Systems and methods for large-scale immune cell proliferation and activation

Abstract

The present invention discloses a three-dimensional (3D) bioreactor for large-scale expansion of immune cells and a method of using the same. The 3D bioreactor includes at least one packed bed chamber including at least one porous scaffold, at least one porous scaffold coated with one or more extracellular matrix proteins (ECMs), at least one container containing a fluid medium, the container being configured such that the fluid medium flows through the packed bed chamber having at least one porous coated scaffold, and at least one population of immune cells suspended in the fluid medium. At least one porous scaffold coated with ECM forms a fixed niche having low shear forces that mimics the natural growth environment of immune cells. This enables large-scale expansion of the population of immune cells flowing through the coated porous scaffold.

Description

Technical Field

[0001] The present invention relates to a system and method for culturing and / or activating immune cells on a large scale. More particularly, the present invention relates to the large-scale culture and / or activation of immune cells in a packed bed bioreactor.

Background Art

[0002] The culture of mammalian cells is in principle complex due to their high sensitivity, relatively slow growth, complex differentiation processes, and the requirement of strict asepsis as a basic condition. Large-scale cell culture is always difficult. Even small-scale cell culture requires attention and specific knowledge because it often faces serious problems. For example, during cell culture preparation and test procedures, cell cultures may be stressed or damaged, so analyses based on cultured cells may show results that are at least partially affected by such damage. Furthermore, applying conclusions derived from results obtained with damaged cells to in vivo situations or the use of cells in immunotherapy can have fatal consequences. Moreover, stressful and damaging situations are not reproducible between individual cell cultures and can affect cell growth. These issues become even more critical when dealing with large-scale cell culture. Over the past few decades, various attempts have been made. Patent Document 1 describes an apparatus and method for culturing cells and / or tissues that mimic the cell structure and immune function of immunologically active tissues, but this system and method are limited to a volume of 4 ml. Further patents and patent applications related to the field of the present invention are Patent Document 2 and Patent Document 3.

[0003] Furthermore, the use of bioreactors for cell and tissue culture is well known. A detailed overview of bioreactor design, prototyping, and process control for reproducible three-dimensional tissue culture is provided in the following link. https: / / www.minerva-kg.de / libraryonline / upload / files / file6400.pdf

[0004] In vitro culture of immune cells is even more difficult. In recent years, the human immune system has been used as the basis for therapeutic techniques that can recognize and kill tumor cells, and has become a central goal of cancer immunotherapy. Therefore, by improving the effectiveness and accessibility of this technology, there is a growing interest in making it widely applicable to adoptive cell therapies (ACT) such as chimeric antigen receptor T (CAR-T) cells, tumor-infiltrating lymphocytes (TIL), dendritic cells (DC), natural killer (NK) cells, and many others. However, in order to implement this technology, the currently available immune cell culture systems do not meet the requirements because they damage cells, have low effectiveness, limited scale-up ability, or are very costly (Reference: Non-Patent Document 1). In this paper, the main culture methods known so far for immune cell culture were examined along with their advantages and disadvantages. More specifically, a comparison was made among stirred flasks, G-Rex flasks, rocking motion bioreactors, stirred tank bioreactors, hollow fiber bioreactors, and CliniMACS Prodigy. Thus, there is an urgent need for an expandable, cost-effective, and GMP-compliant bioreactor for the culture of immune cells.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention aims to provide a system and method for culturing and / or activating an immune cell population on a large scale.

Means for Solving the Problems

[0008] In one main aspect, the present invention relates to a large-scale system and method for culturing and / or activating immune cells. The problems to be solved for the effective large-scale culture of immune cells are to avoid or minimize the high shear stress that would damage the cells in order to obtain a large-scale and homogeneous system, and to create conditions that enable high cell-cell interactions by creating conditions that enable interactions between the cells, activators, and transfection agents to create physical niches and conditions.

