JPWO2021123832A5 - - Google Patents

Description

Having thus described the preferred embodiments of the invention in detail, the invention as defined by the foregoing paragraphs may be construed as many obvious variations of the specific details set forth in the foregoing description. It is to be understood that the invention is not to be limited to these specific details as may be possible without departing from the scope.
The present invention provides, for example, the following items.
(Item 1)
1. A method for preparing a therapeutic population of tumor-infiltrating lymphocytes (TIL), comprising:
(a) aseptically disaggregating a tumor removed from a subject, thereby producing a disaggregated tumor product, wherein the tumor is sufficiently disaggregated to permit cryopreservation of the disaggregated tumor product; Agglomerated step;
(b) cooling the disaggregated tumor product to a suitable cryopreservation temperature;
(c) performing a first expansion to produce a first population of TILs by culturing the cryopreserved disaggregated tumor product in a cell culture medium containing IL-2;
(d) performing a second expansion to produce a second population of TILs by culturing said first population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs); a step of; and
(e) collecting and/or cryopreserving said second population of TILs;
including;
said disaggregation comprises enzymatic disaggregation and/or physical disaggregation, said physical disaggregation comprising repeated physical pressure applied to said resected tumor;
A method, wherein steps (a) to (e) are performed in a closed system.
(Item 2)
A method according to item 1, wherein said disaggregation comprises repeated physical pressures applied at a maximum of 6 N/cm ² , more preferably 3 N/cm ² from 120 to 360 times per minute.
(Item 3)
3. The method of any one of items 1 or 2, wherein the disaggregated tumor product comprises a single cell suspension.
(Item 4)
4. The method of any one of items 1 to 3, wherein the resected tumor is not fragmented prior to disaggregation.
(Item 5)
5. A method according to any one of items 1 to 4, wherein step (a) is carried out at a temperature suitable for enzymatic digestion.
(Item 6)
6. The method of any one of items 1 to 5, wherein step (b) comprises cooling the disaggregated tumor product directly to the cryopreservation temperature.
(Item 7)
7. A method according to any one of items 1 to 6, wherein the period of disaggregation is 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less.
(Item 8)
8. The method according to any one of items 1 to 7, wherein the disaggregation continues or proceeds for at least 1 minute.
(Item 9)
9. The method according to any one of items 1 to 8, wherein the tumor is not macerated.
(Item 10)
10. A method according to any one of items 1 to 9, comprising cooling the disaggregated tumor product in a temperature-controlled device that is programmed to reduce the temperature at a constant rate.
(Item 11)
The cryopreservation temperature is -80°C ± 10°C, and the device controls the temperature at 1°C/min or 1.5°C/min or 2°C/min or 1°C/min ± 0.5°C/min or The method of item 10, wherein the method is programmed to reduce by 1°C/min ± 0.5°C/min or 2°C/min ± 0.5°C/min.
(Item 12)
12. The method of any one of items 1-11, wherein the TIL includes a UTIL or the TIL includes an MTIL.
(Item 13)
13. The method of any one of items 1-12, wherein the first population of TILs is about 1,000,000 to 20,000,000 TILs.
(Item 14)
Any one of items 1 to 13, wherein step (c) comprises growing TILs to produce the first population, and the second expansion of step (d) comprises rapid expansion. the method of.
(Item 15)
15. The method of any one of items 1-14, wherein step (c) is performed for about two weeks and step (d) is performed for about two weeks.
(Item 16)
Items 1 to 15, wherein culturing in step (c) and/or step (d) comprises adding IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof. The method described in any one of the above.
(Item 17)
placing the resected tumor tissue into a flexible container containing a disaggregation solution, sealing the container, subjecting the tumor tissue to physical and/or enzymatic disaggregation, and the disaggregated tumor. 17. The method of any one of items 1-16, comprising cryopreserving the tissue.
(Item 18)
18. A therapeutic population of cryopreserved tumor-infiltrating lymphocytes (TIL) obtained by the method according to any one of items 1 to 17.
(Item 19)
19. The therapeutic population according to item 18, comprising about 5×10 ⁹ to 5×10 ¹⁰ T cells.
(Item 20)
A cryopreservation bag for the therapeutic population according to item 18 or 19.
