NZ741878B2 - Methods for culturing cells and kits and apparatus for same - Google Patents

Methods for culturing cells and kits and apparatus for same Download PDF

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Publication number
NZ741878B2
NZ741878B2 NZ741878A NZ74187816A NZ741878B2 NZ 741878 B2 NZ741878 B2 NZ 741878B2 NZ 741878 A NZ741878 A NZ 741878A NZ 74187816 A NZ74187816 A NZ 74187816A NZ 741878 B2 NZ741878 B2 NZ 741878B2
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New Zealand
Prior art keywords
binding
cells
receptor
streptavidin
agent
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NZ741878A
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NZ741878A (en
Inventor
Keenan Bashour
Lothar Germeroth
Patricia Graf
Christian Stemberger
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Juno Therapeutics Gmbh
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Application filed by Juno Therapeutics Gmbh filed Critical Juno Therapeutics Gmbh
Priority claimed from PCT/IB2016/001618 external-priority patent/WO2017068421A1/en
Publication of NZ741878A publication Critical patent/NZ741878A/en
Publication of NZ741878B2 publication Critical patent/NZ741878B2/en

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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • C12N5/0646—Natural killers cells [NK], NKT cells

Abstract

Provided herein are methods that relate, in some aspects, to the incubation or culturing, such as to induce stimulation of expansion (proliferation), activation, costimulation and/or survival, of a composition of cells, such as a population of lymphocytes. In some aspects, provided are methods and reagents for the stimulation, e.g., of expansion (proliferation), survival or persistence, activation, costimulation, or other effect, of cell populations that involve binding of agents to a molecule on the surface of the cells, thereby providing one or more signals to the cells. In some cases, the reagents are reagents containing a plurality of binding sites for agents, such as multimerization reagents, and thus the one or more agents are multimerized by reversibly binding to the reagent,e.g., thereby creating a stimulatory reagent (multimerized agent), having stimulatory agents multimerized thereon. In some aspects, the multimerized agent can provide for expansion or proliferation or other stimulation of a population of cells, and then such stimulatory agents can be removed by disruption of the reversible bond. Also provided are compositions, apparatus and methods of use thereof.

Claims (101)

1. A method for stimulating T cells, the method comprising: (a) incubating a composition comprising T cells in the presence of: (1) a first receptor-binding agent, n the first receptor-binding agent comprises an anti-CD3 antibody or antibody fragment that specifically binds to a CD3 molecule on the surface of the T cells in a manner that s a primary activation signal in T cells in the composition; and (2) a second receptor-binding agent, wherein the second receptor-binding agent comprises an anti-CD28 antibody or antibody fragment that specifically binds to a CD28 le on the surface of the T cells in a manner that induces a costimulatory signal in T cells in the composition; wherein the first and second or-binding agents are reversibly bound to a reagent comprising a plurality of first binding sites capable of reversibly binding to the first receptorbinding agent and a plurality of second binding sites capable of reversibly binding to the second receptor-binding agent; and wherein the incubation effects the stimulation or expansion of T cells in the ition; (b) within 5 days after initiation of the incubation, disrupting the reversible binding between the first receptor-binding agent and the t and between the second receptorbinding agent and the reagent, thereby generating ated T cells.
2. The method of claim 1, wherein the disruption is carried out greater than 30 minutes after tion of the incubation.
3. The method of claim 1 or claim 2, wherein the disruption is carried out between 1 hour and 4 days after initiation of the incubation, between 6 hours and 3 days after initiation of the incubation, between 12 hours and 2 days after initiation of the tion, or between 1 day and 3 days after initiation of the incubation; or the disruption is carried out between about 1 hour and about 4 days after initiation of the incubation, between about 6 hours and about 3 days after tion of the incubation, between about 12 hours and about 2 days after initiation of the incubation, or between about 1 day and about 3 days after initiation of the incubation.
4. The method of any of claims 1-3, wherein the disruption is carried out greater than or equal to about 1 hour after initiation of the incubation and within 1 day, 2 days, 3 days, or 4 days after initiation of the incubation.
