A method for inducing and preparing TIL cells

Abstract

The present invention relates to an induction medium and an expansion medium for inducing and amplifying TIL cells from tumor tissues, and a method for inducing and / or amplifying TIL cells. The induction medium comprises one or two selected from IL-7 and IL-15, IL-2, TNF-α, CD3 antibody and CD28 antibody. The expansion medium comprises one or two selected from IL-7 and IL-15, IL-2, TNF-α, CD3 antibody, CD28 antibody and CD137 antibody.

Description

Technical Field

[0001] The present invention relates to a method for rapidly inducing and preparing TIL cells from a tumor tissue sample, and in particular to a culture medium for inducing TIL from tumor tissue and a culture medium for rapidly amplifying TIL, and a use method and purpose thereof. Background Art

[0002] Tumor infiltrating lymphocytes (TIL cells) are a group of lymphocytes that migrate from the blood to the tumor to play a role. This group of cells has a specific killing effect on tumor cells. In 1987, Rosenberg et al. studied the specific killing effect of TIL cells on tumor cells in vitro, and developed a method for culturing TIL cells from a variety of tumor tissues in vitro, providing a new adaptive immunotherapy for tumor treatment.

[0003] Through TIL adaptive immunotherapy, the overall response rate of melanoma patients is between 34% and 70%, which controls the progression of melanoma and improves the survival of tumor patients. However, the amount of TIL cells used in clinical practice reaches 6x10 9 More than one cell and multiple courses of treatment are required, which requires obtaining a large number of TILs in a short period of time.

[0004] The in vitro expansion method of TIL cells developed by Rosenberg is the current common TIL cell culture method. This method uses collagenase to digest tumor tissue, separates infiltrating lymphocytes in tumor tissue, and uses a medium containing 2000-6000IU / ml rhIL-2 for in vitro induction. After inducing TIL cells, they are expanded using a medium containing CD3 and CD28 antibodies for clinical use. However, the in vitro induction of TIL cells using a medium containing 2000-6000IU / ml rhIL-2 has a long cycle, and it takes an average of 50 days to expand to the required amount, and the proportion of CD3+CD8+ killer T cells is low, so the timeliness and effectiveness of clinical use are poor. At the same time, more than 50% of patients cannot expand in vitro to the clinical demand. This leads to a small scope of application of TIL cell therapy, and more than 50% of patients cannot be treated with this therapy. In addition, when the amount of tumor tissue is small or the tumor itself is small, Rosenberg's method is difficult to obtain a sufficient amount of cells (Ref: Tumor-infiltratinglymphocytes for the treatment of metastatic cancer). In order to obtain a sufficient amount of tumor tissue, patients must undergo open surgery. However, this treatment method is often not applicable to patients who do not need surgery and therefore cannot obtain a large amount of tumor tissue.

[0005] By adjusting the factors in the TIL cell in vitro culture program, the cell expansion efficiency can be improved compared to the general TIL in vitro culture method, but the culture cycle is still relatively long, with an induction time of about 21 days and expansion to the required amount taking between 30-40 days.

[0006] This situation places high demands on the in vitro expansion system of TIL cells, and it is necessary to develop new induction systems and expansion systems to increase the expansion multiples of TIL cells and shorten the culture cycle. Summary of the invention

[0007] In view of the current state of the art, the present invention optimizes the tissue processing method and culture system, greatly improves the in vitro expansion multiple of TIL cells, shortens the culture cycle, and expands the scope of clinical use.

[0008] In one aspect, the present invention provides a composition for inducing TIL cells from tumor tissue, comprising one or two selected from IL-7 and IL-15, IL-2, TNF-α, CD3 antibody and CD28 antibody.

[0009] In one or more embodiments, the concentration of IL-2 in the composition for inducing TIL cells is 200-1000 U / ml, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000 U / ml, or a range between any two of the above values.

[0010] Preferably, the concentration of IL-2 in the composition for inducing TIL cells is 200-950, 250-850, 350-750, 450-650, or 500-550 U / ml.

[0011] In one or more embodiments, the concentration of IL-7 in the composition for inducing TIL cells is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values.

[0012] Preferably, the concentration of IL-7 in the composition for inducing TIL cells is 30-450, 40-400, 50-350, 60-300, 70-250, 80-200, 90-150 pg / ml.

[0013] In one or more embodiments, the concentration of IL-15 in the composition for inducing TIL cells is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values.

[0014] Preferably, the concentration of IL-15 in the composition for inducing TIL cells is 30-500, 50-500, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, 450-500 pg / ml.

[0015] In one or more embodiments, the concentration of TNF-α in the composition for inducing TIL cells is 10-90 ng / ml, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, or a range between any two of the above values.

[0016] Preferably, the concentration of TNF-α in the composition for inducing TIL cells is 20-80, 30-70, or 40-60 ng / ml.

[0017] In one or more embodiments, the concentration of the CD3 antibody in the composition for inducing TIL cells is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values.

[0018] Preferably, the concentration of the CD3 antibody in the composition for inducing TIL cells is 30-500, 50-450, 100-400, 150-350, or 200-300 pg / ml.

[0019] In one or more embodiments, the concentration of the CD28 antibody in the composition for inducing TIL cells is 1-1000 ng / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ng / ml, or a range between any two of the above values.

[0020] Preferably, the concentration of the CD28 antibody in the composition for inducing TIL cells is 30-950, 50-850, 150-750, 250-650, 350-550, or 450-500 ng / ml.

[0021] In one or more embodiments, the composition for inducing TIL cells comprises 200-1000 U / ml IL-2, 1-500 pg / ml IL-7, 1-500 ng / ml IL-15, 10-90 ng / ml TNF-α, 1-500 pg / ml CD3 antibody, and 100-1000 ng / ml CD28 antibody.

