阅读说明：本技术 用于造血干细胞移植的组合物和方法 (Compositions and methods for hematopoietic stem cell transplantation ) 是由 伊凡·K·迪莫夫 纳撒尼尔·弗恩霍夫 凯文·希恩 于 2018-03-15 设计创作，主要内容包括：本公开内容提供了形成在造血干/祖细胞移植中有用的药物组合物的独特的治疗性细胞群。例如,本公开内容提供了治疗性细胞群,其包括造血干/祖细胞、记忆T细胞、调节性T细胞的富集群,并且其中所述细胞群耗尽幼稚常规αβ-T细胞。本公开内容还提供了使用治疗性细胞群的治疗方法。在其他实施方案中,本公开内容提供了产生治疗性细胞群的方法。(The present disclosure provides unique therapeutic cell populations that form pharmaceutical compositions useful in hematopoietic stem/progenitor cell transplantation. For example, the present disclosure provides a therapeutic cell population comprising an enriched population of hematopoietic stem/progenitor cells, memory T cells, regulatory T cells, and wherein the cell population is depleted of naive conventional α β -T cells. The present disclosure also provides methods of treatment using the therapeutic cell populations. In other embodiments, the present disclosure provides methods of generating a therapeutic cell population.)

1. A pharmaceutical composition comprising a therapeutic cell population enriched for hematopoietic stem/progenitor cells (HSPCs), memory T cells (Tmem), and regulatory T cells (tregs), and wherein the cell population is depleted of naive conventional α β -T cells, wherein the pharmaceutical composition comprises a ratio of naive conventional α β -T cells to tregs of less than 1: 5.

2. The pharmaceutical composition of claim 1, further comprising an enriched population of constant natural killer T cells (inkts).

3. The pharmaceutical composition of claim 1 or 2, comprising one or more unit doses of a cell graft, wherein each unit dose of the cell graft comprises a therapeutic cell population per kilogram (kg) of body weight of a subject receiving the cell graft, and wherein each unit dose of the therapeutic cell population comprises:

more than 3x 10⁵Hematopoietic stem/progenitor cells (HSPC),

more than 3x 10⁵A memory T cell (Tmem),

more than 5x 10⁵A regulatory T cell (Treg), and

less than 3x 10⁵Naive conventional α β -T cells.

4. The pharmaceutical composition of claim 3, wherein the unit dose further comprises more than 0.5x 10³And (4) an iNKT cell.

5. The pharmaceutical composition of any one of claims 1-4, wherein each unit dose of the therapeutic cell population comprises:

1.0x 10⁶to 50x 10⁶Hematopoietic stem/progenitor cells (HSPC),

0.3x 10⁶to 1000x 10⁶A memory T cell (Tmem),

0.5x 10⁶to 1000x 10⁶A regulatory T cell (Treg), and

less than 3x 10⁵Naive conventional α β -T cells.

6. The pharmaceutical composition of any one of the preceding claims, wherein the unit dose further comprises a concentration of 0.5x 10³To 2000x 10³Constant natural killer T cells (iNKT) per kg of cells.

7. The pharmaceutical composition of any one of the preceding claims, wherein the tregs comprise naive populations of tregs, memory populations of tregs, or both.

8. The pharmaceutical composition of any one of the preceding claims, wherein the Tmem comprises a T central memory cell (T)_CM) Population, T-effector memory cells (T)_EM) A population, or any combination thereof.

9. The pharmaceutical composition according to any one of the preceding claims, comprising T central memory stem cells (T)_SCM) And (4) clustering.

10. The pharmaceutical composition according to any of the preceding claims, wherein the HSPC is CD34⁺。

11. The pharmaceutical composition according to any of the preceding claims, wherein the HSPC is CD133⁺、CD90⁺、CD38^-、CD45RA^-、Lin^-Or any of themAnd what combinations are.

12. The pharmaceutical composition according to any of the preceding claims, wherein the HSPCs are cKIT⁺。

13. The pharmaceutical composition according to any of the preceding claims, wherein the HSPC is CD19^-、TCRα^-Or a combination thereof.

14. The pharmaceutical composition of any one of the preceding claims, wherein the Tmem is CD3⁺、CD45RA^-、CD45RO⁺Or any combination thereof.

15. The pharmaceutical composition according to any of the preceding claims, wherein the Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺Or any combination thereof.

16. The pharmaceutical composition according to any of the preceding claims, wherein the naive Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺、CD45RA⁺、CD45RO^-Or any combination thereof.

17. The pharmaceutical composition according to any of the preceding claims, wherein the memory Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺、CD45RA^-、CD45RO⁺Or any combination thereof.

18. The pharmaceutical composition of any one of the preceding claims, wherein the T is_SCMIs CD45RA⁺And CD4⁺Or CD8⁺。

19. The pharmaceutical composition of any one of the preceding claims, wherein the T is_SCMIs CD95⁺、CD122⁺、CXCR3⁺、LFA-1⁺Or any combination thereof.

20. The pharmaceutical composition of any one of the preceding claims, wherein the T is_CMIs CD45RO⁺And CD4⁺Or CD8⁺。

21. The pharmaceutical composition of claim 20, wherein said T is_CMIs CD45RA^-、CD62L⁺、CCR7⁺Or any combination thereof.

22. The pharmaceutical composition of any one of the preceding claims, wherein the T is_EMIs CD4⁺、CD45RO⁺、CD45RA^-、CD62L^-、CCR7^-Or any combination thereof.

23. The pharmaceutical composition of any one of the preceding claims, wherein the iNKT is CD1d-tet⁺、6B11⁺Or both.

24. The pharmaceutical composition of any one of the preceding claims, wherein the iNKT is va24ja18⁺。

25. The pharmaceutical composition according to any one of the preceding claims, wherein the naive conventional α β -T cell is CD25^-、CD127⁺Or both, and TCR α⁺And CD45RA⁺。

26. The pharmaceutical composition according to any of the preceding claims, wherein the naive conventional α β -T cell is TCR α⁺TCRβ⁺CD45RA⁺CD45RO^-CD25^-CD95^-IL-2Rβ^-CD127⁺。

27. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of HSPC to Tmem is 500:1 to 1:1,000.

28. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of HSPCs to tregs is about 100:1 to about 1: 30.

29. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of HSPCs to naive tregs is from 1:500 to 100: 1.

30. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of HSPCs to memory tregs is from 1:500 to 10,000: 1.

31. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of HSPC to iNKT is 1:2 to 500,000: 1.

32. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of naive conventional α β -T cells to HSPCs is less than 1:3, preferably less than 1: 400.

33. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of naive conventional α β -T cells to Tmem is less than 1:30, preferably less than 1: 800.

34. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of naive conventional α β -T cells to naive tregs is less than 1:1, preferably less than 1: 10.

35. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of naive conventional α β -T cells to memory Tregs is less than 1:1, preferably less than 1: 100.

36. The pharmaceutical composition according to any of the preceding claims, wherein the ratio of naive conventional α β -T cells to iNKT is less than 100:1, preferably less than 1: 1.

37. The pharmaceutical composition according to any one of the preceding claims, wherein the ratio of Tmem to Treg is from 2000:1 to 1:10, preferably from 30:1 to 1: 1.

38. The pharmaceutical composition according to any one of the preceding claims, wherein the ratio of Tmem to naive Treg is from 3:1 to 0.1: 1.

39. The pharmaceutical composition according to any one of the preceding claims, wherein the ratio of Tmem to memory Treg is from 27:1 to 0.9: 1.

40. The pharmaceutical composition of any one of the preceding claims, wherein the ratio of iNKT to Tmem is from about 5:1 to about 1:1,000,000.

41. The pharmaceutical composition of any one of the preceding claims, wherein the unit dose comprises 1.0x10⁶To 50x 10⁶And (5) HSPC.

42. The pharmaceutical composition of any one of the preceding claims, wherein the unit dose comprises 0.3x 10⁶To 1000x 10⁶A memory T cell.

43. The pharmaceutical composition of any one of the preceding claims, wherein the unit dose comprises 0.5x 10⁶To 1000x 10⁶And (4) Treg cells.

44. The pharmaceutical composition of any one of the preceding claims, wherein the unit dose comprises 0.2x 10⁶To 500x 10⁶Naive Treg cells.

45. The pharmaceutical composition of any one of the preceding claims, wherein the unit dose comprises 0.5x 10⁶To 500x 10⁶And (4) memory Treg cells.

46. According to any one of the preceding claimsThe pharmaceutical composition of (a), wherein the unit dose comprises 0.5x 10³To 2000x 10³And (4) an iNKT cell.

47. The pharmaceutical composition of any one of the preceding claims, wherein the unit dose comprises less than 3x 10⁵Naive conventional alpha-T cells.

48. The pharmaceutical composition of any one of the preceding claims, wherein the population of cells is provided by treating one or more tissue harvests.

49. The pharmaceutical composition of claim 48, wherein the one or more tissue harvests are from one or more donors.

50. The pharmaceutical composition of claim 48 or 49, wherein the tissue harvest is from an HLA matched sibling donor, an HLA matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA mismatched allogeneic donor, a donor pool, or any combination thereof.

51. The pharmaceutical composition of any one of the preceding claims, wherein the HSPCs are provided by a donor with a haplotype identity to the subject.

52. The pharmaceutical composition of any one of the preceding claims, wherein the Treg, Tmem, iNKT, or any combination thereof is provided by a donor that is an HLA-matched sibling donor or an HLA-matched unrelated donor.

53. The pharmaceutical composition of any one of the preceding claims, wherein the cells are formulated for infusion or injection.

54. The pharmaceutical composition of any one of the preceding claims, wherein the population of cells comprises a formulation for administration to a subject.

55. The pharmaceutical composition of claim 54, wherein the formulation comprises Normosol-R and human serum.

56. The pharmaceutical composition of claim 54 or 55, wherein the human serum comprises 1% of the total formulation.

57. Use of a pharmaceutical composition according to any one of the preceding claims for the treatment of a disease or disorder.

58. The use of claim 57, wherein the disease or disorder is leukemia, lymphoma, chronic infection or autoimmune disease, hematological malignancies, malignant or non-malignant, AML, ALL, CML, CLL, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, MDS, lymphoproliferative diseases, type 1 diabetes, congenital metabolic abnormalities, genetic diseases, sickle cell anemia, beta-thalassemia, multiple sclerosis, solid organ transplantation, Crohn's disease, ulcerative colitis, lupus, hemophagocytic lymphohistiocytosis, glycogen storage disease, mucopolysaccharidosis or any other disease that would benefit from HSPC transplantation.

59. The use of any one of claims 57 or 58, wherein each therapeutic cell population is administered to the subject as a separate pharmaceutical composition.

60. The use of any one of claims 57 or 58, wherein the therapeutic cell population is administered to the subject as a single pharmaceutical composition.

61. The use of any one of claims 57-60, wherein the cells are isolated from a donor that is an HLA matched sibling donor, an HLA matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA mismatched donor, a donor pool, or any combination thereof.

62. The use of any one of claims 57-61, wherein the therapeutic cell population is allogeneic or autologous.

63. The use of any one of claims 57-62, wherein the therapeutic cell population is autologous.

64. The use of any one of claims 57-63, wherein the therapeutic cell population is haploidentical.

65. The use of any one of claims 57-64, wherein the therapeutic cell population is isolated from mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, non-mobilized apheresis product, or any combination thereof.

66. The use of any one of claims 57-65, wherein the therapeutic cell population results from a single tissue harvest.

67. The use of any one of claims 57-66, wherein the therapeutic cell population is derived from one or more tissue harvests.

68. The use of any one of claims 57-67, wherein the therapeutic cell population comprises HSPCs provided by at least a first donor and Tregs and Tmem provided by at least a second donor.

69. The use of claim 68, wherein the therapeutic cell population comprises iNKT cells provided by at least the second donor.

70. The use of claim 68 or 69, wherein the first donor is haploidentical with the subject.

71. The use of any one of claims 68-70, wherein the second donor is an HLA matched sibling donor or an HLA matched or partially matched unrelated donor.

72. The use of any one of claims 57-71, wherein the subject is a human, a non-human primate, a cow, a horse, a sheep, a goat, a pig, a dog, a cat, a mouse, a rabbit, a rat, or a guinea pig.

73. A method of producing the pharmaceutical composition of any one of the preceding claims, comprising processing at least one sample to provide:

a. an enriched population of hematopoietic stem/progenitor cells (HSPCs);

b. an enriched population of regulatory T cells (tregs);

c. an enriched population of memory T cells (Tmem); and

d. formulating said enriched population of HSPCs, memory T cells and tregs as a pharmaceutical composition suitable for administration to a subject, wherein the population of a-c depletes naive conventional α β -T cells.

74. The method of claim 73, further comprising treating the sample to provide depleted Lin⁺A cell population of cells.

75. The method of claim 73 or 74, wherein providing the enriched population of Tregs comprises providing an enriched population of naive Tregs, an enriched population of memory Tregs, or both.

76. The method of any one of claims 73-75, wherein providing the enriched population of Tmem comprises providing enriched T central memory cells (T)_CM) Population, enriched T effector memory cells (T)_EM) A population, or any combination thereof.

77. The method of any one of claims 73-76, further comprising processing the sample to provide an enriched iNKT cell population.

78. The method according to any one of claims 73-77, wherein the enriched population of HSPCs comprises at least 50% CD34⁺HPSC。

79. The method of any one of claims 73-78, wherein the depleted Lin⁺The cell population of the cells comprises 1-30% Lin⁺Cells, preferably less than 1% Lin⁺A cell.

80. The method according to any one of claims 73-79, wherein the enriched population of Tregs comprises 20% -99.9% Tregs.

81. The method of any one of claims 73-80, wherein the enriched population of Tmem comprises 10% -99.9% Tmem.

82. The method of any one of claims 73-81, wherein the enriched population of iNKTs comprises 10% -99.9% iNKTs.

83. The method of any one of claims 73-82, wherein formulating the pharmaceutical composition comprises combining the enriched population of HSPCs, memory T cells, Tregs, iNKT cells, or any combination thereof into a mixed population of enriched cells.

84. The method of claim 83, wherein the mixed population of enriched cells comprises a ratio of HSPC to memory T cells of 500:1 to 1:1,000.

85. The method of claim 83 or 84, wherein the mixed population of enriched cells comprises a ratio of HSPC to naive Treg of 1:500 to 100: 1.

86. The method according to any one of claims 83-85, wherein the mixed population of enriched cells comprises a ratio of HSPC to memory Treg of 1:500 to 10,000: 1.

87. The method of any one of claims 83-86, wherein the mixed population of enriched cells comprises a ratio of HSPCs to inkts from 1:2 to 500,000: 1.

88. The method according to any one of claims 83-87, wherein the mixed population of enriched cells comprises a ratio of naive conventional α β -T cells to HSPCs of less than 1:3, preferably less than 1: 400.

89. The method according to any one of claims 83-88, wherein the mixed population of enriched cells comprises a ratio of naive conventional α β -T cells to Tmem of less than 1:30, preferably less than 1: 800.

90. The method according to any one of claims 83-89, wherein the mixed population of enriched cells comprises a ratio of naive conventional α β -T cells to naive Tregs of less than 1:1, preferably less than 1: 10.

91. The method according to any one of claims 83-90, wherein the mixed population of enriched cells comprises naive conventional α β -T cells to memory Tregs in a ratio of less than 1:1, preferably less than 1: 100.

92. The method of any one of claims 83-91, wherein the mixed population of enriched cells comprises naive conventional α β -T cells to iNKT at a ratio of less than 100:1, preferably less than 1: 1.

93. The method of any one of claims 83-92, wherein the mixed population of enriched cells comprises a Tmem to Treg ratio of 30:1 to 1: 1.

94. The method of any one of claims 83-93, wherein the mixed population of enriched cells comprises a ratio of Tmem to naive tregs in the range of 3:1 to 0.1: 1.

95. The method of any one of claims 83-94, wherein the mixed population of enriched cells comprises a ratio of Tmem to memory tregs in the range of 27:1 to 0.9: 1.

96. The method of any one of claims 83-95, wherein the mixed population of enriched cells comprises less than 0.0014% naive conventional α β -T cells.

97. The method of any one of claims 74-96, wherein Lin⁺The cell expresses CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

98. The method according to any one of claims 73-97, wherein the HSPCs are CD34 +.

99. The method according to any one of claims 73-98, wherein the HSPCs are CD 19-and TCR α/β -.

100. The method of any one of claims 73-99, wherein the HSPCs are CD133+, CD90+, CD38-, CD45RA-, Lin-, or any combination thereof.

101. The method of any one of claims 73-100, wherein the Tmem is CD45RA^-、CD45RO⁺Or any combination thereof.

102. The method of claim 101, wherein the Tmem is T_CM。

103. The method of claim 102, wherein the T is_CMIs CD45RO⁺And CD4⁺Or CD8⁺。

104. The method of claim 102 or 103, wherein said T is_CMIs CD45RA^-、CD62L⁺、CCR7⁺Or any combination thereof.

105. The method of claim 101, wherein the Tmem is T_EM。

106. The method of claim 105, wherein the T is_EMIs CD4⁺、CD45RA⁺、CD45RO^-、CD62L^-、CCR7^-Or any combination thereof.

107. The method of any one of claims 73-106, wherein the Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺Or any combination thereof.

108. The method of claim 107, wherein the Treg is a naive Treg, a memory Treg, or both.

109. The method of claim 108, wherein the naive Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺、CD45RA⁺、CD45RO^-Or any combination thereof.

110. The method of claim 108, wherein the memory Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺、CD45RA^-、CD45RO⁺Or any combination thereof.

111. The method of any one of claims 73-110, wherein the sample comprises mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, non-mobilized apheresis product, or any combination thereof.

112. The method of any one of claims 73-111, wherein the sample is prepared for processing a density gradient, Ficoll, Percoll, erythrocytic hypotonic lysis, ammonium chloride-potassium (ACK) buffer, or any combination thereof.

113. The method of any one of claims 73-111, wherein the sample is provided by a single tissue harvest.

114. The method of any one of claims 73-111, wherein the sample is provided by one or more tissue harvests.

115. The method of any one of claims 73-114, wherein the enriched population of cells is provided by density separation, tetrameric antibody complex-mediated enrichment/depletion, magnetically activated cell sorting, multi-parameter fluorescence based molecular phenotype, or any combination thereof.

116. The method of any one of claims 73-115, comprising:

A. contacting the sample with a molecule that specifically binds CD34 under conditions to form CD34⁺Cell population and CD34^-A population of cells, recovering said CD34 from said sample⁺A population of cells and recovering said CD34 from said sample^-A population of cells; and

B. treating said CD34^-The cell population to provide at least one enriched therapeutic cell population comprising tregs, Tmem, iNKT, or any combination thereof.

117. The method of claim 116, wherein step B comprises performing fine sorting to provide the enriched population of therapeutic cells.

118. The method of claim 116 or 117, wherein step B comprises contacting the CD34^-The population of cells is associated with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, a polypeptide,CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof.

119. The method of claim 116, wherein step B comprises:

i. under conditions such that said CD34^-Contacting the population of cells with at least one molecule that specifically binds to CD45RA to form CD45RA⁺Cell population and CD45RA^-(ii) a population of cells, and recovering said CD45RA^-A population of cells; and

performing fine sorting to separate from the CD45RA⁺Providing the enriched therapeutic cell population in a cell population.

120. The method of claim 119, wherein the fine sorting comprises subjecting the CD45RA to⁺The population of cells is contacted with a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, a CD1d-tet, a 6B11 monoclonal antibody, or a functional fragment thereof, or any combination thereof.

121. The method of claim 120, wherein the fine sorting further comprises subjecting the CD45RA to^-The population of cells is contacted with a molecule that specifically binds CD45RA, a molecule that specifically binds CD45RO, or a combination thereof.