5. The method of any of claims 1-4, n: the first receptor-binding agent ses a first binding partner; and the plurality of first binding sites each are capable of binding to the first binding partner to form the reversible bond between the first receptor-binding agent and the reagent.
6. The method of claim 5, wherein the first binding partner is fused to the C- terminus of a heavy chain of the first receptor-binding agent.
7. The method of any of claims 1-6, wherein: the second receptor-binding agent comprises a second g partner; and the plurality of second binding sites each are capable of g to the second binding partner to form the reversible bond between the second receptor-binding agent and the reagent.
8. The method of claim 7, wherein the second binding r is fused to the C- terminus of a heavy chain of the second receptor-binding agent.
9. The method of any of claims 1-8, wherein the plurality of first g sites are each capable of reversibly binding to the second receptor-binding agent, and the plurality of second binding sites are each capable of reversibly binding to the first or-binding agent.
10. The method of any of claims 7-9, wherein the plurality of first binding sites are each capable of ibly binding to the second binding partner, and the plurality of second binding sites are each capable of reversibly binding to the first binding partner.
11. The method of any of claims 7-10, wherein the first and second g partners are the same or substantially the same.
12. The method of any of claims 1-11, wherein the incubation is further carried out in the presence of a third receptor-binding agent that specifically binds to a third molecule on the surface of one or more of the T cells.
13. The method of claim 12, wherein the third receptor-binding agent specifically binds to the third molecule to induce a further signal in the one or more T cells.
14. The method of claim 12 or claim 13, wherein the third molecule is a cytokine receptor, a chemokine receptor, an adhesion molecule, or a factor that induces cytokine production, chemokine production, and/or expression of an on molecule.
15. The method of any of claims 12-14, wherein the third receptor-binding agent specifically binds to a cytokine or selected from among IL-2R, IL-1R, IL-15R, IFN- gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2.
16. The method of any of claims 12-15, wherein the third receptor-binding agent is a ligand that specifically binds to a ne receptor ed from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2.
17. The method of any of claims 12-16, wherein the third receptor-binding agent is a ligand selected from among IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL- 15, IL-17, and TNF, or is a ically active fragment thereof.
18. The method of any of claims 12-16, wherein the third receptor-binding agent specifically binds to a cytokine receptor selected from among IL-12R, IFN-gammaR, IL-4R, and IL-17R.
19. The method of any of claims 12-14, wherein the third receptor-binding agent is a ligand selected from among IL-2, IL-4, IL-7, IL-10, IL-15, and IL-17, or is a biologically active fragment thereof.
20. The method of any of claims 12-14, wherein the third receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R, and CD132 (IL receptor common gamma chain).
21. The method of any of claims 12-14 and 20, wherein the third or-binding agent is a ligand that specifically binds to a cytokine or selected from among IL-7R, IL-21R, and CD132 (IL receptor common gamma chain).
22. The method of any of claims 12-14, wherein the third receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9, and IL-15, or is a biologically active fragment thereof.
23. The method of any of claims 12-14, wherein the third receptor-binding agent specifically binds to a chemokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4.
24. The method of any of claims 12-14 and 23, wherein the third receptor-binding agent is a ligand that specifically binds to a chemokine receptor ed from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4.
25. The method of any of claims 12-14, 23, and 24, n the third receptorbinding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21, and CCL25, or is a biologically active fragment thereof.
26. The method of any of claims 12-14 and 23-25, n the third receptor-binding agent specifically binds to a ine receptor selected from among CXCR3, CCR7, CXCR1, and CXCR4.
27. The method of any of claims 12-14, wherein the third molecule is an adhesion molecule selected from among CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), CD29/CD49d ), and CD106 (VCAM-1), or is a biologically active fragment thereof.
28. The method of any of claims 12-14 and 27, wherein the third molecule is an adhesion molecule selected from among LFA-1, L-selectin, VCAM-1, and VLA-4, and VLA-4 or is a biologically active fragment thereof.
29. The method of any of claims 12-14, wherein the third molecule is a nuclear factor.
30. The method of any of claims 12-14 and 29, wherein the third molecule is a retinoic acid receptor-related orphan or gamma (RORgamma) or RORalpha.
