.

[0027] 18. The method of aspect 13, wherein said endothelial cells are derived from the chorionic portion of a placenta.

[0028] 19. The method of aspect 18, wherein said endothelial cells are derived from the perivascular area of the chorionic portion of the placenta.

[0029] 20. The method of aspect 13, wherein said endothelial cells are generated from a pluripotent stem cell population.

[0030] 21. The method of aspect 20, wherein said pluripotent stem cell population is selected from a group of cells comprising of: a) embryonic stem cells; b) inducible pluripotent stem cells; c) somatic cell nuclear transfer generated stem cells; and d) parthenogenic stem cells.

[0031] 22. The method of aspect 13, wherein said endothelial cells are generated from endothelial precursor cells.

[0032] 23. The method of aspect 22, wherein said endothelial precursor cells are obtaining from a population of cells selected from a group comprising of: a) peripheral blood mononuclear cells; b) adipose tissue derived stromal vascular fraction; c) umbilical cord blood; e) perivascular tissue obtained from the wharton’s jelly; and f) perivascular tissue obtained from the omentum.

[0033] 24. The method of aspect 22, wherein said endothelial precursor cells possess expression of the marker kdr-1.

[0034] 25. The method of aspect 24, wherein said endothelial cells possess replicative capacity upon isolation.

[0035] 26. The method of aspect 24, wherein said replicative capacity is endowed by culture in a media containing mitogens.

[0036] 27. The method of aspect 26, wherein said mitogens comprise growth factors.

[0037] 2 : 8. The method of aspect 26, wherein said mitogen is fetal calf serum.

[0038] 2 : 9. The method of aspect 26, wherein said mitogen is human serum.

[0039] 30. The method of aspect 26, wherein said mitogen is human umbilical cord blood serum.

[0040] 31. The method of aspect 26, wherein said mitogen is platelet lysate.

[0041] 32. The method of aspect 26, wherein said mitogen is platelet rich plasma.

[0042] 33. The method of aspect 26, wherein said mitogen is selected from a group of mitogens comprising of: a) VEGF; b) IGF-; c) FGF-1; d) FGF-2; e) TGF-alpha; f) FGF-5; g) PDGF; h) EGF; i) IL-13; j) IL-20; k) NGF; 1) BDNF; and m) HGF.

[0043] 34. The method of aspect 1 wherein said immunization is performed with an adjuvant.

[0044] 35. The method of aspect 34, wherein said adjuvant increases generation of cytotoxic T cells capable of suppressing pregnancy onset.

[0045] 36. The method of aspect 34, wherein said adjuvant is capable of stimulating generation of antibodies which inhibit pregnancy.

[0046] 37. The method of aspect 36, wherein said antibodies are antiphospholipid antibodies.

[0047] 38. The method of aspect 1, wherein said stimulation of immunogenicity of said senescent cells is achieved through culture with an agent capable of upregulating HLA expression.

[0048] 39. The method of aspect 1, wherein said stimulation of immunogenicity of said senescent cells is achieved through culture with an agent capable of upregulating expression of costimulatory molecules.

[0049] 40. The method of aspect 38, wherein said agent capable of stimulating upregulation of HLA expression induces activation of NF-kappa B.

[0050] 41. The method of aspect 40, wherein said agent capable of inducing activation of NF-kappa B is an inhibitor of i-kappa B.

[0051] 42. The method of aspect 38, wherein said agent capable of inducing upregulation of HLA is an activator of the JAK-STAT pathway.

[0052] 43. The method of aspect 42, wherein said agent capable of activating theJAK-STAT pathway is interferon gamma.

[0053] 44. The method of aspect 43, wherein said interferon gamma is added to culture of said endothelial cells at a concentration of approximately 100 IU per ml for a period of approximately 48 hours.

[0054] 45. The method of 1, wherein said induction of immunogenicity in said senescent cells is achieved through treatment of said senescent cells with an agent capable of inducing signaling through a toll like receptor.

[0055] 46. The method of 1, wherein said induction of immunogenicity in said senescent cells is achieved through treatment of said senescent cells with an agent capable of inducing signaling through a Pathogen Associated Molecular Pattern (PAMP) receptor.

[0056] 47. The method of aspect 46, wherein said PAMP receptor is selected from a group comprising of: a) MDA5; b) RIG-1; and c) NOD.

[0057] 48. The method of aspect 45, wherein said toll like receptor is TLR-2.

[0058] 49. The method of aspect 48, wherein said TLR-2 is activated by compounds selected from a group comprising of: a) Pam3cys4; b) Heat Killed Listeria monocytogenes (HKLM); and c) FSL-1.

[0059] 50. The method of aspect 45, wherein said toll like receptor is TLR-3.

[0060] 51. The method of aspect 50, wherein said TLR-3 is activated by Poly IC.

[0061] 52. The method of aspect 50, wherein said TLR-3 is activated by double stranded RNA.

[0062] 53. The method of aspect 52, wherein said double stranded RNA is of mammalian origin.

[0063] 54. The method of aspect 52, wherein said double stranded RNA is of prokaryotic origin.

[0064] 55. The method of aspect 53, wherein said double stranded RNA is derived from leukocyte extract.

[0065] 56. The method of aspect 55, wherein said leukocyte extract is a heterogeneous composition derived from freeze-thawing of leukocytes, followed by dialysis for compounds less than 15 kDa.

[0066] 57. The method of aspect 45, wherein said toll like receptor is TLR-4.

[0067] 58. The method of aspect 57, wherein said TLR-4 is activated by lipopolysaccharide.

[0068] 59. The method of aspect 57, wherein said TLR-4 is activated by peptide possessing at least 80 percent homology to the sequenceEFDVILKAAGANKVAVIKAVRGATGLGLKEAKDLVESAPAALKEGVSKDDAEALKKALEEAGAEVEVK (SEQ ID NO: 5).

[0069] 60. The method of aspect 57, wherein said TLR-4 is activated by HMGB-1.

[0070] 61. The method of aspect 57, wherein said TLR-4 is activated by a peptide derived from HMGB-1.

[0071] 62. The method of aspect 61, wherein said HMGB-1 peptide is hp91.

[0072] 63. The method of aspect 45, wherein said toll like receptor is TLR-5.

[0073] 64. The method of aspect 63, wherein said TLR-5 is activated by flagellin.

[0074] 65. The method of aspect 45, wherein said toll like receptor is TLR-7.

[0075] 66. The method of aspect 65, wherein said TLR-7 is activated by imiquimod.

[0076] 67. The method of aspect 45, wherein said toll like receptor is TLR-8.

[0077] 68. The method of aspect 67, wherein said TLR-8 is activated by resmiqiumod.

[0078] 69. The method of aspect 45, wherein said toll like receptor is TLR-9

[0079] 70. The method of aspect 69, wherein said TLR-9 is activated by CpGDNA.

[0080] 71. A method of inducing immunity to senescent cell associated antigens, said method comprising the steps of: a) selecting a patient in need of treatment; b) extracting a somatic cell source from said patient; c) extracting monocytic cells from said patient; d) culturing said monocytic cells in a manner to enhance antigen presentation; e) endowing somatic cells from step “b” with senescent properties; and f) fusing said antigen presenting cells of step “d” with said somatic cells of step “e”.

[0081] 72. The method of aspect 71, wherein said senescent associated antigen is beta galactosidase.

[0082] 73. The method of aspect 71, wherein said senescent associated antigen isPD-L2.

[0083] 74. The method of aspect 71, wherein said senescent associated antigen is pl6INK.

[0084] 75. The method of aspect 71, wherein said senescent associated antigen is beta galactosidase.

[0085] 76. The method of aspect 71, wherein said senescent associated antigen isSignal recognition particle subunit SRP72.

[0086] 77. The method of aspect 71, wherein said senescent associated antigen is rRNA 2'-O-methyltransferase fibrillarin.

[0087] 78. The method of aspect 71, wherein said senescent associated antigen isCLIP-associating protein 1.

[0088] 79. The method of aspect 71, wherein said senescent associated antigen is importin subunit alpha-5.

[0089] 80. The method of aspect 71, wherein said senescent associated antigen is trafficking protein particle complex subunit 6B.

[0090] 81. The method of aspect 71, wherein said senescent associated antigen is nicotinamide phosphoribosyltransferase.

[0091] 82. The method of aspect 71, wherein said senescent associated antigen is calcium / calmodulin-dependent protein kinase type II subunit beta.

[0092] 83. The method of aspect 71, wherein said senescent associated antigen is extended synaptotagmin-2.

[0093] 84. The method of aspect 71, wherein said senescent associated antigen is urokinase-type plasminogen activator.

[0094] 85. The method of aspect 71, wherein said senescent associated antigen is growth-regulated alpha protein;C-X-C motif chemokine 2;C-X-C motif chemokine 3.

[0095] 86. The method of aspect 71, wherein said senescent associated antigen is interleukin-8.

[0096] 87. The method of aspect 71, wherein said senescent associated antigen is proteasome subunit beta type-1.

[0097] 88. The method of aspect 71, wherein said senescent associated antigen is elongation factor Tu, mitochondrial.

[0098] 89. The method of aspect 71, wherein said senescent associated antigen is remodeling and spacing factor 1.

[0099] 90. The method of aspect 71, wherein said senescent associated antigen isATP-dependent Clp protease ATP -binding subunit clpX-like, mitochondrial.

[0100] 91. The method of aspect 71, wherein said senescent said associated antigen is dipeptidyl peptidase 4.

[0101] 92. The method of aspect 71, wherein said senescent associated antigen is matrix metalloproteinase-14.

[0102] 93. The method of aspect 71, wherein said senescent associated antigen isArf-GAP with GTPase, ANK repeat and PH domain-containing protein 3.

[0103] 94. The method of aspect 71, wherein said senescent associated antigen is nucleophosmin.

[0104] 95. The method of aspect 71, wherein said senescent associated antigen is interstitial collagenase.

[0105] 96. The method of aspect 71, wherein said senescent associated antigen is aurora kinase A-interacting protein.

[0106] 97. The method of aspect 71, wherein said senescent associated antigen is oxygen-dependent coproporphyrinogen-III oxidase, mitochondrial.

[0107] 98. The method of aspect 71, wherein said senescent associated antigen is elongation factor 1 -alpha 1.

[0108] 99. The method of aspect 71, wherein said senescent associated antigen is elongation factor G, mitochondrial.

[0109] 100. The method of aspect 71, wherein said senescent associated antigen is leucyl-cystinyl aminopeptidase.

[0110] 101. The method of aspect 71, wherein said senescent associated antigen is haptoglobin.

[0111] 102. The method of aspect 71, wherein said senescent associated antigen is stanniocalcin-1.

[0112] 103. The method of aspect 71, wherein said somatic cell is capable of proliferation.

[0113] 104. The method of aspect 71, wherein said somatic cell is a fibroblast.

[0114] 105. The method of aspect 104 wherein said fibroblast is a dermal fibroblast.

[0115] 106. The method of aspect 104 wherein said fibroblast is a gingival fibroblast.

[0116] 107. The method of aspect 104 wherein said fibroblast is a peripheral blood fibroblast.

[0117] 108. The method of aspect 104 wherein said fibroblast is a menstrual blood fibroblast.

[0118] 109. The method of aspect 104 wherein said fibroblast is an amniotic fluid fibroblast.

[0119] 110. The method of aspect 104 wherein said fibroblast is an amniotic membrane fibroblast.

[0120] 111. The method of aspect 104 wherein said fibroblast is an adipose tissue fibroblast.

[0121] 112. The method of aspect 104 wherein said fibroblast is a bone marrow fibroblast.

[0122] 113. The method of aspect 71, wherein said somatic cell is a leukocyte.

[0123] 114. The method of aspect 113, wherein said leukocyte is a T cell.

[0124] 115. The method of aspect 114, wherein said T cell expresses CD4.

[0125] 116. The method of aspect 115, wherein said CD4 T cell preferentially expresses interferon gamma as compared to interleukin-4.

[0126] 117. The method of aspect 115, wherein said CD4 T cell preferentially expresses interleukin- 1 as compared to interleukin-4.

[0127] 118. The method of aspect 115, wherein said CD4 T cell preferentially expresses interleukin-2 as compared to interleukin-4.

[0128] 119. The method of aspect 115, wherein said CD4 T cell preferentially expresses interleukin-7 as compared to interleukin-4.

[0129] 120. The method of aspect 115, wherein said CD4 T cell preferentially expresses interleukin- 12 as compared to interleukin-4.

[0130] 121. The method of aspect 115, wherein said CD4 T cell preferentially expresses interleukin- 15 as compared to interleukin-4.

[0131] 122. The method of aspect 115, wherein said CD4 T cell preferentially expresses interleukin- 18 as compared to interleukin-4.

[0132] 123. The method of aspect 115, wherein said CD4 T cell is a Thl cell.

[0133] 124. The method of aspect 71, wherein said somatic cell is selected from a group of cells isolated from tissue.

[0134] 125. The method of aspect 124, wherein said cells isolated from tissue are purified for expression of regenerative markers.

[0135] 126. The method of aspect 125, wherein said regenerative marker is CD105.

[0136] 127. The method of aspect 125, wherein said regenerative marker is CD39.

[0137] 128. The method of aspect 125, wherein said regenerative marker is CD73.

[0138] 129. The method of aspect 125, wherein said regenerative marker is c-kit.

[0139] 130. The method of aspect 125, wherein said regenerative marker is c-met.

[0140] 131. The method of aspect 125, wherein said regenerative marker is pim-1.

[0141] 132. The method of aspect 125, wherein said regenerative marker is PD-L1.