122. The method of claim 116, wherein step B comprises:

i. under conditions such that said CD34^-The cell population is combined with at least one specific binding Lin⁺Binding molecule contact of markers to form Lin⁺Cell population and Lin^-Cell population and recovering the Lin^-A population of cells; and

performing fine sorting to separate from the Lin^-Providing the enriched therapeutic cell population in a cell population.

123. The method of claim 122, wherein the Lin⁺The marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

124. The method of claim 122 or 123, wherein the fine sorting comprises subjecting the Lin^-The population of cells is contacted with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, a CD1d-tet molecule, a 6B11 monoclonal antibody, or a functional fragment thereof, or any combination thereof.

125. The method of claim 116, wherein step B comprises:

i. under conditions such that said CD34^-The cell population is specifically combined with at least one Lin⁺Binding molecule contact of markers to form Lin⁺Cell population and Lin^-Cell population and recovering the Lin^-A population of cells; and

subjecting the Lin to conditions^-Contacting the population of cells with a binding molecule that specifically binds to CD25 to form CD25⁺Cell population and CD25^-A cell population, recovering said CD25⁺A population of cells, thereby generating a population of cells comprising tregs, and recovering said CD25^-A population of cells; and

subjecting said CD25 to conditions^-Contacting the population of cells with a binding molecule that specifically binds to CD45RA to form CD45RA⁺Cell population and CD45RA^-(ii) a population of cells, and recovering said CD45RA^-A population of cells.

126. The method of claim 125, wherein the Lin⁺The marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

127. The method of claim 125 or 126, wherein step ii^-Cells and CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or group thereofAnd contacting to form CD1d-tet⁺Cell population, 6B11⁺Cell population or combination thereof and CD1d-tet^-Cell, 6B11^-A population of cells or a combination thereof, and recovering said CD1d-tet⁺A population of cells.

128. The method as set forth in any one of claims 119-127, wherein the CD25 is recovered simultaneously⁺A cell population, and said CD1d-tet⁺Cell, 6B11⁺A cell or a population of both.

129. The method as recited in any one of claims 125-128, further comprising subjecting the CD25 of step ii to⁺The population of cells performs fine sorting to provide a naive population of Treg cells, a memory population of Treg cells, an iNKT cell population, or any combination thereof.

130. The method as set forth in any one of claims 125-129, wherein the CD45RA is recovered⁺A population of cells, and further performing fine sorting to provide a population of tregs, inkts, or both.

131. The method of any one of claims 117-130, wherein the fine sorting comprises purification using a multi-parameter fluorescence based molecular phenotype.

132. The method of any one of claims 73-115, comprising:

A. performing a first rough sort on at least a first sample, thereby providing enriched CD34⁺A population of cells;

B. performing a second rough sort on a second sample, thereby providing Lin^-A population of cells;

C. for the Lin^-The cell population was subjected to a third rough sort to provide CD45RA^-Memory T cell population and provide CD45RA⁺A population of cells; and

D. for the CD45RA⁺The population of cells performs fine sorting, thereby providing a population of tregs.

133. The method of claim 132, wherein the second sample comprises CD34 recovered from step a^-A population of cells.

134. The method of claim 132 or 133, wherein the first sample comprises a sample of at least one haplotype identity.

135. The method of claim 132 or 133, wherein the first sample comprises at least two haploidentical samples.

136. The method of any one of claims 132-135, wherein the first sample comprises mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, unmoved apheresis product, or any combination thereof.

137. The method of any one of claims 132-136, wherein the second sample comprises Peripheral Blood Mononuclear Cells (PBMCs), mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, non-mobilized apheresis product, or any combination thereof.

138. The method of any one of claims 132-137, wherein the first, second, or third rough sorting comprises density separation, tetrameric antibody complex-mediated enrichment/depletion, magnetically activated cell sorting, apheresis, leukopheresis, or any combination thereof.

139. The method of any one of claims 132-138, wherein the fine sorting comprises purification using a multi-parameter fluorescence based molecular phenotype.

140. The method of any one of claims 132-139, wherein the fine sorting provides a naive Treg cell population, a memory Treg cell population, an iNKT cell population, or any combination thereof.

141. The method of any one of claims 132-140 wherein the fine sorting comprises subjecting the CD45RA to conditions⁺Contacting a cell with a binding molecule that specifically binds to CD25 to provide CD25⁺Cell population and CD25^-Cell population and recovering CD25⁺A population of cells, thereby providing a population of tregs.

142. The method of claim 141, wherein said CD25 is further sorted under conditions⁺A population of cells to provide a population of naive Treg cells.

143. The method of any one of claims 132-142 wherein the fine sorting comprises subjecting the CD45RA to conditions⁺Contacting the cell with a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof to provide a CD1d-tet⁺Cell, 6B11⁺Cells or both, and recovering said CD1d-tet⁺Cell, 6B11⁺A cell or a population of both.

144. The method of any one of claims 132-143, wherein the first sample or the second sample is allogeneic, autologous, or a combination thereof.

145. The method of any one of claims 132-144, wherein the second sample is from an HLA-matched unrelated donor, an HLA-matched sibling donor, or a combination thereof.

146. The method as recited in any one of claims 132-145, wherein the Lin is⁺The marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

147. The method of any one of claims 73-115, comprising:

A. contacting the sample with specifically bound Lin under conditions⁺Binding molecule contacting of marker to provide Lin⁺Cell population and Lin^-Cell population and recovering the Lin^-A population of cells; and

B. under certain conditions, the Lin is enabled to be^-Contacting a cell with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25 to provide CD34⁺Cell population, CD25⁺Cell population and CD34^-CD25^-A cell population, and recovering said CD34⁺Cells and CD25⁺Cells, and recovering said CD34^-CD25^-A population of cells; and

C. under conditions such that said CD34^-CD25^-Contacting the population of cells with a binding molecule that specifically binds to CD45RA to provide CD45RA⁺Cell population and CD45RA^-Cell population and recovering CD45RA^-And (4) clustering.

148. The method of claim 147, wherein the Lin⁺The marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

149. The method of claim 147 or 148, wherein step B further comprises conditioning the Lin under conditions^-Contacting the cell with a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof to provide a CD1d-tet⁺Cell, 6B11⁺A population of cells or a combination thereof, and recovering said CD1d-tet⁺Cell, 6B11⁺A cell or a combination thereof.

150. The method as recited in any one of claims 147-149, further comprising pairing CD34⁺Cell, CD25⁺Cell, CD1d-tet⁺Cell, 6B11⁺The cells, or any combination thereof, perform fine sorting, thereby providing CD34⁺Cell, CD25⁺Cell, CD1d-tet⁺Cell, 6B11⁺A cell population of cells or any combination thereof.

151. The method of claim 150, wherein the fine sorting comprises subjecting the CD34 to conditions⁺Cell, CD25⁺Cell, CD1d-tet⁺Cell, 6B11⁺Contacting a cell or any combination thereof with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, to provide a highly enriched CD34⁺Cell, CD4⁺CD25⁺CD127^-/LoCell, CD1d-tet⁺A cell population of cells or a combination thereof.

152. The method of any one of claims 73-115, comprising:

A. performing a rough sort on the sample to provide Lin⁺Cell population and Lin^-Cell population and recovering the Lin^-A population of cells; and

B. for the Lin^-The cell population was roughly sorted to provide a population enriched for HSPC and Tmem and CD45RA⁺(ii) a population of cells and recovering said population of HSPC and Tmem and recovering said CD45RA⁺A population of cells; and

C. for the CD45RA⁺The population of cells is subjected to fine sorting to provide a population of tregs.

153. The method of claim 152, wherein the fine sorting further comprises conditioning the CD45RA under conditions⁺Contacting the population of cells with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, and a binding molecule that specifically binds to CD127 to provide CD34⁺Cell population and CD4⁺CD25⁺CD127^-/LoCell population and recovering CD34⁺Cell population and CD4⁺CD25⁺CD127^-/LoA population of cells.

154. The method of claim 152-153 wherein the fine sorting further comprises subjecting the CD45RA to⁺Contacting the population of cells with CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, and recovering the CD1d-tet⁺Cell, 6B11⁺A population of cells or a combination thereof.

155. The method of any one of claims 152-154, wherein the rough sorting comprises density separation, tetrameric antibody complex-mediated enrichment/depletion, magnetically activated cell sorting, apheresis, leukopheresis, or any combination thereof.

156. The method of any one of claims 152-155, wherein the fine sorting comprises purification using a multi-parameter fluorescence based molecular phenotype.

157. The method of any one of claims 73-115, comprising:

A. contacting the sample with a binding molecule that specifically binds to CD34 under conditions to provide CD34⁺Cell population and CD34^-A cell population, recovering said CD34⁺A cell population, and recovering said CD34^-A population of cells; and

B. under conditions such that said CD34^-Contacting the population of cells with a binding molecule that specifically binds to CD25 to provide CD25⁺Cell population and CD25^-A cell population, recovering said CD25⁺A cell population, and recovering said CD25^-A population of cells; and

C. under conditions such that said CD25^-Contacting the population of cells with a binding molecule that specifically binds to CD45RA to provide CD45RA⁺Cell population and CD45RA^-Cell population and recovering CD45RA^-A population of cells.

158. The method of claim 157, wherein step B further comprises:

i. under conditions such that said CD34^-Contacting the population of cells with a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof to provide a CD1d-tet⁺Cell population, 6B11⁺Cell population or combination thereof and CD1d-tet^-Cell population, 6B11^-Cell population or combination thereof, and recovering CD1d-tet⁺Cell population, 6B11⁺Cell population or combination thereof, and recovering CD1d-tet^-Cell population, 6B11^-A population of cells, or both, thereby providing an iNKT depleted population of cells; and

contacting the iNKT depleted cell population with a binding molecule that specifically binds CD25 under conditions to provide CD25⁺Cell population and CD25^-A cell population, and recovering said CD25⁺Cell population and recovering CD25^-A population of cells.

159. The method of claim 157, wherein step B comprises contacting the CD34 under conditions such that the CD34 is substantially transparent to light^-Contacting the population of cells with a binding molecule that specifically binds to CD25 and a CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, to provide CD25⁺Cell population and CD1d-tet⁺Cell, 6B11⁺A population of cells or combinations thereof and CD34^-CD25^-iNKT depleted cell population and recovering said CD25⁺Cell population and said CD1d-tet⁺Cell, 6B11⁺A population of cells or combinations thereof, and recovering said CD34^-CD25^-iNKT depleted cell populations.

160. The method of claim 157, wherein step B comprises:

i. under conditions such that said CD34^-Contacting the population of cells with a binding molecule that specifically binds to CD25 and a CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, to provide CD25⁺Cell population and CD1d-tet⁺Cell, 6B11⁺A population of cells or a combination thereof, and CD34^-CD25^-iNKT depleted cell population and recovering said CD25⁺Cell population and said CD1d-tet⁺Cell, 6B11⁺A population of cells or combinations thereof, and recovering said CD34^-CD25^-iNKT depleted cell populations; and

by contacting said cells with a specificityA binding molecule that binds CD4 sexually, a binding molecule that binds CD25 specifically, a binding molecule that binds CD127 specifically, a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or any combination thereof, to said CD25⁺Cell population and said CD1d-tet⁺Cell, 6B11⁺Performing fine sorting of the population of cells or combinations thereof to provide enriched CD4⁺CD25⁺CD127^-/LoCell, CD1d-tet⁺Cell, 6B11⁺A cell population of cells or any combination thereof.

161. The method of claim 160, wherein step i^-The cell population was contacted with an anti-CD 25 antibody comprising a label and a biotinylated 6B11 monoclonal antibody, and 6B 11-biotin was contacted with labeled streptavidin conjugated.

162. The method of claim 161, wherein the label is Phycoerythrin (PE).

163. The method of claim 161 or 162, wherein the streptavidin is conjugated to PE/Cy 7.

164. The method as recited in claim 161-163, wherein the CD34^-The cells are then contacted with anti-label magnetic particles.

165. The method according to claim 164, wherein the anti-label magnetic particles are anti-PE magnetic particles.

166. The method as recited in any one of claims 160-165, wherein magnetic sorting is used to separate CD25⁺Cell sum 6B11⁺A cell.

167. The method of any one of claims 160-166, wherein ii comprises contacting the cell with a binding molecule that specifically binds to CD4 and a binding molecule that specifically binds to CD127 under conditionsTo the CD25⁺Cell sum 6B11⁺The population of cells is subjected to fine sorting to provide enriched CD4⁺CD25⁺CD127^-/LoCell population and enrichment of cells 6B11⁺CD127⁺A cell population of cells, or any combination thereof.

168. The method of claim 167, wherein the CD4 binding molecule is an anti-CD 4PerCP labeled antibody, the CD127 binding molecule is an anti-CD 127APC labeled antibody, or a combination thereof.

169. The method of claim 167 or 168, wherein the fine sorting comprises fluorescence activated cell sorting and detection of PE-labeled CD25, PE/Cy 7-labeled 6B11, PerCP-labeled CD4, and APC-labeled CD 127.

170. The method of claim 157, wherein the CD34 recovered in step a⁺Cell population, said CD25 recovered in step B⁺The cell population, or both, is further processed by fine sorting, which comprises subjecting the CD34 to⁺Cell, said CD25⁺The cell or combination thereof is contacted with a binding molecule that specifically binds CD34, a binding molecule that specifically binds CD127, a binding molecule that specifically binds CD45RA, or any combination thereof.

171. The method of any one of claims 73-115, comprising:

A. contacting the sample with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25 under conditions to provide CD34⁺Cell population, CD25⁺Cell population and CD34^-CD25^-A cell population, recovering said CD34⁺Cell population and said CD25⁺A cell population, and recovering said CD34^-CD25^-A population of cells; and

B. under conditions such that said CD34^-CD25^-Contacting the population of cells with a binding molecule that specifically binds to CD45RA to provide CD45RA⁺Cell population and CD45RA^-(ii) a population of cells, and recovering said CD45RA^-A population of cells.

172. The method of claim 171, wherein step a further comprises contacting the sample with CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof under conditions to provide CD1d-tet⁺Cell population, 6B11⁺A cell population or a combination thereof, and recovering said CD1d-tet⁺Cell population, 6B11⁺A population of cells, or a combination thereof.

173. The method of claim 171 or 172, further comprising performing fine sorting on the population of cells provided in step a by contacting the cells with a binding molecule that specifically binds CD34, a binding molecule that specifically binds CD4, a binding molecule that specifically binds CD25, a binding molecule that specifically binds CD127, CD1d-tet, or any combination thereof, to provide enriched CD34⁺Cell, CD4⁺CD25⁺CD127^-/LoCell, CD1d-tet⁺A cell population of cells or any combination thereof.

174. The method of any one of claims 73-115, wherein the method comprises simultaneously treating the sample to provide an enriched cell population comprising HSPCs, Tmem, naive tregs, memory tregs and comprising less than 5% of undesired cell types.

175. The method of claim 174, wherein the sample is contacted with a binding molecule that specifically binds CD34, a binding molecule that specifically binds CD4, a binding molecule that specifically binds CD8, a binding molecule that specifically binds CD25, a binding molecule that specifically binds CD127, a binding molecule that specifically binds CD45RA, a binding molecule that specifically binds CD45RO, or any combination thereof.

176. The method of claim 175, further comprising contacting the sample with a CD1d-tet, 6B11 monoclonal antibodyContacting the body or a functional fragment thereof, or a combination thereof, and recovering the CD1d-tet⁺Cell population, 6B11⁺A population of cells, or a combination thereof.

177. The method of any one of claims 174-176, wherein the sample is contacted with a binding molecule that specifically binds CD34, and CD34 is recovered⁺A population of cells, thereby generating a population of HSPCs.

178. The method of any one of claims 174-177, wherein the sample is contacted with a binding molecule that specifically binds CD3 and does not bind to a binding molecule that specifically binds CD45RA, a binding molecule that specifically binds CD45RO, or a combination thereof, and CD3 is recovered⁺CD45RA^-CD45RO⁺A population of cells.

179. The method of any one of claims 174-178, wherein the sample is contacted with a binding molecule that specifically binds CD4, a binding molecule that specifically binds CD25, a binding molecule that specifically binds CD127, a binding molecule that specifically binds CD45RA, a binding molecule that specifically binds CD45RO, or any combination thereof, and CD4 is recovered⁺CD25⁺CD127^-/loCD45RA⁺CD45RO^-Cell population, CD4⁺CD25⁺CD127^-/lo CD45RA^-CD45RO⁺A population of cells.

180. The method according to any one of claims 73-179, wherein the therapeutic cell population comprises less than 2% naive conventional α β -T cells.

181. The method of any one of claims 73-180, wherein the specific binding to CD34, Lin⁺The molecule of a marker, CD25, CD45RA, CDR45RO, CD4, CD8, CD127, CD90, CD133, CD38, CD95, CD122, CXCR3, LFA-1, CD62L, CCR7 or any other cellular marker is an antibody or antibody fragment.

182. The method of any one of claims 73-181, wherein the antibody or antibody fragment is conjugated to a fluorescent dye, hapten or magnetic particle.

183. The method of any one of claims 73-182, wherein the cells are freshly isolated.

Background

Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) typically involves the transfer of hematopoietic cells from an immune-compatible healthy person (donor) to a patient following a pretreatment regimen. Healthy Hematopoietic Stem Cells (HSCs) can replace damaged hematopoietic tissues in a patient, and immune cells derived from a particular donor can have therapeutic effects on cancer, infections, and immune diseases. However, current allogeneic HSCT methods either involve heterogeneous mixtures of cells, which include contaminated non-therapeutic cells, or purified but limited cell mixtures, which lack the potential therapeutic benefit of an intact graft. In the former case, although many non-therapeutic cells contaminating the treatment-related cells are harmless, even a small fraction of a particular abnormal cell type can have serious adverse consequences for the recipient. For example, residual tumor cells or teratoma initiating cells contaminating the transplanted cell population can be seeded with a tumor in a patient. In another example, a subpopulation of circulating T cells can cause Graft Versus Host Disease (GVHD), a serious and often fatal complication of allogeneic HSCT. In these cases, the pathology caused by contaminating cells supersedes the therapeutic benefit of other T cells introduced during transplantation. In many cases, allogeneic HSCT is a curative treatment for the underlying condition, however, it is indeed a medical treatment that is harmful to the patient when the contaminated transplanted cells react to their new host environment. Therefore, there is an urgent need for HSC graft compositions with fewer harmful cells and an optimal therapeutic cell mixture.

Disclosure of Invention

In some embodiments disclosed herein are pharmaceutical compositions comprising one or more unit doses of a cell graft, wherein each unit dose of the cell graft comprises a therapeutic population of cells per kilogram (kg) of body weight of a subject receiving the cell graft. In some embodiments, each unit dose of the therapeutic cell population comprises more than 3x 10⁵Individual hematopoietic stem/progenitor cells (HSPC), greater than 3x 10⁵Memory T cell (Tmem), more than 5x 10⁵A regulatory T cell (Treg) and less than 3x 10⁵Naive conventional α β -T cells. In some embodiments, the unit dose further comprises 0.5x 10³To 2000x 10³And (3) constant natural killer T cells (iNKT). In some embodiments, the HSPC is CD34⁺Tmem is CD3⁺CD45RA^-CD45RO⁺Treg is CD4⁺CD25⁺CD127^-/lo、CD45RA⁺Or a combination thereof, and the naive conventional alpha beta-T cell is CD3⁺CD45RA⁺CD25^-Va24Ja18^-. In some embodiments, iNKT is CD3⁺Vα24Jα18⁺。

In some embodiments, the pharmaceutical compositions disclosed herein comprise a therapeutic cell population enriched for HSPCs, memory T cells (Tmem), and tregs, and wherein the cell population is depleted of naive conventional α β -T cells, wherein the therapeutic cell population comprises a ratio of naive conventional α β -T cells to tregs of less than 1: 5. In some embodiments, the pharmaceutical composition further comprises a constant iNKT, and the therapeutic cell population comprises a ratio of naive conventional α β -T cells to iNKT of less than 100: 1. In some embodiments, the therapeutic cell population comprises a ratio of naive conventional α β -T cells to HSPCs of less than 1: 400; and a ratio of naive conventional α β -T cells to Tmem of less than 1: 800. In some embodiments, the therapeutic cell population comprises iNKT and the therapeutic cell population comprises a ratio of naive conventional α β -T cells to HSPCs of less than 1: 400; the ratio of naive conventional α β -T cells to Tmem is less than 1: 800; and the ratio of naive conventional α β -T cells to iNKT is less than 100: 1. In some embodiments, the therapeutic cell population comprises a ratio of naive conventional α β -T cells to HSPCs of less than 1: 400. In some embodiments, the therapeutic cell population comprises a ratio of naive conventional α β -T cells to Tmem of less than 1: 3.

