WO2022262805A1 - Method for preparing feeder cell bank - Google Patents

Abstract

Disclosed is a method for building a feeder cell bank. The method comprises: (1) providing a PBMC population; (2) irradiating the PBMC population to obtain a pretreated PBMC population; and (3) sub-packaging and cryopreserving the pretreated PBMC population to obtain the feeder cell bank. The feeder cell bank can be thawed at any time for the proliferation of immune cells low in initial cell quantity, such as TIL cells, NK cells and γδT cells. The provided method for large-batch preparation of feeder cells by building a bank not only reduces the costs of preparation and quality inspection, but also improves the safety of the product and the convenience of use.

Description

A method for preparing a feeder cell bank technical field

The invention relates to the related fields of cell biology and immunology. Specifically, the present invention relates to a method for preparing a feeder cell bank.

Background technique

In recent years, cellular immunotherapy has attracted much attention in the field of tumor treatment. As for the cell therapy method for solid tumors, especially tumor-infiltrating lymphocyte (abbreviated as TIL) therapy is more ideal. Usually, fewer TIL cells can be obtained from tumor samples, and even only ten thousand or several thousand cells can be obtained. Therefore, if rapid and massive expansion of TIL cells in vitro is desired, feeder cells are often relied upon.

Most of the traditional feeder preparations first obtain PBMCs by manually laying cell layers through density gradient centrifugation, and then irradiate them in centrifuge tubes or culture flasks; this method requires high skills and experience for operators, and the whole process is open. The requirements for the preparation environment are also very strict; the stability of cell quality in each batch is difficult to guarantee.

Therefore, there is an urgent need in the art to provide a method for preparing a high-quality feeder layer cell bank suitable for large-scale production.

Contents of the invention

The purpose of the present invention is to provide a method for preparing a high-quality feeder layer cell bank suitable for large-scale production.

Another purpose of the present invention is to provide a better practice for the preparation of enclosed Feeder cells; one-time large batches of Feeder library building operations are performed, and the safety and function are tested and confirmed for use in the production of other types of immune cells. The innovation of the present invention is that it not only reduces the risk and cost, but also provides instant and ready-to-use cells, so as to get rid of the dependence on the availability of equipment, personnel and PBMC sources.

The innovations of the present invention are as follows:

1. Feeders are prepared in large batches at one time, subpackaged into different specifications for cryopreservation, and form a ready-to-use cell bank. Get rid of the traditional tedious small-scale irradiation operation, which reduces the cost and the probability of human error;

2. A variety of additives were added during the preparation process, such as DNase to avoid cell agglomeration and ensure the uniformity of the dose of cells during irradiation; OKT3 can play a pre-activation role to promote the better function of the Feeder;

3. This process is a fully enclosed process, reducing the probability of pollution. In addition, the cells that have been completed in the library must be qualified by quality control before being used for subsequent cell cultures to avoid production failures caused by feeder quality problems.

In a first aspect of the present invention, a method for preparing a feeder cell bank is provided, comprising the steps of:

(1) providing a PBMC cell population;

(2) inactivating the PBMC cell population (such as: irradiation treatment), thereby obtaining a pretreated PBMC cell population; and

(3) Subpackage the pretreated PBMC cell population and freeze it to obtain the feeder layer cell bank, wherein the feeder layer cell bank includes 50-5000 parts of subpackaged PBMC cells, each Aliquots contained 1×10 ⁶ -2×10 ⁹ PBMC cells.

The PBMCs can be inactivated (eg, by irradiation or heat treatment).

In another preferred embodiment, before performing irradiation treatment on the pretreated PBMC cell population, activation pretreatment is also included.

In another preferred example, in step (2), it also includes: performing activation pretreatment on the PBMC cell population with a first medium containing an activator and an anti-aggregation agent, Thereby obtaining activated pretreated PBMC cell population; wherein, the activator includes an activating antibody selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, anti-OX40 antibody, anti-CD137 antibody, or a combination thereof.

In another preferred example, the feeder cell bank includes 100-1000 aliquoted PBMC cells.

In another preferred example, each portion of the packed PBMC cells contains 10 ⁷ -10 ⁹ PBMC cells.

In another preferred example, both steps (2) and (3) are carried out in a closed system.

In another preferred example, the irradiation dose is selected from 40-100Gy.

In another preferred example, the PBMC cell population is from mammals, preferably from humans.

In another preferred example, the PBMC cell population is isolated from human peripheral blood. In certain embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs can be obtained using standard methods, such as Ficoll-Paque gradient separation.

