阅读说明：本技术 用于调节免疫系统的生物工程化支架及其用途 (Bioengineered scaffolds for modulating immune system and uses thereof ) 是由 N·J·沙 A·S·毛 M·D·克尔 D·J·穆尼 D·T·斯卡登 于 2019-12-12 设计创作，主要内容包括：本发明提供了调节受试者中免疫系统的组合物和方法。(The present invention provides compositions and methods for modulating the immune system in a subject.)

1. A composition for modulating the immune system in a subject comprising:

a porous scaffold;

a growth factor present in about 1ng to about 1000ng per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and

Inducing differentiation factors that recruit cells to differentiate into T cell progenitors.

2. The composition of claim 1, wherein the growth factor is present at about 0.03ng/mm³To about 350ng/mm³Support bodyAre accumulated.

3. A composition for modulating the immune system in a subject comprising:

a porous scaffold;

growth factor at about 0.03ng/mm³To about 350ng/mm³And in an amount effective to induce tissue or organ formation within the scaffold and recruit cells into the scaffold; and

inducing differentiation factors that recruit cells to differentiate into T cell progenitors.

4. The composition of claims 1-3, wherein the scaffold comprises a hydrogel.

5. The composition of any one of claims 1-4, wherein the scaffold comprises a cryogel.

6. The composition of any one of claims 1-5, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly (lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazene, poly (vinyl alcohol), poly (alkylene oxide), poly (ethylene oxide), poly (allylamine), poly (acrylate), poly (4-aminomethylstyrene), pluronic polyols, poloxamers, poly (uronic acid), poly (anhydride), poly (vinylpyrrolidone), and any combination thereof.

7. The composition of any one of claims 1-6, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof.

8. The composition of any one of claims 1-7, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof.

9. The composition of any one of claims 1-8, wherein the scaffold comprises pores having a diameter of 1 μ ι η and 100 μ ι η.

10. The composition of any one of claims 1-8, wherein the scaffold comprises macropores.

11. The composition of claim 10, wherein the macropore diameter is about 50 μ ι η and 80 μ ι η.

12. The composition of claim 10 or 11, wherein the scaffold comprises macropores of different sizes.

13. The composition of any one of claims 1-12, wherein the scaffold is injectable.

14. The composition of any one of claims 1-13, wherein the scaffold comprises a methacrylated alginate (MA-alginate).

15. The composition of any one of claims 1-14, wherein the scaffold comprises hyaluronic acid or a hyaluronic acid derivative.

16. The composition of any one of claims 1-15, wherein the scaffold comprises a click hydrogel or a click cryogel.

17. The composition of claim 16, wherein the scaffold comprises click alginate, click gelatin, or click hyaluronic acid.

18. The composition of any one of claims 1-17, wherein the scaffold comprises pore-forming hydrogel microbeads and a bulk hydrogel, wherein the pore-forming hydrogel microbeads degrade at least 10% faster than the bulk hydrogel polymer scaffold upon administration of the scaffold into a subject.

19. The composition of claim 18, wherein the porogenic hydrogel microbeads comprise oxidized alginate.

20. The composition of any one of claims 1-19, wherein the cell is a stem cell.

21. The composition of claim 20, wherein the stem cells are hematopoietic stem cells.

22. The composition of any one of claims 1-19, wherein the cells are progenitor cells.

23. The composition of any one of claims 1-22, wherein the tissue or the organ comprises a bone tissue or a hematopoietic tissue.

24. The composition of any one of claims 1-23, wherein the tissue or the organ is formed about 7-21 days after administration of the composition to the subject.

25. The composition of claim 24, wherein the tissue or the organ is formed about 14 days after administration of the composition to the subject.

26. The composition of any one of claims 1-25, wherein the cells are stromal cells.

27. The composition of any one of claims 1-26, wherein the scaffold is about 100 μ ι η in size³To about 10cm³。

28. The composition of claim 27, wherein the scaffold is about 10mm in size³To about 100mm³。

29. The composition of claim 28, wherein the scaffold is about 30mm in size³。

30. The composition of any one of claims 1-29, wherein the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF- β) superfamily.

31. The composition of claim 30, wherein the growth factor comprises a protein selected from BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, Growth Differentiation Factor (GDF) -1, GDG-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-mullerian hormone (AMH), activin, Nodal, TGF- β 1, TGF- β 2, TGF- β 3, TGF- β 4, and any combination thereof.

32. The composition of claim 30 or 31, wherein the growth factor comprises BMP-2.

33. The composition of claim 30 or 31, wherein the growth factor comprises TGF- β 1.

34. The composition of any one of claims 1-33, wherein the growth factor is present at about 5ng to about 500 ng.

35. The composition of claim 34, wherein the growth factor is present at about 5ng to about 250 ng.

36. The composition of claim 35, wherein the growth factor is present at about 5ng to about 200 ng.

37. The composition of claim 36, wherein the growth factor is present at about 200 ng.

38. The composition of claim 37, whereinThe growth factor is increased by about 6ng/mm³To about 10ng/mm³Are present.

39. The composition of claim 38, wherein the growth factor is present at about 6.5ng/mm³To about 7.0ng/mm³Are present.

40. The composition of any one of claims 1-39, wherein the growth factor retains its biological activity for at least twelve days following incorporation of the growth factor into the scaffold.

41. The composition of any one of claims 1-40, wherein the T cell progenitor cells are capable of differentiating into T cells.

42. The composition of claim 41, wherein the T cells comprise cells selected from the group consisting of CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), and any combination thereof.

43. The composition of claim 41 or 42, wherein the T cells comprise Tregs.

44. The composition of any one of claims 1-43, wherein the differentiation factor binds to a Notch receptor.

45. The composition of claim 44, wherein the Notch receptor is selected from the group consisting of a Notch-1 receptor, a Notch-2 receptor, a Notch-3 receptor, a Notch-4 receptor, and any combination thereof.

46. The composition of claim 44 or 45, wherein the differentiation factor is selected from the group consisting of delta-like 1(DLL-1), delta-like 2(DLL-2), delta-like 3(DLL-3), delta-like 4(DLL-4), Jagged 1, Jagged 2, and any combination thereof.

47. The composition of any one of claims 1-46, wherein the differentiation factor is covalently linked to the scaffold.

48. The composition of claim 47, wherein the differentiation factor is covalently attached to the scaffold using click chemistry.

49. The composition of claim 47 or 48, wherein the differentiation factor is covalently attached to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry, NHS and Dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reactions, azide and dibenzocyclooctyne chemistry, tetrazine and trans-cyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry.

50. The composition of any one of claims 1-49, wherein said differentiation factor is present in an amount of about 1ng to about 1000 μ g per scaffold.

51. The composition of claim 50, wherein said differentiation factor is present in an amount from about 1 μ g to about 100 μ g per scaffold.

52. The composition of claim 51, wherein said differentiation factor is present in an amount from about 1 μ g to about 10 μ g per scaffold.

53. The composition of claim 52, wherein said differentiation factor is present at about 6 μ g per scaffold.

54. The composition of any one of claims 1-53, wherein said differentiation factor retains its biological activity for at least about three months after incorporation of said differentiation factor into said scaffold.

55. The composition of any one of claims 1-54, wherein the recruited cell is a transplanted cell.

56. The composition of any one of claims 1-54, wherein the recruited cell is not a transplanted cell.

57. The composition of claim 55, wherein the recruited cells are autologous.

58. The composition of claim 55, wherein the recruited cells are allogeneic.

59. The composition of claim 55, wherein the recruited cells are xenogeneic.

60. The composition of any one of claims 1-59, wherein the differentiated cells are capable of migrating out of the scaffold.

61. The composition of claim 60, wherein the differentiated cells are capable of homing into the tissue of the subject following administration of the composition to the subject.

62. The composition of any one of claims 1-61, further comprising a homing factor capable of promoting recruitment of the cells to the scaffold.

63. The composition of claim 62, wherein the homing factor comprises stromal cell derived factor (SDF-1).

64. A method of modulating the immune system in a subject, comprising administering to the subject the composition of any one of claims 1-63, thereby modulating the immune system in the subject.

65. A method of reducing immune hyperreactivity in a subject, comprising administering to the subject the composition of any one of claims 1-63, thereby reducing immune hyperreactivity in the subject.

66. A method of increasing donor chimerism in a subject receiving a transplant, comprising administering to the subject the composition of any one of claims 1-63, thereby increasing donor chimerism in the subject.

67. A method of promoting T cell balance remodeling in a subject, comprising administering to the subject the composition of any one of claims 1-63, thereby promoting T cell balance remodeling in the subject.

68. The method of any one of claims 64-67, further comprising administering hematopoietic stem cells or hematopoietic progenitor cells to the subject.

69. The method of claim 68, wherein the composition is administered to the subject simultaneously with or after the administration of hematopoietic stem cells or hematopoietic progenitor cells to the subject.

70. The method of claim 68 or 69, wherein about 1x10 per kilogram body weight of the subject is administered to the subject⁵To about 50x10⁶Hematopoietic stem cells and/or hematopoietic progenitor cells.

71. The method of claim 70, wherein the subject is administered about 1x10 per kilogram body weight of the subject⁵To about 1x10⁶Hematopoietic stem cells or hematopoietic progenitor cells.

72. The method of any one of claims 64-71, wherein the method enhances T cell reconstitution in the subject.

73. The method of claim 72, wherein the method enhances T cell neogenesis.

74. The method of claim 73, wherein enhanced T cell neogenesis is characterized by an enhanced T cell receptor excision cycle (TREC).

75. The method of any one of claims 64-74, wherein the method enhances T cell diversity in the subject.

76. The method of claim 75, wherein the T cell diversity is characterized by an enhanced T Cell Receptor (TCR) repertoire.

77. The method of any one of claims 64-76, wherein the method increases regulatory T cells (T)_reg) The level of (c).

78. The method of any one of claims 64-77, wherein the subject is a human with an impaired immune system.

79. The method of claim 78, wherein the subject has an impaired immune system as a result of immunosenescence.

80. The method of claim 79, wherein the subject is over 30, 40, 50, 60, 70, or 80 years old.

81. The method of any one of claims 78-80, wherein the subject has an impaired immune system due to an innate immune deficiency.

82. The method of any one of claims 78-81, wherein the subject has acquired immunodeficiency.

83. A method of reducing immune hyperreactivity in a subject comprising administering to the subject a composition comprising:

a porous scaffold;

a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and

inducing differentiation factors that recruit cells to differentiate into T cell progenitors,

thereby reducing immune hyperreactivity in the subject.

84. A method of increasing donor chimerism in a subject receiving a transplant comprising administering to the subject a composition comprising:

a porous scaffold;

a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and

inducing differentiation factors that recruit cells to differentiate into T cell progenitors,

thereby increasing donor chimerism in the subject.

85. A method of promoting T cell balance remodeling in a subject comprising administering to the subject a composition comprising:

a porous scaffold;

a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and

Inducing differentiation factors that recruit cells to differentiate into T cell progenitors,

thereby resulting in a reestablishment of T cell balance in the subject.

86. A method of modulating the immune system of a human having an impaired immune system comprising administering to the human a composition comprising:

a porous scaffold;

a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and

inducing differentiation factors that recruit cells to differentiate into T cell progenitors,

thereby modulating the immune system of the human,

wherein the human has an impaired immune system due to immunosenescence, congenital immunodeficiency or acquired immunodeficiency.

87. The method of any one of claims 83-86, wherein the scaffold comprises a hydrogel.

88. The method of any one of claims 83-87, wherein the scaffold comprises a cryogel.

89. The method of any one of claims 83-88, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly (lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazene, poly (vinyl alcohol), poly (alkylene oxide), poly (ethylene oxide), poly (allylamine), poly (acrylate), poly (4-aminomethylstyrene), pluronic polyols, poloxamers, poly (uronic acid), poly (anhydride), poly (vinylpyrrolidone), and any combination thereof.

90. The method of any one of claims 83-89, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof.

91. The method of any one of claims 83-91, wherein the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof.

92. The method of any one of claims 83-91, wherein the scaffold comprises a macropore.

93. The method of claim 92, wherein the macropores are about 1 μ ι η and 100 μ ι η in diameter.

94. The method of claim 93, wherein the macropores have a diameter of about 50 μ ι η and 80 μ ι η.

95. The method of any one of claims 92-94, wherein the scaffold comprises macropores of different sizes.

96. The method of any one of claims 83-95, wherein the scaffold is injectable.

97. The method of any one of claims 83-96, wherein the scaffold comprises a methacrylated alginate (MA-alginate).

98. The method of any one of claims 83-97, wherein the scaffold comprises hyaluronic acid or a hyaluronic acid derivative.

99. The method of any one of claims 83-98, wherein the scaffold comprises a click hydrogel or a click cryogel.

100. The method of claim 99, wherein the scaffold comprises click alginate, click gelatin, or click hyaluronic acid.

101. The method of any one of claims 83-100, wherein the scaffold comprises pore-forming hydrogel microbeads or a bulk hydrogel, wherein said pore-forming hydrogel microbeads degrade at least 10% faster than said bulk hydrogel after administration to a subject.

102. The method of claim 101, wherein the pore-forming hydrogel microbeads comprise oxidized alginate.

103. The method of any one of claims 83-102, wherein the cell is a stem cell or a progenitor cell.

104. The method of claim 103, wherein said cells are selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, recombinant hematopoietic stem cells, recombinant hematopoietic progenitor cells, and any combination thereof.

105. The method of any one of claims 83-102, wherein the cells are selected from the group consisting of hematopoietic bone marrow cells, mobilized peripheral blood cells, reconstituted hematopoietic bone marrow cells, reconstituted mobilized peripheral blood cells, and any combination thereof.

106. The method of any one of claims 83-105, wherein the tissue or the organ comprises a bone tissue or a hematopoietic tissue.

107. The method of claim 106, wherein the tissue or the organ is formed about 7-21 days after administering the composition to the subject.

108. The method of any one of claims 83-107, wherein the tissue or the organ is formed about 14 days after administration of the composition to the subject.

109. The method of claim 107 or 108, wherein at least two compositions are administered to the subject.

110. The method of claim 109, wherein the compositions are similar in size.

111. The method of any one of claims 83-110, wherein the cells are stromal cells.

112. The method of any one of claims 83-111, wherein the scaffold is about 100 μ ι η in size³To about 10cm³。

113. The method of claim 112, wherein the scaffold is about 10mm in size³To about 100mm³。

114. The method of claim 113, wherein the scaffold is about 30mm in size³。

115. The method of any one of claims 83-114, wherein the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF- β) superfamily.

116. The method of claim 115, wherein the growth factor comprises a protein selected from BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, Growth Differentiation Factor (GDF) -1, GDG-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-mullerian hormone (AMH), activin, Nodal, TGF- β 1, TGF- β 2, TGF- β 3, TGF- β 4, and any combination thereof.

117. The method of claim 115 or 116, wherein the growth factor comprises BMP-2.

118. The method of claim 115 or 116, wherein the growth factor comprises TGF- β 1.

119. The method of any one of claims 83-118, wherein the growth factor retains its biological activity for at least twelve days following incorporation of the growth factor into the scaffold.

120. The method according to any one of claims 83-119, wherein the T cell progenitor cells are capable of differentiating into T cells.

121. The method of claim 120, wherein the T cells comprise cells selected from the group consisting of CD4+ T cells, CD8+ T cells, regulatory T cells (tregs), and any combination thereof.

122. The method of claim 119 or 120, wherein the lymphocytes comprise tregs.

123. The method of any one of claims 83-122, wherein the differentiation factor binds to a Notch receptor.

124. The method of claim 123, wherein the Notch receptor is selected from the group consisting of a Notch-1 receptor, a Notch-2 receptor, a Notch-3 receptor, a Notch-4 receptor, and any combination thereof.

125. The method of claim 123 or 124, wherein the differentiation factor is selected from the group consisting of delta-like 1(DLL-1), delta-like 2(DLL-2), delta-like 3(DLL-3), delta-like 4(DLL-4), Jagged 1, Jagged 2, and any combination thereof.

126. The method of any one of claims 83-125, wherein the differentiation factor is covalently linked to the scaffold.

127. The method of claim 126, wherein said differentiation factor is covalently attached to said scaffold using click chemistry.

128. The method of claim 126 or 127, wherein the differentiation factor is covalently attached to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry, avidin-biotin reactions, azide and dibenzocyclooctyne chemistry, tetrazine and trans-cyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry.

129. The method of any one of claims 83-128, wherein the differentiation factor retains its biological activity for at least about three months after incorporation of the differentiation factor into the scaffold.

130. The method of any one of claims 83-129, wherein the recruited cell is a transplanted cell.

131. The method of any one of claims 83-129, wherein the recruited cell is not a transplanted cell.

132. The method of claim 130, wherein the recruited cells are autologous.

133. The method of claim 130, wherein the recruited cells are allogeneic.

134. The method of claim 130, wherein the recruited cells are xenogeneic.

135. The method of any one of claims 83-134, wherein the differentiated cells are capable of migrating out of the scaffold.

136. The method of claim 135, wherein the differentiated cells are capable of homing into the tissue of the subject following administration of the composition to the subject.

137. The method of any one of claims 83-136, wherein the composition further comprises a homing factor capable of promoting recruitment of the cells to the scaffold.

138. The method of claim 137, wherein the homing factor comprises stromal cell derived factor (SDF-1).

139. The method of any one of claims 83-138, wherein the growth factor is present at about 1ng to about 1000 μ g.

140. The method of any one of claims 83-139, wherein the growth factor is present at about 1ng to about 1000 ng.

141. The method of claim 140, wherein said growth factor is present at about 5ng to about 500 ng.

142. The method of claim 141, wherein said growth factor is present at about 5ng to about 250 ng.

143. The method of claim 142, wherein the growth factor is present at about 5ng to about 200 ng.

144. The method of claim 143, wherein said growth factor is present at about 200 ng.

145. The method according to any one of claims 83-144, wherein the growth factor is present at about 0.03ng/mm³To about 350ng/mm³The scaffold volume of (a) exists.

146. The method of claim 145, wherein the growth factor is at about 6ng/mm³To about 10ng/mm³Are present.

147. The method of claim 146, wherein said growth factor is administered at about 6.5ng/mm ³To about 7.0ng/mm³Are present.

148. The method of claims 83-147, wherein the subject is administered about 5x10 per kilogram body weight of the subject⁵To about 50x10⁶Hematopoietic stem cells and/or hematopoietic progenitor cells.

149. The method of claim 148, wherein about 5x10 per kilogram body weight of the subject is administered to the subject⁵Hematopoietic stem cells and/or hematopoietic progenitor cells.

150. The method of claim 148 or 149, wherein the hematopoietic cells are selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, recombinant hematopoietic stem cells, recombinant hematopoietic progenitor cells, and any combination thereof.

151. The method of claim 148 or 149, wherein the hematopoietic cells are selected from the group consisting of hematopoietic bone marrow cells, mobilized peripheral blood cells, recombinant hematopoietic bone marrow cells, recombinant mobilized peripheral blood cells, and any combination thereof.

152. The method of any one of claims 83 and 87-151, wherein the method reduces autoimmunity in the subject.

153. The method of any one of claims 83 and 87-152, wherein the method prevents or treats an autoimmune disease.

154. The method of claim 153, wherein the autoimmune disease is selected from the group consisting of type 1 diabetes, rheumatoid arthritis, psoriasis, arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, addison's disease, graves' disease, sjogren's syndrome, hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, and allergies.

155. The method of any one of claims 83, 84, and 87-151, wherein the method reduces Graft Versus Host Disease (GVHD).

156. The method of claim 155, wherein the GVHD is associated with Hematopoietic Stem Cell Transplantation (HSCT) in the subject.

157. The method of claim 156, wherein said composition is administered concurrently with or after Hematopoietic Stem Cell Transplantation (HSCT).

158. The method of claim 157, wherein the GVHD is associated with a solid organ transplant.

159. The method of claim 158, wherein the composition is administered to the subject prior to said transplantation.

160. The method of any one of claims 155-159, wherein the GVHD is acute GVHD.

161. The method of any one of claims 155-159, wherein the GVHD is chronic GVHD.

162. The method of any one of claims 155-161, wherein the method reduces GVHD associated morbidity, GVHD associated mortality or GVHD associated reduction in long term survival.

163. The method of any one of claims 84 and 87-151, wherein the transplantation is Hematopoietic Stem Cell Transplantation (HSCT).

164. The method of any one of claims 84, 87-151, and 163, wherein the increased donor chimerism comprises a T cell chimerism.

165. The method of claim 164, wherein the T cells comprise CD4+ T cells, CD8+ T cells, or Treg cells.

166. The method of any one of claims 85 and 87-151 wherein the T cell balance reestablishment is characterized by a steady state CD4+: CD8+ T cell ratio in peripheral blood of about 0.9 to about 2.5.

167. The method of any one of claims 86-151, wherein the human has an impaired immune system as a result of immunosenescence.

168. The method of claim 167, wherein the human is over the age of 30, 40, 50, 60, 70, or 80 years.

169. The method of any one of claims 86-151, 167, and 168, wherein the human has an impaired immune system due to an innate immune deficiency.

170. The method of any one of claims 86-151 and 167-169, wherein the human has an impaired immune system due to acquired immunodeficiency.

171. The method of any one of claims 83-170, wherein the method increases regulatory T cells (T) _reg) The level of (c).

172. The method of any one of claims 83-171, wherein the composition is administered by injection.

173. The method of claim 172, wherein the injection is subcutaneous.

174. A syringe, comprising:

a needle head;

a reservoir comprising the composition of any one of claims 1-63; and

and a plunger.

175. A kit, comprising:

the composition of any one of claims 1-63; and

instructions for administering the composition.

176. The method of any one of claims 83-173, further comprising administering a stem cell mobilizing agent or a progenitor cell mobilizing agent to the subject in an amount effective to induce migration of stem cells or progenitor cells from bone marrow into the blood.

177. The method of claim 176, wherein the stem cell mobilizer or the progenitor cell mobilizer is selected from the group consisting of IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF, plerixafor, PDGF, TGF- β, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, and combinations thereof.

178. The method of claim 176 or 177, wherein the stem cell mobilizing agent or the progenitor cell mobilizing agent is administered prior to, concurrently with, or after administration of the composition.

179. The method of any one of claims 176-178, further comprising administering to the subject a therapeutically effective amount of electromagnetic radiation.

Background

Long-term immunodeficiency in patients undergoing Hematopoietic Stem Cell Transplantation (HSCT) remains one of the most serious obstacles to the control of life-threatening blood or bone marrow diseases such as multiple myeloma and leukemia. Prior to transplantation, the recipient will receive cytotoxic radiotherapy and chemotherapy regimens to destroy the diseased cells. A side effect of the regulatory process is severe lymphopenia due to T cell and B cell destruction of the adaptive immune system. Severe post-transplant immunodeficiency, characterized by a dramatic decrease in the number of T and B cells and a decrease in their diversity, can last from one to two years. Immunodeficiency associated severe opportunistic infections (-30%), cancer recurrence (> 50% acute myeloid leukemia) and Graft Versus Host Disease (GVHD) (-40%) are the most common causes of complications and morbidity and mortality in patients receiving HSCT.

There is a need for novel compositions and methods that can be used to improve immune system reconstitution after HSCT. There is also a need for compositions and methods that reduce the risks associated with HSCT and improve patient outcomes.

Disclosure of Invention

Disclosed herein are novel compositions and methods for modulating the immune system in a subject. The compositions and methods disclosed herein provide a means for treating and/or preventing diseases associated with T cell deficiency and/or disorders.

Thus, in one aspect, the invention provides a composition for modulating the immune system in a subject. The composition comprises a porous scaffold; growth factor ofPresent at about 1ng to about 1000ng per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In one embodiment, the growth factor is present at about 0.03ng/mm³To about 350ng/mm³The scaffold volume of (a) exists.

In another aspect, the invention provides a composition for modulating the immune system in a subject. The composition comprises a porous scaffold; a growth factor present in a scaffold volume of about 0.03ng/mm3 to about 350ng/mm3 and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors.

In various embodiments of any of the above aspects or any other aspect of the invention described herein, the scaffold comprises a hydrogel. In one embodiment, the scaffold comprises a cryogel. In another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly (lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazene, poly (vinyl alcohol), poly (alkylene oxide), poly (ethylene oxide), poly (allylamine), poly (acrylate), poly (4-aminomethylstyrene), pluronic polyols, poloxamers, poly (uronic acid), poly (anhydride), poly (vinylpyrrolidone), and any combination thereof. In another embodiment, the scaffold comprises a polymer or copolymer comprising a polymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof. In another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof.

In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises pores having diameters of 1 μm and 100 μm. In one embodiment, the scaffold comprises a large pore. In another embodiment, the macropores have a diameter of about 50 μm to 80 μm. In another embodiment, the scaffold comprises macropores of different sizes.

In various embodiments of any of the above aspects or any other aspect of the invention described herein, the scaffold is injectable.

In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises a methacrylated alginate (MA-alginate).

In various embodiments of the above aspect or any other aspect of the invention described herein, the scaffold comprises hyaluronic acid or a hyaluronic acid derivative.

In various embodiments of any of the above aspects or other aspects of the invention described herein, the scaffold comprises a click hydrogel or a click cryogel. In one embodiment, the scaffold comprises click alginate, click gelatin or click hyaluronic acid.

In various embodiments of the above aspects or any other aspect of the invention described herein, the scaffold comprises pore-forming hydrogel microbeads and a bulk hydrogel, wherein the pore-forming hydrogel microbeads degrade at least 10% faster than the bulk hydrogel polymer scaffold upon administration of the scaffold into a subject. In one embodiment, the pore-forming hydrogel microbeads comprise oxidized alginate.

In various embodiments of the above aspect or any other aspect of the invention described herein, the cell is a stem cell. In one embodiment, the stem cells are hematopoietic stem cells.

In various embodiments of the above aspect or any other aspect of the invention described herein, the cell is a progenitor cell.

In various embodiments of any of the other aspects of the invention described herein or above, the tissue or the organ comprises bone tissue or hematopoietic tissue. In one embodiment, the tissue or the organ is formed about 7-21 days after administration of the composition to the subject. In another embodiment, the tissue or the organ is formed about 14 days after administration of the composition to the subject.

In various embodiments of the above aspect or any other aspect of the invention described herein, the cell is a stromal cell.

In various embodiments of any of the above aspects or any other aspect of the invention described herein, the scaffold has a size of about 100 μm³To about 10cm³. In one embodiment, the size of the stent is about 10mm³To about 100mm³. In another embodiment, the size of the stent is about 30mm ³。

In various embodiments of the above aspects or any other aspect of the invention described herein, the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF- β) superfamily. In one embodiment, the growth factor comprises a protein selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, Growth Differentiation Factor (GDF) -1, GDG-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-Mullerian hormone (AMH), activin, Nodal, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, and any combination thereof. In another embodiment, the growth factor comprises BMP-2. In another embodiment, the growth factor comprises TGF- β 1.

In various embodiments of the above aspect or any other aspect of the invention described herein, the growth factor is present at about 5ng to about 500 ng. In one embodiment, the growth factor is present at about 5ng to about 250 ng. In another embodiment, the growth factor is present at about 5ng to about 200 ng. In another embodiment, the growth factor is present at about 200 ng. In another embodiment, the growth factor is present at about 6ng/mm ³To about 10ng/mm³Are present. In another embodiment, the growth factor is present at about 6.5ng/mm³To about 7.0ng/mm³Are present.

In various embodiments of the above aspects or any other aspect of the invention described herein, the growth factor retains its biological activity for at least twelve days after incorporation of the growth factor into the scaffold.