[0009] In one aspect, the present invention discloses a method and system for culturing immune cells comprising a porous stationary phase located within a flowing medium. The stationary phase may be disposed within a bioreactor or may be disposed in another chamber that is functionally connected to the bioreactor. The porous elements, described in detail below, are disposed within the basket of a packed bed bioreactor, are not movable, do not move with the flow of the liquid surrounding them, and the liquid flows through them. The stationary phase can be implemented in various forms and can be aimed at creating an environment of low flow rate and low shear force.

[0010] As used herein, the terms "porous stationary phase", "porous scaffold", "porous element", and "porous coated scaffold" all refer to the same thing and may be used interchangeably in the following description. In certain embodiments, the porous stationary phase is coated with an extracellular matrix (ECM) and may further be coated with an immune system cell activator to activate and expand immune cells. By combining all the components, a niche is formed for immune system cells that mimics their natural environment within tissues and lymph nodes.

[0011] As used herein, the terms "activator" and "immune cell activator" both refer to the same thing and may be used interchangeably in the following description. In some embodiments, the "activator" and "immune cell activator" are antigen-presenting cells loaded with antigens presented on the cell surface. In another embodiment, the activator is an antibody directed against an activation receptor on the surface of an immune cell. In yet another embodiment, the activator is an antigen conjugated to a molecule capable of being presented to an activation receptor on the surface of an immune cell.

[0012] As used herein, the term "niche" refers to, but is not limited to, a stationary phase having pores through which liquids and particles can pass, such as scaffolds, beads, and carriers. The particles are cells or other components and may be synthetic or natural.

[0013] The formed niche mimics lymph nodes / tissues with respect to the microenvironment in which immune cells naturally grow, resulting in optimal cell proliferation. Further, since the formed niche mimics the natural environment of the cells, it is possible to activate the cells such that only specific cells respond to the activation and a specific selection is made for the desired cells.

[0014] Furthermore, the formed niche enables large-scale proliferation of immune cells while maintaining a relatively low shear force, minimizing cell damage.

[0015] The terms "Media" and "Medium" are synonymous and may be used interchangeably hereinafter.

[0016] The terms "packed bed chamber", "packed bed basket", "basket", and "growth basket" are all intended to be synonymous and may be used interchangeably hereinafter.

[0017] As used herein, "growth" of a cell or cell population referred to herein is intended to be synonymous with proliferation of the cell population and culturing of the cell population, regardless of the presence or absence of cell activation.

[0018] The terms "immune cell", "immune cell population", and "lymphoid cell" as used herein all refer to the same thing and may be used interchangeably in the following description.

[0019] In certain embodiments, lymphoid cells are expanded without significant differentiation. In various embodiments, this expansion is performed on a 2D substrate, on a 3D substrate, or on a 3D substrate after a 2D substrate.

[0020] In some embodiments, lymphoid cells are incubated in a bioreactor, non-limiting examples of which are suspension culture and culture on a 3D carrier. The term "bioreactor culture" refers to culture in an apparatus (bioreactor) that is typically sterile and in which cells are maintained under the controlled conditions described below in connection with FIG. 1.

[0021] As used herein, "activation" of an immune cell or immune cell population referred to herein is intended to be synonymous with exposure of the immune cell to an antigen, which causes a change in cell morphology and elicits an immune response detected by rapid proliferation and secretion of various cytokines and chemokines.

[0022] Accordingly, in one main aspect, the present invention is a three-dimensional (3D) bioreactor for large-scale expansion of immune cells, comprising: a) at least one packed bed chamber containing at least one porous scaffold; b) at least one porous scaffold coated with one or more extracellular matrix proteins (ECM); c) at least one container containing a flowing medium, the flowing medium being configured to flow through the packed bed chamber with at least one porous coated scaffold; and d) at least one population of immune cells suspended in the flowing medium, wherein the at least one porous scaffold coated with the ECM is configured to form a stationary niche with low shear forces that mimics the natural growth environment of immune cells, enabling large-scale expansion of the population of immune cells flowing through the coated porous scaffold. The present invention relates to the 3D bioreactor.