In another preferred example, the PBMC cell population is obtained from human peripheral blood by Sepax C-Pro equipment combined with Ficoll-Hypaque density gradient method, Rotea system or X-Lab closed system. Separation into sealed packaging bags.

In another preference, in step (2), the first medium is selected from NaCl injection, compound electrolyte solution, RPMI-1640 medium, X-Vivo15 medium, AIM-V medium, or a combination thereof .

In certain embodiments, the first medium is a liquid medium.

In certain embodiments, the first medium is complete medium.

In certain embodiments, the first medium comprises basal medium, serum or serum substitute, and/or L-glutamine.

In another preferred example, in step (2), the amount of cells in the obtained pretreated feeder layer cell population is 2×10 ⁶ -200×10 ⁶ cells/ml.

In another preferred example, in step (2), the activator further includes: cytokines.

In another preferred embodiment, in step (2), the cytokine is selected from IL2, IL7, IL15, IL21 or a combination thereof.

In another preferred embodiment, in step (2), the activating antibody is selected from the group consisting of anti-CD3, anti-CD28, or a combination thereof. In certain embodiments, the first culture medium comprises an anti-CD3 antibody or fragment thereof (e.g., an OKT3 antibody or fragment thereof), an anti-CD28 antibody or fragment thereof, an anti-OX40 antibody or fragment thereof, an anti-CD137 antibody or fragment thereof, or their combination.

In another preferred example, in step (2), the concentrations of the cytokines are each independently 2 ng/ml-100 ng/ml (final concentration).

In another preferred example, in step (2), the amount of the IL2 cytokine is 20IU/ml-1000IU/ml. In certain embodiments, the concentration of IL2 in the first culture medium can be about 10 IU/ml to about 2000 IU/ml, about 20 IU/ml to about 2000 IU/ml, about 20 IU/ml to about 1500 IU/ml, about 50 IU/ml ml to about 1000IU/ml, about 100IU/ml to about 500IU/ml, about 100IU/ml to about 500IU/ml, about 100IU/ml to about 300IU/ml, about 50IU/ml to about 500IU/ml, about 200IU/ml ml to about 500 IU/ml, about 200 IU/ml to about 400 IU/ml, about 200 IU/ml to about 300 IU/ml, or about 300 IU/ml to about 1000 IU/ml.

In the first culture medium, IL7, IL15 or IL21 can have about 1 ng/ml to about 200 ng/ml, about 1 ng/ml to about 180 ng/ml, about 1 ng/ml to about 150 ng/ml, about 1 ng/ml to about 120ng/ml, about 1ng/ml to about 100ng/ml, about 1ng/ml to about 80ng/ml, about 1ng/ml to about 50ng/ml, about 2ng/ml to about 200ng/ml, about 2ng/ml to about 180ng/ml, about 2ng/ml to about 150ng/ml, about 2ng/ml to about 120ng/ml, about 2ng/ml to about 100ng/ml, about 2ng/ml to about 80ng/ml, about 2ng/ml to about 50ng/ml, about 5ng/ml to about 200ng/ml, about 5ng/ml to about 180ng/ml, about 5ng/ml to about 150ng/ml, about 5ng/ml to about 120ng/ml, about 5ng/ml to about 100 ng/ml, about 5 ng/ml to about 80 ng/ml, about 5 ng/ml to about 50 ng/ml, about 10 ng/ml to about 200 ng/ml, about 10 ng/ml to about 180 ng/ml, about 10 ng/ml to about 150 ng/ml, about 10 ng/ml to about 120 ng/ml, about 10 ng/ml to about 100 ng/ml, about 10 ng/ml to about 50 ng/ml, about 20 ng/ml to about 200 ng/ml, about 20 ng/ml to About 180 ng/ml, about 20 ng/ml to about 150 ng/ml, about 20 ng/ml to about 120 ng/ml, about 20 ng/ml to about 100 ng/ml, about 20 ng/ml to about 50 ng/ml, or about 3 ng/ml to a concentration range of about 30ng/ml

In certain embodiments, in the first medium, IL7 can have a concentration of about 10 ng/ml to about 100 μg/ml; IL15 can have a concentration of about 10 ng/ml to about 100 μg/ml; IL21 can have a concentration of about 10 ng/ml to about 100 μg/ml; ml to a concentration of about 100 μg/ml.

In another preferred example, in step (2), the amount of the anti-CD3 antibody is 30ng/ml-300ng/ml (final concentration).