(Item 21)
Cryopreservation bag according to item 20 for use in intravenous infusion.
(Item 22)
1. A method for preparing a therapeutic population of tumor-infiltrating lymphocytes (TIL), comprising:
(a) aseptically disaggregating a tumor removed from a subject, thereby producing a disaggregated tumor product, wherein the tumor is sufficiently disaggregated to permit cryopreservation of the disaggregated tumor product; Agglomerated step;
(b) performing a first expansion to produce a first population of TILs by culturing the cryopreserved disaggregated tumor product in cell culture medium containing IL-2;
(c) performing a second expansion to produce a second population of TILs by culturing said first population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs); a step of; and
(d) collecting and/or cryopreserving said second population of TILs;
including;
said disaggregation comprises enzymatic disaggregation and/or physical disaggregation, said physical disaggregation comprising repeated physical pressure applied to the resected tumor;
A method, wherein steps (a) to (d) are performed in a closed system.
(Item 23)
23. A method according to item 22, wherein said deagglomeration comprises repeated physical pressures applied at a maximum of 6 N/cm ² , more preferably 3 N/cm ² from 120 to 360 times per minute.
(Item 24)
24. The method of any one of items 22 or 23, wherein the disaggregated tumor product comprises a single cell suspension.
(Item 25)
25. The method of any one of items 22 to 24, wherein the resected tumor is not fragmented prior to disaggregation.
(Item 26)
26. A method according to any one of items 22 to 25, wherein step (a) is carried out at a temperature suitable for enzymatic digestion.
(Item 27)
27. The method according to any one of items 22 to 26, wherein the period of disaggregation is 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less.
(Item 28)
28. A method according to any one of items 22 to 27, wherein the disaggregation continues or proceeds for at least 1 minute.
(Item 29)
29. The method of any one of items 22 to 28, wherein the tumor is not macerated.
(Item 30)
30. A method according to any one of items 22 to 29, comprising cooling the disaggregated tumor product in a temperature controlled device programmed to reduce the temperature at a constant rate.
(Item 31)
The cryopreservation temperature is -80°C ± 10°C, and the device controls the temperature at 1°C/min or 1.5°C/min or 2°C/min or 1°C/min ± 0.5°C/min or The method of item 30, wherein the method is programmed to decrease by 1°C/min ± 0.5°C/min or 2°C/min ± 0.5°C/min.
(Item 32)
32. The method of any one of items 22-31, wherein the TIL comprises a UTIL or the TIL comprises an MTIL.
(Item 33)
33. The method of any one of items 22-32, wherein the first population of TILs is about 1,000,000 to 20,000,000 TILs.
(Item 34)
according to any one of items 22 to 33, wherein step (b) comprises growing TILs to produce the first population, and the second expansion of step c) comprises rapid expansion. Method.
(Item 35)
35. The method of any one of items 22-34, wherein step (b) is performed for about two weeks and step (c) is performed for about two weeks.
(Item 36)
Items 22 to 35, wherein culturing in step (b) and/or step (c) comprises adding IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof. The method described in any one of the above.
(Item 37)
placing the resected tumor tissue into a flexible container containing a disaggregation solution, sealing the container, subjecting the tumor tissue to physical and/or enzymatic disaggregation, and the disaggregated tumor. 37. The method of any one of items 22 to 36, comprising cryopreserving the tissue.
(Item 38)
A therapeutic population of cryopreserved tumor-infiltrating lymphocytes (TIL) obtained by the method according to any one of items 22 to 37.
(Item 39)
The therapeutic population according to item 38, comprising about 5 x 10 ⁹ to 5 x 10 ¹⁰ T cells.
(Item 40)
A cryopreservation bag for the therapeutic population according to item 38 or 39.
(Item 41)
Cryopreservation bag according to item 40 for use in intravenous infusion.