In some embodiments, disclosed herein are methods of treating a disease or disorder comprising administering to a subject in need thereof a therapeutic cell population, wherein the therapeutic cell population comprises a concentration of 1.0x10⁶To 50x 10⁶HSPC at a concentration of 0.1x 10 per kg subject body weight⁶To 1000x 10⁶Tmem at a concentration of 0.1x 10 per kg subject body weight⁶To 1000x 10⁶Tregs at a concentration of less than 3x 10 per kg body weight of the subject⁵Naive conventional α β -T cells per kg body weight of the subject. In some embodiments, the therapeutic cell population further comprises a concentration of 0.5x 10³To 2000x 10³Individual cells/kg subject body weight iNKT cells.

In some embodiments, disclosed herein are methods of producing a pharmaceutical composition, the method comprising processing at least one sample to provide a therapeutic cell population by:

A. contacting the sample with a binding molecule that specifically binds to CD34 under conditions to provide enriched CD34⁺Recovering enriched CD34 from the population of cells and CD34 depleted cell population⁺A population of cells, and recovering a population of cells depleted in CD 34; and

B. contacting a CD34 depleted cell population with a binding molecule that specifically binds CD25 under conditions to provide enriched CD25⁺Recovering enriched CD25 from the population of cells and CD25 depleted cell population⁺A population of cells, and recovering a population of cells depleted in CD 25; and

C. contacting a CD25 depleted cell population with a binding molecule that specifically binds CD45RA under conditions to provide an enriched CD45RA⁺A population of cells and a CD45RA depleted population of cells, and recovering the CD45RA depleted population of cells; and

D. will enrich CD34⁺Population of cells, enrichment CD25⁺The population of cells and the CD45 depleted cell population are formulated as a pharmaceutical composition suitable for administration to a subject.

Drawings

FIGS. 1A-B: FIG. 1A shows a schematic of a method of producing a molded (sculped) graft cell composition. FIG. 1B shows a schematic of a method of producing a sculpted composition of transplanted cells.

FIGS. 2A-B: fig. 2A shows a schematic of a method of producing a sculptured transplanted cell composition. Fig. 2B shows a schematic of a method of producing a sculptured transplanted cell composition.

FIG. 3 shows a schematic of a method of producing a sculpted composition of transplanted cells.

FIG. 4 shows a schematic of a method of producing a sculpted composition of transplanted cells.

FIG. 5 shows a schematic of a method of producing a sculpted composition of transplanted cells.

FIG. 6 shows a schematic of a method of producing a sculpted composition of transplanted cells.

FIG. 7 shows a schematic of a method of producing a sculpted composition of transplanted cells.

FIG. 8 shows a schematic of a method of producing a sculpted composition of transplanted cells.

Fig. 9 shows a schematic of a method of producing a sculpted transplanted cell composition.

FIG. 10 shows a schematic of a method of producing a sculpted composition of transplanted cells.

FIG. 11 shows a schematic of a method of producing a sculpted composition of transplanted cells.

Fig. 12 shows a schematic of a method of producing a sculpted transplanted cell composition.

Fig. 13 shows a schematic of a method of producing a sculpted transplanted cell composition.

FIG. 14 shows a schematic of a method of producing a sculpted transplanted cell composition.

Fig. 15 shows a schematic of a method of producing a sculpted transplanted cell composition.

Fig. 16 depicts a design of experiments used to evaluate the performance of molded cell grafts in comparison to Bone Marrow Transplant (BMT) compositions and Hematopoietic Stem Cell Transplant (HSCT) compositions.

FIGS. 17A-D show the performance of molded cell grafts in comparison to Bone Marrow Transplant (BMT) compositions and Hematopoietic Stem Cell Transplant (HSCT) compositions. Fig. 17A. Kaplan-Meier curves of survival over time. Fig. 17B. Final results assessed as overall survival, relapse, graft failure and GVHD. FIG. 17C. Exemplary photomicrographs of J774 GPF-Luc tumors in spleens dissected from HSCT mice. Fig. 17D. Representative bioluminescent images of the presence of GFP + cells were compared, which were generated using Xenogen IVIS100 on day + 10.

FIG. 18 depicts a Kaplan-Meier curve of survival of mice treated with molded cell grafts produced by magnetic sorting (MACS) and fluorescence activated sorting (FACS) as compared to molded cell grafts produced by MACS alone.

FIG. 19 depicts a Kaplan-Meier curve showing that mice administered a plastic cell graft are CD34 relative to mice administered with T cells including an isolated post-addback⁺Survival of mice with the cell composition.

Figure 20 depicts a Kaplan-Meier curve showing surviving mice in a xenograft model of adoptive transplantation in which cohorts of mice were administered human PBMC or a sculpted cell graft composition derived from human tissue.

FIGS. 21A-B depict the enrichment of regulatory T cells and iNKT cells from Peripheral Blood Mononuclear Cells (PBMCs) using MACS. Figure 21A shows flow cytometry data showing% purity and% yield of tregs in pre-sorted PBMC and enriched populations. Figure 21B shows flow cytometry data showing% purity and% yield of iNKT cells in pre-sorted PBMC and enriched populations.

FIGS. 22A-B depict enrichment of memory T cells from PBMCs using magnetic sorting. FIG. 22A shows CD3 generated by magnetic sorting using PBMCs⁺CD45RO⁺The% yield of cells and the ratio of naive to memory T cells. Figure 22B shows flow cytometry data showing CD45RO and CD45RA cells in pooled PBMC and CD45RA depleted samples.

FIGS. 23A-B depict CD25⁺Magnetic separation of cells. FIG. 23A shows a magnetically separated CD25⁺Flow cytometric analysis of the cells demonstrated efficient separation of CD25+ CD127+ tregs and characterized subpopulations of memory Treg cells and naive Treg cells. Figure 23B shows an exemplary performance of a magnetic separation strategy for tregs and naive tregs from two donors.

Fig. 24A-B depict biotinylation titrations of the 6B11 antibody. Figure 24A shows binding of 6B11.1, 6B11.2, and 6B11.3 biotinylated antibodies to samples from three independent donors as the concentration of 6B11 antibody increased from zero to 1 μ g/μ L. FIG. 24B shows flow cytometric analysis of samples with 6B11 biotin/streptavidin PE-Cy7 staining and CD127APC staining using either no 6B11 control or 6B11 antibody biotinylated with biotin at 100 μ M (condition 1), 250 μ M (condition 2), or 1mM (condition 3).

Detailed Description

In certain embodiments, the present disclosure provides unique therapeutic cell populations that form pharmaceutical compositions useful in hematopoietic stem/progenitor cell transplantation. For example, some embodiments of the present disclosure provide a therapeutic cell population comprising an enriched population of hematopoietic stem/progenitor cells (HSPCs), memory T cells (Tmem), regulatory T cells (tregs), and wherein the cell population is depleted of naive conventional α β -T cells. In some embodiments, the therapeutic cell population further comprises constant natural killer t (inkt) cells. The present disclosure also provides methods of treatment using the therapeutic cell populations. In other embodiments, the present disclosure provides methods of generating a therapeutic cell population.

The therapeutic cell population is shaped to enrich the number of cells with therapeutic benefit and deplete the cell population that is detrimental to the transplant recipient (e.g., induces secondary disease). HSPCs bring short-term benefits to the reconstitution of blood and immune system function, such as restoring red blood cell, platelet and neutrophil counts. Persistent transplantation of HSPCs reconstitutes adaptive immune function by generating a population of T cells and a population of B cells. Donor T cells as a population are both beneficial and disadvantageous for transplantation. Tmem cells provide benefits because Tmem mediates graft anti-infection as well as graft-versus-leukemia effects, with a lower risk of graft-versus-host disease (GVHD). Naive Treg cells and memory Treg cells provide benefits because tregs help prevent graft rejection and GVHD. In contrast, naive conventional T cells are responsible for GVHD and inappropriate immune reconstitution. Thus, reducing or eliminating naive conventional T cells in a shaped graft disclosed herein enhances therapeutic benefit by reducing the incidence of GVHD in the graft recipient.

Definition of

In this specification, unless otherwise indicated, any concentration range, percentage range, proportion range or integer range should be understood to include any integer value within the stated range and, where appropriate, fractions thereof (such as tenths and hundredths of integers). Moreover, unless otherwise indicated, any numerical range recited herein in relation to any physical characteristic, such as polymer subunit, size, or thickness, is to be understood as including any integer within the range. As used herein, the term "about" refers to ± 20% of the indicated range, value, or structure, unless otherwise specified. The term "consisting essentially of …" limits the scope of the claims to the specified materials or steps or to materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. It is to be understood that, as used herein, the terms "a" and "an" refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to refer to one, both, or any combination thereof. As used herein, the terms "comprising," "having," and "including" are used synonymously, and these terms and their variants are intended to be construed as non-limiting.

As used herein, the term "therapeutic cell" refers to a cell that is selected or administered to a subject based on the ability of the cell to provide a therapeutic benefit to the subject. Exemplary therapeutic cells include hematopoietic stem/progenitor cells, memory T cells, regulatory T cells, and constant natural killer T cells. The therapeutic cell population can include more than one type of therapeutic cell, e.g., HSPC, Tmem, Treg, iNKT, or any combination thereof. The therapeutic cell population can comprise essentially a single therapeutic cell type. In the context of a therapeutic cell population, the percentage (%) of therapeutic cells refers to the percentage of cell types contained in a combination or composition of therapeutic cells, where the total number of therapeutic cells adds to 100%, and a particular therapeutic cell type represents a fraction of the total number of therapeutic cells. For example, a therapeutic cell population comprising 30% HSPCs indicates that about 30% of the total therapeutic cell population is HSPCs. The therapeutic cell population may be present in a larger population of cells that have a neutral effect on the subject, and the neutral cells are not included in the calculation of total therapeutic cells.

As used herein, the term "hematopoietic stem/progenitor cell" or "HSPC" refers to a hematopoietic stem cell and/or hematopoietic progenitor cell that expresses a higher level of a phenotypic marker CD34, CD133, CD90, or any combination thereof, relative to other types of hematopoietic cells (e.g., the cells are positive for expression of the phenotypic marker as determined by flow cytometry, western blotting, or other methods known in the art). Furthermore, HSPCs may be negative for expressed markers relative to other types of hematopoietic cells. For example, such markers include CD19, TCR α, TCR β, CD45RA, Lin, CD38, or any combination thereof. Preferably, HSPC is CD34⁺Cells and/or CD19^-TCRα^-. HSPCs can self-renew or differentiate into (i) myeloid progenitor cells, which ultimately produce monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, or dendritic cells; or (ii) lymphoid progenitor cells that ultimately give rise to T cells, B cells, and lymphoid cells known as natural killer cells (NK cells). For a general discussion of hematopoiesis and HSPC differentiation, see Rowe et al, Cell Stem cell.2016.18: 707-720.

As used herein, the term "naive conventional α β -T cells" or "naive Tcon" refers to expressing the phenotypic markers TCR α/β, CD45RA and expressing moderate to high levels of CD127(CD 127)⁺) But not expressing or low expressing CD45RO and CD 25. In some embodiments, the naive Tcon is characterized by expression of a phenotypic marker of the naive Tcon, including TCR αTCR β, CD3, CD4 or CD8, CD62L, CCR7, CD28, CD127 and CD45 RA. In some embodiments, the naive Tcon is CD3⁺CD25^-CD45RA⁺And includes polymorphic TCR α and polymorphic TCR β. In some embodiments, a naive Tcon is not a naive T regulatory cell, as defined herein. Naive Tcon cells do not express the va24J α 18TCR found on iNKT cells.

As used herein, the term "regulatory T cells" or "tregs" refers to a subset of T cells that are capable of suppressing an autoimmune response and express the phenotypic markers CD4, CD25 but do not express or under express CD 127. Tregs also express FOXP3, however, CD127 expression has been demonstrated with CD4⁺CD25⁺FOXP3 expression on cells was negatively correlated and CD4⁺CD25⁺CD127^-/lowThe phenotype is considered to be an acceptable surrogate marker for Treg and is a practical alternative to intracellular staining of FOXP3 (Cozzo C, et al, J Immunol.2003, 12.1.s.; 171: 5678-82; Liu W, et al, J exp.Med.2006.203(7): 1701-1711; Seddiki N, et al, J exp.Med.2006; 203(7): 1693-1700). Tregs may include at least two subclasses, referred to herein as naive tregs and memory tregs.

As used herein, the term "naive Treg" is a regulatory T cell not subject to an antigen, which as a primary cell expresses the phenotypic markers CD4, CD25 and CD45RA, but does not express or under express CD45RO and CD 127. Naive tregs are advantageous because the cells respond to antigens with a higher plasticity than tregs that have undergone antigens. Furthermore, the life span of naive tregs is longer compared to antigen-experienced tregs.

As used herein, the term "memory Treg" is an antigen-experienced regulatory T cell that is capable of providing an inhibitory effect on autoimmunity and expresses the phenotypic markers CD4, CD25 and CD45RO, but does not express or underexpresses CD127 and CD45 RA.

As used herein, the term "memory T cell" or "Tmem" refers to an antigen-experienced T cell that expresses the phenotypic markers TCR α, TCR β, CD3, CD4 or CD8, CD95, and IL-2R β. Memory T cells provide immunity and can be retained in an inactive state for long periods of time. Memory T cellEffector function can be rapidly acquired after antigen attack again. The memory T cell population may include T central memory cells (T)_CM) Subclass and T-effector memory cells (T)_EM) Any combination of subclasses.

As used herein, a "T central memory cell" or a "T_CM"refers to antigen-experienced T cells that express phenotypic markers CD4 or CD8, CD62L, CD45RO, CCR7, IL-2R β, CD28, CD127 and CD95 but do not express or under express CD45RA compared to naive Tcon cells. After antigen re-challenge, central memory T cells can differentiate into T_EMA cell.

As used herein, a "T effector memory cell" or "T_EMBy "is meant antigen-experienced T cells that express phenotypic markers CD4 or CD8, CD45RO, CD127, IL-2R β and CD95 but do not express or under express CD45RA, CD62L, CCR7 and CD 28. T-effector memory cells eventually differentiate and acquire effector functions after being restimulated by antigens.

As used herein, a "T central memory stem cell" or "T_SCMBy "is meant antigen-bearing T cells that express phenotypic markers CD4 or CD8, CD45RA, CD62L, CD95, IL-2R β, CCR7, CXCR3, CD122, and LFA-1. T is_SCMThe cells have the capacity of memorizing T cells, can rapidly obtain effector functions after antigen re-attack, but are T cells_CMThe cells have enhanced stem cell-like properties, such as long-term persistence, as compared to the cells. T is_SCMThe cells can generate a subset of central memory cells, a subset of effector memory cells, and a subset of effector T cells.

As used herein, "constant Natural killer T cells" or "iNKT" are a subset of CD1 d-restricted Natural Killer T (NKT) cells that express a highly conserved α β -T cell receptor in humans, consisting of a V α 24J α 18TCR α chain (referred to herein as "V α 24J α 18 [. alpha. ]⁺"). iNKT cells can be identified by binding to CD1 d-multimers, such as CD1 d-multimers loaded with α -galactosylceramide (GalCer), PBS-57, PBS-44, or other natural or synthetic glycolipids, and can be found as tetramers, dendrimers and other structures, Fc fusions, or any combination thereof. Another method of identification is specificityAn antibody or combination of antibodies that recognizes the V.alpha.24 J.alpha.18 region. Examples include the V α 24 antibody, the J α 18 antibody, or a monoclonal antibody clone 6B11 that specifically binds to a unique region of the V α 24J α 18TCR and can be used to identify iNKT cells (Montoya et al, immunology.2007.122(1): 1-14). In some embodiments, iNKT cells are loaded with CD1 d-trimer glycolipids⁺(CD1d-tet⁺)、6B11⁺Or both. iNKT cells may be interchangeably referred to herein as CD1d-tet⁺、6B11⁺Or V alpha 24J alpha 18⁺A cell. Without wishing to be bound by a particular mechanism, iNKT cells are believed to promote/accelerate the activity of tregs and HSPCs.

As referred to herein, "lineage positive" or "Lin⁺"the cell expresses a phenotypic marker, such as CD19, CD11c, CD66B, CD14, CD20, or any combination thereof. As referred to herein, "lineage negative" or "Lin" as compared to HSPC, Treg, Tmem or iNKT cells^-"cells do not express or express low expression of the phenotypic markers CD19, CD11c, CD66B, CD14, CD20 or any combination thereof. Lin⁺The cells express phenotypic markers present on mature erythroid cells, granulocytes, macrophages, NK cells, and B and T lymphocytes.

As used herein, "sample" refers to a source of cells (e.g., biological tissue) from which a population of cells can be isolated, enriched, or depleted. In some embodiments, the sample is typically untreated or minimally treated in advance. For example, the sample may be mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, unmoved apheresis product, or any combination thereof. In some embodiments, the sample is prepared or minimally processed by treatment with a density gradient, Ficoll, Percoll, hypotonic lysis of red blood cells, ammonium chloride-potassium (ACK) buffer, washing into a pH-balanced isotonic buffer, or any combination thereof. In some embodiments, the sample is provided by a single tissue harvest. In some embodiments, the sample is provided by one or more tissue harvests.

As used herein, "donor" refers to one or more individuals from which a sample is obtained. For example, a donor may refer to a Human Leukocyte Antigen (HLA) matched sibling, an HLA matched unrelated donor, a partially matched unrelated donor, a haplotype identical related donor, an autologous donor, an HLA mismatched allogeneic donor, a pool of donors, or any combination thereof. In some embodiments, the donor may be a subject. "donor tissue" refers to tissue harvested from a donor. The donor tissue may be a sample. The donor tissue is typically of the same species as the subject.

As used herein, "subject" or "recipient" refers to one or more individuals in need of receiving a treatment, therapy, or cell transplant disclosed herein. The subject treatable by the invention is typically a human. However, other subjects include non-human primates, cows, horses, sheep, goats, pigs, dogs, cats, mice, rabbits, rats or guinea pigs. The subject may be male or female and may be of any suitable age, including infant, juvenile, adolescent, adult and geriatric subjects. During and after treatment, the subject becomes the recipient or transplant recipient.

As used herein, "tissue harvesting" refers to the process of collecting donor tissue or samples from a donor. Non-limiting examples of tissue harvesting include collecting bone marrow, peripheral blood, cord blood, etc., from a donor. Tissue harvesting may be performed by any method known in the art.

