CN114763530A - Method for inducing and preparing TIL cells - Google Patents

Method for inducing and preparing TIL cells Download PDF

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CN114763530A
CN114763530A CN202110054608.2A CN202110054608A CN114763530A CN 114763530 A CN114763530 A CN 114763530A CN 202110054608 A CN202110054608 A CN 202110054608A CN 114763530 A CN114763530 A CN 114763530A
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antibody
concentration
tumor tissue
til
tnf
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孙艳
韩枫
李君�
程静波
刘辉
钱其军
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Shanghai Cell Therapy Group Co Ltd
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Abstract

The present invention relates to an induction medium and an expansion medium for inducing and expanding TIL cells from tumor tissue and a method for inducing and/or expanding TIL cells. The induction medium comprises one or both of IL-7 and IL-15, IL-2, TNF- α, CD3 antibodies, and CD28 antibodies. The amplification medium comprises one or both of IL-7 and IL-15, IL-2, TNF- α, a CD3 antibody, a CD28 antibody, and a CD137 antibody.

Description

Method for inducing and preparing TIL cell
Technical Field
The invention relates to a method for rapidly inducing and preparing TIL cells from a tumor tissue sample, 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 application thereof.
Background
Tumor infiltrating lymphocytes (TIL cells) are a group of lymphocytes that have a role in migrating from the blood to the tumor site, and this group of cells has a specific killing effect on tumor cells. Rosenberg et al, 1987, studied the specific killing effect of TIL cells on tumor cells in vitro, and developed in vitro culture methods of TIL cells derived from various tumor tissues, providing a new adaptive immunotherapy for tumor treatment.
Through TIL adaptive immunotherapy, the overall response rate of melanoma patients is between 34% and 70%, the progression time of melanoma diseases is controlled, and the survival time of tumor patients is improved. However, the amount of cells used clinically in TIL reaches 6x109Cells and multiple courses of treatment are required, which requires large amounts of TIL to be obtained in a short period of time.
The TIL cell in vitro expansion method developed by Rosenberg is the current general TIL cell culture method. The method uses collagenase to digest the tumor tissue, separates out the infiltrating lymphocytes in the tumor tissue, uses rhIL-2 culture medium containing 2000-6000IU/ml to induce in vitro, uses culture medium containing CD3 and CD28 to amplify after inducing TIL cells, and carries out clinical use. However, the use of rhIL-2 medium containing 2000-6000IU/ml in vitro induced TIL cell cycle is longer, the expansion to the required amount takes 50 days on average, and the proportion of CD3+ CD8+ killer T cells is lower, the timeliness and the effectiveness of clinical use are poorer, and more than 50% of patients can not expand to the required amount in vitro. This results in a small range of applicability of TIL cell therapy, and over 50% of patients cannot be treated with this therapy. Furthermore, when the amount of Tumor tissue is small or the Tumor itself is small, it is difficult to obtain a sufficient amount of cells by the Rosenberg method (Ref: Tumor-encapsulating lymphocytes for the treatment of metabolic cancer). In order to obtain a sufficient amount of tumor tissue, the patient must undergo open surgery. For patients who do not have surgery and therefore cannot obtain a large amount of tumor tissue, the treatment method is not applicable.
Compared with the general TIL in-vitro culture method, the method can improve the cell amplification efficiency by adjusting factors in the TIL cell in-vitro culture scheme, but the culture period is still longer, the induction time is about 21 days, and the amplification to the required amount needs 30-40 days.
This situation puts higher demands on the in vitro amplification system of TIL cells, and needs to develop a new induction system and an amplification system to improve the amplification multiple of TIL cells and shorten the culture period.
Disclosure of Invention
Aiming at the current situation of the prior art, the in-vitro amplification multiple of the TIL cells is greatly improved, the culture period is shortened, and the clinical application range is enlarged by optimizing a tissue treatment method and a culture system.
In one aspect, the invention provides a composition for inducing TIL cells from tumor tissue comprising one or both of IL-7 and IL-15, IL-2, TNF- α, CD3 antibodies, and CD28 antibodies.
In one or more embodiments, the concentration of IL-2 in the TIL cell-inducing composition is 200-1000U/ml, such as 200, 300, 400, 500, 600, 700, 800, 900, 1000U/ml, or a range between any two of the foregoing.
Preferably, the concentration of IL-2 in the composition for inducing TIL cells is 200-950, 250-850, 350-750, 450-650, 500-550U/ml.
In one or more embodiments, the concentration of IL-7 in the TIL cell-inducing composition is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing values.
Preferably, the concentration of IL-7 in the TIL cell-inducing composition is from 30-450, 40-400, 50-350, 60-300, 70-250, 80-200, 90-150 pg/ml.
In one or more embodiments, the concentration of IL-15 in the TIL cell-inducing composition is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing.
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.
In one or more embodiments, the concentration of TNF- α in the composition that induces TIL cells is 10-90ng/ml, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90ng/ml, or a range between any two of the foregoing.
Preferably, the concentration of TNF- α in the composition for inducing TIL cells is 20-80, 30-70, 40-60 ng/ml.
In one or more embodiments, the concentration of the CD3 antibody in the TIL cell-inducing composition is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing values.
Preferably, the concentration of the CD3 antibody in the TIL cell-inducing composition is 30-500, 50-450, 100-400, 150-350, 200-300 pg/ml.
In one or more embodiments, the concentration of CD28 antibody in the TIL cell-inducing composition is 1-1000ng/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000ng/ml, or a range between any two of the foregoing.
Preferably, the concentration of the CD28 antibody in the TIL cell-inducing composition is 30-950, 50-850, 150-750, 250-650, 350-550, 450-500 ng/ml.
In one or more embodiments, the TIL cell-inducing composition comprises 200-1000U/ml IL-2, 1-500pg/ml IL-7, 1-500ng/ml IL-15, 10-90ng/ml TNF-. alpha., 1-500pg/ml CD3 antibody, 100-1000ng/ml CD28 antibody.