[0023] The at least one porous scaffold may be further coated or bound with at least one immune cell activator. In some optional embodiments, the immune cell activator is either an antigen-presenting cell (APC) loaded or not loaded with an antigen, or any one of the antigens directly bound to the coated porous scaffold. If the APC is not loaded with an antigen, the antigen may be presented at a later stage according to the requirements for activating immune cells.

[0024] In a further option, the expanded immune cells may be further activated in the packed bed chamber by exposing the population of immune cells to the at least one porous coated scaffold bound to the immune cell activator.

[0025] In another option, the expanded immune cells are further activated in the packed bed chamber by exposure to a soluble immune cell activator suspended therein and may be further expanded by at least one porous ECM-coated scaffold.

[0026] In some embodiments of the present invention, a proliferated and / or activated immune cell population is harvested or reactivated by exposing APCs bound to the at least one coated porous scaffold to an antigen, generating additional activation signals for the immune cell population.

[0027] Furthermore, in some further embodiments, the immune cell population may be harvested or reactivated by transferring the proliferated immune cells to a different bioreactor comprising at least one porous scaffold coated with a different or similar immune cell activator.

[0028] The porous scaffold may be a single porous scaffold matrix expanded within the internal space of the packed bed chamber, or a plurality of mini or micro porous scaffolds filling the packed bed chamber.

[0029] In some further optional embodiments of the present invention, the immune cell population is genetically modified by using a gene modifier incorporated into the bioreactor medium.

[0030] The present invention further relates to a method for large-scale proliferation of immune cells in a three-dimensional (3D) bioreactor, comprising: a) inserting at least one porous scaffold into at least one packed bed chamber; b) coating the at least one porous scaffold with one or more extracellular matrix proteins (ECMs); c) circulating a fluid medium from at least one container, the fluid medium being configured to flow through the packed bed chamber containing the at least one porous coated scaffold; and d) suspending at least one immune cell population in the circulated fluid medium, wherein the at least one porous scaffold coated with the ECM forms a fixed niche having a low shear force that mimics the natural growth environment of immune cells and is configured to enable large-scale proliferation of the immune cell population flowing through the at least one porous scaffold.

[0031] The method may further include, after the step of coating the scaffold with ECM to grow and activate the immune cell population in the packed bed chamber, a step of coating the at least one porous scaffold with at least one immune cell activator; and, after the step of suspending at least one immune cell population in the circulated fluid medium, a step of exposing the immune cell population to the at least one activator.

[0032] The method may further include a step of genetically modifying immune cells in the 3D bioreactor using a gene modifier added to the fluid medium.

[0033] In some optional embodiments, the method further includes harvesting immune cells or a portion of the cells and further growing and reactivating the immune cell population in the same or a different bioreactor.

[0034] Additionally or alternatively, the method described above may further include, after the step of harvesting the immune cells, performing genetic modification outside the system and then reseeding the genetically modified immune cells into the same or a different bioreactor.

[0035] Furthermore, in a further aspect, the present invention is a three-dimensional (3D) bioreactor for large-scale expansion and activation of an immune cell population, comprising: a) at least one packed bed chamber containing at least one porous antigen-presenting cell mimicking scaffold (APC-MS); b) at least one APC-MS coated with one or more extracellular matrix proteins (ECM); c) at least one container containing a flowing medium, wherein the flowing medium is configured to flow through the coated porous APC-MS; and d) at least one immune cell population suspended in the flowing medium, wherein the at least one APC-MS forms a fixed microenvironment with low shear force that mimics the natural growth environment of the immune cell population, enabling large-scale expansion and / or activation of the immune cell population flowing through it, and the object is to provide the 3D bioreactor.

[0036] The immune cell population may be reactivated by exposing the coated porous APC-MS to an antigen, generating additional activation signals in the immune cell population.

[0037] In some optional embodiments, the immune cell population is reactivated by transferring cells to different bioreactors containing at least one coated porous APC-MS having different or similar antigens.