31. The method of any of claims 12-30, wherein the third receptor-binding agent is reversibly bound to the reagent.
32. The method of any of claims 1-31, wherein the tion is carried out at or about 37 ºC ± 2 ºC.
33. The method of any of claims 1-32, wherein the incubation is carried out in the presence of a further agent that is e of delivering a signal to T cells.
34. The method of claim 33, wherein the further agent is capable of enhancing or inducing proliferation of T cells.
35. The method of claim 33 or claim 34, wherein the further agent is a cytokine selected from among IL-2, IL-15, and IL-7.
36. The method of any of claims 1-35, comprising, after the disruption, further incubating the composition comprising the T cells.
37. The method of claim 36, wherein the incubation and further incubation are carried out in the same vessel.
38. The method of claim 36 or claim 37, n the further incubation is carried out at or about 37 ºC ± 2 ºC.
39. The method of any of claims 36-38, wherein the further incubation is carried out in the presence of a further agent that is capable of delivering a signal to T cells.
40. The method of claim 39, wherein the r agent is capable of enhancing or inducing proliferation of T cells.
41. The method of claim 39 or claim 40, wherein the further agent is a cytokine selected from among IL-2, IL-15 and IL-7.
42. The method of any of claims 36-41, wherein the further incubation is carried out for a time that is no more than 14 days, no more than 12 days, no more than 10 days, no more than 8 days, or no more than 6 days.
43. The method of any of claims 1-42, wherein the T cells are y cells from a
44. The method of any of claims 1-43, wherein the T cells are directly isolated from a subject.
45. The method of any of claims 1-44, n the T cells are unfractionated T cells, are ed or isolated CD3+ T cells, are enriched or ed CD4+ T cells, or are enriched or isolated CD8+ T cells.
46. The method of any of claims 1-45, wherein prior to the incubating, the T cells are not enriched for naïve T cells.
47. The method of any of claims 1-46, wherein prior to the incubating, the T cells are not enriched for CD62L+ cells.
48. The method of any of claims 1-47, wherein the T cells are human cells.
49. The method of any of claims 1-48, wherein the reagent is in soluble form.
50. The method of any of claims 1-48, wherein the reagent is bound to a solid support during at least a portion of the incubation, whereby a plurality of the T cells are reversibly immobilized on the solid support during at least a portion of the incubation.
51. The method of claim 50, wherein the solid support is a stationary phase.
52. The method of any of claims 1-51, wherein the anti-CD3 dy or antibody fragment is an anti-CD3 antibody fragment.
53. The method of claim 52, wherein the anti-CD3 antibody fragment is a divalent antibody fragment selected from among a F(ab’)2-fragment and a divalent single-chain Fv (scFv) fragment.
54. The method of claim 52, wherein the anti-CD3 antibody fragment is a monovalent antibody fragment selected from among a Fab fragment, an Fv nt, and an scFv fragment.
55. The method of any of claims 1-54, wherein the anti-CD28 antibody or antibody fragment is an anti-CD28 antibody fragment.
56. The method of claim 55, wherein the anti-CD28 antibody fragment is a nt antibody fragment selected from among a F(ab’)2-fragment and a divalent single-chain Fv (scFv) fragment.
57. The method of claim 55, wherein the anti-CD28 antibody fragment is a monovalent antibody fragment ed from among a Fab fragment, an Fv fragment, and an scFv fragment.
58. The method of any of claims 1-52, 54, 55, and 57, wherein the D3 antibody or antibody fragment is an anti-CD3 Fab, and the anti-CD28 antibody or antibody fragment is an anti-CD28 Fab.
59. The method of any of claims 1-58, wherein the t comprises streptavidin; avidin; a mutein of streptavidin or avidin that reversibly binds to biotin, a biotin analog or a biologically active fragment thereof, or a streptavidin-binding peptide; a t that comprises at least two chelating groups K, wherein the at least two chelating groups are capable of binding to a transition metal ion; an agent capable of binding to an oligohistidine affinity tag; an agent capable of binding to a glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding to calmodulin binding peptide (CBP); an agent e of binding to a FLAG-peptide; an agent capable of binding to an HA-tag; an agent capable of binding to maltose binding protein (MBP); an agent capable of binding to an HSV epitope; an agent e of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.