[0022] In one or more embodiments, the composition for inducing TIL cells comprises

[0023] 400-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 400-600 ng / ml CD28 antibody, or iso-ratio concentrates of these components; or

[0024] 150-250 U / ml IL-2, 1-30 pg / ml IL-7, 1-30 pg / ml IL-15, 40-60 ng / ml TNF-α, 1-30 pg / ml CD3 antibody, 1-30 ng / ml CD28 antibody, or iso-concentrates of these components; or

[0025] 900-1000U / ml IL-2, 450-500pg / ml IL-7, 450-500pg / ml IL-15, 40-60ng / ml TNF-α, 450-500pg / ml CD3 antibody, 900-1000ng / ml CD28 antibody, or iso-concentrates of these components; or

[0026] 400-600 U / ml IL-2, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 450-500 pg / ml CD3 antibody, 900-1000 ng / ml CD28 antibody, or iso-concentrates of these components; or

[0027] 900-1000U / ml IL-2, 450-500pg / ml IL-7, 40-60ng / ml TNF-α, 450-500pg / ml CD3 antibody, 900-1000ng / ml CD28 antibody, or iso-ratio concentrates of these components; or

[0028] 500-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 500-600 ng / ml CD28 antibody, or equal ratio concentrates of these components.

[0029] In one or more embodiments, the composition for inducing TIL cells comprises

[0030] 500U / ml IL-2, 100pg / ml IL-7, 500pg / ml IL-15, 50ng / ml TNF-α, 250pg / ml CD3 antibody, 500ng / ml CD28 antibody, or iso-concentrates of these components; or

[0031] 200U / ml IL-2, 1pg / ml IL-7, 1pg / ml IL-15, 50ng / ml TNF-α, 1pg / ml CD3 antibody, 1ng / ml CD28 antibody, or isomeric concentrates of these components; or

[0032] 1000U / ml IL-2, 500pg / ml IL-7, 500pg / ml IL-15, 50ng / ml TNF-α, 500pg / ml CD3 antibody, 1000ng / ml CD28 antibody, or iso-concentrates of these components; or

[0033] 500U / ml IL-2, 500pg / ml IL-15, 50ng / ml TNF-α, 500pg / ml CD3 antibody, 1000ng / ml CD28 antibody, or iso-concentrates of these components; or

[0034] 1000U / ml IL-2, 500pg / ml IL-7, 50ng / ml TNF-α, 500pg / ml CD3 antibody, 1000ng / ml CD28 antibody, or iso-concentrates of these components; or

[0035] 500U / ml IL-2, 100pg / ml IL-7, 500pg / ml IL-15, 50ng / ml TNF-α, 300pg / ml CD3 antibody, 500ng / ml CD28 antibody, or iso-concentrates of these components.

[0036] In one or more embodiments, the CD3 antibody is an anti-human CD3 antibody or an antigen-binding fragment thereof.

[0037] In one or more embodiments, the CD28 antibody is an anti-human CD28 antibody or an antigen-binding fragment thereof.

[0038] In one or more embodiments, the concentration ratio of CD3 antibody to CD28 antibody is between 3:1 and 1:6, between 2:1 and 1:5, between 1:1 and 1:4, and preferably 1:2.

[0039] In one or more embodiments, the volume of the tumor tissue is 2-40 mm 3 , preferably 5-30mm 3 , more preferably 10-20mm 3 .

[0040] In one or more embodiments, the tumor tissue is solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc.

[0041] The present invention also provides a culture medium for inducing TIL cells from tumor tissue, comprising the composition for inducing TIL cells described herein, a basic culture medium and serum.

[0042] In one or more embodiments, the basal culture medium is selected from the group consisting of X-VIVO, RPMI-1640, and AIM-V.

[0043] In one or more embodiments, the serum is selected from human AB serum, autologous serum of a subject, or serum of animal origin.

[0044] In one or more embodiments, the concentration of serum is 1-10% of the total volume, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the concentration of serum is 1.5-8.5%, 1.5-7.5%, 1.5-6.5%, 1.5-5.5%, 1.5-4.5%, 1.5-3.5%, 1.5-2.5%.

[0045] In one or more embodiments, the volume of the tumor tissue is 2-40 mm 3 , preferably 5-30mm 3 , more preferably 10-20mm 3 .

[0046] In one or more embodiments, the tumor tissue is solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc.

[0047] The present invention also provides a composition for rapidly expanding TIL cells, comprising one or two selected from IL-7 and IL-15, IL-2, TNF-α, CD3 antibody, CD28 antibody and CD137 antibody.

[0048] In one or more embodiments, the concentration of IL-2 in the composition for expanding TIL cells is 200-1000 U / ml, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000 U / ml, or a range between any two of the above values.

[0049] Preferably, the concentration of IL-2 in the composition for expanding TIL cells is 200-950, 250-850, 350-750, 450-650, 500-550 U / ml.

[0050] In one or more embodiments, the concentration of IL-7 in the composition for expanding TIL cells is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values.

[0051] Preferably, the concentration of IL-7 in the composition for expanding TIL cells is 30-450, 40-400, 50-350, 60-300, 70-250, 80-200, 90-150 pg / ml.

[0052] In one or more embodiments, the concentration of IL-15 in the composition for expanding TIL cells is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values.

[0053] Preferably, the concentration of IL-15 in the composition for expanding TIL cells is 30-500, 50-500, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, 450-500 pg / ml.

[0054] In one or more embodiments, the concentration of TNF-α in the composition for expanding TIL cells is 10-90 ng / ml, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, or a range between any two of the above values.

[0055] Preferably, the concentration of TNF-α in the composition for expanding TIL cells is 20-80, 30-70, or 40-60 ng / ml.

[0056] In one or more embodiments, the concentration of the CD3 antibody in the composition for expanding TIL cells is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values.

[0057] Preferably, the concentration of CD3 antibody in the composition for expanding TIL cells is 30-500, 50-450, 100-400, 150-350, 200-300 pg / ml.

[0058] In one or more embodiments, the concentration of CD28 antibody in the composition for expanding TIL cells is 1-1000 ng / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ng / ml, or a range between any two of the above values.

[0059] Preferably, the concentration of CD28 antibody in the composition for expanding TIL cells is 30-950, 50-850, 150-750, 250-650, 350-550, 450-500 ng / ml.

[0060] In one or more embodiments, the concentration of the CD137 antibody in the composition for expanding TIL cells is 100-1100 pg / ml, for example, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 pg / ml, 1100 pg / ml, or a range between any two of the above values.

[0061] Preferably, the concentration of CD137 antibody in the composition for expanding TIL cells is 150-1100, 300-1100, 500-1100, 700-1100, 900-1100, 200-800, 250-750, 300-700, 350-650, 400-600, 450-550 pg / ml.