[0038] According to an embodiment of the present invention, the at least one porous APC-MS may be composed of a single unit expanded within the internal space of the packed bed chamber, or may be a plurality of mini / micro porous APC-MS filling the packed bed chamber.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the practice or testing of the present invention, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0040] The present invention will be described herein by way of example with reference to the accompanying drawings. Referring now to the drawings in detail, it is to be emphasized that the details shown are illustrative and for the purpose of an exemplary description of embodiments of the present invention, and are presented in order to provide a useful and understandable explanation of the principles and conceptual aspects of the present invention. In this regard, no attempt has been made to show the structural details of the present invention in more detail than is necessary for a fundamental understanding of the present invention, and the description in conjunction with the drawings will make apparent to those skilled in the art how some forms of the present invention may be practiced.

Brief Description of the Drawings

[0041]

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Mode for Carrying Out the Invention

[0042] Before detailing at least one embodiment of the present invention, it should be understood that the present invention is not limited to the details described in the following description or illustrated by the examples in its application. The present invention allows for other embodiments or can be implemented or executed in various ways. Also, the expressions and terms used herein are for explanatory purposes and should not be regarded as limiting.

[0043] The present invention aims to provide a system and method for large-scale expansion and activation of various immune cell populations.

[0044] In one main aspect, the present invention provides a 3D bioreactor for large-scale proliferation of immune cells, comprising: a) at least one packed bed chamber containing at least one porous scaffold; b) at least one porous scaffold coated with one or more ECM proteins; c) at least one container containing a flowing medium, wherein the flowing medium is configured to flow through the packed bed chamber with at least one porous coated scaffold; and d) at least one immune cell population suspended in the flowing medium. The at least one porous scaffold coated with ECM is configured to form a fixed niche with low shear force that mimics the natural growth environment of immune cells, enabling large-scale proliferation of the immune cell population flowing through the coated porous scaffold.

[0045] In a further aspect, the present invention provides a method comprising: a) inserting at least one porous scaffold into at least one packed bed chamber; b) coating the at least one porous scaffold with one or more extracellular matrix proteins (ECMs); c) circulating a fluid medium from at least one container, the fluid medium being configured to flow through a packed bed chamber containing the at least one porous coated scaffold; and d) suspending at least one population of immune cells in the circulated fluid medium, wherein the at least one porous scaffold coated with ECM forms a fixed niche having a low shear force that mimics the natural growth environment of immune cells and is configured to enable large-scale proliferation of the population of immune cells flowing through the at least one porous scaffold.

[0046] In yet a further aspect, the present invention provides a bioreactor comprising: a) at least one packed bed chamber containing at least one porous APC-MS; b) at least one APC-MS coated with one or more ECM proteins; c) at least one container containing a fluid medium, the fluid medium being configured to flow through the coated porous APC-MS; and d) at least one population of immune cells suspended in the fluid medium, wherein the at least one APC-MS forms a fixed microenvironment having a low shear force that mimics the natural growth environment of the population of immune cells and enables large-scale proliferation and / or activation of the population of immune cells flowing through it.

[0047] The main aspects of the present invention and options for carrying out the present invention will be better understood from the following detailed description of various illustrative and non-limiting drawings and examples set forth below. Reference is now made to the drawings.

[0048] FIG. 1 is a schematic cross-sectional view of an optional packed bed bioreactor 100 for the proliferation, activation and harvesting of a population of immune cells, according to an embodiment of the present invention.