In one or more embodiments, the composition for inducing TIL cells comprises
400-600U/ml IL-2, 90-150pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF- α, 200-300pg/ml CD3 antibody, 400-600ng/ml CD28 antibody, or an equal ratio concentrate of these components; or
150-U/ml IL-2, 1-30pg/ml IL-7, 1-30pg/ml IL-15, 40-60ng/ml TNF-. alpha.1-30 pg/ml CD3 antibody, 1-30ng/ml CD28 antibody, or an equal-ratio concentrate of these components; or
900-1000U/ml IL-2, 450-500pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 450-500pg/ml CD3 antibody, 900-1000ng/ml CD28 antibody, or an equal ratio concentrate of these components; or
400-600U/ml IL-2, 450-500pg/ml IL-15, 40-60ng/ml TNF- α, 450-500pg/ml CD3 antibody, 900-1000ng/ml CD28 antibody, or an isocratic concentrate of these components; or
900-1000U/ml IL-2, 450-500pg/ml IL-7, 40-60ng/ml TNF-. alpha.450-500 pg/ml CD3 antibody, 900-1000ng/ml CD28 antibody, or an isocratic concentrate of these components; or
500-600U/ml IL-2, 90-150pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 200-300pg/ml CD3 antibody, 500-600ng/ml CD28 antibody, or an equal ratio concentrate of these components.
In one or more embodiments, the composition for inducing TIL cells comprises
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 an equal ratio concentrate of these components; or
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 an equal ratio concentrate of these components; or
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 an equal ratio concentrate of these components; or
500U/ml IL-2, 500pg/ml IL-15, 50ng/ml TNF- α, 500pg/ml CD3 antibody, 1000ng/ml CD28 antibody, or an isocratic concentrate of these components; or
1000U/ml IL-2, 500pg/ml IL-7, 50ng/ml TNF- α, 500pg/ml CD3 antibody, 1000ng/ml CD28 antibody, or an isocratic concentrate of these components; or
500U/ml IL-2, 100pg/ml IL-7, 500pg/ml IL-15, 50ng/ml TNF-. alpha., 300pg/ml CD3 antibody, 500ng/ml CD28 antibody, or an equivalent concentrate of these components.
In one or more embodiments, the CD3 antibody is an anti-human CD3 antibody or antigen-binding fragment thereof.
In one or more embodiments, the CD28 antibody is an anti-human CD28 antibody or antigen-binding fragment thereof.
In one or more embodiments, the ratio of the concentration 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, preferably 1: 2.
In one or more embodiments, the tumor tissue has a volume of 2-40mm3Preferably 5-30mm3More preferably 10 to 20mm3。
In one or more embodiments, the tumor tissue is a solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, and the like.
The invention also provides a culture medium for inducing TIL cells from tumor tissue, comprising the TIL cell-inducing composition described herein, a basal medium, and serum.
In one or more embodiments, the basal medium is selected from the group consisting of X-VIVO, RPMI-1640, and AIM-V.
In one or more embodiments, the serum is selected from human AB serum, subject autologous serum, or animal derived serum.
In one or more embodiments, the concentration of serum is 1-10% of the total volume, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the foregoing 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%.
In one or more embodiments, the tumor tissue has a volume of 2-40mm3Preferably 5-30mm3More preferably 10 to 20mm3。
In one or more embodiments, the tumor tissue is a solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, and the like.
The present invention also provides a composition for rapidly expanding TIL cells comprising one or both of IL-7 and IL-15, IL-2, TNF- α, a CD3 antibody, a CD28 antibody, and a CD137 antibody.
In one or more embodiments, the concentration of IL-2 in the composition that expands TIL cells is 200-1000U/ml, such as 200, 300, 400, 500, 600, 700, 800, 900, 1000U/ml, or a range between any two of the foregoing.
Preferably, the concentration of IL-2 in the composition for amplifying TIL cells is 200-950, 250-850, 350-750, 450-650, 500-550U/ml.
In one or more embodiments, the concentration of IL-7 in the TIL cell expanding composition is from 1 to 500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing values.
Preferably, the concentration of IL-7 in the TIL cell expanding composition is 30-450, 40-400, 50-350, 60-300, 70-250, 80-200, 90-150 pg/ml.
In one or more embodiments, the concentration of IL-15 in the TIL cell expanding composition is from 1 to 500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing values.
Preferably, the concentration of IL-15 in the composition for amplifying 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.
In one or more embodiments, the concentration of TNF- α in the composition that expands TIL cells is 10-90ng/ml, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90ng/ml, or a range between any two of the foregoing.
Preferably, the concentration of TNF- α in the composition for expanding TIL cells is 20-80, 30-70, 40-60 ng/ml.
In one or more embodiments, the concentration of the CD3 antibody in the TIL cell-expanding composition is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing.
Preferably, the concentration of the CD3 antibody in the composition for expanding TIL cells is 30-500, 50-450, 100-400, 150-350, 200-300 pg/ml.
In one or more embodiments, the concentration of CD28 antibody in the composition that expands TIL cells is 1-1000ng/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000ng/ml, or a range between any two of the foregoing.
Preferably, the concentration of the CD28 antibody in the composition for amplifying TIL cells is 30-950, 50-850, 150-750, 250-650, 350-550, 450-500 ng/ml.
In one or more embodiments, the concentration of the CD137 antibody in the composition for expanding TIL cells is 100-1100pg/ml, such as 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000pg/ml, 1100pg/ml, or a range between any two of the foregoing values.
Preferably, the concentration of the CD137 antibody in the composition for amplifying the 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.
In one or more embodiments, the composition for expanding TIL cells comprises 200-1000U/ml IL-2, 1-500pg/ml IL-7, 1-500ng/ml IL-15, 10-90ng/ml TNF-. alpha., 1-500pg/ml CD3 antibody, 100-1000ng/ml CD28 antibody, 100-1100pg/ml CD137 antibody.