In certain embodiments, the anti-CD3 antibody (e.g., OKT3) or fragment thereof may have about 3 ng/ml to about 100 ng/ml, about 1 ng/ml to about 100 ng/ml, about 5 ng/ml in the first culture medium. ml to about 100ng/ml, about 10ng/ml to about 100ng/ml, about 15ng/ml to about 100ng/ml, about 20ng/ml to about 100ng/ml, about 30ng/ml to about 100ng/ml, about 40ng/ml ml to about 100ng/ml, about 50ng/ml to about 100ng/ml, about 60ng/ml to about 100ng/ml, about 3ng/ml to about 80ng/ml, about 3ng/ml to about 60ng/ml, about 3ng/ml ml to about 50ng/ml, about 5ng/ml to about 50ng/ml, about 10ng/ml to about 50ng/ml, about 10ng/ml to about 300ng/ml, about 10ng/ml to about 200ng/ml, about 20ng/ml ml to about 100ng/ml, about 50ng/ml to about 200ng/ml, about 50ng/ml to about 100ng/ml, about 30ng/ml to about 100ng/ml, about 50ng/ml to about 150ng/ml, about 30ng/ml ml to about 200 ng/ml, or about 3 ng/ml to about 30 ng/ml concentration range. In some embodiments, the cell culture medium comprises about 30 ng/mL of an anti-CD3 antibody (eg, OKT3) or a fragment thereof. In one embodiment, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 pg/mL anti-CD3 antibody (eg, OKT3) or a fragment thereof. In one embodiment, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL , between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL anti-CD3 antibody (eg, OKT3) or its fragment.

In another preferred embodiment, the anti-cell aggregation agent is selected from: DNase (DNase), RNase, protease, or a combination thereof.

In another preferred embodiment, the anti-aggregation agent is DNase.

In another preferred example, in step (2), the anti-aggregation agents are each independently 10U-100U/ml (final concentration). In some embodiments, in the first culture medium, the anti-cell aggregation agent has about 10U/ml to about 200U/ml, about 10U/ml to about 150U/ml, about 10U/ml to about 100U/ml, about 15U/ml to about 200U/ml, about 15U/ml to about 150U/ml, about 15U/ml to about 100U/ml, about 15U/ml to about 80U/ml, about 15U/ml to about 50U/ml, about 20 U/ml to about 200 U/ml, about 20 U/ml to about 150 U/ml, about 20 U/ml to about 100 U/ml, about 20 U/ml to about 80 U/ml, about 20 U/ml to about 50 U/ml, or A concentration range of about 20 U/ml to about 40 U/ml.

In another preference, in step (3), the freezing includes:

(3.1) In the second culture vessel, the pretreated PBMC cell population in (2) is mixed with the cell cryopreservation solution to obtain the PBMC cell mixture;

(3.2) Preserve the PBMC cell mixture in the previous step at low temperature to obtain the feeder layer cell bank.

In another preferred embodiment, in step (3.1), the mixing of the pretreated PBMC cell population and cell cryopreservation solution is carried out on Sepax C-Pro equipment, RoTea system, Sefia or FINIA filling equipment.

In another preferred example, the cryopreservation refers to storage in liquid nitrogen.

In another preferred embodiment, the cell freezing solution is selected from DMSO, CS10 cell freezing solution, cell freezing medium, or a combination thereof.

In certain embodiments, the cryopreservation solution comprises glycerol, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), or combinations thereof.

The final concentration of DMSO in the cell sample after adding the cryopreservation solution can be in the range of 0% to about 10% (v/v). In some embodiments, the final concentration of DMSO may range from about 7% to 10% (v/v).

In another preferred example, in step (3), the feeder layer cells are frozen at a density of 2×10 ⁶ -200×10 ⁶ cells/ml.

In certain embodiments, the cells are present at about 10 ⁴ cells/ml to about 2×10 ⁸ cells/ml, about 10 ⁴ cells/ml to about 10 ⁸ cells/ml, about 10 ⁵ cells/ml to about 10 ⁷ cells/ml, about 10 ⁵ cells/ml to about 10 ⁸ cells/ml, about 10 ⁴ cells/ml to about 10 ⁷ cells/ml, about 10 ⁵ cells/ml, about 10 ⁶ cells/ml, or a concentration range of about ¹⁰⁷ cells/ml are present in the cryopreservation composition. The concentration of cells in the cryopreservation composition may be higher than ^2x108 cells/ml or lower than ¹⁰⁴ cells/ml.

In another preferred example, the cryopreservation time is ≥ 30 days, preferably ≥ 60 days, preferably ≥ 120 days, more preferably ≥ 210 days.