[0049] In the illustrated embodiment, the growth and vibration chamber 116 (hereinafter referred to as the "basket") is equipped with at least one porous scaffold 10. The terms "carrier" and "scaffold" may be used interchangeably and are both coated as described in detail with reference to FIGS. 2(1) and 2(2) and are configured to form a fixed niche within this basket, referring to a porous element that reduces shear forces and mimics the natural environment of the immune cell population. The basket wall 1161 is preferably separated from the inner wall 40 of the bioreactor and can move up and down. The bioreactor 100 is a liquid medium via an inlet pipe 120 that is configured and operable to supply various media to the bioreactor and then optionally be autoclaved. In other embodiments, after sterilization, the liquid is replaced with a growth medium that saturates the basket 116 and its contents. The basket 116 divides the fluid within the bioreactor 100 into substantially three main sections. An upper section 122 mainly containing fresh medium inserted via the inlet pipe 120, an intermediate section 124 containing the medium within the basket 116 surrounding the coated scaffold, and a lower section 126 mainly containing the medium that has flowed through the basket. Compared to the fresh medium in the upper section 122, the medium flowing through the middle section typically has fewer nutrients consumed by the immune cells, a reduced flow rate as it flows through at least one porous-coated scaffold, interacts with and / or is activated by other cells, and becomes enriched with compounds and debris secreted by the immune cells. In the embodiments described herein, the medium in the upper section 122 is agitated by an impeller 119 that creates fluid movement, as indicated by arrow 109. In still further embodiments, various parameters, such as temperature, pH, dissolved oxygen concentration, etc., are set at the start of the process as part of the system setup procedure (see the flow in FIGS. 2(1) and 2(2) below) and are constantly adapted to suspension conditions as needed. In still further embodiments, the initial agitation rate of the medium may be set low to promote cell adhesion to the coated scaffold and then the agitation rate may be increased. As needed, cells can be harvested from the medium for the manufacture of the final product or for further expansion of the immune cells described in detail below (FIGS. 2(1) and 2(2)). In some embodiments, the rotation of the impeller 119 creates a negative pressure in the draft tube 150, drawing the medium along with the cells from the lower section 126 through the draft tube 150 and then through the ports of the impeller 119 into the upper section 122, circulating the medium and the immune cells in a uniform continuous loop in the direction indicated by arrow 109. In still further embodiments of the present invention, various parameters of the medium can be controlled by adjusting the medium by monitoring various parameters through the electrodes 106. In some optional embodiments, a ring sparger (not visible) is disposed within the impeller aeration chamber 11 to oxygenate the medium flowing through the ports of the impeller 119 via gas added from the external port 103, which medium may be retained within the housing 5 and the sparger line 7. In some other optional embodiments, gas may be added via the inlet 120. Alternatively, the sparged gas may be trapped in a remote chamber and absorbed into the nutrient medium, flushing the entire system. In some optional embodiments, the water jacket 117 covers the media area within the bioreactor 100, and is provided with a port 13 for inflowing jacket water and a port 14 for outflowing. The removal pipe 110 is arranged along the bioreactor, has an opening in the lower media section 126, and can harvest immune cells from the media under the basket 116 as needed. The removal pipe 110 can also be used for debris removal and media refreshment by removing a portion of the used media and adding fresh media.

[0050] In some embodiments, a continuous stirred tank bioreactor may be used, where the culture medium is continuously supplied to the bioreactor, the product is continuously withdrawn, and a steady state with a constant time is maintained within the bioreactor. A stirred tank bioreactor equipped with a fibrous bed basket is available, for example, from New Brunswick Scientific Co. (Edison, NJ). Further bioreactors that can be used include, for example, fixed bed bioreactors, perfusion bioreactors using polyactive foam, radial flow perfusion bioreactors including tubular poly-L-lactic acid (PLLA) porous scaffolds, and other bioreactors known in the art suitable for the purposes of the present invention, but not limited thereto. A "fixed bed bioreactor" refers to a bioreactor in which a cell growth substrate does not normally lift from the bottom of the incubation container in the presence of a growth medium. For example, the substrate can be made not to lift by having a sufficient density, and / or can be filled with mechanical pressure so as not to lift. The substrate may be a single mass or multiple masses. Usually, the substrate remains substantially in place during the standard stirring speed of the bioreactor. In some embodiments, a plurality of carriers are loosely packed, for example, to form a loose packed bed and are immersed in a nutrient medium.