In various embodiments of any of the above aspects or any other aspect of the invention described herein, the T cell progenitor is capable of differentiating into a T cell. In one embodiment, the T cell comprises a peptide selected from the group consisting of CD4⁺T cell, CD8⁺T cell, regulatory T cell (T)_reg) And any combination thereof. In another embodiment, the T cell comprises T_reg。

In various embodiments of any of the above aspects or any other aspect of the invention described herein, the differentiation factor binds to a Notch receptor. In one embodiment, the Notch receptor is selected from the group consisting of a Notch-1 receptor, a Notch-2 receptor, a Notch-3 receptor, a Notch-4 receptor, and any combination thereof. In another embodiment, the differentiation factor is selected from the group consisting of delta-like 1(DLL-1), delta-like 2(DLL-2), delta-like 3(DLL-3), delta-like 4(DLL-4), Jagged 1, Jagged 2, and any combination thereof.

In various embodiments of the above aspect or any other aspect of the invention described herein, the differentiation factor is covalently linked to the scaffold. In one embodiment, the differentiation factor is covalently linked to the scaffold using click chemistry. In another embodiment, the differentiation factor is covalently attached to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry, NHS and Dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reactions, azide and dibenzocyclooctyne chemistry, tetrazine and trans cyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry.

In various embodiments of the above aspects or any other aspect of the invention described herein, the differentiation factor is present in an amount from about 1ng to about 1000 μ g per scaffold. In one embodiment, the differentiation factor is present in an amount from about 1 μ g to about 100 μ g per scaffold. In another embodiment, the differentiation factor is present in an amount from about 1 μ g to about 10 μ g per scaffold. In another embodiment, the differentiation factor is present at about 6 μ g per scaffold.

In various embodiments of the above aspects or any other aspect of the invention described herein, the differentiation factor retains its biological activity for at least about three months after incorporation into the scaffold.

In various embodiments of any of the above aspects or any other aspect of the invention described herein, the recruited cell is a transplanted cell. In one embodiment, the recruited cells are autologous. In another embodiment, the recruited cells are allogeneic. In another embodiment, the recruited cells are xenogeneic.

In various embodiments of the above aspect or any other aspect of the invention described herein, the recruited cell is not a transplanted cell.

In various embodiments of the above aspect or any other aspect of the invention described herein, the differentiated cells are capable of migrating out of the scaffold. In one embodiment, the differentiated cells are capable of homing into the tissue of the subject following administration of the composition to the subject.

In various embodiments of the above aspects or any other aspect of the invention described herein, the composition further comprises a homing factor capable of promoting recruitment of the cells to the scaffold. In one embodiment, the homing factor comprises stromal cell derived factor (SDF-1).

In one aspect, the present invention provides a method of modulating the immune system in a subject comprising administering to the subject the aforementioned composition of the invention, thereby modulating the immune system in the subject.

In another aspect, the present invention provides a method of reducing immune hyperreactivity in a subject, comprising administering to the subject the aforementioned composition of the invention, thereby reducing immune hyperreactivity in the subject.

In another aspect, the present invention provides a method of increasing donor chimerism in a subject receiving a transplant, comprising administering to the subject the aforementioned composition of the invention, thereby increasing donor chimerism in the subject.

In another aspect, the present invention provides a method of promoting T cell balance remodeling in a subject, comprising administering to the subject the aforementioned composition of the invention, thereby promoting T cell balance remodeling in the subject.

In various embodiments of the above aspects or any other aspect of the invention described herein, the method further comprises administering hematopoietic stem cells or hematopoietic progenitor cells to the subject. In one embodiment, wherein the composition is administered to the subject simultaneously with or after administration of hematopoietic stem cells or hematopoietic progenitor cells to the subject. In another embodiment, about 1x10 per kilogram body weight of the subject is administered to the subject ⁵To about 50x10⁶Hematopoietic stem cells and/or hematopoietic progenitor cells. In another embodiment, about 1x10 per kilogram body weight of the subject is administered to the subject⁵To about 1x10⁶Hematopoietic stem cells or hematopoietic progenitor cells.

In various embodiments of the above aspects or any other aspect of the invention described herein, the method enhances T cell reconstitution in a subject. In one embodiment, the method enhances T cell neogenesis. In another embodiment, the enhanced T cell neogenesis is characterized by an enhanced T cell receptor excision loop (TREC).

In various embodiments of the above aspects or any other aspect of the invention described herein, the method enhances T cell diversity in the subject. In one embodiment, the T cell diversity is characterized by an enhanced T Cell Receptor (TCR) repertoire.

In various embodiments of the above aspects or any other aspect of the invention described herein, the method increases regulatory T cells (T)_reg) The level of (c).

In various embodiments of the above aspect or any other aspect of the invention described herein, the subject is a human with an impaired immune system. In one embodiment, the subject has an impaired immune system as a result of immunosenescence. In another embodiment, the subject is over 30, 40, 50, 60, 70, or 80 years of age. In one embodiment, the subject has an impaired immune system due to an innate immune deficiency. In another embodiment, the subject has acquired immunodeficiency.

In another aspect, the present invention provides a method of reducing immune hyperreactivity in a subject comprising administering to the subject a composition comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing a differentiation factor that recruits cells to differentiate into T cell progenitors, thereby reducing immune hyperreactivity in the subject.

In another aspect, the invention provides a method of increasing donor chimerism in a subject receiving a transplant, comprising administering to the subject a composition comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing a differentiation factor that recruits cells to differentiate into T cell progenitors, thereby increasing donor chimerism in the subject.

In another aspect, the invention provides a method of promoting T cell balance remodeling in a subject comprising administering to the subject a composition comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into T cell progenitors, thereby causing a reestablishment of T cell balance in the subject.

In another aspect, the present invention provides a method of modulating the immune system of a human with an impaired immune system comprising administering to the human a composition comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into T cell progenitors, thereby modulating the immune system of a human, wherein the human has an impaired immune system due to immunosenescence, congenital immunodeficiency, or acquired immunodeficiency.

In various embodiments of the above aspect, the scaffold comprises a hydrogel. In one embodiment, the scaffold comprises a cryogel. In another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly (lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazene, poly (vinyl alcohol), poly (alkylene oxide), poly (ethylene oxide), poly (allylamine), poly (acrylate), poly (4-aminomethylstyrene), pluronic polyols, poloxamers, poly (uronic acid), poly (anhydride), poly (vinylpyrrolidone), and any combination thereof. In another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of alginate, alginate derivatives, and combinations thereof. In another embodiment, the scaffold comprises a polymer or copolymer selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and combinations thereof.

In various embodiments of the above aspect, the scaffold comprises a hole. In one embodiment, the pores have a diameter of about 1 μm to about 100 μm. In another embodiment, the pores have a diameter of about 50 μm to about 80 μm. In another embodiment, the scaffold comprises pores of different sizes.

In various embodiments of the above aspect, the scaffold is injectable. In one embodiment, the scaffold comprises a methacrylated alginate (MA-alginate). In another embodiment, the scaffold comprises hyaluronic acid or a hyaluronic acid derivative.

In various embodiments of the above aspect, the scaffold comprises a click hydrogel or a click cryogel. In one embodiment, the scaffold comprises click alginate, click gel or click hyaluronic acid.

In various embodiments of the above aspects, the scaffold comprises pore-forming hydrogel microbeads or a bulk hydrogel, wherein the pore-forming hydrogel microbeads degrade at least 10% faster than the bulk hydrogel polymer scaffold upon administration of the scaffold into a subject. In one embodiment, the pore-forming hydrogel microbeads comprise oxidized alginate.

In various embodiments of the above aspect, the cell is a stem cell or a progenitor cell. In one embodiment, the cell is selected from the group consisting of a hematopoietic stem cell, a hematopoietic progenitor cell, a recombinant hematopoietic stem cell, a recombinant hematopoietic progenitor cell, and any combination thereof. In another embodiment, the cell is selected from the group consisting of a hematopoietic bone marrow cell, a mobilized peripheral blood cell, a recombinant hematopoietic bone marrow cell, a recombinant mobilized peripheral blood cell, and any combination thereof.

In various embodiments of the above aspect, the tissue or the organ comprises bone tissue or hematopoietic tissue. In one embodiment, the tissue or the organ is formed about 7-21 days after administration of the composition to the subject. In another embodiment, the tissue or the organ is formed about 14 days after administration of the composition to the subject. In another embodiment, at least two compositions are administered to the subject. In another embodiment, the compositions are similar in size.

In various embodiments of the above aspect, the cell is a stromal cell.

In various embodiments of the above aspect, the scaffold has a size of about 100 μm ³To about 10cm³. In one embodiment, the size of the stent is about 10mm³To about 100mm³. In another embodiment, the size of the stent is about 30mm³。

In various embodiments of the above aspects, the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF- β) superfamily. In one embodiment, the growth factor comprises a protein selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, Growth Differentiation Factor (GDF) -1, GDG-2, GDF-3, GDF-5, GDF-6, GDF-8, GDF-9, GDF-10, GDF-11, GDF-15, anti-Mullerian hormone (AMH), activin, Nodal, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, and any combination thereof. In another embodiment, the growth factor comprises BMP-2. In another embodiment, the growth factor comprises TGF- β 1.

In various embodiments of the above aspect, the growth factor retains its biological activity for at least twelve days following incorporation of the growth factor into the scaffold.

In various embodiments of the above aspects, the T cell progenitor cells are capable of differentiating into T cells. In one embodiment, the T cell comprises a peptide selected from the group consisting of CD4 ⁺T cell, CD8⁺T cell, regulatory T cell (T)_reg) And any combination thereof. In another embodiment, the T cell comprises T_reg。

In various embodiments of the above aspects, the differentiation factor binds to a Notch receptor. In one embodiment, the Notch receptor is selected from the group consisting of a Notch-1 receptor, a Notch-2 receptor, a Notch-3 receptor, a Notch-4 receptor, and any combination thereof. In another embodiment, the differentiation factor is selected from the group consisting of delta-like 1(DLL-1), delta-like 2(DLL-2), delta-like 3(DLL-3), delta-like 4(DLL-4), Jagged 1, Jagged 2, and any combination thereof.

In various embodiments of the above aspect, the differentiation factor is covalently linked to the scaffold. In one embodiment, the differentiation factor is covalently linked to the scaffold using click chemistry. In another embodiment, the differentiation factor is covalently attached to the scaffold using N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry, avidin-biotin reactions, azide and dibenzocyclooctyne chemistry, tetrazine and trans-cyclooctene chemistry, tetrazine and norbornene chemistry, or disulfide chemistry.

In various embodiments of the above aspects, the differentiation factor retains its biological activity for at least about three months after incorporation into the scaffold.

In various embodiments of the above aspect, the recruited cell is a transplanted cell. In one embodiment, the recruited cells are autologous. In another embodiment, the recruited cells are allogeneic. In another embodiment, the recruited cells are xenogeneic.

In various embodiments of the above aspect, the recruited cell is not a transplanted cell.

In various embodiments of the above aspect, the differentiated cells are capable of migrating out of the scaffold. In one embodiment, the differentiated cells are capable of homing into the tissue of the subject following administration of the composition to the subject.

In various embodiments of the above aspect, the composition further comprises a homing factor capable of promoting recruitment of the cells to the scaffold. In one embodiment, the homing factor comprises stromal cell derived factor (SDF-1).

In various embodiments of the above aspects, the growth factor is present in an amount from about 1ng to about 1000 μ g. In one embodiment, the growth factor is present in an amount from about 1ng to about 1000 ng. In another embodiment, the growth factor is present at about 5ng to about 500 ng. In another embodiment, the growth factor is present in an amount from about 5ng to about 250 ng. In another embodiment, the growth factor is present in an amount from about 5ng to about 200 ng. In another embodiment, the growth factor is present at about 200 ng.

In various embodiments of the above aspects, the growth factor is at about 0.03ng/mm³To about 350ng/mm³The scaffold volume of (a) exists. In one embodiment, the growth factor is present at about 6ng/mm³To about 19ng/mm³Are present. In another embodiment, the growth factor is present at about 6.5ng/mm³To about 7.0ng/mm³Are present.

In various embodiments of the above aspects, about 5x10 per kilogram body weight of the subject is administered to the subject⁵To about 50x10⁶Hematopoietic stem cells and/or hematopoietic progenitor cells. In one embodiment, about 5x10 per kilogram body weight of the subject is administered to the subject⁵Hematopoietic stem cells and/or hematopoietic progenitor cells. In another embodiment, the hematopoietic cells are selected from hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem cellsHematopoietic stem cells, recombinant hematopoietic progenitor cells, and any combination thereof. In another embodiment, the hematopoietic cells are selected from the group consisting of hematopoietic bone marrow cells, mobilized peripheral blood cells, recombinant hematopoietic bone marrow cells, recombinant mobilized peripheral blood cells, and any combination thereof.

In various embodiments of the above aspects, the method reduces autoimmunity in the subject.

In various embodiments of the above aspects, the method prevents or treats an autoimmune disease. In one embodiment, the autoimmune disease is selected from the group consisting of type 1 diabetes, rheumatoid arthritis, psoriasis, arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, addison's disease, graves' disease, sjogren's syndrome, hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, and allergies.

In various embodiments of the above aspects, the method reduces Graft Versus Host Disease (GVHD). In one embodiment, the GVHD is associated with Hematopoietic Stem Cell Transplantation (HSCT) in the subject. In another embodiment, the composition is administered simultaneously with or after Hematopoietic Stem Cell Transplantation (HSCT). In another embodiment, the GVHD is associated with a solid organ transplant. In another embodiment, the composition is administered to the subject prior to said transplantation. In another embodiment, the GVHD is acute GVHD. In one embodiment, the GVHD is chronic GVHD. In another embodiment, the method reduces GVHD-related morbidity, GVHD-related mortality, or GVHD-related reduction in long-term survival.

In various embodiments of the above aspect, the transplantation is Hematopoietic Stem Cell Transplantation (HSCT).

In various embodiments of the above aspects, the increased donor chimerism comprises a T cell chimerism. In one embodiment, the T cell comprises CD4⁺T cell, CD8⁺T cells or T_regA cell.

In various embodiments of the above aspects, the T cell balance remodeling is characterized by steady state CD4 in peripheral blood ⁺:CD8⁺The T cell ratio is about 0.9 to about 2.5.

In various embodiments of the above aspects, the human has an impaired immune system as a result of immunosenescence. In one embodiment, the human is over the age of 30, 40, 50, 60, 70 or 80 years.

In various embodiments of the above aspects, the human has an impaired immune system due to an innate immune deficiency.

In various embodiments of the above aspects, the human has an impaired immune system due to acquired immunodeficiency.

In various embodiments of the above aspects, the method increases regulatory T cells (T)_reg) The level of (c).

In various embodiments of the above aspects, the composition is administered by injection. In one embodiment, the injection is subcutaneous.

In yet another aspect of the present invention, a syringe is provided. The syringe comprises a needle; a reservoir comprising the composition of the various embodiments of the above aspects or any other aspect of the invention described herein; and a plunger.

In yet another aspect of the invention, the invention provides a kit. The kit comprises the compositions of the various embodiments of the above aspects or any other aspect of the invention described herein, and instructions for administering the compositions.

In various embodiments of the above aspects, the method further comprises administering to the subject a stem cell mobilizing agent or a progenitor cell mobilizing agent in an amount effective to induce migration of stem cells or progenitor cells from the bone marrow into the blood. In one embodiment, the stem or progenitor cell mobilizer is selected from the group consisting of IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF, plerixafor, PDGF, TGF- β, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, or a combination thereof. In another embodiment, the stem cell mobilizer or progenitor cell mobilizer is administered prior to, concurrently with, or after administration of the composition. In another embodiment, the method further comprises administering to the subject a therapeutically effective amount of electromagnetic radiation.

Other features and advantages of the present invention will be apparent from the following detailed description and the accompanying drawings.

Drawings

FIGS. 1A-1E, 1J and 1M show that alginate-PEG-DLL 4-based Bone Marrow Cryogels (BMCs) display DLL4 and BMP-2 and preferentially expand common lymphoid progenitor Cells (CLPs). FIGS. 1F-1I, 1K and 1L show an extended characterization of BMC bioactivity.

FIG. 1A is a schematic of the preparation of covalently crosslinked BMC.

Fig. 1B is a representative cross-sectional Scanning Electron Micrograph (SEM) image of BMC. Scale bar, 1 mm.

Fig. 1C is a representative SEM of pore shape and structure within the BMC cross-section. Scale bar 200 μm.

Fig. 1D shows the release kinetics of encapsulated BMP-2 and covalently linked DLL4 (n-5 per group).

Fig. 1E shows the binding kinetics of DLL4 before and after surface plasmon resonance measurement of methacrylate linker modification.

FIG. 1F shows the combined BMP-2 release bioactivity measured using alkaline phosphatase activity in MC3T3-E1 preosteoblasts compared to BMP-2 in media without incorporation of BMC (native BMP-2) and without addition of BMP-2 (growth media).

FIG. 1G shows the use of alkaline phosphatase activity in MC3T3-E1 preosteoblasts to quantify BMP-2 biological activity at discrete time intervals after release.

FIG. 1H shows the in vitro biological activity of the Notch ligand DLL-4 measured using a colorimetric method.

FIG. 1I shows representative fluorescence microscopy images of Citrine expression in different time intervals on twin BMCs (top row) and on blank BMCs (bottom row) for CHO-K1+2xHS4-UAS-H2B-Citrine-2xHS4cH1+ hNECD-Gal4esn c9 Notch acceptor cell lines.

Figure 1J shows the in vitro differentiation of ex vivo mouse and human hematopoietic stem and progenitor cells into CLP as a function of the degree of methacrylate group functionalization on the polymer backbone (n ═ 5).

FIGS. 1K and 1L show the fold expansion and survival of mouse (FIG. 1K) and human (FIG. 1L) hematopoietic cells after 7 days in vitro culture.

The data in fig. 1F-1I, 1K and 1L are mean ± standard deviation (n ═ 5) and representative from 3 independent experiments. (. P <0.05,. P <0.01,. P <0.001, analysis of variance (ANOVA) using Tukey post-hoc test).

FIG. 1M shows the ratio of Lin-common lymphoid and myeloid mouse progenitor cells quantified in growth medium, blank, single-factor and two-factor BMC.

The image and aperture quantization in fig. 1B and 1C represent ten independent repetitions. The data in fig. 1D, 1J, and 1M are the mean ± standard deviation of five 337 experimental replicates and are representative from 3 independent experiments. Different samples were analyzed separately.

Figures 2B-2K show in vivo deployment and host integration of BMCs. FIGS. 2A, 2L and 3B-3F show extended in vivo characterization of BMC.

Figure 2A shows representative flow cytometry plots of pre-and post-lineage depleted bone marrow cells for transplantation (5 independent experiments).

FIG. 2B shows the schedule of L-TBI, HSCT and simultaneous BMC injection. B6 h irradiated with 1000cGy (dose 1) and subsequently implanted with 5X10 within 49 h after L-TBI⁵Lineage depleted syngeneic GFP BM cells.

Figure 2C shows the volume of BMC bar in vivo as a function of time post delivery in BMC for various combinations comprising BMP-2 and DLL-4.

Figure 2D shows confocal microscope images of donor GFP + cells (green) identified within BMC (red).

Fig. 2E and 2F show representative microcomputerized tomographic (microCT, scale bar 1mm) imaging (fig. 2E) and histology (scale bar 1mm) (fig. 2F) of the bi-functionalized BMC 3 weeks after injection of bone shells (green arrow) and hematopoietic tissues (yellow arrow).

Figure 2G shows images of BMC (blue) in subcutaneous tissue at different time points after injection.

FIGS. 2H and 2I show BMC sections (blue arrows) of identified vessels (FIG. 2H) histologically Verhoeff-Van Giesen stained at days 10, 30, 40, and 90 post-transplantation, and quantification of vessel density within these sections (FIG. 2I).

FIGS. 2J and 2K show histological Safranin-O stained sections of BMC with alginate (red line-like staining) at days 10, 20, 40, 60, and 90 post-transplantation (FIG. 2J) and quantification of accessible regions of alginate in these sections (FIG. 2K).

Figure 2L shows BMC border images extracted from subcutaneous tissue at predetermined time intervals after transplantation, with BMC borders identified with collagen (blue-green) and cells (black) and alginate (red) in some sections, observed using Safranin-O staining (10 x objective magnification).

The data in fig. 2C represent the mean ± standard deviation of five experimental replicates, representing two independent experiments (. P <0.05,. P <0.01,. P <0.001, analysis of variance (ANOVA) using Tukey post hoc testing). The images in fig. 2D-2G and 2J represent four independent samples. Data in fig. 2I and 2K represent mean ± standard deviation from eight samples, representing four independent experiments (. P <0.05,. P <0.01, no significant difference, Tukey post-test ANOVA). Different samples were analyzed separately. The data in fig. 2L represent the mean ± standard deviation of n-5, representing 2 independent experiments.

FIGS. 3A and 3G-3P show in vivo recruitment of donor cells to BMC and enhanced seeding of thymic progenitor cells.

FIG. 3A shows the total number and types of donor-derived GFP + cells in BMC containing the BMP-2 and DLL-4 and blank BMC combinations.

FIG. 3B shows representative flow cytometry plots of bone marrow and BMC (Dual and BMP-2 only) 28 days post-transplantation. Donors GFP, bone marrow, HSC, lymphoid-activated pluripotent progenitor cells (LMPP), CLP and bone marrow progenitor cells were identified.

Figure 3C shows a representative flow cytometry plot and host mesenchymal stromal cells in BMC and endogenous bone marrow. Sca-1⁺Progenitor cells are represented as part of CD 45-cells. CD44, CD73, CD29, CD105 and CD106 expressing cells are indicated as Sca-1 ⁺A fraction of progenitor cells.

Fig. 3D shows the quantification of bone alkaline phosphatase (bALP) and oil red-o (oro) in bone marrow and BMC on day 20 post subcutaneous injection (n ═ 6-7).

Figure 3E shows colony formation experiments performed using bone marrow cells from mice treated with only transplantation and dual BMC at days 10, 35, and 70 post-transplantation.

FIG. 3F shows the concentration of homing factor SDF-1 α and lymphoid progenitor cells supporting cytokine IL-7 in collected BMC. Mice post-hematopoietic stem cell transplantation treated with BMP-2BMC and mice post-hematopoietic stem cell transplantation treated with dual BMC were analyzed and compared with cytokine concentrations in bone marrow of the same group.

The data in fig. 3C-3F are the mean ± standard deviation of n-4, n-7, n-4 and n-4, respectively, and represent 2 independent experiments. The data in fig. 3B are from n-10, representing 2 independent experiments (. P <0.05,. P <0.01,. P <0.001, analysis of variance (ANOVA) using Tukey post hoc testing).

FIG. 3G shows the absolute number of donors GFP + CLP and the percentage of Ly6D-CLP in BMP-2 and two-factor BMC.

Figure 3H shows a schematic of the experimental setup for surgical transplantation of harvested BMC from post HSCT mice to sublethal irradiated mice.

FIG. 3I shows Double Positive (DP), Single Positive (SP) CD4+ and SP CD8+ cells quantified in the thymus 20 days after BMC surgical transplantation.

FIG. 3J shows quantification of total number of early T lineage progenitor cells (ETP; CD44+ CD25-c-kit +) as a function of lineage depleted transplant cell dose, compared to BMC treatment at the lowest cell dose at multiple time points post-transplant with a representative FACS profile (5 experimental replicates, 2 independent experiments per time point).

FIGS. 3K-3P show the total number of early T lineage progenitor cell (ETP; CD44+ CD25-c-kit +), DN2(CD44+ CD25-), DN3(CD44+ CD25-), DP, SP4, SP8 thymocyte subpopulations compared at various time points after transplantation under different treatment conditions.

For FIGS. 3A, 3G and 3K, 3P, mice were transplanted with 5x10 within 48 hours after L-TBI 375(1x1000cGy)⁵Lineage depleted syngeneic GFP BM cells. In FIG. 3H and 3I, one initial group of mice was transplanted with 5X10 within 48 hours after L-TBI⁵Lineage depleted syngeneic GFP BM cells. The subsequent group of mice received SL TBI (1x500cGy) without subsequent cell transplantation. In FIG. 3J, the mouse was transplanted with 5X10⁴To 5x10⁵Lineage depleted GFP cells. In fig. 3A, all groups were compared to transplant-only controls (. P) <0.05,**P<0.01,***P<0.001, analysis of variance (ANOVA) using Tukey post hoc test). The data in figure 3A represent the mean ± standard deviation of ten mice per group and represent two independent experiments. The data in FIGS. 3G and 3I-3P represent the mean. + -. standard deviation of five mice per group (each time point in FIGS. 3I-3P) and represent two independent experiments. Comparison with the lowest cell dose group in FIG. 3J and the transplant-only group in FIGS. 3I-3P (. about.P)<0.05,**P<385 0.01,***P<0.001, analysis of variance (ANOVA) using Tukey post hoc test). Different samples were analyzed separately.

FIGS. 3Q-3T show the expanded characteristics of thymocyte structure and body weight after transplantation. B6 mice were irradiated with a 1x1000cGy L-TBI dose, followed by a 5x10 transplant within 48 hours after L-TBI⁵Lineage depleted syngeneic GFP BM cells and processed as shown.

Fig. 3Q shows total thymocytes quantitated 32 days and 42 days post-transplantation.

Figure 3R shows the quantitative thymus weights from 12 days to 42 days post HSCT.

Fig. 3S shows quantitation of mTEC, cTEC, fibroblasts, and endothelial cells 22 days after HSCT.

FIG. 3T shows early T lineage progenitor cells (ETP; CD 44) compared to 22 days post HSCT under different treatment conditions⁺CD25^-c-kit⁺)、DN2(CD44⁺CD25^-)、DN3(CD44⁺CD25^-) DP, SP4, SP8 total thymocyte subpopulation.

The thymus of mice post HSCT without BMC (transplant only), mice post HSCT treated with BMP-2BMC and mice post HSCT treated with dual BMC were collected and weighed and compared to non-irradiated mice. All groups in fig. 3Q, 3S and 3T were compared to the transplant-only control group. 10 days after HSCT, BMCs were removed and surgically placed in the subcutaneous pockets of a second group of B6 mice irradiated with 500cGy SL-TBI. The values are expressed as absolute numbers. The data in figures 3Q-3T represent the mean ± standard deviation of 5 mice per group at each time point and represent at least two independent experiments (. P <0.05,. P <0.01,. P <0.001, analysis of variance (ANOVA) using Tukey post hoc testing).

FIGS. 4A and 4D-4L show enhanced BMC-mediated T cell reconstitution. Fig. 4B, 4C, 4M and 4N show expanded characterization of the blood cell analysis after HSCT.

Fig. 4A shows the sum of CD3+ CD4+ and CD3+ CD8+ in the peripheral blood of mice after HSCT. B6 mice were irradiated with a 1x1000cGy L-TBI dose. Mice were then transplanted with 5x10 within 48 hours after L-TBI⁵Lineage depleted syngeneic GFP BM cells and processed as shown.

Figure 4B shows a representative FACS gating strategy for measuring immune cell reconstitution following HSCT in C57BL/6J mice transplanted with GFP + donor hematopoietic stem and progenitor cells from 5 independent experiments.

Figure 4C shows reconstitution of B cells and bone marrow cells in vivo. B6 mice were irradiated with a 1x1000cGy L-TBI dose, followed by a 5x10 transplant within 48 hours after L-TBI⁵Lineage depleted syngeneic GFP BM cells. Peripheral blood was analyzed for BMC-free post-HSCT mice (transplant only), BMC-2 BMC treated post-HSCT mice, and dual BMC treated post HSCT mice, and the measured amounts were compared to pre-irradiation immune cell concentrations.

