# US 2021/0275591 A1 · ALS Treatment Using Induced Regulatory T (iTREG) Cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0275591 A1 - Document ID: US20210275591A1 - Status: Published application - Kind: A1 - Application number: 17/320,891 - Filed: 2021-05-14 - Published: 2021-09-09 - Earliest claimed: 2018-11-16 - Assignees: Rapa Therapeutics, LLC - Applicants: Rapa Therapeutics, LLC - Inventors: Daniel Harding FOWLER - Web page: https://patentgrep.com/patent/US20210275591A1 ## CPC - A61K 31/513: having oxo groups directly attached to the heterocyclic ring, e.g. cytosine - A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)} - A61K 31/675: having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate - A61K 31/7056: containing five-membered rings with nitrogen as a ring hetero atom - A61K 38/1793: {for cytokines; for lymphokines; for interferons} - A61K 39/0008: {Antigens related to auto-immune diseases; Preparations to induce self-tolerance} - A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells - A61K 40/22: Immunosuppressive or immunotolerising - A61K 40/414: {Nervous system antigens} - A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia - C12N 5/0637: {Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg} - G01N 33/5088: {of vertebrates} - A61K 2039/577: {tolerising response} - A61K 2239/31: characterized by the route of administration - A61K 2239/38: characterised by the dose, timing or administration schedule - A61K 2239/47: Brain; Nervous system - C12N 2500/38: Vitamins - C12N 2501/04: Immunosuppressors, e.g. cyclosporin, tacrolimus - C12N 2501/06: Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide - C12N 2501/15: Transforming growth factor beta (TGF-β) - C12N 2501/165: Vascular endothelial growth factor [VEGF] - C12N 2501/2302: Interleukin-2 (IL-2) - C12N 2501/2304: Interleukin-4 (IL-4) - C12N 2501/51: B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand) - C12N 2501/515: CD3, T-cell receptor complex - C12N 2501/999: Small molecules not provided for elsewhere - C12N 2506/11: from blood or immune system cells - G01N 2333/46: from vertebrates - G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present disclosure provides methods for treating ALS using pentostatin and cyclophosphamide treatment followed by TREG and/or TREG/Th2 hybrid cells from de-differentiated T cells. The present disclosure further provides methods for producing TREG and TREG/Th2 hybrid cells from de-differentiated T cells, said TREG and TREG/Th2 hybrid cells, populations thereof and compositions thereof. Methods for producing de-differentiated T cells, said de-differentiated T cells, populations thereof and compositions thereof are also provided. ## Claims ### Claim 1 A method for treating amyotrophic lateral sclerosis in a subject in need thereof, comprising: subjecting said subject to one or more primary treatment cycles, each of said one or more primary treatment cycles comprising: administering to said subject pentostatin; and/or administering to said subject cyclophosphamide; and subjecting said subject to one or more immune therapy treatment cycles comprising: administering to said subject a composition comprising a therapeutically effective amount of manufactured TREG cells. ### Claim 2 (depends on 1) The method of claim 1, wherein each of said one or more immune therapy treatment cycles further comprise administering to said subject a nucleoside reverse transcriptase inhibitor. ### Claim 3 (depends on 2) The method of claim 2, wherein said nucleoside reverse transcriptase inhibitor is an inhibitor of the NLRP3 inflammasome. ### Claim 4 (depends on 2) The method of claim 2, wherein said nucleoside reverse transcriptase inhibitor is lamivudine. ### Claim 5 (depends on 1) The method of claim 1, wherein each of said one or more primary treatment cycles is between 2 weeks and 5 weeks long. ### Claim 6 (depends on 1, 2, 3, 4, 5) The method of any one of claims 1-5, wherein each of said one or more immune therapy treatment cycles further comprise: administering to said subject pentostatin; and/or administering to said subject cyclophosphamide. ### Claim 7 (depends on 2) The method of claim 2, wherein each of said one or more immune therapy treatment cycles is 18 weeks long. ### Claim 8 (depends on 1) The method of claim 1, wherein pentostatin is administered to said subject at a dose of between 0.5 mg/m2 and 4 mg/m2. ### Claim 9 (depends on 1) The method of claim 1, wherein said pentostatin is administered on day 1 or days 1 and 4 of each of said one or more primary treatment cycles. ### Claim 10 (depends on 1) The method of claim 1, wherein a dose of said cyclophosphamide is between 50 mg and 400 mg. ### Claim 11 (depends on 1) The method of claim 1, wherein said cyclophosphamide is administered on days 1, 2 and 3 or days 1, 2, 3, 4, and 5 of each of said one or more primary treatment cycles. ### Claim 12 (depends on 1) The method of claim 1, wherein said method comprises two or more primary treatment cycles, wherein each of said two or more primary treatment cycles is separated by 0 to 2 