In certain embodiments, the mixture or combination of cryopreservation composition and cells is frozen at a temperature ranging from about -70°C to about -200°C. In certain embodiments, the cells are at or below about 8°C, at or below about 4°C, at or below about 0°C, at or below about -20°C, at or below about -50°C. at or below about -60°C, at or below about -70°C, at or below about -80°C, at or below about -90°C, at or below about -100°C, at or below Store or freeze at about -110°C, at or below about -120°C, at or below about -135°C, at or below about -196°C, or in liquid nitrogen.

In another preferred example, the preparation method is carried out in a closed system throughout the process.

In the second aspect of the present invention, a feeder cell bank is provided, and the feeder cell bank is prepared by the method as described in the first aspect of the present invention.

In another preferred example, the cryopreservation density of cells in the feeder cell bank is 2×10 ⁶ -200×10 ⁶ cells/ml.

In another preferred example, the survival rate of the revived feeder cells in the cell bank is ≥80%, preferably ≥90%, more preferably ≥95%, and most preferably ≥99%.

In certain embodiments, the cells have at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% Survival after resuscitation.

In another preferred embodiment, the recovery rate of feeder cells in the cell bank is ≥70%, preferably ≥80%, more preferably ≥85%, and more preferably ≥90%.

In another preferred example, the "yield" is the ratio of the number E1 of living cells obtained by thawing the feeder cells after cryopreservation to the number E0 of the feeder cells before cryopreservation.

In the third aspect of the present invention, a use of the feeder cell bank according to the second aspect of the present invention is provided for culturing immune cells.

In a fourth aspect of the present invention, a method for culturing immune cells is provided, comprising the steps of:

(1) providing an initial cell population of immune cells, wherein the number of immune cells in the initial cell population is n0;

(2) revive the feeder cell bank as described in the second aspect of the present invention, and obtain the revived feeder cells;

(3) co-culture the recovered feeder cells obtained in the previous step with the initial cell population of immune cells in (1) for a period of time t1, so as to obtain prepared immune cells, wherein the number of cells in the immune cells is n1 .

In another preferred embodiment, the immune cells include tumor infiltrating lymphocytes (TIL), NK cells, and γδT cells, or a combination thereof.

In another preferred example, t1 is 4-16 days, preferably 9-14 days.

In another preferred example, n1/n0 is 10-100000, preferably 100-50000, more preferably 1000-10000.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Fig. 1 shows the process flow diagram of Feeder cell bank building in the present invention.

Figure 2 shows the comparison of the cell viability of the Feeder cell bank in the present invention before and after cryopreservation for 4 months.

Figure 3 shows the comparison of the recovery yield of the Feeder cell bank in the present invention before and after freezing for 4 months.

Fig. 4 has shown the growth curve of the Feeder cells cultured alone after the Feeder cell bank of the present invention has been cryopreserved for 4 months and revived.

Figure 5 shows the growth curve of the co-culture of Feeder cells and TIL cells after the Feeder cell bank of the present invention was frozen for 4 months and revived.

Figure 6 shows the comparison of the recovery yield and viability of the Feeder cell bank in the present invention before and after cryopreservation for different periods of time.

Fig. 7 shows the change of the growth curve of the co-culture of Feeder cells and TIL cells after the Feeder cell bank of the present invention was frozen for 7 months and revived.

detailed description

After extensive and in-depth research, the inventors first developed a method for building a feeder cell bank (Feeder cells). The obtained Feeder cell bank has stable quality, is suitable for cryopreservation, and is suitable for immune cells with a very low initial cell mass ( Such as rapid mass production of TIL cells). Specifically, the isolated PBMC cells are pretreated in the presence of activating antibodies, cytokines, and DNase to prepare a feeder layer cell bank. The library building method of the present invention is carried out in a closed system of the whole process, and is particularly suitable for large-scale industrial production. The cryopreserved feeder layer cell bank is available immediately, and has good viability and cell yield after recovery. The present invention has been accomplished on this basis.

The key innovations of the present invention are as follows: Feeders are prepared in large batches at one time, subpackaged into different specifications for cryopreservation, and form a ready-to-use cell bank. Get rid of the traditional tedious small-scale irradiation operation, reduce the cost and the probability of human error; add a variety of additives in the preparation process, such as DNase to avoid cell clumping, and ensure the uniformity of the dose of cell irradiation; OKT3 can play a role in predicting The role of activation promotes the better function of the Feeder; this process is a fully enclosed process, which reduces the probability of pollution. In addition, the cells that have been completed in the library will be used for subsequent cell culture only after being qualified by quality control, so as to avoid production failures caused by feeder quality problems.

the term

As used herein, "feeder cell bank of the present invention", "Feeder cell bank of the present invention", "cell bank of the present invention", and "feeder cell bank" can be used interchangeably, all of which are based on the first aspect of the present invention Preparation method to obtain a feeder cell bank.