FIGS. 4D-4F show the recovery over time of the ratio of CD4+ to CD8+ T cells in measured blood (FIG. 4D), spleen (FIG. 4E), and bone marrow (FIG. 4F) compared to the same group in FIG. 4A in non-irradiated mice. In FIGS. 4A and 4D-4F, mice post-HSCT without BMC (transplant only), injected rapidly with BMP-2 and DLL-4, BMC with BMP-2 (BMP-2BMC), or BMC with BMP-2 and DLL4 (Dual BMC) were analyzed,

In FIGS. 4G-4L, B6 mice were irradiated with 500cGy SL-TBI and subsequently implanted with 5X10 within 48 hours after irradiation⁵Lineage depleted bone marrow cells. DP (FIG. 4G) SP4 (FIG. 4H) and SP8 (FIG. 4I) thymocytes and peripheral CD4 in the spleen of SL-TBI syn-HSCT mice+ (FIG. 4J) and total CD8+ T cells (FIG. 4K) and B cells (FIG. 4L), the mice received and did not receive dual BMC treatment 28 days after transplantation. The data in fig. 4A, 4D-4F are mean ± standard deviation of each group of 8 groups at each time point and represent at least 3 independent experiments. The data in fig. 4G-4L are mean ± standard deviation of n-10 mice and represent 2 independent experiments. (. P) <0.05,**P<0.01,***P<0.001, analysis of variance (ANOVA) using Tukey post hoc test).

Fig. 4M shows representative FACS plots of donor and host chimeras after HSCT in thymocytes (DP, SP4, SP8) and splenocytes (CD4+, CD8+, B220+) at day 28 post BM treatment and transplantation of mice transplanted only.

Figure 4N shows a representative flow cytometry plot of host (CD45.2) and donor (CD45.1) chimerism in sublethally irradiated mice 28 days post-transplantation.

In FIGS. 4M and 4N, B6 mice were irradiated with 500cGy SL-TBI, followed by transplantation of 5X10 within 48 hours after irradiation⁵Lineage depleted bone marrow cells. The data in figure 4C represent the mean ± standard deviation of five mice per group at each time point. The data in fig. 4C, 4M and 4N are representative of two independent experiments. (. P)<0.05,**P<0.01,***P<0.001, analysis of variance (ANOVA) using Tukey post hoc test).

FIGS. 5A, 5B, 5D-5G, 5I, 5J and 5L-5P show enhanced T cell reconstitution and reduced GVHD in NSG-BLT mice and mice after allogeneic HST. Fig. 5H, 5K and 5Q show expanded flow cytometry characterization of BMC-generated T cells and culture-generated T cell progenitors.

Figures 5A, 5B and 5D show reconstitution of CD3+ T cells (figure 5A) and CD19+ B cells (figure 5B) with a typical flow cytometry plot at day 75 in humanized NSG-BLT mice along with CD4 +: CD8+ (fig. 5D).

FIG. 5C shows expanded characterization of NSG-BLT mouse blood cell analysis. FIG. 5C shows the quantification of pre-B CFU from bone marrow of NSG-BLT mice receiving and not receiving BMC treatment at two time points post-transplantation. Data are mean ± standard deviation of n-4 from a single donor in one experiment. (. P <0.05,. P <0.01,. P <0.001, analysis of variance (ANOVA) using Tukey post-hoc test).

Fig. 5E shows an exemplary flow cytometry plot at day 75.

Fig. 5F and 5G show survival in NSG-BLT mice (n ═ 10 per group) (fig. 5F) and reconstitution of human regulatory T cells in the thymus and spleen of NSG-BLT mice (fig. 5G).

In FIGS. 5A, 5B and 5D-5G, xenogenic humanized BLT (marrow-liver-thymus) mice (Brainer, D.M. et al, Induction of robust cellular and human viral-induced humanized immune responses in human immunogenic viral-induced BLT mice. journal of virology 83,7305-7321(2009)) were generated and used as described previously. BMC-free mice (NSG-BLT) and mice with Dual BMC (NSG-BLT + Dual BMC) from human donor tissue from the same source were analyzed.

FIG. 5H shows a scheme for identifying T in thymus and spleen_regCD4 of cells⁺FoxP3 in cells and isoforms ⁺Representative flow cytometry profiles of cells (3 independent experiments).

FIGS. 5I and 5J show survival (FIG. 5I) and reconstitution (FIG. 5J) of donor-derived regulatory T cells in the thymus and spleen of allo-suppressed Balb/c mice. In FIGS. 5I and 5J, BALB/cJ receptor mice received 850cGy of L-TBI. Within 48 hours after irradiation, the allogenic GFP 5x10 was administered⁵Lineage depleted GFB BM cells and 10⁶GFP splenocytes were transplanted onto mice. One group received dual BMC treatment simultaneously.

FIG. 5K shows HSC (Lin) sorted 14 days after co-culture with OP9-DL1 cells^-ckit⁺Sca-1⁺) And representative FACS profiles of T cell progenitors expressing CD44/CD25 (2 independent experiments).

FIGS. 5L-5P show a comparison of T cell reconstitution in mice treated with BMC or OP9-DL1 derived pro-T cells. Balb/cJ recipient mice received 850Gy L-TBI and provided OP9-DL1 culture-derived 5x10⁶Allogeneic GFP T cell progenitor cell +10³Isogenic HSC or dual-BMC +5x10⁵Lineage depleted allogeneic GFP BM cells. DP (FIG. 5L) and SP4 (FIG. 5M) and SP8 thymocytes in the thymus of mice 424 transplanted 28 days post-transplantation(FIG. 5N) and total number of peripheral CD4+ (FIG. 5O) and CD8+ T cells (FIG. 5P) in the spleen. Data in a-d are mean ± standard deviation of n-10 mice at the start of the 425 study, representing 3 donors.

The data in fig. 5G and 5J are mean ± standard deviation of n ═ 7 mice. The data in figure 5I are mean ± standard deviation of n-10 mice. The data in fig. 5G, 5I and 5J represent 2 independent experiments. The data in fig. 5L-5P are mean ± standard deviation of n-10 mice, representing 2 independent experiments. Different samples were analyzed separately. (. P <0.05,. P <0.01,. P <0.001, analysis of variance (ANOVA)429) using Tukey post-hoc testing).

Figure 5Q shows a representative flow cytometry profile for identifying ckit and isoforms of ETP in the thymus.

Figures 6A-6H show quantitative analysis of T cell export, immune repertoire and vaccination in mice with regenerative T cells.

FIGS. 6A and 6B show signal binding T cell receptor deletion loop (sTREC) analysis from (a) isolated thymus (FIG. 6A) and spleen (FIG. 6B) of mice.

Figure 6C shows the diversity of T cell antigen receptors analyzed by sequencing V and J segments of CDR3 β chains in BMC and transplanted mice. Each band represents a clone. The figure provides the depth (length of bars) and diversity (number of bars) of mouse T cells. Samples were collected from five mice per group and combined data is presented.

Figure 6D shows the corresponding schedule for antigen-specific donor T cell analysis by vaccination.

Fig. 6E and 6F show SIINFEKL-tetramer + donor CD8+ T cells counted in vaccinated mice after syn-HSCT (fig. 6E) and allo-HSCT (fig. 6F).

Fig. 6G and 6H show stimulation of 440OP9-DL1T cell precursor and dual BMC treated splenocytes at days 22 and 42 after HSCT and staining of surface markers and intracellular cytokines using antibodies specific for CD45.1, CD4, IFN- γ, and TNF- α. Cells were gated on CD4+ or CD8+ donor cells and analyzed for IFN-. gamma. -and TNF-. alpha. -positive cells. In FIGS. 6A-6C and 6E, the B6 receptor accepts1000cGy L-TBI and 5x10⁵Lineage depleted allogeneic GFP BM cells. Mice post HSCT without BMC (transplant only), mice post HSCT treated with BMP-2BMC, and mice post HSCT treated with dual BMC were analyzed and compared to non-irradiated mice that did not receive a transplant or inoculation. In FIG. 6B, sjTREC is normalized to 10⁵CD4+ splenocytes. In FIGS. 6F-6H, Balb/cJ recipient mice received 850Gy L-TBI and were provided with OP9-DL1 culture-derived 5x10⁶Allogeneic GFP T cell progenitor cell +10³Isogenic HSC or dual-BMC +5x10⁵Lineage depleted allogeneic GFP BM cells. The data in fig. 6A and 6B are mean ± standard deviation of n-10 mice, the data in fig. 6E and 6F are mean ± standard deviation of n-5 mice, and the data in fig. 6G and 6H are mean ± standard deviation of n-7 mice. All experiments are representative of two independent experiments. (. P) <0.05,**P<0.01,***P<0.001, analysis of variance (ANOVA) using Tukey post hoc test).

Detailed Description

I. Definition of

In order that the invention may be more readily understood, certain terms are first defined.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the case of any potential ambiguity, the definition provided herein takes precedence over any dictionary or external definition.

The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural (i.e., one or more) unless otherwise indicated herein or clearly contradicted by context. Unless otherwise specified, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"). Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

The term "about" or "approximately" generally means within 5%, or more preferably within 1%, of a given value or range.

In general, the terms "treat" or "treatment" are defined as the application or use of a therapeutic agent to a patient, or to a tissue or cell line isolated from a patient, who is suffering from a disease, disease symptom, or disease predisposition, with the aim of curing, treating, ameliorating, relieving, altering, remediating, ameliorating, improving, or affecting the disease, disease symptom, or disease predisposition. Thus, treatment may include inhibition, restraint, prevention, treatment, or a combination thereof. Treatment refers in particular to increasing the time to progression, accelerating remission, inducing remission, increasing remission, accelerating recovery, increasing the efficacy of or decreasing resistance to alternative therapy, or a combination thereof. "inhibiting" or "inhibiting" refers to, inter alia, delaying the onset of symptoms, preventing disease recurrence, reducing the number and frequency of relapses, increasing latency between symptom episodes, reducing the severity of symptoms, reducing the severity of episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing latency of symptoms, ameliorating symptoms, reducing secondary infection, prolonging patient survival, or a combination thereof. In one embodiment, the symptoms are primary, and in another embodiment, the symptoms are secondary. "Primary" refers to symptoms resulting directly from a disease, such as diabetes, while secondary refers to symptoms arising from or due to a primary cause. A symptom may be any manifestation of a disease or pathological condition.

Thus, as used herein, the term "treatment" or "treatment" includes any administration of the compositions described herein, and includes: (i) preventing the disease from occurring in a subject who may be predisposed to the disease but does not yet experience or exhibit the pathology or symptomatology of the disease; (ii) inhibiting a disease in a subject experiencing or exhibiting a pathology or symptom of the disease (i.e., arresting further development of the pathology and/or symptom); or (iii) ameliorating the disease (i.e., reversing the pathology and/or symptoms) in a subject experiencing or exhibiting the pathology or symptoms of the disease.

"treating," "preventing," or "ameliorating" of a disease or disorder refers to delaying or preventing the onset, reversal, alleviation, amelioration, inhibition, slowing or stopping the progression, worsening or regression of such disease or disorder, the progression or severity of the disorder associated with such disease or disorder. In one embodiment, the symptoms of the disease or disorder are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

The efficacy of the treatment is determined in conjunction with any known method for diagnosing a condition. Alleviation of one or more symptoms of the condition indicates that the composition is of clinical benefit. Any of the methods of treatment described above may be applied to any suitable subject, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably humans.

As used herein, the term "subject" includes any subject that may benefit from a hydrogel or implantable drug delivery device of the present invention. The term "subject" includes animals, e.g., vertebrates, amphibians, fish, mammals, non-human animals, including humans and primates, e.g., chimpanzees, monkeys, and the like. In one embodiment of the invention, the subject is a human.

The term "subject" also includes livestock of agricultural production, such as cattle, sheep, goats, horses, pigs, donkeys, camels, buffalo, rabbits, chickens, turkeys, ducks, geese and bees; and domestic animals such as dogs, cats, caged birds and ornamental fish, and so-called laboratory animals such as hamsters, guinea pigs, rats and mice.

Compositions for modulating the immune system

The invention features compositions and methods for modulating the immune system in a subject. The compositions of the present invention comprise a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing differentiation of the recruited cells into T cell progenitors.

The compositions and methods of the present invention provide advantages over the prior art. For example, the compositions and methods described herein enhance T cell progenitor seeding, T cell neogenesis, and broadening of the T cell receptor repertoire of the thymus. In addition, the compositions and methods described herein promote the production of donor CD4+ regulatory T cells and improve survival after allogeneic HSCT. The compositions and methods described herein increase donor chimerism, T cell production and induce a more robust antibody-specific T cell response to stimulation compared to adoptive transfer of T cell progenitors. The compositions of the invention may represent an easy to administer, ready-to-use method to achieve T cell production and reduced GVHD in HSCT.

In certain embodiments, the compositions and methods described herein also reduce toxicity resulting from the use of significantly reduced levels of growth factor, such as 1ng to about 1000 μ g growth factor or 1ng to about 1000ng growth factor, as compared to what is directed in the art. Furthermore, the compositions and methods of the present invention exhibit surprisingly enhanced activity, allowing for comparable efficacy in transplanting a reduced number of cells.

Support frame

The compositions of the present invention comprise a scaffold, such as a polymeric scaffold. The scaffold may comprise one or more biomaterials. Preferably, the biomaterial is a biocompatible material that is non-toxic and/or non-immunogenic. As used herein, the term "biocompatible material" refers to any material that, when implanted or placed in proximity to a biological tissue of a subject, does not elicit a significant immune response or deleterious tissue response over time, such as a unique shadow or significant irritation.

The scaffold may comprise a non-biodegradable or biodegradable biomaterial. In certain embodiments, the biomaterial may be a non-biodegradable material. Exemplary non-biodegradable materials include, but are not limited to, metals, plastic polymers, or silk polymers. In certain embodiments, the polymeric scaffold comprises a biodegradable material. Biodegradable materials can be degraded by physical or chemical action, such as by the combined action of heat, oxidation, or the level of ion exchange, or by cellular action, such as the processing of enzymes, peptides, or other compounds by nearby or resident cells. In certain embodiments, the polymeric scaffold comprises both non-degradable and degradable materials.

In some embodiments, the scaffold composition can degrade based on a predetermined rate selected from the group consisting of temperature, pH, hydration state and porosity, crosslink density, type, and susceptibility of chemical or backbone linkers to degradation. Alternatively, the scaffold composition degrades at a predetermined rate based on the chemical polymer ratio. For example, high molecular weight polymers consisting of lactide alone degrade within a few years, e.g. 1-2 years, while low molecular weight polymers consisting of a 50:50 mixture of lactide and glycolide degrade within weeks, e.g. 1, 2, 3, 4, 6 or 10 weeks. Calcium cross-linked gels consisting of high molecular weight, high guluronic acid alginate will degrade in vivo over a period of months (1, 2, 4, 6, 8, 10 or 12 months) to years (1, 2 or 5 years), whereas gels consisting of low molecular weight alginate and/or partially oxidized alginate will degrade within weeks.

In certain embodiments, one or more compounds or proteins disclosed herein (e.g., growth factors, differentiation factors, and homing factors) are covalently or non-covalently attached or attached to the scaffold composition. In various embodiments, one or more compounds or proteins disclosed herein are incorporated into or present in the structure or pore of the scaffold composition.

In some embodiments, the scaffold comprises a biomaterial that is modified, e.g., oxidized or reduced. The degree of modification, e.g., oxidation, can vary from about 1% to about 100%. As used herein, the degree of modification refers to the mole percentage of sites on the biomaterial that are modified by the functional groups. For example, the degree of modification can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, (ii), 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Intermediate values and ranges of recited values are intended to be part of the present invention. Exemplary modified biomaterials, such as hydrogels, include, but are not limited to, reduced alginate, oxidized alginate, MA-alginate (methacrylated alginate), or MA-gelatin.

Exemplary biomaterials suitable for use as scaffolds according to the present invention include glycosaminoglycans, silk, fibrin, collagen, and collagen, and collagen,Polyethylene glycol (PEG), polyhydroxyethylmethacrylate, polyacrylamide, poly (N-vinylpyrrolidone), (PGA), polylactic-co-glycolic acid (PLGA), Polyepsilon Caprolactone (PCL), polyethylene oxide, polypropylene fumarate (PPF), polyacrylic acid (PAA), polyhydroxybutyric acid, hydrolyzed polyacrylonitrile, polymethacrylic acid, polyvinylamine, alginate, pectinic acid; and alginate, fully or partially oxidized alginate, hyaluronic acid, carboxymethylcellulose, heparin sulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan gum, xanthan gum, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic starch, and combinations thereof. In certain embodiments, the biomaterial is selected from the group consisting of alginate, fully or partially oxidized alginate, and combinations thereof.

The stent of the present invention may comprise an outer surface. Alternatively or additionally, the stent may comprise an inner surface. The outer or inner surface of the stent of the present invention may be solid or porous. The pore size of the scaffold may be less than about 10nm, from about 100nm to 20 μm, or greater than 20 μm, for example up to and including 1000 μm in diameter. For example, the pores may be nanopores, micropores, or macropores. For example, the diameter of the nanopore is less than about 10 nm; the diameter of the micropores is in the range of about 100nm to 20 μm; and, the macropores have a diameter of greater than about 20 μm, such as greater than about 50 μm, such as greater than about 100 μm, such as greater than about 400 μm, such as greater than 600 μm or greater than 800 μm. In some embodiments, the pore size is about 50 μm to about 80 μm.

In some embodiments, the stents of the present invention are organized into various geometric shapes (e.g., discs, beads, pellets), niches, planar layers (e.g., sheets). For example, a disc having a diameter of about 0.1-200 millimeters (e.g., 5, 10, 20, 40, or 50 millimeters) may be implanted subcutaneously. The disc may have a thickness of 0.1 to 10 mm, for example 1, 2 or 5 mm. The disc can be easily compressed or lyophilized for administration to a patient. Exemplary discs for subcutaneous administration have the following dimensions: diameter 8 mm and thickness 1 mm.

In some embodiments, the scaffold may include multiple components and/or compartments. In other embodiments, the multi-compartment device is assembled in vivo by applying successive layers of similarly or differently doped gel or other scaffold material to the target site. For example, the device is formed by using a needle to sequentially inject the next inner layer into the center of the previously injected material, forming concentric spheres. In some embodiments, the non-concentric compartments are formed by injecting material into different locations in a previously injected layer. A multi-headed injection device extrudes the compartments in parallel and simultaneously. These layers are made of similar or different biomaterials differently doped with pharmaceutical compositions. Alternatively, the compartments self-organize according to their hydrophilic/hydrophobic properties or secondary interactions within each compartment. In other embodiments, the multi-component scaffold is optionally constructed in concentric layers, each layer having different physical properties, such as the percentage of polymer, the percentage of cross-linking of the polymer, the chemical composition of the hydrogel, pore size, porosity and pore structure, stiffness, toughness, ductility, viscoelasticity, growth factors, differentiation factors and/or homing factors incorporated therein, and/or other compositions incorporated therein.

Hydrogel and frozen gel scaffolds

In certain embodiments, the scaffold of the present invention comprises one or more hydrogels. Hydrogels are polymer gels that contain a network of pendant compound chains. Hydrogels are generally compositions comprising hydrophilic polymer chains. The network structure of hydrogels makes them capable of absorbing large amounts of water. Some hydrogels are highly stretchable and elastic; others are viscoelastic. Hydrogels are sometimes found to be a colloidal gel in which water is the dispersion medium. In certain embodiments, hydrogels are highly absorbent (they may contain more than 99% water (v/v)) natural or synthetic polymers that have a degree of flexibility very similar to natural tissue due to their significant water content. In certain embodiments, the hydrogel may have the property that when an appropriate shear stress is applied, the deformable hydrogel is significantly and reversibly compressed (up to 95% of its volume), resulting in an injectable macroporous preformed scaffold. Hydrogels have been used in therapeutic applications, for example as carriers for in vivo delivery of therapeutic agents (e.g., small molecules, cells, and biologics). Hydrogels are typically made from polysaccharides such as alginate. Polysaccharides can be chemically manipulated to adjust their properties and the properties of the resulting hydrogels.

The hydrogels of the present invention may be porous or non-porous. Preferably, the compositions of the present invention are formed from a porous hydrogel. For example, the hydrogel may be nanoporous, wherein the pores are less than about 10nm in diameter; micropores, wherein the diameter of the pores is preferably in the range of about 100nm to 20 μm; or macropores, wherein the pores have a diameter greater than about 20 μm, more preferably greater than about 100 μm, and even more preferably greater than about 400 μm. In certain embodiments, the hydrogel is macroporous, the pores having a diameter of about 50-80 μm. In some embodiments, the hydrogel is macroporous, having aligned pores with a diameter of about 400-500 μm. Methods for preparing porous hydrogels are known in the art. (see, e.g., U.S. patent No. 6,511,650, incorporated herein by reference).

Hydrogels can be composed of a variety of different rigid, semi-rigid, flexible, gel, self-assembling, liquid crystal, or fluid compositions, such as peptide polymers, polysaccharides, chorus polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals, and metal alloys. The composition is assembled into a hydrogel using methods known in the art, such as injection molding, lyophilization of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, foaming, fiber forming/processing, particle leaching, or combinations thereof. The assembled device is then implanted or applied to the body of the individual to be treated.

Compositions comprising hydrogels can be assembled in vivo in a variety of ways. Hydrogels are made of a gelling material and are introduced into the body in an ungelled form, where they gel in situ. Exemplary methods of delivering the components of the composition to the site where assembly occurs include methods of injection, spraying, printing, or precipitation at the tissue site through a needle or other extrusion tool (e.g., delivery using an applicator inserted through a cannula). In some embodiments, the non-gelled or non-shaped hydrogel material is mixed with at least one pharmaceutical composition prior to or at the time of introduction into the body. The resulting in vivo/in situ assembled device, e.g., hydrogel, contains a mixture of at least one pharmaceutical composition.

In situ assembly of the hydrogel may occur as a result of association or concerted or chemically catalyzed polymerization of the polymers. The concerted or chemical catalysis is initiated by a number of endogenous factors or conditions at or near the assembly site (e.g., body temperature, in vivo ions or pH), or exogenous factors or conditions provided to the assembly site by the operator (e.g., photon, thermal, electrical, acoustic or other radiation directly irradiated after introduction of the ungelled material). Energy is directed to the hydrogel material by a radiation beam or by a thermal or optical conductor, such as a wire or fiber optic cable or an ultrasonic transducer. Alternatively, shear thinning materials such as amphiphiles are used which re-crosslink after shear forces applied thereto are relieved, for example by passing them through a needle.

In some embodiments, the hydrogel can be assembled ex vivo. In some embodiments, the hydrogel is injectable. For example, hydrogels are macroporous scaffolds made in vitro. After injection into the body, the hole collapses causing the gel to become very small and allowing it to pass through the needle. See, e.g., WO 2012/149358; and Bencherif et al, 2012, proc.natl.acad.sci.usas 109.48:19590-5, the contents of which are incorporated herein by reference).

Suitable hydrogels for in vivo and ex vivo assembly of hydrogel devices are well known in the art and are described, for example, in Lee et al, 2001, chem. Rev.7: 1869-1879. Methods of self-assembling peptide amphiphiles are described, for example, in Hartgenink et al, 2002, Proc. Natl. Acad. Sci. USA 99: 5133-. The method of reversible gels after shear thinning is exemplified in Lee et al, 2003, adv.Mat.15: 1828-1832.

In certain embodiments, an exemplary hydrogel is composed of materials compatible with the encapsulating material, including polymers, nanoparticles, polypeptides, and cells. Exemplary hydrogels are made from alginate, polyethylene glycol (PEG), PEG-acrylate, agarose, hyaluronic acid, or synthetic proteins such as collagen or engineered proteins (i.e., peptide-based self-assembled hydrogels). For example, commercially available hydrogels include BD ^TM PuraMatrix^TM。BD^TM PuraMatrix^TMPeptide hydrogels are a synthetic matrix used to create a defined three-dimensional (3D) microenvironment for cell culture.

In some embodiments, the hydrogel is a biocompatible polymer matrix, which is biodegradable in whole or in part. Examples of hydrogel-forming materials include alginates and alginate derivatives, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid (PLGA) polymers, gelatin, collagen, agarose, hyaluronic acid derivatives, natural and synthetic polysaccharides, polyaminoacids such as polypeptides, in particular poly (lysine), polyesters such as polyhydroxybutyrate and poly-e-caprolactone, polyanhydrides; polyphosphazenes, polyvinyl alcohols, poly (alkylene oxides) especially poly (ethylene oxide), poly (allylamine) (PAM), poly (acrylates), modified styrene polymers such as poly (4-aminomethylstyrene), pluronic polyols, polyoxas, poly (uronic acids), poly (vinylpyrrolidone) and copolymers of the above, including graft copolymers. Synthetic polymers and naturally occurring polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels may also be used. As used herein, the term "derivative" refers to a compound that is derived from an analogous compound by a chemical reaction. For example, oxidized alginates are those obtained by oxidation of an alginate and are derivatives of alginate.

The implantable composition can have virtually any regular or irregular shape, including but not limited to spheres, cubes, polyhedra, prisms, cylinders, rods, discs, or other geometric shapes. Thus, in some embodiments, the implant is in the form of a cylinder having a diameter of about 0.5 to about 10mm and a length of about 0.5 to about 10 cm. Preferably, it is about 1 to about 5mm in diameter and about 1 to 5cm in length.

In some embodiments, the compositions of the present invention are in the form of spheres. When the composition is in spherical form, in some embodiments, its diameter ranges from about 0.5 to about 50 mm. In some embodiments, the spherical implant is about 5 to about 30mm in diameter. In an exemplary embodiment, the diameter is about 10 to about 25 mm.

In certain embodiments, the scaffold comprises a click hydrogel or a click cryogel. A click hydrogel or cryogel is a gel in which cross-linking between hydrogel or cryogel polymers is facilitated by a click reaction between the polymers. Each polymer may contain one or more functional groups that can be used for a click reaction. In view of the high specificity of the functional group pairs in the click reaction, the active compound may be added to the pre-device prior to or simultaneously with the formation of the hydrogel device by click chemistry. Non-limiting examples of click reactions that may be used to form a click hydrogel include copper I catalyzed azidoyne cycloaddition reactions, strain promoted homo-alkyne cycloaddition reactions, thiol-ene photo-coupling, diels-alder reactions, anti-electron demand diels-alder reactions, tetrazole-alkene light click reactions, oxime reactions, thiol-michael additions, and aldehyde-hydrazide couplings. Non-limiting aspects of click hydrogels are described in Jiang et al, 2014, Biomaterials,35: 4969-.

In various embodiments, click alginates are utilized (see, e.g., PCT international patent application publication No. WO 2015/154078 published on 10/8 of 2015, which is incorporated herein by reference in its entirety).

In some embodiments, a hydrogel (e.g., cryogel) system delivers one or more agents (e.g., growth factors such as BMP-2, and/or differentiation factors such as DLL-4, while creating space for cells (e.g., stem cells for Hematopoietic Stem Cell (HSC) infiltration and trafficking, etc.) in some embodiments, a hydrogel system according to the invention delivers BMP-2, which acts as a Hematopoietic Stem Cell (HSC) and/or hematopoietic progenitor cell enhancing/recruiting factor, and DLL-4 as a differentiation factor that promotes the T cell lineage specific differentiation of hematopoietic stem cells and/or hematopoietic progenitor cells.

In some embodiments, a cryogel composition (e.g., a MA-alginate formed cryogel composition) can serve as a delivery platform by creating local niches (e.g., specific niches for enhancing directed differentiation of T lineages). In some embodiments, the cryogel creates a local niche where cells (e.g., recruited stem or progenitor cells) and various exemplary agents of the invention (e.g., growth factors and/or differentiation factors) can be controlled. In certain embodiments, the cells and exemplary agents of the present invention are positioned in a small volume and the contact of the cells and agents can be quantitatively controlled in space and time.