[0142] 133. The method of aspect 125, wherein said tissue is selected from a group comprising of: : a) nephrotic tissue; b) hepatic tissue; c) stomach tissue; d) dermal tissue; e) thymic tissue; f) neural tissue; g) hair tissue; h) ocular tissue; i) lymphatic tissue; j) musculoskeletal tissue; k) bone tissue; 1) endocrine tissuee; m) vascular tissue; n) hematopoietic tissue; o) colonic tissue; p) cardiac tissue; q) muscle tissue; r) nail tissue; s) cartilage tissue; t) pancreatic tissue; u) bladder tissue; v) penile tissue; w) testicular tissue; x) prostatic tissue; y) ovarian tissue; z) fallopian tissue; aa) digestive tissue; and ab) glandular tissue.

[0143] 134. The method of aspect 71, wherein said somatic cells are induced to undergo senescence by growth factor induced proliferation in vitro,

[0144] 135. The method of aspect 134, wherein said growth factors are selected from a group comprising of; bone morphogenic protein (BMP)-l, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein- 10, bone morphogenic protein-11, bone morphogenic protein- 12, bone morphogenic protein- 13, bone morphogenic protein-14, bone morphogenic protein-15, brain derived neurotrophic factor, ciliary neurotrophic factor, cytokine-induced neutrophil chemotactic factor 1, cytokine- induced neutrophil chemotactic factor 2a, cytokine-induced neutrophil chemotactic factor 2p, p endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial- derived neutrophil attractant, fibroblast growth factor (FGF) 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor (acidic), fibroblast growth factor (basic), growth related protein, growth related protein a, growth related protein p, growth related protein y, heparin binding epidermal growth factor, hepatocyte growth factor, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor BB, pre-B cell growth stimulating factor, stem cell factor, transforming growth factor a, transforming growth factor P, transforminggrowth factor pi, transforming growth factor [31.2, transforming growth factor P2, transforming growth factor P3, latent transforming growth factor i, transforming growth factor P binding protein I, transforming growth factor P binding protein II, transforming growth factor P binding protein III, and vascular endothelial growth factor, interleukin (IL)-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL- 15, IL- 16, IL- 17, IL- 18, interferon (IFN), IFN-y, tumor necrosis factor (TNF), TNF1, TNF2, TNF-a, macrophage colony stimulating factor (M-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), megakaryocyte colony stimulating factor (Meg-CSF)-thrombopoietin, stem cell factor, and erythropoietin.

[0145] 136. The method of aspect 71, wherein one or more senescence-associated antigens are administered prior to, subsequent with, or concurrently with autologous senescent cells and autologous dendritic cells.

[0146] 137. The method of aspect 136, wherein said senescence associated antigens are selected from a group comprising of: cleavage stimulation factor subunit 2 tau variant, vesicle-trafficking protein SEC22b, geranylgeranyl transferase type-1 subunit beta, glyceraldehyde-3 -phosphate dehydrogenase, histone H2B type 1-K, TRPM8 channel- associated factor 1, macrophage migration inhibitory factor, complement factor H-related protein 2, dystonin, glucocorticoid receptor, dihydropyrimidinase-related protein 2, metallothionein-2;Metallothionein-lE;Metallothionein-lG;Metallothionein- lX;Metallothionein-lM, mRNA turnover protein 4 homolog, tumor protein D54, E3 ubiquitin-protein ligase UBR4, insulin-like growth factor-binding protein 2, pyruvate kinase PKM, serine beta-lactamase-like protein LACTB, mitochondrial, TBC1 domain family member 4, elongation factor 1-beta, proteasome activator complex subunit 1, EPM2A-interacting protein 1, insulin-like growth factor-binding protein 5, phosphatidylinositol-glycan-specific phospholipase D, prefoldin subunit 2, nardilysin, biotinidase, stress-induced-phosphoprotein 1, tubulin beta-2A chain;Tubulin beta-2B chain, adenylate kinase 2, mitochondrial, ATP -binding cassette sub-family F member 1, unconventional myosin-Ib, glutaredoxin-1, T-complex protein 1 subunit gamma, heat shock protein beta-1, cullin-associated NEDD8-dissociated protein 2, talin-1, exportin- 1, complement C3, regulator of nonsense transcripts 2, elongation factor 1 -delta, heat shock protein HSP 90-beta, mitogen-activated protein kinase kinase kinase kinase 4;Misshapen- like kinase 1;TRAF2 and NCK-interacting protein kinase, heat shock protein HSP 90- alpha, ribose-phosphate pyrophosphokinase 2, myosin regulatory light chain 12B,RAD50, prostaglandin E synthase 3, plasminogen activator inhibitor 1, ladinin-1, coronin- 1C, utrophin, N-alpha-acetyl transferase 25, NatB auxiliary subunit, pyruvate carboxylase, mitochondrial, hexokinase-l;Putative hexokinase HKDC1, general transcription factor 3C polypeptide 5, ubiquinone biosynthesis monooxygenase COQ6, mitochondrial, heterogeneous nuclear ribonucleoprotein K, matrin-3, proto-oncogene serine / threonine-protein kinase, tetraspanin-9, histone H2A type 1-BZE, procollagenlysine, 2-oxoglutarate 5-dioxygenase 3, lysyl oxidase homolog 2, tripeptidyl-peptidase 1, protein ENL, neutral alpha-glucosidase AB, hemoglobin subunit alpha, rab GTPase- binding effector protein 2, tubulin beta-4B chain, dihydropyrimidinase-related protein 1, PDZ and LIM domain protein 1, thioredoxin domain-containing protein 17, mth938 domain-containing protein, thioredoxin-like protein 1, serpin Hl, MICOS complex subunit MIC60, carboxypeptidase Q, kinesin-like protein KIFlB;Kinesin-like protein KIF1A, small ubiquitin-related modifier 1, actin, cytoplasmic 1; Actin, cytoplasmic 2, proteasome subunit alpha type- 1, 26S proteasome non-ATPase regulatory subunit 8, exportin-2, lysosomal Pro-X carboxypeptidase, nuclear pore complex protein Nup93, eukaryotic initiation factor 4A-I, coatomer subunit alpha, protein SDA1 homolog, glutathione peroxidase 1, 6-phosphogluconolactonase, RNA-binding protein 7, FH1 / FH2 domain-containing protein 1, protein ecdysoneless homolog, propionyl-CoA carboxylase alpha chain, mitochondrial, sorting nexin-9, WD repeat-containing protein 1, serine / threonine-protein kinase Nekl, vacuolar protein sorting-associated protein 33B, polyhomeotic-like protein 2, acyl-coenzyme A, thioesterase 9, mitochondrial, SH3 domain-binding protein 4, peroxidasin homolog, pro-epidermal growth factor, and septin- 11.

[0147] 138. The method of aspect 71, wherein said monocytic cell is plastic adherent.

[0148] 139. The method of aspect 71, wherein said monocytic cell expresses CD14.

[0149] 140. The method of aspect 71, wherein said monocytic cell expresses CD16.

[0150] 141. The method of aspect 71, wherein said monocytic cell is enriched from peripheral blood by use of a density gradient.

[0151] 142. The method of aspect 71, wherein said monocytic cell is enriched from peripheral blood by use of a size exclusion means.

[0152] 143. The method of aspect 71, wherein said monocytic cell is enriched from peripheral blood by use of a size inclusion means.

[0153] 144. The method of aspect 71, wherein monocyte is harvested from mobilized peripheral blood.

[0154] 145. The method of aspect 144, wherein said blood is mobilized by treatment of the patient with G-CSF.

[0155] 146. The method of aspect 144, wherein said blood is mobilized by treatment of the patient with GM-CSF.

[0156] 147. The method of aspect 144, wherein said blood is mobilized by treatment of the patient with M-CSF.

[0157] 148. The method of aspect 144, wherein said blood is mobilized by treatment of the patient with an antagonist of SDF-1.

[0158] 149. The method of aspect 71, wherein said monocytes are transformed into dendritic cells.

[0159] 150. The method of aspect 149, wherein said dendritic cells are capable of activating naive T cells.

[0160] 151. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of regenerative cell therapy.

[0161] 152. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of endocrine organ replacement cell therapy.

[0162] 153. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of T cell therapy.

[0163] 154. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of B cell therapy.

[0164] 155. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of Treg cell therapy.

[0165] 156. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of dendritic cell therapy.

[0166] 157. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of NK cell therapy.

[0167] 158. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of NKT cell therapy.

[0168] 159. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of monocytic cell therapy.

[0169] 160. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of CAR-T cell therapy.

[0170] 161. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of gamma delta T cell therapy.

[0171] 162. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of CAR-NK cell therapy.

[0172] 163. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of CAR-gamma delta cell therapy.

[0173] 164. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of tumor infiltrating lymphocyte T cell therapy.

[0174] 165. The method of aspect 71, wherein said immunity to said cells expressing said senolytic antigens is used to augment efficacy of checkpoint inhibitor cancer therapy.

[0175] 166. The method of aspect 165, wherein said checkpoint inhibitor blocksCTLA4.

[0176] 167. The method of aspect 165, wherein said checkpoint inhibitor blocksPD-L1.

[0177] 168. The method of aspect 165, wherein said checkpoint inhibitor blocksPD-L2.

[0178] 169. The method of aspect 165, wherein said checkpoint inhibitor blocks

[0179] 170. The method of aspect 165, wherein said checkpoint inhibitor blocks indolamine 2,3 dioxygenase.

[0180] 171. The method of aspect 71, wherein said immunity to said senescent cells is utilized to enhance activity of regenerative cells.

[0181] 172. The method of aspect 71, wherein mesenchymal stem cells are administered subsequent to induction of immunity towards senescent cells in order to enhance therapeutic activity of said mesenchymal stem cells.

[0182] 173. The method of aspect 172, wherein lymphotoxin is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0183] 174. The method of aspect 172, wherein said lymphotoxin is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0184] 175. The method of aspect 172, wherein said lymphotoxin is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0185] 176. The method of aspect 172, wherein said mesenchymal stem cell is exposed to TRANCE.

[0186] 177. The method of aspect 176, wherein said TRANCE is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0187] 178. The method of aspect 176, wherein said TRANCE is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0188] 179. The method of aspect 176, wherein said TRANCE is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0189] 180. The method of aspect 172, wherein said mesenchymal stem cell is exposed to interleukin- 1.

[0190] 181. The method of aspect 180, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0191] 182. The method of aspect 180, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0192] 183. The method of aspect 181, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0193] 184. The method of aspect 172, wherein said mesenchymal stem cell is exposed to interleukin- 1.

[0194] 185. The method of aspect 184, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0195] 186. The method of aspect 185, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0196] 187. The method of aspect 185, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0197] 188. The method of aspect 172, wherein said mesenchymal stem cell is exposed to interleukin- 1.

[0198] 189. The method of aspect 188, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0199] 190. The method of aspect 188, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0200] 191. The method of aspect 188, wherein said interleukin- 1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0201] 192. The method of aspects 172, wherein said mesenchymal stem cell is exposed to interleukin-6.

[0202] 193. The method of aspect 192, wherein said interleukin-6 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0203] 194. The method of aspect 192, wherein said interleukin-6 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0204] 195. The method of aspect 192, wherein said interleukin-6 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0205] 196. The method of aspect 172, wherein said mesenchymal stem cell is exposed to interleukin-8.

[0206] 197. The method of aspect 196, wherein said interleukin-8 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0207] 197. The method of aspect 196, wherein said interleukin-8 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0208] 198. The method of aspect 196, wherein said interleukin-8 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0209] 199. The method of aspect 172, wherein said mesenchymal stem cell is exposed to interleukin- 17.

[0210] 200. The method of aspect 199, wherein said interleukin- 17 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0211] 202. The method of aspect 199, wherein said interleukin- 17 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0212] 203. The method of aspect 199, wherein said interleukin- 17 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0213] 204. The method of aspect 172, wherein said mesenchymal stem cell is exposed to interleukin- 18.

[0214] 205. The method of aspect 204, wherein said interleukin- 18 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0215] 206. The method of aspect 204, wherein said interleukin- 18 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0216] 207. The method of aspect 204, wherein said interleukin- 18 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baseline.

[0217] 208. The method of aspect 172, wherein said mesenchymal stem cell is exposed to HMGB 1.

[0218] 209. The method of aspect 208, wherein said HMGB1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 25% as compared to baseline.

[0219] 210. The method of aspect 208, wherein said HMGB1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 50% as compared to baseline.

[0220] 211. The method of aspect 208, wherein said HMGB1 is applied to said mesenchymal stem cell at a concentration and duration sufficient to increase production of LIF by 100% as compared to baselineDETAILED DESCRIPTION

[0221] The invention teaches means and methods of stimulating immunity towards autologous senescent cells. In one embodiment the invention provides a composition of matter comprised of cells made senescent by genotoxic damage that are fused with autologous derived antigen presenting cells such as dendritic cells.

[0222] Unless defined differently, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. In particular, the following terms and phrases have the following meaning.

[0223] The Term "Angiogenesis" means any alteration of an existing vascular bed or the formation of new vasculature which benefits tissue perfusion. This includes the formation of new vessels by sprouting of endothelial cells from existing blood vessels or the remodeling of existing vessels to alter size, maturity, direction or flow properties to improve blood perfusion of tissues. As used herein the terms, "angiogenesis," "revascularization," "increased collateral circulation," and "regeneration of blood vessels" are considered as synonymous.

[0224] The terms "antigenic precursor" or "precursor" relative to immunogenic epitopes, as used herein refer to peptides capable of being processed to immunogenic peptides by the cells of the subject.

[0225] The terms "Class II major histocompatability complex", "Class II MHC" and "Class II", as used herein refer to molecules that are expressed on various cell types and which play an essential role in the recognition of protein antigens by T cells. Class II MHC molecules typically bind peptides of from about 7 to 30 or more amino acids and form complexes that are recognized by antigen-specific CD4+ T cells. Such peptide / CD4+ T cell complexes facilitate antibody production against the peptide antigen by an immunocompetent subject.

