A method for generating a cell population with increased nucleic acid uptake.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- ZEON CORP
- Filing Date
- 2026-02-06
- Publication Date
- 2026-06-09
AI Technical Summary
【0005】 一態様では、遺伝子操作された細胞組成物を得るための方法であって、(a)1つまたは複数の標的細胞を含む生体試料を提供すること;(b)1つまたは複数の標的細胞を含む生体試料から、7マイクロメートル以下である所定の直径の細胞成分を除去して、濃縮された標的細胞集団を得ること;および(c)濃縮された標的細胞集団を外因性核酸と接触させ、これにより遺伝子操作された標的細胞集団を提供することを含む、方法が本明細書に記載される。一部の実施形態では、所定の直径は4マイクロメートル以下である。ある特定の実施形態では、所定の直径は約5マイクロメートル以下である。ある特定の実施形態では、所定の直径は約4マイクロメートル以下である。ある特定の実施形態では、生体試料は、1つまたは複数の細胞を含む液体である。ある特定の実施形態では、1つまたは複数の細胞はヒト細胞である。ある特定の実施形態では、生体試料は、血液関連試料、骨髄試料、および脂肪試料、ならびにその組合せからなるリストから選択される。ある特定の実施形態では、生体試料はヒト生体試料である。ある特定の実施形態では、生体試料は血液関連試料である。ある特定の実施形態では、血液関連試料は、約2%よりも多い血球容量を含む。ある特定の実施形態では、血液関連試料は、約4%よりも多い血球容量を含む。ある特定の実施形態では、血液関連試料は、約30%未満の血球容量を含む。ある特定の実施形態では、血液関連試料は、白血球アフェレーシス産物である。ある特定の実施形態では、外因性核酸はウイルスの成分である。ある特定の実施形態では、ウイルスは、レンチウイルス、アデノウイルス、またはアデノ随伴ウイルス、およびその組合せからなるリストから選択される。ある特定の実施形態では、ウイルスはレンチウイルスである。ある特定の実施形態では、ウイルスはアデノウイルスである。ある特定の実施形態では、ウイルスはアデノ随伴ウイルスである。ある特定の実施形態では、アデノ随伴ウイルスは、AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、またはAAV9である。ある特定の実施形態では、ウイルスはシュードタイプ化したウイルス(pseudotyped virus)である。ある特定の実施形態では、ウイルスは非ウイルス核酸を含む。ある特定の実施形態では、非ウイルス核酸は遺伝子編集用のガイドRNAを含む。ある特定の実施形態では、非ウイルス核酸は、遺伝子編集システムのポリペプチド成分をコードする配列を含む。当技術分野で公知のそのようなシステムは、TALENs、CRISPR-Cas9、CRISPR-Cas12および同類のものを含む。ある特定の実施形態では、非ウイルス核酸は、標的鎖およびガイド鎖を含むCRISPRコンストラクトである。ある特定の実施形態では、非ウイルス核酸はポリペプチドをコードする。ある特定の実施形態では、ポリペプチドは、免疫グロブリン、キメラ抗原受容体、T細胞受容体、サイトカイン、またはケモカインを含む。ある特定の実施形態では、非ウイルス核酸はキメラ抗原受容体を含む。ある特定の実施形態では、ウイルスを濃縮された標的細胞集団と接触させることは、10:1以上の感染多重度で行われる。ある特定の実施形態では、ウイルスを濃縮された標的細胞集団と接触させることは、25:1以上の感染多重度で行われる。ある特定の実施形態では、ウイルスを濃縮された標的細胞集団と接触させることは、50:1以上の感染多重度で行われる。ある特定の実施形態では、濃縮された標的細胞集団を外因性核酸と接触させることは、エレクトロポレーションにより生じる。ある特定の実施形態では、濃縮された標的細胞集団を外因性核酸と接触させることは、細胞圧縮により生じる。ある特定の実施形態では、濃縮された標的細胞集団は造血幹細胞を含む。ある特定の実施形態では、濃縮された標的細胞集団は免疫細胞を含む。ある特定の実施形態では、免疫細胞はCD45+免疫細胞を含む。ある特定の実施形態では、免疫細胞は、Bリンパ球またはTリンパ球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、CD3+Tリンパ球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、CD3+、CD4+Tリンパ球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、CD3+、CD8+Tリンパ球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、CD3+、CD8+Tリンパ球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、CD3+、CD8+、CD4+Tリンパ球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、CD3+、CD8-、CD4-Tリンパ球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、少なくとも約35%のCD3+Tリンパ球を含む細胞の集団を含む。ある特定の実施形態では、濃縮された標的細胞集団は、少なくとも約40%のCD3+Tリンパ球を含む細胞の集団を含む。ある特定の実施形態では、濃縮された標的細胞集団はナチュラルキラー細胞を含む。ある特定の実施形態では、濃縮された標的細胞集団は脂肪由来幹細胞を含む。ある特定の実施形態では、濃縮された標的細胞集団は骨髄由来幹細胞を含む。ある特定の実施形態では、濃縮された標的細胞集団は間葉系幹細胞を含む。ある特定の実施形態では、濃縮された標的細胞集団は、約500:1以下の血小板対標的細胞の比で血小板を含む。ある特定の実施形態では、濃縮された標的細胞集団は、約100:1以下の血小板対標的細胞の比で血小板を含む。ある特定の実施形態では、濃縮された標的細胞集団は、約10:1以下の血小板対標的細胞の比で血小板を含む。ある特定の実施形態では、濃縮された標的細胞集団は、約5:1以下の血小板対標的細胞の比で血小板を含む。