In certain embodiments, hydrogels (e.g., cryogels) can be designed to coordinate the delivery of both growth and differentiation factors spatially and temporally, potentially enhancing overall immunomodulatory performance by modulating differentiation and/or dedifferentiation of recruited cells (e.g., hematopoietic stem or progenitor cells). In certain embodiments, the cells and growth factors/differentiation factors are localized in a small volume and the delivery of the factors in time and space can be quantitatively controlled. Since growth/differentiation factors are released locally, there is expected to be little systemic effect compared to systemic delivery of agents (e.g., growth factors).

Examples of polymer compositions for preparing cryogels or hydrogels are described throughout this disclosure and include alginates, hyaluronic acid, gelatin, heparin, dextran, carob bean gum, PEG derivatives (including PEG-co-PGA and PEG-peptide conjugates). The technique can be used with any biocompatible polymer, such as collagen, chitosan, carboxymethyl cellulose, pullulan, polyvinyl alcohol (PVA), poly (2-hydroxyethyl methacrylate) (PHEMA), poly (N-isopropylacrylamide) (PNIPAAm), or poly (acrylic acid) (PAAc). For example, in one particular embodiment, the composition comprises an alginate-based hydrogel/cryogel. In another embodiment, the scaffold comprises a gelatin-based hydrogel/cryogel.

Cryogels are a class of materials with highly porous interconnected structures that are produced using low temperature gelation (or cryogelation) techniques. Cryogels also have a highly porous structure. Typically, the active compound is added to the cryogel apparatus after the pore/wall structure of the cryogel has been cryogenically formed. The cryogels are characterized by high porosity, e.g., at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% pores, with thin pore walls characterized by high density polymer cross-linking. As used herein, the term "porosity" refers to the percentage of pore volume to scaffold volume. Intermediate values and ranges of recited values are intended to be part of the present invention. The walls of cryogels are typically dense and highly cross-linked, allowing them to be compressed through a needle into a subject without permanent deformation or significant structural damage.

In various embodiments, the pore walls comprise at least about 10, 15, 20, 25, 30, 35, or 40% (w/v) polymer. Intermediate values and ranges of recited values are intended to be part of the present invention. In other embodiments, the pore walls comprise about 10-40% polymer. In some embodiments, a polymer concentration of about 0.5-4% (w/v) is used (prior to cryogelation), and the concentration increases significantly after cryogelation is complete. Non-limiting aspects of cryogel gelation and increase in polymer concentration after cryogelation are discussed in Beduer et al, 2015Advanced Healthcare Materials 4.2:301-312, the entire contents of which are incorporated herein by reference.

In certain embodiments, cryogelation includes techniques for performing a polymerization crosslinking reaction in a reaction solution to be frozen. Non-limiting examples of freeze-gelation techniques are described in U.S. patent publication No. 20140227327, published 2014, 8, 14, which is incorporated herein by reference in its entirety. One advantage of cryogels is their high reversible deformability compared to conventional macroporous hydrogels obtained by phase separation. Frozen gels may be very soft, but may deform and reform into a shape. In certain embodiments, the cryogel may be very tough, may withstand high deformation, such as elongation and torsion, and may also be squeezed under mechanical force to exclude its solvent content. The improved deformability of the alginate cryogels results from the high cross-linking density of the unfrozen liquid channels of the reaction system.

During cryogelation, during freezing of the macromer (e.g., methacrylated alginate) solution, the macromer and initiator system (e.g., APS/TEMED) are expelled from the frozen concentrate within the channels between the ice crystals, and thus the reaction only occurs in these unfrozen liquid channels. After polymerization and ice melting, a porous material is produced whose microstructure is a negative replica of the ice formed. The ice crystals act as porogens. The desired pore size is achieved in part by varying the temperature of the cryogelation process. For example, the process of cryogelation is typically performed by rapidly freezing the solution at-20 ℃. Lowering the temperature to, for example, -80 ℃ will result in more ice crystals and smaller pores. In some embodiments, the cryogel is produced by low temperature polymerization of methacrylated (MA-) -alginate and MA-PEG. In some embodiments, the cryogel is produced by low temperature polymerization of at least MA-alginate, growth factors, differentiation factors, and MA-PEG.

In some embodiments, the invention also features a gelatin scaffold, e.g., a gelatin hydrogel, e.g., a gelatin cryogel, that is a cellular correspondence platform for biomaterial-based therapy. Gelatin is a mixture of polypeptides extracted from collagen by partial hydrolysis. These gelatin stents have significant advantages over other types of stents and hydrogels/cryogels. For example, the gelatin scaffolds of the present invention support the attachment, proliferation and survival of cells, and are degraded by cells, e.g., by reaction with enzymes such as Matrix Metalloproteinases (MMPs), e.g., recombinant matrix metalloproteinase-2 and-9.

In certain embodiments, the preformed gelatin cryogel rapidly regains its substantially original shape ("shape memory") when injected subcutaneously into a subject (e.g., a mammal such as a human, dog, cat, pig, or horse) and causes little or no adverse host immune response (e.g., immune rejection) after injection.

In some embodiments, a hydrogel (e.g., cryogel) comprises a polymer that is modified, e.g., sites on the polymer molecule are modified with methacrylic groups (methacrylate (MA) or acrylic groups (acrylate). an exemplary modified hydrogel/cryogel is MA-alginate (methacrylated alginate) or MA-gelatin.in the case of MA-alginate or MA-gelatin, 50% corresponds to the degree of methacrylation of the alginate or gelatin.

In certain embodiments, the polymer may also be modified with acrylate groups instead of methacrylate groups. This product is then referred to as an acrylate polymer. The degree of methacrylation (or acrylation) of most polymers can vary. However, some polymers (e.g., PEG) retain their water solubility even at 100% chemical modification. After crosslinking, the polymer typically reaches near complete methacrylate group conversion, indicating a crosslinking rate of about 100%. As used herein, the term "crosslinking rate" refers to the percentage of macromers that are covalently linked. For example, the polymers in the hydrogel are 50-100% crosslinked (covalent bonds). The degree of crosslinking correlates with the durability of the hydrogel. Thus, high levels of crosslinking (90-100%) of the modified polymer are desirable.

For example, highly crosslinked hydrogel/cryogel polymer compositions can be characterized as having at least about 50% polymer crosslinking (e.g., about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%; values and ranges intermediate to those stated are intended to be part of the invention). High levels of crosslinking impart mechanical robustness to the structure. Preferably, the percentage of crosslinking is less than about 100%. The composition is formed using a free radical polymerization process and a freeze gelation process. For example, cryogels are formed by low temperature polymerization of methacrylated gelatin, methacrylated alginate or methacrylated hyaluronic acid. In some embodiments, the cryogel comprises a concentration of methacrylated gelatin macromer or methacrylated alginate macromer of about 1.5% (w/v) or less (e.g., about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or less; intermediate values and ranges of said values are intended to be part of the present invention). In some embodiments, the methacrylated gelatin or alginate macromer is at a concentration of about 1% (w/v).

In certain embodiments, the cryogel comprises at least about 75% (v/v) wells, e.g., about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (v/v) or more wells. Intermediate values and ranges of recited values are intended to be part of the present invention. In some embodiments, the pores are interconnected. Interconnectivity is important to the functionality of hydrogels and/or cryogels because without interconnectivity, water can become trapped in the gel. The interconnectivity of the pores allows water (and other compositions such as cells and compounds) to enter and exit the structure. In certain embodiments, in a fully hydrated state, the hydrogel (e.g., cryogel) comprises at least about 90% water (volume of water/volume of scaffold) (e.g., about 90 to 99%, at least about 92%, 95%, 97%, 99%, or more). For example, at least about 90% (e.g., at least about 92%, 95%, 97%, 99% or more) of the volume of the frozen gel is made up of the liquid (e.g., water) contained in the pores. Intermediate values and ranges of recited values are intended to be part of the present invention. In certain embodiments, up to about 50%, 60%, 70% water is not present in the compressed or dehydrated hydrogel, e.g., a cryogel comprises less than about 25% (e.g., about 20%, 15%, 10%, 5% or less) water.

In certain embodiments, the cryogels of the invention comprise pores large enough for cells to pass through. For example, the cryogel comprises pores having a diameter of about 20-500 μm, such as about 20-30 μm, about 30-150 μm, about 50-500 μm, about 50-450 μm, about 100-400 μm, about 200-500 μm. In some embodiments, the hydrated pore size is about 1-500 μm (e.g., about 10-400 μm, about 20-300 μm, about 50-250 μm). In certain embodiments, the cryogel comprises pores having a diameter of about 50-80 μm.

In some embodiments, the injectable hydrogel or cryogel is further functionalized by the addition of functional groups selected from the group consisting of: amino, vinyl, aldehyde, thiol, silane, carboxyl, azide, or alkyne. Alternatively or additionally, the cryogel is further functionalized by the addition of additional cross-linking agents (e.g., multi-armed polymers, salts, aldehydes, etc.). The solvent may be aqueous, in particular acidic or basic. The aqueous solvent may include water-miscible solvents (e.g., methanol, ethanol, DMF, DMSO, acetone, dioxane, etc.).

For cryogels, low temperature crosslinking may occur in the mold and the cryogel (injectable) may be degradable. The pore size can be controlled by the choice of the primary solvent used, the incorporation of the porogen, the freezing temperature and rate of application, the crosslinking conditions (e.g., polymer concentration), and the type and molecular weight of the polymer used. The shape of the cryogel can be determined by the mold and can therefore assume any shape desired by the manufacturer, for example by preparing various sizes and shapes (discs, cylinders, squares, wires, etc.) by low temperature polymerization.

Injectable cryogels can be prepared at micron to centimeter scales. An exemplary volume may be from a few hundred μm³(e.g., about 100-³) To about 10cm³Are not equal. In certain embodiments, an exemplary scaffold composition is about 100 μm in size³To about 100mm³. In various embodiments, the scaffold size is about 10mm³To about 100mm³. In certain embodiments, the scaffold has a size of about 30mm³。

In some embodiments, the cryogel is hydrated, loaded with the compound and loaded into a syringe or other delivery device. For example, syringes are pre-filled and refrigerated until use. In another embodiment, the frozen gel is dehydrated, e.g., lyophilized, optionally with a compound (e.g., a growth factor or differentiation factor) loaded in the gel and dried or stored under refrigeration. Prior to administration, the syringe or device containing the cryogel may be contacted with a solution containing the compound to be delivered. For example, the syringe of a cryogel pre-filled syringe is filled with a physiologically compatible solution, such as Phosphate Buffered Saline (PBS). Alternatively, the cryogel will be administered to the desired anatomical site, followed by administration of a physiologically compatible solution, optionally containing other ingredients, such as growth factors and/or differentiation factors or together with one or more compounds disclosed herein. The frozen gel is then rehydrated and its shape integrity is restored in situ. In certain embodiments, the volume of PBS or other physiological solution administered after the cryogel is placed is typically about 10 times the volume of the cryogel itself.

Cryogels also have the following advantages: upon compression, the frozen gel composition maintains structural integrity and shape memory properties. For example, the cryogel may be injected through a hollow needle. For example, the cryogel returns to its approximately original geometry after passing through a needle (e.g., a 16 gauge (G) needle), e.g., having an inner diameter of 1.65 mm). Other exemplary needle sizes are 16 gauge, 18 gauge, 20 gauge, 22 gauge, 24 gauge, 26 gauge, 28 gauge, 30 gauge, 32 gauge, or 34 gauge needles. Injectable cryogels are designed to pass through hollow structures, such as very thin needles, e.g., 18-30 gauge G needles. In certain embodiments, the cryogel returns to approximately the original geometry within a short time period, e.g., less than about 10 seconds, less than about 5 seconds, less than about 2 seconds, or less than about 1 second, after passing through the needle.

Any suitable injection device can be used to inject the cryogel into a subject. For example, the cryogel may be injected through a needle using a syringe. The syringe may comprise a plunger, a needle and a composition comprising the present invention. Injectable cryogels can also be injected into a subject using a catheter, cannula, or stent.

Injectable cryogels can be molded to the desired properties in the form of rods, squares, discs, spheres, cubes, fibers, foams. In some cases, the cryogel is in the form of a disk, cylinder, square, rectangle, or wire. For example, the cryogel composition may be about 100 μm in size ³To 10cm³E.g. 10mm³To about 100mm³. For example, the frozen gel composition can have a diameter of about 1mm to about 50mm (e.g., about 5 mm). Optionally, the thickness of the cryogel is from about 0.2mm to about 50mm (e.g., about 2 mm).

Three exemplary frozen gel material systems are described below.

a) Methacrylated gelatin cryogel (CryoGeIMA) -an exemplary cryogel using methacrylated gelatin, and the results are described in U.S. patent application publication No. 2014-0227327, published 8-14-2014, which is incorporated by reference herein in its entirety.

b) Methacrylated alginate cryogel (CryoMAAlginate) -an exemplary cryogel using methacrylated alginate, and the results are described in U.S. patent application publication No. 2014-0227327, published 8-14-2014, which is incorporated by reference herein in its entirety.

c) Click alginate cryogels (cryociks) -substrates of laponite nanosheets are click alginates (PCT international patent application No. WO2015/154078 published 10/8/2015, which is incorporated herein by reference in its entirety). In some embodiments, the substrate comprises a lithiated diatomaceous earth (a commercially available silicate clay used in many consumer products such as cosmetics). Laponite has a large surface area and a high negative charge density, which enables it to adsorb positively charged moieties on various proteins and other bioactive molecules through electrostatic interactions, thus enabling drug loading. When placed in an environment with a low drug concentration, the adsorbed drug is released from the laponite in a sustained manner. The system allows for more flexible release of various agents, such as growth factors, than the substrate alone.

Various embodiments of the present subject matter include a delivery vehicle comprising a pore-forming scaffold composition. For example, following injection of the hydrogel into a subject, pores (e.g., macropores) are formed in situ within the hydrogel. The pores formed in situ by degradation of the sacrificial porogen hydrogel within the surrounding hydrogel (bulk hydrogel) facilitate recruitment and trafficking of cells, as well as release of any composition or agent described herein, such as a growth factor (e.g., BMP-2), differentiation factor, or homing factor, or any combination thereof. In some embodiments, the sacrificial porogen hydrogel, the bulk hydrogel, or both the sacrificial porogen hydrogel and the bulk hydrogel may comprise any composition or agent described herein, such as a growth factor, a differentiation factor, and/or a homing factor, or any combination thereof.

In various embodiments, the pore-forming composition becomes macroporous over time when present in the body of a recipient animal (e.g., a mammalian subject). For example, the pore-forming composition can comprise a sacrificial porogen hydrogel and a bulk hydrogel, wherein the sacrificial porogen hydrogel degrades at least about 10% faster than the bulk hydrogel (e.g., at least about 15% faster, at least about 20% faster, at least about 25% faster, at least about 30% faster, at least about 35% faster, at least about 40% faster, at least about 45% faster, at least about 50% faster). Intermediate values and ranges of recited values are intended to be part of the present invention. The sacrificial porogen hydrogel may degrade leaving large pores in its place. In certain embodiments, the macropores are open interconnected macropores. In some embodiments, the sacrificial porogen hydrogel may degrade faster than the bulk hydrogel because the sacrificial porogen hydrogel is (i) more soluble in water (including lower solubility index), (ii) cross-linked with a protease-mediated degradation motif, described in U.S. patent application publication No. 2005-0119762 (which is incorporated herein by reference in its entirety) published 6/2/2005, (iii) comprises a shorter polymer that degrades faster than the longer bulk hydrogel polymer, (iv) is modified to make it more susceptible to hydrolysis (e.g., by oxidation) than the bulk hydrogel, and/or (v) is more susceptible to enzymatic degradation than the bulk hydrogel.

In various embodiments, the scaffold is loaded (e.g., impregnated) with one or more active compounds after polymerization. In certain embodiments, the device or scaffold polymer-forming material is mixed with one or more active compounds prior to polymerization. In some embodiments, the device or scaffold polymer-forming material is mixed with one or more active compounds prior to polymerization and then loaded with more of the same or one or more additional active compounds after polymerization.

In some embodiments, the pore size or total pore volume of the composition or scaffold is selected to affect the release of the compound from the device or scaffold. Exemplary porosities (e.g., nanoporous, microporous, and macroporous scaffolds and devices) and total pore volumes (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more of the scaffold volume) are described herein. Intermediate values and ranges of recited values are intended to be part of the present invention. The increased pore size and total pore volume increases the amount of compound that can be delivered into or near a tissue, such as bone marrow. In some embodiments, the pore size or total pore volume is selected to increase the speed at which the active ingredient exits the composition or scaffold. In various embodiments, the active ingredient may be incorporated into the scaffold material of the hydrogel or cryogel, e.g., to achieve continuous release of the active ingredient from the nail or device over a longer period of time than the active ingredient may diffuse from the lumen.

Porosity affects the recruitment of cells to and release of substances from the device and scaffold. The pores may be, for example, nanopores, micropores, or macropores. For example, the diameter of the nanopore is less than about 10 nm. The micropores have a diameter in the range of about 100nm to about 20 μm. The macropores have a diameter greater than about 20 μm (e.g., greater than about 100 μm or greater than about 400 μm). Exemplary macropore sizes include diameters of about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, and about 600 μm. Intermediate values and ranges of recited values are intended to be part of the present invention. The macropores are of a size that allows eukaryotic cells to enter or leave the composition. In one embodiment, the macroporous composition has pores with a diameter of about 400 μm to about 500 μm. The preferred pore size depends on the application. In certain embodiments, the pores have a diameter of about 50 μm to about 80 μm.

In various embodiments, the composition is manufactured in one stage, wherein a layer or compartment is manufactured and infused or coated with one or more compounds. Exemplary biologically active compositions comprise polypeptides and polynucleotides. In certain embodiments, the composition is manufactured in two or more (3, 4, 5, 6 … … 10 or more) stages, wherein one layer or compartment is manufactured and infused or coated with one or more compounds, then a second, third, fourth or more layers are constructed, then one or more compounds are infused or coated sequentially. In some embodiments, each layer or compartment is the same as each other or is distinguished from each other by the amount or mixture of bioactive compositions and different chemical, physical and biological properties. By controlling the molecular weight, degradation rate, and scaffold formation process, polymers can be formulated for specific applications. Coupling reactions can be used to covalently attach a bioactive agent (e.g., a differentiation factor) to a polymer backbone.

In some embodiments, one or more compounds are added to the scaffold composition using known methods, including surface adsorption, physical immobilization, e.g., using a phase change to entrap a substance in the scaffold material. For example, when the scaffold composition is in an aqueous or liquid phase, the growth factors are mixed with the scaffold composition and upon a change in environmental conditions (e.g., pH, temperature, ionic concentration), the liquid gels or coagulates to entrap the bioactive substance. In some embodiments, covalent coupling, e.g., using an alkylating or acylating agent, is used to provide stable, long-term presentation of the compound in a defined conformation on the scaffold. Exemplary reagents for covalent coupling of such species are provided in the table below.

Table 1: method for covalent coupling of peptides/proteins to polymers

[a] EDC, i.e. l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride;

DCC dicyclohexylcarbodiimide

Alginate stent

In certain embodiments, the compositions of the present invention comprise alginate hydrogels. The alginates are polysaccharide-based polymers that can be formulated for specific applications by controlling molecular weight, degradation rate, and scaffold formation methods. The alginate polymer is composed of two different monomer units, (1-4) -linked β -D-mannuronic acid (M units) and α L-guluronic acid (G units) monomers, which can vary in proportion and order distribution along the polymer chain. The alginate polymer is a polyelectrolyte system that is resistant to divalent cations (e.g., Ca) ⁺²、Mg⁺²、Ba⁺²) Have strong affinity and form stable hydrogels when exposed to these molecules. See Martinsen A. et al, 1989, Biotech.&Bioeng, 33: 79-89). For example, calcium cross-linked alginate hydrogels can be used in dental applications, wound dressings, chondrocyte transfer, and as matrices for other cell types. Without wishing to be bound by theory, it is believed that the G units are preferably crosslinked using calcium crosslinking, whereas crosslinking based on click reactions is less distinguishable on G units or M units (i.e. both G and M units can be crosslinked by click chemistry). Such alginate hydrogels and methods for their manufacture are known in the art. See, e.g., international patent application publication No. WO2017/075055a1, published on 5/4/2017, which is incorporated herein by reference in its entirety.

Alginate polymers useful in the context of the present invention may have an average molecular weight of from about 20kDa to about 500kDa, such as from about 20kDa to about 40kDa, from about 30kDa to about 70kDa, from about 50kDa to about 150kDa, from about 130kDa to about 300kDa, from about 230kDa to about 400kDa, from about 300kDa to about 450kDa, or from about 320kDa to about 500 kDa. In one embodiment, alginate polymers useful in the present invention may have an average molecular weight of about 32 kDa. In another embodiment, alginate polymers useful in the present invention can have an average molecular weight of about 265 kDa. In some embodiments, the alginate polymer has a molecular weight of less than about 1000kDa, such as less than about 900kDa, less than about 800kDa, less than about 700kDa, less than about 600kDa, less than about 500kDa, less than about 400kDa, less than about 300kDa, less than about 200kDa, less than about 100kDa, less than about 50kDa, less than about 40kDa, less than about 30kDa, or less than about 25 kDa. In some embodiments, the alginate polymer has a molecular weight of about 1000kDa, such as about 900kDa, about 800kDa, about 700kDa, about 600kDa, about 500kDa, about 400kDa, about 300kDa, about 200kDa, about 100kDa, about 50kDa, about 40kDa, about 30kDa, or about 25 kDa. In one embodiment, the alginate polymer has a molecular weight of about 20 kDa.

The coupling reaction can be used to covalently attach a biologically active agent (e.g., an atom, chemical group, nucleoside, nucleotide, nucleobase, sugar, nucleic acid, amino acid, peptide, polypeptide, protein, or protein complex) to the polymer backbone.

The term "alginate" is used interchangeably with the term "alginate polymer" and includes unmodified alginates or modified alginates. Modified alginates include, but are not limited to, oxidized alginates (e.g., comprising one or more alginate monomeric units) and/or reduced alginates (e.g., comprising one or more alginate monomeric units). In some embodiments, the oxidized alginate comprises an alginate comprising one or more aldehyde groups, or an alginate comprising one or more carboxylate groups. In other embodiments, the oxidized alginate comprises a highly oxidized alginate, e.g., comprising one or more alginate units. The oxidized alginate may also contain relatively small amounts of aldehyde groups (e.g., less than 15%, e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% aldehyde groups or oxidation (on a molar basis)). Intermediate values and ranges of recited values are intended to be part of the present invention. The term "alginate" or "alginate polymer" may also include alginates, such as unmodified alginates, oxidized alginates or reduced alginates, or methacrylated alginates or acrylated alginates. The alginate may also refer to any number of alginic acid derivatives (e.g., calcium, sodium or potassium salts, or propylene glycol alginate). See, e.g., WO1998012228a1, incorporated herein by reference.

Hyaluronic acid

In certain embodiments, the compositions of the present invention comprise a hyaluronic acid hydrogel. Hyaluronic acid (HA; conjugate base hyaluronate) is an anionic non-sulfated glycosaminoglycan and is widely distributed in connective, epithelial or neural tissue. As one of the main components of the extracellular matrix, hyaluronic acid significantly contributes to cell proliferation and migration. Natural hyaluronic acid is an important component of articular cartilage, muscle connective tissue and skin.

Hyaluronic acid is a disaccharide polymer composed of D-glucuronic acid and N-acetyl-D-glucosamine linked by alternating β - (1 → 4) and β - (1 → 3) glycosidic linkages. Hyaluronic acid may be 25,000 disaccharide repeats in length. The size range of the hyaluronic acid polymer is 5,000 to 20,000,000 Da. Hyaluronic acid may also contain silicon.

Hyaluronic acid is stable energetically, in part due to the stereochemistry of its constituent disaccharides. The bulky groups on each sugar molecule are in sterically favored positions, while the smaller hydrogens occupy less favored axial positions.

Hyaluronic acid can be degraded by a family of enzymes known as hyaluronidases, which are present in many mammals, such as humans. Hyaluronic acid can also be degraded by non-enzymatic reactions. These include acidic and basic hydrolysis, ultrasonic decomposition, thermal decomposition and oxidant degradation.

Due to its high biocompatibility and its ubiquitous presence in the extracellular matrix of tissues, hyaluronic acid can be used to form hydrogels, such as cryogels, as a scaffold for biomaterials in tissue engineering studies. The hyaluronic acid hydrogel is formed by crosslinking. Hyaluronic acid can form hydrogels, such as cryogels, into a desired shape to deliver therapeutic molecules into a host. Hyaluronic acid used in the present compositions may be crosslinked by linking thiols, methacrylates, cetyl amide, and tyramine. Hyaluronic acid may also be directly crosslinked with formaldehyde or divinyl sulfone.

The term "hyaluronic acid" includes unmodified hyaluronic acid or modified hyaluronic acid. Modified hyaluronic acid includes, but is not limited to, oxidized hyaluronic acid and/or reduced hyaluronic acid. The term "hyaluronic acid" or "hyaluronic acid polymer" may also include hyaluronic acid, such as unmodified hyaluronic acid, oxidized hyaluronic acid or reduced hyaluronic acid, or methacrylated hyaluronic acid or acrylated hyaluronic acid. Hyaluronic acid also refers to any number of hyaluronic acid derivatives.

Porous and pore-forming scaffold

The scaffold of the present invention may be non-porous or porous. In certain embodiments, the scaffold of the present invention is porous. The porosity of the scaffold composition affects the migration of cells through the device. The pores may be nanoporous, microporous, or macroporous. For example, the diameter of the nanopore is less than about 10 nm. The micropores have a diameter in the range of about 100nm to about 20 μm. The macropores have a diameter greater than about 20 μm (e.g., greater than about 100 μm or greater than about 400 μm). Exemplary macropore sizes include diameters of about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, and about 600 μm. Intermediate values and ranges of recited values are intended to be part of the present invention. The macropores are of a size that allows eukaryotic cells to enter or leave the composition. In certain embodiments, the macroporous composition has pores with a diameter of about 400 μm to about 500 μm. The size of the holes can be adjusted for different purposes. For example, for cell recruitment and cell release, the pore size may be greater than 50 μm. In certain embodiments, the macroporous composition has pores with a diameter of about 50 μm to about 80 μm.

In some embodiments, the stent contains pores prior to administration to a subject. In some embodiments, the scaffold comprises a pore-forming scaffold composition. Pore-forming scaffolds and methods of making pore-forming scaffolds are known in the art. See, for example, U.S. patent publication US2014/0079752a1, which is incorporated by reference herein in its entirety. In certain embodiments, the pore-forming scaffold is not initially porous, but rather becomes macroporous over time while residing in a recipient animal, such as a mammalian subject. In certain embodiments, the pore-forming scaffold is a hydrogel scaffold. The pores may be formed at different times, for example, after about 12 hours of administration, i.e., residence in the subject, or after 1, 3, 5, 7, or 10 days or more.

In certain embodiments, the pore-forming scaffold comprises a first hydrogel and a second hydrogel, wherein the first hydrogel degrades at least about 10% faster (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 2% faster, or at least about 5 times faster) than the second hydrogel. Intermediate values and ranges of recited values are intended to be part of the present invention. In certain embodiments, the first hydrogel comprises a porogen that degrades to leave pores in its place. For example, the first hydrogel is a porogen and the resulting pores after in situ degradation are within 25% of the original porogen size, such as within 20%, within 15%, or within 10% of the original porogen size. Preferably, the resulting pores are within 5% of the original porogen size. Intermediate values and ranges of recited values are intended to be part of the present invention. Due to differences in their physical, chemical and/or biological properties, the first hydrogel may degrade faster than the second hydrogel. In certain embodiments, the first hydrogel degrades faster than the second hydrogel because the first hydrogel is more soluble in water (including a lower solubility index). In certain embodiments, the first hydrogel degrades faster because it crosslinks with a protease-mediated degradation motif, as described in U.S. patent publication No. US2005/0119762a1, the contents of which are incorporated herein by reference.