[0226] The term "immune response" as used herein refers to a cellular immune response such as a cytotoxic T cell response and / or a humoral immune response such as production of antibodies against an immunogenic epitope.

[0227] The term "immunocompetent subject", as used herein refers to a subject having immune response cells which upon exposure to an immunogenic epitope, is capable of mounting a cellular and / or humoral immune response against the immunogenic epitope. The invention is useful for both the human and other mammalian subjects.

[0228] The term immunogenic "epitope" or "antigenic determinant", as used herein refers to a portion of the amino acid sequence which will generate a T- and / or B-cell mediated immune response. It is preferred that the epitope be unique; that is, an immune response generated to the specific epitope show little or no cross-reactivity with other antigens.

[0229] The term "active immunization", as used herein means the administration of a vaccine which induces an immune response by the immune response cells of the subject. "Active immunization" may be achieved by exposure of the immune response cells of the subject to a nucleic acid sequence or an amino acid sequence.

[0230] The term "exposing", as used herein means bringing the immune response cells of the subject in contact with a nucleic acid construct. Such "exposing", may take place in vitro, e.g., by introduction of the construct into a host cell by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation, or in vivo, e.g., by introduction of a "naked" nucleic acid construct into a host by injection into muscle or other tissue.

[0231] The term "passive immunization", as used herein is meant the direct administration of antibodies to a subject, as an immunization approach.

[0232] The term "immune response cells", as used herein refers to the cells of a subject which are capable of processing antigens and presenting them in conjunction with Class I or Class II MHC.

[0233] The term "polynucleotide" as used herein refers to a polymeric molecule having a backbone which supports bases capable of hydrogen bonding to typical polynucleotides, where the polymer backbone presents the bases linked by phosphodiester bonds in a manner to permit such hydrogen bonding in a sequence specific fashion between the polymeric molecule and a typical polynucleotide (e.g.,single-stranded DNA). "Polynucleotides" include polymers having modifications, e.g., those involving phosphodiamidate morpholine (PMO) chemistry.

[0234] The term "recombinant nucleic acid", as used herein refers to a nucleic acid sequence originally formed in vitro, generally by the manipulation of the nucleic acid by endonucleases, in a form not normally found in nature.

[0235] The term "homology" or "homologue" as used herein refers to the level of identity between two sequences, i.e., 70% homology means the same thing as 70% sequence identity when determined by the algorithms described below, and accordingly a homologue of a given sequence has at least about 70% or 80%, preferably about 80%, 85%, 90% or 95% sequence identity over a given length of the sequence.

[0236] In one embodiment the invention provides a means of generating a population of cells with ability to kill senescent cells. In one embodiment approximately 50 ml of peripheral blood is extracted from a patient in which inhibition of fertility is desired and peripheral blood monoclear cells (PBMC) are isolated using the Ficoll Method. PBMC are subsequently resuspended in approximately 10 ml RPMI media with 10% fetal calf serum and allowed to adhere onto a plastic surface for 2-4 hours. The adherent cells are then cultured at 37°C in RPMI media supplemented with 1,000 U / mL granulocyte-monocyte colony-stimulating factor and 500 U / mL IL-4. This procedure, or a procedure similar to it, can be utilized for the generation of dendritic cells. Half of the volume of the GM-CSF and IL-4 supplemented media is changed every other day. Immature DCs are harvested on day 7. In one embodiment said generated DC are treated with senescent cell extracts. Said extracts are added to said immature dendritic cells on day 7. Senescent cell pulsed dendritic cells may be administered directly as a vaccine, or may be utilized to stimulate autologous patient T cell clones in vitro. Said T cell clones may be selected for specificity to proliferating endothelial cells. Additionally, in some embodiments, whether for in vitro stimulation of T cells, or for direct use as a senescent cell vaccine, the senescent cell pulsed dendritic cells may be further purified from culture through use of flow cytometry sorting or magnetic activated cell sorting (MACS), or may be utilized as a semi-pure population. In one embodiment DC are exposed to agents capable of stimulating maturation in vitro subsequent to pulsing with senescence associated antigens or cell extracts. Specific means of stimulating in vitro maturation include culturing DC or DC containing populations with a toll like receptor agonist. Another means of achieving DC maturation involves exposure of DC to TNF-alpha at a concentration of approximately 20 ng / mL. In another embodiment, a mixture ofsenescent cells together with immature dendritic cells is used as a combination cellular vaccine. In another embodiment, senescent cells (live or extracts or fixed) are administered in combination with dendritic cells together with activated T cells and / or NK cells. In order to activate T cells and / or NK cells in vitro, cells are cultured in media containing approximately 1000 lU / ml of interferon gamma. Incubation with interferon gamma may be performed for the period of 1 hour to the period of 14 days. Preferably, incubation is performed for approximately 48 hours, after which T cells and / or NK cells may be further stimulated via the CD3 and CD28 receptors. One means of accomplishing this is by addition of antibodies capable of activating these receptors. In one embodiment approximately, 3 ug / ml of anti-CD3 antibody is added, together with approximately 2 ug / ml anti-CD28. In order to promote survival of T cells and NK cells, was well as to stimulate proliferation, a T cell / NK mitogen may be used. In one embodiment the cytokine IL-2 is utilized. Specific concentrations of IL-2 useful for the practice of the invention are approximately 400 u / mL IL-2. Media containing IL-2 and antibodies may be changed every two days for approximately 7-24 days. In one particular embodiment DC are included to said T cells and / or NK cells in order to endow cytotoxic activity towards tumor cells. In a particular embodiment, inhibitors of caspases are added in the culture so as to reduce rate of apoptosis of T cells and / or NK cells. Generated cells can be administered to a subject intradermally, intramuscularly, subcutaneously, intraperitoneally, intraarterially, intravenously (including a method performed by an indwelling catheter), intratumorally, or intralymphatically.

[0237] In one embodiment, a method is provided for evoking an immune response specific for a senescent cell in a subject, with the purpose of enhancing regenerative medicine therapeutic efficacy. In another embodiment killing of senescent cells is utilized to induce augmentation of therapeutic effects of chemotherapy and / or immunotherapy.

[0238] The invention provides stimulation of immunity through immunogenic composition comprising: (a) a pharmaceutically acceptable excipient, and (b) an immunogen. In particular embodiments, the immunogen is selected from (i) an isolated senescent cell-associated antigen or an antigenic fragment thereof, wherein the senescent cell-associated antigen is selected from (A) pl6INK4a, (B) a senescent cell-associated antigen, and (C) a senescent cell-associated antigen that is encoded by a nucleic acid sequence and wherein the antigenic fragment comprises at least 20 contiguous amino acids of the senescent cell-associated antigen; (ii) an isolated polynucleotide encoding atleast two senescent cell-associated antigens of (i) or antigenic fragments thereof; (iii) at least two isolated polynucleotides, wherein a first isolated polynucleotide encodes a first senescent cell-associated antigen or an antigenic fragment thereof, and wherein the first senescent cell-associated antigen is selected from (A) pl6INK4a, (B) a senescent cell- associated antigen selected from and (C) a senescent cell-associated antigen that is encoded by a nucleic acid sequence selected and wherein the antigenic fragment comprises at least 20 contiguous amino acids of the first senescent cell-associated antigen, and a second polynucleotide encodes a second senescent cell-associated antigen, wherein the second senescent cell-associated antigen is selected from (A) pl6INK4a, (B) a senescent cell-associated antigen selected from 1, and (C) a senescent cell-associated antigen that is encoded by a nucleic acid sequence selected from 3, and wherein the antigenic fragment comprises at least 20 contiguous amino acids of the second senescent cell-associated antigen; (iv) a recombinant expression vector that is a viral vector comprising a polynucleotide that encodes the senescent cell-associate antigen or antigenic fragment thereof of (i); (v) a senescent cell membrane preparation, a senescent cell organelle preparation, or an exosome; (vi) a fusion polypeptide comprising at least two senescent cell-associated antigens, wherein each of the at least two senescent cell- associated antigens are different and each is selected from (A) pl6INK4a, (B) a senescent cell-associated antigen selected, and (C) a senescent cell-associated antigen that is encoded by a nucleic acid sequence selected from; (vii) a fusion polypeptide comprising at least two antigenic fragments wherein each of the at least two antigenic fragments comprises at least 20 contiguous amino acids of a senescent cell-associated antigen selected from (A) pl6INK4a, (B) a senescent cell-associated antigen selected from , and (C) a senescent cell-associated antigen that is encoded by a nucleic acid sequence selected; (viii) a fusion polypeptide comprising at least one senescent cell-associated antigen of (i) or an antigenic fragment thereof and a co-stimulatory polypeptide; and (ix) a modified dendritic cell wherein a dendritic cell is isolated from the subject and is modified by (A) introducing a senescent cell-associated antigen, or an antigenic fragment that comprises at least 20 contiguous amino acids of the senescent cell-associated antigen, wherein the senescent cell-associated antigen is selected from (A) pl6INK4a, (B) a senescent cell-associated antigen selected from, and (C) a senescent cell -associated antigen that is encoded by a nucleic acid sequence selected (B) introducing a polynucleotide encoding the senescent cell -associated antigen, or an antigenic fragment of (A), into the dendritic cell ex vivo to provide a modified dendritic cell, and wherein themodified dendritic cell is administered to the subject. In certain particular embodiments for use in the methods described above and herein, the senescent cell-associated antigen that is encoded by a nucleic acid sequence selected from any one of ADAMTS7, APLP2, ATP6V0D2, BCHE, Cl lorf87, CD46, CYB5D2, FBXL7, GPR137B, IFI27L1, IL15RA, LAMP2, MYOIO, NEU1, NHSL2, NPAS2, OR1F1, PEA15, RAB23, RARB, RNPC3, SELO, SELT, SEMA5B, SERP1, SERPINE1, SLC9A7, SNX3, TBC1D1, TBRG1, TCEANC, TFPI, TNFAIP1, TUBG2, USP18, or ZNF419. In other more specific embodiments, the senescent cell-associated antigen that is encoded by a nucleic acid sequence selected from is any one of NEU1, SELO, SERP1, SERPINE1, or SNX3. In other specific embodiments, the senescent cell-associated antigen is pl6INK4a. In other certain particular embodiments, the at least first and the at least second senescent cell- associated antigen encoded by a nucleic acid sequence selected from are different and selected from any one of ADAMTS7, APLP2, ATP6V0D2, BCHE, Cl lorf87, CD46, CYB5D2, FBXL7, GPR137B, IFI27L1, IL15RA, LAMP2, MYOIO, NEU1, NHSL2, NPAS2, OR1F1, PEA15, RAB23, RARB, RNPC3, SELO, SELT, SEMA5B, SERP1, SERPINE1, SLC9A7, SNX3, TBC1D1, TBRG1, TCEANC, TFPI, TNFAIP1, TUBG2, USP18, and ZNF419 is selected from any one of NEU1, SELO, SERP1, SERPINE1, and SNX3. In still other particular embodiments, the at least first senescent cell-associated antigen or the at least second senescent cell-associated antigen is pl6INK4a.

[0239] In certain other embodiments of the method described above and herein, the immunogen comprises at least two isolated senescent cell-associated antigens or antigenic fragments thereof, wherein (a) a first isolated senescent cell -associated antigen, or an antigenic fragment thereof that comprises at least 20 contiguous amino acids of the first senescent cell-associated antigen, and (b) a second isolated senescent cell-associated antigen or an antigenic fragment thereof that comprises at least 20 contiguous amino acids of the second senescent cell-associated antigen, are different and each independently is selected from (A) pl6INK4a, (B) a senescent cell-associated antigen selected from In certain particular embodiments, the at least first and the at least second isolated senescent cell -associated antigen are different and encoded by a nucleic acid sequence selected, wherein the at least first and the at least second isolated senescent cell- associated antigens are selected from ADAMTS7, APLP2, ATP6V0D2, BCHE, Cl lorf87, CD46, CYB5D2, FBXL7, GPR137B, IFI27L1, IL15RA, LAMP2, MYOIO, NEU1, NHSL2, NPAS2, OR1F1, PEA15, RAB23, RARB, RNPC3, SELO, SELT, SEMA5B, SERP1, SERPINE1, SLC9A7, SNX3, TBC1D1, TBRG1, TCEANC, TFPI,TNFAIP1, TUBG2, USP18, and ZNF419. In other more specific embodiments, the at least first and the at least second isolated senescent cell-associated antigens encoded by a nucleic acid sequence from 2 are selected from NEU1, SELO, SERP1, SERPINE1, and SNX3. In still other particular embodiments, the at least first senescent cell-associated antigen or the at least second senescent cell-associated antigen is pl6INK4a.

[0240] In a more particular embodiment, the senescent cell-associated antigen is present on the cell surface of the senescent cell. In still another embodiment, when the immunogen comprises a first and the second senescent cell-associated antigen, the first and the second senescent cell-associated antigen are each present on the cell surface of the senescent cell.

[0241] In certain other embodiments, the immunogenic composition comprises a recombinant expression vector that comprises the polynucleotide of (b)(ii) operatively linked to at least one regulatory expression sequence. In a more particular embodiment, the recombinant expression vector is a viral vector. In still another specific embodiment, the viral vector is selected from an adenovirus vector, lentivirus vector, a herpes virus vector, adenovirus-associated vector, or a poxvirus vector. In another particular embodiment, the adenoviral vector is a replication-defective adenovirus. In certain specific embodiment, the replication-defective adenovirus is a recombinant human adenovirus having a serotype selected from Adi l, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49, and Ad50.