As used herein, with respect to a cell population or a cell type in a mixture, "enriched" refers to a cell population that has been treated to increase the relative amount or proportion of the enriched cell type relative to other cells in the mixture (e.g., calculating the cell type). Thus, depending on the source of the initial population of cells subjected to the enrichment process, the mixture or composition may comprise 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more (in number or count) of the "enriched" population of cells (relative to the other cells in the mixture). In some embodiments, the enrichment process can result in an "enriched" cell population that is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more relative to other cells in the mixture. For example, in some embodiments, the cell mixture enriched for iNKT cells may comprise about 0.03 to 1% iNKT cells, 0.05 to 0.5% iNKT cells, 0.1 to 1% iNKT cells, or any combination thereof. Exemplary methods of enriching cell populations include Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS).

As used herein, with respect to a cell population or a cell type in a mixture, "depleted" refers to a cell population that has been treated to reduce the relative amount or proportion of the depleted cell type relative to other cells in the mixture (e.g., calculating the cell type). In some embodiments, cells subjected to a depletion process can result in a mixture or composition comprising 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%, 0.0000001%, 0.00000001%, or less (number or count) of "depleted" cell populations. In some embodiments, cells subjected to a depletion process can result in a mixture or composition comprising 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or less depleted population relative to an untreated sample. In some embodiments, after the treatment step of depleting the cell type, the depleted cell type can no longer be detected using conventional methods.

In certain embodiments, the amount of a certain cell type in the mixture is enriched, while the amount of a different cell type is depleted, e.g., enriched CD34⁺Cell depletion of CD34^-A cell.

A cell population that is "positive" for a marker refers to a cell population that stains uniformly above the level found on an isotype control. In some embodiments, a reduction or low expression of one or more markers refers to a loss or measurement of Mean Fluorescence Intensity (MFI) that is at least 1log10 lower compared to a reference control. In some embodiments, increased or high expression of one or more markers refers to an increase or measurement of MFI that is at least 1log10 higher compared to an isotype control or a reference control. In some embodiments, an at least 2-fold increase in MFI relative to a reference population indicates that the cell is positive for expression of the marker. For example, a population of cells positive for a marker may exhibit an MFI that is 2-fold to 4-fold, 4-fold to 10-fold, 10-fold to 100-fold, and 100-fold to 1,000-fold, 1,000-fold to 10,000-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more as compared to an isotype control. In some embodiments, a population of cells positive for one or more markers refers to the percentage of cells expressing the marker, which can be at least 50% cells, 55% cells, 60% cells, 65% cells, 70% cells, 75% cells, 80% cells, 85% cells, 90% cells, 95% cells, and 100% cells, and any between 50% and 100% as compared to a reference population of cells.

A cell population that is "negative" for a marker refers to a cell population that has not been significantly stained with a specific antibody compared to an isotype control. In some embodiments, a decrease in MFI by at least 2-fold relative to a reference population indicates that the cell is negative for expression of the marker. For example, a population of cells that are negative for a marker can exhibit an MFI that is 2-fold to 4-fold, 4-fold to 10-fold, 10-fold to 100-fold lower, as compared to a positive control, as well as 100-fold to 1,000-fold, 1,000-fold to 10,000-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 5,000-fold, 10,000-fold, or less. In some embodiments, reduced or low expression of one or more markers refers to a reduction in the percentage of cells in the population that express the marker as compared to a reference population of cells that is at least 20% cells, 25% cells, 30% cells, 35% cells, 40% cells, 45% cells, 50% cells, 55% cells, 60% cells, 65% cells, 70% cells, 75% cells, 80% cells, 85% cells, 90% cells, 95% cells, and 100% cells, and any between 20% and 100%.

As used herein, "percent purity" or "% purity" refers to the number of target cells multiplied by 100 and then divided by the number of counted cellular events, as measured on a flow cytometer, hemocytometer, coulter counter, microscopy or other cell counting method (target cell number x 100/cell event number).

As used herein, "total percent yield" or "total% yield" refers to the number of target cells after a treatment step multiplied by 100 and then divided by the number of target cells in the initial population (number of target cells after a treatment step x 100/number of target cells in the initial population). "percent yield of treatment step" or "percent yield of treatment step" refers to the number of target cells after a treatment step multiplied by 100 and then divided by the number of target cells in the pretreatment population (number of target cells after a treatment step x 100/number of target cells in the pretreatment population).

As used herein, "rough sorting" refers to a method of enriching or depleting a cell population, wherein the resulting population may comprise at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of a particular cell population, as compared to the starting cell mixture, depending on the source of the initial cell population subjected to rough sorting. Methods for performing rough sorting are well known in the art and may include density separation, apheresis/leukopheresis, tetrameric antibody complex-mediated enrichment/depletion, and Magnetically Activated Cell Sorting (MACS), such asOr EASYSEP^TM/ROBOSEP^TM。

As used herein, "fine sorting" refers to a method of enriching or depleting a cell population, wherein the resulting population may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more of a particular cell population as compared to the starting cell mixture. Methods of performing fine sorting are well known in the art and may include multi-parameter fluorescence based molecular phenotypes such as Fluorescence Activated Cell Sorting (FACS) and microfluidic based sorting. For example, additional methods of performing fine sorting are provided in U.S. provisional patent application No. 62/421,979, which is incorporated by reference herein in its entirety.

As used herein, the term "binding molecule" can be any of a number of different molecules or aggregates, and the terms are used interchangeably. Proteins, polypeptides, peptides, nucleic acids (nucleotides, oligonucleotides and polynucleotides), antibodies, sugars, polysaccharides, lipids, receptors, test compounds (particularly compounds produced by combinatorial chemistry) can be individually or in combination (alone or by binding of target ligands to each other) as binding molecules.

As used herein, "specifically binds" or "specific for …" refers to a binding molecule (e.g., an antibody) or binding domain and a target (molecule or complex) at or above 10⁵M^-1Affinity or K of_a(i.e., the equilibrium binding constant for a particular binding interaction, in units of 1/M) (equals the association rate for that association reaction [ k ]_on]And dissociation rate [ k ]_off]Ratio) to be associated or associated with no significant association or association with any other molecule or component in the sample. Binding molecules or binding domains can be classified as "high affinity" binding molecules or binding domains or "low affinity" binding molecules or binding domains. A "high affinity" binding molecule or binding domain refers to K_aIs at least 10⁷M^-1At least 10⁸M^-1At least 10⁹M^-1At least 10¹⁰M^-1At least 10¹¹M^-1At least 10¹²M^-1Or at least 10¹³M^-1Those binding molecules or binding domains of (a). A "low affinity" binding molecule or binding domain refers to K_aIs at most 10⁷M^-1At most 10⁶M^-1At most 10⁵M^-1Those binding molecules or binding domains of (a).Alternatively, affinity can be defined as the equilibrium dissociation constant (K) for a particular binding interaction_d) In units of M (e.g., 10)^-5M to 10^-13M)。

As used herein, "shaped cell graft" or "shaped graft" refers to a cell population that is treated from a starting cell population to provide a number of specific cell types within a specified range and to reduce or eliminate undesirable cell types to a specified range. Ranges are typically provided in the number of cells per kg of patient body weight for a particular range of variation, but may also be expressed as total number in the graft. A shaped cell graft may comprise a mixture of cells that includes a ratio of target cells to counted cells that are not found in nature, or a percentage representation that is not found in nature.

As used herein, "unit dose" refers to a specified minimum number, specified number, or range of therapeutic cell populations per kilogram (kg) body weight of a subject receiving a molded cell graft. It is recognized that the number of unit doses will vary depending on the size of the subject. The unit dose may be divided into portions of the unit dose according to the body weight of the subject. In some embodiments, the therapeutic cell population (e.g., HPSC, Tmem, Treg, iNKT, etc.) can be divided into separate containers for administration to the subject.

Therapeutic cell compositions

In certain embodiments, the present disclosure provides unique therapeutic cell populations that form pharmaceutical compositions useful in hematopoietic stem/progenitor cell transplantation. The therapeutic cell population can include an enriched population of hematopoietic stem/progenitor cells (HSPCs), memory T cells (Tmem), regulatory T cells (tregs), and wherein the cell population is depleted of naive conventional α β -T cells. In some embodiments, the tregs comprise naive tregs, memory tregs, or both. In some embodiments, Tmem comprises T central memory stem cells (T)_SCM) T central memory cell (T)_CM) Population, T-effector memory cells (T)_EM) A population, or any combination thereof. In some embodiments, the therapeutic population comprises an enriched population of constant natural killer T cells (inkts).

In some embodiments, the therapeutic composition can comprise HSPC and Tmem. In some cases, the therapeutic composition can comprise HSPCs, Tmem, and tregs. In some cases, the therapeutic composition may comprise HSPCs and tregs. In some cases, the therapeutic composition may comprise HSPC and iNKT cells. In some cases, the therapeutic composition can comprise HSPC, Treg, Tmem, and iNKT cells.

In certain embodiments of the disclosure, the HSPC is CD34⁺. HSPC can further or alternatively be described as CD133⁺、CD90⁺、CD38^-、CD45RA^-、Lin^-Or any combination thereof. In some embodiments, the HSPC is CD19^-、TCRα/β^-Or a combination thereof. In some embodiments, the Treg is CD25⁺、CD4⁺And CD127^-/loA cell or any combination thereof. In some embodiments, the Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺Or any combination thereof. In some embodiments, the naive Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺、CD45RA⁺、CD45RO^-Or any combination thereof. In some embodiments, the memory Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺、CD45RA^-、CD45RO⁺Or any combination thereof. In some embodiments, Tmem is CD25^-、CD45RA^-A cell or any combination thereof. In some embodiments, Tmem is CD3⁺、CD45RA^-、CD45RO⁺Or any combination thereof. In some embodiments, T_SCMIs CD45RA⁺And CD4⁺Or CD8⁺。T_SCMCan also be described as CD95⁺、CD122⁺、CXCR3⁺、LFA-1⁺Or any combination thereof. In some embodiments, T_CMIs CD45RO⁺And CD4⁺Or CD8⁺。T_CMCan also be described as CD45RA^-、CD62L⁺、CCR7⁺Or any combination thereof. In some embodiments, T_EMIs CD4⁺、CD45RO⁺、CD45RA^-、CD62L^-、CCR7^-Or any combination thereof. In some embodiments, the iNKT is CD1d-tet⁺、6B11⁺Or both. In some embodiments, iNKT is va24ja18⁺. In some embodiments, the iNKT cell may be CD25⁺、6B11⁺、CD4⁺、Vα24Jα18⁺、CD127^dim/-A cell or any combination thereof. In any of the embodiments described herein, the naive conventional α β -T cell is CD25^-、CD45RA⁺Or any combination thereof. In some embodiments, the naive conventional α β -T cell may be a TCR α/β⁺CD45RA⁺And CD25^-、CD127⁺Or both. Naive conventional α β -T cells can also be described as TCR α⁺TCRβ⁺CD45RA⁺CD45RO^-CD25^-CD95^-IL-2Rβ^-CD127⁺Vα24Jα18^-。

In certain embodiments, the concentration of therapeutic cells is described as the ratio of HSPCs to another cell type. In some embodiments, the ratio of HSPC to Tmem includes from 500:1 to 1:1,000, 400:1 to 1:1,000, 300:1 to 1:1,000, 200:1 to 1:1,000, 100:1 to 1:1,000, 50:1 to 1:1,000, 10:1 to 1:1,000, 5:1 to 1:1,000, 4:1 to 1:1,000, 3:1 to 1:1,000, 2:1 to 1:1,000, 1:1 to 1:1,000, 500:1 to 1:900, 500:1 to 1:800, 500:1 to 1:700, 500:1 to 1:600, 500:1 to 1:500, 500:1 to 1:400, 500:1 to 1:300, 500:1 to 1:200, 500:1 to 1:100, 500:1 to 1:50, 500:1 to 1:20, 500:1 to 1:1, 500:1, 1:1 to 1:100, 500:1, 1:1, 1:8, 1:1, 1 500:1 to 1:3, 500:1 to 1:2, 500:1 to 1:1, 400:1 to 1:900, 300:1 to 1:800, 200:1 to 1:700, 100:1 to 1:600, 50:1 to 1:500, 10:1 to 1:400, 5:1 to 1:300, 4:1 to 1:200, 3:1 to 1:100, 2:1 to 1:50, or 1:1 to 1: 20.

In some embodiments, the ratio of HSPC to Tmem includes a range from 10:1 to 1:200, 100:1 to 1:2,000, or 1,000:1 to 1:20,000.

The ratio of HSPCs to tregs may include a range from 20:1 to 1:3, 100:1 to 1:30, or 200:1 to 1: 300. The ratio of HSPCs to naive tregs may include a range from 1:500 to 100:1, 1:400 to 100:1, 1:300 to 100:1, 1:200 to 100:1, 1:100 to 100:1, 1:50 to 100:1, 1:20 to 100:1, 1:10 to 100:1, 1:5 to 100:1, 1:1 to 100:1, 1:200 to 50:1, 1:200 to 20:1, 1:200 to 10:1, 1:200 to 5:1, 1:100 to 1:1, 40:1 to 1:3, 200:1 to 1:15, or 400:1 to 1: 150. The ratio of HSPCs to memory tregs may include a range from 1:500 to 10,000:1, 1:400 to 10,000:1, 1:300 to 10,000:1, 1:200 to 10,000:1, 1:100 to 10,000:1, 1:50 to 10,000:1, 1:20 to 10,000:1, 1:10 to 10,000:1, 1:5 to 10,000:1, 1:1 to 10,000:1, 1:500 to 5,000:1, 1:500 to 1,000:1, 1:500 to 900:1, 1:500 to 800:1, 1:500 to 700:1, 1:500 to 600:1, 1:500 to 500:1, 1:500 to 400:1, 1:500 to 300:1, 1:500 to 200:1, 1:500 to 100:1, 1:500 to 50:1, 1:500 to 20:1, 1:500 to 1:1, 1:500 to 500:1, or 1:500 to 500:1, 1:500 to 10:1, 1:1, or 1:500 to 500: 1.

The ratio of HSPCs to inkts may include a range from 1:2 to 1,000,000:1, 1:2 to 500,000:1, 1:1 to 500,000:1, 100:1 to 1,000,000:1, 100:1 to 500,000:1, 100:1 to 100,000:1, 500:1 to 1,000,000:1, 500:1 to 500,000:1, 500:1 to 100,000:1, 1,000:1 to 1,000,000:1, 1,000:1 to 500,000:1, 1,000:1 to 100,000:1, 10,000:1 to 1:2, 100,000:1 to 1:20, or 1,000,000:1 to 1: 200.

In certain embodiments, the concentration of therapeutic cells is described as the ratio of naive conventional α β -T cells to therapeutic cell types. The ratio of naive conventional α β -T cells to HSPCs may be less than 1:3, less than 1:50, less than 1:100, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1,000, less than 1:1,500, less than 1:2,000, less than 1:3,000, less than 1:4,000, less than 1:5,000, less than 1:6,000, less than 1:7,000, less than 1:8,000, less than 1:9,000, less than 1:10,000, less than 1:50,000, less than 1:100,000, less than 1:200,000, less than 1:300,000, less than 1:400,000, less than 1:500,000, less than 1:600,000, less than 1:700,000, less than 1:800,000, less than 1:900,000, less than 1:500,000, less than 1,000.

The ratio of naive conventional α β -T cells to Tmem may be less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1: 50000.

The ratio of naive conventional α β -T cells to tregs may be less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1: 50000. The ratio of naive conventional α β -T cells to naive tregs may be less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1: 50000. The ratio of naive conventional α β -T cells to memory tregs may be less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1: 50000.

The ratio of naive conventional α β -T cells to iNKT may be less than 100:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, less than 1: 50000.

In some embodiments of the present disclosure, the ratio of Tmem to Treg is independent of (e.g., varies greatly from) the concentration of the starting cells. This provides an advantage because the concentration of Tmem can be controlled independently of the concentration of tregs (e.g., dose escalation). The ratio of Tmem to Treg may be from 30:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1: 1. In certain embodiments, the ratio of Tmem to Treg may be from 1:1 to 200:1, 1:10 to 2000:1, or 1:100 to 20,000: 1. The ratio of Tmem to naive tregs may be from 5:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, or 1:1 to 1: 10. The ratio of Tmem to memory Treg may be from 27:1 to 0.9:1, 30:1 to 1:10, 25:1 to 1:10, 20:1 to 1:10, 15:1 to 1:10, 10:1 to 1:10, 30:1 to 1:9, 30:1 to 1:8, 30:1 to 1:7, 30:1 to 1:6, 30:1 to 1:5, 30:1 to 1:4, 30:1 to 1:3, 30:1 to 1:2, or 30:1 to 1: 1.

In some embodiments of the disclosure, the ratio of Treg to iNKT may be from 20,000:1 to 1:5, 200,000:1 to 1:50, or 2,000,000:1 to 1: 500.

In some embodiments of the disclosure, the ratio of iNKT to Tmem may be from 2:1 to 1:100,000, 5:1 to 1:1,000,000, or 10:1 to 1:10,000,000.

In some embodiments, the number of naive conventional a β -T cells in the composition may be less than about 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 27%, 30%, 32% or 35% of the number of HSPCs. In some embodiments, the number of naive conventional a β -T cells in the composition may be about 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 27%, 30%, 32% or 35% of the number of HSPCs. In some embodiments, the number of naive conventional a β -T cells in the composition may be from about 2% to about 7%, from about 2% to about 10%, from about 2% to about 15%, from about 2% to about 20%, from about 2% to about 25%, from about 2% to about 30%, from about 2% to about 35%, from about 7% to about 10%, from about 7% to about 15%, from about 7% to about 20%, from about 7% to about 25%, from about 7% to about 30%, from about 7% to about 35%, from about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 35%, about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 25% to about 30%, about 25% to about 35%, or about 30% to about 35%.

In some embodiments, the number of naive conventional a β -T cells in the composition can be less than about 0.1%, 1%, 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 22%, or 25% of the number of Tmem cells. In some embodiments, the number of naive conventional a β -T cells in the composition may be up to about 20%. In some embodiments, the number of naive conventional a β -T cells in the composition can be about 0.1% to about 2%, about 0.1% to about 5%, about 0.1% to about 10%, about 0.1% to about 15%, about 0.1% to about 20%, about 2% to about 5%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 10% to about 15%, about 10% to about 20%, or about 15% to about 20% of the number of Tmem cells.

In some embodiments, the number of naive conventional a β -T cells in the composition may be less than about 0.05%, 0.5%, 1%, 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 22%, 25% or 30% of the number of Treg cells. In some embodiments, the number of naive conventional a β -T cells in the composition may be from about 0.05% to about 1%, from about 0.05% to about 5%, from about 0.05% to about 10%, from about 0.05% to about 15%, from about 0.05% to about 20%, from about 0.05% to about 30%, from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 15%, from about 1% to about 20%, from about 1% to about 30%, from about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 30%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 30%, from about 15% to about 20%, from about 15% to about 30%, or from about 20% to about 30% of the number of Treg cells.

In some embodiments, the number of naive conventional a β -T cells in the composition can be less than about 1%, 10%, 20%, 50%, 70%, 80%, or 90% of the number of iNKT cells. In some embodiments, the number of naive conventional a β -T cells in the composition may be from about 1% to about 10%, from about 1% to about 20%, from about 1% to about 50%, from about 1% to about 70%, from about 1% to about 80%, from about 1% to about 90%, from about 10% to about 20%, from about 10% to about 50%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, from about 20% to about 50%, from about 20% to about 70%, from about 20% to about 80%, from about 20% to about 90%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, from about 70% to about 80%, from about 70% to about 90%, or from about 80% to about 90% of the number of iNKT cells.