weeks. ### Claim 13 (depends on 2) The method of claim 2, wherein a first of said one or more immune therapy treatment cycles and a last of said one or more primary treatment cycles are separated by 0 to 2 weeks. ### Claim 14 (depends on 4) The method of claim 4, wherein said lamivudine is administered to said subject at a dose between 150 mg daily and 150 mg twice daily. ### Claim 15 (depends on 1) The method of claim 1, wherein said pentostatin and cyclophosphamide are administered to said subject in a single composition. ### Claim 16 (depends on 1) The method of claim 1, wherein said steps of administering to said subject pentostatin and cyclophosphamide comprise: administering to said subject a first composition comprising pentostatin; and administering to said subject a second composition comprising cyclophosphamide. ### Claim 17 (depends on 14) The method of claim 14, wherein said single composition is administered to said subject intravenously. ### Claim 18 (depends on 6) The method of claim 6, wherein said step of administering to said subject pentostatin during each of said one or more immune therapy treatment cycles is performed on days 1 and 4 of each of said one or more immune therapy treatment cycles. ### Claim 19 (depends on 6, 18) The method of claim 6 or claim 18, wherein said step of administering to said subject pentostatin during each of said one or more immune therapy treatment cycles is performed on days 1, 2, 3, 4 and 5 of each of said one or more immune therapy treatment cycles. ### Claim 20 (depends on 6, 18, 19) The method of any one of claims 6 and 18-19, wherein pentostatin is administered to said subject at a dose of between 1 mg/m2 and 2 mg/m2 during each of said one or more immune therapy treatment cycles. ### Claim 21 (depends on 20) The method of claim 20, wherein pentostatin is administered to said subject at a dose of 2 mg/m2 during each of said one or more immune therapy treatment cycles. ### Claim 22 (depends on 6, 18, 19, 20, 21) The method of any one of claims 6 and 18-21, wherein cyclophosphamide is administered to said subject at a dose of between 100 mg and 200 mg during each of said one or more immune therapy treatment cycles. ### Claim 23 (depends on 22) The method of claim 22, wherein cyclophosphamide is administered to said subject at a dose of 100 mg. ### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) The method of any one of claims 1-23, wherein said manufactured TREG cells are administered to said subject on day 8 of each of said one or more immune therapy treatment cycles. ### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) The method of any one of claims 1-24, wherein said method comprises two or more immune therapy treatment cycles and each of said two or more immune therapy treatment cycles are separated by 0 to 4 weeks. ### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) The method of any one of claims 1-24, wherein said method comprises two or more immune therapy treatment cycles and each of said two or more immune therapy treatment cycles are separated by at least 4 weeks. ### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) The method of any one of claims 1-26, wherein said manufactured TREG cells are administered to said subject during each of said one or more immune therapy treatment cycles at a dose between 1×106 cells per kg of the subject's body weight and 5×106 cells per kg of the subject's body weight. ### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) The method of any one of claims 1-27, wherein said manufactured TREG cells comprise a ratio of central memory to effector memory cells selected from 1:1, 3:1, 10:1, 1:3 and 1:10. ### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) The method of any one of claims 1-28, wherein said composition comprising manufactured TREG cells further comprises natural thymus-derived TREG cells. ### Claim 30 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) The method of any one of claims 1-29, further comprising, prior to said one or more primary treatment cycles: harvesting peripheral lymphocytes from said subject. ### Claim 31 (depends on 30) The method of claim 30, further comprising, after harvesting peripheral lymphocytes from said subject: culturing said peripheral lymphocytes to yield said manufactured TREG cells. ### Claim 32 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) The method of any one of claims 1-31, further comprising, after said one or more primary treatment cycles: harvesting peripheral lymphocytes from said subject. ### Claim 33 (depends on 32) The method of claim 32, further comprising, after harvesting peripheral lymphocytes from said subject after said one or more primary treatment cycles: culturing said peripheral lymphocytes to yield said manufactured TREG cells. ### Claim 34 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) The method of any one of claims 1-33, further comprising, after each of said one or more primary treatment cycles: measuring absolute lymphocyte count (ALC) in said subject and, if ALC<750 per μl proceeding to said one or