ある特定の実施形態では、濃縮された標的細胞集団は、約100:1以上の赤血球対標的細胞の比で赤血球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、約250:1以上の赤血球対標的細胞の比で赤血球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、約500:1以上の赤血球対標的細胞の比で赤血球を含む。ある特定の実施形態では、濃縮された標的細胞集団は、約1000:1以下の赤血球対標的細胞の比で赤血球を含む。ある特定の実施形態では、方法は、濃縮された標的細胞集団を活性化剤と接触させることをさらに含む。ある特定の実施形態では、方法は、所定の直径または所定の密度の細胞成分を1つまたは複数の標的細胞を含む生体試料から除去した後であるが、1つまたは複数の標的細胞を外因性核酸と接触させる前に、濃縮された標的細胞集団を活性化剤と接触させることが生じることを含む。ある特定の実施形態では、活性化剤は、抗CD3抗体、抗CD28抗体、抗CD137抗体、抗CD2抗体、抗CD35抗体、インターロイキン-2、インターロイキン-7、もしくはインターロイキン-15、インターロイキン-21、インターロイキン-6、TGFベータ、CD40リガンド、PMA/イオノマイシン、コンカナバリンA、ポークウィードマイトジェン、またはフィトヘマグルチニンのうちの1つまたは複数を含む。ある特定の実施形態では、活性化剤は、抗CD3抗体、抗CD28抗体、インターロイキン-2、インターロイキン-7、もしくはインターロイキン-15のうちの1つまたは複数を含む。ある特定の実施形態では、遺伝子操作された標的細胞は、1つまたは複数の標的細胞を外因性核酸と接触させた3日後に、密度勾配分離法よりも大きな形質導入効率を示す。ある特定の実施形態では、遺伝子操作され濃縮された標的細胞集団は、1つまたは複数の標的細胞を外因性核酸と接触させた3日後に、少なくとも約50%の形質導入効率を示す。ある特定の実施形態では、遺伝子操作され濃縮された標的細胞集団は、1つまたは複数の標的細胞を外因性核酸と接触させた3日後に、少なくとも約60%の形質導入効率を示す。ある特定の実施形態では、遺伝子操作された標的細胞は、1つまたは複数の標的細胞を外因性核酸と接触させた6日後に、密度勾配分離法よりも大きな形質導入効率を示す。ある特定の実施形態では、遺伝子操作され濃縮された標的細胞集団は、1つまたは複数の標的細胞を外因性核酸と接触させた6日後に、少なくとも約60%の形質導入効率を示す。ある特定の実施形態では、遺伝子操作され濃縮された標的細胞集団は、1つまたは複数の標的細胞を外因性核酸と接触させた6日後に、少なくとも約70%の形質導入効率を示す。ある特定の実施形態では、方法は、濃縮された標的細胞集団を外因性核酸と接触させた後4日目までにまたはそれよりも早期に、遺伝子操作され濃縮された標的細胞集団を収穫することをさらに含む。ある特定の実施形態では、方法は、濃縮された標的細胞集団を外因性核酸と接触させた後5日目までにまたはそれよりも早期に、遺伝子操作され濃縮された標的細胞集団を収穫することをさらに含む。ある特定の実施形態では、方法は、濃縮された標的細胞集団を外因性核酸と接触させた後6日目までにまたはそれよりも早期に、遺伝子操作され濃縮された標的細胞集団を収穫することをさらに含む。ある特定の実施形態では、方法は、濃縮された標的細胞集団を外因性核酸と接触させた後7日目までにまたはそれよりも早期に、遺伝子操作され濃縮された標的細胞集団を収穫することをさらに含む。ある特定の実施形態では、方法は、濃縮された標的細胞集団を外因性核酸と接触させた後8日目までにまたはそれよりも早期に、遺伝子操作され濃縮された標的細胞集団を収穫することをさらに含む。ある特定の実施形態では、細胞は、濃縮された標的細胞集団を外因性核酸と接触させた3日から8日後に収穫される。ある特定の実施形態では、細胞は、濃縮された標的細胞集団を外因性核酸と接触させた3日から7日後に収穫される。ある特定の実施形態では、細胞は、濃縮された標的細胞集団を外因性核酸と接触させた3日から6日後に収穫される。ある特定の実施形態では、細胞は、濃縮された標的細胞集団を外因性核酸と接触させた3日から5日後に収穫される。ある特定の実施形態では、少なくとも1×108個の遺伝子操作された標的細胞が収穫される。ある特定の実施形態では、少なくとも1×107個の遺伝子操作された標的細胞が収穫される。ある特定の実施形態では、生体試料から所定の直径の細胞成分を除去することは、密度勾配媒体の使用を用いない。ある特定の実施形態では、生体試料から所定の直径の細胞成分を除去することは、決定論的横置換法(deterministic lateral displacement)を用いる。
Smart Images
Figure 2026094180000001_ABST
Abstract
Claims
1. A cell population isolated from a sample derived from a target, including cells containing heterologous DNA, compared to a buffy coat cell population isolated from the sample by density gradient centrifugation, a) The number of white blood cells in the cell population is at least twice the number of white blood cells in the buffy coat cell population; b) The number of T cells in the cell population is at least twice the number of T cells in the buffy coat cell population; c) The ratio of red blood cells to T cells in the cell population is less than or equal to one-fifth of the ratio of red blood cells to T cells in the buffy coat cell population; d) The platelet-to-T cell ratio in the cell population is less than or equal to one-fifth of the platelet-to-T cell ratio in the buffy coat cell