In certain embodiments, the therapeutic cell population comprises 10% to 65% HSPCs. In certain embodiments, the therapeutic cell population comprises between 2% and 20% tregs. In certain embodiments, the therapeutic cell population comprises 25% to 90% Tmem. In certain embodiments, the therapeutic cell population comprises less than 2% naive, conventional α β -T cells. In certain embodiments, the therapeutic cell population comprises: 10% to 65% HSPC; 2% to 20% tregs; 25% to 90% Tmem; and less than 2% naive conventional α β -T cells. In some embodiments, the population of tregs comprises 2% to 5%, 2% to 10%, 2% to 20%, 2% to 30%, 2% to 40%, 2% to 50%, 5% to 10%, 5% to 20%, 5% to 30%, 5% to 40%, 5% to 50%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 15% to 20%, 15% to 30%, 15% to 40%, or 15% to 50% naive tregs. In some embodiments, the population of tregs comprises between 75% and 95% memory tregs. In some embodiments, the naive T cell population comprises 0.1% to 10% T_SCM. In some embodiments, the population of Tmem comprises 0% to 99% T_CM. In some embodiments, the population of Tmem comprises 0% to 99% T_EM. In certain embodiments, the group further comprises 0.01% to 5%, 0.05% to 5%,0.1% to 5%, 0.5% to 5%, 1% to 5%, 0.01% to 1.5%, 0.05% to 1.5%, 0.1% to 1.5%, 0.5% to 1.5%, or 1% to 1.5% of iNKT.

In any of the embodiments disclosed herein, the population of cells can be provided by processing a sample from one or more tissue harvests. In some embodiments, the sample or tissue is harvested from a single donor. The one or more tissue harvests may be from one or more donors. For example, the tissue harvest may be from an HLA-matched sibling donor, an HLA-matched unrelated donor, a partially matched unrelated donor, a haplotype identical related donor, an autologous donor, a fully HLA-mismatched allogeneic donor, a donor pool, or any combination thereof.

In any of the embodiments disclosed herein, the cell population can be a formulation for administration to a subject. In some cases, the cell population can be formulated using excipients. In some embodiments, the cells are formulated for infusion or injection. Excipients may include Normosol-R and human serum. Human serum may comprise 0.2% of the total formulation. Human serum may comprise 0.5% of the total formulation. Human serum may comprise 1% of the total formulation. Human serum may comprise 2% of the total formulation. In addition, the formulation may comprise a pH buffer, such as 0.1 mM-100 mM phosphate pH 6.0-9.0, 0.1-100mM HEPES pH 6.0-9.0, 0.1 mM-100 mM bicarbonate pH 6.0-9.0, 0.1 mM-100 mM citrate pH 6.0-9.0, 0.1-100mM acetate pH 4.0-8.0, or any combination thereof. The formulation may comprise an electrolyte, such as 5 mM-400 mM NaCl, 0.5 mM-50 mM KCl, 0.05 mM-50 mM CaCl2, 0.05 mM-50 mM MgCl2, 0.05 mM-50 mM LiCl2, 0.05 mM-50 mM MnCl2, or any combination thereof. The formulation may comprise an energy source, such as 0.1 mM-100 mM glucose, 0.1 mM-100 mM pyruvate, 0.1 mM-100 mM fructose, 0.1-100mM sucrose, 0.1-50 mM glycerol, 0.1 mM-100 mM gluconolactone, 0.1-100mM gluconic acid, or any combination thereof. The formulation may comprise an antioxidant, such as 0.05-10 mM glutathione (reduced), 0.05-10 mM glutathione (oxidized), 0.001 mM-10mM beta-mercaptoethanol, 0.001 mM-10mM dithiothreitol, 0.01-100 mM ascorbate, 0.001-10 mM tris (2-carboxyethyl) phosphine, or any combination thereof. The formulation may comprise a stabilizer such as 0.01% -10% human serum albumin, 0.01% -10% bovine serum albumin, 0.1% -99% human serum, 0.1% -99% fetal bovine serum, 0.01% -10% IgG, 0.1% -10% immunoglobulin, 0.06% -60% trehalose, or a molecular polymer such as 0.1% -20% polyethylene glycol (MW 200-.

In some embodiments disclosed herein, the population of cells administered to the subject is formulated based on the weight of the subject (e.g., number of cells/kg weight of the subject). The cell population may be formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a unit dose of a cell transplant (e.g., a cell population), wherein each unit dose of a cell transplant comprises a therapeutic cell population per kilogram (kg) body weight of a subject receiving the cell transplant. In some embodiments, the population or unit dose of cells comprises about 1x10 per kg of subject⁶HSPC to about 20x 10⁶And (5) HSPC. In some embodiments, the population or unit dose of cells comprises at least about 1x10 per kg of subject⁶And (5) HSPC. In some embodiments, the population or unit dose of cells comprises at most about 20x 10 per kg of subject⁶And (5) HSPC. In some embodiments, the population or unit dose comprises HSPC cells ranging from about 0.5x 10⁶To 50x 10⁶、1.0x 10⁶To 20x 10⁶Or 2.0x 10⁶To 10x 10⁶Individual cells/kg subject weight.

In some embodiments, the population or unit dose of cells comprises about 1x10⁶HSPC to about 2x 10⁶HSPC, about 1X10⁶HSPC to about 5x 10⁶HSPC, about 1X10⁶HSPC to about 7x 10⁶HSPC, about 1X10⁶HSPC to about 10x 10⁶HSPC, about 1X10⁶HSPC to about 15x 10⁶HSPC, about 1X10⁶HSPC to about 20x 10⁶HSPC, about 2x 10⁶HSPC to about 5x 10⁶HSPC, about 2x 10⁶HSPC to about 7x 10⁶HSPC, about 2x 10⁶HSPC to about 10x 10⁶HSPC, about 2x 10⁶HSPC toAbout 15x 10⁶HSPC, about 2x 10⁶HSPC to about 20x 10⁶HSPC, about 5x 10⁶HSPC to about 7x 10⁶HSPC, about 5x 10⁶HSPC to about 10x 10⁶HSPC, about 5x 10⁶HSPC to about 15x 10⁶HSPC, about 5x 10⁶HSPC to about 20x 10⁶HSPC, about 7X 10⁶HSPC to about 10x 10⁶HSPC, about 7X 10⁶HSPC to about 15x 10⁶HSPC, about 7X 10⁶HSPC to about 20x 10⁶HSPC, about 10X 10⁶HSPC to about 15x 10⁶HSPC, about 10X 10⁶HSPC to about 20x 10⁶HSPC or about 15x 10⁶HSPC to about 20x 10⁶Per kilogram subject HSPCs. In some embodiments, the population of cells comprises about 1x10⁶HSPC, about 2x 10⁶HSPC, about 5x 10⁶HSPC, about 7X 10⁶HSPC, about 10X 10⁶HSPC, about 15X 10⁶HSPC or about 20x 10⁶Per kilogram subject HSPCs.

In some embodiments, the population or unit dose of cells comprises about 1x10⁶Tmem cell to about 100x 10⁶One Tmem cell per kilogram subject. In some embodiments, the population or unit dose of cells comprises at least about 1x10⁶One Tmem cell per kilogram subject. In some embodiments, the population or unit dose of cells comprises up to about 100x 10⁶One Tmem cell per kilogram subject. In some embodiments, the population or unit dose of cells comprises about 1x10⁶Tmem cell to about 10x 10⁶Tmem cell, about 1x10⁶Tmem cell to about 20x 10⁶Tmem cell, about 1x10⁶Tmem cell to about 50x 10⁶Tmem cell, about 1x10⁶Tmem cell to about 75x 10⁶Tmem cell, about 1x10⁶Tmem cell to about 100x 10⁶Tmem cell, about 10x 10⁶Tmem cell to about 20x 10⁶Tmem cell, about 10x 10⁶Tmem cell to about 50x 10⁶Tmem cell, about 10x 10⁶Tmem cell to about 75x 10⁶Tmem cell, about 10x 10⁶Tmem cell to about 100x 10⁶Tmem cell, about 20x 10⁶Tmem cell to about 50x 10⁶Tmem cell, about 20x 10⁶Tmem cell to about 75x 10⁶Tmem cell, about 20x 10⁶Tmem cell to about 100x 10⁶Tmem cell, about 50x 10⁶Tmem cell to about 75x 10⁶Tmem cell, about 50x 10⁶Tmem cell to about 100x 10⁶Tmem cell or about 75x 10⁶Tmem cell to about 100x 10⁶One Tmem cell per kilogram subject. In some embodiments, the population or unit dose of cells comprises about 1x10⁶Tmem cell, about 10x 10⁶Tmem cell, about 20x 10⁶Tmem cell, about 50x 10⁶Tmem cells, about 75x 10⁶Tmem cell or about 100x 10⁶One Tmem cell per kilogram subject.

In some embodiments, the population or unit dose of cells comprises about 0.5x 10⁶Treg to about 2.5x 10⁶One Treg cell per kilogram of subject. In some embodiments, the population or unit dose of cells comprises at least about 0.5x 10⁶One Treg cell per kilogram of subject. In some embodiments, the population or unit dose of cells comprises at most about 2.5x 10⁶One Treg cell per kilogram of subject. In some embodiments, the population or unit dose of cells comprises about 0.5x 10⁶Treg cells to about 1x10⁶About 0.5x 10 Treg cells⁶Treg to about 1.5x 10⁶About 0.5x 10 Treg cells⁶Treg cells to about 2x 10⁶About 0.5x 10 Treg cells⁶Treg to about 2.5x 10⁶About 1x10 Treg cells⁶Treg to about 1.5x 10⁶About 1x10 Treg cells⁶Treg cells to about 2x 10⁶About 1x10 Treg cells⁶Treg to about 2.5x 10⁶Treg cells, about 1.5X 10⁶Treg cells to about 2x 10⁶Treg cells, about 1.5X 10⁶Treg to about 2.5x 10⁶Treg cells or about 2x 10⁶Treg to about 2.5x 10⁶One Treg cell per kilogram of subject. In some embodiments, the population or unit dose of cells comprises about 0.5x 10⁶About 1x10 Treg cells⁶Treg cells, about 1.5X 10⁶About 2x 10 Treg cells⁶Treg cells or about 2.5x 10⁶One Treg cell per kilogram of subject. In some embodiments, the population or unit dose of cells comprises naive tregs ranging from about 0.1x 10⁶To about 500x 10⁶About 0.2x 10⁶To about 500x 10⁶About 0.3x 10⁶To about 500x 10⁶About 0.4x 10⁶To about 500x 10⁶About 0.5x 10⁶To about 500x 10⁶About 0.6x 10⁶To about 500x 10⁶About 0.7x 10⁶To about 500x 10⁶About 0.8x 10⁶To about 500x 10⁶About 0.9x 10⁶To about 500x 10⁶Or about 1x10⁶To about 500x 10⁶Individual cells/kg subject weight. In some embodiments, the population or unit dose of cells comprises memory tregs ranging from 0.005x 10⁶To 500x 10⁶Individual cells/kg subject weight.

In some embodiments, the population or unit dose of cells comprises iNKT cells ranging from 0.5x 10³To 2000x 10³0.5x 10 cells/kg subject body weight³To 1x10⁷Individual cells/kg subject body weight or 1.0x10⁴To 2.5x 10⁶Individual cells/kg subject weight. In some embodiments, the population or unit dose of cells comprises about 0.01x 10⁶To about 3x 10 iNKT cells⁶One iNKT cell per kg subject. In some embodiments, the population or unit dose of cells comprises at least about 0.01x 10⁶One iNKT cell per kg subject. In some embodiments, the population or unit dose of cells comprises up to about 3x 10⁶One iNKT cell per kg subject. In some embodiments, the population or unit dose of cells comprises about 0.01x 10⁶Individual iNKT cells to about 0.1x 10⁶iNKT cell, about 0.01X 10⁶To about 1x10 iNKT cells⁶An iNKT cell,About 0.01x 10⁶To about 1.5x 10 iNKT cells⁶iNKT cell, about 0.01X 10⁶To about 2x 10 iNKT cells⁶iNKT cell, about 0.01X 10⁶To about 3x 10 iNKT cells⁶Individual iNKT cells, about 0.1X 10⁶To about 1x10 iNKT cells⁶Individual iNKT cells, about 0.1X 10⁶To about 1.5x 10 iNKT cells⁶Individual iNKT cells, about 0.1X 10⁶To about 2x 10 iNKT cells⁶Individual iNKT cells, about 0.1X 10⁶To about 3x 10 iNKT cells⁶Individual iNKT cells, about 1X10⁶To about 1.5x 10 iNKT cells⁶Individual iNKT cells, about 1X10⁶To about 2x 10 iNKT cells⁶Individual iNKT cells, about 1X10⁶To about 3x 10 iNKT cells⁶Individual iNKT cells, about 1.5X 10⁶To about 2x 10 iNKT cells⁶Individual iNKT cells, about 1.5X 10⁶To about 3x 10 iNKT cells⁶Individual iNKT cell or about 2x 10⁶To about 3x 10 iNKT cells⁶One iNKT cell per kg subject. In some embodiments, the population or unit dose of cells comprises about 0.01x 10⁶Individual iNKT cells, about 0.1X 10⁶Individual iNKT cells, about 1X10⁶Individual iNKT cells, about 1.5X 10⁶Individual iNKT cells, about 2X 10⁶Individual iNKT cell or about 3x 10⁶One iNKT cell per kg subject.

In some embodiments, the population or unit dose of cells comprises less than 1x10⁶A range of naive conventional α β -T cells per kg body weight of the subject. In some embodiments, the population or unit dose of cells comprises less than 3x 10⁵A range of naive conventional α β -T cells per kg body weight of the subject. In some embodiments, the population or unit dose of cells comprises less than 7.5x 10⁴Less than 5x 10 cells/kg subject body weight⁴Per cell/kg, less than 1x10⁴Individual cell/kg, less than 0.5x 10⁴Individual cell/kg or less than 1x10³A range of naive conventional α β -T cells per kg body weight of the subject.

In some embodiments, each unit dose of the therapeutic cell population comprises: 1.0x10⁶To 50x 10⁶Hematopoietic stem/progenitor cells (HSPC), 0.1X 10⁶To 1000x 10⁶A memory T cell (Tmem), 0.1x 10⁶To 1000x 10⁶A regulatory T cell (Treg) and less than 3x 10⁵Naive conventional α β -T cells. In some embodiments, each unit dose of the therapeutic cell population comprises: 3.0x 10⁶To 50x 10⁶Hematopoietic stem/progenitor cells (HSPC), 0.3X 10⁶To 1000x 10⁶A memory T cell (Tmem), 0.5x 10⁶To 1000x 10⁶A regulatory T cell (Treg) and less than 3x 10⁵Naive conventional α β -T cells. In some embodiments, each unit dose of the therapeutic cell population comprises: 1.0x10⁶To 50x 10⁶Hematopoietic stem/progenitor cells (HSPC), 0.3X 10⁶To 1000x 10⁶A memory T cell (Tmem), 0.5x 10⁶To 1000x 10⁶A regulatory T cell (Treg) and less than 3x 10⁵Naive conventional α β -T cells.

In any of the embodiments disclosed herein, the HSPCs may be provided by a donor with haploidenticity to the subject. In some embodiments, the Treg, Tmem, iNKT, or any combination thereof is provided by a donor that is an HLA matched sibling donor or an HLA matched unrelated donor or an HLA partially matched unrelated donor. In some embodiments, the HSPCs are provided by a donor that is haploidentical to the subject and the Treg, Tmem, iNKT, or any combination thereof is provided by a donor that is an HLA-matched sibling donor or an HLA-matched unrelated donor.

Method of treatment

The present disclosure provides methods of performing cell transplantation therapy in a subject having a disease, condition, or disorder, comprising: administering to the subject any of the therapeutic cell graft compositions described herein. For example, a therapeutic composition comprising cells can be infused into a subject in need of the composition.

Subjects that can be treated include those suffering from leukemia, lymphoma, chronic infections or autoimmune diseases, hematological malignancies, malignant or non-malignant, AML, ALL, CML, CLL, multiple myeloma, hodgkin's lymphoma, non-hodgkin's lymphoma, MDS, lymphoproliferative diseases, type 1 diabetes, inborn errors of metabolism, genetic diseases, severe combined immunodeficiency, sickle cell anemia, beta-thalassemia, multiple sclerosis, solid organ transplantation, crohn's disease, ulcerative colitis, lupus, hemophagocytic lymphohistiocytosis, glycogen storage disease, breast cancer, other solid tumors, leukodystrophy, mucopolysaccharidosis or any other disease that would benefit from HSPC transplantation. In some embodiments, the subject suffers from relapsed ALL or AML or primary refractory ALL or AML and fewer than 10% blasts. In some embodiments, the subject suffers from high risk AML that is ≧ CR1 or minimal residual disease positive. In some embodiments, the subject suffers from > CR1 or high risk ALL positive for minimal residual disease. In some embodiments, the subject is suffering from high risk CML. In some embodiments, the subject is suffering from a high risk myeloproliferative disorder. In some embodiments, the subject suffers from relapsed non-hodgkin lymphoma that is responsive to therapy. In some embodiments, the subject is afflicted with MDS, which at the time of transplantation has a blast cell count of less than 10% blast cells. In certain embodiments, the subject has one or more of the following characteristics: age 18-65, carnivosyl score ≧ 60 or ECOG ≦ 2, HCT complication index ≦ 4, creatinine <1.5mg/dL, cardiac ejection fraction > 45%, DLCO > 60% after correction, total bilirubin <3 times Upper Limit of Normal (ULN) (unless due to gilbert syndrome), AST and ALT <3 times ULN, not pregnant or lactating, HIV negative, and no co-morbid conditions that would limit life expectancy below 6 months. In certain embodiments, the subject has one or more of the following characteristics: age patients 0-3, 3-6, 6-12, 12-14, 12-18, 18-65, 65-70, 70-75, 75-80, 80-90 or more, or any range therebetween, a carnowski score of 60 or 80 or more, or ECOG of 2 or less, HCT complication index of 4 or less, creatinine <1.5mg/dL, cardiac ejection fraction > 45%, DLCO > predicted 60% post correction, total bilirubin <3 times the upper limit or <1.5 times the Upper Limit of Normal (ULN) (unless due to gilbert syndrome), AST and ALT <3 times or <1.5 times ULN, not pregnant or not lactating, HIV negative, and no co-morbid that would limit life expectancy below 6 months.

The therapeutic cell compositions described herein can be administered in place of traditional HCT. Exemplary therapeutic cell compositions are compatible with Reduced Intensity Conditioning (RIC) and Myeloablative (MA) regimens. Because of the reduced risk of GVHD, MA pretreatment can be used in place of RIC in some patients, especially patients with potential malignancies. Because of the reduced GVHD of the therapeutic cellular compositions disclosed herein, alloreactivity may be beneficial in the case of multiple myeloma where the risk of GVHD is greater than alloreactivity and autotransplantation is currently used clinically. Non-malignant conditions may benefit from enhanced immune reconstitution, so infection rates are reduced, transplant rates are increased and the chimerism persists.

The therapeutic cell compositions described herein are administered to a subject according to known techniques or variants thereof that will be apparent to those skilled in the art.

An "effective amount" or "therapeutically effective amount" refers to an amount of a composition described herein that, when administered to a subject (e.g., a human), is sufficient to aid in the treatment of a disease. The amount of a composition that constitutes a "therapeutically effective amount" will vary depending on the cell preparation, the condition and its severity, the mode of administration, and the age of the subject to be treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and the present disclosure. When referring to an individual active ingredient or composition administered alone, a therapeutically effective dose refers to that ingredient or composition alone. When referring to a combination, a therapeutically effective dose refers to the combined amounts of the active ingredients, the composition, or both that produce the therapeutic effect, whether administered sequentially, concurrently, or simultaneously.