more immune therapy treatment cycles. ### Claim 35 A method for the treatment of amyotrophic lateral sclerosis in a subject in need thereof, comprising: a first treatment cycle, a second treatment cycle, optionally, one or more additional treatment cycles, and one or more immune therapy treatment cycles, said first treatment cycle comprising: administering pentostatin to said subject, and/or administering cyclophosphamide to said subject; said second treatment cycle comprising: administering pentostatin to said subject, and/or administering cyclophosphamide to said subject; each of said one or more additional treatment cycles comprising: administering pentostatin to said subject, and/or administering cyclophosphamide to said subject each of said one or more immune therapy treatment cycles comprising: administering pentostatin to said subject, and/or administering cyclophosphamide to said subject, and administering manufactured TREG cells to said subject. ### Claim 36 (depends on 35) The method of claim 35, wherein said step of administering pentostatin to said subject is performed on day 1 of said first treatment cycle. ### Claim 37 (depends on 36) The method of claim 36, wherein pentostatin is administered to said subject at a dose of 1 mg/m2 during said first treatment cycle. ### Claim 38 (depends on 35, 36, 37) The method of any one of claims 35-37, wherein said step of administering cyclophosphamide to said subject is repeated during said first treatment cycle. ### Claim 39 (depends on 38) The method of claim 38, wherein said step of administering cyclophosphamide to said subject is performed on days 1, 2, and 3 of said first treatment cycle. ### Claim 40 (depends on 35, 36, 37, 38, 39) The method of any one of claims 35-39, wherein cyclophosphamide is administered to said subject at a dose of 100 mg during said first treatment cycle. ### Claim 41 (depends on 35) The method of claim 35, wherein said first treatment cycle is 14 days long. ### Claim 42 (depends on 35) The method of claim 35, wherein said second treatment cycle is 14 days long. ### Claim 43 (depends on 35) The method of claim 35, wherein each of said one or more additional treatment cycles are each 14 days long. ### Claim 44 (depends on 35) The method of claim 35, wherein said first treatment cycle, said second treatment cycle and each of said one or more additional treatment cycles are separated by 0 to 2 weeks. ### Claim 45 (depends on 35) The method of claim 35, further comprising prior to administering pentostatin to said subject in said first treatment cycle: measuring the creatinine clearance (CrCl) of said subject and adjusting a dose of pentostatin to be administered to said subject based on the CrCl, wherein pentostatin is administered at 1 mg/m2 when CrCl≥60 mL/min/1.73 m2, wherein pentostatin is administered at 0.5 mg/m2 when 60 mL/min/1.73 m2>CrCl≥30 mL/min/1.73 m2, and wherein pentostatin is not administered when CrCl<30 mL/min/1.73 m2. ### Claim 46 (depends on 35) The method of claim 35, further comprising prior to administering cyclophosphamide to said subject in said first treatment cycle: measuring absolute neutrophil count (ANC) and adjusting a dose of cyclophosphamide to be administered to said subject based on the ALC and ANC, wherein cyclophosphamide is administered at a dose of 100 mg when ANC>1000, wherein cyclophosphamide is administered at a dose of 50 mg when ANC is 500-999, and wherein cyclophosphamide is not administered when ANC<500. ### Claim 47 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46) The method of any one of claims 35-46, wherein said step of administering pentostatin to said subject during said second treatment cycle is performed on day 1 of said second treatment cycle. ### Claim 48 (depends on 35) The method of claim 35, wherein pentostatin is administered to said subject at a dose of 2 mg/m2 during said second treatment cycle. ### Claim 49 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48) The method of any one of claims 35-48, wherein said step of administering cyclophosphamide to said subject during said second treatment cycle is repeated during said second treatment cycle. ### Claim 50 (depends on 49) The method of claim 49, wherein said step of administering cyclophosphamide to said subject during said second treatment cycle is performed on days 1, 2, and/or 3 of said second treatment cycle. ### Claim 51 (depends on 35, 36, 37, 38, 39, 40) The method of any one of claims 35-40, wherein cyclophosphamide is administered to said subject at a dose of 100 mg during said second treatment cycle. ### Claim 52 (depends on 35) The method of claim 35, further comprising prior to administering pentostatin to said subject during said second treatment cycle: measuring the creatinine clearance (CrCl) of said subject and adjusting a dose of pentostatin to be administered to said subject based on the CrCl, wherein pentostatin is administered at 2 mg/m2 when CrCl≥60 mL/min/1.73 m2, wherein pentostatin is administered at 1 mg/m2 when 60 mL/min/1.73 m2>CrCl>30 mL/min/1.73 m2, and wherein pentostatin is not