In one or more embodiments, a composition for expanding TIL cells comprises:
400-600U/ml IL-2, 90-150pg/ml IL-7, 450-550pg/ml IL-15, 40-60ng/ml TNF- α, 200-300pg/ml CD3 antibody, 400-600ng/ml CD28 antibody, 400-600pg/ml CD137 antibody, or an isocratic concentrate of these components; or
400-600U/ml IL-2, 90-150pg/ml IL-7, 450-550pg/ml IL-15, 40-60ng/ml TNF- α, 1-30pg/ml CD3 antibody, 1-30ng/ml CD28 antibody, 100-200pg/ml CD137 antibody, or an equal ratio concentrate of these components; or
900-1000U/ml IL-2, 450-500pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 450-500pg/ml CD3 antibody, 900-1000ng/ml CD28 antibody, 900-1000pg/ml CD137 antibody, or an isocratic concentrate of these components; or
900-1000U/ml IL-2, 450-500pg/ml IL-15, 40-60ng/ml TNF- α, 200-300pg/ml CD3 antibody, 900-1000ng/ml CD28 antibody, 900-1000pg/ml CD137 antibody, or an equal ratio concentrate of these components; or
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, 900-1000pg/ml CD137 antibody, or an equal ratio concentrate of these components; or
400-600U/ml IL-2, 90-150pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 200-300pg/ml CD3 antibody, 400-600ng/ml CD28 antibody, 900-1100pg/ml CD137 antibody, or an isocratic concentrate of these components; or
500-600U/ml IL-2, 90-150pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 200-300pg/ml CD3 antibody, 500-600ng/ml CD28 antibody, 900-1100pg/ml CD137 antibody, or an isocratic concentrate of these components.
In one or more embodiments, a composition for expanding TIL cells comprises:
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 an equal ratio concentrate of these components; or
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 an equal ratio concentrate of these components; or
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 an equal ratio concentrate of these components; or
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 an equal ratio concentrate of these components; or
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 an equal ratio concentrate of these components; or
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 an equal ratio concentrate of these components; or
500U/ml IL-2, 100pg/ml IL-7, 500pg/ml IL-15, 50ng/ml TNF-. alpha.250 pg/ml CD3 antibody, 500ng/ml CD28 antibody, 1100pg/ml CD137 antibody, or an equivalent concentrate of these components.
In one or more embodiments, the CD3 antibody is an anti-human CD3 antibody or antigen-binding fragment thereof.
In one or more embodiments, the CD28 antibody is an anti-human CD28 antibody or antigen-binding fragment thereof.
In one or more embodiments, the CD137 antibody is an anti-human CD28 antibody or antigen-binding fragment thereof.
In one or more embodiments, the ratio of the concentration 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, preferably 1: 2.
In one or more embodiments, the CD3 antibody to CD137 antibody concentration ratio is between 3:1 and 1:6, between 2:1 and 1:5, between 1:1 and 1:4, preferably 1: 2.
A TIL cell rapid expansion medium comprising a composition for expanding TIL cells described herein, a basal medium, and serum.
In one or more embodiments, the basal medium is selected from the group consisting of X-VIVO, RPMI-1640, and AIM-V.
In one or more embodiments, the serum is selected from human AB serum, subject autologous serum, or animal derived serum.
In one or more embodiments, the concentration of serum is 1-10% of the total volume, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the foregoing 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%.
The present invention also provides a method for obtaining TIL cells from tumor tissue, comprising the steps of:
(1) pre-treating tumor tissue; and
(2) incubating the 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 medium is a medium described herein for inducing TIL cells from tumor tissue and the second medium is a TIL cell rapid expansion medium described herein.
In one or more embodiments, the tumor tissue is not treated with an enzyme prior to the incubation of step (2).
In one or more embodiments, the pre-treating of tumor tissue of step (1) comprises fragmenting and/or dissociating tumor tissue, preferably comprises
(1.1) cleaning and immersing tumor tissues by using a treatment solution;
(1.2) fragmenting tumor tissue; and
optionally (1.3) washing the tumor tissue with the first medium.
In one or more embodiments, the pretreatment does not include the use of an enzyme treatment.
In one or more embodiments, the treatment fluid does not contain an enzyme.
In one or more embodiments, the treatment fluid is the basal medium used in the first culture medium.
In one or more embodiments, the incubation of step (2) is for 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.
In one or more embodiments, the incubation of step (2) comprises renewal of the medium on alternate days, e.g., on days 3, 5, 7, 9, 11 of incubation.
In one or more embodiments, the incubation of step (3) is 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.
In one or more embodiments, the incubation of step (3) comprises renewal of the medium on alternate days, e.g., on days 3, 5, 7, 9, 11, 13, 15, 17 of incubation.
In one or more embodiments, the tumor tissue has a volume of 2-40mm3Preferably 5-30mm3More preferably 10 to 20mm3。
In one or more embodiments, the tumor tissue is a solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, and the like.
The invention also provides a method of expanding TIL cells comprising incubating TIL cells with a second medium as described herein.
In one or more embodiments, the TIL cells are the first TIL cell population and/or the second TIL cell population of any of the embodiments described herein.
The present invention also provides a method of inducing and expanding TIL cells, comprising the steps of:
(1) pre-treating tumor tissue;
(2) incubating the tumor tissue with a first culture medium to obtain a first TIL cell population;
(3) incubating the first TIL cell population with a second culture medium to obtain a second TIL cell population,
wherein the first medium is a medium described herein for inducing TIL cells from tumor tissue and the second medium is a TIL cell rapid expansion medium described herein.
In one or more embodiments, the tumor tissue is not treated with an enzyme prior to the incubation of step (2).