(Item 42)
1. A method for preparing a therapeutic population of tumor-infiltrating lymphocytes (TIL) in a closed system, comprising:
(a) (i) cryopreserving the resected tumor and disaggregating the cryopreserved tumor; or
(ii) disaggregating the removed tumor and cryopreserving the disaggregated tumor, or
(iii) cryopreserving the resected tumor and processing the tumor into a plurality of tumor fragments; or
(iv) processing the resected tumor into a plurality of tumor fragments and cryopreserving said tumor fragments;
(b) performing a first expansion to produce a first population of TILs by culturing the cryopreserved disaggregated tumor product in cell culture medium containing IL-2;
(c) performing a second expansion to produce a second population of TILs by culturing said first population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs); a step of; and
(d) collecting and/or cryopreserving said second population of TILs;
method including.
(Item 43)
43. The method of item 42, wherein the disaggregation comprises physical disaggregation, enzymatic disaggregation, or physical and enzymatic disaggregation.
(Item 44)
44. The method of any one of items 42 or 43, wherein the disaggregation comprises repeated physical pressure applied to the removed tumor.
(Item 45)
45. A method according to any one of items 42 to 44, wherein said deagglomeration comprises repeated physical pressures applied at a maximum of 6 N/cm ² , more preferably 3 N/cm ² from 120 to 360 times per minute.
(Item 46)
46. A method according to any one of items 42 to 45, wherein the physical disaggregation comprises grinding and shearing.
(Item 47)
47. The method of any one of items 42-46, wherein the cryopreserved disaggregated tumor comprises a single cell suspension.
(Item 48)
48. A method according to any one of items 42 to 47, wherein the step of disaggregating is performed at a temperature suitable for enzymatic digestion.
(Item 49)
49. The method of any one of items 42-48, wherein cryopreserving comprises directly cooling the resected or disaggregated tumor to a set cryopreservation temperature.
(Item 50)
50. The method of any one of items 42 to 49, comprising cooling the ablated or disaggregated tumor in a temperature controlled device that is programmed to reduce temperature at a constant rate.
(Item 51)
The cryopreservation temperature is -80°C ± 10°C, and the device controls the temperature at 1°C/min or 1.5°C/min or 2°C/min or 1°C/min ± 0.5°C/min or 51. The method of item 50, wherein the method is programmed to decrease by 1°C/min ± 0.5°C/min or 2°C/min ± 0.5°C/min.
(Item 52)
A method for isolating a therapeutic population of TILs from tumor tissue removed from a subject, the method comprising:
(a) placing the resected tumor tissue into an automated device for semi-automated sterile disaggregation of tumor tissue comprising a programmable processor and a disposable sterile kit, the sterile kit comprising a closed system; The sterile kit includes:
a disaggregation module for receiving and processing material including tumor tissue;
an optional enrichment module for filtration of disaggregated solid tissue material and separation of non-disaggregated tissue and filtrate;
a stabilization module for further processing and/or storage of the disaggregated product material as required;
including;
Each of the modules includes one or more flexible containers connected by one or more conduits adapted to allow flow of the tissue material therebetween. Ori,
each of said modules comprising one or more ports for allowing sterile loading of media and/or reagents into said one or more flexible containers;
(b) aseptically disaggregating the removed tumor, thereby producing a disaggregated tumor;
(c) performing a first expansion to produce a first population of UTILs by culturing the disaggregated tumor in a cell culture medium containing IL-2;
(d) performing a second expansion to produce a second population of TILs by culturing said first population of UTILs with additional IL-2, OKT-3, and antigen presenting cells (APCs); a step of; and
(f) collecting and/or cryopreserving the second population of UTILs;
method including.
(Item 53)
33. The method of item 32, wherein said disaggregation comprises repeated physical pressure applied to said removed tumor.
(Item 54)
54. A method according to any one of items 52 or 53, wherein said deagglomeration comprises repeated physical pressures applied at a maximum of 6 N/cm ² , more preferably 3 N/cm 2 from ¹²⁰ to 360 times per minute.
(Item 55)
55. The method of any one of items 52-54, wherein the resected tumor is not fragmented prior to disaggregation.
(Item 56)
56. The method of any one of items 52-55, wherein the tumor tissue is not macerated.
(Item 57)
57. A method according to any one of items 52 to 56, wherein the disaggregation is carried out for 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less.