60. The method of claim 59, wherein the reagent ses an er or polymer of streptavidin, avidin, or the streptavidin or avidin mutein.
61. The method of claim 60, wherein the oligomer or polymer comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 tetramers of the streptavidin, avidin, or streptavidin or avidin mutein.
62. The method of any of claims 8-61, wherein the first binding partner ses biotin or a biologically active fragment thereof, and the reagent comprises a streptavidin or avidin mutein that ibly binds to biotin.
63. The method of any of claims 8-61, n the first g partner comprises a biotin analog or a biologically active fragment thereof, and the reagent comprises streptavidin, avidin, or a streptavidin or avidin mutein that reversibly bind to the biotin analog.
64. The method of claim 63, wherein the biotin analog of the first binding partner is iotin, lipoic acid, desthiobiotin, diaminobiotin, hydroxyazobenzene-benzoic acid (HABA), or dimethyl-HABA.
65. The method of any of claims 8-61, wherein the first g partner comprises a streptavidin-binding peptide, and the reagent comprises streptavidin, avidin, or a streptavidin or avidin mutein that reversibly binds to the streptavidin-binding peptide.
66. The method of claim 65, wherein the avidin-binding peptide of the first binding partner comprises the sequence of amino acids set forth in any of SEQ ID NO: 7, 8, and 15-19.
67. The method of any of claims 7-66, wherein the second binding partner comprises biotin, and the reagent comprises a streptavidin or avidin mutein that reversibly binds to biotin.
68. The method of any of claims 7-66, wherein the second binding r ses a biotin analog or a biologically active fragment f, and the reagent comprises streptavidin, avidin, or a streptavidin or avidin mutein that reversibly binds to the biotin analog.
69. The method of claim 68, wherein the biotin analog of the second binding partner is iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, hydroxyazobenzene-benzoic acid (HABA), or dimethyl-HABA.
70. The method of any of claims 7-66, wherein the second binding partner comprises a streptavidin-binding e, and the reagent comprises streptavidin, avidin, or a streptavidin or avidin mutein that ibly binds to the streptavidin-binding e.
71. The method of claim 70, wherein the streptavidin-binding peptide of the second binding partner comprises the sequence of amino acids set forth in any of SEQ ID NO: 7, 8, and 15-19.
72. The method of any of claims 59-71, wherein the reagent ses an oligomer or polymer of the streptavidin mutein.
73. The method of any of claims 60-72, wherein individual molecules of the oligomer or polymer are crosslinked by a polysaccharide.
74. The method of any of claims 60-72, wherein individual molecules of the oligomer or polymer are crosslinked by a bifunctional linker.
75. The method of claim 74, wherein the bifunctional linker is a amine-to-thiol linker.
76. The method of any of claims 59-75, wherein the streptavidin mutein comprises the amino acid sequence Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 1.
77. The method of any of claims 59-76, wherein the streptavidin mutein ses: a) the sequence of amino acids set forth in any of SEQ ID NOS: 3-6, 27, and 28; b) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence ty to any of SEQ ID NOS: 3-6, 27, and 28; contains the amino acid sequence corresponding to Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 1; and reversibly binds to biotin or a ically active form thereof, a biotin analog or a biologically active nt thereof, or a streptavidin-binding e; or c) a functional fragment of a) or b) that reversibly binds to biotin or a biologically active form thereof, a biotin analog or a biologically active fragment thereof, or a streptavidin-binding peptide.
78. The method of any of claims 59-77, wherein the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NO: 3-6, 27, and 28.
79. The method of any of claims 59-78 n the streptavidin mutein begins N- terminally in the region of amino acid positions 10 to 16 of SEQ ID NO: 1 and terminates C- terminally in the region of amino acid positions 133 to 142 of SEQ ID NO: 1.
80. The method of any of claims 76-79, wherein the streptavidin mutein further comprises an amino acid replacement or replacements at a position corresponding to position 117, 120,, and/or 121 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 1.
81. The method of claim 80, wherein the amino acid replacement or replacements are selected from among Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121, and Phe121.
82. The method of claim 80 or claim 81, n the amino acid replacement or replacements are selected from Glu117, Gly120, and Tyr121.