In one or more embodiments, the pre-treating of tumor tissue of step (1) comprises fragmenting and/or dissociating tumor tissue, preferably comprises
(1.1) cleaning and immersing tumor tissues by using a treatment solution;
(1.2) fragmenting tumor tissue; and
optionally (1.3) washing the tumor tissue using a first culture medium.
In one or more embodiments, the pretreatment does not include the use of an enzyme treatment.
In one or more embodiments, the treatment fluid does not contain an enzyme.
In one or more embodiments, the treatment fluid is the basal medium used in the first culture medium.
In one or more embodiments, the incubation of step (2) is for 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.
In one or more embodiments, the incubation of step (2) comprises renewal of the medium on alternate days, e.g., on days 3, 5, 7, 9, 11 of incubation.
In one or more embodiments, the incubation of step (3) is 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.
In one or more embodiments, the incubation of step (3) comprises renewal of the medium on alternate days, e.g., on days 3, 5, 7, 9, 11, 13, 15, 17 of incubation.
In one or more embodiments, the tumor tissue has a volume of 2-40mm3Preferably 5-30mm3More preferably 10 to 20mm3。
In one or more embodiments, the tumor tissue is a solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, and the like.
The invention also provides for the use of a composition for inducing TIL cells as described herein in the preparation of a culture medium for inducing TIL cells from a tumor tissue.
In one or more embodiments, the tumor tissue has a volume of 2-40mm3Preferably 5-30mm3Is more excellentSelecting 10-20mm3。
In one or more embodiments, the tumor tissue is a solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, and the like.
The invention also provides the use of a composition for inducing TIL cells as described herein and optionally basal medium and/or serum, in inducing TIL cells from a tumor tissue.
In one or more embodiments, the basal medium is selected from the group consisting of X-VIVO, RPMI-1640, and AIM-V.
In one or more embodiments, the serum is selected from human AB serum, subject autologous serum, or animal derived serum.
In one or more embodiments, the concentration of serum is 1-10% of the total volume, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the foregoing values. Preferably, the serum concentration 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%.
In one or more embodiments, the tumor tissue has a volume of 2-40mm3Preferably 5-30mm3More preferably 10 to 20mm3。
In one or more embodiments, the tumor tissue is a solid tumor tissue such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, and the like.
The invention also provides for the use of a composition for expanding TIL cells as described herein in the preparation of a culture medium for expanding TIL cells or a pharmaceutical composition comprising TIL cells.
The invention also provides for the use of the compositions and optionally the basal medium and/or serum described herein for expanding TIL cells to expand TIL cells.
In one or more embodiments, the basal medium is selected from the group consisting of X-VIVO, RPMI-1640, and AIM-V.
In one or more embodiments, the serum is selected from human AB serum, subject autologous serum, or animal derived serum.
In one or more embodiments, the serum is at a concentration of 1-10% of the total volume, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the foregoing values. Preferably, the serum concentration 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%.
The invention also provides the use of TIL cells obtained by any of the methods of the invention in the preparation of a medicament for the treatment of cancer.
The invention has the advantages that:
the invention solves the technical problems of low amplification factor of TIL cells in vitro, long culture period and low proportion of CD3+ CD8+ cells. By using the current general TIL in-vitro culture technology, more than 60 percent of TIL cells have low in-vitro amplification times and cannot meet the current clinical treatment requirements, and the proportion of CD3+ CD8+ cells is low, so that the clinical effect is poor. The technical scheme of the invention relates to tumor tissue treatment and preparation of a TIL cell in-vitro culture medium, which can obviously improve the in-vitro amplification multiple of TIL cells, shorten in-vitro amplification time, and simultaneously can obviously improve the proportion of CD3+ CD8+ cells, wherein in some embodiments, the proportion of CD3+ CD8+ cells is as high as 80%, so that the application range of a TIL treatment method can be effectively improved, and the clinical effect is improved.
The invention can shorten the induction time to about 7 days (more than 2 x 10)6The cells grow out) and can be expanded to the quantity required by clinic within 25 to 30 days, thereby greatly shortening the expansion period of TIL cells in vitro, increasing the timeliness of the therapy, simultaneously obtaining CD3+ CD8+ cells with higher proportion and improving the clinical curative effect.
Drawings
FIG. 1 shows a comparison of the fold expansion of TIL cells between the method of the present invention and conventional TIL culture methods.
FIG. 2 shows a comparison of cell expansion data between the methods of the present invention and conventional TIL culture methods for 15-27 days.
FIG. 3 shows a comparison of cell expansion data between the present invention and existing TIL culture methods for 15-27 days.
FIG. 4 shows the ratio of CD3+ CD4+ and CD3+ CD8+ cells in TIL cells cultured by the methods of the invention.
Detailed Description
To increase the timeliness of TIL therapy and shorten the time of in vitro TIL cell culture, the inventors developed induction and expansion media for inducing, expanding, and methods of inducing and/or expanding TIL cells from tumor tissue. The TIL cells treated by the culture medium do not need enzyme treatment on tumor tissues, the induction time is shortened to about 7 days, the TIL cells can be expanded to the clinically required number in 30 days, and the in-vitro expansion multiple of the TIL cells and the proportion of CD3+ CD8+ cells can be obviously improved by up to 80%.
The present invention is suitable for isolating TIL from any tumor tissue, particularly from small volumes of tumor tissue (e.g., small amounts of tumor tissue obtained by minimally invasive surgery). The volume of the tumor tissue is 2-40mm3Preferably 5-30mm3More preferably 10-20mm3. Exemplary tumor tissues include solid tumor tissues such as thyroid tumor, gallbladder cancer, lung cancer, breast cancer, liver cancer, and the like.
The present invention provides compositions and media for inducing TIL cells. The inducing composition comprises one or both of IL-7 and IL-15, IL-2, TNF-alpha, CD3 antibodies, and CD28 antibodies. The induction medium comprises one or two selected from IL-7 and IL-15, IL-2, TNF-alpha, CD3 antibody, CD28 antibody, basal medium and serum. The induction medium may alternatively comprise IL-7 and IL-15, or may be free of IL-7 and IL-15.