As used herein, the terms "comprising", "comprising" and "containing" are used interchangeably to include not only open definitions, but also semi-closed, and closed definitions. In other words, the terms include "consisting of", "consisting essentially of".

As used herein, the term "OKT3" is an anti-human CD3 monoclonal antibody.

PBMC cells, Feeder cells and TIL cells

PBMC cells: Peripheral blood mononuclear cells refer to mononuclear cells isolated from human peripheral blood. In the present invention, PBMC cells are used to prepare feeder layer cells. In the present invention, automation can be accomplished through various approaches, including but not limited to Sepax C-Pro equipment combined with Ficoll-Hypaque density gradient method (Ficoll-Hypaque density gradient method), Rotea system or X-Lab closed system The PBMC cell isolation procedure.

Feeder cells: feeder cells. Usually small or very low-density starting cells are difficult to grow in vitro, and auxiliary cells need to be added to help maintain a high growth density and biological activity, thereby promoting the expansion of a small number of cells. These auxiliary cells are called feeder cells. The Feeder cells in the present invention are derived from PBMC cells, and are frozen after pretreatment such as radiation irradiation to prepare the Feeder cell bank of the present invention.

TIL cells: Tumor infiltrating lymphocytes, which are white blood cells that leave the bloodstream and enter the tumor. The TCR receptors on the surface of such cells are usually mutated and can specifically recognize tumor surface antigens.

The preparation method of Feeder cell bank of the present invention

The invention utilizes different equipment, disposable pipeline consumables and transfer bags to realize the closed Feeder preparation process. Including the whole process of separation of PBMC, cell irradiation, Feeder subpackage, cryopreservation, and recovery (Figure 1).

The present invention adopts a closed experimental design, separates PBMCs from 2-3 parts of fresh leukocyte apheresis products in advance, and directly irradiates them under high-dose X-ray conditions (40-100Gy); The donor's PBMC cells are mixed, subpackaged and cryopreserved; a large-scale (≥2E10) and quality-stable Feeder cell bank is established.

The present invention adopts Sepax C-Pro equipment in combination with Ficoll-Hypaque density gradient method (Ficoll-Hypaque density gradient method), Rotea system or X-Lab closed system, etc., to extract from human peripheral blood PBMCs were isolated into sealed bags. Then paste the irradiation indication label strip, transfer to the X-ray device to complete the irradiation according to the specified dose. Then use closed consumables such as Sepax C-Pro equipment, RoTea system, Sefia or FINIA filling to mix the feeder cells with the cell freezing solution, and divide the feeder cells into cryopreservation bags, and transfer them to liquid nitrogen after gradient cooling save.

In a specific embodiment of the present invention, the method for building a library of feeder cells of the present invention includes:

a. Separate the PBMCs from the freshly collected white blood cell apheresis, add activating antibodies and cytokines to stimulate, then add DNase and irradiate under high-dose X-ray conditions;

b. Mix the irradiated PBMC with the cell cryopreservation solution according to a certain ratio, and cryopreserve in various specifications as the feeder layer cell bank.

The feeder cell bank in the present invention can be revived at any time, and is used for the expansion of immune cells with very low initial cell mass, such as TIL cells, NK cells and γδT cells.

The method of preparing feeder cells in large quantities in the present invention not only reduces the cost of preparation and quality inspection, but also improves the safety of the product and the convenience of use.

In the present invention, there is no particular requirement on the medium used, as long as it is a medium for culturing immune cells. Including but not limited to RPMI-1640, X-Vivo15 or AIM-V and other media.

Activating antibodies include but are not limited to one or a mixture of anti-CD3, anti-CD28, anti-OX40, etc. Cytokines include, but are not limited to, one or a mixture of IL2, IL7, IL15, IL21, and the like.

The types of cryopreservation solutions include but are not limited to various commercial or self-made cell cryopreservation protection solutions such as CS10 cell cryopreservation medium and cell cryopreservation medium.

In the present invention, common culture containers such as culture flasks, cell culture bags, G-Rex, etc. can be used without limitation. Those skilled in the art can easily select culture containers suitable for different instruments and practice the present invention. In particular, transfer bags of appropriate size can be selected for cell irradiation, subpackaging, and cryopreservation.

Cell cryopreservation

Cell cryopreservation is an important means of biological preservation of species. If the cells are directly frozen without adding any conditions, the water in the cells and the external environment will form ice crystals, resulting in mechanical damage, electrolyte increase, osmotic pressure changes, dehydration, pH changes, protein denaturation, etc. in the cells, which are serious. cause cell death. If a protective agent is added to the culture medium, the freezing point can be lowered. Under slow freezing conditions, the water in the cells can penetrate the cells before freezing, and storage at low temperatures can reduce the formation of ice crystals.