[0062] In one or more embodiments, the composition for expanding TIL cells comprises 200-1000 U / ml IL-2, 1-500 pg / ml IL-7, 1-500 ng / ml IL-15, 10-90 ng / ml TNF-α, 1-500 pg / ml CD3 antibody, 100-1000 ng / ml CD28 antibody, and 100-1100 pg / ml CD137 antibody.

[0063] In one or more embodiments, the composition for expanding TIL cells comprises:

[0064] 400-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-550 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 400-600 ng / ml CD28 antibody, 400-600 pg / ml CD137 antibody, or iso-concentrates of these components; or

[0065] 400-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-550 pg / ml IL-15, 40-60 ng / ml TNF-α, 1-30 pg / ml CD3 antibody, 1-30 ng / ml CD28 antibody, 100-200 pg / ml CD137 antibody, or isomeric concentrates of these components; or

[0066] 900-1000U / ml IL-2, 450-500pg / ml IL-7, 450-500pg / ml IL-15, 40-60ng / mlTNF-α, 450-500pg / ml CD3 antibody, 900-1000ng / ml CD28 antibody, 900-1000pg / ml CD137 antibody, or iso-concentrates of these components; or

[0067] 900-1000U / ml IL-2, 450-500pg / ml IL-15, 40-60ng / ml TNF-α, 200-300pg / mlCD3 antibody, 900-1000ng / mlCD28 antibody, 900-1000pg / mlCD137 antibody, or iso-ratio concentrates of these components; or

[0068] 900-1000U / ml IL-2, 450-500pg / ml IL-7, 40-60ng / ml TNF-α, 450-500pg / mlCD3 antibody, 900-1000ng / mlCD28 antibody, 900-1000pg / mlCD137 antibody, or iso-concentrates of these components; or

[0069] 400-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 400-600 ng / ml CD28 antibody, 900-1100 pg / ml CD137 antibody, or iso-ratio concentrates of these components; or

[0070] 500-600U / ml IL-2, 90-150pg / ml IL-7, 450-500pg / ml IL-15, 40-60ng / ml TNF-α, 200-300pg / ml CD3 antibody, 500-600ng / ml CD28 antibody, 900-1100pg / ml CD137 antibody, or equal ratio concentrates of these components.

[0071] In one or more embodiments, the composition for expanding TIL cells comprises:

[0072] 500U / ml IL-2, 100pg / ml IL-7, 500pg / ml IL-15, 50ng / ml TNF-α, 250pg / ml CD3 antibody, 500ng / ml CD28 antibody, 500pg / ml CD137 antibody, or iso-concentrates of these components; or

[0073] 500U / ml IL-2, 100pg / ml IL-7, 500pg / ml IL-15, 50ng / ml TNF-α, 1pg / ml CD3 antibody, 1ng / ml CD28 antibody, 100pg / ml CD137 antibody, or concentrates of these components in equal proportions; or

[0074] 1000U / ml IL-2, 500pg / ml IL-7, 500pg / ml IL-15, 50ng / ml TNF-α, 500pg / ml CD3 antibody, 1000ng / ml CD28 antibody, 1000pg / ml CD137 antibody, or concentrates of these components in equal proportions; or

[0075] 1000U / ml IL-2, 500pg / ml IL-15, 50ng / ml TNF-α, 250pg / ml CD3 antibody, 1000ng / ml CD28 antibody, 1000pg / ml CD137 antibody, or concentrates of these components in equal proportions; or

[0076] 1000U / ml IL-2, 500pg / ml IL-7, 50ng / ml TNF-α, 500pg / ml CD3 antibody, 1000ng / ml CD28 antibody, 1000pg / ml CD137 antibody, or concentrates of these components in equal proportions; or

[0077] 500U / ml IL-2, 100pg / ml IL-7, 500pg / ml IL-15, 50ng / ml TNF-α, 250pg / ml CD3 antibody, 500ng / ml CD28 antibody, or iso-concentrates of these components; or

[0078] 500U / ml IL-2, 100pg / ml IL-7, 500pg / ml IL-15, 50ng / ml TNF-α, 250pg / ml CD3 antibody, 500ng / ml CD28 antibody, 1100pg / ml CD137 antibody, or iso-concentrates of these components.

[0079] In one or more embodiments, the CD3 antibody is an anti-human CD3 antibody or an antigen-binding fragment thereof.

[0080] In one or more embodiments, the CD28 antibody is an anti-human CD28 antibody or an antigen-binding fragment thereof.

[0081] In one or more embodiments, the CD137 antibody is an anti-human CD28 antibody or an antigen-binding fragment thereof.

[0082] In one or more embodiments, the concentration ratio of CD3 antibody to CD28 antibody is between 3:1 and 1:6, between 2:1 and 1:5, between 1:1 and 1:4, and preferably 1:2.

[0083] In one or more embodiments, the concentration ratio of CD3 antibody to CD137 antibody is between 3:1 and 1:6, between 2:1 and 1:5, between 1:1 and 1:4, and preferably 1:2.

[0084] A TIL cell rapid expansion medium comprises the composition for expanding TIL cells described herein, a basal medium and serum.

[0085] In one or more embodiments, the basal culture medium is selected from the group consisting of X-VIVO, RPMI-1640, and AIM-V.

[0086] In one or more embodiments, the serum is selected from human AB serum, autologous serum of a subject, or serum of animal origin.

[0087] In one or more embodiments, the concentration of serum is 1-10% of the total volume, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the concentration of serum is 1.5-8.5%, 1.5-7.5%, 1.5-6.5%, 1.5-5.5%, 1.5-4.5%, 1.5-3.5%, 1.5-2.5%.

[0088] The present invention also provides a method for obtaining TIL cells from tumor tissue, comprising the steps of:

[0089] (1) pre-treating tumor tissue; and

[0090] (2) incubating the tumor tissue with a first culture medium to obtain a first TIL cell population; and

[0091] Optionally (3) incubating the first TIL cell population with a second culture medium to obtain a second TIL cell population,

[0092] Wherein, the first culture medium is the culture medium for inducing TIL cells from tumor tissue described herein, and the second culture medium is the TIL cell rapid expansion culture medium described herein.

[0093] In one or more embodiments, the tumor tissue is not treated with an enzyme before incubation in step (2).