In some embodiments, the HSPC cells are CD34⁺The concentration of cells was applied at 1.0x10⁶To 50x 10⁶1.0x10 cells/kg subject body weight⁶To 20x 10⁶Individual cells/kg subject body weight or 2.0x 10⁶To 10x 10⁶Individual cells/kg subject weight. In some embodiments, the HSPC cells are administered at about 1x10⁶HSPC to about 20x 10⁶Concentration of individual HSPCs per kilogram of subject. In some embodiments, the HSPC cells are administered at a dose of at least about 1x10⁶Concentration of individual HSPCs per kilogram of subject. In some embodiments, the HSPC cells are administered at a dose of up to about 20x 10⁶Concentration of individual HSPCs per kilogram of subject. In some embodiments, the HSPC cells are administered at about 1x10⁶HSPC to about 2x 10⁶HSPC, about 1X10⁶HSPC to about 5x 10⁶HSPC, about 1X10⁶HSPC to about 7x 10⁶HSPC, about 1X10⁶HSPC to about 10x 10⁶HSPC, about 1X10⁶HSPC to about 15x 10⁶HSPC, about 1X10⁶HSPC to about 20x 10⁶HSPC, about 2x 10⁶HSPC to about 5x 10⁶HSPC, about 2x 10⁶HSPC to about 7x 10⁶HSPC, about 2x 10⁶HSPC to about 10x 10⁶HSPC, about 2x 10⁶HSPC to about 15x 10⁶HSPC, about 2x 10⁶HSPC to about 20x 10⁶HSPC, about 5x 10⁶HSPC to about 7x 10⁶HSPC, about 5x 10⁶HSPC to about 10x 10⁶HSPC, about 5x 10⁶HSPC to about 15x 10⁶HSPC, about 5x 10⁶HSPC to about 20x 10⁶HSPC, about 7X 10⁶HSPC to about 10x 10⁶HSPC, about 7X 10⁶HSPC to about 15x 10⁶HSPC, about 7X 10⁶HSPC to about 20x 10⁶HSPC, about 10X 10⁶HSPC to about 15x 10⁶HSPC, about 10X 10⁶HSPC to about 20x 10⁶HSPC or about 15x 10⁶HSPC to about 20x 10⁶Concentration of individual HSPCs per kilogram of subject. In some embodiments, the HSPC cells are administered at about 1x10⁶HSPC, about 2x 10⁶HSPC, about 5x 10⁶HSPC, about 7X 10⁶HSPC, about 10X 10⁶HSPC, about 15X 10⁶HSPC or about 20x 10⁶Concentration of individual HSPCs per kilogram of subject.

In some embodiments, Tmem is at 0.1x 10⁶To 1000x 10⁶1.0x10 cells/kg subject body weight⁶To 250x 10⁶Individual cells/kg subject body weight or 2.9x 10⁶To 10.1x 10⁶10.1X 10 cells/kg subject body weight⁶To 30.1x 10⁶30.1X 10 cells/kg subject body weight⁶To 101x 10⁶1.0x10 cells/kg subject body weight⁶To 100x 10⁶The concentration of individual cells/kg body weight of the subject. In some embodiments, the Tmem cell is at about 1x10⁶Tmem cell to about 100x 10⁶Concentration of one Tmem cell per kilogram subject. In some embodiments, the Tmem cell is at least about 1x10⁶Concentration of one Tmem cell per kilogram subject. In some embodiments, Tmem cells are at most about 100x 10⁶Concentration of one Tmem cell per kilogram subject. In some embodiments, the Tmem cell is at about 1x10⁶Tmem cell to about 10x 10⁶Tmem cell, about 1x10⁶Tmem cell to about 20x 10⁶Tmem cell, about 1x10⁶Tmem cell to about 50x 10⁶Tmem cell, about 1x10⁶Tmem cell to about 75x 10⁶Tmem cells, about 1X10⁶Tmem cell to about 100x 10⁶Tmem cell, about 10x 10⁶Tmem cell to about 20x 10⁶Tmem cell, about 10x 10⁶Tmem cell to about 50x 10⁶Tmem cell, about 10x 10⁶Tmem cell to about 75x 10⁶Tmem cell, about 10x 10⁶Tmem cell to about 100x 10⁶Tmem cell, about 20x 10⁶Tmem cell to about 50x 10⁶Tmem cell, about 20x 10⁶Tmem cell to about 75x 10⁶Tmem cell, about 20x 10⁶Tmem cell to about 100x 10⁶Tmem cell, about 50x 10⁶Tmem cell to about 75x 10⁶Tmem cell, about 50x 10⁶Tmem cell to about 100x 10⁶Tmem cell or about 75x 10⁶Tmem cell to about 100x 10⁶Concentration of one Tmem cell per kilogram subject. In some embodiments, the Tmem cell is at about 1x10⁶Tmem cell, about10x 10⁶Tmem cell, about 20x 10⁶Tmem cell, about 50x 10⁶Tmem cells, about 75x 10⁶Tmem cell or about 100x 10⁶Concentration of one Tmem cell per kilogram subject.

In some embodiments, the Treg is at 0.1x 10⁶To 1000x 10⁶0.1x 10 cells/kg subject body weight⁶To 5x 10⁶Individual cells/kg subject body weight or 0.5x 10⁶To 2.5x 10⁶The concentration of individual cells/kg body weight of the subject. In some embodiments, the tregs are on the order of 0.5x 10⁶Treg to about 2.5x 10⁶The concentration of individual Treg cells per kilogram of subject is administered. In some embodiments, the tregs are on the order of at least about 0.5x 10⁶The concentration of individual Treg cells per kilogram of subject is administered. In some embodiments, the tregs are on the order of up to about 2.5x 10⁶The concentration of individual Treg cells per kilogram of subject is administered. In some embodiments, the tregs are on the order of 0.5x 10⁶Treg cells to about 1x10⁶About 0.5x 10 Treg cells⁶Treg to about 1.5x 10⁶About 0.5x 10 Treg cells⁶Treg cells to about 2x 10⁶About 0.5x 10 Treg cells⁶Treg to about 2.5x 10⁶About 1x10 Treg cells⁶Treg to about 1.5x 10⁶About 1x10 Treg cells⁶Treg cells to about 2x 10⁶About 1x10 Treg cells⁶Treg to about 2.5x 10⁶Treg cells, about 1.5X 10⁶Treg cells to about 2x 10⁶Treg cells, about 1.5X 10⁶Treg to about 2.5x 10⁶Treg cells or about 2x 10⁶Treg to about 2.5x 10⁶The concentration of individual Treg cells per kilogram of subject is administered. In some embodiments, the tregs are on the order of 0.5x 10⁶About 1x10 Treg cells⁶Treg cells, about 1.5X 10⁶About 2x 10 Treg cells⁶Treg cells or about 2.5x 10⁶The concentration of individual Treg cells per kilogram of subject is administered.

In some embodiments, the naive Treg is at about 0.1x 10⁶To about 500x 10⁶About 0.2x 10⁶To about 500x 10⁶About 0.3x 10⁶To about 500x 10⁶About 0.4x 10⁶To about 500x 10⁶About 0.5x 10⁶To about 500x 10⁶About 0.6x 10⁶To about 500x 10⁶About 0.7x 10⁶To about 500x 10⁶About 0.8x 10⁶To about 500x 10⁶About 0.9x 10⁶To about 500x 10⁶Or about 1x10⁶To about 500x 10⁶The concentration of individual cells/kg body weight of the subject.

In some embodiments, the memory Treg is at 0.005x 10⁶To 500x 10⁶The concentration of individual cells/kg body weight of the subject.

In some embodiments, the iNKT cells are at 0.5x 10²To 2000x 10³0.5x 10 cells/kg subject body weight²To 1x10⁴0.5x 10 cells/kg subject body weight³To 1x10⁵0.5x 10 cells/kg subject body weight⁴To 1x10⁶0.5x 10 cells/kg subject body weight⁵To 1x10⁷0.5x 10 cells/kg subject body weight²To 1x10⁷Individual cells/kg subject body weight or 1.0x10⁴To 2.5x 10⁶The concentration of individual cells/kg body weight of the subject. In some embodiments, the iNKT cells are administered at about 0.01x 10⁶To about 3x 10 iNKT cells⁶Individual iNKT cells were administered per kilogram subject concentration. In some embodiments, the iNKT cells are administered at a dose of at least about 0.01x 10⁶Individual iNKT cells were administered per kilogram subject concentration. In some embodiments, the iNKT cells are at most about 3x 10⁶Individual iNKT cells were administered per kilogram subject concentration. In some embodiments, the iNKT cells are administered at about 0.01x 10⁶Individual iNKT cells to about 0.1x 10⁶iNKT cell, about 0.01X 10⁶To about 1x10 iNKT cells⁶iNKT cell, about 0.01X 10⁶To about 1.5x 10 iNKT cells⁶iNKT cell, about 0.01X 10⁶To about 2x 10 iNKT cells⁶iNKT cell, about 0.01X 10⁶To about 3x 10 iNKT cells⁶An iNKT cellAbout 0.1x 10⁶To about 1x10 iNKT cells⁶Individual iNKT cells, about 0.1X 10⁶To about 1.5x 10 iNKT cells⁶Individual iNKT cells, about 0.1X 10⁶To about 2x 10 iNKT cells⁶Individual iNKT cells, about 0.1X 10⁶To about 3x 10 iNKT cells⁶Individual iNKT cells, about 1X10⁶To about 1.5x 10 iNKT cells⁶Individual iNKT cells, about 1X10⁶To about 2x 10 iNKT cells⁶Individual iNKT cells, about 1X10⁶To about 3x 10 iNKT cells⁶Individual iNKT cells, about 1.5X 10⁶To about 2x 10 iNKT cells⁶Individual iNKT cells, about 1.5X 10⁶To about 3x 10 iNKT cells⁶Individual iNKT cell or about 2x 10⁶To about 3x 10 iNKT cells⁶Individual iNKT cells were administered per kilogram subject concentration. In some embodiments, the iNKT cells are administered at about 0.01x 10⁶Individual iNKT cells, about 0.1X 10⁶Individual iNKT cells, about 1X10⁶Individual iNKT cells, about 1.5X 10⁶Individual iNKT cells, about 2X 10⁶Individual iNKT cell or about 3x 10⁶Individual iNKT cells were administered per kilogram subject concentration.

In some embodiments, the administered cells comprise less than 3x 10⁵Naive conventional α β -T cells per kg body weight of the subject. In some embodiments, the administered cells comprise less than 2x 10⁵Less than 1x10 cells/kg subject body weight⁵Less than 7.5x 10 cells/kg subject body weight⁴Individual cell/kg, less than 5x 10⁴Per cell/kg, less than 1x10⁴Individual cell/kg, less than 0.5x 10⁴Individual cell/kg or less than 1x10³Naive conventional α β -T cells per kg body weight of the subject.

As further discussed in the examples, the purity of the therapeutic cell composition may be important to the clinical outcome of the treatment. For example, treatment of a cell fraction that may contain naive Tcon-contaminated cells using a method that results in low purity may compromise therapeutic efficacy by resulting in higher levels of GVHD, grade 3-4 acute GVHD, grade 3-4 steroid-resistant acute GVHD, chronic GVHD, graft failure, graft rejection, severe infection, organ failure, VOD/SOS, and relapse, as compared to therapeutic cell compositions prepared using high fidelity sorting techniques (e.g., FACS).

Without wishing to be bound by theory, it is believed that the therapeutic cell compositions disclosed herein provide superior therapeutic benefit in myeloablative HSPC transplantation procedures, as the provision of Treg, Tmem and iNKT cells with HSPCs allows earlier rescue of the subject's immune system compared to traditional myeloablative HSPC transplantation procedures. When subjects receive traditional myeloablative HSPC transplants, the immune system may take up to a year to begin significant recovery and provide protective immunity. In contrast, it is believed that in the therapeutic compositions disclosed herein, the introduction of Treg, Tmem, or iNKT cells along with HSPCs provides for the recruitment of ablated immune cells in the subject. These supplemented cells begin to provide significant immune function shortly after administration. Thus, the therapeutic cell compositions disclosed herein provide superior therapeutic benefits compared to traditional HSPC transplantation. It is also believed that regulatory T cells may assist in transplantation and prevent GVHD by inhibiting xenoreactive T cells in solid tissues. It is also believed that iNKT cells provide a positive feedback signal to tregs to promote an inhibitory environment, particularly in solid tissues. It is also believed that HSPCs will reconstitute the blood and immune system of myeloablative patients, including reconstitution of NK cells, which in turn is believed to provide graft versus leukemia effects. It is also believed that memory T cells provide anti-infective and anti-leukemic effects for a limited period of time (less than 5 years). Due to their limited lifespan, these cells cannot withstand the long-term graft-versus-host disease response.

In some embodiments, the therapeutic cell population is administered to the subject as a separate pharmaceutical composition. For example, the enriched HSPC, Tmem, Treg, or iNKT cell populations may be administered sequentially. In some embodiments, the therapeutic cell population is administered in multiple doses. In some cases, the dose of the population of cells may comprise HSPCs. In some cases, the dose of the population of cells may comprise Treg cells. In some cases, the dose of the cell population may comprise Tmem cells. In some cases, the dose of the cell population may comprise iNKT cells. In some embodiments, the therapeutic cell population is administered to the subject simultaneously as a single pharmaceutical composition. The dosage may be administered as a course of treatment. A course of treatment can include administering doses to a subject at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days. A course of treatment may include administering doses to a subject at intervals of 1 week, 2 weeks, 3 weeks, or 4 weeks. A course of treatment may include administering doses to a subject at intervals of 1 month, 2 months, 3 months, or 4 months.

In some embodiments, the therapeutic compositions described herein can be administered as a separate population of cells. For example, a first dose of a therapeutic composition can include administration of any one of the HSPC, Treg, or Tmem cell populations alone or in any combination. The subsequent dose may then comprise any of the above cell types not present in the first dose. The dose may further comprise iNKT cells. For example, the first dose may comprise HSPCs and the second dose may comprise tregs and Tmem cells. Other dosage combinations may also be administered to the subject.

In some embodiments, a complete dose of a therapeutic composition can include multiple doses. The complete therapeutic composition described herein can be administered in at least 1 dose. The complete therapeutic compositions described herein can be administered in up to 30 doses. In some cases, a complete therapeutic composition described herein can be administered in about 2 doses, 5 doses, 10 doses, 15 doses, 20 doses, 25 doses, or 30 doses. In some cases, a dose of the complete therapeutic composition may comprise HSPCs, Treg cells, Tmem cells. In some cases, each dose of the complete therapeutic composition may comprise a different population of cells.

In some embodiments, a unit dose can comprise at least 3x 10⁵Per kilogram subject HSPCs. In some embodiments, a unit dose can comprise at least 3x 10⁵One Treg cell per kilogram of subject. In some embodiments, a unit dose can comprise at least 3x 10⁵One Tmem cell per kilogram subject. In some embodiments, a unit dose can comprise at least 3x 10⁵One iNKT cell per kg subject. In some implementationsIn the regimen, the unit dose may comprise less than 3x 10⁵Each Tcon cell per kg subject.

In some embodiments, the entire therapeutic composition can be administered within about 1 day. In some cases, the entire therapeutic composition may be administered within up to 30 days. For example, a first dose of a therapeutic composition described herein can be administered within 1 day, while a second dose is administered after 10 days. In some cases, the entire therapeutic composition can be administered within about 1 day, 2 days, 5 days, 10 days, 15 days, 20 days, or 30 days.

In some embodiments, the cells are isolated from a donor that is an HLA-matched sibling donor, an HLA-matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA-mismatched donor, a donor pool, or any combination thereof. In some embodiments, the therapeutic cell population is allogeneic. In some embodiments, the therapeutic cell population is autologous. In some embodiments, the therapeutic cell population is haploidentical. In some embodiments, the therapeutic cell population is isolated from mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, unmoved apheresis product, or any combination thereof.

In some embodiments, the therapeutic cell population is derived from a single tissue harvest. In some embodiments, the therapeutic cell population is derived from one or more tissue harvests. In some embodiments, the therapeutic cell population comprises HSPCs provided by at least a first donor and tregs and Tmem provided by at least a second donor. In some embodiments, the therapeutic cell population comprises iNKT cells provided by at least a second donor. In certain embodiments, the first donor is haploidentical with the subject. In certain embodiments, the second donor is an HLA-matched sibling donor or an HLA-matched or partially matched unrelated donor.

In some embodiments, prior to treatment, the subject has been pretreated with radiation, chemotherapy, recombinant proteins, antibodies or toxin-conjugated antibodies, or any combination thereof. In some embodiments, for cell transplantation therapy, the subject is pre-treated by first treating the subject with myeloablative therapy. Exemplary myeloablative therapies include chemotherapy or radiation therapy. It is believed that myeloablative therapy provides a therapeutic benefit by debulking a tumor. Cancer cells are generally more susceptible to chemotherapy/radiation than many normal cells. However, if the tumor initiating cells survive the chemotherapy/radiotherapy process, the subject is at risk of relapse. Thus, high levels of chemotherapy can help to eliminate the tumor initiating population; however, at these concentrations, toxicity to normal cells occurs. Although some susceptible normal cells are not necessary, hematopoietic stem cells are lethal from high levels of chemotherapy. Myeloablative therapy eradicates a sufficient amount of HSCs, otherwise the patient will die without transplantation. When HSPCs are injected into a myelocleared subject, the donor cells can rescue the subject and reconstitute the subject's blood and immune system for life. In some embodiments, the myeloablative therapy comprises administration of busulfan, cyclophosphamide, TBI, fludarabine, etoposide, or any combination thereof. In some embodiments, the myeloablative therapy comprises administration of an anti-cKIT antibody. In some embodiments, the myeloablative therapy comprises administration of an antibody drug conjugate. The antibody drug conjugate may be, for example, an anti-CD 45-saporin therapeutic antibody or an anti-cKit-saporin therapeutic antibody. In some embodiments, the myeloablative therapy is a pretreatment therapy of reduced intensity.

In certain embodiments, the therapeutic cell population is administered to the subject as a combination therapy comprising an immunosuppressive agent. Exemplary immunosuppressive agents include sirolimus, tacrolimus, cyclosporine, mycophenolate mofetil, antithymocyte globulin, corticosteroids, calcineurin inhibitors, antimetabolites such as methotrexate, post-transplant cyclophosphamide, or any combination thereof. In some embodiments, the subject is pre-treated with sirolimus or tacrolimus alone as a prophylactic against GVHD. In some embodiments, the therapeutic cell population is administered to the subject prior to the immunosuppressive agent. In some embodiments, the therapeutic cell population is administered to the subject after the immunosuppressive agent. In some embodiments, the therapeutic cell population is administered to the subject concurrently with the immunosuppressive agent. In some embodiments, the therapeutic cell population is administered to the subject in the absence of an immunosuppressive agent. In some embodiments, the patient receiving the therapeutic cell population receives the immunosuppressive agent for less than 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, or 1 week.

In some embodiments, a subject receiving Bu/Flu and/or Bu/Cy begins an intravenous infusion of tacrolimus at an initial dose of 0.03 mg/kg/day beginning on day +3 with a target of 4-8 ng/ml. In some embodiments, subjects being vaccinated with Cy/TBI (also for TBI/VP-16 or TBI/VP-16/Cy) began sirolimus on day +3 with an initial dose of 6mg loading dose, followed by 2mg per day, with a target of 3-8 ng/ml. If the subject is determined by the treating physician to become intolerant to their particular GVHD prophylaxis or have other causes of alteration, the treating physician can judge whether to alter the prophylaxis and recommend the use of tacrolimus, sirolimus, or mycophenolate mofetil. If GVHD occurs, the appropriate treatment plan and dosage will begin. Recipients who develop acute GVHD will be treated at the discretion of the treating physician.