population; e) The percentage of senescent cells in the cell population is at least 10% less than the percentage of senescent cells in the buffy coat cell population; f) The percentage of exhausted cells in the cell population is at least 10% less than the percentage of exhausted cells in the buffy coat cell population; g) The percentage of T effector memory cells expressing CD45Ra in a cell population is at least 10% less than the percentage of T effector memory cells expressing CD45Ra in a buffy coat cell population; h) The percentage of T central memory cells in the cell population is at least 10% greater than the percentage of T central memory cells in the buffy coat cell population; i) The percentage of T central memory cells or T effector memory cells in the cell population is at least 10% greater than the percentage of T central memory cells or T effector memory cells in the buffy coat cell population; j) The percentage of cells containing heterologous DNA in the cell population is at least 20% greater than the percentage of cells containing heterologous DNA in the buffy coat cell population; the cell population and the buffy coat cell population have been transduced with a viral vector containing heterologous DNA; k) The cell population can grow to contain at least 2 × 10 e⁹ T cells containing heterologous DNA in at least 30% less time than the buffy coat cell population; the cell population and the buffy coat cell population have been transduced with a viral vector containing heterologous DNA; l) The cell population expresses more interferon-gamma than the buffy coat cell population; m) The cell population expresses more GM-CSF than the buffy coat cell population; n) The amount of IL-6 secreted by the cell population is less than that secreted by the buffy coat cell population; o) The amount of MCP-1 secreted by the cell population is less than that of the buffy coat cell population; p) The amount of IL-1Ra secreted by the cell population is less than that secreted by the buffy coat cell population; q) The cell population contains a larger average absolute telomere length than the buffy coat cell population; or r) The cell population contains T cells with a larger average absolute telomere length than T cells purified from the buffy coat cell population. A group of cells.
2. The cell population according to claim 1, wherein the number of white blood cells in the cell population is at least twice the number of white blood cells in the buffy coat cell population.
3. The cell population according to claim 1, wherein the number of T cells in the cell population is at least twice the number of T cells in the buffy coat cell population.
4. The cell population according to claim 1, wherein the ratio of red blood cells to T cells in the cell population is one-fifth or less of the ratio of red blood cells to T cells in the buffy coat cell population.