[0094] In one or more embodiments, the pretreatment of tumor tissue in step (1) comprises fragmenting and / or dissociating tumor tissue, preferably comprising

[0095] (1.1) Use treatment solution to clean and immerse tumor tissue;

[0096] (1.2) fragmented tumor tissue; and

[0097] Optionally (1.3) wash the tumor tissue using the first culture medium.

[0098] In one or more embodiments, pretreatment does not include the use of enzyme treatment.

[0099] In one or more embodiments, the treatment fluid is enzyme-free.

[0100] In one or more embodiments, the treatment fluid is the basal medium used in the first culture medium.

[0101] In one or more embodiments, the incubation of step (2) lasts for at most 15 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days.

[0102] In one or more embodiments, the incubation in step (2) comprises refreshing the culture medium every other day, for example, refreshing the culture medium on the 3rd, 5th, 7th, 9th, 11th day of incubation.

[0103] In one or more embodiments, the incubation of step (3) lasts for up to 30 days, up to 25 days, up to 20 days, up to 19 days, up to 18 days, up to 17 days, up to 16 days, up to 15 days, up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days.

[0104] In one or more embodiments, the incubation of step (3) comprises refreshing the culture medium every other day, for example, refreshing the culture medium on day 3, 5, 7, 9, 11, 13, 15, or 17 of incubation.

[0105] In one or more embodiments, the volume of the tumor tissue is 2-40 mm 3 , preferably 5-30mm 3 , more preferably 10-20mm 3 .

[0106] In one or more embodiments, the tumor tissue is solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc.

[0107] The present invention also provides a method for expanding TIL cells, comprising incubating the TIL cells using the second culture medium described herein.

[0108] In one or more embodiments, the TIL cells are the first TIL cell population and / or the second TIL cell population described in any embodiment herein.

[0109] The present invention also provides a method for inducing and amplifying TIL cells, comprising the steps of:

[0110] (1) Pretreatment of tumor tissue;

[0111] (2) incubating the tumor tissue with a first culture medium to obtain a first TIL cell population;

[0112] (3) incubating the first TIL cell population with a second culture medium to obtain a second TIL cell population,

[0113] Wherein, the first culture medium is the culture medium for inducing TIL cells from tumor tissue described herein, and the second culture medium is the TIL cell rapid expansion culture medium described herein.

[0114] In one or more embodiments, the tumor tissue is not treated with an enzyme before incubation in step (2).

[0115] In one or more embodiments, the pretreatment of tumor tissue in step (1) comprises fragmenting and / or dissociating tumor tissue, preferably comprising

[0116] (1.1) Use treatment solution to clean and immerse tumor tissue;

[0117] (1.2) fragmented tumor tissue; and

[0118] Optionally (1.3) wash the tumor tissue using the first culture medium.

[0119] In one or more embodiments, pretreatment does not include the use of enzyme treatment.

[0120] In one or more embodiments, the treatment fluid is enzyme-free.

[0121] In one or more embodiments, the treatment fluid is the basal medium used in the first culture medium.

[0122] In one or more embodiments, the incubation of step (2) lasts for at most 15 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days.

[0123] In one or more embodiments, the incubation in step (2) comprises refreshing the culture medium every other day, for example, refreshing the culture medium on the 3rd, 5th, 7th, 9th, 11th day of incubation.

[0124] In one or more embodiments, the incubation of step (3) lasts for up to 30 days, up to 25 days, up to 20 days, up to 19 days, up to 18 days, up to 17 days, up to 16 days, up to 15 days, up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days.

[0125] In one or more embodiments, the incubation of step (3) comprises refreshing the culture medium every other day, for example, refreshing the culture medium on day 3, 5, 7, 9, 11, 13, 15, or 17 of incubation.

[0126] In one or more embodiments, the volume of the tumor tissue is 2-40 mm 3 , preferably 5-30mm 3 , more preferably 10-20mm 3 .

[0127] In one or more embodiments, the tumor tissue is solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc.

[0128] The present invention also provides use of the composition for inducing TIL cells described herein in preparing a culture medium for inducing TIL cells from tumor tissue.

[0129] In one or more embodiments, the volume of the tumor tissue is 2-40 mm 3 , preferably 5-30mm 3 , more preferably 10-20mm 3 .

[0130] In one or more embodiments, the tumor tissue is solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc.

[0131] The present invention also provides the use of the composition for inducing TIL cells described herein and optionally a basal culture medium and / or serum in inducing TIL cells from tumor tissue.

[0132] In one or more embodiments, the basal culture medium is selected from the group consisting of X-VIVO, RPMI-1640, and AIM-V.

[0133] In one or more embodiments, the serum is selected from human AB serum, autologous serum of a subject, or serum of animal origin.

[0134] In one or more embodiments, the concentration of serum is 1-10% of the total volume, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the concentration of serum is 1.5-8.5%, 1.5-7.5%, 1.5-6.5%, 1.5-5.5%, 1.5-4.5%, 1.5-3.5%, 1.5-2.5%.

[0135] In one or more embodiments, the volume of the tumor tissue is 2-40 mm 3 , preferably 5-30mm 3 , more preferably 10-20mm 3 .

[0136] In one or more embodiments, the tumor tissue is solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc.

[0137] The present invention also provides the use of the composition for expanding TIL cells described herein in preparing a culture medium for expanding TIL cells or a pharmaceutical composition containing TIL cells.

[0138] The present invention also provides the composition for expanding TIL cells as described herein and the use of optional basal culture medium and / or serum in expanding TIL cells.

[0139] In one or more embodiments, the basal culture medium is selected from the group consisting of X-VIVO, RPMI-1640, and AIM-V.

[0140] In one or more embodiments, the serum is selected from human AB serum, autologous serum of a subject, or serum of animal origin.

[0141] In one or more embodiments, the concentration of serum is 1-10% of the total volume, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the concentration of serum is 1.5-8.5%, 1.5-7.5%, 1.5-6.5%, 1.5-5.5%, 1.5-4.5%, 1.5-3.5%, 1.5-2.5%.

[0142] The present invention also provides the use of TIL cells obtained by any method of the present invention in the preparation of drugs for treating cancer.