The concentration of IL-2 in the composition or medium for inducing TIL cells is 200-1000U/ml, such as 200, 300, 400, 500, 600, 700, 800, 900, 1000U/ml, or a range between any two of the foregoing values; the concentration of IL-7 is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing values; the concentration of IL-15 is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing values; the concentration of TNF- α is from 10ng/ml to 90ng/ml, such as 10, 20, 30, 40, 50, 60, 70, 80, 90ng/ml, or a range between any two of the foregoing; the concentration of CD3 antibody is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing; the concentration of CD28 antibody is 1-1000ng/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000ng/ml, or a range between any two of the foregoing.
The present invention provides compositions and media for expanding TIL cells. The inducing composition comprises one or both of IL-7 and IL-15, IL-2, TNF-alpha, a CD3 antibody, a CD28 antibody, and a CD137 antibody. The amplification medium comprises one or two selected from IL-7 and IL-15, IL-2, TNF-alpha, CD3 antibody, CD28 antibody, CD137 antibody, basal medium and serum. The amplification medium may alternatively comprise IL-7 and IL-15, or may be free of IL-7 and IL-15.
The concentration of IL-2 in the composition or medium for expanding TIL cells is 200-1000U/ml, such as 200, 300, 400, 500, 600, 700, 800, 900, 1000U/ml, or a range between any two of the foregoing values; the concentration of IL-7 is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing values; the concentration of IL-15 is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the foregoing values; a concentration of TNF- α of 10-90ng/ml, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90ng/ml, or a range between any two of the foregoing; the concentration of the CD3 antibody is 1-500pg/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500pg/ml, or a range between any two of the above values; the concentration of CD28 antibody is 1-1000ng/ml, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000ng/ml, or a range between any two of the foregoing; the concentration of the CD137 antibody is 100-1100pg/ml, such as 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100pg/ml, or a range between any two of the above values.
The CD3, CD28, CD137 antibodies described herein can be the corresponding antibodies from any species or recombinant antibodies. Such antibodies or variants thereof are well known in the art. In the inducing composition or the culture medium, the concentration ratio of the CD3 antibody to the CD28 antibody is 3:1 to 1:6, 2:1 to 1:5, or 1:1 to 1: 4. In the amplification composition or the culture medium, the concentration ratio of the CD3 antibody to the CD28 antibody is 3:1 to 1:6, 2:1 to 1:5, 1:1 to 1: 4; the concentration ratio of the CD3 antibody to the CD137 antibody is between 3:1 and 1:6, between 2:1 and 1:5, and between 1:1 and 1: 4.
The serum described herein can be any serum known in the art to be useful in the culture of animal cells (e.g., TIL cells), such as human AB serum, subject autologous serum, or animal derived serum. The concentration of serum is 1-10% of the total volume, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the foregoing values.
The "basal medium" as used 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, X-VIVO. The composition and content of these media are well known in the art. Illustratively, AIM-V medium contains L-glutamine, streptomycin sulfate, and gentamicin sulfate; the RPMI-1640 culture medium contains various amino acids, vitamins, inorganic salts and other components required by cell growth; the X-VIVO culture medium contains L-glutamine and gentamicin sulfate. A preferred embodiment uses AIM-V medium as the basal medium.
The present invention also provides a method for obtaining TIL cells from tumor tissue using the above induction and/or expansion medium, comprising the steps of: (1) pre-treating tumor tissue; and (2) incubating the 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 described herein for inducing TIL cells from tumor tissue and the second culture medium is a TIL cell rapid expansion medium described herein. In the methods described herein, the tumor tissue is not treated with an enzyme prior to the incubation of step (2). Thus, the pretreatment does not include the use of an enzyme treatment. Pretreatment includes any one or more of the treatments for tumor tissue known in the art, except that no enzyme treatment is used, so long as the treatment can be used for subsequent TIL cell induction. Illustratively, pre-treating the tumor tissue includes fragmenting and/or dissociating the tumor tissue, preferably including (1.1) washing, submerging the tumor tissue with a treatment fluid; (1.2) fragmenting tumor tissue; and optionally (1.3) washing the tumor tissue using the first culture medium. The treatment solution does not contain an enzyme. The treatment liquid may be a basal medium used in the above-mentioned first medium, for example, AIM-V. The invention also provides a method of expanding TIL cells alone, comprising incubating TIL cells, which are the first and/or second TIL cell populations of any embodiment described herein, with a second culture medium as described herein. The TIL cell obtained by any method of the present invention can be used for treating cancer such as thyroid cancer, gallbladder cancer, lung cancer, breast cancer, liver cancer, etc., or for preparing a medicament for treating cancer.
Herein, the time for inducing TIL cells (i.e. incubation of step (2)) is 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 medium needs to be renewed during induction incubation.
Herein, the time for expanding 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 medium also needs to be refreshed during the amplification incubation.
The invention also provides for the use of a TIL cell-inducing composition as described herein in the preparation of a medium for inducing TIL cells from tumor tissue, and the use of a TIL cell-inducing composition as described herein and optionally a basal medium and/or serum, in inducing TIL cells from tumor tissue. The invention also provides for the use of a composition for expanding TIL cells as described herein in the preparation of a medium for expanding TIL cells or a pharmaceutical composition comprising TIL cells, and the use of a composition for expanding TIL cells and optionally a basal medium and/or serum as described herein in expanding TIL cells. The basal medium, serum, is as described elsewhere herein.
The invention will be elucidated hereinafter by means of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise indicated, all those materials and methods conventional in the art.
Examples
Example 1
The experimental group differs from the control group in that: two media used in the experimental group, one being the rapid induction TIL cell medium: a culture medium A; one medium for rapid expansion of TIL cells: a culture medium B; the control group used TIL universal medium.