In certain embodiments, the molecular weight of the polymer used to form the first hydrogel composition (porogen) is about 50 kilodaltons (kDa), and the molecular weight of the polymer used to form the second hydrogel composition (bulk) is about 250 kDa. Shorter polymers (e.g., polymers of porogens) degrade faster than longer polymers (e.g., polymers of bulk compositions). In certain embodiments, the composition is modified to be more hydrolytically degradable due to the presence of saccharide groups (e.g., about 3-10% of the saccharides of the alginate composition). In certain embodiments, the porogen hydrogel is chemically modified, e.g., oxidized, to make it more susceptible to degradation. In some embodiments, the porogen hydrogel is more susceptible to enzymatic degradation than the bulk hydrogel. The composite (first and second hydrogel) compositions are permeable to bodily fluids, for example, comprising an enzyme that is exposed to the composition and degrades the porogen hydrogel. In some embodiments, the second hydrogel is crosslinked around the first hydrogel, i.e., the porogen (first hydrogel) is completely physically embedded in the bulk (second) hydrogel.

The click reagents disclosed herein may be provided in a bulk hydrogel or a porogen hydrogel. In an exemplary embodiment, the click agents, such as polymers and nanoparticles, are provided in a bulk hydrogel.

In certain embodiments, hydrogel microbeads ("porogens") are formed. The porogen is encapsulated in a "bulk" hydrogel that is not or is degraded at a slower rate than the porogen. The composite material is devoid of pores immediately after the hydrogel is formed or injected into the desired site in the body. Subsequently, porogen degradation results in the formation of pores in situ. The size and distribution of the pores is controlled during porogen formation and mixed with the polymer of the bulk hydrogel being formed.

In some embodiments, the polymer used for the pore-forming scaffold is naturally occurring or synthetic. In one embodiment, both the porogen and the bulk hydrogel are formed from alginate.

In certain embodiments, alginate polymers suitable for use in porogen formation have a molecular weight of 5,000 to 500,000 daltons. The polymer is optionally further modified (e.g., by oxidation with sodium periodate) (Bouhadir et al, 2001, Biotech.prog.17: 945-. In certain embodiments, the polymer is extruded through a sparger with coaxial gas flow to a divalent cation (e.g., Ca)²⁺Or Ba²⁺) Cross-linked in the bath to form hydrogel microbeads. Higher gas flow rates result in lower porosity The diameter of the dose.

In some embodiments, the porogen hydrogel microbeads contain oxidized alginate. For example, the porogen hydrogel may comprise about 1-50% (w/v) oxidized alginate. In exemplary embodiments, the porogen hydrogel may comprise about 1-10% (w/v) oxidized alginate. In one embodiment, the porogen hydrogel may comprise about 7.5% oxidized alginate.

In certain embodiments, the concentration of dipotassium ion used to form the porogen can vary from about 5 to about 500mM, and the concentration of polymer is about 1% to about 5% (weight/volume). However, any method that produces a porogen that is significantly smaller than the bulk phase is suitable. Porogen chemistry can be further manipulated to produce porogens that interact with or inhibit interaction with host proteins and/or cells.

Alginate polymers suitable for forming block hydrogels have a molecular weight of about 5,000 to about 500,000 Da. The polymer may be further modified (e.g., by oxidation with sodium periodate) to promote degradation, so long as the bulk hydrogel degrades more slowly than the porogen. The polymer may also be modified to present biological cues that control cellular response (e.g., integrin binding adhesion peptides, such as RGD). The porogens or bulk hydrogels may also encapsulate bioactive factors, such as oligonucleotides, growth factors, or drugs to further control cellular response. The concentration of divalent ions used to form the bulk hydrogel can vary from about 5 to about 500mM, and the concentration of polymer is from about 1% to about 5% (weight/volume). The elastic modules of the block polymer are adapted according to their purpose, for example to recruit stem or progenitor cells.

Methods related to producing the hydrogels described herein include the following. Bouhadir et al, 1999, Polymer,40:3575-84 (incorporated herein by reference in its entirety) described the oxidation of alginate with sodium periodate and characterized the reaction. Bouhadir et al, 2001, Biotechnol.prog.,17:945-50 (incorporated herein by reference in its entirety) describe the oxidation of high molecular weight alginates to form alginic acid aldehyde (alginic acid dialdehyde is high molecular weight (M)_w) Alginate, wherein the alginate is present in a percentage, for example 5%The sugar of (a) is oxidized to form an aldehyde), and applications that allow rapid degradation of the hydrogel. Kong et al, 2002, Polymer,43:6239-46 (incorporated herein by reference in their entirety) describe the use of gamma radiation to reduce the weight average molecular weight (M) of Guluronic Acid (GA) -rich alginates_w) Without significantly reducing GA content (e.g., gamma radiation selectively attacks mannuronic acid, the MA block of alginate). The alginate consists of GA blocks and MA blocks, it is the GA blocks that give the alginate rigidity (elastic modules). Kong et al, 2002, Polymer,43:6239-46 (incorporated herein by reference in its entirety) showed a binary combination of high molecular weight, GA-rich alginate crosslinked with irradiated, low molecular weight, high GA alginate with calcium to form a rigid hydrogel, but with a faster degradation rate and lower solution viscosity, GA-rich alginate compared to hydrogels made from the same total weight concentration of only high molecular weight. Alsberg et al, 2003, J Dent Res,82(11):903-8 (incorporated herein by reference in its entirety) describe the degradation profile of hydrogels made from irradiated, low molecular weight, GA-rich alginate for use in bone tissue engineering. Kong et al, 2004, adv.Mater,16(21):1917-21 (incorporated herein by reference in its entirety) describe the control of the hydrogel degradation curve by combining a gamma irradiation program with an oxidation reaction and apply to cartilage engineering.

Techniques for controlling the degradation of hydrogen biomaterials are well known in the art. For example, Lutolf MP et al, 2003, Nat Biotechnol.,21:513-8 (incorporated herein by reference in its entirety) describe poly (ethylene glycol) -based materials that are engineered for degradation by mammalian enzymes (MMPs). Bryant SJ et al, 2007, Biomaterials,28(19):2978-86(US 7,192,693B 2; incorporated herein by reference in its entirety) describe a method of producing hydrogels with macroporosity. Pore templates (e.g., polymethylmethacrylate beads) are encapsulated in the bulk hydrogel, and the porogen is then extracted using acetone and methanol while leaving the bulk hydrogel intact. Silva et al, 2008, Proc.Natl.Acad.Sci USA,105(38):14347-52 (incorporated herein by reference in its entirety; US 2008/0044900) describe the deployment of endothelial progenitor cells from alginate sponges. Sponges are made by forming alginate hydrogels and then freeze drying (ice crystal to pore). Ali et al, 2009, Nat Mater (incorporated herein by reference in its entirety) describe the use of porous scaffolds to recruit dendritic cells and edit them to elicit an anti-tumor response. Huebsch et al, 2010, Nat Mater,9:518-26 (incorporated herein by reference in its entirety) describe the use of hydrogel elastic molds to control the differentiation of encapsulated mesenchymal stem cells.

In some embodiments, the scaffold composition comprises open interconnected macropores. Alternatively or additionally, the scaffold composition comprises a pore-forming scaffold composition. In certain embodiments, the pore-forming scaffold composition can comprise a sacrificial porogen hydrogel and a bulk hydrogel, wherein the pore-forming scaffold composition is free of macropores. For example, upon administration of the pore-forming scaffold to a subject, the sacrificial porogen hydrogel degrades at least 10% faster than the bulk hydrogel leaving macropores. In some embodiments, the sacrificial porogen hydrogel is in the form of a porogen that degrades to form the macropores. For example, the macropores may include pores having a diameter of, for example, about 10-400 μm.

Growth factor

The compositions of the present invention may comprise a growth factor. The term "growth factor" as used herein refers to an agent capable of stimulating cell growth, proliferation, healing and/or cell differentiation. In certain embodiments, the growth factor is a polypeptide. Growth factor polypeptides generally act as signal molecules. In certain embodiments, the growth factor polypeptide is a cytokine.

In certain embodiments, upon administration of the composition to a subject, the growth factor may recruit cells to the scaffold. The recruited cells are autologous. For example, the recruited cells may be stromal cells from the subject. In certain embodiments, the autologous cells can be stem cells of the subject (e.g., umbilical cord stem cells). The recruited cells may also be syngeneic, allogeneic or xenogeneic. The term "syngeneic" as used herein refers to genetically identical, or sufficiently identical and immunologically compatible to allow transplantation. For example, syngeneic cells may include transplanted cells obtained from an oogonial twin. The term "allogeneic gene" as used herein refers to a cell that is genetically distinct although from an individual of the same species. The term "xenogeneic" as used herein refers to cells derived from different species and thus genetically distinct.

For example, the recruited cells may be donor cells in a transplant. In certain embodiments, the transplantation is Hematopoietic Stem Cell Transplantation (HSCT). As used herein, HSCT refers to the transplantation of pluripotent hematopoietic stem cells or hematopoietic progenitor cells, typically derived from bone marrow, peripheral blood or umbilical cord blood. HSCT can be autologous (using the patient's own stem or progenitor cells), allogeneic (stem or progenitor cells from a donor), syngeneic (from an egg twin) or allogeneic (from a different species).

The growth factors of the present invention may induce the formation of tissues or organs within or around the administered composition. In certain embodiments, the tissue or the organ is a bone tissue or a hematopoietic tissue. Tissue formation may be limited to scaffolds of the composition.

Methods of incorporating polypeptides (e.g., growth factor polypeptides) are known in the art. See U.S. patent No. 8,728,456; 8,067,237 and 10,045,947; U.S. patent publication nos. US 20140079752; international patent publication nos. WO 2017/136837; incorporated herein by reference in its entirety. The release of the growth factor polypeptide can be controlled. Methods for controlled release of polypeptides (e.g., growth factor polypeptides) are known in the art. See U.S. patent No. 8,728,456; 8,067,237, respectively; 10,045,946, incorporated by reference in their entirety. In certain embodiments, the growth factor (e.g., BMP-2) may be released over an extended period of time, e.g., 7-30 days or longer. Controlled release of growth factors may affect the time of tissue or organ formation within the scaffold. In certain embodiments, the release of growth factors is controlled in order to produce functional, active bone nodules or tissue within one or two weeks after subcutaneous injection of the compositions of the invention.

In certain embodiments, the growth factor retains its biological activity for an extended period of time. The term "biological activity" as used herein refers to a beneficial or adverse reaction of an agent, such as a growth factor. The biological activity of the growth factor may be measured by any suitable means. For example, the biological activity of BMP-2 can be measured by its ability to induce bone nodule or tissue formation and/or recruit cells into the scaffold. In certain embodiments, the growth factor retains its biological activity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the incorporation of the biological factor into the scaffold.

Exemplary growth factors include, but are not limited to, Bone Morphogenetic Protein (BMP), Epidermal Growth Factor (EGF), transforming growth factor beta (TGF- β), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (GM-CSF), Nerve Growth Factor (NGF), neurotrophic factor, Platelet Derived Growth Factor (PDGF), Erythropoietin (EPO), Thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Insulin Growth Factor (IGF), and interleukins.

In some embodiments, the growth factor comprises a protein belonging to the transforming growth factor protein beta (TGF- β) superfamily. As used herein, the TGF- β superfamily is a large group of structurally related cell regulatory proteins. The TGF- β superfamily includes four major subfamilies: the TGF- β subfamily, the bone morphogenic proteins and growth differentiation factors, the activation and inhibition subfamilies, and a subfamily comprising various members. Proteins from the TGF-. beta.subfamily are active as homodimers or heterodimers, with the two chains linked by a disulfide bond. TGF- β subfamily proteins interact with a conserved family of cell surface serine/threonine-specific protein kinase receptors and use the conserved protein family known as SMAD to generate intracellular signals. TGF-beta subfamily proteins play an important role in the regulation of basic biological processes such as growth, development, tissue homeostasis, and immunoregulation.

Exemplary TGF- β subfamily proteins include, but are not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, MSTN, NODAL, NRTN, PSPN, TGF- β 1, TGF- β 2, TGF- β 3, and TGF- β 4. In a particular embodiment, the growth factor is BMP2.

In a certain embodiment, the growth factor comprises a Bone Morphogenetic Protein (BMP). As used herein, a BMP is a protein of a group of growth factors, also referred to by the term cytokines and metabogens. BMPs can induce the formation of bone and cartilage and constitute an important set of morphogenetic signals that coordinate the organization of the tissues throughout the body. Loss or lack of BMP signaling may be an important factor in a disease or disorder.

In certain embodiments, the BMP is selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, and any combination thereof. In certain embodiments, the BMP is BMP-2. BMP-2 plays an important role in the development of bone and cartilage. BMP-2 is effective in inducing osteoblast differentiation of a variety of cell types.

In certain embodiments, the growth factor comprises a TGF- β subfamily protein. As used herein, a TGF- β subfamily protein or TGF- β is a multifunctional cytokine, including four different subtypes (TGF- β 1, TGF- β 2, TGF- β 3, and TGF- β 4). Activated TGF- β is complexed with other factors to form a serine/threonine kinase complex, binds to the TGF- β receptor, and consists of type 1 and type 2 receptor subunits. Upon binding to TGF- β, type 2 receptor kinases phosphorylate and activate type 1 receptor kinases, thereby activating a signaling cascade. This results in the activation of different downstream substrates and regulatory proteins, inducing the transcription of different target genes that play a role in the differentiation, chemotaxis, proliferation and activation of many immune cells.

In certain embodiments, the growth factor comprises TGF- β 1. TGF-beta 1 induces Tregs (iTregs) with regulatory function and T secreting pro-inflammatory cytokines on CD4+ T cells_h17 in the cells. TGF- β 1 alone precipitated expression of Foxp3 and activated T helper differentiated tregs.

Growth factors (e.g., BMP-2 or TGF-. beta.1) may be isolated from endogenous sources or synthesized in vitro or in vivo. Endogenous growth factor polypeptides can be isolated from healthy human tissue. The synthetic growth factor polypeptides are synthesized in vivo following transfection or transformation of the template DNA into a host organ or cell (e.g., a mammalian or human cell line). Alternatively, synthetic growth factor polypeptides are synthesized in vitro by cell-free translation or other art-recognized methods (Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol.1,2,3(1989), which is incorporated herein by reference).

In certain embodiments, the growth factor (e.g., BMP-2 or TGF-. beta.1) polypeptide is recombinant. In some embodiments, the growth factor polypeptide is a humanized derivative of a mammalian growth factor polypeptide. Exemplary mammalian species from which the growth factor polypeptide is derived include, but are not limited to, mice, rats, hamsters, guinea pigs, ferrets, cats, dogs, monkeys, or primates. In some embodiments, the growth factor is a recombinant human protein. In some embodiments, the growth factor is a recombinant murine (mouse) protein. In some embodiments, the growth factor is a humanized derivative of a recombinant mouse protein.

In certain embodiments, the growth factor polypeptide is modified to increase in vivo protein stability. In certain embodiments, the growth factor polypeptide may be engineered to be more or less immunogenic. The terms "immunogenic" and "immunogenicity" refer to the ability of a particular substance (e.g., a protein, antigen, or epitope) to elicit an immune response in a human or other animal.

In certain embodiments, the growth factor may be present at about 0.001nmol to about 1000nmol per scaffold, or 0.001 to about 100nmol per scaffold, or about 0.001nmol to about 1nmol per scaffold.

In some embodiments, the growth factor may be present in an amount of about 1ng to 1000 micrograms per stent. For example, the growth factor may be present in an amount of about 1 μ g to about 1000 μ g, about 1 μ g to about 500 μ g, about 1 μ g to about 200 μ g, about 1 μ g to about 100 μ g, about 1 μ g to about 50 μ g, or about 1 μ g to about 10 μ g.

In certain embodiments, the compositions of the present invention comprise nanogram amounts of growth factor (e.g., about 1ng to about 1000ng of BMP-2). For example, the growth factor may be present in an amount of about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, about 10ng to about 200ng, about 50ng to about 200ng, about 100ng to about 200ng, and about 200 ng. Nanogram amounts of growth factors are also released in a controlled manner. Nanogram amounts of growth factors and/or controlled release can help to reduce toxicity of the compositions and methods of the invention compared to other delivery systems that use high doses of growth factors and have suboptimal release kinetics.

In various embodiments, the amount of growth factor present in the scaffold may vary depending on the size of the scaffold. For example, the growth factor may be present at about 0.03ng/mm³(weight ratio of amount of growth factor to scaffold volume) to about 350ng/mm³Present, e.g., about 0.1ng/mm³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150ng/mm³About 3ng/mm³To about 100ng/mm³About 4ng/mm³To about 50ng/mm³About 5ng/mm³To about 25ng/mm³About 6ng/mm³To about 10ng/mm³Or about 6.5ng/mm³To about 7.0ng/mm³。

In some embodiments, the amount of growth factor is at about 300ng/mm³To about 350. mu.g/mm³Is present in an amount of, for example, about 400ng/mm³To about 300. mu.g/mm³About 500ng/mm³To about 200. mu.g/mm³About 1. mu.g/mm³To about 100. mu.g/mm³About 5. mu.g/mm³To about 50. mu.g/mm³About 10. mu.g/mm³To about 25. mu.g/mm³。

Differentiation factor

The compositions of the invention may comprise a differentiation factor. As used herein, the differentiation factor is an agent that can induce differentiation of cells, e.g., recruit cells. In certain embodiments, the differentiation factor is a polypeptide. As used herein, "differentiation," "cell differentiation," or other similar terms refer to the process by which a cell changes from one cell type to another. In certain embodiments, the cells become more specialized types, such as stem cells or progenitor cells to T cell progenitor cells. Differentiation occurs many times during the development of multicellular organisms as it changes from a simple fertilized egg to a complex system of tissue and cell types. Differentiation will continue in adulthood as adult stem cells divide during tissue repair and normal cell renewal and produce fully differentiated daughter cells. Differentiation may alter cell size, morphology, membrane potential, metabolic activity, and response to signals. These changes may be due to highly controlled modification of gene expression.

In dividing cells, there are multiple levels of cellular potency, i.e., the ability of the cell to differentiate into other cell types. Greater potency indicates that more cell types can be derived. Cells that can differentiate into all cell types, including placental tissue, are referred to as totipotent cells. Cells of all cell types that can differentiate into adult organisms are called pluripotent cells. In mammals, such as humans, pluripotent cells may include embryonic stem cells and adult pluripotent cells. Induced pluripotent stem cells (iPS) can be produced by fibroblasts through the inducible expression of certain transcription factors such as Oct4, Sox2, c-Myc, and KIF 4. A pluripotent cell is a cell that can differentiate into a variety of different, but closely related cell types. Oligopotent cells are more restricted than pluripotent cells, but can still differentiate into a number of closely related cell types. Eventually, a unipotent cell can differentiate into only one cell type, but is capable of self-renewal.

In certain embodiments, the differentiation factors of the present invention induce differentiation of stem or progenitor cells into T cell progenitors. As used herein, the term "T cell progenitor" refers to a progenitor cell that can eventually differentiate into T lymphocytes (T cells). As used herein, the term "lymphocyte" refers to a subset of white blood cells in the immune system of a vertebrate (e.g., a human). Lymphocytes include natural killer cells, T cells and B cells. Lymphocytes originate from a common lymphoid progenitor during the hematopoietic process, during which the stem cells differentiate into a variety of blood cells within the bone marrow before differentiating into different lymphocyte types.

In some embodiments, the T cell progenitors comprise common lymphoid progenitors. As used herein, the term "common lymphoid progenitor" refers to the earliest lymphoid progenitor cell that produces lymphocytes, including T lineage cells, B lineage cells, and Natural Killer (NK) cells. In various embodiments, the T cell lineage includes T cell competent common lymphoid progenitor cells. As used herein, the term "T cell competent common lymphoid progenitor" refers to a common lymphoid progenitor that differentiates into a T lineage progenitor. T cell competent common lymphoid progenitor cells are generally characterized by the absence of the biomarker Ly 6D. The compositions of the invention can produce ectopic niches that mimic important features of bone marrow and induce differentiation of stem or progenitor cells into T cell progenitors.

In certain embodiments, the lymphocytes comprise T cells. In some embodiments, the T cell is a naive T cell. As used herein, naive T cells are T cells that have differentiated in bone marrow. Naive T cells can include CD4⁺T cell, CD8⁺T cells and regulatory T cells (T)_reg)。

In certain embodiments, the differentiation factor induces the differentiation of recruited cells into T cell progenitors. In certain embodiments, the differentiation factor induces the differentiation of recruited cells into T cell progenitors via the Notch signaling pathway. The Notch signaling pathway is a highly conserved cellular signaling system, found in many multicellular organisms. Mammals possess four different Notch receptors, designated Notch1, Notch2, Notch3 and Notch 4. Notch signaling plays an important role in the differentiation of T cell lineages from common lymphoid progenitor cells. In certain embodiments, the differentiation factors bind to one or more Notch receptors and activate Notch signaling pathways. In certain embodiments, the differentiation factor is selected from the group consisting of delta-like 1(DLL-1), delta-like 2(DLL-2), delta-like 3(DLL-3), delta-like 4(DLL-4), Jagged 1, Jagged2, and any combination thereof. In certain embodiments, binding of the differentiation factors to one or more Notch receptors activates the Notch signaling pathway and induces T cell lineage differentiation.

In certain embodiments, the differentiation factor is delta-like 4 (DLL-4). DLL-4 is a homolog of Drosophila Delta protein. The Delta protein family includes Notch ligands, characterized by a DSL domain, EGF repeats, and a transmembrane domain.

In certain embodiments, the differentiation factor polypeptide is isolated from an endogenous source or synthesized in vivo or in vitro. Endogenous differentiation factor polypeptides can be isolated from healthy human tissue. The synthetic differentiation factor polypeptide is synthesized in vivo after transfection or transformation of the template DNA into a host organism or cell (e.g., a mammalian or cultured human cell line). Alternatively, synthetic differentiation factor polypeptides are synthesized in vitro by cell-free translation or other art-recognized methods (Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol.1,2,3(1989), which is incorporated herein by reference).

In certain embodiments, the differentiation factor polypeptide may be recombinant. In some embodiments, the differentiation factor polypeptide is a humanized derivative of a mammalian differentiation factor polypeptide. Exemplary mammalian species from which the differentiation factor polypeptide is derived include, but are not limited to, mice, rats, hamsters, guinea pigs, ferrets, cats, dogs, monkeys, or primates. In some embodiments, the differentiation factor is a recombinant human protein. In some embodiments, the differentiation factor is a recombinant murine (mouse) protein. In some embodiments, the differentiation factor is a humanized derivative of a recombinant mouse protein.

In certain embodiments, the differentiation factor polypeptide is modified to achieve a desired activity, such as increasing protein stability in vivo. In certain embodiments, the differentiation factor polypeptide may be engineered to be more or less immunogenic.

In certain embodiments, the differentiation factor (e.g., DLL-4) can be covalently linked to a scaffold according to the present invention. For example, rather than being released from the scaffold material, the differentiation factors may be covalently bound to the local compound backbone and retained within a composition that is formed upon implantation of the composition into a subject. By covalently binding or coupling a differentiation factor to the scaffold material, such differentiation factor will be retained within the scaffold formed upon administration of the composition to a subject and thus may be used to promote differentiation of hepatocytes or progenitor cells, as contemplated herein. In certain embodiments, the differentiation factor is chemically coupled to the scaffold material using N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Any method known in the art for covalently binding or coupling differentiation factors may be used without limitation. See "Bioconjugate Techniques (Third Addition)", Greg t. hermanson, Academic Press, 2013. In some embodiments, the differentiation factor is covalently linked to the scaffold using click chemistry. Methods of covalently binding or coupling differentiation factors include, but are not limited to, avidin-biotin reactions, azide and dibenzocyclooctyne chemistries, tetrazine and trans-cyclooctene chemistries, tetrazine and norbornene chemistries, or disulfide bonds.

In certain embodiments, differentiation factors of the invention (e.g., DLL-4) further comprise a tether (e.g., PEG)_2k) And a methacrylate group (MA). In certain embodiments, the differentiation factor is methacrylated DLL-4-PEG_2k。

In certain embodiments, the covalent linkage retains the differentiation factor within the scaffold to provide a differentiation signal to recruit cells in the scaffold. For example, less than 1% of the total differentiation factor was detected outside the scaffold. The biological activity of the differentiation factors can be maintained for a longer period of time, for example at least three months after incorporation into the scaffold. The biological activity of the differentiation factor may be measured by any suitable method, such as colorimetric determination of DLL-4.

In certain embodiments, the differentiation factor is present at about 0.01nmol to about 1000nmol, about 0.1nmol to about 100nmol, or about 1nmol to about 10nmol per scaffold.

In some embodiments, the differentiation factor is present in an amount from about 1ng to about 1000 μ g per scaffold. For example, the differentiation factor may be present in an amount of about 10ng to about 500 μ g, about 50ng to about 250 μ g, about 100ng to about 200 μ g, about 1 μ g to about 100 μ g, about 1 μ g to about 50 μ g, about 1 μ g to about 25 μ g, about 1 μ g to about 10 μ g, about 2 μ g to about 10 μ g, or about 6 μ g.

In various embodiments, the amount of differentiation factor present in the scaffold may vary depending on the size of the scaffold. For example, the differentiation factor may be at about 0.03ng/mm³(weight ratio of amount of differentiation factor to scaffold volume) to about 350. mu.g/mm³Present, e.g., about 0.1ng/mm³To about 300. mu.g/mm³About 1ng/mm³To about 250. mu.g/mm³About 10ng/mm³To about 200. mu.g/mm³About 0.1. mu.g/mm³To about 100. mu.g/mm³About 0.1. mu.g/mm³To about 50. mu.g/mm³About 0.1. mu.g/mm³To about 20. mu.g/mm³About 0.1. mu.g/mm³To about 10. mu.g/mm³About 0.1. mu.g/mm³To about 5. mu.g/mm³About 0.1. mu.g/mm³To about 1. mu.g/mm³About 0.1. mu.g/mm³To about 0.5. mu.g/mm³Or about 0.2. mu.g/mm³。

In certain embodiments, DLL-4 is present at about 6 μ g per scaffold.

Homing factor

In certain embodiments, the compositions of the present invention further comprise a homing factor. As used herein, the term "homing factor" refers to an agent capable of inducing directional movement of a cell, such as a stem cell or progenitor cell. In certain embodiments, a homing factor of the invention is a signaling protein that induces directional chemotaxis in nearby responsive cells. In various embodiments, the homing factor is a cytokine and/or a chemokine.

In certain embodiments, such homing factors in the compositions of the invention promote homing of cells (e.g., transplanted stem and/or progenitor cells) to a scaffold composition administered to a subject. In certain aspects, such homing factors promote infiltration of cells (e.g., transplanted stem or progenitor cells) into a scaffold composition administered to a subject. In some embodiments, the homing factor comprises stromal cell derived factor (SDF-1). In certain embodiments, the homing factor is encapsulated in a material. In certain embodiments, the homing factor is released from the material over an extended period of time (e.g., 7-30 days or longer, about 17-18 days).

In certain embodiments, the homing factor retains its biological activity for an extended period of time. The biological activity of the growth factor may be measured by any suitable means. In certain embodiments, the homing factor retains its biological activity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after incorporation of the homing factor into the scaffold.