[0143] Advantages of the invention:

[0144] The present invention solves the technical problems of low in vitro expansion multiples of TIL cells, long culture period and low CD3+CD8+ cell ratio. Using the currently common TIL in vitro culture technology, more than 60% of TIL cells have low in vitro expansion multiples, which cannot meet the current clinical treatment needs, and the CD3+CD8+ cell ratio is low, and the clinical effect is poor. The technical scheme of the present invention relates to a method for processing tumor tissue and preparing an in vitro culture medium for TIL cells, which can significantly increase the in vitro expansion multiples of TIL cells, shorten the in vitro expansion time, and significantly increase the CD3+CD8+ cell ratio. In some embodiments, the CD3+CD8+ cell ratio is as high as 80%, which can effectively increase the scope of use of TIL treatment methods and improve clinical effects.

[0145] The present invention can shorten the induction time to about 7 days (more than 2*10 6 The cells can grow and expand to the clinically required number within 25-30 days, which greatly shortens the in vitro expansion cycle of TIL cells and increases the timeliness of therapy. At the same time, a higher proportion of CD3+CD8+ cells can be obtained, thereby improving clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1 The figure shows the comparison of TIL cell expansion folds between the method of the present invention and the conventional TIL culture method.

[0147] Figure 2 The cell expansion data of the method of the present invention and the conventional TIL culture method for 15-27 days are compared.

[0148] Figure 3 The data of cell expansion at 15-27 days are compared between the method of the present invention and the existing TIL culture method.

[0149] Figure 4 The ratio of CD3+CD4+ and CD3+CD8+ cells in TIL cells cultured by the method of the present invention is shown. DETAILED DESCRIPTION

[0150] In order to increase the timeliness of TIL therapy and shorten the in vitro culture time of TIL cells, the inventors have developed an induction medium and an expansion medium for inducing and amplifying TIL cells from tumor tissues, as well as a method for inducing and / or amplifying TIL cells. Using these culture media to treat TIL cells does not require enzyme treatment of tumor tissues, shortens the induction time to about 7 days, and can be expanded to the number required for clinical use within 30 days, and can significantly increase the in vitro expansion multiples of TIL cells and increase the proportion of CD3+CD8+ cells by up to 80%.

[0151] The present invention is suitable for isolating TIL from any tumor tissue, and is particularly suitable for isolating TIL from small-volume tumor tissue (e.g., a small amount of tumor tissue obtained by minimally invasive surgery). The volume of the tumor tissue is 2-40 mm 3 , preferably 5-30mm 3 , more preferably 10-20mm 3 Exemplary tumor tissues include solid tumor tissues such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc.

[0152] The present invention provides a composition and culture medium for inducing TIL cells. The induction composition comprises one or two selected from IL-7 and IL-15, IL-2, TNF-α, CD3 antibody and CD28 antibody. The induction culture medium comprises one or two selected from IL-7 and IL-15, IL-2, TNF-α, CD3 antibody, CD28 antibody, basal culture medium and serum. The induction culture medium may contain either IL-7 or IL-15, or may not contain IL-7 or IL-15.

[0153] In the composition or culture medium for inducing TIL cells, the concentration of IL-2 is 200-1000 U / ml, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000 U / ml, or a range between any two of the above values; the concentration of IL-7 is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values; the concentration of IL-15 is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values; the concentration of TNF-α is 10-90 ng / ml, for example 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, or a range between any two of the above values; the concentration of CD3 antibody is 1-500 pg / ml, for example 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values; the concentration of CD28 antibody is 1-1000 ng / ml, for example 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ng / ml, or a range between any two of the above values.

[0154] The present invention provides a composition and culture medium for amplifying TIL cells. The induction composition comprises one or two selected from IL-7 and IL-15, IL-2, TNF-α, CD3 antibody, CD28 antibody and CD137 antibody. The amplification culture medium comprises one or two selected from IL-7 and IL-15, IL-2, TNF-α, CD3 antibody, CD28 antibody, CD137 antibody, basal culture medium and serum. The amplification culture medium may contain either IL-7 and IL-15 or may not contain IL-7 and IL-15.

[0155] In the composition or culture medium for expanding TIL cells, the concentration of IL-2 is 200-1000 U / ml, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000 U / ml, or a range between any two of the above values; the concentration of IL-7 is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values; the concentration of IL-15 is 1-500 pg / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values; the concentration of TNF-α is 10-90 ng / ml, for example 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, or a range between any two of the above values; the concentration of CD3 antibody is 1-500 pg / ml, for example 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg / ml, or a range between any two of the above values; the concentration of the CD28 antibody is 1-1000 ng / ml, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ng / ml, or a range between any two of the above values; the concentration of the CD137 antibody is 100-1100 pg / ml, for example, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100 pg / ml, or a range between any two of the above values.

[0156] The CD3 antibody, CD28 antibody, and CD137 antibody described herein can be corresponding antibodies or recombinant antibodies from any species. These antibodies or variants thereof are well known in the art. In the above-mentioned induction composition or culture medium, the concentration ratio of CD3 antibody to CD28 antibody is between 3:1 and 1:6, between 2:1 and 1:5, and between 1:1 and 1:4. In the above-mentioned amplification composition or culture medium, the concentration ratio of CD3 antibody to CD28 antibody is between 3:1 and 1:6, between 2:1 and 1:5, and between 1:1 and 1:4; the concentration ratio of CD3 antibody to CD137 antibody is between 3:1 and 1:6, between 2:1 and 1:5, and between 1:1 and 1:4.

[0157] Serum described herein can be any serum known in the art that can be used for animal cell (e.g., TIL cell) culture, such as human AB serum, subject autologous serum or animal-derived serum. The concentration of serum is 1-10% of the total volume, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or the range between any two of the above values.

[0158] The "basal medium" described herein is any medium known in the art that can be used for TIL cell culture, including but not limited to AIM-V, RPMI-1640, and X-VIVO. The components and contents of these culture media are well known in the art. Exemplarily, AIM-V culture medium contains L-glutamine, streptomycin sulfate and gentamicin sulfate; RPMI-1640 culture medium contains various amino acids, vitamins, inorganic salts and other ingredients required for cell growth; X-VIVO culture medium contains L-glutamine and gentamicin sulfate. The preferred embodiment uses AIM-V culture medium as the basal culture medium.