administered when CrCl<30 mL/min/1.73 m2. ### Claim 53 (depends on 35) The method of claim 35, further comprising prior to administering cyclophosphamide to said subject during said second treatment cycle: measuring absolute neutrophil count (ANC) and adjusting a dose of cyclophosphamide to be administered to said subject based on the ANC, wherein cyclophosphamide is administered at a dose of 100 mg when ANC>1000, wherein cyclophosphamide is administered at a dose of 50 mg when ANC is 500-999, and wherein cyclophosphamide is not administered when ANC<500. ### Claim 54 (depends on 35) The method of claim 35, further comprising, prior to each of said one or more additional treatment cycles: measuring absolute lymphocyte count (ALC) in said subject and adjusting the treatment of said subject based on the ALC, wherein if ALC<750, administering to said subject a maintenance treatment cycle comprising subjecting said patient to said one or more immune therapy treatment cycles, and wherein if ALC>750 subjecting patient to said one or more additional treatment cycles. ### Claim 55 (depends on 54) The method of claim 54, wherein each of said one or more immune therapy treatment cycles are 18 weeks long. ### Claim 56 (depends on 54) The method of claim 54, wherein ALC<750, and wherein said manufactured TREG cells are administered to said subject a dose between 1×106 cells per kg of the subject's body weight and 5×106 cells per kg of the subject's body weight during each of said one or more immune therapy cycles. ### Claim 57 (depends on 56) The method of claim 56, wherein said manufactured TREG cells are administered to said subject on day 8 of each of said one or more immune therapy treatment cycles. ### Claim 58 (depends on 35) The method of claim 35, wherein pentostatin is administered to said subject on days 1 and 4 of each of said one or more immune therapy treatment cycles at a dose of 2 mg/m2. ### Claim 59 (depends on 35) The method of claim 35, wherein cyclophosphamide is administered to said subject on days 1, 2, 3, 4, and/or 5 of each of said one or more immune therapy treatment cycles at a dose of 100 mg. ### Claim 60 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) The method of any one of claims 35-53, wherein each of said one or more immune therapy treatment cycles further comprises: administering to said subject a nucleoside reverse transcriptase inhibitor. ### Claim 61 (depends on 60) The method of claim 60, wherein said nucleoside reverse transcriptase inhibitor is lamivudine. ### Claim 62 (depends on 61) The method of claim 61, wherein said lamivudine is administered to said subject during each of said one or more immune therapy treatment cycles at a dose of 150 mg daily to 150 mg twice daily. ### Claim 63 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62) The method of any one of claims 35-62, wherein said subject is subjected to said one or more additional treatment cycles. ### Claim 64 (depends on 63) The method of claim 63, wherein said step of administering pentostatin to said subject during each of said one or more additional treatment cycles is performed on days 1 and/or 4 of said one or more additional treatment cycles. ### Claim 65 (depends on 64) The method of claim 64, wherein pentostatin is administered to said subject at a dose of 2 mg/m2 during each of said one or more additional treatment cycles. ### Claim 66 (depends on 63, 64, 65) The method of any one of claims 63-65, wherein said step of administering cyclophosphamide to said subject is repeated during each of said one or more additional treatment cycles. ### Claim 67 (depends on 66) The method of claim 66, wherein said step of administering cyclophosphamide to said subject during each of said one or more additional treatment cycles is performed on days 1, 2, 3, 4 and/or 5 of each of said one or more additional treatment cycles. ### Claim 68 (depends on 63, 64, 65, 66, 67) The method of any one of claims 63-67, wherein cyclophosphamide is administered to said subject at a dose of 100 mg to 200 mg during each of said one or more additional treatment cycles. ### Claim 69 (depends on 63) The method of claim 63, further comprising prior to administering pentostatin to said subject during each of said one or more additional treatment cycles: measuring the creatinine clearance (CrCl) of said subject and adjusting a dose of pentostatin to be administered to said subject based on the CrCl, wherein pentostatin is administered at 2 mg/m2 when CrCl≥60 mL/min/1.73 m2, wherein pentostatin is administered at 1 mg/m2 when 60 mL/min/1.73 m2>CrCl≥30 mL/min/1.73 m2, and wherein pentostatin is not administered when CrCl<30 mL/min/1.73 m2. ### Claim 70 (depends on 63) The method of claim 63, further comprising prior to administering cyclophosphamide to said subject during each of said one or more additional treatment cycles: measuring absolute neutrophil count (ANC) and adjusting a dose of cyclophosphamide to be administered to said subject based on ANC, wherein cyclophosphamide is administered at a dose of 100 mg when ANC>1000, wherein