In some embodiments, the homing factor is present at about 0.01nmol to about 1000nmol, about 0.1nmol to about 100nmol, or about 1nmol to about 10nmol per scaffold.

In some embodiments, the homing factor is present in an amount of about 1ng to about 1000 μ g per scaffold. For example, the homing factor can be present in an amount of about 10ng to about 500 μ g, about 50ng to about 250 μ g, about 100ng to about 200 μ g, about 1 μ g to about 100 μ g, about 1 μ g to about 50 μ g, about 1 μ g to about 25 μ g, about 1 μ g to about 10 μ g, about 2 μ g to about 10 μ g, or about 6 μ g.

In various embodiments, the amount of differentiation factor present in the scaffold may vary depending on the size of the scaffold. For example, the differentiation factor may be at about 0.03ng/mm³(weight ratio of amount of differentiation factor to scaffold volume) to about 350. mu.g/mm³Present, e.g., about 0.1ng/mm³To about 300. mu.g/mm³About 1ng/mm³To about 250. mu.g/mm³About 10ng/mm³To about 200. mu.g/mm³About 0.1. mu.g/mm³To about 100. mu.g/mm³About 0.1. mu.g/mm³To about 50. mu.g/mm³About 0.1. mu.g/mm³To about 20. mu.g/mm³About 0.1. mu.g/mm³To about 10. mu.g/mm³About 0.1. mu.g/mm³To about 5. mu.g/mm³About 0.1. mu.g/mm³To about 1. mu.g/mm³About 0.1. mu.g/mm³To about 0.5. mu.g/mm³Or about 0.2. mu.g/mm³。

Exemplary stent compositions

The present invention provides scaffold compositions for modulating the immune system in a subject. The compositions of the present invention comprise a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors.

In one aspect, the invention provides a composition for modulating the immune system in a subject comprising a porous scaffold; a growth factor present at about 1ng to about 1000 μ g per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. For example, the growth factor may be present in an amount of about 1 μ g to about 500 μ g, about 1 μ g to about 200 μ g, about 1 μ g to about 100 μ g, about 1 μ g to about 50 μ g, or about 1 μ g to about 10 μ g.

In another aspect, the invention provides a composition for modulating the immune system in a subject comprising a porous scaffold; a growth factor present in about 1ng to about 1000ng per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In more specific embodiments, the growth factor is present in an amount of about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200 ng.

In yet another aspect, the present invention relates to a composition for modulating the immune system in a subject comprising a porous scaffold; growth factor at about 0.03ng/mm ³To about 350ng/mm³And in an amount effective to induce tissue or organ formation within the scaffold and recruit cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In various embodiments, the growth factor may be at about 0.1ng/mm³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150ng/mm³About 3ng/mm³To about 100ng/mm³About 4ng/mm³To about 50ng/mm³About 5ng/mm³To about 25ng/mm³About 6ng/mm³To about 10ng/mm³Or about 6.5ng/mm³To about 7.0ng/mm³Are present.

The composition may be designed to release the growth factor in a controlled manner. The reduced amount of growth factor and/or controlled release provides advantages over the prior art, such as reduced toxicity associated with the use of high levels of growth factor, such as BMP-2, and suboptimal release kinetics.

In various embodiments, the growth factor is a bone morphogenic protein, such as BMP-2, BMP-4, BMP-7, BMP-12, BMP-14, or any combination thereof. In some embodiments, the growth factor is BMP-2. In certain embodiments, the growth factor is a TGF- β, such as TGF- β 1, TGF- β 2, TGF- β 3, TGF- β 4, or any combination thereof. In a particular embodiment, the growth factor comprises TGF-. beta.1.

The porous scaffold of the composition according to the invention may be any biocompatible and biodegradable scaffold. In certain embodiments, the porous scaffold comprises a hydrogel and a cryogel. In various embodiments, the hydrogel or cryogel comprises an alginate or alginate derivative, a gel or gelatin derivative, or hyaluronic acid derivative.

In certain embodiments, the differentiation factors of the present invention comprise polypeptides that bind to Notch receptors. In various embodiments, the differentiation factor is selected from the group consisting of delta-like 1(DLL-1), delta-like 2(DLL-2), delta-like 3(DLL-3), delta-like 4(DLL-4), Jagged 1, Jagged2, and any combination thereof. In some embodiments, the differentiation factor comprises DLL-4. In certain embodiments, the differentiation factor is covalently linked to a porous scaffold.

In a particular embodiment, the family of compounds of the invention comprises injectable cryogels comprising an alginate or alginate derivative, such as a methacrylated alginate, or hyaluronic acid derivative; a growth factor for bone morphogenetic proteins such as BMP-2; and is the Delta-like familyDifferentiation factors for proteins, such as DLL-4. The growth factor may be present at about 200ng or about 6.5ng/mm per scaffold ³To about 7.0ng/mm³Are present.

Methods of modulating the immune system

The invention features methods of modulating the immune system in a subject. In certain embodiments of the invention, the method for modulating the immune system in a subject comprises administering to the subject one or more compositions of the invention. The composition may comprise a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In a particular embodiment, the composition comprises a porous scaffold; a growth factor present in about 1ng to about 1000ng per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors.

In certain embodiments, the method further comprises administering hematopoietic stem cells or hematopoietic progenitor cells to the subject.

In certain embodiments, the cell is a stem cell or a progenitor cell. As used herein, the term "stem cell" refers to a biological cell that is capable of differentiating into other types of cells and is capable of dividing to produce more stem cells of the same type. Stem cells include embryonic stem cells isolated from the inner cell mass of blastocysts and adult stem cells present in various tissues. In adult organisms, stem and progenitor cells act as a repair system for the body, replenishing adult tissue. In certain embodiments, the stem cell is an embryonic stem cell, a fetal stem cell, an amniotic membrane stem cell, an umbilical cord stem cell, an adult stem cell, or an induced pluripotent stem cell. In certain embodiments, the stem cells are hematopoietic stem cells. Hematopoietic stem cells are stem cells that produce other blood cells, including the myeloid and lymphoid lineages of blood cells.

As used herein, the term "progenitor cell" refers to a biological cell that can differentiate into a particular type of cell. Progenitor cells are generally more differentiated than stem cells. Typically, progenitor cells divide only a limited number of times.

In certain embodiments, the progenitor cell is a blast, such as a lymphoblast, bone marrow cell, or bone marrow precursor cell. In certain embodiments, the progenitor cell is a cell capable of differentiating into a T cell progenitor cell. In certain embodiments, the lymphocytes comprise T cells, such as naive T cells.

In certain embodiments, the recruited cells are hematopoietic bone marrow cells, or mobilized peripheral blood cells.

In certain embodiments, the cell is a recombinant cell. As used herein, the term "recombinant cell" refers to a cell into which a genetic modification has been introduced. The genetic modification may be at the chromosomal level or extrachromosomal. "genetic modification at the chromosomal level" refers to a genetic modification in the genome of a cell, such as an insertion, deletion and/or substitution on the chromosome of the cell. An extrachromosomal genetic modification refers to a genetic modification that is not located in the genome of a cell. For example, a plasmid containing a gene encoding a protein can be introduced into a cell. The plasmid can replicate and be transmitted from the parent cell to the progeny cell.

In various embodiments, the genetic modification introduces a gene into the cell. The introduced gene may compensate for the function of the cell defect gene. For example, the cell may contain a mutated defective gene. Genetic modification can introduce a wild-type functional gene into a cell to restore the function of the gene. In some embodiments, the genetic modification may increase or decrease the expression of certain genes. For example, genetic modifications may introduce small interfering rnas (sirnas) specific for a gene to inhibit expression of the gene.

Methods of genetically modifying cells are well known in the art, for example, the methods described in Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Vol.1,2,3(1989), which is incorporated herein by reference.

In certain embodiments, the genetic modification can be introduced by gene editing, also known as genome editing. Gene editing is a group of techniques that enables skilled artisans to alter the DNA of an organism. These techniques allow for the addition, removal, or alteration of genetic material at specific locations in the genome. Gene editing techniques include, but are not limited to, meganuclease systems, Zinc Finger Nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, and CRISPR-Cas systems. The CRISPR-Cas system is an abbreviation for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein systems, in particular CRISP-Cas9, that is faster, cheaper, more accurate and more efficient than other existing genome editing methods.

In certain embodiments, the invention features methods of modulating the immune system in a subject after the subject receives a transplant. For example, the subject may receive a hematopoietic stem cell transplant. In certain embodiments, the composition of the invention is administered to the subject simultaneously with or following hematopoietic stem cell transplantation.

In certain embodiments, at least two compositions are administered to the subject. The compositions may be similar.

In certain embodiments, the methods of the invention modulate an immune response in a human over the age of 30. For example, the human may be over 40 years old, over 50 years old, over 60 years old, over 70 years old, over 80 years old.

In some embodiments, one or more compositions of the invention (e.g., bone marrow cryogels) may be administered in conjunction with stem cell mobilization techniques. Stem Cell Mobilization is a process of moving Stem cells from the bone marrow into the Blood using certain Cell mobilizing agents, described, for example, in Hopman and Dipersio, Advances in Stem Cell mobilisation, Blood Rev.,2014,28(1):31-40, the contents of which are incorporated herein by reference. Such techniques can also be used for mobilization of progenitor cells.

Thus, in certain embodiments, the stem cell and/or progenitor cell mobilizing agent is administered to the subject in an amount effective to induce migration of stem cells and/or progenitor cells from the bone marrow into the blood. The released stem and/or progenitor cells are then recruited into the compositions of the invention (e.g., bone marrow cryogel) to differentiate into T cell progenitors. The stem cell and/or progenitor cell mobilizer is administered prior to, concurrently with, or after use of the composition (e.g., bone marrow cryogel).

In various embodiments, a composition (e.g., bone marrow cryogel) described herein can be administered to a subject in combination with a stem cell and/or progenitor cell mobilizer. In particular embodiments, the subject is an elderly human, e.g., a human may be over 30, 40, 50, 60, 70, or 80 years of age. The stem cell and/or progenitor cell mobilizing agent mobilizes the subject's own stem cells and/or progenitor cells from the bone marrow so that these cells can home to the compositions of the invention, thereby enhancing T cell production in the subject without involving other conditioning or stem cell transplantation.

In certain embodiments, the compositions described herein (e.g., bone marrow cryogels) can be administered to a subject in conjunction with stem cell and/or progenitor cell mobilization techniques and stem cell transplantation. The transplantation may be autologous, allogeneic or xenogeneic.

In some embodiments, a therapeutically effective amount of one or more cell mobilizing agents that can stimulate mobilization into the peripheral blood stream, produce and/or improve function of one or more cell types is administered. The agents may be administered by any desired route of administration, for example, orally, rectally, intravenously, intramuscularly, subcutaneously or by aerosol. Some non-limiting embodiments of agents that can stimulate the function of mobilizing into the peripheral blood stream, producing and/or improving cell types include IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF, plerixafor, PDGF, TGF- β, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, and the like. In addition to naturally occurring growth factors, growth factor analogs and growth factor derivatives such as fusion proteins may also be used. In some embodiments, the method comprises administering a therapeutically effective amount of G-CSF and a therapeutically effective amount of electromagnetic radiation. In some embodiments, the method comprises administering a combination of a therapeutically effective amount of plerixafor and a therapeutically effective amount of electromagnetic radiation. In some embodiments, a therapeutically effective amount of electromagnetic radiation is combined with another agent, which in some embodiments may be a hematopoietic stem cell mobilizer. In some embodiments, a therapeutically effective amount of electromagnetic radiation is combined with a combination of two or more of GM-CSF, G-CSF, plerixafor, IL-1, IL-2, IL-3, IL-6PDGF, TGF- β, NGF, IGF, growth hormone, erythropoietin, thrombopoietin, or another agent.

Recruitment and differentiation of cells to T cell progenitors

In one aspect, the invention features a method of recruiting cells to a scaffold and inducing the recruited cells to differentiate into T cell progenitors. In particular, the method comprises administering to a subject one or more compositions of the invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes. In a particular embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; a growth factor present in about 1ng to about 1000ng (e.g., about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng) per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes. In another specific embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; a growth factor present in about 1ng to about 1000ng (e.g., about 1ng to about 500 μ g, about 5 μ g to about 250 μ g, about 10 μ g to about 100 μ g) per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes. In another embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; growth factor at about 0.03ng/mm ³(ratio of weight of growth factor to volume of scaffold) to about 350ng/mm³(e.g., about 0.1 ng/mm)³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150 ng-mm³About 3ng/mm³To about 100ng/mm³About 4ng/mm³To about 50ng/mm³Or about 5ng/mm³To about 25ng/mm³) Present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In certain embodiments, the growth factor and/or homing factor is used to recruit cells to a scaffold according to the invention. The differentiation and optionally growth factors of the composition subsequently induce differentiation of the cells into T cell progenitors.

In some embodiments, the method further comprises administering hematopoietic stem cells or hematopoietic progenitor cells to the subject.

In certain embodiments, the growth factor (e.g., BMP-2) and/or homing factor (e.g., SDF-1) recruits stem or progenitor cells capable of differentiating into T cell progenitors. Differentiation factors (e.g., DLL-4) induce differentiation of stem or progenitor cells into T cell progenitors. In certain embodiments, the lymphocytes comprise naive T cells. In certain embodiments, the lymphocytes comprise T cells, e.g., CD4 ⁺T cell, CD8⁺T cells and/or regulatory T cells (T)_reg)。

In certain embodiments, the recruited cell is a transplanted cell. The transplanted cells (e.g., hematopoietic stem cells or progenitor cells) may be autologous. For example, the transplanted cells may be the subject's own umbilical cord stem cells or stem cells obtained from the subject prior to treatment, e.g., radiation therapy. The transplanted cells may be syngeneic, e.g., hematopoietic stem or progenitor cells from a homozygotic twin of the subject. The transplanted cells may also be allogeneic, e.g., hematopoietic stem or progenitor cells obtained from a donor of the same species. The transplanted cells may also be xenogeneic, such as hematopoietic stem or progenitor cells obtained from a different species.

In certain embodiments, the cell may be a bone marrow stromal cell from a subject. The stromal cells refer to connective tissue cells of an organ, such as bone marrow. The stromal cells support the function of the parenchymal cells of the organ (e.g., bone marrow). Bone marrow stromal cells produce DLL-4, which provides an important functional environment for the generation of T cell competent common lymphoid progenitor cells. Growth factors (e.g., BMP-2) may also promote differentiation of the stromal cell's bone lineage.

In certain embodiments, the recruited cell may not be a stem cell or a progenitor cell.

Reduction of immune hyperreactivity

In one aspect, the invention provides a method of reducing an immune hyper-response in a subject by administering to the subject one or more compositions of the invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold (e.g., about 1ng to about 1000ng, about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng per scaffold); and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors.

In another aspect, the invention provides a method of reducing an immune hyper-response in a subject by administering to the subject one or more compositions of the invention comprising a porous scaffold; a growth factor present in about 1ng to about 1000ng (e.g., about 1ng to about 500 μ g, about 5 μ g to about 250 μ g, about 10 μ g to about 100 μ g) per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In another aspect, the invention provides a method of reducing an immune hyper-response in a subject by administering to the subject one or more compositions of the invention comprising a porous scaffold; growth factor at about 0.03ng/mm³(ratio of weight of growth factor to volume of scaffold) to about 350ng/mm³(e.g., about 0.1 ng/mm)³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150ng/mm³About 3ng/mm³To about 100ng/mm³About 4ng/mm³To about 50ng/mm³Or about 5ng/mm³To about 25ng/mm³) Present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In some embodiments, the method further comprises administering hematopoietic stem cells or hematopoietic progenitor cells to the subject.

The immune system is a strictly regulated network, and can maintain the balance of immune homeostasis under normal physiological price regulation. In general, when challenged with foreign antigens, a specific appropriate response is initiated aimed at restoring homeostasis. However, in certain cases, this balance cannot be maintained and the immune response is inadequate or excessive. When the immune response is overreactive, autoimmune diseases, allergies and/or GVHD diseases may result.

In certain embodiments, the invention features increasing regulatory T cells (T) in a subject_reg) The method of (1). T is_regCells, also known as suppressor T cells, are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent and/or treat autoimmune diseases. T is_regThe cells have immunosuppressive effects and induce and proliferate by inhibiting or down regulating effector T cells. T is_regThe cell cells carried the biomarkers CD4, FOXP3, and CD 25. Without wishing to be bound by any theory, the T increased by the method of the invention_regThe cellular level is thought to result in a reduction in over-reactivity.

Autoimmune diseases

In a particular aspect, the present invention provides a method of preventing and/or treating an autoimmune disease by administering to a subject one or more compositions of the present invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold (e.g., about 1ng to about 1000ng, about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng per scaffold); and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. As used herein, the term "autoimmune disease" refers to a disease, disorder or disease in which the subject's immune system attacks and/or damages its own tissues. Exemplary immune system disorders include, but are not limited to, type 1 diabetes, rheumatoid arthritis, psoriasis, arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and celiac disease.

In certain embodiments, the subject lacks T_regCells and develop autoimmune diseases. The compositions of the invention and hematopoietic stem or progenitor cells can be administered to a subject. Hematopoietic stem or progenitor cells can be obtained from a subject. The compositions of the invention may contain growth factors (e.g., TGF- β family proteins) to induce differentiation of hematopoietic stem cells to T_regA cell.

Allergy (S)

In certain embodiments, the invention features preventing and/or treating allergy by administering to a subject one or more compositions of the invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold (e.g., about 1ng to about 1000ng, about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng per scaffold); and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors.

Allergy, also known as allergic disease, is a variety of conditions caused by hypersensitivity of the immune system to substances that are normally harmless in the environment. Without wishing to be bound by any theory, the T increased by the method of the invention_regThe cellular level may prevent and/or treat allergy.

GVHD

In another aspect, the present methods provide a method of alleviating symptoms associated with Graft Versus Host Disease (GVHD) by administering to a subject one or more compositions of the invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold (e.g., about 1ng to about 1000ng, about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng per scaffold); and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. GVHD is the medical condition after receiving transplanted tissue from a genetically different donor. GVHD is commonly associated with stem cell transplantation, such as hematopoietic stem cell transplantation. GVHD has also been demonstrated in other forms of transplanted tissue, such as solid organ transplantation.

In certain embodiments, the GVHD is associated with hematopoietic stem cell transplantation. In certain embodiments, the GVHD is associated with a solid organ transplant.

In certain embodiments, the invention features increasing T_regThe level of cells. In certain embodiments, T_regThe cells are differentiated from transplanted hematopoietic stem cells (e.g., donor hematopoietic stem cells). Without wishing to be bound by any theory, in view of the donor T _regThe cells play an important role in GVHD inhibition, donor T in hematopoietic stem cell transplantation_regThe enhancement of cells may contribute to the reduction of symptoms of GVHD in subjects undergoing hematopoietic stem cell transplantation. In certain embodiments, the invention features increasing autologous T in a subject_regMethods at the cellular level.

Enhancement of donor chimerism in a subject receiving a transplant

In another aspect, the present invention provides a method of enhancing donor chimerism in a subject receiving a transplant by administering to the subject one or more compositions of the present invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In a particular embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; growth factors, which are present per scaffold1ng to about 1000ng (e.g., about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng) and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In another specific embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; a growth factor present at about 1ng to about 1000 μ g per scaffold (e.g., about 1ng to about 500 μ g, about 5 μ g to about 250 μ g, about 10 μ g to about 100 μ g) and in an amount effective to induce formation of a tissue or organ within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes. In another embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; growth factor at about 0.03ng/mm ³(ratio of weight of growth factor to volume of scaffold) to about 350ng/mm³(e.g., about 0.1 ng/mm)³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150ng/mm³About 3ng/mm³To about 100ng/mm³About 4ng/mm³To about 50ng/mm³Or about 5ng/mm³To about 25ng/mm³) Present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In some embodiments, the method further comprises administering hematopoietic stem cells or hematopoietic progenitor cells to the subject.

Donor chimerism typically occurs when a subject receives a hematopoietic stem cell transplant. As used herein, the term "chimerism" refers to the presence of lymphohematopoietic cells of non-host origin. Complete or complete chimerism generally refers to complete replacement of the host by donor lymphohematopoietic cells. Mixed chimerism indicates the presence of both donor and recipient cells, e.g., lymphocytes, in a given cellular compartment. In allogeneic hematopoietic stem or progenitor cell transplantation, low levels of donor chimerism are often associated with insufficient T cell production, which predisposes the patient to infection by pathogens and may lead to GVHD.

T cell balance reconstitution

In another aspect, the invention provides a method of causing T cell balance to be reestablished in a subject by administering to the subject one or more compositions of the invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In a particular embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; a growth factor present in about 1ng to about 1000ng (e.g., about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng) per scaffold and in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In another specific embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; a growth factor present at about 1ng to about 1000 μ g per scaffold (e.g., about 1ng to about 500 μ g, about 5 μ g to about 250 μ g, about 10 μ g to about 100 μ g) and in an amount effective to induce formation of a tissue or organ within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes. In another embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; growth factor at about 0.03ng/mm ³(ratio of weight of growth factor to volume of scaffold) to about 350ng/mm³(e.g., about 0.1 ng/mm)³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150ng/mm³About 3ng/mm³To about 100ng/mm³About 4ng/mm³To about 50ng/mm³Or about 5ng/mm³To about 25ng/mm³) Exist and are effective to induce tissue or organ formation within the scaffold and to thin the tissue or organ(ii) an amount of cells recruited into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In some embodiments, the subject is further administered hematopoietic stem or progenitor cells. The compositions of the invention are administered simultaneously with or after administration of the hematopoietic stem or progenitor cells.

The term "T cell balance reconstitution" as used herein refers to the reconstitution of CD4 with CD⁺:CD8⁺Reconstitution of T cells characterized by a ratio within the normal range over a specified period of time (e.g., 30 days). For example, CD4⁺Reconstitution of cells is often delayed in HSCT recipients. The method of the present invention can accelerate CD4⁺T cell reconstitution and leads to T cell balance reconstitution.

Hematopoietic Stem Cell Transplantation (HSCT) is a curative treatment for a variety of diseases, but allogeneic HSCT is limited by T cell defects and dysregulation. CD4 in subjects receiving allogeneic HSCT ⁺T cell recovery is often delayed, resulting in inversion of the normal CD4/CD8 ratio, which is about 0.9 to about 2.5 in peripheral blood. This ratio may be different in other tissues or organs.

In certain embodiments, the methods of the invention incorporate CD4⁺:CD8⁺Ratio stabilized to normal range, while CD4 in subjects receiving HSCT only⁺The T cell compartment has not been completely reconstituted. In certain embodiments, the balanced T cell reconstitution is characterized by steady state CD4 within the normal range 30 days or less after transplantation of hematopoietic stem cells and administration of the compositions of the invention⁺:CD8⁺A ratio.

In certain embodiments according to the invention, a subject, e.g., a human, receives about 1x10 per kilogram body weight of the subject in hematopoietic stem cell transplantation⁵To about 50x10⁶Hematopoietic stem or progenitor cells. In certain embodiments, the subject receives about 1x10 per kilogram body weight of the subject⁵Hematopoietic stem cells.

The methods of the invention result in a similar or better cure and/or therapeutic effect as compared to subjects receiving only hematopoietic stem cell or T cell progenitor infusion (i.e., not treated with the compositions of the invention). For example, treatment with the compositions of the invention may result in a greater number of T cell progenitors and functional T cells in the thymus and periphery, e.g., even when lower doses are used relative to T cell progenitor infusion alone. In some embodiments, a similar or better cure and/or therapeutic effect may be achieved when less than ten percent (10%) of the hematopoietic stem or progenitor cells used alone for HSCT are administered to a subject in combination with a composition of the invention.

In various embodiments, the T cell balance reconstitution is further characterized by enhanced T cell neogenesis. As used herein, the term "neogenesis" refers to the production of new cells. In various embodiments, enhanced T cell neogenesis is characterized by an enhanced T cell receptor excision loop (TREC). In certain embodiments, T cell neogenesis using the compositions and methods of the invention achieves a baseline or normal number of TRECs. As used herein, the term "baseline number of TRECs" refers to the number of TRESCs in a subject prior to the subject receiving any treatment that damages the immune system in the subject. As used herein, the term "normal amount of TREC" refers to the amount of TREC in an individual with an intact immune system. The normal amount of TREC may be within a certain range. In certain embodiments, TREC can be assessed in humans in a quantitative and noninvasive manner by estimating TREC in peripheral blood cells.

Antigen specific T cell responses

In another aspect, the present invention provides a method of inducing an antigen-specific T cell response in a subject by administering to the subject one or more compositions of the present invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In a particular embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; a growth factor in an amount of about 1ng to about 1000ng (e.g., about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 2 ng) per scaffold 00ng) and in an amount effective to induce formation of a tissue or organ within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In another specific embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; a growth factor present at about 1ng to about 1000 μ g per scaffold (e.g., about 1ng to about 500 μ g, about 5 μ g to about 250 μ g, about 10 μ g to about 100 μ g) and in an amount effective to induce formation of a tissue or organ within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes. In another embodiment, a composition is administered to a subject, the composition comprising a porous scaffold; growth factor at about 0.03ng/mm³(ratio of weight of growth factor to volume of scaffold) to about 350ng/mm³(e.g., about 0.1 ng/mm)³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150ng/mm³About 3ng/mm³To about 100ng/mm3, about 4ng/mm³To about 50ng/mm³Or about 5ng/mm³To about 25ng/mm³) Present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In some embodiments, the method further comprises administering to the subject hematopoietic liver cells and hematopoietic progenitor cells.

In certain embodiments, the subject has an impaired immune system. As used herein, the term "compromised immune system" refers to a state in which the immune system's ability to fight infections and cancer is compromised or completely absent. Many instances of immune system impairment are acquired in the acquired ("secondary") due to extrinsic factors affecting the immune system in the subject. Examples of such extrinsic factors include HIV infection, age, and environmental factors such as nutrition. In certain embodiments, immunosuppression of certain drugs, such as steroids, may be a side effect or the intended purpose of treatment. Examples of such uses include (i) as an anti-rejection measure in organ transplant surgery and (ii) in patients with excessive or generalized immune systems such as autoimmune diseases. In certain embodiments, some anti-cancer therapies, such as radiation therapy and/or chemotherapy, result in an impairment of the immune system. The condition in which the immune system is compromised in a subject may be referred to as an "immunodeficiency".

In certain embodiments, the subject has received a hematopoietic stem cell transplant. In certain embodiments, the subject has a hematological or myeloid cancer, e.g., myeloid cancer or leukemia. The subject may undergo regulatory cytotoxic radiation and/or chemotherapy to destroy tumor cells, which may result in severe lymphopenia due to T cell and B cell destruction by the adaptive immune system.

In certain embodiments, the methods of inducing an antigen-specific response comprise administering to a subject a composition of the invention and a vaccine comprising an antigen.

Modulation of the immune system in an immunocompromised subject

In one aspect, the invention provides a method of modulating the immune system of an immunocompromised subject by administering to the subject one or more compositions of the invention comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold (e.g., about 1ng to about 1000ng, about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng per scaffold); and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors.

In another aspect, the present invention provides a method of modulating the immune system of an immunocompromised subject by administering to the subject one or more compositions of the present invention comprising a porous scaffold; a growth factor present at about 1ng to about 1000 μ g per scaffold (e.g., about 1ng to about 500 μ g, about 5 μ g to about 250 μ g, about 10 μ g to about 100 μ g) and in an amount effective to induce formation of a tissue or organ within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In another aspect, the present invention provides a method of modulating the immune system of an immunocompromised subject by administering to the subject one or more compositions of the present invention comprising a porous scaffold; growth factor at about 0.03ng/mm³(ratio of weight of growth factor to volume of scaffold) to about 350ng/mm³(e.g., about 0.1 ng/mm)³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150ng/mm³About 3ng/mm³To about 100ng/mm³About 4ng/mm³To about 50ng/mm³Or about 5ng/mm³To about 25ng/mm³) Present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In some embodiments, the subject is further administered hematopoietic stem or progenitor cells.