[0159] The present invention also provides a method for obtaining TIL cells from tumor tissue using the above-mentioned induction and / or expansion culture medium, comprising the steps of: (1) pre-treating tumor tissue; and (2) incubating tumor tissue with a first culture medium to obtain a first TIL cell population; and optionally (3) incubating the first TIL cell population with a second culture medium to obtain a second TIL cell population, wherein the first culture medium is a culture medium for inducing TIL cells from tumor tissue as described herein, and the second culture medium is a TIL cell rapid expansion culture medium as described herein. In the method described herein, the tumor tissue is not treated with an enzyme before incubation in step (2). Therefore, the pretreatment does not include the use of an enzyme treatment. In addition to not using an enzyme treatment, the pretreatment includes any one or more treatment methods for tumor tissue in the art, as long as the treatment method can be used for subsequent TIL cell induction. Exemplarily, pre-treating tumor tissue includes fragmenting and / or dissociating tumor tissue, preferably including (1.1) washing and immersing tumor tissue with a treatment solution; (1.2) fragmenting tumor tissue; and optionally (1.3) washing tumor tissue with a first culture medium. The treatment solution does not contain an enzyme. The treatment liquid can be the basal medium used in the above-mentioned first culture medium, such as AIM-V. The present invention also provides a method for amplifying TIL cells alone, comprising incubating TIL cells with the second culture medium described herein, wherein the TIL cells are the first TIL cell population and / or the second TIL cell population described in any embodiment of the present invention. The TIL cells obtained by any method of the present invention can be used to treat cancer or to prepare a drug for treating cancer, such as thyroid cancer, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc.

[0160] Herein, the time for inducing TIL cells (i.e., incubation in step (2)) is very short, lasting up to 15 days, up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days. Typically, the culture medium needs to be renewed during the induction incubation.

[0161] Herein, the time of amplifying TIL cells (incubation of step (3)) lasts for at most 30 days, at most 25 days, at most 20 days, at most 19 days, at most 18 days, at most 17 days, at most 16 days, at most 15 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days. Typically, the culture medium also needs to be updated during the amplification incubation period.

[0162] The present invention also provides the use of the composition for inducing TIL cells described herein in the preparation of a culture medium for inducing TIL cells from tumor tissue, and the use of the composition for inducing TIL cells described herein and optional basal culture medium and / or serum in inducing TIL cells from tumor tissue. The present invention also provides the use of the composition for amplifying TIL cells described herein in the preparation of a culture medium for amplifying TIL cells or a pharmaceutical composition containing TIL cells, and the use of the composition for amplifying TIL cells described herein and optional basal culture medium and / or serum in amplifying TIL cells. The basal culture medium and serum are as described elsewhere herein.

[0163] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, materials and methods conventional in the art.

[0164] Example

[0165] Example 1

[0166] The difference between the experimental group and the control group is that the experimental group used two culture media, one was a rapid induction TIL cell culture medium: culture medium A; the other was a TIL cell rapid expansion culture medium: culture medium B; the control group used TIL universal culture medium.

[0167] Experimental Materials

[0168] The tumor tissue was obtained from the resected tumor tissue of a female patient with gallbladder cancer, and its volume was 10 mm 3 ; AIM-V culture medium was purchased from Gibco; gentamicin sulfate and interleukin-2 (IL-2) were purchased from Shandong Quangang Pharmaceutical Co., Ltd.; TNF-α was purchased from Jinan Protein Technology Co., Ltd.; interleukin-7 (IL-7) was purchased from Chimerigen; interleukin-15 (IL-15) was purchased from Primegene; CD3 antibody (catalog number: 10981-MM08) was purchased from Sino Biological Technology Co., Ltd., CD28 antibody (catalog number: 11524-H001) was purchased from Sino Biological Technology Co., Ltd.; CD137 antibody (catalog number: 10041-T16) was purchased from Sino Biological Technology Co., Ltd.

[0169] Experimental procedures

[0170] 1. Solution Preparation

[0171] (1) Treatment solution: 50 ml of AIM-V medium plus 12 mg of 2 million IU / ml gentamicin sulfate to prepare 100 ml;

[0172] (2) Medium A: AIM-V medium supplemented with 500 U / ml IL-2, 100 pg / ml IL-7, 500 pg / ml IL-15, 50 ng / ml TNF-α, 250 pg / ml CD3 antibody, 500 ng / ml CD28 antibody, and 2% human AB serum;

[0173] (3) Medium B: AIM-V medium supplemented with 500 U / ml IL-2, 100 pg / ml IL-7, 500 pg / ml IL-15, 50 ng / ml TNF-α, 250 pg / ml CD3 antibody, 500 ng / ml CD28 antibody, 500 pg / ml CD137 antibody, and 2% human AB serum;

[0174] (4) TIL universal culture medium: Add 3000 U / ml IL-2 to AIM-V culture medium.

[0175] 2. Tissue Processing

[0176] (1) Place the tumor tissue sample in a 50 ml centrifuge tube and wash it three times with treatment solution, using 30 ml of treatment solution each time;

[0177] (2) After washing, the tumor tissue sample was transferred to a 100 mm dish and the treatment solution was added to immerse the tissue specimen;

[0178] (3) Use sterile scissors and forceps to carefully remove fat and blood clots on the tumor tissue. After removal, use scissors to cut the tumor tissue into 2-4 mm 3 Size of tissue blocks;

[0179] (4) After the tumor tissue is sheared, the treatment solution is removed by aspiration and the tissue is washed once with culture medium A.

[0180] 3. Experimental Grouping

[0181] (1) Place the processed tissue blocks in a six-well plate and add 6 tissue blocks. Add 3 ml of culture medium A;

[0182] (2) Place the processed tissue blocks in a six-well plate and add 6 tissues. Add 3 ml of TIL universal culture medium. This group is the control group.

[0183] (3) The two groups of cells treated as above were cultured in a 37°C, 5% CO2 incubator and recorded as day 0.

[0184] 4. TIL culture

[0185] (1) On day 5, take the cells out of the incubator and observe them under a microscope to see if any cells have grown. Use a pipette to take the culture medium and centrifuge. For the cells obtained by centrifugation, resuspend the cells in the experimental group with culture medium B and transfer them to a new six-well plate, and resuspend the cells in the control group with universal culture medium. Add the corresponding culture medium A and universal culture medium to the original six-well plate. After treatment, place the plate in a 37°C, 5% CO2 incubator for culture.

[0186] (2) Day 7 and day 9 follow the same procedure as (1). After resuspending, use a K2 counter to count the samples.