(Item 58)
58. The method of any one of items 52 to 57, comprising, after step (b), (b') cryopreserving the disaggregated tumor in the stabilization module.
(Item 59)
59. The method of any one of item 58, wherein step (b') comprises cooling the disaggregated tumor product directly to a cryopreservation temperature.
(Item 60)
The cryopreservation temperature is -80°C ± 10°C, and the device controls the temperature at 1°C/min or 1.5°C/min or 2°C/min or 1°C/min ± 0.5°C/min or The method of item 59, wherein the method is programmed to decrease by 1°C/min ± 0.5°C/min or 2°C/min ± 0.5°C/min.
(Item 61)
The automated device comprises:
a sensor capable of recognizing whether the disaggregation process is completed in the disaggregation module prior to transfer of the disagglomeration solid tissue to the optional enrichment module;
determining the amount of medium required within one or more vessels of said disaggregation module, said enrichment module and/or said stabilization module and controlling the transfer of material between the respective vessels; weight sensor;
a sensor for controlling the temperature within the vessel of one or more of the deagglomeration module, the enrichment module and/or the stabilization module;
at least one air bubble sensor for controlling the transfer of medium between the inlet and outlet of each container in the module;
at least one pump, optionally a peristaltic pump, for controlling the transfer of medium between the input port and the output port;
a pressure sensor for assessing the pressure within the enrichment module;
one or more valves for controlling a tangential flow filtration process within said enrichment module; and/or
one or more clamps for controlling the transfer of medium between the inlet and the outlet of each module;
61. The method of any one of items 52-60, further comprising one or more of: in any combination.
(Item 62)
the programmable processor controls the deagglomeration module to enable physical and enzymatic degradation of the solid tissue material; and/or the programmable processor controls the deagglomeration module to enable physical and enzymatic degradation of the solid tissue material; 62. The method of any one of items 52 to 61, wherein the stabilization module is controlled to cryopreserve in the container.
(Item 63)
The disaggregation module repeatedly grinds and shears the tumor tissue at a maximum of 6N/cm ² , more preferably 3N/cm ² from 120 to 360 times per minute, and the stabilization module repeatedly crushes and shears the disaggregation tumor tissue to -80°C ± 10°C, and the device cools the temperature to 1°C/min or 1.5°C/min or 2°C/min or 1°C/min ± 0.5°C/min or 1°C/min. 63. The method of any one of items 52 to 62, wherein the method is programmed to decrease at ±0.5°C/min or 2°C/min ±0.5°C/min.
(Item 64)
A flexible container suitable for use in a closed system for isolating a therapeutic population of TIL, comprising:
To process the tissue,
one or more layers made of a sealable polymer, wherein at least three edges of the flexible container are sealed during manufacture;
an open edge of the flexible container through which tissue material is inserted in use;
one or more fittings configured to connect the flexible container to at least one element by means of a tube;
including;
A flexible container, wherein a section proximate the opening edge is sealed to form a seal after tissue material is placed within the flexible container.
(Item 65)
65. The flexible container of item 64, wherein the seal is formed using a heat sealer operating at a predetermined pressure, a predetermined temperature, and a predetermined time frame.
(Item 66)
66. A flexible container according to any one of items 64 or 65, configured for use in a device for mechanically comminuting tissue material contained in the flexible container.
(Item 67)
Flexible container according to any one of items 64 to 66, suitable for repeated physical pressure applications of up to 6 N/cm ² , more preferably 3 N/cm ² from 120 to 360 times per minute.
(Item 68)
A system for extracting TIL from tumor tissue, the system comprising:
deagglomerated flexible container,
a stabilized flexible container, and
at least one located on at least one of the deagglomerating flexible container or the stabilizing flexible container, which is capable of providing at least one of a source of tissue, a condition of the tissue, or an identifier. two indicator tags
A kit including;
a disagglomerating element capable of treating at least a portion of the tissue within the disagglomerating flexible container to form a treated fluid;
an enrichment element capable of enriching at least a portion of the treated fluid to form a desired material;
a stabilizing element capable of storing a portion of said desired material within said stabilizing flexible container and optionally controlling freezing;
at least one indicator tag reader located on at least one of the disaggregation element or the stabilizing element, capable of providing at least one of a source of tissue or a state of the tissue in the stabilizing element;
system, including.