5. The cell population according to claim 1, wherein the platelet-to-T cell ratio in the cell population is one-fifth or less of the platelet-to-T cell ratio in the buffy coat cell population.
6. The cell population according to claim 1, wherein the percentage of senescent cells in the cell population is at least 10% less than the percentage of senescent cells in the buffy coat cell population.
7. The cell population according to claim 1, wherein the percentage of exhausted cells in the cell population is at least 10% less than the percentage of exhausted cells in the buffy coat cell population.
8. The cell population according to claim 1, wherein the percentage of T effector memory cells expressing CD45Ra in the cell population is at least 10% less than the percentage of T effector memory cells expressing CD45Ra in the buffy coat cell population.
9. The cell population according to claim 1, wherein the percentage of T central memory cells in the cell population is at least 10% greater than the percentage of T central memory cells in the buffy coat cell population.
10. The cell population according to claim 1, wherein the percentage of cells that are T central memory cells or T effector memory cells in the cell population is at least 10% greater than the percentage of cells that are T central memory cells or T effector memory cells in the buffy coat cell population.
11. The cell population according to claim 1, wherein the percentage of cells containing heterologous DNA in the cell population is at least 20% greater than the percentage of cells containing heterologous DNA in the buffy coat cell population; and the cell population and the buffy coat cell population are transduced with a viral vector containing heterologous DNA.
12. The cell population contains heterologous DNA in at least 2 × 10¹⁶ times less than the buffy coat cell population, at least 30% less time. ９ The cell population according to claim 1, which can grow to contain 1 T cell; the cell population and the buffy coat cell population are transduced with a viral vector containing heterologous DNA.
13. The cell population according to claim 1, which expresses more interferon-gamma than the buffy coat cell population.
14. The cell population according to claim 1, which expresses more GM-CSF than the buffy coat cell population.
15. The cell population according to claim 1, wherein the amount of IL-6 secreted by the cell population is less than that of the buffy coat cell population.
16. The cell population according to claim 1, wherein the amount of MCP-1 secreted by the cell population is less than that of the buffy coat cell population.
17. The cell population according to claim 1, wherein the amount of IL-1Ra secreted by the cell population is less than that of the buffy coat cell population.
18. The cell population according to claim 1, comprising a larger mean absolute telomere length than the buffy coat cell population.
19. The cell population according to claim 1, comprising T cells having a larger mean absolute telomere length than T cells purified from a buffy coat cell population.
20. A cell population according to any one of claims 1 to 19, wherein the heterologous DNA includes an inverted terminal repeat sequence or a long terminal repeat sequence.
21. The cell population according to any one of claims 1 to 20, wherein density gradient centrifugation comprises layering the sample onto an aqueous solution containing sodium diatrizoate, disodium calcium EDTA, and a neutral, highly branched, high-mass, hydrophilic polysaccharide [e.g., Ficol] having a density of about 1.078 g / ml.
22. A cell population according to any one of claims 1 to 21, wherein the sample is LeucoPak.
23. The cell population according to any one of claims 1 to 21, wherein the sample is residual leukocytes derived from donated platelets.
24. The cell population according to any one of claims 1 to 21, wherein the sample is a blood sample.
25. The cell population according to claim 24, wherein the blood cell volume of the blood sample is >2%.
26. The cell population according to claim 24, wherein the blood cell volume of the blood sample is >4%.
27. The cell population according to any one of claims 24 to 26, wherein the blood cell volume of the blood sample is <30%.
28. The cell population according to any one of claims 1 to 21, wherein the sample is a leukocyte ferresis or apheresis sample.
29. The cell population according to any one of claims 1 to 21, wherein the sample is a fatty sample or a bone marrow sample.
30. A cell population according to any one of claims 1 to 29, wherein the subject is human.
31. A cell population according to any one of claims 1 to 30, wherein the subject is a healthy individual.
32. A cell population according to any one of claims 1 to 30, wherein the subject has cancer.
33. The cell population according to claim 32, wherein the cancer is leukemia.
34. The cell population according to any one of claims 1 to 33, wherein the viral vector is a lentiviral vector.
35. The cell population according to any one of claims 1 to 33, wherein the viral vector is an adenovirus vector.
36. The cell population according to any one of claims 1 to 33, wherein the viral vector is an adeno-associated virus vector.