Experimental Material
The tumor tissue is obtained from excised tumor tissue of female gallbladder cancer patient, and has a volume of 10mm3(ii) a AIM-V medium was purchased from Gibco; gentamicin sulfate and interleukin-2 (IL-2) were purchased from Shandong spring harbor pharmaceutical Co., Ltd; TNF- α was purchased from near shore protein science, Inc.; interleukin-7 (IL-7) was purchased from Chimerigen; interleukin-15 (IL-15) was purchased from Primegene; CD3 antibody (cat # 10981-MM08) was purchased from Yi Qiao Shen Tech Co., Ltd, and CD28 antibody (cat # 11524-H001) was purchased from Yi Qiao Shen Tech Co., Ltd; CD137 antibody (cat # 10041-T16) was obtained from Chinesota technology, Inc.
Experimental procedure
1. Solution preparation
(1) Treating fluid: adding 200 ten thousand IU/ml gentamicin sulfate 12mg into 50ml AIM-V culture medium to prepare 100 ml;
(2) culture medium A: adding 500U/ml IL-2, 100pg/ml IL-7, 500pg/ml IL-15, 50ng/ml TNF-alpha, 250pg/ml CD3 antibody, 500ng/ml CD28 antibody and 2% human AB serum into AIM-V culture medium;
(3) and (3) a culture medium B: adding 500U/ml IL-2, 100pg/ml IL-7, 500pg/ml IL-15, 50ng/ml TNF-alpha, 250pg/ml CD3 antibody, 500ng/ml CD28 antibody, 500pg/ml CD137 antibody and 2% human AB serum into AIM-V culture medium;
(4) TIL universal medium: IL-2 was added to AIM-V medium at 3000U/ml.
2. Tissue treatment
(1) Placing the tumor tissue sample in a 50ml centrifuge tube, and cleaning for 3 times by using 30ml of treatment solution each time;
(2) after cleaning, transferring the tumor tissue sample to a 100mm flat dish, and adding a treatment solution to immerse the tissue sample;
(3) using sterilized scissors and forceps, the tumor tissue was carefully removed of fat and blood clots. After removal, the tumor tissue was cut to 2-4mm with scissors3A size tissue mass;
(4) after the tumor tissue was sheared, the treatment fluid was aspirated off and washed once with medium a.
3. Experiment grouping
(1) The processed tissue blocks were placed in a six-well plate and 6 tissues were placed. Adding 3ml of culture medium A;
(2) the processed tissue blocks were placed in a six-well plate and 6 tissues were placed. Adding 3ml of TIL universal culture medium, and taking the group as a control group;
(3) placing the above two groups of treated cells at 37 deg.C and 5% CO2Cultured in an incubator and recorded as day 0.
TIL culture
(1) day5 cells were removed from the incubator, observed under a microscope, and observed for cell growth, and the medium was removed using a pipette and centrifuged. For cells obtained by centrifugation, experimental cells were resuspended using medium B and transferred to new six-well plates, and control group was resuspended using universal medium. The original six-well plate was added with the corresponding medium a and universal medium. After the treatment is finished, the mixture is placed at 37 ℃ and 5% CO2Culturing in an incubator。
(2) day7 and day9 are operated in the same manner as in (1). The resuspended samples were counted using a K2 counter.
(3) day11 medium was aspirated and centrifuged, and the pellet was transferred to a flask using medium B; the control group was as above. After the treatment is finished, the temperature is increased to 37 ℃ and 5 percent CO2Culturing in an incubator. Six-well plates containing tumor tissue were discarded.
(4) day13 TIL cells without tissue were removed to be observed under microscope, pipetted to homogenize the cells, transferred to a 50ml centrifuge tube for sampling and counting, then centrifuged at 1500rpm for 5min, and the experimental group was resuspended to 1 × 10 with medium B6Cells/ml, transferred to T75 flasks; control group was resuspended 1 x10 in TIL general medium6Cells/ml, transferred to the corresponding culture vessel.
(5) day15, day17 was performed as in (4), and fold expansion of cells was calculated from the primary count and day17 counts.
(6) Cells were cultured to day27 and sampled and counted every other day.
Example 2
1. Solution preparation
(1) Culture medium A: adding 200U/ml IL-2, 1pg/ml IL-7, 1pg/ml IL-15, 50ng/ml TNF-alpha, 1pg/ml CD3 antibody, 1ng/ml CD28 antibody and 2% human AB serum into AIM-V culture medium;
(2) and (3) a culture medium B: adding 500U/ml IL-2, 100pg/ml IL-7, 500pg/ml IL-15, 50ng/ml TNF-alpha, 1pg/ml CD3 antibody, 1ng/ml CD28 antibody, 100pg/ml CD137 antibody and 2% human AB serum into AIM-V culture medium;
2. experimental procedures were as in example 1, cultured to day 17.
Example 3
1. Solution preparation
(1) Culture medium A: adding 1000U/ml IL-2, 500pg/ml IL-7, 500pg/ml IL-15, 50ng/ml TNF-alpha, 500pg/ml CD3 antibody, 1000ng/ml CD28 antibody and 2% human AB serum into AIM-V culture medium;
(2) and (3) a culture medium B: adding 1000U/ml IL-2, 500pg/ml IL-7, 500pg/ml IL-15, 50ng/ml TNF-alpha, 500pg/ml CD3 antibody, 1000ng/ml CD28 antibody, 1000pg/ml CD137 antibody and 2% human AB serum into AIM-V culture medium;
2. experimental procedures were as in example 1, and culturing was carried out until day 17.