cyclophosphamide is administered at a dose of 50 mg when ANC is 500-999, and wherein cyclophosphamide is not administered when ALC<50 or ANC<500. ### Claim 71 (depends on 63) The method of claim 63, wherein said one or more additional treatment cycles comprise at least two additional treatment cycles, a final of said at least two additional treatment cycles comprising prior to administering cyclophosphamide to said subject during said final of said at least two treatment cycles: measuring absolute lymphocyte count (ALC) and absolute neutrophil count (ANC) and adjusting a dose of cyclophosphamide to be administered to said subject based on the ALC and ANC, wherein cyclophosphamide is administered at a dose of 200 mg when ALC≥1250, wherein cyclophosphamide is administered at a dose of 100 mg when ANC>1000 and 750750 continuing to subject patient to said one or more additional treatment cycles. ### Claim 74 (depends on 73) The method of claim 73, wherein each of said one or more immune therapy treatment cycles is 4 weeks long. ### Claim 75 (depends on 60, 61) The method of any one of claims 60-61, further comprising during each of said one or more immune therapy treatment cycles: measuring the creatinine clearance (CrCl) of said subject and adjusting a dose of lamivudine to be administered to said subject based on the CrCl, wherein lamivudine is administered at 150 mg twice daily when CrCl≥50 mL/min/1.73 m2, wherein lamivudine is administered at 150 mg daily when 50 mL/min>CrCl≥30 mL/min/1.73 m2, and wherein lamivudine is not administered when CrCl<30 mL/min/1.73 m2. ### Claim 76 A method for treating amyotrophic lateral sclerosis in a subject in need thereof, comprising: one or more treatment cycles comprising: administering to said subject a therapeutically effective amount of manufactured TREG cells. ### Claim 77 (depends on 76) The method of claim 76, wherein said manufactured TREG cells are administered at a dose of 1×106 cells per kg of the subject's body weight and 5×106 cells per kg of the subject's body weight. ### Claim 78 (depends on 76) The method of claim 76, wherein said manufactured TREG cells comprise a ratio of central memory to effector memory cells selected from 1:1, 3:1, 10:1, 1:3 and 1:10. ### Claim 79 (depends on 76) The method of claim 76, wherein each of said one or more treatment cycles further comprise: administering to said subject pentostatin; and/or administering to said subject cyclophosphamide. ### Claim 80 (depends on 79) The method of claim 79, wherein pentostatin is administered to said subject at a dose of between 1 mg/m2 and 2 mg/m2. ### Claim 81 (depends on 79) The method of claim 79, wherein a dose of said cyclophosphamide is between 100 mg and 200 mg. ### Claim 82 (depends on 79) The method of claim 79, wherein said pentostatin is administered to said subject on days 1 and 4 of each of said one or more treatment cycles. ### Claim 83 (depends on 79) The method of claim 79, wherein said cyclophosphamide is administered to said subject on days 1, 2, 3, 4 and/or 5 of each of said one or more treatment cycles. ### Claim 84 (depends on 79) The method of claim 79, wherein said subject was previously treated with pentostatin and cyclophosphamide. ### Claim 85 (depends on 76, 77, 78, 79, 80, 81, 82, 83, 84) The method of any one of claims 76-84, wherein each of said one or more treatment cycles further comprises administering to said subject a nucleoside reverse transcriptase inhibitor. ### Claim 86 (depends on 85) The method of claim 85, wherein said nucleoside reverse transcriptase inhibitor is an inhibitor of the NLRP3 inflammasome. ### Claim 87 (depends on 86) The method of claim 86, wherein said nucleoside reverse transcriptase inhibitor is lamivudine. ### Claim 88 (depends on 87) The method of claim 87, wherein said lamivudine is administered to said subject at a dose between 150 mg daily and 150 mg twice daily. ### Claim 89 (depends on 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88) The method of any one of claims 76-88, further comprising administering to said subject normal TREG cells simultaneously with said manufactured TREG cells. ### Claim 90 (depends on 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89) The method of any one of claims 76-89, wherein each of said one or more treatment cycles is 4 weeks apart. ### Claim 91 (depends on 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90) The method of any one of claims 76-90, further comprising, prior to said one or more treatment cycles: harvesting peripheral lymphocytes from said subject. ### Claim 92 (depends on 91) The method of claim 91, further comprising, after harvesting peripheral lymphocytes from said subject: culturing said peripheral lymphocytes to yield said manufactured TREG cells. ### Claim 93 A method for treating amyotrophic lateral sclerosis in a subject in need thereof, comprising: administering to said subject a therapeutically effective amount of manufactured TREG cells. ### Claim 94 (depends on 93) The method of claim 93, wherein said manufactured TREG cells are administered at a dose of 