37. A cell population according to any one of claims 1 to 36, wherein heterologous DNA encodes CRISPR guide RNA.
38. A cell population according to any one of claims 1 to 36, wherein heterologous DNA encodes siRNA or miRNA.
39. A cell population according to any one of claims 1 to 36, wherein heterologous DNA encodes a polypeptide.
40. The cell population according to claim 39, wherein the polypeptide is a chimeric antigen receptor.
41. The cell population according to claim 40, wherein the chimeric antigen receptor is selected from a list consisting of tisagenlecleucel, axicapbutagensilolucel, brexcapbutagenautolucel, lysocabbutagenmaralucel, idekabutagenbiculucel, and combinations thereof.
42. The cell population according to claim 39, wherein the polypeptide is an immunoglobulin, a T cell receptor, a cytokine, or a chemokine.
43. A cell population according to any one of claims 1 to 42, wherein at least 90% of the cells in the cell population are viable.
44. A method for obtaining a genetically modified leukocyte composition, (a) Concentrating large cell populations from biological samples containing leukocytes without performing density gradient centrifugation; (b) bringing a large group of cells into contact with the activator; and (c) Transduction of a large group of cells using a viral vector containing polynucleotides; A method that includes this.
45. The method according to claim 44, wherein the large cells have a diameter of at least 4 μm.
46. The method according to claim 44, wherein the large cells have a diameter of at least 5 μm.
47. The method according to claim 44, wherein the large cells have a diameter of at least 7 μm.
48. A method for obtaining a genetically modified leukocyte composition, (a) Removing components smaller than a predetermined size from a biological sample containing leukocytes, without performing density gradient centrifugation, to produce a larger population of cells; (b) bringing a large group of cells into contact with the activator; and (c) Transduction of a large group of cells using a viral vector containing polynucleotides; A method that includes this.
49. The method according to claim 48, wherein the predetermined size is 4 μm.
50. The method according to claim 48, wherein the predetermined size is 5 μm.
51. The method according to claim 48, wherein the predetermined size is 7 μm.
52. The method according to any one of claims 44 to 51, wherein the biological sample includes human cells.
53. The method according to any one of claims 44 to 52, wherein the biological sample is LeucoPak.
54. The method according to any one of claims 44 to 52, wherein the biological sample is residual leukocytes derived from donated platelets.
55. The method according to any one of claims 44 to 52, wherein the biological sample is a blood sample.
56. The method according to claim 55, wherein the blood sample has a blood cell volume of >2%.
57. The method according to claim 55, wherein the blood sample has a blood cell volume of >4%.
58. The method according to claim 55, wherein the blood sample has a blood cell volume of <30%.
59. The method according to any one of claims 44 to 52, wherein the biological sample is a leukocyte ferresis or apheresis sample.
60. The method according to any one of claims 44 to 52, wherein the biological sample is a fat sample or a bone marrow sample.
61. The method according to any one of claims 44 to 60, wherein the subject is a human.
62. The method according to any one of claims 44 to 61, wherein the subject is a healthy individual.
63. The method according to any one of claims 44 to 61, wherein the subject has cancer.
64. The method according to claim 63, wherein the cancer is leukemia.
65. The method according to any one of claims 44 to 64, wherein the viral vector is a lentiviral vector.
66. The method according to any one of claims 44 to 64, wherein the viral vector is an adenovirus vector.
67. The method according to any one of claims 44 to 64, wherein the viral vector is an adeno-associated virus vector.
68. The method according to any one of claims 44 to 67, wherein the polynucleotide is heterologous DNA or heterologous RNA.
69. The method according to any one of claims 44 to 67, wherein the polynucleotide encodes a CRISPR guide RNA.
70. The method according to any one of claims 44 to 67, wherein the polynucleotide encodes siRNA or miRNA.
71. The method according to any one of claims 44 to 67, wherein the polynucleotide encodes a polypeptide.
72. The method according to claim 71, wherein the polypeptide is a chimeric antigen receptor.
73. The method according to claim 72, wherein the chimeric antigen receptor is selected from a list consisting of tisagenlecleucel, axicapbutagensilolucel, brexcapbutagenautolucel, lysocabbutagenmaralucel, idekabutagenbiculucel, and combinations thereof.
74. The method according to claim 71, wherein the polypeptide is an immunoglobulin, a T cell receptor, a cytokine, or a chemokine.
75. The method according to any one of claims 44 to 74, wherein at least 90% of the cells of the genetically modified leukocyte composition are viable.
76. The method according to any one of claims 44 to 75, wherein the concentration comprises array-based separation, acoustic foreforetic isolation, or affinity separation.