[0242] In yet another embodiment of the method described above and herein, the immunogen comprises at least one polynucleotide that encodes (a) a first senescent cell- associated antigen, or an antigenic fragment thereof that comprises at least 20 contiguous amino acids of the first senescent cell-associated antigen, and (b) a second senescent cell- associated antigen or an antigenic fragment thereof that comprises at least 20 contiguous amino acids of the second senescent cell-associated antigen, wherein the first and second senescent cell-associated antigens are different and each independently is selected from (A) pl6INK4a, (B) a senescent cell-associated antigen selected from. In certain particular embodiments, the at least first and the at least second isolated senescent cell-associated antigen are encoded by a nucleic acid sequence selected from, wherein the at least first and the at least second isolated senescent cell-associated antigens are selected from ADAMTS7, APLP2, ATP6V0D2, BCHE, Cl lorf87, CD46, CYB5D2, FBXL7, GPR137B, IFI27L1, IL15RA, LAMP2, MYO10, NEU1, NHSL2, NPAS2, OR1F1, PEA15, RAB23, RARB, RNPC3, SELO, SELT, SEMA5B, SERP1, SERPINE1,SLC9A7, SNX3, TBC1D1, TBRG1, TCEANC, TFPI, TNFAIP1, TUBG2, USP18, and ZNF419. In other more specific embodiments, the at least first and the at least second isolated senescent cell -associated antigens encoded by a nucleic acid sequence from are selected independently from NEUl, SELO, SERP1, SERPINE1, and SNX3. In still other particular embodiments, the at least first senescent cell-associated antigen or the at least second senescent cell-associated antigen is pl6INK4a. In still another embodiment, the immunogenic composition comprises a recombinant expression vector that comprises the at least one polynucleotide operatively linked to at least one regulatory expression sequence. In certain embodiments, the recombinant expression vector is a viral vector. In still another specific embodiment, the viral vector is selected from an adenovirus vector, lentivirus vector, a herpes virus vector, adenovirus-associated vector, or a poxvirus vector. In another particular embodiment, the adenoviral vector is a replication-defective adenovirus. In certain specific embodiment, the replication-defective adenovirus is a recombinant human adenovirus having a serotype selected from Adi 1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49, and Ad50.

[0243] In yet another embodiment of the method described above and herein, the immunogen comprises at least two polynucleotides wherein a first polynucleotide encodes the first senescent cell -associated antigen, or an antigenic fragment thereof, and a second polynucleotide encodes the second senescent cell-associated antigen, or an antigenic fragment thereof. In a specific embodiment, the immunogenic composition comprises (a) a recombinant expression vector that comprising the at least two polynucleotides wherein each of the at least two polynucleotides is operatively linked to at least one regulatory expression sequence; or (b) a first recombinant expression vector that comprises the first polynucleotide operatively linked to at least one regulatory expression sequence and a second recombinant expression vector that comprises the first polynucleotide operatively linked to at least one regulatory expression sequence. In certain embodiments, the recombinant expression vector is a viral vector. In still more particular embodiments, the recombinant expression vector of (a) the first recombinant expression vector of (b) and the second recombinant expression vector of (b) are each a viral vector. In still another specific embodiment, the viral vector is selected from an adenovirus vector, lentivirus vector, a herpes virus vector, adenovirus-associated vector, or a poxvirus vector. In another particular embodiment, the adenoviral vector is a replication-defective adenovirus. In certain specific embodiment, the replication-defective adenovirus is a recombinant human adenovirus having a serotype selected from Adi 1,Ad24, Ad26, Ad34, Ad35, Ad48, Ad49, and Ad50. In one particular embodiment, the first recombinant expression vector and the second recombinant expression vector are each the same or different recombinant human adenoviral vector having a serotype independently selected from Adi l, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49, and Ad50.

[0244] In yet another embodiment of the method described above and herein, when the immunogen comprises a dendritic cell, the dendritic cell is modified by introducing a recombinant expression vector comprising the polynucleotide. In certain embodiments, the recombinant expression vector is a viral vector selected from an adenovirus vector, lentivirus vector, a herpes virus vector, adenovirus-associated vec, or a poxvirus vector. In another particular embodiment, the adenoviral vector is a replication-defective adenovirus. In certain specific embodiment, the replication-defective adenovirus is a recombinant human adenovirus having a serotype selected from Adi 1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49, and Ad50.

[0245] In particular embodiments, with respect to the methods described above and herein, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant. In other particular embodiments, with respect to the methods described above and herein, the immunogenic composition further comprises (a) a co-stimulatory polypeptide that enhances the adaptive immune response to the immunogen; (b) a polynucleotide encoding the co-stimulatory polypeptide; or (c) a recombinant expression vector that comprises the polynucleotide sequence, which is operatively linked to at least one regulatory expression sequence. In still other specific embodiments, the subject has or is at risk of developing a disease or disorder treatable by clearing senescent cells from a tissue of the subject. In still more specific embodiments, of the methods described above and herein, the disease or disorder is an age-related disease or disorder.

[0246] Also provided herein is an immunogenic composition that comprises a pharmaceutically acceptable excipient and at least one immunogenic preparation selected from: (a) an immunogenic preparation comprising a first isolated senescent cell- associated antigen, or an antigenic fragment thereof that comprises at least 20 contiguous amino acids of the first senescent cell-associated antigen, and a second isolated senescent cell-associated antigen or an antigenic fragment thereof that comprises at least 20 contiguous amino acids of the second senescent cell-associated antigen, wherein the first and second senescent cell -associated (b) an immunogenic preparation comprising at least one polynucleotide that encodes the first senescent cell-associated antigen, or an antigenic fragment thereof, and the second senescent cell-associated antigen or an antigenicfragment thereof; (c) an immunogenic preparation comprising at least two polynucleotides wherein a first polynucleotide encodes the first senescent cell-associated antigen, or an antigenic fragment thereof, and a second polynucleotide encodes the second senescent cell-associated antigen, or an antigenic fragment thereof; (d) an immunogenic preparation comprising a senescent cell membrane preparation, a senescent cell organelle preparation, or an exosome; (e) an immunogenic preparation comprising a fusion polypeptide comprising at least two senescent cell-associated antigens, wherein each of the at least two senescent cell -associated antigens are different and selected independently from pl6INK4a, any one of ADAMTS7, APLP2, ATP6V0D2, BCHE, Cl lorf87, CD46, CYB5D2, FBXL7, GPR137B, IFI27L1, IL15RA, LAMP2, MYO 10, NEU1, NHSL2, NPAS2, OR1F1, PEA15, RAB23, RARB, RNPC3, SELO, SELT, SEMA5B, SERP1, SERPINE1, SLC9A7, SNX3, TBC1D1, TBRG1, TCEANC, TFPI, TNFAIP1, TUBG2, USP18, or ZNF419. In other more specific embodiments, the senescent cell-associated antigen that is encoded by a nucleic acid sequence is any one of NEU1, SELO, SERP1, SERPINE1, or SNX3. In other specific embodiments, the senescent cell-associated antigen is pl6INK4a. In other certain particular embodiments, the at least first and the at least second senescent cell-associated antigen encoded by a nucleic acid sequence selected are different and selected from any one of ADAMTS7, APLP2, ATP6V0D2, BCHE, Cl lorf87, CD46, CYB5D2, FBXL7, GPR137B, IFI27L1, IL15RA, LAMP2, MYO10, NEU1, NHSL2, NPAS2, OR1F1, PEA15, RAB23, RARB, RNPC3, SELO, SELT, SEMA5B, SERP1, SERPINE1, SLC9A7, SNX3, TBC1D1, TBRG1, TCEANC, TFPI, TNFAIP1, TUBG2, USP18, and ZNF419. In other more specific embodiments, the at least first and the at least second senescent cell-associated antigens encoded by a nucleic acid sequence selected from

[0247] is selected from any one of NEU1, SELO, SERP1, SERPINE1, and SNX3. In still other particular embodiments, the at least first senescent cell-associated antigen or the at least second senescent cell-associated antigen is pl6INK4a.

[0248] In particular embodiments, with respect to the immunogenic composition described above and herein, the senescent cell-associated antigen is present on the cell surface of the senescent cell. In another particular embodiment, the first and the second senescent cell-associated antigen are each present on the cell surface of the senescent cell. In yet another specific embodiment, (i) a recombinant expression vector comprises the at least one polynucleotide of (b) operatively linked to at least one regulatory expression sequence; or (ii) a recombinant expression vector comprises the at least twopolynucleotides of (c), wherein each polynucleotide is operatively linked to at least one regulatory expression sequence; or (iii) the dendritic cell is modified by introducing a recombinant expression vector comprising the polynucleotide; or (iv) a first recombinant expression vector comprises the first polynucleotide of (c) and a second recombinant expression vector comprises the second polynucleotide of (c). In particular embodiments, the recombinant expression vector of (i), (ii), and (iii), and the first and second recombination vectors of (iv) are each a viral vector. In certain embodiments, the recombinant expression vector is a viral vector selected from an adenovirus vector, lentivirus vector, a herpes virus vector, adenovirus-associated vector, or a poxvirus vector. In another particular embodiment, the adenoviral vector is a replication-defective adenovirus. In certain specific embodiment, the replication-defective adenovirus is a recombinant human adenovirus having a serotype selected from Adi 1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49, and Ad50.

[0249] In certain particular embodiments described above and herein, the immunogenic composition further comprises (a) a co-stimulatory polypeptide that enhances the adaptive immune response to the immunogen; (b) a polynucleotide encoding the co-stimulatory polypeptide; or (c) a recombinant expression vector that comprises the polynucleotide sequence encoding the co-stimulatory polypeptide, which is operatively linked to at least one regulatory expression sequence. In still other embodiments, the immunogenic compositions described above and herein further comprise a pharmaceutically acceptable adjuvant.

[0250] In yet another embodiment, a recombinant antibody is provided that comprises (a) at least one immunoglobulin variable region domain that specifically binds to a senescent cell -associated antigen selected from (A) pl6INK4a, (B) a senescent cell- associated antigen ; and (b) a modified human Fc region that exhibits enhanced affinity for an Fey receptor. In a particular embodiment, the recombinant antibody further comprises a second immunoglobulin variable region (Fv), wherein the second variable region specifically binds to the same or a different senescent cell-associated antigen selected from pl6INK4a, a senescent cell-associated antigen selected. Also provided is an immunogenic composition that comprises the recombinant antibody described above and herein and a pharmaceutically acceptable carrier. In still another embodiment, a method is provided for facilitating clearance of a senescent cell from a subject, comprising administering to the subject the immunogenic composition comprising the recombinant antibody described above and herein. In certain particular embodiments, thesenescent cell-associated antigen that is encoded by a nucleic acid sequence selected from is any one of ADAMTS7, APLP2, ATP6V0D2, BCHE, Cl lorf87, CD46, CYB5D2, FBXL7, GPR137B, IFI27L1, IL15RA, LAMP2, MYOIO, NEU1, NHSL2, NPAS2, OR1F1, PEA15, RAB23, RARB, RNPC3, SELO, SELT, SEMA5B, SERP1, SERPINE1, SLC9A7, SNX3, TBC1D1, TBRG1, TCEANC, TFPI, TNFAIP1, TUBG2, USP18, or ZNF419); In other more specific embodiments, the senescent cell-associated antigen that is encoded by a nucleic acid sequence selected from any one of NEU1, SELO, SERP1, SERPINE1, or SNX3. In still other specific embodiments, the senescent cell-associated antigen is pl6INK4a.

[0251] In one embodiment, a process is provided for formulating the immunogenic composition described above and herein, comprising (a) producing the immunogenic preparation that comprises the first isolated senescent cell-associated antigen, or an antigenic fragment thereof, and the second isolated senescent cell-associated antigen or an antigenic fragment thereof, each as described above and herein, by (i) culturing a first host cell into which a recombinant expression vector comprising at least one regulatory expression sequence operatively linked to a nucleotide sequence that encodes the first senescent cell-associated antigen, or an antigenic fragment thereof in a medium and for a time sufficient to produce the first senescent cell-associated antigen; and (ii) culturing a second host cell into which a recombinant expression vector comprising at least one regulatory expression sequence operatively linked to a nucleotide sequence that encodes the second senescent cell -associated antigen, or an antigenic fragment thereof in a medium and for a time sufficient to produce the second senescent cell-associated antigen; (iii) isolating the first senescent cell-associated antigen from the first host cell culture, and isolating the second senescent cell-associated antigen from the second host cell culture; and (c) formulating the first and the second cell-associated antigens with a pharmaceutically acceptable excipient. In a particular embodiment, the medium is a serum-free medium.

[0252] Uses of the immunogenic compositions described above are also provided for evoking an immune response specific for a senescent cell in a subject, wherein the immune response comprises clearance of the senescent cell by the immune system of the subject, and for the manufacture of a medicament for evoking an immune response specific for a senescent cell in a subject, wherein the immune response comprises clearance of the senescent cell by the immune system.