1×106 cells per kg of the subject's body weight and 5×106 cells per kg of the subject's body weight. ### Claim 95 (depends on 93) The method of claim 93, wherein said manufactured TREG cells comprise a ratio of central memory to effector memory cells selected from 1:1, 3:1, 10:1, 1:3 and 1:10. ### Claim 96 (depends on 93) The method of claim 93, further comprising, prior to administering said manufactured TREG cells to said subject: administering to said subject pentostatin; and/or administering to said subject cyclophosphamide. ### Claim 97 (depends on 96) The method of claim 96, wherein pentostatin is administered to said subject at a dose of between 0.5 mg/m2 and 4 mg/m2. ### Claim 98 (depends on 96) The method of claim 96, wherein a dose of said cyclophosphamide is between 50 mg and 400 mg. ### Claim 99 (depends on 93) The method of claim 93, wherein said subject was previously treated with pentostatin and cyclophosphamide. ### Claim 100 (depends on 93, 94, 95, 96, 97, 98, 99) The method of any one of claims 93-99, further comprising administering to said subject a nucleoside reverse transcriptase inhibitor. ### Claim 101 (depends on 100) The method of claim 100, wherein said nucleoside reverse transcriptase inhibitor is an inhibitor of the NLRP3 inflammasome. ### Claim 102 (depends on 101) The method of claim 101, wherein said nucleoside reverse transcriptase inhibitor is lamivudine. ### Claim 103 (depends on 102) The method of claim 102, wherein said lamivudine is administered to said subject at a dose between 150 mg daily and 150 mg twice daily. ### Claim 104 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103) The method of any one of claims 93-103, further comprising administering to said subject normal TREG cells simultaneously with said manufactured TREG cells. ### Claim 105 (depends on 93, 94, 95) The method of any one of claims 93-95, further comprising, prior to administering said manufactured TREG cells to said subject: harvesting peripheral lymphocytes from said subject; and culturing said peripheral lymphocytes to yield said manufactured TREG cells. ### Claim 106 (depends on 1, 105) The method of any one of claims 1-105, further comprising administering to said subject an anti-TNF-α therapy. ### Claim 107 A method for differentiating de-differentiated T cells to TREG/Th2 cells, comprising: culturing de-differentiated T cells in a culture medium comprising IL-2, IL-4 and TGF-β; adding anti-CD3/anti-CD28 coated magnetic beads at a ratio of 3:1 (bead:T cell ratio); incubating said de-differentiated T cells for a period of time to yield TREG/Th2 cells. ### Claim 108 (depends on 107) The method of claim 107, wherein said culture medium further comprises pemetrexed. ### Claim 109 (depends on 107, 108) The method of any one of claims 107-108, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU/mL. ### Claim 110 (depends on 107, 108, 109) The method of any one of claims 107-109, wherein said IL-4 is present in said culture medium at a concentration of about 1000 IU/mL. ### Claim 111 (depends on 107, 108, 109, 110) The method of any one of claims 107-110, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng/mL. ### Claim 112 (depends on 108) The method of claim 108, wherein said pemetrexed is present in said culture medium at a concentration of up to 100 nM. ### Claim 113 (depends on 108) The method of claim 108, wherein said pemetrexed is present in said culture medium at a concentration of about 10 nM. ### Claim 114 (depends on 112, 113) The method of any one of claims 112-113, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU/mL. ### Claim 115 (depends on 112, 113, 114) The method of any one of claims 112-114, wherein said IL-4 is present in said culture medium at a concentration of about 1000 IU/mL. ### Claim 116 (depends on 112, 113, 114, 115) The method of any one of claims 112-115, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng/mL. ### Claim 117 A method for differentiating de-differentiated T cells to TREG/Th2 cells, comprising: culturing de-differentiated T cells, wherein said de-differentiated T cells express RAPTOR and RICTOR at a level that is at least 10% reduced relative to control T cells in a culture medium comprising IL-2, IL-4 and TGF-β; adding anti-CD3/anti-CD28 coated magnetic beads at a ratio of 3:1 (bead:T cell ratio); incubating said de-differentiated T cells for a period of time to yield TREG/Th2 cells. ### Claim 118 (depends on 117) The method of claim 117, wherein said culture medium further comprises pemetrexed. ### Claim 119 (depends on 117, 118) The method of any one of claims 117-118, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU/mL. ### Claim 120 (depends on 117, 118, 119) The method of any one of claims 117-119, wherein said IL-4 is present in said culture medium at a concentration of about 1000 IU/mL. ### Claim 121 (depends on 117, 118, 119, 120) The method of any one of claims 117-120, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng/mL. ### Claim 122 (depends on 118) The method of claim 118, wherein said pemetrexed is present in said