77. The method according to claim 76, wherein the array-based separation includes a microfluidic device designed for deterministic transverse displacement.
78. The method according to claim 77, wherein the microfluidic device includes a plurality of arrays of obstacles arranged in rows substantially perpendicular to the direction of fluid flow and in columns substantially parallel to the direction of fluid flow, the columns being offset from the direction of fluid flow by an angle of inclination.
79. The method according to claim 78, wherein the device includes at least 50 arrays of obstacles.
80. The method according to claim 79, wherein the device includes at least 50 parallel arrays of obstacles.
81. The method according to claim 78, wherein the plurality of obstacles include at least 50 rows of obstacles.
82. The method according to claim 78 or 79, wherein the multiple obstacles include at least 50 rows of obstacles.
83. The method according to claim 78, wherein the inclination angle is approximately 1 / 100.
84. The method according to any one of claims 77 to 83, wherein each of the multiple obstacles has the shape of a diamond, a circle, an ellipsoid, or a hexagon.
85. The method according to any one of claims 77 to 84, wherein each of the multiple obstacles has a length P1 that is substantially parallel to the direction of fluid flow and is longer than a length P2 that is substantially perpendicular to the direction of fluid flow.
86. The method according to claim 85, wherein each of the multiple obstacles has an elongated hexagonal shape.
87. The method according to any one of claims 77 to 86, wherein P1 is approximately 10 μm to approximately 60 μm and P2 is approximately 10 μm to approximately 30 μm.
88. The method according to any one of claims 77 to 86, wherein P1 is approximately 40 μm and P2 is approximately 20 μm.
89. The method according to any one of claims 77 to 88, wherein P1 is 50% to 150% longer than P2.
90. The method according to any one of claims 77 to 88, wherein obstacles in a row of obstacles are separated by a G1 gap of approximately 22 μm, and obstacles in a row of obstacles are separated by a G2 gap of approximately 17 μm.
91. The method according to any one of claims 77 to 90, wherein the buffer flows continuously through a microfluidic device.
92. The method according to any one of claims 77 to 91, wherein the flow rate in the microfluidic device is at least about 1000 mL per hour.
93. The method according to any one of claims 77 to 92, wherein the microfluidic device functions in an oscillating flow state.
94. The method according to any one of claims 44 to 93, wherein the activator comprises one or more of anti-CD3 antibody, anti-CD28 antibody, interleukin-2, interleukin-7, or interleukin-15.
95. The method according to claim 94, wherein an anti-CD3 antibody or an anti-CD28 antibody is conjugated to a solid support.
96. The method according to claim 95, wherein the solid support is a magnetic bead.
97. The method according to any one of claims 95 to 96, further comprising contacting a large population of cells with an anti-CD3 antibody or anti-CD28 antibody conjugated on a solid support to further enrich the affinity of leukocytes expressing CD3 or CD28.
98. The method according to any one of claims 44 to 97, wherein transduction involves contacting a large population of cells with a viral vector containing polynucleotides at an infection multiplicity of at least 5.
99. The method according to any one of claims 44 to 98, further comprising treating a biological sample with a nuclease prior to (a).
100. The method according to any one of claims 44 to 99, further comprising freezing a large group of cells and thawing a large group of cells.
101. (a) The method according to any one of claims 44 to 100, further comprising culturing a large population of cells.
102. The method according to claim 101, wherein the culturing is performed for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
103. The method according to any one of claims 101 to 102, wherein the incubation period is 15, 10, 9, 8, 7, 6, 5, 4, or 3 days or less.
104. The method according to any one of claims 101 to 103, wherein at least 70% of the T cells express polynucleotides / polypeptides.
105. The method according to claim 104, wherein the percentage of cells expressing the polypeptide is determined by flow cytometry.
106. A genetically modified leukocyte composition containing at least 1 × 10 ９ The method according to any one of claims 101 to 105, comprising 1 T cell.
107. The method according to any one of claims 101 to 106, wherein at least 75% of the T cells in the genetically modified leukocyte composition are T central memory cells or T effector memory cells after 6 days of culture.
108. The method according to any one of claims 101 to 106, wherein at least 85% of the T cells in the genetically modified leukocyte composition are T central memory cells or T effector memory cells after 9 days of culture.
109. The method according to any one of claims 44 to 108, further comprising freezing a genetically modified leukocyte population and thawing a genetically modified leukocyte population.
110. The method according to any one of claims 44 to 109, further comprising administering a genetically modified population of white blood cells to an individual suffering from a tumor or cancer.