Example 4
1. Solution preparation
(1) Culture medium A: adding 500U/ml IL-2, 500pg/ml IL-15, 50ng/ml TNF-alpha, 500pg/ml CD3 antibody, 1000ng/ml CD28 antibody and 2% human AB serum into AIM-V culture medium;
(2) and (3) a culture medium B: adding 1000U/ml IL-2, 500pg/ml IL-15, 50ng/ml TNF-alpha, 250pg/ml CD3 antibody, 1000ng/ml CD28 antibody, 1000pg/ml CD137 antibody and 2% human AB serum into AIM-V culture medium;
2. experimental procedures were as in example 1, cultured to day 17.
Example 5
1. Solution preparation
(1) Culture medium A: adding 1000U/ml IL-2, 500pg/ml IL-7, 50ng/ml TNF-alpha, 500pg/ml CD3 antibody, 1000ng/ml CD28 antibody and 2% human AB serum into AIM-V culture medium;
(2) and (3) a culture medium B: adding 1000U/ml IL-2, 500pg/ml IL-7, 50ng/ml TNF-alpha, 500pg/ml CD3 antibody, 1000ng/ml CD28 antibody, 1000pg/ml CD137 antibody and 2% human AB serum into AIM-V culture medium;
2. experimental procedures were as in example 1, cultured to day 17.
Example 6
1. Solution preparation
Medium a was the same as medium B: adding 500U/ml IL-2, 100pg/ml IL-7, 500pg/ml IL-15, 50ng/ml TNF-alpha, 250pg/ml CD3 antibody, 500ng/ml CD28 antibody and 2% human AB serum into AIM-V culture medium;
2. experimental procedures were as in example 1, incubated to day27, and samples were taken for flow analysis.
Example 7
According to the published patent CN110462027A, the tumor treatment and culture methods are adopted, the culture is carried out till the 27 th day, and samples are taken and counted every 1 day after the 15 th day. Wherein the first cell culture medium is AIM-V containing IL-2 with concentration of 3000IU/ml, IL-15 with concentration of 10ng/ml, and IL21 with concentration of 5 ng/ml; the second cell culture medium was AIM-V containing IL-2 at a concentration of 3000IU/ml, IL-15 at a concentration of 10ng/ml, IL21 at a concentration of 5ng/ml, CD3 at a concentration of 30ng/ml, and 41BB at a concentration of 30 ug/ml.
Example 8
1. Solution preparation
(1) Culture medium A: 680U/ml IL-2, 500pg/ml IL-7, 50ng/ml TNF-alpha, 40pg/ml CD3 antibody, 1ng/ml CD28 antibody, 2% human AB serum were added to AIM-V medium;
(2) and (3) a culture medium B: 680U/ml IL-2, 500pg/ml IL-7, 50ng/ml TNF-alpha, 40pg/ml CD3 antibody, 1ng/ml CD28 antibody, 900pg/ml CD137 antibody, 2% human AB serum were added to AIM-V medium;
2. experimental procedures were as in example 1, and culturing was carried out until day 17.
Example 9
1. Solution preparation
(1) Culture medium A: 680U/ml IL-2, 20pg/ml IL-7, 20pg/ml IL-15, 50ng/ml TNF-alpha, 40pg/ml CD3 antibody, 1ng/ml CD28 antibody, 2% human AB serum were added to AIM-V medium;
(2) and (3) a culture medium B: 680U/ml IL-2, 20pg/ml IL-7, 20pg/ml IL-15, 50ng/ml TNF-alpha, 40pg/ml CD3 antibody, 1ng/ml CD28 antibody, 900pg/ml CD137 antibody, 2% human AB serum were added to AIM-V medium;
2. experimental procedures were as in example 1, cultured to day 17.
Example 10
Proliferation fold of TIL cells
TiL cell expansion fold (day17/day5) was plotted based on day5 and day17 counts, as shown in FIG. 1. As shown in FIG. 1, the proliferation times of TIL cells cultured by the method of the present invention are significantly higher than those of the TIL cells cultured by the conventional method, which suggests that the method of the present invention can significantly improve the proliferation ability of TIL cells.
Example 11
TIL cell expansion curve
TIL cell expansion curves were plotted according to the cell densities of day15 to day27 in examples 6 and 7, as shown in FIGS. 2 and 3, in the experimental and control groups of example 1. The expansion curve shows that the combination of factors used in the present invention can significantly improve the expansion efficiency of TIL cells.
Example 12
TIL phenotype identification
The TIL cells were cultured according to the TIL cell preparation method of example 1 and example 6, and the TIL cells were analyzed for CD3+ CD4+ and CD3+ CD8+ clustering by flow cytometry using APC Mouse Anti-human CD3 (purchased from Biolegend), FITC Anti-human CD4 (purchased from BD), BV510 Anti-human CD8a (purchased from Biolegend) flow antibodies, as shown in FIG. 4. By adopting the culture system disclosed by the invention in example 1, the TIL cell of the CD3+ CD8+ population reaches 79.46%, and the TIL cell of the CD3+ CD8+ population in example 6 only reaches 32.67%, so that the proportion of the CD3+ CD8+ cells in the TIL cell can be obviously improved.

Claims (10)

1. A composition for inducing TIL cells from tumor tissue comprising one or both of IL-7 and IL-15, IL-2, TNF- α, a CD3 antibody, and a CD28 antibody,
preferably, the first and second electrodes are formed of a metal,
the concentration of IL-2 in the composition for inducing TIL cells is 200-1000U/ml, the concentration of IL-7 is 1-500pg/ml, the concentration of IL-15 is 1-500pg/ml, the concentration of TNF-alpha is 10-90ng/ml, the concentration of CD3 antibody is 1-500pg/ml, and the concentration of CD28 antibody is 1-1000 ng/ml;
the concentration ratio of the CD3 antibody to the CD28 antibody is between 3:1 and 1: 6;
the volume of the tumor tissue is 2-40mm3；
The tumor tissue is solid tumor tissue.