Selection and classification of cell populations

Prior to formulation or administration of the therapeutic cells, a source of cells is obtained from a donor (e.g., peripheral blood mononuclear cells, bone marrow, umbilical cord blood) from which the therapeutic cells are enriched or depleted. Methods for enriching or depleting a particular subcellular population in a cell mixture are well known in the art. For example, the cell population can be enriched or depleted by density separation, rosette-bound tetrameric antibody complex-mediated enrichment/depletion, Magnetic Activated Cell Sorting (MACS), molecular phenotypes based on multi-parameter fluorescence, such as Fluorescence Activated Cell Sorting (FACS), or any combination thereof. Additional methods of enriching or depleting a cell population are provided, for example, U.S. provisional application 62/421,979; U.S. patent application publication numbers 2014/0011690; and U.S. patent application publication No. 2016/0245805, which is incorporated by reference herein in its entirety. In general, these methods of enriching or depleting a cell population are generally referred to herein as "sorting" the cell population or "contacting the cells under conditions" to form or produce an enriched (+) or depleted (-) cell population.

Accordingly, embodiments of the present disclosure include a method for producing a pharmaceutical composition, the method comprising processing at least one sample to provide: (a) an enriched population of hematopoietic stem/progenitor cells (HSPCs); (b) an enriched population of regulatory T cells (tregs); (c) an enriched population of memory T cells (Tmem); and (d) formulating the enriched population of HSPCs, memory T cells and tregs as a pharmaceutical composition suitable for administration to a subject, wherein the population of (a) - (c) depletes naive conventional α β -T cells. In some embodiments, the method may further comprise treating the sample to provide an enriched iNKT cell population.

In some embodiments, the method further comprises treating the sample to provide depleted Lin⁺A cell population of cells. The method may comprise providing an enriched population of naive tregs, an enriched population of memory tregs, or both. In some embodiments, providing an enriched population of Tmem comprises providing an enriched population of T central memory stem cells (T)_SCM) Population, enriched T central memory cells (T)_CM) Population, enriched T effector memory cells (T)_EM) A population, or any combination thereof. In some embodiments, the method may further comprise treating the sample to provide an enriched iNKT cell population.

In some embodiments, the enriched HSPC population comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more CD34⁺HSPC. In some embodiments, depleted Lin⁺The cell population of the cells comprises 1-30% Lin⁺Cells, preferably less than 1% Lin⁺A cell. In some embodiments, the enriched population of tregs comprises 20% -99.9% tregs. In some embodiments, the enriched population of Tmem comprises 10% to 99.9% of Tmem. In some embodiments, the enriched iNKT population comprises 10% -99.9% inkts.

In some embodiments, formulating the pharmaceutical composition comprises combining the enriched population of HSPCs, memory T cells, tregs, inkts, or any combination thereof into a mixed population of enriched cells.

In some embodiments, the mixed population of enriched cells comprises a ratio of HSPCs to Tmem of from 500:1 to 1:1,000, 400:1 to 1:1,000, 300:1 to 1:1,000, 200:1 to 1:1,000, 100:1 to 1:1,000, 50:1 to 1:1,000, 10:1 to 1:1,000, 5:1 to 1:1,000, 4:1 to 1:1,000, 3:1 to 1:1,000, 2:1 to 1:1,000, 1:1 to 1:1,000, 500:1 to 1:900, 500:1 to 1:800, 500:1 to 1:700, 500:1 to 1:600, 500:1 to 1:500, 500:1 to 1:400, 500:1 to 1:300, 500:1 to 1:200, 500:1 to 1:100, 500:1 to 1:600, 500:1 to 1:1, 500:1 to 1:1, 1:1, 1:1, 1 500:1 to 1:4, 500:1 to 1:3, 500:1 to 1:2, 500:1 to 1:1, 400:1 to 1:900, 300:1 to 1:800, 200:1 to 1:700, 100:1 to 1:600, 50:1 to 1:500, 10:1 to 1:400, 5:1 to 1:300, 4:1 to 1:200, 3:1 to 1:100, 2:1 to 1:50, or 1:1 to 1: 20. In some embodiments, the mixed population of enriched cells comprises a ratio of HSPCs to Tmem in a range from 10:1 to 1:200, 100:1 to 1:2,000, or 1,000:1 to 1:20,000.

In some embodiments, the mixed population of enriched cells comprises a ratio of HSPCs to tregs that can range from 20:1 to 1:3, 100:1 to 1:30, or 200:1 to 1: 300. The ratio of HSPCs to naive tregs may include a range from 1:500 to 100:1, 1:400 to 100:1, 1:300 to 100:1, 1:200 to 100:1, 1:100 to 100:1, 1:50 to 100:1, 1:20 to 100:1, 1:10 to 100:1, 1:5 to 100:1, 1:1 to 100:1, 1:200 to 50:1, 1:200 to 20:1, 1:200 to 10:1, 1:200 to 5:1, 1:100 to 1:1, 40:1 to 1:3, 200:1 to 1:15, or 400:1 to 1: 150.

In some embodiments, the ratio of HSPCs to memory tregs comprised by the mixed population of enriched cells may comprise a ratio ranging from 1:500 to 10,000:1, 1:400 to 10,000:1, 1:300 to 10,000:1, 1:200 to 10,000:1, 1:100 to 10,000:1, 1:50 to 10,000:1, 1:20 to 10,000:1, 1:10 to 10,000:1, 1:5 to 10,000:1, 1:1 to 10,000:1, 1:500 to 5,000:1, 1:500 to 1,000:1, 1:500 to 900:1, 1:500 to 800:1, 1:500 to 700:1, 1:500 to 600:1, 1:500 to 500:1, 1:500 to 400:1, 1:500 to 300:1, 1:500 to 200:1, 1:500 to 100:1, 1:500 to 500:1, 1: 500:1, 1:500 to 500:1, 1: 500:1, or 1: 500: 1:500 to 500:1, 1: 500:1, 1.

In some embodiments, the enriched mixed population of cells comprises a ratio of HSPCs to inkts that can range from 1:2 to 1,000,000:1, 1:2 to 500,000:1, 1:1 to 500,000:1, 100:1 to 1,000,000:1, 100:1 to 500,000:1, 100:1 to 100,000:1, 500:1 to 1,000,000:1, 500:1 to 500,000:1, 500:1 to 100,000:1, 1,000:1 to 1,000,000:1, 1,000:1 to 500,000:1, 1,000:1 to 100,000:1, 10,000:1 to 1:2, 100,000:1 to 1:20, or 1,000,000:1 to 1: 200.

In some embodiments, the mixed population of enriched cells comprises a ratio of naive conventional α β -T cells to HSPCs of less than 1:3, less than 1:50, less than 1:100, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1,000, less than 1:1,500, less than 1:2,000, less than 1:3,000, less than 1:4,000, less than 1:5,000, less than 1:6,000, less than 1:7,000, less than 1:8,000, less than 1:9,000, less than 1:10,000, less than 1:50,000, less than 1:100,000, less than 1:200,000, less than 1:300,000, less than 1:400,000, less than 1:500,000, less than 1:600,000, less than 1:800,000, less than 1:900, or less than 1:300,000.

In some embodiments, the mixed population of enriched cells comprises a ratio of naive conventional α β -T cells to Tmem of less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1: 50000.

The ratio of naive conventional α β -T cells to tregs may be less than 1:1, 1:10, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1: 50000. The ratio of naive conventional α β -T cells to naive tregs may be less than 1:1, 1:10, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1: 50000. The ratio of naive conventional α β -T cells to memory tregs may be less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1: 50000. The ratio of naive conventional α β -T cells to iNKT may be less than 100:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, less than 1: 50000.

In some embodiments of the present disclosure, the ratio of Tmem to Treg is independent of (e.g., varies greatly from) the concentration of the starting cells. This provides an advantage because the concentration of Tmem can be controlled independently of the concentration of tregs (e.g., dose escalation). The ratio of Tmem to Treg may be from 30:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1: 1. In certain embodiments, the ratio of Tmem to Treg may be from 1:1 to 200:1, 1:10 to 2000:1, or 1:100 to 20,000: 1. The ratio of Tmem to naive tregs may be from 5:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, or 1:1 to 1: 10. The ratio of Tmem to memory Treg may be from 27:1 to 0.9:1, 30:1 to 1:10, 25:1 to 1:10, 20:1 to 1:10, 15:1 to 1:10, 10:1 to 1:10, 30:1 to 1:9, 30:1 to 1:8, 30:1 to 1:7, 30:1 to 1:6, 30:1 to 1:5, 30:1 to 1:4, 30:1 to 1:3, 30:1 to 1:2, or 30:1 to 1: 1.

In some embodiments of the disclosure, the ratio of Treg to iNKT may be from 20,000:1 to 1:5, 200,000:1 to 1:50, or 2,000,000:1 to 1: 500.

In some embodiments of the disclosure, the ratio of iNKT to Tmem may be from 2:1 to 1:100,000, 5:1 to 1:1,000,000, or 10:1 to 1:10,000,000.

In some embodiments, the HSPC is CD34⁺. HSPC can also be described as CD133⁺、CD90⁺、CD38^-、CD45RA^-、Lin^-Or any combination thereof. In some embodiments, the HSPC is CD19^-、TCRα/β^-Or a combination thereof. In some embodiments, Lin⁺The cell expresses CD19, CD11c, CD66B, CD14, CD20, or any combination thereof. In some embodiments, the Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺Or any combination thereof. In some embodiments, the naive Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺、CD45RA⁺、CD45RO^-Or any combination thereof. In some embodiments, the memory Treg is CD4⁺、CD25⁺、CD127^-/lo、FoxP3⁺、CD45RA^-、CD45RO⁺Or any combination thereof. In some embodiments, Tmem is CD3⁺、CD45RA^-、CD45RO⁺Or any combination thereof. In some embodiments, T_SCMIs CD45RA⁺And CD4⁺Or CD8⁺。T_SCMCan also be described as CD95⁺、CD122⁺、CXCR3⁺、LFA-1⁺Or any combination thereof. In some embodiments, T_CMIs CD45RO⁺And CD4⁺Or CD8⁺。T_CMCan also be described as CD45RA^-、CD62L⁺、CCR7⁺Or any combination thereof. In some embodiments, T_EMIs CD4⁺、CD45RO⁺、CD45RA^-、CD62L^-、CCR7^-Or any of themAnd what combinations are. In some embodiments, the iNKT is CD1d-tet⁺、6B11⁺、Va24Ja18⁺Or any combination thereof. In any of the embodiments described herein, the naive conventional α β -T cell is TCR α/β⁺CD45RA⁺And CD25^-、CD127⁺Or both. Naive conventional α β -T cells can also be described as TCR α⁺TCRβ⁺CD45RA⁺CD45RO^-CD25^-CD95^-IL-2Rβ^-CD127⁺。

In some embodiments, the biological sample can be sorted to isolate populations of interest, such as HSPCs, Treg cells, Tmem cells, or combinations thereof. In some cases, a biological sample may be contacted with a molecule that specifically binds CD34 to isolate HSPCs. The sample may be directed against CD34⁺Enrichment was performed to generate CD34⁺Cell population and CD34^-A population of cells. In some cases, CD34 may be used^-The population of cells is contacted with a molecule that specifically binds CD 25. The sample can then be tested for CD34^-Cell population for CD25⁺Sorting of cell populations to enrich for CD25⁺Cells, thereby producing CD34^-CD25^-Cell population and CD34^-CD25⁺A population of cells. CD34 may also be used^-CD25⁺The cell population is sorted to generate a population of Treg cells. Can make CD34^-CD25⁺The cell is contacted with a molecule that specifically binds to CD4 and a molecule that specifically binds to CD 127. The cells can then be sorted to produce CD34^-CD25⁺CD4⁺CD127^dim/-A population of Treg cells. In some cases, CDs 34 may also be sorted^-CD25^-The cell population was enriched for Tmem cells. The cell may be contacted with a molecule that specifically binds CD45 RA. Cell samples can be sorted to produce CD34^-CD25^-CD45RA^-Tmem cell population. CD45RA can be discarded⁺Naive conventional α β -T cells to deplete the naive conventional α β -T cells.

In some embodiments, the biological sample can be sorted to isolate a population of interest, such as HSPCs, Treg cells, Tmem cells, iNKT cells, or a combination thereof. In some casesIn some cases, the biological sample may be contacted with a molecule that specifically binds CD34 to isolate HSPCs. The sample may be directed against CD34⁺Enrichment was performed to generate CD34⁺Cell population and CD34^-A population of cells. In some cases, CD34 may be used^-The population of cells is contacted with a molecule that specifically binds CD25 and a molecule that specifically binds 6B11. The sample can then be tested for CD34^-Cell population for CD25⁺Sorting of cell populations to enrich for CD25⁺And 6B11⁺Cells, thereby producing CD34^-CD25^-6B11^-Cell population and CD34^-CD25⁺6B11⁺A population of cells. CD34 may also be sorted^-CD25⁺6B11⁺Cell populations to generate Treg and iNKT cell populations. Can make CD34^-CD25⁺6B11⁺The cell is contacted with a molecule that specifically binds to CD4 and a molecule that specifically binds to CD 127. The cells can then be sorted to produce CD34^-CD25⁺6B11⁺CD4⁺CD127^dim/-A population of Treg cells. In some cases, CDs 34 may also be sorted^-CD25^-6B11^-The cell population was enriched for Tmem cells. The cell may be contacted with a molecule that specifically binds CD45 RA. Cell samples can be sorted to produce CD34^-CD25^-6B11^-CD45RA^-Tmem cell population. CD45RA can be discarded⁺Naive conventional α β -T cells to deplete the naive conventional α β -T cells. In some embodiments, the method of producing a therapeutic composition described herein can comprise separately sorting different cell populations. For example, HSPCs, Treg cells, Tmem cells, and/or iNKT cells may be sorted individually. The separately sorted populations can be mixed to form a therapeutic composition. In some cases, separate cell populations may be isolated from different donors. For example, HSPCs can be isolated from donor 1, and Treg and Tmem cells can be sorted from donor 2. Alternatively, all cell populations may be isolated from the same donor. In some embodiments, the sample comprises mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, unmoved apheresis product, or any combination thereof. In some embodimentsWherein the sample comprises cultured cells derived from PBMCs. In some embodiments, the sample comprises cultured cells derived from induced pluripotent stem cells (ipscs). In some embodiments, the sample is prepared for processing a density gradient, Ficoll, Percoll, erythrocytic hypotonic lysis, ammonium chloride-potassium (ACK) buffer, or any combination thereof. In some embodiments, the sample is provided by a single tissue harvest. In some embodiments, the sample is provided by one or more tissue harvests.

Example sorting scheme 1

In certain embodiments, a method for producing a pharmaceutical composition comprises: A. contacting the sample with a molecule that specifically binds to CD34 under conditions to form CD34⁺Cell population and CD34^-Cell population, recovering CD34 from the sample⁺Cell population and recovering CD34 from the sample^-A population of cells; processing CD34^-The cell population to provide at least one enriched therapeutic cell population comprising tregs, Tmem, iNKT, or any combination thereof, (see fig. 1A and 1B). In some embodiments, step B comprises performing fine sorting to provide an enriched population of therapeutic cells (see fig. 1A). For example, step B may include having CD34^-The population of cells is contacted with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, a CD1d-tet, a 6B11 monoclonal antibody, or a functional fragment thereof, or any combination thereof.

In certain embodiments, step B comprises: i. under conditions such that CD34 is not present^-Contacting the population of cells with at least one molecule that specifically binds to CD45RA to form CD45RA⁺Cell population and CD45RA^-Cell population and recovering CD45RA^-A population of cells; performing fine sorting to separate from CD45RA⁺An enriched therapeutic cell population is provided in the cell population. Fine sorting may include sorting CD45RA⁺The cell population is combined with a molecule specifically binding to CD4, a molecule specifically binding to CD8, a molecule specifically binding to CD25, a molecule specifically binding to CD127, a CD1d-tet, a 6B11 monoclonal antibody or a functional sheet thereofSegments, or any combination thereof. Fine sorting may also include refining CD45RA^-The population of cells is contacted with a molecule that specifically binds CD45RA, a molecule that specifically binds CD45RO, or a combination thereof.

In certain embodiments, step B comprises: i. under conditions such that CD34 is not present^-The cell population is combined with at least one specific binding Lin⁺Binding molecule contact of markers to form Lin⁺Cell population and Lin^-Cell population and Lin recovery^-A population of cells; performing fine sorting to separate from Lin^-An enriched therapeutic cell population is provided in the cell population. In some embodiments, fine sorting comprises contacting Lin^-The population of cells is contacted with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, a CD1d-tet molecule, a 6B11 monoclonal antibody, or a functional fragment thereof, or any combination thereof.

In certain embodiments, step B comprises: i. under conditions such that CD34 is not present^-The cell population is specifically combined with at least one Lin⁺Binding molecule contact of markers to form Lin⁺Cell population and Lin^-Cell population and Lin recovery^-A population of cells; under certain conditions, Lin^-Contacting the population of cells with a binding molecule that specifically binds to CD25 to form CD25⁺Cell population and CD25^-Recovering CD25 from the cell population⁺A population of cells, thereby generating a population of cells comprising tregs, and recovering CD25^-A population of cells; subjecting CD25 to conditions^-Contacting the population of cells with a binding molecule that specifically binds to CD45RA to form CD45RA⁺Cell population and CD45RA^-Cell population and recovering CD45RA^-Cell population (see FIG. 1A). In some embodiments, step ii^-Contacting the cell with a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof to form a CD1d-tet⁺Cell population, 6B11⁺Cell population or combination thereof and CD1d-tet^-Cell, 6B11^-Cells or combinations thereofAnd recovering CD1d-tet⁺A population of cells. In some embodiments, CD25 is recovered simultaneously⁺Cell population and CD1d-tet⁺Cell, 6B11⁺A cell or a population of both. In some embodiments, the method further comprises CD25 for step ii⁺The population of cells performs fine sorting to provide a naive population of Treg cells, a memory population of Treg cells, an iNKT cell population, or any combination thereof. In some embodiments, CD45RA is recovered⁺A population of cells, and further performing fine sorting to provide a population of tregs, inkts, or both.

Example sorting scheme 2

In certain embodiments, a method for producing a pharmaceutical composition comprises: A. performing a first rough sort on at least a first sample, thereby providing enriched CD34⁺A population of cells; B. performing a second rough sort on a second sample, thereby providing Lin^-A population of cells; C. to Lin^-The cell population was subjected to a third rough sort to provide CD45RA^-Memory T cell population and provide CD45RA⁺A population of cells; to CD45RA⁺The cell population is subjected to fine sorting, thereby providing a population of tregs (see fig. 2 and 3).

In some embodiments, the second sample comprises CD34 recovered from step a^-Cell population (see FIG. 3). In some embodiments, the first sample comprises at least one haploidentical sample (see fig. 2A). In some embodiments, the first sample comprises at least two haploidentical samples (see fig. 2B). In some embodiments, the first sample comprises mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, unmoved apheresis product, or any combination thereof. In some embodiments, the second sample comprises Peripheral Blood Mononuclear Cells (PBMCs), mobilized peripheral blood, mobilized apheresis product, bone marrow, cord blood, non-mobilized apheresis product, or any combination thereof. In some embodiments, the first sample or the second sample is allogeneic, autologous, or a combination thereof. In some embodiments, the second sample is from an HLA-matched unrelatedDonors, HLA-matched sibling donors, or combinations thereof (see fig. 2A and B).

In some embodiments, the first, second, or third rough sorting comprises density separation, tetrameric antibody complex-mediated enrichment/depletion, magnetically activated cell sorting, apheresis, leukopheresis, or any combination thereof.