[0253] In one embodiment of the invention, provided is a cellular vaccine generated from senescent endothelial progenitor cells. Said progenitor cells may be extracted from a variety of tissues, one specific embodiment teaches derivation of endothelial cells extracted from placental tissue, isolated into a homogeneous or semi-homogeneous mixture, treated with agents capable of augmenting immunogenicity, and subsequently administered into a recipient in which immune response to proliferating endothelium is desired. In one specific example, endothelial cells are purified from a human placenta according to the following steps: a) Fetal membranes are manually peeled back and the villous tissue is isolated from the placental structure, with caution being used not to extract the deciduas or fibrous elements of the placental structure; b) The fetal villous tissue is subsequently washed with cold saline to remove blood and scissors are used to mechanically digest the tissue into pieces as small as possible; c) The minced tissue is then enzymatically digested. Specifically, about 25 grams of minced tissue is incubated with approximately 56 ml of liquid solution which has been pre-warmed to a temperature of 37 Celsius. Said solution comprised of Hanks Buffered Saline Solution (HBSS) supplemented with 25mM of HEPES and containing Calcium and Magnesium, said solution containing 0.28% collagenase, 0.25% dispase, and 0.01% DNAse (added during the incubation periods as described below); d) The mixture of minced placental villus tissue and digesting solution is incubated under stirring conditions for three incubation periods of 20 minutes each. Ten minutes after the first incubation period and immediately after the second and third incubation periods, the DNAse is added to make up a total concentration of DNase, by volume, of 0.01%; e) In the first and second incubations, the incubation flask is set at an angle, and the tissue fragments are allowed to settle for approximately 1 minute, with 35 ml of the supemantant cell suspension being collected and replaced by 38 ml (after the first digestion) or 28 ml (after the second digestion) of fresh digestion solution. After the third digestion the whole supernatant is collected; f) The supernatant collected from all three incubations is pooled and is poured through approximately four layers of sterile gauze and through one layer of 70 micro meter polyester mesh. The filtered solution is then centrifuged for 1000g for 10 minutes through diluted new born calf serum, said new born calf serum diluted at a ratio of 1 volume saline to 7 volumes of new born calf serum; g) The pooled pellet is then resuspended in 35 ml of warm DMEM with 25 mM HEPES containing 5 mg DNase I; h) The suspension is then mixed with 10 ml of 90% Percoll to give a final density of 1.027 g / ml and is centrifuged at 550 g for 10 minutes with the centrifuge brake off; i) The pelletis then collected and resuspended in 15 ml of DMEM with 25 mM HEPES that is layered over a discontinuous Percoll gradient comprising of 20%-70% Percoll in 10% steps and centrifuged at 1900 g for 20 minutes; j) The cells found at the 1.037 g / ml and 1.048 g / ml are collected.

[0254] The collected cells are subsequently expanded in vitro and exposed to conditions of senescence induction. One means of inducing senescence involves administration of genotoxic conditions. Preferably said genotoxic conditions are provided at sublethal levels to induce senescence but not apoptosis. In one embodiment said senescence is induced by exposure to chemotherapeutic agents such as alkylating agents. In one specific embodiment said senescent endothelial cells are generated by exposure to doxorubicin. Other drugs that may be used to induce senescence include but are not limited to: : Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; A. metantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Camptothecin; Caracemide;Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Combretestatin A-4; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA (N-[2-(Dimethyl- amino)ethyl]acridine-4-carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate;Diaziquone; Docetaxel; Dolasatins; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Ellipticine; Elsamitrucin; Enloplatin; Enpromate;Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil 1131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Homocamptothecin; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; PeploycinSulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Rhizoxin; Rhizoxin D; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Tel oxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP53; Topotecan Hydrochloride;Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinl eurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; 2- Chlorodeoxyadenosine; 2' Deoxyformycin; 9-aminocamptothecin; raltitrexed; N- propargyl-5,8-dideazafolic acid; 2chloro-2'-arabino-fluoro-2'-deoxyadenosine; 2-chloro- 2 '-deoxy adenosine; anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea (MNU); N,N'-Bis(2- chloroethyl)-N-nitrosourea (BCNU); N-(2-chloroethyl)-N' cyclohexyl-N-nitrosourea (CCNU); N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU); N- (2-chloroethyl)-N'-(diethyl) ethylphosphonate-N-nitrosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin; Carboplatin; Ormaplatin; Oxaliplatin; Cl -973; DWA 2114R; JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6- Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide 9-amino camptothecin;Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone;losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F- ara-AMP); or 2-chlorodeoxyadenosine (2-Cda).

[0255] In other embodiments radiation is provided to the cells for generation of said senescent phenotype.

[0256] Senescent cells may be fused with dendritic cells, or may be lyzed and said lysate used to pulse dendritic cells.

[0257] Antigen presenting cells (APC) are particularly important in eliciting an effective immune response. By definition, APC not only can present antigens to T cells with antigen-specific receptors, but can provide all the signals necessary for T cell activation. Such signals are incompletely defined, but probably involve a variety of cell surface molecules as well as cytokines or growth factors. Further, the factors necessary for the activation of naive or unprimed T cells may be different from those required for the re-activation of previously primed memory T cells. The ability of APC to both present antigens and deliver signals for T cell activation is commonly referred to as an accessory cell function. Although monocytes and B cells have been shown to be competent APC, their antigen presenting capacities in vitro appear to be limited to the re-activation of previously sensitized T cells. Hence, they are not capable of directly activating functionally naive or unprimed T cell populations. The term "dendritic cells" refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues (Steinman, 1991, Ann. Rev. Immunol. 9: 271-296). These cells include lymphoid DC of the spleen, Langerhans cells of the epidermis, and veiled cells in the blood circulation. Although they are collectively classified as a group based on their morphology, high levels of surface MHC-class II expression, and absence of certain other surface markers expressed on T cells, B cells, monocytes, and natural killer cells, it is presently not known whether they derive from a common precursor or can all function as APC in the same manner.

[0258] Human DCs are obtained from any tissue where they reside including non- lymphoid tissues such as the epidermis of the skin (Langerhans cells) and lymphoid tissues such as the spleen, bone marrow, lymph nodes and thymus as well as the circulatory system including blood (blood DCs) and lymph (veiled cells). Human peripheral blood is an easily accessible ready source of human DCs and is used as a source according to a preferred embodiment of the invention. Cord blood is anothersource of human DCs and in cases where a male is born into a family known to be at high risk for cancer, cord blood can be used as a source of DCs which can be cryopreserved for later use, if needed.

[0259] Because DCs occur in low numbers in any tissues in which they reside, including human peripheral blood, DCs must be enriched or isolated for use. Any of a number of procedures entailing repetitive density gradient separation, positive selection, negative selection or a combination thereof are used to obtain enriched populations or isolated DCs. Examples of such methods for isolating DCs from human peripheral blood include: O'Doherty et al, 1993, J. Exp. Med. 178: 1067-1078; Young and Steinman, 1990, J. Exp. Med. 171 : 1315-1332; Freudenthal and Steinman, 1990, PNAS USA 57: 7698- 7702; Macatonia et al., 1989, Immunol. 67: 285-289; and Markowicz and Engleman, 1990, J. Clin. Invest. 85: 955-961. A method for isolating DCs from human peripheral blood which avoids exposure of the cells to sheep red blood cells and / or fetal calf serum is described in PCT Publication WO94 / 02156. An example of a method for isolating DCs from lymphoid tissue is described in Macatonia et al., 1989, J. Exp. Med. 169: 1255- 1264. Once the DCs are obtained, they are cultured in appropriate culture medium to expand the cell population and / or maintain the DCs in a state for optimal antigen uptake, processing and presentation. Particularly advantageous for maintenance of the proper state of "maturity" of DCs in in vitro culture is the presence of both granulocyte / macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4). Preferred is a combination of GM-CSF : IL-4 in concentration of about 500 units / ml of each. A recent study reveals optimal antigen presentation by "immature" vs. mature DC (Koch et al., 1995, J. Immunol. 155: 93-100). Immature DCs may be preferred according to certain embodiments of the present invention. According to a preferred embodiment of the invention, DCs are obtained from a patient to be treated. The DCs are used to activate autologous T cells of the patient, either in vitro or in vivo, for immunotherapy.

[0260] According to an alternate embodiment, DCs are obtained from a healthy individual. The relevant HLA antigens (both class I and II, e.g., HLA-A, B, C and DR) on the individual's PBMC's are identified and DCs which match the patient, in terms of HLA antigens, are isolated and expanded as described above. According to another embodiment of the invention, "extended life span dendritic cells" are used. Human cells have a finite life span in vitro usually limited to approximately 50-70 population doublings before undergoing apoptosis. As used herein, the term "extended life span dendritic cells" is intended to mean DCs that have been genetically modified so that theycan be expanded in in vitro cell culture medium for an extended period of time, including but not limited to at least about 100 additional population doublings. Extended life span DCs are obtained, for example, by EBV-transformation of DCs obtained from peripheral blood of patients, or by insertion into DCs, using techniques known to those skilled in the art, of a specific cell cycle regulatory gene including but not limited to a gene which encodes cyclin A, B, D or E or retinoblastoma protein. According to yet another embodiment of the invention, DCs can be preserved, e.g., by cryopreservation either before exposure or following exposure to a senescence antigen. A controlled slow cooling rate is critical. Different cryoprotective agents (Rapatz et al., 1968, Cryobiology 5(1): 18- 25) and different cell types have different optimal cooling rates (see, e.g., Rowe and Rinfret, 1962, Blood 20: 636; Rowe, 1966, Cryobiology 3(1): 12-18; Lewis et al., 1967, Transfusion 7(1): 17-32; and Mazur, 1970, Science 168939-949 for effects of cooling velocity on survival of marrow-stem cells and on their transplantation potential). The heat of fusion phase where water turns to ice should be minimal. The cooling procedure can be carried out by use of, e.g., a programmable freezing device or a methanol bath procedure. Programmable freezing apparatuses allow determination of optimal cooling rates and facilitate standard reproducible cooling. Programmable controlled-rate freezers such as Cryomed or Planar permit tuning of the freezing regimen to the desired cooling rate curve. After thorough freezing, cells can be rapidly transferred to a long-term cryogenic storage vessel. In a preferred embodiment, samples can be cryogenically stored in liquid nitrogen (-196° C.) or its vapor (-165° C ). Such storage is greatly facilitated by the availability of highly efficient liquid nitrogen refrigerators, which resemble large Thermos containers with an extremely low vacuum and internal super insulation, such that heat leakage and nitrogen losses are kept to an absolute minimum.

[0261] Considerations and procedures for the manipulation, cryopreservation, and long term storage of hematopoietic stem cells, particularly from bone marrow or peripheral blood, is largely applicable to the DCs of the invention. Such a discussion can be found, for example, in the following references, incorporated by reference herein: Gorin, 1986, Clinics in Haematology 15(l)"19-48; Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, Jul. 22-26, 1968, International Atomic Energy Agency, Vienna, pp. 107-186. Other methods of cry opreservation of viable cells, or modifications thereof, are available and envisioned for use (e.g., cold metal-mirror techniques; Livesey and Linner, 1987, Nature 327: 255; Linner et al., 1986,J. Histochem. Cytochem. 34(9): 1123-1135; see also U.S. Pat. No. 4,199,022 by Senken et al., U.S. Pat. No. 3,753,357 by Schwartz, U.S. Pat. No. 4,559,298 by Fahy.

[0262] Frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37°-41° C.) and chilled immediately upon thawing.lt may be desirable to treat the cells in order to prevent cellular clumping upon thawing. To prevent clumping, various procedures can be used, including but not limited to the addition before and / or after freezing of Dnase

[0263] For example, it may be desired to culture purified CD8+T cells with antigen exposed DCs to elicit senescence specific CTL. In addition, early elimination of CD4+T cells may prevent the overgrowth of CD4+cells in a mixed culture of both CD8+and CD4+T cells. T cell purification may be achieved by positive, or negative selection, including but not limited to, the use of antibodies directed to CD2, CD3, CD4, CD5, and CD8.

[0264] On the other hand, it may be desired to use a mixed population of CD4+and CD8+T cells to elicit a senescence specific response encompassing both a cytotoxic and TH immune response.

[0265] According to a preferred embodiment, the T cells are obtained from the same patient from which the DCs were obtained. After stimulation or activation in vitro, the autologous T cells are administered to the patient to provoke and afford an immunoresponse. For example, T cells are administered, by intravenous infusion, at doses of about 108-109cells / m2of body surface area (see, Ridell et al., 1992, Science 257: 238-241). Infusion can be repeated at desired intervals, for example, monthly. Recipients are monitored during and after T cell infusions for any evidence of adverse effects. According to another embodiment, the T cells are obtained from a patient and the DCs which are used to stimulate the cells are obtained from an HLA-matched healthy donor. According to yet another embodiment, both the T cells and the DCs are obtained from an HLA-matched healthy donor, e.g., a sibling of the patient. This embodiment may be particularly advantageous, for example, when the patient is a late stage

[0266] patient who has been treated with radiation and / or chemotherapy agents and may not be able to provide sufficient or efficient DCs. The T cells after stimulation, are administered as described above.

[0267] According to another embodiment of the invention, DCs isolated from a patient are cultured, exposed in vitro to a senescence antigen and after expansion and / or cry opreservation are administered back to the patient to stimulate an immune response,including T cell activation, against the patient's senescence cells in vivo. Using this approach with the patient's own dendritic cells provides the following advantages: (1) no foreign DNA is utilized; (2) infection of cells for purposes of cDNA expression using various viral vectors are eliminated; (3) antigen is presented to dendritic cells in the form of soluble protein which will be taken into the dendritic cells and processed for MHC / peptide presentation of the cell surface; (4) dendritic cells express B7's on their surface alleviating the necessity to transfect this cDNA into dendritic cells; (5) the use of endogenous B7's on dendritic cell surface eliminates the need to provide T cells with 11-2 or other cytokines either in the form of the cytokine itself or transfection of the cDNA into specific cells; (6) all procedures are carried out using the patient's own cells.

[0268] In practice, DCs, are exposed in vitro to senescent cell antigen(s), washed and administered to elicit an immune response or to augment an existing, albeit weak, response. As such, the DCs constitute an anti -senescence vaccine and / or immunotherapeutic agent. DCs presenting a senescence specific antigen are administered, via intravenous infusion, at a dose of about 106- 108cells. The immune response of the patient can be monitored. Infusion can be repeated at desired intervals based upon the patient's immune response.

[0269] Pharmaceutical formulations of a therapeutically effective amount of an agent of the invention (e.g., peptides, polypeptides, proteins, small molecules, antibodies, or antibody fragments that target senescent cells), or pharmaceutically acceptable salt- thereof, can be administered orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous, or subcutaneous injection, inhalation, intradermally, optical drops, or implant), nasally, vaginally, rectally, sublingually, or topically. The pharmaceutical formulation can include the agent of the invention in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.