The most important part of cell freezing is the cell freezing solution. Traditional cell freezing solutions contain animal serum, mainly fetal bovine serum and/or calf serum. Serum is an extremely complex mixture formed by removing fibrin from plasma. Some of its components are still unclear, and the composition and content of serum often vary with the sex, age, physiological conditions and nutritional conditions of blood-donating animals. Serum contains various plasma proteins, polypeptides, fats, carbohydrates, hormones, inorganic substances, etc.

Cell recovery

As used herein, the term "cell rejuvenation" refers to the process by which dormant cells are reactivated. Generally, a procedure well known to those skilled in the art, that is, rapid recovery method, is used to quickly transfer the frozen tube from liquid nitrogen to a warm water bath, preferably at 37°C-40°C, and stir occasionally to accelerate thawing; after the cells are completely thawed, the Sterilize the frozen tube; wash and resuspend the cells after thawing, transfer to a cell culture bottle, and culture in a CO ₂ incubator; check the cell viability and viability.

cell bank

As used herein, the term "cell bank" refers to a type of cell population that has been stored in a specific cryopreservation medium for a long time after a certain program of cooling. The cells in the cell bank can be derived from humans; they can also be derived from (non-human) mammals, such as rats, mice, monkeys, cats, sheep, etc.; they can also be derived from birds, such as chickens. Cells can be derived from different organs and tissues, such as: oral cavity, kidney, liver, lymph, muscle, ovary, etc. Cells can be kept in cell banks for decades or more.

The cell bank in the present invention refers to the feeder layer cell bank obtained by the preparation method of the first aspect of the present invention, which is especially suitable for rapid and large-scale cultivation of immune cells (such as TIL cells). Specifically, co-cultivating the feeder cells obtained after recovery of the feeder cell bank of the present invention and TIL cells can achieve high-efficiency expansion of TIL cells, and the best expansion can be achieved by 100,000 times in 2 weeks.

The main advantages of the present invention include:

(1) The feeder cell bank construction method of the present invention can not only maintain the appropriate growth density of TIL cells in the early stage, but also secrete certain growth-promoting factors to improve the growth activity and proliferation rate of TIL cells in vitro.

(2) The Feeder cell bank of the present invention is to pre-frozen the prepared Feeder cells into a certain specification according to the requirements, and after the safety and effectiveness are tested by QC, it is used for other low-initial products such as TIL and NK. The production of immune cell therapy products is ready-to-use.

(3) The process design and quality control in the Feeder library building method in the present invention all reflect the requirements of GMP norms. The entire process of long-term storage, collection and use of the Feeder cells established in the library is strictly controlled.

(4) The Feeder cell bank of the present invention has good recovery survival rate and recovery yield.

(5) The Feeder cell library construction method of the present invention has strict specifications and controls in the selection of donors, blood sample collection, and logistics transportation; The closed process system can reduce the probability of Feeder cells being polluted by other batches of cells, personnel and the environment.

Below in conjunction with specific embodiment, further state the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate detailed conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

The preparation of embodiment 1 Feeder cell bank

1.1PBMC separation:

The closed PBMC separation equipment is used to obtain mononuclear cells by removing plasma, platelets and red blood cells through the separation method of Sepax combined with Ficoll density gradient liquid, Rotea reverse flow centrifugal system, X-Lab and other mononuclear cell separation systems.

1.2 Cell irradiation process:

Resuspend the isolated PBMC (about 1×10 ¹⁰ /100ml) in a medium containing activating antibody OKT3 (an anti-CD3 antibody) (50ng/ml), cytokine IL2 (300IU/ml) and DNase (20U/ml). In the serum-containing AIM-V medium (30-100ml), after pretreatment for 0.5-10 hours, conduct irradiation treatment, and the irradiation dose is set between 40-100Gy. Freezing process.

1.3 Cryopreservation of Feeder cells

Cryopreservation method: Place the Feeder cells packaged in the cryopreservation bag in the programmed cooling equipment such as Thermo Scientific Forma CryoMed Controlled Rate Freezer, Via Freeze, etc., cool the cells to <-80°C according to the gradient of 1°C/min, and then transfer to ≤-130°C liquid nitrogen environment for long-term storage.