(Item 69)
A method for treating cancer in a subject, the method comprising:
(a) aseptically disaggregating a tumor removed from said subject, thereby producing a disaggregated tumor product, wherein said tumor is sufficiently disaggregated to permit cryopreservation of said disaggregated tumor product; a step of being disaggregated;
(b) cooling the disaggregated tumor product to a suitable cryopreservation temperature;
(c) performing a first expansion to produce a first population of TILs by culturing the cryopreserved disaggregated tumor product in cell culture medium containing IL-2;
(d) performing a second expansion to produce a second population of TILs by culturing said first population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs); step;
(e) collecting and/or cryopreserving said second population of TILs; and
(f) administering said second population of TILs to said subject.
including;
said disaggregation comprises enzymatic disaggregation and/or physical disaggregation, said physical disaggregation comprising repeated physical pressure applied to the resected tumor;
A method, wherein steps (a) to (e) are performed in a closed system.
(Item 70)
70. A method according to item 69, wherein said deagglomeration comprises repeated physical pressures applied at a maximum of 6 N/cm ² , more preferably 3 N/cm ² from 120 to 360 times per minute.
(Item 71)
71. The method of any one of items 69 or 70, wherein the disaggregated tumor product comprises a single cell suspension.
(Item 72)
72. The method of any one of items 69-71, wherein the resected tumor is not fragmented prior to disaggregation.
(Item 73)
73. A method according to any one of items 69 to 72, wherein step (a) is carried out at a temperature suitable for enzymatic digestion.
(Item 74)
74. The method of any one of items 69-73, wherein step (b) comprises cooling the disaggregated tumor product directly to the cryopreservation temperature.
(Item 75)
75. The method of any one of items 69 to 74, wherein the period of disaggregation is 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less.
(Item 76)
76. A method according to any one of items 69 to 75, wherein the disaggregation continues or proceeds for at least 1 minute.
(Item 77)
77. The method of any one of items 69-76, wherein the tumor is not macerated.
(Item 78)
78. The method of any one of items 69-77, comprising cooling the disaggregated tumor product in a temperature-controlled device that is programmed to reduce temperature at a constant rate.
(Item 79)
The cryopreservation temperature is -80°C ± 10°C, and the device controls the temperature at 1°C/min or 1.5°C/min or 2°C/min or 1°C/min ± 0.5°C/min or 79. The method of item 78, wherein the method is programmed to decrease by 1°C/min ± 0.5°C/min or 2°C/min ± 0.5°C/min.
(Item 80)
79. The method of any one of items 69-79, wherein the TIL includes a UTIL or the TIL includes an MTIL.
(Item 81)
81. The method of any one of items 69-80, wherein the first population of TILs is about 1,000,000 to 20,000,000 TILs.
(Item 82)
82, wherein step (c) comprises growing TILs to produce the first population, and wherein the second expansion of step (d) comprises rapid expansion. the method of.
(Item 83)
83. The method of any one of items 69-82, wherein step (c) is performed for about two weeks and step (d) is performed for about two weeks.
(Item 84)
Any of items 69 to 83, wherein culturing in step (c) and/or step (d) comprises adding IL7, IL-12, IL-15, IL-18, IL-21, or a combination thereof. The method described in paragraph (1).
(Item 85)
placing the resected tumor tissue into a flexible container containing a disaggregation solution, sealing the container, subjecting the tumor tissue to physical and/or enzymatic disaggregation, and the disaggregated tumor. 85. The method of any one of items 69-84, comprising cryopreserving the tissue.
(Item 86)
86. The method of any one of items 69-85, wherein said second population of TILS comprises ^about 5x109 ^to 5x1010 T cells.
(Item 87)
The cancer is bladder cancer, breast cancer, cancer caused by human papillomavirus, cervical cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), lung cancer, melanoma, ovarian cancer, 87. The method according to any one of items 69 to 86, which is non-small cell lung cancer (NSCLC), renal cancer or renal cell carcinoma.

Claims (1)

Invention described in the specification.