[0270] Methods well known in the art for making pharmaceutical formulations can be found, for example, in Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions. The compositions may optionally contain sweetening, flavoring, coloring, perfuming, and / or preserving agents in order to provide a more palatable preparation. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient. Thesemay include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and / or lubricating agents (e.g., magnesium stearate) may also be used. Tablets and pills can additionally be prepared with enteric coatings.

[0271] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules. These forms contain inert diluents commonly used in the art, such as water or an oil medium. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents.

[0272] Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, or emulsions. Examples of suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphthalenes, and injectable organic esters, such as ethyl oleate. Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents.Biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or polyoxy ethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the agents of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

[0273] Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.

[0274] Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to active substances, excipients such as coca butter or a suppository wax. Compositions for nasal or sublingual administration are also prepared with standard excipients known in the art. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or spray, or as a gel.

[0275] The amount of active ingredient in the compositions of the invention can be varied. One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the agent beingadministered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient. In addition, the severity of the condition targeted by an agent of the invention will also have an impact on the dosage level. Generally, dosage levels of an agent of the invention of between 0.1 pg / kg to 100 mg / kg of body weight are administered daily as a single dose or divided into multiple doses. Preferably, the general dosage range is between 250 pg / kg to 5.0 mg / kg of body weight per day. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of administration. For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, which are well known in the art. In general, the precise therapeutically effective dosage can be determined by the attending physician in consideration of the above-identified factors.

[0276] An agent of the invention (e.g., a peptide, polypeptide, protein, small molecule, antibody, or antibody fragment that targets senescent cells) can be administered in a sustained release composition, such as those described in, for example, U.S. Pat. Nos. 5,672,659, 5,595,760. The use of immediate or sustained release compositions depends on the type of condition being treated. If the condition consists of an acute or over-acute disorder, a treatment with an immediate release form will be preferred over a prolonged release composition. Alternatively, for preventative or long-term treatments, a sustained released composition will generally be preferred.

[0277] An agent of the invention (e.g., a peptide, polypeptide, protein, small molecule, antibody, or antibody fragment that targets senescent cells) can be prepared in any suitable manner. The agent may be isolated from naturally-occurring sources, recombinantly produced, or produced synthetically, identified from a library of small molecules, or produced by a combination of these methods. The synthesis of short peptides is well known in the art. See e.g., Stewart et al., Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984). A peptide portion of any of the agents of the invention can be synthesized according to standard peptide synthesis methods known in the art.

[0278] In some embodiments, the therapeutic compositions are administered in a “therapeutically effective amount.” Such a therapeutically effective amount is an amount sufficient to obtain the desired physiological effect, e.g., treatment of a condition,disorder, disease and the like or reduction in symptoms of the condition, disorder, disease and the like. For example, the therapeutic agents can be administered to treat any of the conditions, disorders, or diseases described herein. Examples include congestive heart failure, myocardial infarction, cardiac ischemia myocarditis, arrhythmia or any combination thereof.

[0279] To achieve the desired effect(s), the composition can be formulated in single or divided dosages. For example, a GSK3 inhibitor, a WNT agonist, a TGF-beta inhibitor, an inhibitor of extracellular signal -regulated kinase 1 (ERK1), an inhibitor of Ras GTPase-activating protein (Ras-GAP), an Oct-4 activator, a pl60ROCK inhibitor (where pl60ROCK is a rho-associated protein kinase), an iron chelator and / or KDM5B inhibitor, an inhibitor of G9a histone methyltransferase, an inhibitor of various growth factor receptors such as PDGF receptor beta, a protein kinase receptor inhibitor, an inhibitor of PDGF-BB receptor, and / or a growth factor (e.g., any of the CIM1 growth factors) can present in the composition in amounts specified above or in dosages of at least about 0.01 mg / kg to about 500 to 750 mg / kg, of at least about 0.01 mg / kg to about 300 to 500 mg / kg, at least about 0.1 mg / kg to about 100 to 300 mg / kg or at least about 1 mg / kg to about 50 to 100 mg / kg of body weight, although other dosages may provide beneficial results. The amount administered will vary depending on various factors including, but not limited to the combination of compounds chosen for administration, the disease, the weight, the physical condition, the health, the age of the mammal, as well as other physiological factors. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.

[0280] For example, a reprogramming composition can include CHIR99021 (e.g., at about 10-20 pM). A83-01 (e.g., at about 0.5-1.0 pM), SCI (e.g., at about 0.5-1.0 pM), OAC (e.g., at about 1-10 pM), Y27632 (e.g., at about 5-10 pM), BIX-01294 (e.g., at about 0.5-2.0 pM), AS8351 (e.g., at about 1.0-3.0 pM), SU16f (e.g., at about 2-5 pM), and JNJ-10198409 (e.g., at about 0.05-0.2 pM). As described herein PBIT (e.g., at about 10 pM).

[0281] Reprogrammed cells can be included in the compositions in varying amounts depending upon the disease or injury to be treated. For example, the compositions can be prepared in liquid form for local or systemic administration containing about 103 to about 1012 reprogrammed cells, or about 104 to about 1010 reprogrammed cells, or about 105 to about 108 reprogrammed cells. One or more of the following types of compounds can also be present in the composition with the cells: a GSK3 inhibitor, a WNT agonist, aTGF-beta inhibitor, an inhibitor of extracellular signal-regulated kinase 1 (ERK1), an inhibitor of Ras GTPase-activating protein (Ras-GAP), an Oct-4 activator, a pl60ROCK inhibitor (where pl60ROCK is a rho-associated protein kinase), an iron chelator and / or KDM5B inhibitor, an inhibitor of G9a histone methyltransferase, an inhibitor of various growth factor receptors such as PDGF receptor beta, a protein kinase receptor inhibitor, an inhibitor of PDGF -BB receptor, and / or one or more growth factors (e.g., any of the CIM1 growth factors).

[0282] Administration of the composition may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is for response to traumatic injury or for more sustained therapeutic purposes, and other factors known to skilled practitioners. Similarly, cell(s) can be contacted with the composition in a continuous manner, or intermittently, depending upon the need for reprogrammed cells, the manufacturing schedule, the convenience of workers, and / or the selected recipient's physiological condition. The administration or contacting of the cells with compositions of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration to recipients and / or subjects is contemplated.

[0283] To prepare the composition, the compounds are synthesized and / or the cells are generated, and the components are purified as necessary or desired. The compounds, cells, and / or other agents can be suspended in a pharmaceutically acceptable carrier. If the composition contains only compounds, without cells, the composition can be lyophilized. These compounds and cells can be adjusted to an appropriate concentration, and optionally combined with other agents. The absolute weight of a given compound and / or other agent included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least one compound can be administered.Alternatively, the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.

[0284] Daily doses of the compounds can vary as well. Such daily doses can range, for example, from about 0.1 g / day to about 50 g / day, from about 0.1 g / day to about 25 g / day, from about 0.1 g / day to about 12 g / day, from about 0.5 g / day to about 8 g / day, from about 0.5 g / day to about 4 g / day, and from about 0.5 g / day to about 2 g / day.

[0285] It will be appreciated that the amount of compounds and cells for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately, the attendant health care provider may determine proper dosage. A pharmaceutical composition may be formulated with the appropriate ratio of each compound in a single unit dosage form for administration with or without cells. Cells can be separately provided and either mixed with a liquid solution of the compound composition, or administered separately. The compounds can also be formulated for sustained release (for example, using microencapsulation, see WO 94 / 07529, and U.S. Pat. No. 4,962,091). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system. One or more suitable unit dosage forms containing the compounds and / or the reprogrammed cells can be administered by a variety of routes including parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), intracranial, intraspinal, oral, rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes.

[0286] In one embodiment iPSC are generated from various sources and utilized to generate insulin producing cells in vitro. In other embodiments the process is performed in vivo. Methods for generation of iPSCs from somatic cells involves forced expression of a set of polypeptides or induction factors (IFs). IFs currently known to the art include but are not limited to polypeptides encoded by the genes: c-Myc, Oct3 / 4, Sox2, and Klf4. In addition, small molecule compounds such as histone deacetylace inhibitors may be used or a combination of IFs and small molecules may be used to generate iPSCs. The somatic cells may be used directly, i.e., without culturing or passaging, in the referenced induction methods; or, the somatic cells may be cultured and / or passaged prior to their use in the referenced induction methods. The induced cells may be induced from the somatic cells of a postnatal donor or non-pluripotent donor as described in U.S. application Ser. No. 12 / 157,967, filed Jun. 13, 2008; First Inventor Kazuhiro Sakurada, which is herein incorporated by reference in its entirety. The induced stem cells may be generated from any cell-type including but not limited to those described.

[0287] IPSCs or iSCs may be used directly for differentiation or regenerative medicine. In other cases, iPSCs or iSCs may be stored by the regenerative medicine business, stem cell technology business or a third party. Alternatively, iPSCs or iSCs may be expanded using culturing methods described in U.S. application Ser. No. 12 / 157,967, filed Jun. 13, 2008; First Inventor Kazuhiro Sakurada, which is herein incorporated by reference in its entirety, prior to or after storage. iSCs may be stored in any manner which preserves their multipotent or pluripotent capabilities including cryogenic storage, and culturing. In some cases the donor, potential recipient of the iPSCs or derivatives thereof, or payee may be billed for generation and or delivery of iPSCs or differentiated cells or tissues. In some cases a kit may be marketed and sold which includes a means for generation of iPSCs. During the induction process, forced expression of certain polypeptides is carried out in cultured cells for a period of time, after which the induced cells are screened for a number of morphological and gene expression properties that characterize multipotent and pluripotent stem cells. Induced cells that meet these screening criteria may then be subcloned and expanded. In some cases, the cells to be induced may be cultured for a period of time prior to the induction procedure.Alternatively, the cells to be induced may be used directly in the induction process without a prior culture period. In some embodiments, the type of cell culture medium used is the same or very similar before, during, and after the induction process. In other cases, different cell culture media are used at different points. For example, one type of culture medium may be used directly before the induction process, while a second type of media is used during the induction process. At times, a third type of culture medium is used during the induction process. Cells may be cultured in medium supplemented with a particular serum. In some embodiments, the serum is fetal bovine serum (FBS). The serum can also be fetal calf serum (FCS). In some cases, the serum may be Human AB serum. Mixtures of serum may also be used, e.g. mixture of FBS and Human AB, FBS and FCS, or FCS and Human AB. Culture of cells may be carried out under a low serum culture conditions prior to, during, or following induction. A “low serum culture condition” refers to the use of a cell culture medium containing a concentration of serum ranging from 0% (v / v) (i.e., serum-free) to about 5% (v / v), e.g., 0% to 2%, 0% to 2.5%, 0% to 3%, 0% to 4%, 0% to 5%, 0.1% to 2%, 0.1% to 5%, 0.1%, 0.5%, 1%, 1.2%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4%. In some embodiments, the serum concentration is from about 0% to about 2%. In some cases, the serum concentration is about 2%. In some cases, the serum concentration is preferably 2% or less. In other embodiments, cells arecultured under a “high serum condition,” i.e., greater than 5% serum to about 20% serum, e.g., 6%, 7%, 8%, 10%, 12%, 15%, or 20%. Culturing under high serum conditions may occur prior to, during, and / or after induction. Some representative media that the cells can be cultured in include: MAPC, FBM, ES, MEF-conditioned ES (MC-ES), and mTeSR™ (available, e.g., from StemCell Technologies, Vancouver, Canada), See Ludwig et al (2006), Nat Biotechnol, 24(2): 185-187. In other cases, alternative culture conditions for growth of human ES cells are used, as described in, e.g., Skottman et al (2006), Reproduction, 132(5):691-698. In some embodiments, the cells are cultured in MAPC, FBM, MC-ES, or mTeSR™ prior to and / or during the introduction of induction factors to the cells; and the cells are cultured in MC-ES or mTeSR™ medium later in the induction process. MAPC (2% FBS) Medium may comprise: 60% Dulbecco's Modified Eagle's Medium -low glucose, 40% MCDB 201, Insulin Transferrin Selenium supplement, (0.01 mg / ml insulin; 0.0055 mg / ml transferrin; 0.005 pg / ml sodium selenite), lx linolenic acid albumin (1 mg / mL albumin; 2 moles linoneic acid / mole albumin), 1 nM dexamethasone, 2% fetal bovine serum, 1 nM dexamethasone, 10-4 M ascorbic acid, and 10 pg / ml gentamycin. FBM (2% FBS) Medium may comprise: MCDB202 modified medium, 2% fetal bovine serum, 5 pg / ml insulin, 50 mg / ml gentamycin, and 50 ng / ml amphotericin-B. ES Medium may comprise: 40% Dulbecco's Modified Eagle's Medium (DMEM) 40% F12 medium, 2 mM L-glutamine, l x non-essential amino acids (Sigma, Inc., St. Louis, Mo.), 20% Knockout Serum Replacement™ (Invitrogen, Inc., Carlsbad, Calif.), and 10 pg / ml gentamycin. MC-ES medium may be prepared as follows. ES medium is conditioned on mitomycin C-treated murine pluripotent fibroblasts (MEFs), harvested, filtered through a 0.45-pM filter, and supplemented with about 0.1 mM P mercaptoethanol, about 10 ng / ml bFGF or FGF-2, and, optionally, about 10 ng / ml activin A. In some cases, irradiated MEFs are used in place of the mitomycin C-treated MEFs. When either low or high serum conditions are used for culturing the cells, one or more growth factors such as fibroblast growth factor (FGF)-2; basic FGF (bFGF); platelet- derived growth factor (PDGF), epidermal growth factor (EGF); insulin-like growth factor (IGF); or insulin can be included in the culture medium. Other growth factors that can be used to supplement cell culture media include, but are not limited to one or more: Transforming Growth Factor n-1 (TGF n-1), Activin A, Noggin, Brain-derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Neurotrophin (NT)-l, NT-2, or NT 3. In some cases, one or more of such factors is used in place of the bFGF or FGF- 2 in the MC-ES medium or other cell culture medium. In some cases, the concentrationof growth factors in the culture media described (e.g., MAPC, FBM, MC-ES, mTeSR™) is from about 2 ng / ml to about 20 ng / ml, e.g., about 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 10 ng / ml, 12 ng / ml, 14 ng / ml, 15 ng / ml, 17 ng / ml, or 20 ng / ml. In some embodiments, the concentration of bFGF or FGF2 is from about 2 ng / ml to about 5 ng / ml; from about 5 ng / ml to about 8 ng / ml; from about 9 ng / ml to about 11 ng / ml; from about 11 ng / ml to about 15 ng / ml; or from about 15 ng / ml to about 20 ng / ml. The growth factors may be used alone or in combination. For example, FGF-2 may be added alone to the medium; in another example, both PDGF and EGF are added to the culture medium. In some examples, following initiation of the forced expression of genes or polypeptides (e.g., immediately after a retroviral infection period) in cells, the “induced cells” are maintained in MC-ES medium as described herein.