Add the irradiated PBMCs to the CS10 cell cryopreservation solution containing 5-20% DMSO at a volume ratio of 1:1 to obtain the feeder layer cell bank of the present invention. The number of cryopreserved cells is 100×10 ⁶ /bag, or 1000×10 ⁶ /bag; the duration of cryopreservation is >180 days. The cell freezing density is 2×10 ⁶ -200×10 ⁶ cells/ml, and the freezing specification is 10-50ml/bag.

(1) Feeder cells frozen for 4 months

From Figures 2, 3 and Table 1, it can be seen that compared with the cells before cryopreservation, the viability of the recovered Feeder cells after 4 months of cryopreservation was maintained above 90%, and the cell yield after recovery (cell yield after recovery =The number of viable cells obtained after recovery/the number of viable cells of the feeder layer cells before cryopreservation)>70%, meeting the requirements of subsequent functional experiments.

Table 1 Differences in viability and cell number of Feeder cells cryopreserved for 4 months before cryopreservation and after recovery

(2) Long-term stability of frozen Feeder cells

It can be seen from Figure 4 and Table 2 that the Feeder cells (batch number: M046) prepared according to this patented process flow have a survival rate of >90% and a recovery rate of >70% after cryopreservation for different periods of time.

Table 2 Feeder cryopreservation stability data

Freezing days (Day)	Cell viability after recovery (%)	Actual recovery rate of Feeder cells (%)
99	96.3	85
117	96.9	78
157	96.7	77
190	97.1	82

1.4 Culture of Feeder cells after recovery

Use culture medium to adjust the revived Feeder cells to a density of 1×10 ⁶ -10×10 ⁶ cells/ml, inoculate them into square flasks, culture bags, G-Rex and other culture containers, and add activating antibody OKT3 and cytokines IL2, monitors the growth of Feeder cells.

The traditional way to prepare Feeder cells is usually an open manual operation; PBMCs are separated by ficoll density gradient centrifugation, and then placed in centrifuge tubes or culture dishes for irradiation; after irradiation is completed, they are directly used for downstream cell culture. In addition, the traditional method does not add activating antibody OKT3, cytokine IL2 and DNase.

The experimental results are shown in Figure 5 and Table 3: during the separate culture process, the amount of Feeder cells gradually decreased, and no division and proliferation occurred. This feature illustrates that the feeder cells of the present invention can provide a good nutritional environment without affecting the growth of other cells in co-culture, while avoiding the probability of graft-versus-host disease (GVHD) reactions .

Table 3 The growth data of two batches of Feeder prepared by the new process and cultured alone for two weeks in vitro (× ¹⁰⁶ )

number of days	traditional way	Database building process-1	Database construction process-2
D0	10.00	10.00	10.00
D3	3.31	3.315	2.63
D4	n/a	3.03	n/a
D5	1.94	n/a	1.840
D6	n/a	2.525	n/a
D8	1.230	n/a	1.100
D10	1.050	1.975	1.020
D12	0.603	n/a	0.595
D14	0.334	1.458	0.458

Note, n/a refers to the unsampled count of this sampling point.

Example 2 Functional Verification of Feeder Cell Bank

(1) Feeder cells frozen for 4 months

Inoculate TIL and feeder cells recovered after 4 months of cryopreservation into the culture square flask according to the ratio of 1:10-1:1000, add 30ng/ml activation antibody OKT3 and 6000IU/ml cytokine IL2, from the cultured From day 4 or day 5, 10-90% fresh medium was replaced every 2-3 days and the cell density was adjusted to 0.1×10 ⁶ -5×10 ⁶ cells/ml. As a control, other culture conditions were kept the same, but no Feeder cells were added, and TIL cells were cultured alone (the initial cell volume was 0.1×10 ⁶ cells).

As can be seen from Figure 6 and Table 4, compared with the traditional method (using the traditional method described in Example 1.4 of the present invention to prepare Feeder cells), the feeder cells of the present invention (banking batch 1) can better stimulate TIL Cell Proliferation. On the 10th day, the proliferation rate of TIL cells is significantly different between the culture process of the present invention and the traditional culture method.

Table 4. The amount of TIL cells under different culture methods (× ¹⁰⁶ )

number of days	Database construction process	traditional way	control
D0	0.1	0.1	0.1
D4	8.4	9.8	0.154
D6	11.8	7.8	0.470
D8	25.2	14.4	0.570
D10	56.9	26.8	0.529
D14	179.9	70.9	0.440

(2) Feeder cell bank frozen for 7 months

It can be seen from Figure 7 that the Feeder cell bank prepared according to this patented process flow, after 7 months of cryopreservation, the revived Feeder cells still have a good ability to promote the proliferation of TIL cells. When cultured for 15 days, the expansion factor of TIL cells can reach 5000-6000.