culture medium at a concentration of up to 100 nM. ### Claim 123 (depends on 118) The method of claim 118, wherein said pemetrexed is present in said culture medium at a concentration of about 10 nM. ### Claim 124 (depends on 122, 123) The method of any one of claims 122-123, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU/mL. ### Claim 125 (depends on 122, 123, 124) The method of any one of claims 122-124, wherein said IL-4 is present in said culture medium at a concentration of about 1000 IU/mL. ### Claim 126 (depends on 122, 123, 124, 125) The method of any one of claims 122-125, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng/mL. ### Claim 127 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126) The method of any one of claims 107-126, wherein said culture medium is X-Vivo 20 supplemented with 5% human AB serum. ### Claim 128 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127) The method of any one of claims 107-127, wherein said period of time is between 3 days and 40 days. ### Claim 129 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128) A TREG/Th2 cell produced by the method of any one of claims 107-128. ### Claim 130 A method for differentiating de-differentiated T cells to TREG cells, comprising: culturing de-differentiated T cells having reduced expression of RAPTOR and RICTOR relative to a control population of T cells in a culture medium comprising IL-2 and TGF-β; adding anti-CD3/anti-CD28 coated magnetic beads at a ratio of 3:1 (bead:T cell ratio); incubating said de-differentiated T cells for a period of time to yield TREG cells. ### Claim 131 (depends on 130) The method of claim 130, wherein said culture medium further comprises pemetrexed. ### Claim 132 (depends on 130, 131) The method of any one of claims 130-131, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU/mL. ### Claim 133 (depends on 130, 131, 132) The method of any one of claims 130-132, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng/mL. ### Claim 134 (depends on 131) The method of claim 131, wherein said pemetrexed is present in said culture medium at a concentration of up to 100 nM. ### Claim 135 (depends on 131) The method of claim 131, wherein said pemetrexed is present in said culture medium at a concentration of about 10 nM. ### Claim 136 (depends on 134, 135) The method of any one of claims 134-135, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU/mL. ### Claim 137 (depends on 134, 135, 136) The method of any one of claims 134-136, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng/mL. ### Claim 138 A method for differentiating de-differentiated T cells to TREG cells, comprising: culturing de-differentiated T cells, wherein said de-differentiated T cells express RAPTOR and RICTOR at a level that is at least 10% reduced relative to control T cells in a culture medium comprising IL-2 and TGF-β; adding anti-CD3/anti-CD28 coated magnetic beads at a ratio of 3:1 (bead:T cell ratio); incubating said de-differentiated T cells for a period of time to yield TREG cells. ### Claim 139 (depends on 138) The method of claim 138, wherein said culture medium further comprises pemetrexed. ### Claim 140 (depends on 138, 139) The method of any one of claims 138-139, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU/mL. ### Claim 141 (depends on 138, 139, 140) The method of any one of claims 138-140, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng/mL. ### Claim 142 (depends on 139) The method of claim 139, wherein said pemetrexed is present in said culture medium at a concentration of up to 100 nM. ### Claim 143 (depends on 139) The method of claim 139, wherein said pemetrexed is present in said culture medium at a concentration of about 10 nM. ### Claim 144 (depends on 142, 143) The method of any one of claims 142-143, wherein said IL-2 is present in said culture medium at a concentration of about 100 IU/mL. ### Claim 145 (depends on 142, 143, 144) The method of any one of claims 142-144, wherein said TGF-β is present in said culture medium at a concentration of about 10 ng/mL. ### Claim 146 (depends on 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145) The method of any one of claims 130-145, wherein said culture medium is X-Vivo 20 media supplemented with 5% AB serum. ### Claim 147 (depends on 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146) The method of any one of claims 130-146, wherein said period of time is between 3 days and 40 days. ### Claim 148 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147) The method of any one of claims 107-147, wherein said de-differentiated T cells have a reduced expression of RAPTOR and RICTOR relative to a control population of T cells ### Claim 149 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148) A TREG cell produced by the method of any one of claims 107-128 and 130-148. ### Claim 150 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148) A TREG cell or hybrid TREG/Th2 cell produced by the method of any one of claims 107-128 and 130-148, wherein lymphocytes collected by apheresis for subsequent T cell culture