[0187] (3) On day 11, the culture medium was aspirated and centrifuged, and the precipitate was transferred to a culture bottle using culture medium B. The control group was treated in the same manner. After treatment, the cells were placed in a 37°C, 5% CO2 incubator for culture. The six-well plate containing the tumor tissue was discarded.

[0188] (4) On day 13, the TIL cells without tissue were taken out and observed under a microscope. The cells were evenly blown with a pipette and transferred to a 50 ml centrifuge tube for sampling and counting. Then, they were centrifuged at 1500 rpm for 5 min. The experimental group was resuspended in medium B to 1×10 6 cells / ml, and transferred to T75 culture flasks; the control group was resuspended in TIL universal medium to 1*10 6 cells / ml and transferred to corresponding culture containers.

[0189] (5) Day 15 and day 17 operations are the same as (4), and the cell proliferation multiples are calculated based on the first counting result and the day 17 counting result.

[0190] (6) Cells were cultured until day 27 and samples were taken and counted every other day.

[0191] Example 2

[0192] 1. Solution preparation

[0193] (1) Culture medium A: AIM-V culture medium supplemented with 200 U / ml IL-2, 1 pg / ml IL-7, 1 pg / ml IL-15, 50 ng / ml TNF-α, 1 pg / ml CD3 antibody, 1 ng / ml CD28 antibody, and 2% human AB serum;

[0194] (2) Medium B: AIM-V medium supplemented with 500 U / ml IL-2, 100 pg / ml IL-7, 500 pg / ml IL-15, 50 ng / ml TNF-α, 1 pg / ml CD3 antibody, 1 ng / ml CD28 antibody, 100 pg / ml CD137 antibody, and 2% human AB serum;

[0195] 2. The experimental steps are the same as those in Example 1, and the cells are cultured until day 17.

[0196] Example 3

[0197] 1. Solution preparation

[0198] (1) Medium A: AIM-V medium supplemented with 1000 U / ml IL-2, 500 pg / ml IL-7, 500 pg / ml IL-15, 50 ng / ml TNF-α, 500 pg / ml CD3 antibody, 1000 ng / ml CD28 antibody, and 2% human AB serum;

[0199] (2) Medium B: AIM-V medium supplemented with 1000 U / ml IL-2, 500 pg / ml IL-7, 500 pg / ml IL-15, 50 ng / ml TNF-α, 500 pg / ml CD3 antibody, 1000 ng / ml CD28 antibody, 1000 pg / ml CD137 antibody, and 2% human AB serum;

[0200] 2. The experimental steps are the same as those in Example 1, and the cells are cultured until day 17.

[0201] Example 4

[0202] 1. Solution preparation

[0203] (1) Medium A: AIM-V medium supplemented with 500 U / ml IL-2, 500 pg / ml IL-15, 50 ng / ml TNF-α, 500 pg / ml CD3 antibody, 1000 ng / ml CD28 antibody, and 2% human AB serum;

[0204] (2) Medium B: AIM-V medium supplemented with 1000 U / ml IL-2, 500 pg / ml IL-15, 50 ng / ml TNF-α, 250 pg / ml CD3 antibody, 1000 ng / ml CD28 antibody, 1000 pg / ml CD137 antibody, and 2% human AB serum;

[0205] 2. The experimental steps are the same as those in Example 1, and the cells are cultured until day 17.

[0206] Example 5

[0207] 1. Solution preparation

[0208] (1) Medium A: AIM-V medium supplemented with 1000 U / ml IL-2, 500 pg / ml IL-7, 50 ng / ml TNF-α, 500 pg / ml CD3 antibody, 1000 ng / ml CD28 antibody, and 2% human AB serum;

[0209] (2) Medium B: AIM-V medium supplemented with 1000 U / ml IL-2, 500 pg / ml IL-7, 50 ng / ml TNF-α, 500 pg / ml CD3 antibody, 1000 ng / ml CD28 antibody, 1000 pg / ml CD137 antibody, and 2% human AB serum;

[0210] 2. The experimental steps are the same as those in Example 1, and the cells are cultured until day 17.

[0211] Example 6

[0212] 1. Solution preparation

[0213] Medium A was the same as medium B: 500 U / ml IL-2, 100 pg / ml IL-7, 500 pg / ml IL-15, 50 ng / ml TNF-α, 250 pg / ml CD3 antibody, 500 ng / ml CD28 antibody, and 2% human AB serum were added to AIM-V medium;

[0214] 2. The experimental steps were the same as those in Example 1. The cells were cultured until day 27 and samples were taken for flow cytometry analysis.

[0215] Example 7

[0216] According to the embodiment of the patent CN110462027A, the tumor treatment method and culture method in the patent are adopted, and the culture is carried out until the 27th day, and the samples are counted every 1 day after the 15th day. The first cell culture medium is AIM-V containing IL-2 concentration of 3000IU / ml, IL-15 concentration of 10ng / ml, and IL21 concentration of 5ng / ml; the second cell culture medium is AIM-V containing IL-2 concentration of 3000IU / ml, IL-15 concentration of 10ng / ml, IL21 concentration of 5ng / ml, CD3 antibody concentration of 30ng / ml, and 41BB antibody of 30ug / ml.

[0217] Example 8

[0218] 1. Solution preparation

[0219] (1) Medium A: AIM-V medium supplemented with 680 U / ml IL-2, 500 pg / ml IL-7, 50 ng / ml TNF-α, 40 pg / ml CD3 antibody, 1 ng / ml CD28 antibody, and 2% human AB serum;

[0220] (2) Medium B: AIM-V medium supplemented with 680 U / ml IL-2, 500 pg / ml IL-7, 50 ng / ml TNF-α, 40 pg / ml CD3 antibody, 1 ng / ml CD28 antibody, 900 pg / ml CD137 antibody, and 2% human AB serum;

[0221] 2. The experimental steps are the same as those in Example 1, and the cells are cultured until day 17.

[0222] Example 9

[0223] 1. Solution preparation

[0224] (1) Medium A: AIM-V medium supplemented with 680 U / ml IL-2, 20 pg / ml IL-7, 20 pg / ml IL-15, 50 ng / ml TNF-α, 40 pg / ml CD3 antibody, 1 ng / ml CD28 antibody, and 2% human AB serum;

[0225] (2) Medium B: AIM-V medium supplemented with 680 U / ml IL-2, 20 pg / ml IL-7, 20 pg / ml IL-15, 50 ng / ml TNF-α, 40 pg / ml CD3 antibody, 1 ng / ml CD28 antibody, 900 pg / ml CD137 antibody, and 2% human AB serum;

[0226] 2. The experimental steps are the same as those in Example 1, and the cells are cultured until day 17.