In certain embodiments, the subject is further administered a stem cell and/or progenitor cell mobilizing agent such that the released stem cells and/or progenitor cells are recruited to a composition of the invention (e.g., a bone marrow cryogel). The stem/progenitor cell mobilizer is administered prior to, concurrently with, or subsequent to administration of the composition of the invention (e.g., bone marrow cryogel).

In some embodiments, the impairment of the immune system is caused by immunosenescence. As used herein, the term "immunosenescence" refers to the deterioration of the immune system due to natural age. It relates to the ability of the host to respond to infection and the development of long-term immunological memory, particularly by vaccination. Immune aging is a multifactorial disease that causes many pathologically significant health problems in the elderly population. Immunosenescence is associated with a decrease in the ability of hematopoietic stem cells to self-renew. Immunosenescent subjects may exhibit a decreased ratio of CD4+/CD8+, a decreased antigen recognition repertoire of T Cell Receptor (TCR) diversity, and/or impaired proliferation in response to antigen stimulation. Immunosenescence can even begin very rarely, for example, in humans 30 years of age.

In some embodiments, the subject has an impaired immune system due to an innate immune deficiency. As used herein, "congenital immunodeficiency" also referred to as "primary immunodeficiency" refers to a defect, deletion, or deficiency of one or more major components of the immune system. These diseases are genetically determined and often manifest as frequent, chronic or opportunistic infections in infancy and childhood. The classification is based on whether the main components of the immune system are defective, absent or defective. Diagnosis can be determined by tests such as differential WBC counts, absolute lymphocyte counts, quantitative immunoglobulin (Ig) measurements, and antibody titers. Treatment typically involves prophylactic antibiotics to control and prevent infection. The prognosis of primary immunodeficiency disease is variable and depends on the particular disease.

Exemplary congenital immunodeficiency disorders include, but are not limited to, Bsimoo immunodeficiency, e.g., Bruton's agammaglobulinemia, Selective IgA deficiency, common variant immunodeficiency disorders, congenital t-cell immunodeficiency, e.g., DiGeorge's syndrome, autosomal dominant hyperimmunoglobulin E syndrome, IL-12 receptor deficiency, chronic cutaneous mucosal candidiasis, IPEX syndrome (immune dysregulation, multiple endocrinopathies, enteropathy, X-linked), a mixture of congenital immunodeficiences, e.g., Severe complex immunodeficiency (SCID, bubby boy's disease, Glanzmann-Rinker syndrome, lymphocyte deficiency), Wiscott-Aldrich syndrome, hyper IgM syndrome, ataxia telangiectasia, congenital neutrophilic and phagocytic diseases, e.g., Chronic Granulomatous Disease (CGD), leukocyte adhesion deficiency type 1, Chediak-Higashi syndrome, myeloperoxidase deficiency, severe congenital neutropenia, congenital deficiencies such as terminal deficiency, C3 deficiency.

In certain embodiments, the subject has an impaired immune system due to acquired immunodeficiency. As used herein, the term "acquired immunodeficiency" refers to an immunodeficiency caused by an external factor that affects the immune system in a subject. Acquired immunodeficiency, also known as secondary immunodeficiency, can be caused by a variety of immunosuppressive agents, such as malnutrition, aging, specific drugs or treatments (e.g., chemotherapy (cytotoxic drugs), disease-modifying antirheumatic drugs, immunosuppressive drugs after organ transplantation, glucocorticoids, radiotherapy) and environmental toxins (e.g., mercury and other heavy metals), pesticides and petrochemicals (e.g., styrene, dichlorobenzene, xylene and ethylphenol). For drugs, the term "immunosuppression" generally refers to beneficial and potential adverse effects that reduce immune system function, while "immunodeficiency" generally refers only to adverse effects that result from an increase in the direction of wind from infection.

Many specific diseases cause immunosuppression either directly or indirectly. This includes many types of cancer, particularly bone marrow and blood cell cancers (leukemia, lymphoma, multiple myeloma) and certain chronic infections. Immunodeficiency is also a hallmark of acquired immunodeficiency syndrome (AIDS), caused by the Human Immunodeficiency Virus (HIV). HIV directly infects a small number of T helper cells and also indirectly impairs other immune system responses. Various hormonal and metabolic disorders may also lead to immune deficiencies, including but not limited to anemia, hypothyroidism, diabetes and hypoglycemia. Smoking, alcohol abuse and drug abuse also suppress immune responses.

Without wishing to be bound by any theory, the T cell balance reconstitution and antigen-specific T cell responses provided by the compositions and methods of the invention may modulate the immune system of a subject with an impaired immune system.

IV. reagent kit

Any of the compositions described herein can be included in a kit. In non-limiting embodiments, the kit comprises a composition comprising a porous scaffold; a growth factor present in an amount effective to induce tissue or organ formation within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In a particular embodiment, the kit comprises a composition comprising a porous scaffold; a growth factor present at about 1ng to about 1000ng (e.g., about 5ng to about 500ng, about 5ng to about 250ng, about 5ng to about 200ng, or about 200ng) per scaffold, And is present in an amount effective to induce formation of a tissue or organ within the scaffold and recruitment of cells into the scaffold; and a differentiation factor that induces the recruitment of cells to differentiate into T cell progenitors. In another particular embodiment, the kit comprises a composition comprising a porous scaffold; a growth factor present at about 1ng to about 1000 μ g per scaffold (e.g., about 1ng to about 500 μ g, about 5 μ g to about 250 μ g, about 10 μ g to about 100 μ g) and in an amount effective to induce formation of a tissue or organ within the scaffold and recruitment of cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes. In another embodiment, the kit comprises a composition comprising a porous scaffold; growth factor at about 0.03ng/mm³(ratio of weight of growth factor to volume of scaffold) to about 350ng/mm³(e.g., about 0.1 ng/mm)³To about 300ng/mm³About 0.5ng/mm³To about 250ng/mm³About 1ng/mm³To about 200ng/mm³About 2ng/mm³To about 150ng/mm³About 3ng/mm³To about 100ng/mm³About 4ng/mm³To about 50ng/mm³Or about 5ng/mm³To about 25ng/mm³) Present in an amount effective to induce tissue or organ formation within the scaffold and recruit cells into the scaffold; and inducing differentiation factors that recruit cells to differentiate into lymphocytes.

In some embodiments, the kit comprises a composition described elsewhere herein.

In a particular embodiment, the kit comprises a syringe or alternative injection device for administering the composition. In a specific embodiment, the pre-filled syringe or pre-filled injection device is pre-filled with the composition.

The kit may further comprise reagents or instructions for administering the compositions of the invention to a subject. It may also include one or more reagents.

The components of the kit may be packaged in aqueous media or in lyophilized form. The container means of the kit will generally be at least one vial, test tube, flask, bottle, syringe or other container means into which the components can be placed and preferably aliquoted appropriately. When more than one component is present in the kit, the kit will typically further comprise a second, third or other additional container into which additional components may be separately placed. The kit may further comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent. However, a combination of components may be included in the vial. The kits of the invention will also typically include a means for containing the compositions of the invention, such as compositions for modulating the immune system, and any other sealed reagent containers for commercial sale.

When the components of the kit are provided as one and/or more liquid solutions, the liquid solution is an aqueous solution, with sterile aqueous solutions being particularly preferred. However, the components of the kit may be provided as dry powders. When the kit and/or components are provided in dry powder form, the powder may be reconstituted by the addition of a suitable solvent. It is envisaged that the solvent may also be provided in another container means.

The invention is further illustrated by the following examples, which should not be construed as limiting. All sources of citation, for example, references, publications, databases, database entries, and techniques cited herein are also incorporated by reference into this application even if not explicitly recited in the citation. In case of conflict between a source of a reference and a statement in this application, the statement in this application controls.

The section and table headings are not intended to be limiting.

Examples

Abstract

Allogeneic hematopoietic stem cell suppression (HSCT) is a curative treatment for a variety of diseases, but the lack and dysregulation of T cells limits its use. Here is reported a biomaterial-based scaffold that mimics the characteristics of T cell lymphopoiesis in bone marrow. Bone Marrow Cryogels (BMCs) release bone morphogenic protein 2 to recruit stromal cells and provide Notch ligand Delta-like ligand 4 to promote the directed differentiation of T cell lineages of mouse and artificial blood progenitor cells. BMC injected subcutaneously into mice at HSCT enhances T cell progenitor seeding, T cell neogenesis and diversification of T cell receptor repertoire of the thymus. Peripheral T cell reconstitution increased about 6-fold in mouse HSCT and about 2-fold in human xenogeneic HSCT. In addition, BMC promoted the production of donor CD4+ regulatory T cells and increased survival after allogeneic HSCT. Compared to adoptive transfer of T cell progenitors, BMC increased donor chimerism, T cell production, and antigen-specific T cell response to vaccination. BMC can provide a ready method to enhance T cell regeneration and alleviate graft versus host disease in HSCT.

Introduction to

T cells are important helper, effector and regulator cells for antigen-specific immunity, which is important for life. Reduced T-cell number and functional deficiencies are associated with diseases such as aunt congenital deficiency, autoimmune and immune surveillance disorders (Goronzy, J.J. & Weyand, C.M. Successful and maladaptive T cell imaging. immunity 46,364- & 378 (2017); Liston, A., Enders, A. & Siggs, O.M. Unraveling the association of partial T-cell 883 immunological and immune regulation. Nature Reviews immunity 8,545- & 558884 (2008)). In allogeneic HSCT, T cell production is significantly deficient, which predisposes patients to infection with pathogens and may lead to Graft Versus Host Disease (GVHD) (Blazar, b.r., Murphy, W.J. & Abedi, m.advance in graft-cover-host disease biology and therapy.nature Reviews Immunology 12, 443-. These complications can be fatal and limit the use of HSCT in curable environments. The balanced reconstitution of initial helper and effector T cell subsets, as well as the restoration of T cell receptor banks, remains an important unmet clinical need (Krenger, w., Blazar, b.r. & Hollnder, g.a.thymic T-cell depletion in adaptive stem cell transplantation. blood 117,6768-6776 (2011)).

Regeneration of new T cells from transplanted hematopoietic cells requires a sufficient pool of bone marrow derived T cell progenitors (ZLottoff, D.A. et al, Delivery of prognostitors to the thymus limits T-linkage recompatibility after bone marrow translation, Blood 118, 1962-.&Brink, M.R.Thymus: the next generation, Immunological reviews 271,56-71 (2016)). While there are currently no clinical criteria for enhancing T cell production in vivo, most work has focused on the use of cytokines and cell-based therapies from the post-myeloid stages of T cell lymphopoiesis. However, in clinical trials, T cells expanded the cytokines IL-7 and IL-2(Mohtashami, m., Shukla, s., Zandstra, P).&Pfl ü cker, J.C.in Synthetic Immunology 95-120(Springer,2016)) increases the major mature T cell subpopulation (Perales, M.A., et al, Recombinant human interleukin-7(CYT107) proteins T-cell recovery after inductive cell transfer, Blood 120, 4882-cell 4891(2012), and IL-2 is further limited by toxicity (Skrombolas, D).&Freeinger, J.G. Challenges and horizontal solutions for creating the discovery of IL-2treatment in tumor therapy, Expert review of clinical immunology 10, 207-. In contrast, in preclinical mouse studies, administration of IL-22 has been shown to increase early thymocyte recovery (Dudakov, J.A. et al, Interleukin-22drive end genetic regeneration in micro. science 336,91-95 (2012)) or alternatively, Adoptive donor T cell Infusion has been used to provide antigen-specific T cell protection against common pathogens (cobbed, M. et al, adaptive transfer of cytotoxic CTL-specific CTL cell transfer after selection of HLA-peptide receptors; Journal of Experimental Medicine 202,379-386 (2005); Rooney, C.M. et al, Infusion of cytotoxic T cells for depletion of the thymic and expression promoter expression) but is limited by the increased risk of transient T cell expansion and expression in response to progenitor T cells 15592, 1555, supra, to promote the production of thymus-dependent T cells. Activation of T cell precursors by Notch signaling Produced in vitro and co-administration of these cells with HSCT can improve thymogenesis and thymic structure without exogenous co-administration of cytokines (Zakrzewski, J.L. et al, Tumor immunothergy MHC barriers using allogenic T-cell precursors, Nature biotechnology 26,453 (2008); Van Coppernolle, S. et al, functional matrix CD4 and CD8 TCR α β cell gene, in 9-DL1 cells from Human CD34+ heterologous cells, The Journal of immunological cell 183,4859 cell 4870 (2009); Awoong, G. et al, Human T-cell gene in vitro tissue and thymic tissue culture medium T-42122, 4210. et al, blood tissue culture medium T-cell 4219, blood culture medium T-cell 423, and 4210). However, in vitro cell culture to produce sufficient progenitor cells is laborious and only demonstrates a transient enhancement of the thymopoiesis of the donor cells. Thus, extensive clinical transformation of this approach can be complicated.

In order to develop a widely applicable technology, common lymphoid progenitor Cells (CLPs) that reside in the anterior medulla of the thymus are emphasized, which have the ability to differentiate into primary T lymphocytes when Notch signaling is activated, and are the main source of thymopoiesis (Love, P.E. & Bhandolas, A. signal integration and cross stem differentiation and infection, Nature Reviews Immunology 11,469 (2011.; Radike, F., MacDonald, H.R. & Tarchini-cotter, F.Regulation of origin and adaptive immunity by Notch, Nature Reviews Immunology 13,427 (2013); Serwold, T.Ehrh., L.I.R. R., I.R., I.I. Pat. No. 807. and milk production of thymus, III. and milk production, III. A. production of thymus, III. A. 3, B. A. production of thymus, III. A. 3, A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. Matrix components that enhance the niche of bone marrow dedicated to T cell lineage differentiation consist of osteocalcin-expressing bone marrow stromal cells that produce delta-like ligand-4 (DLL-4), which provides a functional microenvironment critical to the production of T cell competent CLP (Vionnie, w. et al, Specific bone cells product DLL4 to production of gene from human breast. journal of Experimental media 2014em. jem. 1843(2015)) these stromal cells are damaged by pretreatment processes, which may affect their T cell lineage guiding function. Furthermore, clinical experience in AIDS patients indicates that The adult thymus has The capacity to significantly improve cellular composition and T cell neogenesis despite previous dysfunction and atrophy (Smith, K.Y., et al, Thymic size and lymphocyte restriction in tissues with human immunodeficiency virus after 48 wells of Zidovudine, lamivudine, and ritonavir therapy, The Journal of infectious diseases 181,141-147 (2000).) these previous findings support The development of niches based on specific biological aspects of T cell lymphopoiesis in The bone marrow.

It is hypothesized that T cell lymphocytes can be designed to generate bone marrow niches to promote the production of in vivo T cell progenitors that migrate into the native thymus, undergoing host-driven selection to produce a more balanced and broader immune repertoire. To test this hypothesis, an injectable biomaterial-based cryogel (BMC) scaffold was created. BMC scaffolds promote T cell development in vivo by integrating molecular signals that appear at bone marrow niches. BMC comprises a macroporous hydrogel-based scaffold that allows cellular infiltration. It releases bone morphogenic protein 2(BMP-2) to promote recruitment of host stromal cells and differentiation of their osteocytes lineage, and provides bioactive Notch ligand DLL-4 to the infiltrating hematopoietic cells at a predefined density. These T lineage cues enhance thymic vaccination of progenitor cells and allow donor T cells to be reconstituted following isogenic (syn) and allogenic (allo) HSCT in mice. BMC-reconstituted T cells are functional, have distinct T Cell Receptor (TCR) repertoires, and reduce induction of GVHD.

Example 1: bioactive macroporous Bone Marrow Cryogel (BMC) for in vitro differentiation of hematopoietic progenitor cells into progenitor T cells

The scaffold-based alginate PEG BMC was a macroporous hydrogel with interconnected pores of 50-80 μm diameter (FIGS. 1A-1C). DLL-4 is integrated into the polymer backbone to facilitate T cell lineage programs in hematopoietic progenitor cells (radcke, f., MacDonald, h.r. & Tacchini-cotter, f. regulation of both origin and adaptive immunity by Notch, Nature Reviews Immunology 13,427 (2013)). To achieve bone formation from new bone formation (Wozney, J.M. et al, Novel regulators of bone formation: molecular bonds and activities, Science 242,1528-1534(1988)), BMP-2 is added to the reaction mixture prior to low temperature polymerization for subsequent release in soluble form in vivo. These frozen agricultural crosses have both immobilized (DLL-4) and soluble (BMP-2) signals, unlike cryogels previously designed for controlled protein release (Koshy, s.t., Zhang, d.k., grilman, j.m., Stafford, A.G. & Mooney, d.j.Injectable nanocomposite gels for versatille protein drug delivery, Acta biomateriala 65,36-43 (2018)).

In this work, BMC also supports the growth of bone and hematopoietic tissues. In vitro BMP-2 release (encapsulation efficiency 90%) showed an initial burst of approximately 5% of loading followed by release in a sustained manner (fig. 1D). Less than 1% of the total loaded DLL4 was detected in the supernatant and modified DLL-4 had similar binding kinetics to the unmodified protein (fig. 1E). In the concentrated released samples, more than 90% of the bioactivity of the released BMP-2 was retained relative to freshly reconstituted BMP-2 (FIG. 1F). The bioactivity of BMP-2 ranged from 95% on day 3 to 85% on day 12, confirming the high activity of the released BMP-2 (FIG. 1G). The highest in vitro biological activity of DLL-4 was found at early time points on day 0 and day 10 (fig. 1H and 1I). The biological activity declined at subsequent time points, but was still above baseline after 3 months. To measure the ability of BMC to induce hematopoietic progenitor mouse and human cell differentiation by Notch signaling, primary lineage depleted bone marrow cells and umbilical cord blood derived human CD34+ hematopoietic cells from mice were cultured in BMC (fig. 1J).

Expansion of common lymphoid progenitor cells saturated at 1% functionalization of the MA-COOH groups on the polymer backbone with MA-DLL4, corresponding to about 6 μ g of MA-DLL4 per gel, and this condition was selected for further evaluation. There was no significant difference in fold expansion and viability numbers for total human and mouse cells under any of the experimental conditions analyzed (fig. 1K and 1L). However, only when DLL-4 was incorporated into BMC, either alone or in combination with BMP-2, was the fraction of lymphoid progenitor cells enhanced (fig. 1M).

Example 2: BMC creates bone nodules with hematopoietic tissue characteristics in vivo

BMC was next analyzed for its ability to induce host and graft cell trafficking in the HSCT mouse model. Lineage-depleted hematopoietic cells isolated from donor mouse bone marrow were transplanted intravenously in lethal whole-body irradiated (L-TBI) mice (5X 10)⁴(ii) a 93% lineage depletion, fig. 2A), and BMC (cell free) was simultaneously injected into the dorsal subcutaneous tissue (fig. 2B). To fully quantify cellular infiltration in BMC, the size of each subcutaneous nodule was measured over a 6-week period (fig. 2C). In BMC with BMP-2, the nodule size rapidly increased to 3 times the initial volume by 10 days post-implantation and was well infiltrated by donor hematopoietic cells (fig. 2D) and localized bone nodules were formed over a period of about 2 weeks (fig. 2E and 2F). Notably, bone formation was accompanied by vascularization, DLL-4 could still be cascade-en, and bone was restricted to the BMC scaffold, suggesting that BMC provided control of this process at ectopic sites (fig. 2G-2L).

Hematopoietic tissue was visible in the bone nodules on the inner surface of the BMC around the lamellar bone regions (fig. 2F). Infiltration into the capillary lattice of BMC was noted as early as 2 days post-implantation (fig. 2G) and quantification was initiated on day 10. Histomorphometry was used to assess vascular density in BMC at various time intervals until 3 months post-transplantation (fig. 2F). By day 10, the vessels were at about 25 vessels/mm ²Present (fig. 2I). Vascular density increased to 70 vessels/mm after day 30²And remain unchanged. To quantify alginate/DLL-4 distribution and accessibility in BMC, histomorphological analysis was performed using safranin-O staining. Approximately 85% of the alginate was accessible at the earliest time point on day 10 and gradually decreased to 25% by day 90 (fig. 2I-2K). At any point in time, the alginate was not sequestered in any particular region within the BMC.

Example 3: BMC recruitment and expansion of host matrices and transplanted hematopoietic cells

The infiltration and cellular components of the transplanted hematopoietic cells in BMCs were evaluated at various time points post-transplantation. Donor GFP + cells expanded when BMP-2 was included but DLL-4 alone was not present (FIGS. 3A and 3B). BMC-filled stromal cells were found to be similar to native bone marrow, there was no difference in hematopoietic cell engraftment in native bone marrow of BMC-treated and untreated mice, and concentrations of SDF-1 α and interleukin 7 were similar in BMC and native bone marrow (fig. 3C-3G).

BMC-filled stromal cells were identified using immunophenotypic markers (Sca-1, CD29, CD44, CD73, CD105, CD106) that are commonly associated with mouse mesenchymal stromal cells and compared to bone marrow of 496 transplanted mice (fig. 3C). Sca-1 in BMC versus bone marrow ⁺Matrix subpopulations were moderately elevated, but the overall repopulation kinetics of these matrix subpopulations were similar in both tissues. In BMC with BMP-2, Bone Alkaline Phosphatase (BAP) was comparable to native bone (FIG. 3D). Oil red o (oro) was used to quantify adipose tissue, which was found to be present in BMC in lower amounts than whole natural bone. Higher ORO was quantified in BMP-2 containing BMC relative to BMP-2 free BMC (FIG. 3D). To measure whether BMC affected the transplantation of cells in transplanted endogenous bone marrow, colony formation assays were performed at 3 time points (fig. 3E). No difference in total number or type of CFU produced by cells from bone marrow of mice treated with or without dual BMCs was noted. The concentrations of homing factor stromal cell-derived factor-1 alpha (SDF-1 alpha) and lymphoprogenitor supporting cytokine interleukin-7 (IL-7) in the harvested BMC (Brainard, D.M. et al, Induction of robust cellular and human viral-induced immune responses BLT mice, Journal of virology 83, 7305-treated human 7321(2009)) were also similar or higher in irradiated bone marrow from the same mice (FIG. 2F). ETP, DN2 and DN 3-like cells were not detected in BMC.

At later time points (> 5 days post-transplantation), more primitive donor hematopoietic cells (HSCs) and lymphoid-initiated pluripotent progenitor cells (LMPP) were quantitated in BMC. At day 14, 6 to 700 million total GFP were quantified in BMC containing either one-factor BMP-2 or two-factor BMP-2 and DLL-4⁺A cell. Over 80% of the cells in both groups were CD11b⁺Bone marrow cells. However, the CLP moiety in the transplanted donor cells was only amplified within the two-factor BMC, resulting in an approximately 100-fold increase at 2 weeks post-transplantation relative to BMC containing BMP-2 alone (fig. 3A). At 6 weeks post-transplantation, CLP in the two-factor BMC was about 10-fold higher, with about 30% and 70% dividedAre located in a T cell competent Ly6D subgroup (fig. 3G).

Progenitor T cells from bone marrow migrate to the thymus to differentiate into naive T cells. To directly assess whether cells from BMC migrated to the thymus, dual-factor BMC was delivered to a set of naive lethally irradiated mice along with stem cell therapy (fig. 3H and 3I). Dual BMCs were then removed from these mice on day 10 and surgically implanted under the dorsal skin of recipients receiving sub-lethal radiation. At day 20 after BMC transplantation, donor GFP in the thymus of these mice was quantified⁺And a host cell. GFP (green fluorescent protein) ⁺DP、SP CD4⁺And SP CD8⁺Cells were quantified in the thymus of BMC-transplanted recipient mice, which confirmed the migration of T cell progenitors from BMC to the thymus. In a separate study, dual BMC treatment resulted in a greater increase in the number of ETPs in the thymus compared to a 10-fold increase in the dose of transplanted cells without BMC (fig. 3J). Over time, dual BMC also outperformed bolus delivery of factors in BMC and BMP-2BMC only (fig. 3K-3P) in producing thymocyte subpopulations.

To analyze the effect of cell dose on T cell progenitor seeding in the thymus, a subset of early thymic progenitor cells (ETP) was identified in L-TBI (5X 10)⁴–5x10⁵Individual cells) are quantitated as the dose of transplanted lineage-depleted cells increases. Dose-dependent enhancement of ETP was found within this dose range (fig. 3A and 3H). When administered with the lowest cell dose (5X 10)⁴Individual cells), ETP in the thymus increased 5-fold compared to the transplant only group with the highest cell dose. Dual BMC treatment initially enhanced ETP (3-fold on days 12 and 42), and at subsequent time points, DN2, DN3, DP, and SP thymocyte subpopulations were increased (fig. 3I-3N and fig. 5Q). Thymocyte structure and body weight from dual BMC treated mice were significantly higher at 12 to 42 days than from the transplanted group only. There were no significant differences in the number of thymic stromal subsets (mTEC, cTEC, fibroblasts, and endothelial cells; FIGS. 3S and 3T) at 22 days post HSCT.

Example 4: BMC enhanced T cell regeneration and reduced GVHD following HSCT

In-use dual functionalizationAcceleration of T cell reconstitution was observed in peripheral blood of BMC treated mice approximately 4 weeks post-transplantation, but no significant difference was observed in B cell or bone marrow cell reconstitution (fig. 4A-4C). Analysis of T cell subsets in blood, spleen and bone marrow revealed that mice with bifunctional BMC were homeostatic for CD4 in spleen and bone marrow 30 days after transplantation and peripheral blood 40 days after transplantation⁺:CD8⁺T cell ratios recovered (FIGS. 4D-4F). T cell reconstitution was enhanced when BMC treatment was used in the context of HSCT following sub-lethal whole-body irradiation (SL-TBI) (fig. 4G-4N). At 28 days post-transplantation, the absolute numbers of donor chimeric and DP donor thymocytes were 1.5-fold and 2-fold higher, respectively, in BMC-treated mice than in mice receiving transplantation alone (fig. 4G). There was also a higher donor chimerism and donor-derived single positive CD4 in BMC treated mice⁺(2-fold and 3-fold, respectively) and single positive CD8⁺Absolute number of thymocytes (1.7-fold and 15-fold, respectively) (fig. 4H and 4I). Peripherally, the donor is chimeric in CD4⁺(3.5 times) and CD8⁺(2.5 fold) higher in T cells. No difference was observed in chimerism or absolute number of B cells.

The rate of human T cell reconstitution was next measured using the established xenogeneic NSG-BLT mouse model (Branard, D.M. et al, Induction of robust cellular and human viral-specific adaptive immune responses in human immunogenic viral-induced BLT mice, Journal of virology 83,7305-7321 (2009)). In BMC-treated NSG-BLT mice, the initial rate of T cell reconstitution was early enhanced, the B cell reconstitution rate was moisture, transiently decreased (fig. 5A and 5B), and pre-B cell CFU was temporarily decreased in BMC-treated NSG-BLT mouse bone marrow (fig. 5C). Peripheral CD4 in BMC-treated mice 50-60 days post-transplantation⁺:CD8⁺T cell ratio was stable, while CD4 in control NSG-BLT⁺The compartments were not completely reconstituted (fig. 5D). Strikingly, the increase in the rate of T cell reconstitution did not accelerate GVHD-related mortality. In contrast, NSG-BLT mice that received the bifunctional BMC survived longer than NSG-BLT mice (FIG. 5F). In BMC-treated NSG-BLT mice, CD4 in thymus and spleen of BMC-treated mice was 50 days after transplantation in this model⁺FoxP3⁺Regulatory T cells (T)_reg) 2 times higher (fig. 5G and 5H). Similar survival improvements were observed in allogeneic MHC mismatched HSCT mouse model receiving BMC (fig. 5I), and donor-derived CD4+ FoxP3+ T _regBMC treated mice in this model were 5-fold and 4-fold higher in thymus and spleen, respectively, 15 days after transplantation (fig. 5J).