2. The composition of claim 1, wherein the composition comprises:
400-600U/ml IL-2, 90-150pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF- α, 200-300pg/ml CD3 antibody, 400-600ng/ml CD28 antibody, or an equal ratio concentrate of these components; or
150U/ml IL-2, 1-30pg/ml IL-7, 1-30pg/ml IL-15, 40-60ng/ml TNF-. alpha., 1-30pg/ml CD3 antibody, 1-30ng/ml CD28 antibody, or an equivalent concentrate of these components; or
900-1000U/ml IL-2, 450-500pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 450-500pg/ml CD3 antibody, 900-1000ng/ml CD28 antibody, or an equal ratio concentrate of these components; or
400-600U/ml IL-2, 450-500pg/ml IL-15, 40-60ng/ml TNF- α, 450-500pg/ml CD3 antibody, 900-1000ng/ml CD28 antibody, or an isocratic concentrate of these components; or
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 an isocratic concentrate of these components; or
500-600U/ml IL-2, 90-150pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 200-300pg/ml CD3 antibody, 500-600ng/ml CD28 antibody, or an equal ratio concentrate of these components.
3. A culture medium for inducing TIL cells from a tumor tissue, comprising the TIL cell-inducing composition of claim 1 or 2, a basal medium, and serum,
preferably, the first and second electrodes are formed of a metal,
the basic culture medium is selected from X-VIVO, RPMI-1640 and AIM-V;
the concentration of serum is 1-10% of the total volume.
4. A composition for rapidly expanding TIL cells comprising one or both of IL-7 and IL-15, IL-2, TNF- α, a CD3 antibody, a CD28 antibody, and a CD137 antibody,
preferably, the first and second electrodes are formed of a metal,
the concentration of IL-2 in the composition for amplifying TIL cells is 1000U/ml, the concentration of IL-7 is 1-500pg/ml, the concentration of IL-15 is 1-500pg/ml, the concentration of TNF-alpha is 10-90ng/ml, the concentration of CD3 antibody is 1-500pg/ml, the concentration of CD28 antibody is 1-1000ng/ml, and the concentration of CD137 antibody is 100 pg/ml;
the ratio of the concentration of CD3 antibody to CD28 antibody is between 3:1 and 1: 6;
the concentration ratio of the CD3 antibody to the CD137 antibody is between 3:1 and 1:6,
more preferably, the composition for expanding TIL cells comprises:
400-600U/ml IL-2, 90-150pg/ml IL-7, 450-550pg/ml IL-15, 40-60ng/ml TNF- α, 200-300pg/ml CD3 antibody, 400-600ng/ml CD28 antibody, 400-600pg/ml CD137 antibody, or an isocratic concentrate of these components; or
400-600U/ml IL-2, 90-150pg/ml IL-7, 450-550pg/ml IL-15, 40-60ng/ml TNF- α, 1-30pg/ml CD3 antibody, 1-30ng/ml CD28 antibody, 100-200pg/ml CD137 antibody, or an equal ratio concentrate of these components; or
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, 900-1000pg/ml CD137 antibody, or an equal ratio concentrate of these components; or
900-1000U/ml IL-2, 450-500pg/ml IL-15, 40-60ng/ml TNF- α, 200-300pg/ml CD3 antibody, 900-1000ng/ml CD28 antibody, 900-1000pg/ml CD137 antibody, or an isocratic concentrate of these components; or
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, 900-1000pg/ml CD137 antibody, or an isocratic concentrate of these components; or
400-600U/ml IL-2, 90-150pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 200-300pg/ml CD3 antibody, 400-600ng/ml CD28 antibody, 900-1100pg/ml CD137 antibody, or an isocratic concentrate of these components; or
500-600U/ml IL-2, 90-150pg/ml IL-7, 450-500pg/ml IL-15, 40-60ng/ml TNF-alpha, 200-300pg/ml CD3 antibody, 500-600ng/ml CD28 antibody, 900-1100pg/ml CD137 antibody, or an isocratic concentrate of these components.
5. A TIL cell rapid expansion medium comprising the TIL cell expansion composition of claim 4, a basal medium, and serum,
preferably, the first and second liquid crystal display panels are,
the basic culture medium is selected from X-VIVO, RPMI-1640 and AIM-V;
the concentration of serum is 1-10% of the total volume.
6. A method for obtaining TIL cells from tumor tissue comprising the steps of:
(1) pre-treating tumor tissue; and
(2) incubating the 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 medium is the medium for inducing TIL cells from tumor tissue according to claim 3, and the second medium is the TIL cell rapid expansion medium according to claim 5,
preferably, the first and second electrodes are formed of a metal,
the pretreatment of step (1) does not include the use of enzyme treatment;
the pre-treatment of the tumor tissue of step (1) comprises fragmenting and/or dissociating the tumor tissue;
the tumor tissue is not treated by enzyme before incubation in the step (2);
the incubation of step (2) is continued for up to 15 days;
the incubation of step (3) lasts for up to 30 days;
the volume of the tumor tissue is 2-40mm3；
The tumor tissue is solid tumor tissue.
7. Use of the composition for inducing TIL cells according to claim 1 or 2 for preparing a culture medium for inducing TIL cells from tumor tissue,
preferably, the first and second liquid crystal display panels are,
the volume of the tumor tissue is 2-40mm3；
The tumor tissue is solid tumor tissue.
8. Use of the composition for rapid expansion of TIL cells according to claim 4 for preparing a culture medium for expanding TIL cells or a pharmaceutical composition comprising TIL cells.
9. The use of the composition for rapid expansion of TIL cells and optionally a basal medium and/or serum according to claim 4 for expanding TIL cells,
the basic culture medium is selected from X-VIVO, RPMI-1640 and AIM-V;
the concentration of serum is 1-10% of the total volume.
10. Use of TIL cells obtained by the method of claim 6 in the preparation of a medicament for the treatment of cancer.
CN202110054608.2A 2021-01-15 2021-01-15 Method for inducing and preparing TIL cells Pending CN114763530A (en)

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