83. The method of any of claims 80-82, wherein the amino acid replacements are Glu117, Gly120, and Tyr121.
84. The method of any of claims 59-83, wherein the streptavidin mutein comprises: a) the sequence of amino acids set forth in SEQ ID NO: 27 or 28; b) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ce identity to SEQ ID NOS: 27 or 28; contains the amino acid sequence ponding to Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and ; and reversibly binds to biotin or a ically active fragment, a biotin analog or a biologically active fragment thereof, or a avidin-binding peptide; or c) a functional fragment of a) or b) that reversibly binds to biotin or a biologically active fragment, a biotin analog or a biologically active fragment thereof, or a streptavidin-binding peptide.
85. The method of any of claims 1-84, wherein the disruption comprises introducing to the T cells a composition sing a substance capable of reversing the bond between the first receptor-binding agent and the reagent and between the second or-binding agent and the reagent.
86. The method of claim 85, wherein the substance is a competition agent.
87. The method of claim 85 or claim 86, wherein: the reagent comprises the streptavidin mutein; and the substance comprises biotin or a biologically active fragment thereof or a biotin analog or a biologically active fragment thereof.
88. The method of claim 87, wherein the substance comprises D-biotin.
89. The method of claim 87, wherein the substance comprises a biotin analog that is iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, hydroxyazobenzene-benzoic acid (HABA), or dimethyl-HABA.
90. The method of any of claims 5-89, wherein the dissociation nt (KD) for the reversible binding n the plurality of first binding sites and the first g partner is in the range of 10-2 M to 10-13 M.
91. The method of any of claims 7-90, wherein the dissociation constant (KD) for the reversible binding between the plurality of second binding sites and the second binding partner is in the range of 10-2 M to 10-13 M.
92. The method of any of claims 1-91, wherein the stimulated T cells comprise greater than 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% of a T cell subset comprising a phenotype that is surface positive for CD62L (CD62L+) as a percentage of the total T cells in the composition or the total cells in the composition.
93. The method of claim 92, wherein the T cell subset further comprises a phenotype comprising: a) CD127+; and/or b) any one or more of +, CD45RO-, CCR7+, and CD27+ and any one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rß+, CXCR3+, and LFA-1+.
94. The method of claim 92 or claim 93, wherein the T cell subset: a) comprises a low level of TCR rearrangement excisions circles (TREC); b) expresses a proliferation ; c) exhibits the capacity to proliferate in the presence of a stimulatory agent; and/or d) exhibits the capacity to produce a cytokine ed from among IFN-gamma, TNF, and IL-2 in the presence of a stimulatory agent.
95. The method of any of claims 92-94, wherein the T cell subset is long-lived memory T cells.
96. The method of any of claims 92-95, wherein the T cell subset is T memory stem cells (TSCM).
97. The method of any of claims 1-96, further comprising introducing a recombinant nucleic acid molecule into T cells in the composition, which nucleic acid molecule encodes a recombinant protein, whereby T cells in the ition s the recombinant protein.
98. The method of claim 97, wherein the recombinant receptor is a chimeric antigen receptor or transgenic T cell or (TCR).
99. The method of any of claims 1-98, wherein the method is performed ex vivo.
100. A composition, comprising a plurality of stimulated T cells produced by the method of any of claims 1-99.
101. The composition of claim 100, r comprising a pharmaceutically acceptable excipient. W0 68421 W0 68421 3.m. 5A start of 2&3 e analysis R} {H {‘12. <33 68 ami‘iw?,¥.}3:'m}§i$.82-x.» mu?'imerixeé agent (1028 Maxis R6. 58 3.3x1398 26.ea .1. muMm uuwuuvowwe- § § :me {mini {21‘ {EB-Swan addition FiG‘ SC c,» {:3 (1821‘ Z‘na?s M m?h?cc «\um 0338‘ Y (#85 agmagxw Q “89s.“ u??ggv , Thismmcf8«33min gééi?m FIG‘ 6A {Egnfmg a: 3; maéizmx makings amaéyséx , > w m me. {ii} iii 8‘3. {1? 113;: I§:}“§3§0§§W 3" ’ ‘ B \\‘
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