[0227] Example 10

[0228] TIL cell proliferation

[0229] According to the counting results of day5 and day17, the TIL cell expansion multiple (day17 / day5) was plotted. Figure 1 As shown. Figure 1 It can be seen that when the method of the present invention is used to culture TIL cells, the expansion multiple of TIL cells is significantly higher than that of the conventional TIL culture method, indicating that the method of the present invention can significantly improve the proliferation ability of TIL cells.

[0230] Embodiment 11

[0231] TIL cell expansion curve

[0232] The TIL cell expansion curve was drawn based on the cell density of the experimental group and the control group in Example 1 and the cell density from day 15 to day 27 in Examples 6 and 7. Figure 2 , 3 The amplification curve shows that the factor combination used in the present invention can significantly improve the TIL cell amplification efficiency.

[0233] Example 12

[0234] TIL phenotype identification

[0235] The TIL cells were cultured according to the preparation methods of Examples 1 and 6, and flow cytometry was used to analyze the CD3+CD4+ and CD3+CD8+ populations of TIL cells using APC Mouse Anti-human CD3 (purchased from Biolegend), FITC anti-human CD4 (purchased from BD), and BV510anti-human CD8a (purchased from Biolegend). The results are as follows: Figure 4 In Example 1 of the culture system of the present invention, the CD3+CD8+ population TIL cells reached 79.46%, while in Example 6, the CD3+CD8+ population was only 32.67%. Therefore, the present invention can significantly increase the proportion of CD3+CD8+ cells in TIL cells.

Claims

1. A TIL cell rapid expansion medium, comprising a composition for expanding TIL cells, a basal medium and serum, wherein the composition is composed of any of the following: (1) 400-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-550 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 400-600 ng / ml CD28 antibody, 400-600 pg / ml CD137 antibody; or (2) 400-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-550 pg / ml IL-15, 40-60 ng / ml TNF-α, 1-30 pg / ml CD3 antibody, 1-30 ng / ml CD28 antibody, 100-200 pg / ml CD137 antibody; or (3) 900-1000 U / ml IL-2, 450-500 pg / ml IL-7, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 450-500 pg / ml CD3 antibody, 900-1000 ng / ml CD28 antibody, 900-1000 pg / ml CD137 antibody; or (4) 900-1000 U / ml IL-2, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 900-1000 ng / ml CD28 antibody, 900-1000 pg / ml CD137 antibody; or (5) 900-1000 U / ml IL-2, 450-500 pg / ml IL-7, 40-60 ng / ml TNF-α, 450-500 pg / ml CD3 antibody, 900-1000 ng / ml CD28 antibody, 900-1000 pg / ml CD137 antibody; or (6) 400-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 400-600 ng / ml CD28 antibody, 900-1100 pg / ml CD137 antibody; or (7) 500-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 500-600 ng / ml CD28 antibody, 900-1100 pg / ml CD137 antibody.

2. The culture medium according to claim 1, characterized in that The basal culture medium was selected from X-VIVO, RPMI-1640 and AIM-V.

3. The culture medium according to claim 1, characterized in that The concentration of serum is 1-10% of the total volume.

4. A method for obtaining TIL cells from tumor tissue, comprising the steps of: (1) pre-treatment of tumor tissue; and (2) incubating the tumor tissue with a first culture medium to obtain a first TIL cell population; and (3) incubating the first TIL cell population with a second culture medium to obtain a second TIL cell population, in, The first culture medium comprises a composition for inducing TIL cells, a basal culture medium and serum, and the second culture medium is a TIL cell rapid expansion culture medium according to any one of claims 1 to 3, Wherein, the composition for inducing TIL cells is composed of any of the following composition: (a) 400-600 U / ml IL-2, 90-150 pg / ml IL-7, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300 pg / ml CD3 antibody, 400-600 ng / ml CD28 antibody; or (b) 150-250 U / ml IL-2, 1-30 pg / ml IL-7, 1-30 pg / ml IL-15, 40-60 ng / ml TNF-α, 1-30 pg / ml CD3 antibody, 1-30 ng / ml CD28 antibody; or (c) 900-1000 U / ml IL-2, 450-500 pg / ml IL-7, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 450-500 pg / ml CD3 antibody, 900-1000 ng / ml CD28 antibody; or (d) 400-600 U / ml IL-2, 450-500 pg / ml IL-15, 40-60 ng / ml TNF-α, 450-500 pg / ml CD3 antibody, 900-1000 ng / ml CD28 antibody; or (e) 900-1000 U / ml IL-2, 450-500 pg / ml IL-7, 40-60 ng / ml TNF-α, 450-500 pg / ml CD3 antibody, 900-1000 ng / ml CD28 antibody; or (f) 500-600 U / ml IL-2, 90-150pg / ml IL-7, 450-500pg / ml IL-15, 40-60 ng / ml TNF-α, 200-300pg / ml CD3 antibody, 500-600ng / ml CD28 antibody.

5. The method according to claim 4, characterized in that The basal medium in the first culture medium is selected from X-VIVO, RPMI-1640 and AIM-V.

6. The method according to claim 4, characterized in that The concentration of serum in the first culture medium is 1-10% of the total volume.

7. The method according to any one of claims 4 to 6, characterized in that The method also has one or more features selected from the following: The pretreatment in step (1) does not include enzyme treatment; The pretreatment of the tumor tissue in step (1) includes fragmenting and / or dissociating the tumor tissue; The tumor tissue is not treated with enzymes before incubation in step (2); The incubation in step (2) lasts for up to 15 days; The incubation in step (3) lasts for up to 30 days; The volume of the tumor tissue is 2-40mm 3 ; Tumor tissue is solid tumor tumor tissue.

8. Use of the TIL cell rapid expansion medium according to any one of claims 1 to 3 in the preparation of a pharmaceutical composition containing TIL cells.

9. Use of the TIL cell rapid expansion culture medium described in any one of claims 1-3 in expanding TIL cells.

Images