[0288] In some embodiments, cells are maintained in the presence of a rho, or rho- associated, protein kinase (ROCK) inhibitor to reduce apoptosis. In some cases, an inhibitor of Rho associated kinase is added to the culture medium. For example, the addition of Y-27632 (Calbiochem; water soluble) or Fasudil (HA1077: Calbiochem), an inhibitor of Rho associated kinase (Rho associated coiled coil-containing protein kinase) may be used to culture the human pluripotent and multipotent stem cells of the present invention. In some cases the concentration of Y-27632 or Fasudil, is from about 5 pM to about 20 pM, e.g., about 5 pM, 10 pM, 15 pM, or 20 pM. The cells may be cultured for about 1 to about 12 days e.g., 2 days, 3 days, 4.5 days, 5 days, 6.5 days, 7 days, 8 days, 9 days, 10 days, or any other number of days from about 1 day to about 12 days prior to undergoing the induction methods described herein. In some cases, the induced cells are cultured in complete ES medium in a 37nC, 5% CO2 incubator, with medium changes about every 1 to 2 days. In some embodiments, induced the induced cells are cultured and observed for about 14 days to about 40 days, e.g., 15, 16, 17, 18, 19, 20, 23, 24, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38 days, or any other period from about 14 days to about 40 days prior to identifying and selecting clones comprising “induced cells” based on morphological characteristics. Morphological characteristics for identifying induced cell clones include, but are not limited to, a small cell size with a high nucleus-to-cytoplasm ratio; formation of small monolayer colonies within the space between parental cells (e.g., between fibroblasts). The cells may be plated at a cell density of about 1 ^ 103 cells / cm2 to about 1 x 104 cells / cm2, e.g., 2x 103 cells / cm2, 3.5x 103 cells / cm2, 6x 103 cells / cm2, 7x 103 cells / cm2, 9x 103 cells / cm2, or any other cell density from about 1 x 103 cells / cm2 to about 1 x 104 cells / cm2. The cells can be plated and cultured directly on tissue culture-grade plastic. Alternatively, cells are plated and cultured on a coated substrate, e.g., a substrate coated with fibronectin, gelatin, matrigel™, collagen, or laminin. Suitable cell culture vessels include, e.g., 35 mm, 60 mm, 100 mm, and 150 mm cell culture dishes, 6- well cell culture plates, and other size-equivalent cell culture vessels. In some cases, the cells are cultured with feeder cells. For example, the cells may be cultured on a layer, or carpet, of MEFs.

[0289] Media with low concentrations of serum may be particularly useful to enrich for undifferentiated stem cells. The undifferentiated cells cultured under low serum conditions may or may not share certain properties with MSCs, MAPCs, and / or MIAMI cells. Differences in phenotype may be due, in part, to culture methods used to obtain MSCs, MAPCs and MIAMI cells. For example, MSCs are often obtained by isolating the non-hematopoeitic cells (e.g., interstitial cells) adhering to a plastic culture dish when tissue, e.g., bone marrow, fat, muscle, or skin etc., is cultured in a culture medium containing a high-concentration serum (5% or more). However, even under these culture conditions, a very small number of undifferentiated cells can be maintained, especially if the cells were passaged under certain culture conditions (e.g., low passage number or low-density culturing). In some embodiments, in order to culture and grow human pluripotent stem cells induced from the undifferentiated stem cells of the present invention present in a human postnatal tissue, it is preferred that the cells are subcultured every 5 to 7 days in a culture medium containing the additives described herein on a MEF-covered plastic culture dish or a matrigel-coated plastic culture dish. In some cases, the cells may be cultured at a low density, which may be accomplished by splitting the cells from about 1 :6 to 1 :3 or by plating the cells at 103 cells / cm2 to 3^ 104 cells / cm2. Primary culture ordinarily occurs immediately after the cells are isolated from a donor, e.g., human. The primary cells can be subjected to a second subculture, a third subculture, a fourth subculture, and greater than four subcultures. A “second” subculture describes primary culture cells subcultured once, a “third” subculture describes primary cultures subcultured twice, a “fourth” subculture describes primary cells subcultured three times, etc. The culture techniques described herein may generally include culturing from the period between the primary culture and the fourth subculture, but other culture periods may also be employed. Preferably, cells are cultured from primary culture to second subculture.

[0290] Inducing a cell to become multipotent or pluripotent can be accomplished in numerous ways. In some embodiments, the methods for induction of pluripotency ormultipotency in one or more cells include forcing expression of a set of induction factors (IFs). In some cases, the set of IFs includes one or more: an Oct3 / 4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, or a c-Myc polypeptide. In some cases, the set does not include a c-Myc polypeptide. For example, the set of IFs can include: an Oct3 / 4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, but not a c-Myc polypeptide. In some cases, the set of IFs does not include polypeptides that might increase the risk of cell transformation. In some cases, the set may include a c-Myc polypeptide. In certain cases, the c-Myc polypeptide is a constitutively active variant of c-Myc. In some instances, the set includes a c-Myc polypeptide capable of inducible activity, e.g., a c- Myc-ER polypeptide,

[0291] In other cases, the set of IFs may include: an Oct3 / 4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, but not a TERT polypeptide, a SV40 Large T antigen polypeptide, HPV16 E6 polypeptide, a HP VI 6 E7 polypeptide, or a Bmil polypeptide. In some cases, the set of IFs does not include a TERT polypeptide. In some cases, the set of IFs does not include a SV40 Large T antigen. In other cases, the set of IFS does not include a HPV 16 E6 polypeptide or a HPV 16 E7 polypeptide.

[0292] In some cases, the set of IFs includes three IFs, wherein two of the three IFs are an Oct3 / 4 polypeptide and a Sox2 polypeptide. In other cases, the set of IFs includes two IFs, wherein the two polypeptides are a c-Myc polypeptide and a Sox2 polypeptide In some cases, the set of induction factors is limited to Oct 3 / 4, Sox2, and Klf4 polypeptides. In other cases, the set of induction factors may be limited to a set of four IFs: an Oct3 / 4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide. A set of IFs may include IFs in addition to an Oct 3 / 4, a Sox2, and a Klf4 polypeptide. Such additional IFs include, but are not limited to Nanog, TERT, LIN28, CYP26A1, GDF3, FoxD3, Zfp42, Dnmt3b, Ecatl, and Tell polypeptides. In some cases, the set of additional IFs does not include a c Myc polypeptide. In some cases, the set of additional IFs does not include polypeptides that might increase the risk of cell transformation. Forced expression of IFs may be maintained for a period of at least about 7 days to at least about 40 days, e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 33 days, or 37 days. In a first step, a cell population (the starting cell population) comprising at least one cell capable of differentiation is provided. In some embodiments, the cell capable of differentiation is a pancreatic progenitor cell expressing PDX1 and NKX6.1.

[0293] In some embodiments, the starting cell population comprises at least 5% pancreatic progenitor cells, such as at least 10% pancreatic progenitor cells, such as at least 15% pancreatic progenitor cells, such as at least 20% pancreatic progenitor cells, such as at least 25% pancreatic progenitor cells, such as at least 30% pancreatic progenitor cells, such as at least 35% pancreatic progenitor cells, such as at least 40% pancreatic progenitor cells, such as at least 45% pancreatic progenitor cells, such as at least 50% pancreatic progenitor cells, such as at least 55% pancreatic progenitor cells, such as at least 60% pancreatic progenitor cells, such as at least 65 pancreatic progenitor cells, such as at least 70% pancreatic progenitor cells, such as at least 75% pancreatic progenitor cells, such as at least 80% pancreatic progenitor cells, such as at least 85% pancreatic progenitor cells, such as at least 90% pancreatic progenitor cells, such as at least 95% pancreatic progenitor cells. In order to determine the fraction of progenitor cells comprised in a cell population, for example in the starting population, methods known in the art can be employed, such as, but not limited to, immunostaining or flow cytometry methods.

[0294] Without being bound by theory, the percentage of pancreatic progenitor cells in the starting cell population can be estimated by the expression of GP2. Thus in some embodiments, the starting cell population comprises at least 5% cells expressing GP2, such as at least 10% cells expressing GP2, such as at least 15% cells expressing GP2, such as at least 20% cells expressing GP2, such as at least 25% cells expressing GP2, such as at least 30% cells expressing GP2, such as at least 35% cells expressing GP2, such as at least 40% cells expressing GP2, such as at least 45% cells expressing GP2, such as at least 50% cells expressing GP2, such as at least 55% cells expressing GP2, such as at least 60% cells expressing GP2, such as at least 65% cells expressing GP2, such as at least 70% cells expressing GP2, such as at least 75% cells expressing GP2, such as at least 80% cells expressing GP2, such as at least 85% cells expressing GP2, such as at least 90% pancreatic progenitor cells, such as at least 95% pancreatic progenitor cells. GP2 expression can be determined by methods known in the art, such as immunostaining methods, flow cytometry methods or quantitative measurements of transcription levels. Likewise, without being bound by theory, the percentage of PDX1+NKX6.1+ cells in the starting cell population can be estimated by the expression of GP2. Thus in some embodiments, the starting cell population comprises at least 5% cells expressing GP2, such as at least 10% cells expressing GP2, such as at least 15% cells expressing GP2, such as at least 20% cells expressing GP2, such as at least 25%cells expressing GP2, such as at least 30% cells expressing GP2, such as at least 35% cells expressing GP2, such as at least 40% cells expressing GP2, such as at least 45% cells expressing GP2, such as at least 50% cells expressing GP2, such as at least 55% cells expressing GP2, such as at least 60% cells expressing GP2, such as at least 65% cells expressing GP2, such as at least 70% cells expressing GP2, such as at least 75% cells expressing GP2, such as at least 80% cells expressing GP2, such as at least 85% cells expressing GP2, such as at least 90% pancreatic progenitor cells, such as at least 95% pancreatic progenitor cells. GP2 expression can be determined by methods known in the art, such as immunostaining methods, flow cytometry methods or quantitative measurements of transcription levels.Example 1 Preparation of Clinical Grade Senescence Targeting Immunotherapy

[0295] Dendritic cells targeting senescent cells are prepared from 200 ml of peripheral blood obtained from the patient to be treated. Subsequent to ficoll purification of peripheral blood mononuclear cells, monocytes are isolated by CD14 selection using magnetic activated cell sorting (MACS). CD14 positive cells are cultured for 5 days in 50 ng / ml GM-CSF and 500 ng / ml IL-4, with media changed once every two days. On Day 5 cells are pulsed with 10 ug / ml of autologous tumor lysate and subsequently transfected with siRNA pool specific for IDO silencing.

[0296] Senescent cell lysate is generated as follows: peripheral blood lymphocytes are obtained under aseptic conditions. Cells are stimulated to proliferate by treatment with 5 ug / ml of PHA and genotoxically stressed with 40 ng. ml of doxorubicin Samples are washed in sterile saline solution, weighed, and snap frozen in liquid nitrogen for storage until use. After successful initiation of dendritic cell culture, samples are thawed in 37 Celsius water bath, and dissociated by use of syringes with decreasing needle sizes, starting with 18G, then 20G and finally 21G. The signal cell suspension is diluted in sterile phosphate buffered saline and centrifuged at 300g for 5 minutes to obtain a pellet. Subsequently, the pellet is resuspended in PBS and filtered through 70 and 30 micrometer filters (Corning Cell Strainers, Carlsbad, California). The resulting suspension is subsequently centrifuged at 300g for 5 minutes and sonicated for 1 hour to generate the lysate. The lysate is assessed microscopically with trypan blue staining to detect whether any viable cells remain. In the situation of identification of viable cells, sonication is again performed. The protein concentration of the lysate is assessed by using the bicinchoninic acid method (BCA, Pierce Biotechnlogy / ThermoFischer Scientific).Loading of DC is performed by administration of a final concentration of 100 micrograms per ml of senescent cell lysate onto DC for a period of 12 hours, subsequently to which the DC are washed and replated. Washing of DC is performed in RPMI media. Cells are subsequently replated for transfection.

[0297] Transfection is performed using 2 micrograms per ml of siRNA premixed with 5 microliters per ml of GeneSilencer transfection reagent. Procedures from Zheng et al. Int. J. Cancer: 132, 967-977) are applied to human CD14 derived monocytes for the generation of DC, pulsing with tumor antigen from surgical sample, and gene silencing.