In some embodiments, fine sorting may include purification using a molecular phenotype based on multi-parameter fluorescence. The fine sorting may provide a naive population of Treg cells, a memory population of Treg cells, an iNKT cell population, or any combination thereof. Fine sorting may involve subjecting CD45RA to conditions⁺Contacting a cell with a binding molecule that specifically binds to CD25 to provide CD25⁺Cell population and CD25^-Cell population and recovering CD25⁺A population of cells, thereby providing a population of tregs. In some embodiments, the CD25 is further sorted under conditions⁺A population of cells to provide a population of naive Treg cells. In some embodiments, fine sorting comprises subjecting CD45RA to conditions⁺Contacting the cell with a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, to provide a CD1d-tet⁺Cell, 6B11⁺Cells or both, and recovering CD1d-tet⁺Cell, 6B11⁺A cell or a population of both.

Example sorting scheme 3

In certain embodiments, a method for producing a pharmaceutical composition comprises: A. enabling the sample to be specifically combined with Lin under certain conditions⁺Binding molecule contacting of marker to provide Lin⁺Cell population and Lin^-Cell population and Lin recovery^-A population of cells; B. under certain conditions, Lin is made^-Contacting a cell with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25 to provide CD34⁺Cell population, CD25⁺Cell population and CD34^-CD25^-Cell population and recovering CD34⁺Cells and CD25⁺Cells, and recovering CD34^-CD25^-A population of cells; under certain conditions, CD34^-CD25^-Cell population and specific binding CD45RA to provide CD45RA⁺Cell population and CD45RA^-Cell population and recovering CD45RA^-Clusters (see fig. 4).

In some embodiments, step B further comprises contacting Lin under conditions^-Contacting the cell with a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof to provide a CD1d-tet⁺Cell, 6B11⁺A population of cells or a combination thereof, and recovering CD1d-tet⁺Cell, 6B11⁺Cells or combinations thereof (see fig. 4). In some embodiments, the method further comprises screening for CD34⁺Cell, CD25⁺Cell, CD1d-tet⁺The cells, 6B11+ cells, or any combination thereof perform fine sorting, thereby providing CD34⁺Cell, CD25⁺Cell, CD1d-tet⁺Cell, 6B11⁺A population of cells or any combination thereof (see figure 4). In some embodiments, fine sorting comprises subjecting CD34 to conditions⁺Cell, CD25⁺Cell, CD1d-tet⁺Cell, 6B11⁺Contacting a cell or any combination thereof with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, to provide a highly enriched CD34⁺Cell, CD4⁺CD25⁺CD127^-/LoCell, CD1d-tet⁺A cell population of cells or a combination thereof (see figure 4).

Example sorting scheme 4

In certain embodiments, a method for producing a pharmaceutical composition comprises: A. performing rough sorting on a sample to provide Lin⁺Cell population and Lin^-Cell population and Lin recovery^-A population of cells; B. to Lin^-The cell population was roughly sorted to provide a population enriched for HSPC and Tmem and CD45RA⁺Recovering a population of cells, and recovering a population of HSPC and Tmem and recovering CD45RA⁺A population of cells; to CD45RA⁺The cell population was subjected to fine sorting to provide a population of tregs (see figure 5). In some embodiments, fine sorting is further included at certain intervalsConditions such that CD45RA⁺Contacting the population of cells with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, and a binding molecule that specifically binds to CD127 to provide CD34⁺Cell population and CD4⁺CD25⁺CD127^-/LoCell population and recovering CD34⁺Cell population and CD4⁺CD25⁺CD127^-/LoA population of cells. In some embodiments, fine sorting further comprises subjecting CD45RA to⁺Contacting the population of cells with CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, and recovering the CD1d-tet⁺Cell, 6B11⁺A population of cells or a combination thereof. In some embodiments, rough sorting comprises density separation, tetrameric antibody complex-mediated enrichment/depletion, magnetically activated cell sorting, apheresis, leukopheresis, or any combination thereof.

Example sorting scheme 5

In certain embodiments, a method for producing a pharmaceutical composition comprises: A. contacting the sample with a binding molecule that specifically binds to CD34 under conditions to provide CD34⁺Cell population and CD34^-Recovering CD34 from the cell population⁺Cell population and recovering CD34^-A population of cells; B. under conditions such that CD34 is not present^-Contacting the population of cells with a binding molecule that specifically binds to CD25 to provide CD25⁺Cell population and CD25^-Recovering CD25 from the cell population⁺Cell population and recovering CD25^-A population of cells; under certain conditions, CD25^-Contacting the population of cells with a binding molecule that specifically binds to CD45RA to provide CD45RA⁺Cell population and CD45RA^-Cell population and recovering CD45RA^-Cell populations (see fig. 6, 10, 11, 12).

In some embodiments, step B further comprises: i. under conditions such that CD34 is not present^-Contacting the population of cells with a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof to provide a CD1d-tet⁺Cell population, 6B11⁺Cell population or combination thereof and CD1d-tet^-Cell population, 6B11^-Cell population or combination thereof, and recovering CD1d-tet⁺Cell population, 6B11⁺A cell population or a combination thereof, thereby providing an iNKT depleted cell population and recovering CD1d-tet^-Cell population, 6B11^-A population of cells, or both, thereby providing an iNKT depleted population of cells; contacting the iNKT depleted cell population with a binding molecule that specifically binds CD25 under conditions to provide CD25⁺Cell population and CD25^-Cell population and recovering CD25⁺Cell population and recovering CD25^-Cell population (see figure 6).

In some embodiments, step B comprises contacting CD34 under conditions^-Contacting the population of cells with a binding molecule that specifically binds to CD25 and a CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, to provide CD25⁺Cell population and CD1d-tet⁺Cell, 6B11⁺A population of cells or combinations thereof and CD34^-CD25^-iNKT depleted cell population and recovering CD25⁺Cell population and CD1d-tet⁺Cell, 6B11⁺A population of cells or combinations thereof, and recovering CD34^-CD25^-iNKT depleted cell populations (see figure 10).

In some embodiments, step B comprises: i. under conditions such that CD34 is not present^-Contacting the population of cells with a binding molecule that specifically binds to CD25 and a CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, to provide CD25⁺Cell population and CD1d-tet⁺Cell, 6B11⁺A population of cells or a combination thereof, and CD34^-CD25^-iNKT depleted cell population and recovering CD25⁺Cell population and CD1d-tet⁺Cell, 6B11⁺A population of cells or combinations thereof, and recovering CD34^-CD25^-iNKT depleted cell populations; treating CD25 by contacting the cell with a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or any combination thereof⁺Cell population and CD1d-tet⁺Cell, 6B11⁺Performing fine sorting of the population of cells or combinations thereof to provide enriched CD4⁺CD25⁺CD127^-/LoCell, CD1d-tet⁺Cell, 6B11⁺A cell population of cells or any combination thereof (see figure 11).

In some embodiments, step B comprises: i. make CD34^-The cell population is contacted with an anti-CD 25 antibody comprising a label (e.g., fluorescent phycoerythrin) and a biotinylated 6B11 monoclonal antibody (6B 11-biotin). 6B 11-biotin was then contacted with streptavidin conjugated with the same label as the anti-CD 25 antibody. In some embodiments, streptavidin is conjugated to phycoerythrin/Cy 7(SAv-PE/Cy 7). The cells are then contacted with anti-label magnetic particles. In some embodiments, the magnetic particles are anti-PE magnetic particles (e.g., magnetic beads). CD25 was then isolated using MACS⁺Cells and 6B11 bound cells to produce CD25⁺Cell sum 6B11⁺A population of cells, and CD34^-CD25^-iNKT depleted cell populations (see figure 15). In some embodiments, step B further comprises: targeting CD25 by contacting the cell with a binding molecule that specifically binds CD4 and a binding molecule that specifically binds CD127 under conditions⁺Cell sum 6B11⁺The population of cells is subjected to fine sorting to provide enriched CD4⁺CD25⁺CD127^-/LoCell population and enrichment of cells 6B11⁺CD127⁺A cell population of cells, or any combination thereof. In some embodiments, the CD4 binding molecule is an anti-CD 4PerCP labeled antibody. In some embodiments, the CD127 binding molecule is an anti-CD 127 APC-labeled antibody. Fine sorting may include FACS in which CD25-PE, 6B 11-biotin-SAv-PE/Cy 7, CD4-PerCP and CD127-APC are detected.

In some embodiments, the CD34 recovered in step a⁺Cell population, CD25 recovered in step B⁺The cell population, or both, is further processed by fine sorting, which comprises subjecting CD34 to⁺Cell, CD25⁺The cell or combination thereof is contacted with a binding molecule that specifically binds CD34, a binding molecule that specifically binds CD127, a binding molecule that specifically binds CD45RA, or any combination thereof (see fig. 12).

Example sorting scheme 6

In certain embodiments, a method for producing a pharmaceutical composition comprises: A. contacting the sample with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25 under conditions to provide CD34⁺Cell population, CD25⁺Cell population and CD34^-CD25^-Recovering CD34 from the cell population⁺Cell population and CD25⁺Cell population and recovering CD34^-CD25^-A population of cells; under certain conditions, CD34^-CD25^-Contacting the population of cells with a binding molecule that specifically binds to CD45RA to provide CD45RA⁺Cell population and CD45RA^-Cell population and recovering CD45RA^-Cell population (see FIG. 7). In some embodiments, step A further comprises contacting the sample with a CD1d-tet, a 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, under conditions to provide CD1d-tet⁺Cell population, 6B11⁺Cell population or combination thereof, and recovering CD1d-tet⁺Cell population, 6B11⁺A population of cells or a combination thereof (see figure 7). In some embodiments, the method further comprises performing fine sorting on the population of cells provided in step a by contacting the cells with a binding molecule that specifically binds CD34, a binding molecule that specifically binds CD4, a binding molecule that specifically binds CD25, a binding molecule that specifically binds CD127, CD1d-tet, or any combination thereof, to provide enriched CD34⁺Cell, CD4⁺CD25⁺CD127^-/LoCell, CD1d-tet⁺A cell population of cells or any combination thereof (see figure 8).

Example sorting scheme 7

In certain embodiments, the methods for producing a pharmaceutical composition comprise simultaneously treating a sample to provide an enriched cell population comprising HSPCs, Tmem, naive tregs, memory tregs and comprising less than 5% of undesired cell types (see fig. 13). In some embodiments, the sample is contacted with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD8, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45ROOr any combination thereof. In some embodiments, the method further comprises contacting the sample with CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, and recovering the CD1d-tet⁺Cell population, 6B11⁺A population of cells, or a combination thereof. In some embodiments, the sample is contacted with a binding molecule that specifically binds CD34, and CD34 is recovered⁺A population of cells, thereby generating a population of HSPCs. In some embodiments, the sample is contacted with a binding molecule that specifically binds CD3 and does not bind to a binding molecule that specifically binds CD45RA, a binding molecule that specifically binds CD45RO, or a combination thereof, and CD3 is recovered⁺CD45RA^-CD45RO⁺A population of cells. In some embodiments, the sample is contacted with a binding molecule that specifically binds CD4, a binding molecule that specifically binds CD25, a binding molecule that specifically binds CD127, a binding molecule that specifically binds CD45RA, a binding molecule that specifically binds CD45RO, or any combination thereof, and CD4 is recovered⁺CD25⁺CD127^-/lo CD45RA⁺CD45RO^-Cell population, CD4⁺CD25⁺CD127^-/lo CD45RA^-CD45RO⁺A population of cells.

In any of the preceding embodiments, the therapeutic cell population comprises less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001% naive conventional α β -T cells.

In any of the above embodiments, Lin⁺The marker may be CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

In any of the above embodiments, the specific binding to CD34, Lin⁺The molecule of the marker, CD25, CD45RA, CDR45RO, CD4, CD8, CD127, CD90, CD133, CD38, CD95, CD122, CXCR3, LFA-1, CD62L, CCR7 or any other cellular marker is an antibody or antibody fragment.

As used herein, the term "antibody" is a broad term and is used in its ordinary sense, including but not limited to, reference to naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional and humanized antibodies and antigen binding fragments thereof. It will be understood that the choice of the molecular epitope or region against which an antibody is directed will determine its specificity, e.g., for each molecular form (if present) or for the totality (e.g., all or substantially all of the molecules).

Methods for producing antibodies are well known. One skilled in the art will recognize that a number of procedures may be used to generate Antibodies, for example, as described in Antibodies, a Laboratory Manual, Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, n.y. It will also be appreciated by those skilled in the art that binding fragments or Fab fragments of the mimobodies can be prepared from genetic information by various procedures (Antibody Engineering: A Practical Approach (Borreboeck, C., eds.), 1995, Oxford University Press, Oxford; J.Immunol.149,3914-3920 (1992)). Monoclonal and polyclonal antibodies to molecules such as proteins and markers are also commercially available (R and D Systems, Minneapolis, Minn.; HyTest Ltd., Turk, Finland; Abcam Inc., Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass., USA; BiosParafic, Emeryville, Calif.).

In some embodiments, the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody. In embodiments, the antibodies of the present disclosure are compatible with downstream applications of cell populations extracted according to the present methods. For example, the antibodies of the present disclosure can be non-immunogenic humanized antibodies. In some embodiments, the antibodies of the present disclosure comprise an epitope tag for immobilizing the antibody prior to or after extraction of the sample, thereby depleting the antibody from the extracted cell population.

In some embodiments, the antibody is a biotinylated 6B11 antibody. 6B11 is an antibody that binds to constant TCR Va24Ja 18. In some embodiments, the 6B11 antibody is biotinylated at pH 5.5-6.0, 6.0-6.5, 6.5-7, 7.5-8, 8-8.5, 8.5-9, 9-9.5, or 9.5-10. In some embodiments, the 6B11 antibody is biotinylated in phosphate, borate, or hepes buffers. In some embodiments, the buffer is an amine-free buffer. BiotinThe reaction can be carried out at about 0mM NaCl, 50mM NaCl, 100mM NaCL, 150mM NaCl, 300mM NaCl or 500mM NaCl or an intermediate value. Modification sites for proteins (e.g., antibodies) include free amines, free thiols and carbohydrates, artificially introduced azides, and artificially introduced alkynes. The concentration of biotin may vary between 10. mu.M-50. mu.M, 50. mu.M-100. mu.M, 100. mu.M-250. mu.M, 250. mu.M-500. mu.M, 500. mu.M-1 mM, 1mM-2mM, 2mM-5mM, 5mM-10 mM. The reaction time may vary from 10 to 30 minutes, 30 minutes to 1 hour, 1 hour to 1.5 hours, 1.5 hours to 2.5 hours, 2.5 hours to 6 hours, 6 hours to 10 hours, 10 hours to 18 hours. The biotinylated 6B11 antibodies disclosed herein may be used in any of the methods disclosed herein to sort, purify, or isolate Va24Ja18⁺Cell (iNKT cell) population.

Capture and detection binding partner pairs, e.g., capture and detection antibody pairs, may be used in embodiments of the present disclosure. Thus, in some embodiments, a sorting and purification scheme is used in which two binding partners, e.g., two antibodies, are typically used. One binding partner is a capture partner, typically immobilized on a particle, and the other binding partner is a detection binding partner, typically attached to a detectable label. Such antibody pairs are available from several commercial sources, such as BiosPacific, Emeryville, calif. Antibody pairs can also be designed and prepared by methods well known in the art. In particular embodiments, the antibody is biotinylated or biotinylated.

In some embodiments, there is a second imaging component that non-specifically binds all members of the starting cell population. Thus, the signal can be read to normalize the amount of fluorescence between the chambers. One example is an antibody that will bind to one or more proteins that are ubiquitously expressed on the cell surface of the starting cell population.

In some embodiments, the antibody or antibody fragment is conjugated to or labeled with a fluorescent dye, hapten or magnetic particle.

Several strategies are known in the art that can be used to label the binding partners to enable their detection or discrimination in a mixture of particles. The tag may be attached by any known means, including methods that utilize non-specific or specific interactions. In addition, labeling can be accomplished directly or via a binding partner.

The emission from the moiety, e.g., fluorescence, should be sufficient to allow detection using the detector described herein. In general, the compositions and methods of the present disclosure utilize a highly fluorescent moiety, e.g., a moiety capable of emitting electromagnetic radiation when excited by an electromagnetic radiation source at the excitation wavelength of the moiety. Several portions are suitable for use in the compositions and methods of the present disclosure.

In addition to electromagnetic radiation, tags that can be activated by energy are also useful in the present disclosure. Such labels may be activated by, for example, electrical, thermal, or chemical reaction (e.g., chemiluminescent labels). Likewise, many enzymatically activated tags are well known to those skilled in the art.

In general, the fluorescence of the moiety involves a combination of quantum efficiency and lack of sufficient photobleaching (to make the moiety detectable above background levels in the disclosed detector) with the consistency required for the detection limits, accuracy and precision desired for the assay.

Furthermore, the nature of the moiety is consistent with its use in the chosen assay. In some embodiments, the assay is an immunoassay, wherein a fluorescent moiety is attached to an antibody; the moiety must have properties such that it does not aggregate with other antibodies or proteins, or that it does not undergo aggregation beyond that which is consistent with the required assay accuracy and precision. In some embodiments, the fluorescent moiety dye molecule has a combination of: 1) high absorption coefficient; 2) high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with labeling of a molecule of interest (e.g., a protein) such that analysis can be performed using the analytical instruments and systems of the present disclosure (e.g., without causing precipitation of the protein of interest, or of the protein to which the moiety is attached).

The fluorescent moiety may comprise a single entity (quantum dot or fluorescent molecule) or multiple entities (e.g., multiple fluorescent molecules). It is to be understood that when the term "moiety" is used herein to refer to a group of fluorescent entities (e.g., a plurality of fluorescent dye molecules), each individual entity may be attached to a binding partner separately, or the entities may be attached together, so long as the entities as a group provide sufficient fluorescence for detection.

In some embodiments, the fluorescent dye molecule comprises at least one substituted indole ring system wherein the substituent on the 3-carbon of the indole ring comprises a chemically reactive group or conjugate. Examples include Alexa Fluor molecules.

In some embodiments, the tags include tags of a first type and a second type, e.g., two different ALEXA' sA dye (Invitrogen), wherein the dye molecules of the first type and the second type have different emission spectra.

A non-inclusive list of useful fluorescent entities for the fluorescent moiety includes: ALEXA488、ALEXA532、ALEXA555、ALEXA647、ALEXA700、ALEXA750. Fluorescein, B-phycoerythrin, allophycocyanin, PBXL-3, Atto 590 and Qdot 605.

The tag may be attached to the particle or binding partner by any method known in the art, including adsorption, covalent binding, biotin/streptavidin, or other binding pairs. Further, the tag may be attached by a connector. In some embodiments, the tag is cleaved by the analyte, thereby releasing the tag from the particle. Alternatively, the analyte may prevent cleavage of the linker.

The isolated cell population may be used immediately or may be cultured in vitro after isolation using methods well known in the art. The cell population may be cultured in a medium such as RPMI-1640, DMEM, X-Vivo 10, X-Vivo 15, or variants and combinations thereof. The culture medium may contain 1-20% human serum. The culture medium may comprise a receptor-activating cytokine or molecule, such as rapamycin, SCF, SDF-1, TPO, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-15, IL-17, IL-18, IL-23, IL-33, TGF-b, IFNg, IFNa, and combinations thereof. The cell population can be stimulated with an agonist such as a particle (microparticle, nanoparticle, protein, or cell) comprising a multivalent display of a binding molecule such as anti-CD 3, anti-CD 28, CD64, CD86, anti-IL-21R, CD137, or a combination thereof.

In addition, the cells may be frozen, or may be frozen before or after isolation. In case the cells are to be stored for a longer time, storage at around-80 ℃ or in liquid nitrogen is preferred to ensure that the cells can be reused after thawing. For this purpose, the cells are usually stored in DMSO and/or FCS/HS together with culture medium, glucose, etc. After thawing the cells, they can be used directly for therapeutic purposes or in vitro experiments, or expanded and/or differentiated using growth factors, antigens, cells, etc.