BMC treatment of the invention was next compared to an extensively studied T cell progenitor infusion method consisting essentially of Double Negative (DN)2 and Double Negative (DN)3 precursors (> 90%) generated ex vivo by administration of OP9-DL1 feeder cells (fig. 5K). Donor chimerism was significantly higher in DP, SP4 and SP8 thymocyte populations 28 days post HSCT in BMC-treated mice (fig. 5L-5N). In the spleen, CD4⁺And CD8⁺Donor chimerism of T cells was all higher and donor CD4 was found in BMC treatment⁺And CD8⁺The absolute number of T cells was higher (fig. 5O and 5P).

Example 5: BMC enhances the diversity and function of regenerative T cells

T cell receptor diversity (TCR diversity) is produced by random somatic recombination of gene segments in the thymus. In dual BMC treated mice, thymocyte structures and thymus weights were significantly higher than those of the control group transplanted alone, about 6 weeks after transplantation (fig. 3Q and 3R). To characterize whether the increase in thymocyte structure also enhanced the function and diversity of T cells regenerated in isogenic transplantation, TCR excision loops (TRECs) were quantified. TREC is a marker of TCR rearrangement. TCR repertoire analysis was also performed. The TREC numbers in the thymus of dual-BMC treated mice were similar to those of non-irradiated control mice 1 month after transplantation and were both greater than those of mice transplanted only and BMP-2BMC treated mice (fig. 6A). In the spleen, a lower number of overall TRECs was noted in the same group compared to the non-irradiated control group, but mice treated with dual BMCs still had a higher TREC count compared to the transplanted and single factor BMP-2BMC only group (fig. 6B). The diversity of TCR V and J segments in the CDR3 β chain was assessed in BMC treated and transplanted mice 30 days after HSCT using the Simpson Index (SI) which takes into account the number of T cell clones present and the relative abundance of each clone. The SI of mice with bifunctional BMCs was 40% of that of the non-irradiated control group, whereas the SI of mice transplanted or BMP-2BMC only was lower (16% and 8%, respectively) (fig. 6C). In HSCT, The lack of naive T cells with extensive TCR repertoires is associated with an increased risk of immune complications and opportunistic infections (Douek, D.C. et al, Assessment of genetic output in additives after clinical laboratory cell translation and diagnosis of T-cell retrieval, The Lance 355, 1875-18811076 (2000)). The recovery of a large number of TRECs in the thymus and periphery of dual BMC treated mice reflects an increase in thymopoiesis. A relatively higher frequency of specific TCR clones was also observed in mice treated with dual BMC, but greater overall diversity indicates that the thymic-derived reconstitution is more balanced.

To measure the ability of regenerative T cells to respond in an antigen-specific manner, isogenic grafts were inoculated and subsequently challenged with the model protein Ovalbumin (OVA) 30 days post-transplantation (fig. 6D). OVA epitope (SIINFEKL) -tetramer compared to transplanted mice receiving no or only BMP-2⁺CD8+ T cells were significantly higher in mice receiving the bifunctional BMCs (fig. 6E). Similarly, in dual BMC-treated mice receiving allogeneic xenografts, the donor antigen-specific T cell response was about 3-fold higher than that produced by naive T cell therapy (fig. 6F). The production of Interferon (IFN) -gamma and tumor necrosis factor alpha (TNF-alpha) after in vitro stimulation was found to be in comparison with the donor CD4 on day 22 in the BMC treatment group and the T cell progenitor treatment group⁺And CD8⁺T cells were comparable, but a significantly greater proportion of T cells from the BMC treated group produced these factors at day 42 (fig. 6G and 6H). Post-vaccination CD8 in post-HSCT BMC-treated mice⁺The powerful antigen-specific generation of T cells suggests that BMC treatment may be used in conjunction with post-HSCT vaccination.

Example 6: materials and methods

The invention was carried out using the following materials and methods.

General methods and statistics

The sample size of animal studies was based on previous work without additional statistical estimation (Branard, D.M. et al, Induction of both road cellular and road video-specific adaptive immune responses in human engineered graphics BLT-mice, Journal of virology 83,7305-7321 (2009); Bencherif, S.A. et al, Injectable Cryogel-based book-cell vehicles. Nature communications 6,7556 (201556); Palchaudhurri, R. et al, Non-genetic conditioning for biological samples 1066, biological simulation 2016, 2016). Results were analyzed using GraphPad Prism software by using one-way contrast analysis and Tukey post hoc tests. In the case of analysis of variance, differences between groups were found to be similar by the Bartlett test. The survival curves were analyzed by using the log rank (Mantel-Cox) test. Word number coding is used for blind samples and blood counts.

Material

UP LVG sodium alginate with high gulonate content was purchased from ProNova biomedicalal; 2-Morpholinoethanesulfonic acid (MES), sodium chloride (NaCl), sodium hydroxide (NaOH), N-hydroxysuccinimide (NHS), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC), 2-aminoethyl methacrylate hydrochloride (AEMA) and acetone were purchased from Sigma-Aldrich. ACRL-PEG-NH2(3.5kDa) and 4arm PEG acrylate (10kDa) were purchased from JenKem Technology.

Bone Marrow Cryogel (BMC) manufacture

Bone marrow cryogels were made according to some modifications of the previously described techniques (Bencherif, S.A. et al, objective expressed scans with shape-memory properties. proceedings of the National Academy of Sciences of the United States of America 109,19590-19595 (2012). The alginate was reacted with AEMA to prepare alginate methacrylate (MA-alginate). sodium alginate was dissolved in a buffer solution of 100mM MES buffer (0.6% (wt/vol), pH 6.5) NHS and EDC was added to the mixture to activate the carboxylic acid groups on the alginate backbone, then AEMA (molar ratio of NHS: EDC: AEMA 1:1.3:1.1) was added and the solution was stirred at Room Temperature (RT) for 24 hours Salt: 4arm PEG acrylate 4: 1) then Tetramethylethylenediamine (TEMED) (0.5% (wt/vol)) and Ammonium Persulfate (APS) (0.25% (wt/vol)) were added to synthesize alginate-PEG BMC. ACRYL-PEG-NH2 was conjugated with Delta-like ligand 4(DLL-4) (R & DSystems) using carbodiimide chemistry (NHS: EDC: DLL4 molar ratio 1:1.3: 1.1). BMP-2(R & D Systems) was added to the polymer solution prior to low temperature polymerization. All precursor solutions were pre-cooled to 4 ℃ prior to freezing to reduce the polymerization rate. After the initiator was added to the prepolymer solution, the solution was quickly transferred to a pre-chilled (-20 ℃) polytetrafluoroethylene mold. After overnight incubation, the gels were thawed and collected in petri dishes on ice.

For Scanning Electron Microscopy (SEM), BMC was incubated in freshly prepared ethanol solutions (30, 50, 70, 90 and 100%) at increasing concentrations for 20 minutes each. BMC was then incubated in hexamethyldisilazane (electron microscopy science) for 10 minutes and dried in a desiccator/vacuum chamber for at least 1 hour before they were mounted for SEM. The dried BMC was adhered to a sample stub using a carbon tape and coated with platinum/palladium in a sputter coater. The samples were imaged using secondary electron detection on a Carl Zeiss Supra 55VP field emission Scanning Electron Microscope (SEM).

Quantitation of biomolecule release

Stock concentrations of BMP-2 were known from the manufacturer and verified using ELISA. To determine the release kinetics encapsulation efficiency of BMP-2 and confirm stable binding of DLL-4, BMC was incubated in 1ml sterile PBS at 37 ℃ with shaking. The media is replaced periodically. The released agent in the supernatant was detected by elisa (peprotech). The sample is released until BMP-2 is no longer detectable in the release medium. Subsequently, the cryogel is digested with at least 1000U of alginate lyase. Digestion products were analyzed for BMP-2 using ELISA. The amounts of BMP-2 and DLL-4 in the cryogel and the release medium were compared to the known amounts of loaded BMP-2 and DLL-4 to calculate the encapsulation efficiency.

Biomolecule activity assay

Alkaline phosphatase Activity assay for BMP-2 bioactivity

MC3T3-E1 subclone 4 cells were used to perform alkaline phosphatase assays as described in Macdonald, M.L. et al, Tissue integration of growth factor-analyzing layer-by-layer polyelectrolyte coated animals, Biomaterials 32,1446-1453 (2011). Cells were cultured under different experimental conditions: (1) growth medium, (2) differentiation medium supplemented with BMP-2 released from BMC and (3) native BMP-2. Notch activation assay for DLL-4 biological Activity

To quantify the in vitro bioactivity of DLL-4 after exposure to serum proteins, which could inactivate this morphogen in vivo, the previously characterized Notch reporter cell line CHO-K1+2xHS4-UAS-H2B-Citrine-2xHS4 cH1+ hNECD-Gal4esn c9 was used, being a gift from M.Elowitz (Caltech) (Sprinzak, D. et al, Cis-interactions between Notch and Delta gene mutuallyextrinsic knowledge sites Nature 465,86 (2010); Nandagopanal, N. et al, Dynamic ligand degradation in the Notch signaling pathway 172, 819 869-. These cells were grown in the presence of 5% CO2 in a humidified environment at 37 ℃ in Alpha MEM Earle's salts (Irvine scientific) supplemented with 10% Tet System applied FBS (Clontech), 100U/ml penicillin-100. mu.g streptomycin-0.292 mg/ml L-glutamine (Gibco). BMCs with or without DLL-4 were incubated with complete cell culture medium in 96-well plates, without cells. At predetermined time intervals (up to 3 months), 2 million Notch reporter cells were seeded into the wells of BMC. After 24 hours, confocal microscopy was performed using a Zeiss LSM 710 confocal system. The colorimetric output in response to binding of the Notch ligand DLL-4 was quantified and used as an indicator of DLL-4 bioactivity in the scaffold (FIG. 1H). In particular, the total YFP fluorescence per cell in the field of view (50-100; 4-5 fields, including more than 80% of the gel surface) was calculated and the background fluorescence was subtracted. The median YFP fluorescence was calculated and divided by the median fluorescence of cells seeded on BMC without DLL-4 and reported.

Affinity determination by surface plasmon resonance

Dissociation constants of wild-type DLL4 and MA-DLL4 for Notch1 were determined by surface plasmon resonance using a BIAcore T200 instrument (GE Healthcare) as previously described (bHeliotis,M.,Lavery,K.,Ripamonti,U.,Tsiridis,E.&Di Silvery, L.transformation of a predefined hydrophilic/osteopenic protein-1 imprinted into a modulated specific bone flap in the human chemical, International journal of oral and maxillofacial surgery 35, 265. the material 269 (2006)). Briefly, biotinylated recombinant Notch1 was immobilized on a streptavidin-coated sensor chip (GE Healthcare). Wild-type methacrylated DLL4 protein, which was present in increasing concentrations in the buffer, was flowed through the chip at 20 ℃. The binding and dissociation phases were performed at 10. mu.l/min for 120 seconds and 60 seconds, respectively. Steady-state binding curves were fitted to the 1:1Langmuir model using BIAcore evaluation software to determine K_d。

In vitro cell culture in Bone Marrow Cryogels (BMCs)

Mouse BM cells were harvested from the extremities. The comminuted tissue and cells were filtered through a 70 micron screen. The cells were passed through a 20 gauge needle to prepare a single cell suspension. The total cell number was determined by counting the cells using a hemocytometer. Mature immune cells (expressing CD 3-beta, CD45R/B220, Ter-119, CD11B, or Gr-1) were removed from BM cells by magnetic selection (BD Biosciences). Cells were incubated with Pacific Blue-coupled lineage antibodies (antibodies to CD3, NK1.1, Gr-1, CD11b, CD19, CD4 and CD 8) and a mixture of Sca-1 and c-kit specific antibodies. Hematopoietic cell (Lin) isolation Using FacsAria cell sorter (BD) ^-Sca-1^hic-kit^hi). The purity of the sorted cells is more than or equal to 95 percent. Human cord blood-derived CD34+ cells (Allcells) were purchased and expanded for 7 days using expansion supplements (Stemcell Technologies). Isolation of CD34 Using Positive selection kit (StemCell Technologies)⁺A cell. 96-well plates were precoated with Pluronic F127 (Sigma). Each BMC was placed individually in a well of a 96-well plate. Ten thousand of the mouse or human cells isolated as described above were added to a volume of 200 μ L RPMI (containing L-glutamine) 1640 containing 10% Fetal Bovine Serum (FBS) and 1% antibiotic and antibacterial solution (containing penicillin, streptomycin and amphotericin B) in the same well. For mouse cells, 10ng/ml stem cell factor (SCF; R) was added to the medium&D Systems), 10ng/mL FMS-like tyrosine kinase 3 ligand (Flt 3L;R&d Systems) and 1ng/mL interleukin 7 (IL-7; r&D Systems) were performed with 50% medium replacement steps on days 2, 4 and 6. For human cells, 100ng/mL SCF, 100ng/mL Flt3L, 100ng/mL TPO (R)&D Systems). After one week of culture, cells were isolated by digesting BMC with 1mg/ml alginate lyase (Sigma). The solution was passed through a 70 μm filter and the cells were treated as described below for FACS analysis.

BM transplantation and blood analysis

All animal work was approved by the animal care and use committee of the harvard institute and followed the guidelines and associated ethical regulations of the national institutes of health. C57BL/6(B6, H-2)^b)、BALB/c(H-2^d)、C57BL/6(CD 45.1⁺) B6-Tg (UBC-GFP)30Scha/J (GFP) and NSG mice (Jackson Laboratories) are female and aged 6 to 8 weeks at the start of the experiment. All mice in each experiment were age matched and were not randomized. The pre-established missing criteria for animals were the failure to inject the required cell dose in the transplanted mice and death due to post-operative complications of the humanized mice. Health issues not related to surgery (e.g., malocclusion, severe dermatitis) are the criteria for omission and euthanasia.

Transplantation model

All TBI experiments were performed with a Cs-137 gamma radiation source. Sublethal TBI (SL-TBI, B6 receptor), 1x500cGy +5x10⁵Lineage depleted bone marrow cells; isogenic HSCT (syn-HSCT, B6 receptor) 1x1000cGy +5x10⁴To 5x10⁵(as shown) lineage depleted GFP BM cells; allogeneic HSCT with GVHD (BALB/cMHC mismatch receptor) 1x850cGy +5x10⁵Lineage depleted GFP BM cells +1x10⁶GFP splenocytes; allogeneic HSCT (BALB/c MHC mismatch receptor) 1x850cGy +5x10 without GVHD ⁵Lineage depleted GFP BM cells or 5X10⁶GFP T cell progenitors +103 syngeneic HSCs generated in vitro, as described elsewhere, were unmodified 48. Humanized BLT (bone marrow-liver-thymus) mouse studies were performed by the MGH and Ragon institute human immune system mouse project and were approved by the IACUC institute as previously described (brain, d.m., et al, InThe description of the manufacture of the specimen cellular and the human viral-induced synthesized BLT microorganisms in the human immunological viral-induced BLT microorganisms, Journal of virology 83,7305 and 7321 (2009). BM cells for transplantation or analysis were harvested by crushing all limbs or one femur, respectively, and processed as described above. When mice were anesthetized, two BMCs suspended in 0.2ml sterile PBS were injected subcutaneously into the dorsal side through a 16-gauge needle. One BMC was injected on each side of the spine and approximately midway between the hind and anterior limbs. Subcutaneous nodule size is quantified over time by measuring the length, width, and height of the nodule using calipers. Mice from all groups (typically 10 mice/group) were continuously bled. The white blood cell, hemoglobin, red blood cell, platelet and hematocrit levels were quantified by CBC analysis (Abaxis VetScan HM 5). .

To directly assess whether cells from BMC migrated to the thymus, the two-factor BMC was delivered to a set of naive, lethally irradiated mice (receiving 1000cGy L-TBI and 5x 10) along with stem cell therapy⁵Lineage depleted GFP BM cells and B6 receptor for two-factor BMC). 10 days after HSCT, the dual factor BMC was removed and immediately surgically transplanted into the subcutaneous pocket of a second group of B6 mice that received 500cGy SL-TBI 48 hours ago without any additional cell transplantation. 20 days after surgery, mice were sacrificed and thymocytes were analyzed from these mice.

Flow cytometry (FACS) analysis

Anti-mouse antibodies against CD 8-alpha (53-6.7), CD 3-alpha (145-2C11), B220(RA3-6B2), CD11B (M1/70), CD25(PC61), CD117(2B8), Sca-1(D7), CD127(A7R34), and anti-human antibodies against CD45(H130), CD3(HIT3a), CD4(SK3), CD19(HIB19), CD34(581), CD38(HB-7), and CD7(CD7-6B7), IFN-gamma (XMG1.1), TNF-alpha (MP6-XT22), and the corresponding isotypes were purchased from BioLegend. Anti-human CD8(RPA-T8) was purchased from BD Biosciences. CD44(IM7) was purchased from eBioscience. SIINFEKL tetramer (Alexa Fluor 647H-2K)^bOVA) was obtained from NIH Tetramer Core Facility. All cells were gated based on forward and side scatter properties to limit debris including dead cells. Diluting the antibody according to the manufacturer's recommendations And (3) a body. Cells were gated based on fluorescence minus one control and the frequency of cells staining positive for each marker was recorded. To quantify T, B and bone marrow cells, blood samples were lysed with red blood cells and stained with anti-CD 45, -B220, -CD3, -CD4, -CD8, and-CD 11B antibodies, and the absolute numbers of T, B and bone marrow cells were calculated using flow cytometry frequency and leukocyte values obtained by CBC analysis. Donor events within the blood cell-based CD45+ and stromal cell CD45 gates were analyzed.

Bone, fat quantification and histology

After euthanasia, BMC and tissues were removed. To quantify bone using bone alkaline phosphatase (BALP), BMC and femur were crushed and homogenized and filtered through a 70 μm filter. Subsequently, BALP was quantified using a BALP ELISA kit (Creative Diagnostics) according to the manufacturer's protocol. Oil red O staining kit (Biovision) was used for lipid quantification. Harvested BMC and bone were cleaned, fixed, processed and stained according to the manufacturer's protocol. In measuring the absorbance (OD)₄₉₂) Previously, BMC and bone were then crushed, deformed and resuspended in equal volume. For histological staining, the tissues were fixed in 4% Paraformaldehyde (PFA). The PFA-immobilized samples were partially decalcified using a rapid decalcification formic acid/hydrochloric acid mixture (decalcification solution, VWR) for about 4 hours and embedded in paraffin. Sample sections (5 μm) were stained with conventional trichrome, safranin-O or Verhoeff-Van Giesen.

Quantification of thymic T cell receptor excising cycle (TREC)

TREC quantification was performed as described previously (Warnke, P. et al, Growth and transfer of a custom designed bone grain in man, The Lancet364,766-770 (2004)). Briefly, thymus was harvested from unirradiated C57BL/6 mice, transplanted mice, and transplanted mice injected with BMC (30 days post conditioning). After homogenizing in a Bullet Blender Storm BBX24 instrument (Next Advance, Inc.), total DNA was extracted using TRIZOL. DNA was quantified by UV-Vis, and 1. mu.g of DNA per sample was used as input for real-time PCR. The absolute number of signal-joining trecs (sjtrecs) per sample was calculated using a standard curve of mouse sjTREC plasmid.

TCR analysis

Extracted lymphocyte RNA was quantified using UV-Vis. Equimolar amounts of RNA from each sample were submitted to irpertoire for sequencing and bioinformatic analysis, where samples were reverse transcribed and amplified using a primer set that specifically amplifies β TCR RNA. The sequencing results give the total read range and number of unique CDRs 3 for each sample.

Vaccination and non-specific T cell stimulation studies

30 days after transplantation, animals were immunized with a bolus vaccine containing 100 μ g Ovalbumin (OVA), 100 μ g CpG-ODN, and 1 μ g GM-CSF. After 10 days, the animals were challenged with intravenous ovalbumin. At day 12, spleens were collected from euthanized mice in the vaccination study. Splenocytes were isolated by mechanically disrupting the spleen against a 70 μm cell filter. Erythrocytes in the collected tissue were lysed and leukocytes were prepared for analysis. For non-specific stimulation, cells were incubated with PMA (10ng/mL) + ionomycin (2. mu.M) for 5 hours. After 2 hours of incubation, brefeldin A (10. mu.g/mL) was added. Cells were then harvested, washed, and stained with fluorochrome-conjugated T cell surface antigen antibodies. Subsequently, cells were fixed and permeabilized with the fixing/permeabilizing solution kit reagent (BD) and stained with IFN-. gamma.TNF-. alpha.specific antibodies.

Discussion of the related Art

Here, it was demonstrated that BMC based on acellular biomaterials mimics the key features of T cell lymphocyte production bone marrow niches and promotes regeneration of immunocompetent cells after hematopoietic stem cell transplantation. Subcutaneous injection of BMC links with host vasculature to form a host-device interface and provide lineage-guiding cues to progenitor cells recruited to the donor in vivo. It is well known that BMP-2 induces differentiation of Bone lineage of recruited mesenchymal cells and indirectly promotes rapid neovascularization (Smadja, D.M. et al Bone morphogenetic proteins 2and 4. optionally expressed by Bone tissue formation in rapid neovascularization. and vascular biology 28, 2137. 2143 (2008). in this work, early cardiovascular formation was observed, followed by maturation of The vasculature to a density consistent with that observed in The endogenous Bone marrow (Lafage-industry, M.H. et al Association of Bone differentiation and tissue modification. 4. found in The quantitative and quantitative hematopoietic stem cell of hematopoietic tissue and tissue differentiation of Bone tissue differentiation in The same PTH-peptide and tissue differentiation of Bone tissue differentiation in the bone marrow, J Cell Biol 167,1113-1122 (2004)); song, J.et al, An in vivo model to study and manipulating the biochemical stem cell niche, Blood 115, 2592-. Incorporation of The biologically active Notch ligand DLL-4 on a polymer scaffold promoted early enhancement of T cell progenitor production in BMC and resulted in a significant increase in The number of thymic progenitors relative to control groups receiving lineage-depleted bone marrow transplants, and The control groups were consistent with established isogenic and allogeneic HSCT models (Wils, E. -J. et al, Flt3 ligand ex vivo precursors to recovery of thymic and acelerogenic T cell recovery after bone marrow transplantation, The Journal of Immunology 178,3551-3557 (2007); Maillard, I. et al, Notch-dependent amino acid sequences ex vivo cloning of bone graft transplantation, Blood 107, 3511. complexity (TCR 19), and a growing library of TCRs generating antibodies.

The BMC approach is conceptually and practically different from other strategies that promote T cell regeneration after HSCT, and its relevance in HSCT is supported by preclinical studies in this work. When a dose 10-fold lower than the T cell progenitor infusion is used, it results in an increase in the number of T cell progenitors and functional T cells in the thymus and periphery. BMC treatment differs from other methods in that it can be administered simultaneously at HSCT. In contrast, the T cell progenitors are generated in vitro from donor hematopoietic cells within 2-4 weeks, have complex cell culture requirements in preclinical models, and are patient-specific. By providing T cell promoting cues for HSCT transplanted in vivo, without ex vivo culture, the BMC approach may be a ready product, avoiding the extensive infrastructure required for cell manufacturing (Garber, K. (Nature Publishing Group,2018) and possibly supplementing the activities of cytokine therapy.

Enhancement of human T cell reconstitution in xenogeneic NSG-BLT mice was accompanied by a modest and transient reduction in B cell reconstitution, consistent with a corresponding reduction in pre-B CFU. Although this humanized mouse model is widely accepted by human immune cells, it is well known that key mouse cytokines are inefficient in inducing hematopoiesis, including in this model from human CD34⁺Cells develop human B cells (Jangalwe, s., Shultz, l.d., Mathew, a.&Brehm, M.A. improved B cell reduction in manipulated NOD-scid IL2R gamma null ceramic transforming human stem cell factor, gradient-gradient gel-stabilizing factor and interleukin-3, Immunity, inflammation and disease 4,427-440 (2016)). It is likely that Notch activation of the transplanted cell fraction, with its seed BMC enhanced T cell dedifferentiation at the expense of B cell dedifferentiation. However, the transient reduction in B cell production is modest and unlikely to be of clinical significance.

The formation of bone nodules is limited to the geometry of the scaffold, consistent with previous reports on scaffold-induced bone formation, which is well tolerated in many species, including the non-human primates (Ripamonti, U.S. bone indication by recombinant human osteoprogenic protein-1(hOP-1, BMP-7) in the host Papio disease with expression of mRNA of gene products of the TGF- β surfactant, Journal of cellular and molecular medium 9,911-928(2005) and in humans (Helioti, M., Lavery, K., Ripapaya, U.S. iriridis, E.S. Silviaio, L.transformation of expression of fibrous/osteoprogenic protein-1, tissue of tissue, the Lancet 364,766-770 (2004)). Past clinical experience with other stent-based systems has shown that the size of the device may remain unchanged from species to species (Hodi, S. (2012)). Even though larger growth factor doses may be required for human use, it is expected that The controlled release provided by such polymer-based hydrogel systems will allow The use of doses of BMP-2 several orders of magnitude lower than The large doses currently used clinically, delivered as bolus injections and associated with adverse side effects (Carragee, E.J., Hurwitz, E.L. & Weiner, B.K. anterior review of recombinant human bone pathological protein-2 tertiary in physiological supply: emissive safety controls and less ions left, The Spine Journal 11,471 (2011)). After T-cell regeneration, the BMC can be easily removed, similar to other devices commonly used in HSCT or made of biodegradable materials for resorption (Biffi, R. et al, Use of portable implantable central venous access ports for high-dose therapy and peripheral blood culture cell transplantation: resources of anaerobic series of tissues, antibiotics of oncology 15,296-300 (2004)).

Allogeneic post-HSCT CD4⁺Recovery of T cells is often delayed, resulting in inversion of the normal CD4/CD8 ratio (Li, M.O.&Rudensky, A.Y.T. cell receiver signalling in the control of regulation T-cell differentiation and function. Nature Reviews Immunology 16,220 (2016). In BMC treated mice, T cell reconstitution in thymus and spleen of humanized and allogeneic xenograft mice was more balanced and donor CD4 was present⁺Regulatory T cells (T)_reg) And (4) enhancing. In view of the donor T_regA key role in GVHD inhibition (Hoffmann, p., Ermann, j., Edinger, m., fatman, C.G.&Strober,S.Donor-type CD4⁺CD25⁺regulatory T cells present in a least squares approach and later analogue bone mapping, Journal of Experimental Medicine 196,389-399(2002)), BMC mediated donor T cells_regEnhanced production may contribute to the reduction of GVHD-like pathology and to the increase in mouse survival, possibly through TGF-beta family proteins (a key to Treg expansion)Bond regulatory factor) ((Wan, Y.Y).&'Yin-Yang' functions of transforming growth factor-beta and T-regulatory cells in immune regulation, Immunological functions 220,199-213 (2007)). Furthermore, the time course in the allogeneic GVHD model is consistent with at least some BMC effects due to effects on pre-existing T-type or mature T cells.

Taken together, these findings indicate that BMC represents an easy-to-use, ready-to-use system that can enhance T cell regeneration after HSCT. If the BMC system behaves similarly in a human environment, it may be a means to eliminate immune complications and opportunistic infections that limit the clinical application of potentially curative HSCT.

Is incorporated by reference

All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents of the formula

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.