are obtained in the steady-state or after subject treatment with an anti-TNF-α therapeutic that is relatively selective in terms of neutralizing the serum, cell-free form of TNF-α, most notably the recombinant receptor molecule etanercept or the monoclonal antibody adalimumab. ### Claim 151 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148) A TREG cell or hybrid TREG/Th2 cell produced by the method of any one of claims 107-128 and 130-148, wherein the TREG cell or hybrid TREG/Th2 cell or a population therof has increased expression by flow cytometry of at least one the following molecules relative to control Th1/Tc1 cells: CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, OX40, and combinations thereof. ### Claim 152 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148) A TREG cell or hybrid TREG/Th2 cell produced by the method of any one of claims 107-128 and 130-148, wherein the TREG cell or hybrid TREG/Th2 cell or a population thereof has reduced secretion of inflammatory cytokines relative to control Th1/Tc1 cells, including IFN-γ and TNF-α. ### Claim 153 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148) A TREG cell or hybrid TREG/Th2 cell produced by the method of any one of claims 107-128 and 130-148, wherein said TREG cell or hybrid TREG/Th2 cell or a population thereof has altered expression of T cell fate transcription factors relative to control Th1/Tc1 cells, most notably a decrease in TBET and an increase in FOXP3. ### Claim 154 (depends on 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148) A TREG cell or hybrid TREG/Th2 cell produced by the method of any one of claims 107-128 and 130-148, wherein said TREG cell or hybrid TREG/Th2 cell or a population thereof has additional phenotypic traits relative to control Th1/Tc1 cells, including: increased secretion of the Th2 cytokine IL-4; and increased expression of the Th2 transcription factor GATA3. ### Claim 155 A population of TREG or TREG/Th2 cells having at least 5% of CD4+ or CD8+ T cells that express GATA3. ### Claim 156 A population of TREG or TREG/Th2 cells having at least 5% of CD4+ or CD8+ T cells that express FoxP3 ### Claim 157 A population of TREG or TREG/Th2 cells having at least 10% of CD4+ or CD8+ T cells that express CD73. ### Claim 158 A population of TREG or TREG/Th2 cells having at least 10% of CD4+ or CD8+ T cells that express CD103. ### Claim 159 A population of TREG or TREG/Th2 cells having at least 20% of CD4+ or CD8+ T cells that express CD150. ### Claim 160 A population of TREG or TREG/Th2 cells that expresses at least 5 pg/mL of IL-4 after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1. ### Claim 161 A population of TREG or TREG/Th2 cells that expresses at least 100 pg/mL of IL-2 after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1. ### Claim 162 A population of TREG or TREG/Th2 cells that expresses less than 100 μg/mL of IFN-γ or GM-CSF after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1. ### Claim 163 A population of TREG or TREG/Th2 cells that expresses less than 100 μg/mL of TNF-α or IL-17F after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1. ### Claim 164 (depends on 1, 106) The method of any one of claims 1-106, wherein the manufactured TREG cells have one or more of the following properties: at least 10% increased expression of one or more of: CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, OXO40, and combinations thereof, as measured by flow cytometry relative to control Th1/Tc1 cells; at least 10% decreased secretion of IFN-γ relative to control Th1/Tc1 cells; at least 10% decreased secretion of TNF-α relative to control Th1/Tc1 cells; at least 10% decreased expression of TBET relative to control Th1/Tc1 cells; at least 10% increased expression of FOXP3 relative to control Th1/Tc1 cells; at least 5% of CD4+ or CD8+ T cells that express GATA3 as measured by flow cytometry; at least 5% of CD4+ or CD8+ T cells that express FOXP3 as measured by flow cytometry; at least 5% of CD4+ or CD8+ T cells that express CD73 as measured by flow cytometry; at least 5% of CD4+ or CD8+ T cells that express CD103 as measured by flow cytometry; at least 5% of CD4+ or CD8+ cells that express both FOXP3 and GATA3 as measured by flow cytometry; at least 20% of CD4+ or CD8+ T cells that express CD150 as measured by flow cytometry; expression of at least 5 pg/mL/1×106 cells/day of IL-4 after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1; expression of at least 100 pg/mL/1×106 cells/day of IL-2 after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1; expression of less than 100 pg/mL/1×106 cells/day of IFN-γ after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1; expression of less than 100 pg/mL/1×106 cells/day of GM-CSF after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1; expression of less than 10 pg/mL/1×106 cells/day of TNF-α after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1; expression of less than 10 pg/mL/1×106 cells/day of IL-17 after co-stimulation with anti-CD3/anti-CD28 beads at a bead:T cell ratio of 3:1; and combinations thereof.