[0298] Specifically, transfection of siRNA is performed as follows: Two micrograms of double-stranded siRNA is mixed with 5 microliters of GeneSilencerTM transfection reagent

[0299] (Gene Therapy Systems, San Diego, CA). The mixture is suspected by 10 inversions and added to 1 x 10(5) DC progenitors per 6 well plate in a volume of 2 ml media. After 4 hours of incubation at 37 Celsius in fully humidified atmosphere, an equal volume of RPMI-1640 supplemented with 20% FCS and 50 ng / ml GM-CSF and 500 ng / ml IL-4 is added to the transfected DC. Viability of cells is assessed subsequent to transfection using trypan blue staining with an acceptable cut-off of >70%.Sequences of siRNA utilized are as follows:

[0300] UUAUAAUGACUGGAUGUUC (SEQ ID NO: 1)

[0301] GUCUGGUGUAUGAAGGGUU (SEQ ID NO: 2)

[0302] CUCCUAUUUUGGUUUAUGC (SEQ ID NO: 3)

[0303] GCAGCGUCUUUCAGUGCUU (SEQ ID NO: 4)Vaccine Characterization2. Reagents

[0304] a. Tabulation of Reagents Used in Manufactureb. Qualification Program

[0305] Reagent qualification occurs as part of our current manufacturing practice at General BioTechnology. The qualification process begins with the vender of the reagent. The vender is qualified through our standard operating procedure. A corresponding form is completed and approval gained before a vender can be used. The Criteria identified as important in qualifying a supplier include quality of product, services offered, competitive pricing, communication, availability, how complaints are handled and the overall fit to our systems. This list is not all inclusive. Quality Systems reviews each qualification form and will approve based on the criteria stated above. Once the vender is approved, they are added to the Supplies and Services List. Associates ordering supplies including reagents use the list. Only approved venders on the list are used by associates ordering supplies involving reagents.

[0306] Once the reagent arrives, it is logged on the Supplies Receipt, Inspection and Inventory Log. The form instructs the associate to complete certain information for the incoming reagent. These fields are date received, initials of receiver, name of the item, manufacturer, lot number, expiration date, package passed visual inspection, product passed visual inspection, date available for use and quantity. The COA is examined for reagents and placed in the applicable COA binder under that reagent name. These binders are retained per the record retention procedure. Once this is completed the reagent is released from quarantine and placed in the applicable area. If the reagent needs refrigerated or is to remain frozen, it is placed in the applicable storage environment. FDA approved reagents are used if available per the Material Management program at General BioTechnology. c. Determination of Removal of Reagents from Final Product

[0307] No testing for residual reagents will be performed. d. Other Concerns

[0308] Penicillin: Penicillin and streptomycin will be used to prevent bacterial contamination of initial MSC cultures; antibiotics will not be used after the first passage. The patient who will receive the MSC generated in this protocol will have no known drug allergies, and thus it will be safe if they receive a small residual amount of penicillin.3. Excipients

[0309] An excipient used in the cry opreservation of the cells is Dimethyl Sulfoxide (DMSO). Each dose of vaccine is cryopreserved using 10% DMSO, or 2 mL of DMSOin a total volume of 10 mL of final product. Infusion of this amount of DMSO is well within the safety parameters for a 30 kg child; Pediatric Stem Cell Transplant SOP states that the maximum dose of DMSO is 15 mg / kg / dose.4. Additional Considerations a. Combination Products

[0310] This IND does not include a device or additional drug as part of the final product.B. Product Manufacturing- Procedures1. Preparation of Vaccines a. Method of Cell Processing / Culture Conditions

[0311] Manufacturing procedures take place in class 10,000 clean production suite. Each technician properly gowns when entering in the GMP room. Before entry into the clean lab area, the technician obtains a bunny suit in the ante room. After the hood of the bunny suit is placed on, a mouth covering is put on, making sure that all hair is fully covered under the hood and mouth covering. The technician puts on a pair of sterile powder free gloves, and enters the clean lab space with the sample. Environmental monitoring is performed in the Class 10,000 clean room. The clean room uses Biological Safety Cabinets (BSC) which maintains a Class 5 environment. BSC are certified annually by an outside qualified vender. Settling plates are performed every time the BSC is in use for processing and evaluated for acceptable criteria based on USP. One settling plate is placed in the BSC during processing for a minimum of 30 minutes. Once per package, as a negative control, one covered settling plate is placed inside the BCS at the same time. After the settling plate is in the BSC, evaluate the plate for presence of bacterial colonies, Colony Forming Units (cfu), by allowing the plate to incubate for 48 hours at 37°C. Levels requiring alert are more than 1 colony per plate. Incubator temperature should be 36-38° C. TSA plates are used to evaluate the wide spectrum of possible bacteria present. Prepared plates are in their original wrapping at 2 - 8°C and are warmed to room temperature prior to use. The product is validated from the time of manufacture to be stable at room temperature (25°C) for 192 h (8 days).

[0312] Additionally the clean room is monitored for room temperature and particle counts. Acceptable room temperature is between 15 and 30 degrees Celsius. A MetOne Aerocet 531 particle counter is used to evaluate the particles in the air. The particle counter is used to detect and count the number of particles found in the air of the clean room. It is used to confirm that the number of loose particles in the air is less than 10,000 0.5 micron particles per ft3. The particle counter is run on a weekly basis in the three major areas of the clean room space. It is run for 30 minutes each in the gowning area, on the counter inside the clean room space and inside the hood. A settle plate is placed each time the particle counter is in use, next to the counter for the 30 minutes it is being run. After each use of the clean room, the BSC is wiped down with 5.25% bleach then followed by a 70% isopropyl alcohol. Countertops inside the clean room space are wiped down with 70% isopropyl alcohol each day. Once a week all surfaces inside the clean room, including floor, are wiped down with enzymatic cleaner LpH using a dry disposable cloth. Yearly, all walls and ceiling are clean with a lint roller, and all soft walls are cleaned with 70% isopropyl alcohol.

[0313] Before laboratory technicians are allowed into the clean room, a gowning competency must be passed. ROD AC plates are utilized to assess the competency of the technician. The acceptable limits of CFU / plate are listed in the table below. This is again repeated annually for all qualified technicians.

[0314]

[0315] The vaccine is prepared from 200 ml of peripheral blood obtained from the patient to be treated. Subsequent to ficoll purification of peripheral blood mononuclear cells, monocytes are isolated by CD14 selection using magnetic activated cell sorting (MACS). CD14 positive cells are cultured for 5 days in 50 ng / ml GM-CSF and 500 ng / ml IL-4, with media changed once every two days. On Day 5 cells are pulsed with 10ug / ml of autologous senescent cell lysate and subsequently transfected with siRNA pool specific for IDO silencing. Transfection is performed using 2 micrograms per ml of siRNA premixed with 5 microliters per ml of GeneSilencer transfection reagent. Procedures from Zheng et al. Int. J. Cancer: 132, 967-977) are applied to human CD14 derived monocytes for the generation of DC, pulsing with tumor antigen from surgical sample, and gene silencing).

[0316] Sequences of siRNA utilized are as follows:

[0317] a) UUAUAAUGACUGGAUGUUC (SEQ ID NO: 1)

[0318] b) GUCUGGUGUAUGAAGGGUU (SEQ ID NO: 2)

[0319] c) CUCCUAUUUUGGUUUAUGC (SEQ ID NO: 3)

[0320] d) GCAGCGUCUUUCAGUGCUU (SEQ ID NO: 4) b. IrradiationCells are not irradiated during any step in the process nor before use. c. Final Harvest

[0321] Cells are re-suspended in ImL of Isolyte S Multi -Electrolyte Solution. Then, 3 milliliters of a 10% DMSO made with Isolyte S is added at a controlled rate over 5 minutes to the cells for a total of 4ml of final product. The cell dose is packaged in a Charter CF-50 freezing bag, placed in a box in box freezing case and put in a validated - 85°C freezer. All processes in the generation, expansion, and product production are performed under conditions and testing that is compliant with current Good Manufacturing Processes and appropriate controls. Guidance issued by the FDA in 1998 Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy, the 2008 Guidance for FDA Reviewers and Sponsors Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs), and the 1993 FDA points-to-consider document for master cell banks are all followed for the generation of the cell products described.2. Process Timing and Intermediate Storage

[0322] The time elapsed from cell collection to storage is variable. A typical sample will take 2 weeks from time of collection until freezing for quarantine. Time cannot becalculated through final harvest because storage time is unknown. Storage time is based on need for the cells.

[0323] The Vaccine samples are stored as frozen cells according to the validated instructions for use for the Charter CF-50 bags. Some data concerning freezing and recovery are presented in the validation procedure for cell products. This is in agreement with other industry standards for storage of cell therapy products. Prior to patient administration, doses will be sent to the administering facility frozen. The dose(s) will be sent in a dry shipper that will continuously monitor the temperature in route.Temperature data from shipment will be downloaded upon return of the dry shipper to GBT. Data will be shared with administering facility upon request. The facility will be responsible for the thawing of the cells. Once the cells are thawed a time limit of 6 hours has been established by which the cells must be administered. The temperature of 4 degrees Celsius must be maintained during storage of the thawed cells prior to administration.3. Final Formulation

[0324] The vaccine will be provided with a certificate of analysis for each batch certifying purity and lack of contaminants according to WHO blood banking regulations and 21CFR1271 regulations. Charter CF-50 bags will be filled with cellular product from cell bank that has been generated and tested. Filling of the bags will be performed with cells previously generated from autologous patients. Cells are resuspended in 4 mL of Isolyte S Multi -Electrolyte Solution (B. Braun Medical) containing 10% DMSO.

[0325] Each Charter CF-50 bag will contain 10 million cells in a volume of 4 ml. Four intradermal injections of 1 ml each (2.5 million cells) will be performed for each injection session.II. PRODUCT TESTINGA. Microbiological Testing1. Sterility Testing (Bacterial and Fungal Testing)

[0326] a. A 2mL aliquot of expended media from the culture is collected and placed into 2 SPS collection tubes (each tube containing ImL of the expended media). One tube is labeled for “Aerobic” and one tube is labeled for “Anaerobic” with a uniqueidentifier for the sample. Samples are shipped to LABS, Inc for sterility testing. The USP / CFR 610.12 GMP (BASIC STERILITY) testing method is used for sterility. Bacteriostatic / Fungistatic activity uses the direct inoculation method.

[0327] b. Cultures are incubated at LABS, Inc for two weeks for sterility screeningwe received results within 3 weeks of shipment.2. Mycoplasma

[0328] In process testing will be completed using expended media from the dendritic cell cultures will be tested for mycoplama using the Lonza Lucetta™ Luminometer with MycoAlert® Mycoplasma Detection Assay at General BioTechnology. MCB and final release testing is completed at Labs, Inc. Testing at Labs, Inc will test for the presence of agar cultivable and non-agar cultivable mycoplasma.C. Purity1. Residual Contaminants

[0329] The final product contains dendritic cell senescent vaccine are-suspended in Isolyte S Multi -Electrolyte Solution (B. Braun Medical, Irvine, CA) with 10% dimethyl sulfoxide (DMSO) as a cryopreservative. Any small amounts of medium which may remain in the product are safe for infusion. No testing for residual reagents is performed. Testing is performed using the US Pharmacopeia standard for basic sterility USP / CFR 610.12 GMP.

[0330] Flow cytometry is used to validate the potency by marking CD83 expression (>80%)2. Pyrogenicity / Endotoxin

[0331] A 2mL aliquot of expended media from the culture is shipped to LABS, Inc for endotoxin testing. The USP / CFR 610.12 GMP (BASIC STERILITY) testing method is used for sterility.D. Potency

[0332] Potency is tested by ability to stimulate mixed lymphocyte reaction.

[0333] III. FINAL PRODUCT RELEASE CRITERIA TESTING

Claims

WHAT IS CLAIMED IS:

1. A method for reducing senescent cells in a mammal experiencing senescence with immune competency comprising: immunizing said mammal with immune competency with a vaccine composition capable of stimulating immunity towards senescent cells and / or antigens of senescent cells in an amount sufficient in reducing senescent cells in said mammal.

2. The method of claim 1, wherein said senescent cells are host cells from said mammal treated with a genotoxin.

3. The method of claim 2, wherein said genotoxin is a chemotherapy or a radiotherapy.

4. The method of claim 1, wherein said senescence is associated with production of enhanced levels of TNF-alpha, YLK-40, IL-11 or IL-23 receptor as compared to a nonsenescent cell.

5. The method of claim 1, wherein said senescent cells are autologous expanded fibroblasts treated with a genotoxin and lysate of said senescent cells is administered to an antigen presenting cell.

6. The method of claim 5, wherein said antigen presenting cell is selected from the group consisting of: a) neutrophils; b) monocytes; c) macrophages; d) endothelial cells; and e) dendritic cells.

7. The method of claim 6, wherein said antigen presenting cell is treated with an inhibitor of an immunological checkpoint.

8. The method of claim 7, wherein said immunological checkpoint is indolamine 2,3 dioxygenase.

9. The method of claim 8, wherein said immunological checkpoint is suppressed by administration of one or more agents capable of inducing RNA interference and / or gene editing.

10. The method of claim 8, wherein said antigen presenting cell is a dendritic cell.

11. The method of claim 10, wherein said dendritic cell is activated.

12. The method of claim 11, wherein said dendritic cell is activated by contact with a toll like receptor agonists.

13. The method of claim 12, wherein said toll like receptor is selected from the group consisting of: a) TLR2; b) TLR3; c) TLR4; d) TLR5; e)TLR 7 / 8; and f) TLR9.

14. The method of claim 12, wherein said TLR agonist is selected from the group consisting of: a) LPS; b) Poly IC; c) beta glucan; and d) neutrophil extracellular traps.

15. The method of claim 1, wherein said senescence is associated with enhanced angiogenesis and said vaccine concurrently targets angiogenic antigens.

16. The method of claim 1, wherein said vaccine composition capable of stimulating immunity towards senescent cells contains beta galactosidase as well as senescent endothelial cells.

17. The method of claim 16, wherein said endothelial cells are derived from the umbilical vein.

18. The method of claim 16, wherein said umbilical vein cells express CD 133.

19. The method of claim 16, wherein said umbilical vein cells express CD34.

20. The method of claim 16, wherein said umbilical vein cells express c-kit.

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