In addition, the feeder cell bank obtained in the present invention can not only maintain the appropriate growth density of TIL cells in the early stage, but also secrete certain growth-promoting factors (such as IL2, IL7) to improve the growth activity and proliferation rate of TIL cells in vitro .

Comparative example C1

The preparation method in Example 1 is adopted, wherein only the activation antibody is added.

Cells clumped when pretreated or irradiated. The cell viability of PBMC gradually decreased with the prolongation of the treatment time in vitro. Adding DNase can prevent the uneven distribution of cells caused by the agglutination of dead cells and affect the irradiation effect.

discuss:

Compared with the existing traditional preparation method, the library building method of the present invention has the following points of improvement:

The present invention has strict specifications and controls in the process of donor selection, blood sample collection, and logistics transportation; and the subsequent PBMC separation, cell irradiation, freezing storage, and packaging are all designed as a closed system for the entire process, which is safe for personnel and The environment is less dependent.

In the present invention, the prepared Feeder is pre-frozen and stored into a certain specification according to the requirements, and after the safety and effectiveness are tested by QC, it is then used in the production of other immune cell therapy products with low initial quantities such as TIL and NK. Provide instant availability guarantee.

The process design and quality control of Feeder library construction mentioned in the present invention all embody the requirements of GMP norms. The entire process of long-term storage, collection and use of the Feeder cells established in the library is strictly controlled. And after 4 months of cryopreservation, the viability of the recovered cells was >80%, and the recovered recovery rate was >70%.

Before irradiation, activating antibodies and cytokines will be added in advance to initially stimulate PBMC, and then mediate the relevant immune response mechanism and improve the function of Feeder cells.

A low dose of DNase will also be added during irradiation to avoid aggregation caused by some terminal effector cell death in the blood. The irradiated blood is in the state of single-cell homogeneous suspension to ensure the uniform quality of the irradiated cells.

During the in vitro culture process of the feeder cells prepared by the method of the present invention, as time goes on, the cell mass decreases continuously and does not divide and proliferate; subsequent use in the production process of TIL cells can also reduce the occurrence of product transplantation The probability of a graft-versus-host disease (GVHD) response.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A method for preparing a feeder cell bank, comprising the steps of:

   (1) providing a PBMC cell population;

   (2) irradiating the PBMC cell population to obtain a pretreated PBMC cell population; and

   (3) Subpackage the pretreated PBMC cell population and freeze it to obtain the feeder layer cell bank, wherein the feeder layer cell bank includes 50-5000 parts of subpackaged PBMC cells, each Aliquots contained 1×10 ⁶ -2×10 ⁹ PBMC cells.
2. The method according to claim 1, characterized in that, in step (2), also comprising: carrying out activation pretreatment to described PBMC cell population with the first culture medium, containing activator in the first culture medium And anti-cell aggregation agent, thereby obtain the PBMC cell population through activating pretreatment; Wherein, described activator comprises the activation antibody selected from the following group: the antibody of anti-CD3, the antibody of anti-CD28, the antibody of anti-OX40, anti-CD137 Antibodies, or combinations thereof.
3. The method according to claim 1, wherein the PBMC cell population is from a mammal, preferably from a human.
4. The method according to claim 1, characterized in that, in step (2), the activator further comprises: cytokines.
5. The method according to claim 1, characterized in that, the anti-cell aggregation agent is selected from the group consisting of: DNase (DNase), RNase, protease, or a combination thereof.
6. The method according to claim 1, characterized in that, the frozen storage time is ≥ 30 days, preferably ≥ 60 days, preferably ≥ 120 days, more preferably ≥ 210 days.
7. A feeder cell bank, characterized in that the feeder cell bank is prepared by the method as claimed in claim 1.
8. A method for cultivating immune cells, comprising the steps of:

   (1) providing an initial cell population of immune cells, wherein the number of immune cells in the initial cell population is n0;

   (2) resuscitating the feeder layer cell bank as claimed in claim 2, obtaining the resuscitated feeder layer cells;

   (3) Co-cultivate the recovered feeder cells obtained in the previous step with the initial cell population of immune cells in (1) for a time t1 to obtain prepared immune cells, wherein the number of prepared immune cells is n1.
9. The method of claim 8, wherein the immune cells comprise tumor infiltrating lymphocytes (TIL), NK cells, and γδT cells, or combinations thereof.
10. The method according to claim 8, characterized in that, the ratio of the initial cell population to the number of prepared immune cells, n1/n0, is 10-100000, preferably 100-50000, more preferably 1000-10000 .

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