[0051] Furthermore, in certain embodiments, a perfusion bioreactor is used and the perfusion chamber contains a 3D substrate. In certain embodiments, the 3D substrate is in the form of a porous scaffold 10. The porous scaffold may be made as a single large unit that encompasses the entire or most of the volume of the packed bed chamber. Alternatively, the porous scaffold in use may be a plurality of small particles. In some further optional embodiments, the porous scaffold may be, for example, a macrocarrier, a microcarrier, or a mixture thereof. Non-limiting examples of commercially available carriers include alginate-based (GEM, Global Cell Solutions), dextran-based (Cytodex®, GE Healthcare), collagen-based (Cultispher®, Percell Biolytica), and polystyrene-based (SoloHill Engineering) microcarriers.

[0052] In certain embodiments, the T cells are those referred to as "APC-MS" and are incubated within the bioreactor. This term refers to a scaffold that binds or is associated with a lymphocyte activation component (in more specific embodiments, may include any scaffold referred to herein), and these more specific embodiments are fibrous carriers or mesoporous silica micro-rods that are bound to or coated with the activation component.

[0053] Unless otherwise specified, the term packed bed bioreactor refers to a bioreactor in which a cell growth substrate does not normally lift off the bottom of the incubation vessel in the presence of a growth medium. For example, the substrate can have a sufficient density so as not to lift, and / or can be packed with mechanical pressure so as not to lift. The substrate may be a single mass or multiple masses. Typically, the substrate remains substantially in place during perfusion at the standard perfusion rate of the bioreactor. In certain embodiments, the definition does not exclude the possibility that the substrate may be lifted at abnormally high perfusion rates, for example, above 200 rpm.

[0054] In other embodiments, the biocontainer is used to grow cells and, in further embodiments, is adapted for suspension culture. In various embodiments, the biocontainer is used and / or adapted for batch, fed-batch, or continuous culture.

[0055] Figures 2(1) and 2(2) are flowchart diagrams that illustrate at a high level the main sequence of steps for preparing and using a packed bed bioreactor system for the growth, activation, and harvesting of an immune cell population according to an embodiment of the present invention.

[0056] The system setup step 310 includes assembling the system, filling the bioreactor basket with a desired amount of porous scaffold, connecting the desired electrodes for monitoring and controlling culture parameters (e.g., pH, dissolved oxygen, temperature) during system use, connecting the tubes necessary for fresh nutrient supply and by-product removal, sealing the system and performing a integrity test, and sterilizing the system by steam sterilization in an autoclave. After sterilization, the system is connected to a bioreactor control station and the electrodes are calibrated.

[0057] The porous scaffolds hereinafter referred to as "carriers" may be made of natural or synthetic materials and may have various dimensions. Some non-limiting examples of commercially available carriers include alginate-based (GEM, Global Cell Solutions), dextran-based (Cytodex®, GE Healthcare), collagen-based (Cultispher®, Percell Biolytica), and polystyrene-based (SoloHill Engineering) carriers. Alternatively, the porous scaffold may include a fibrous material, optionally an adhesive fibrous material, and may be, for example, either a woven fiber matrix or a non-woven fiber matrix. Non-limiting examples of fibrous scaffolds include those commercially available from Eppendorf TM AG of Germany (Eppendorf TM AG), including the Fibra-Cel® disk of New Brunswick Scientific Co., Inc. containing a polyester mesh, and the BioNOC TM II carrier commercially available from CESCO BioProducts of Atlanta, GA, made of PET (polyethylene terephthalate). TM II carrier. In certain embodiments, the fibrous matrix referred to comprises polyester, polypropylene, polyalkylene, polyfluorochloroethylene, polyvinyl chloride, polystyrene, or polysulfone. In more specific embodiments, the fibrous matrix is selected from polyester and polypropylene.

[0058] To create an environment that mimics the natural environment of immune cells, an ECM coating step 312 is performed. In this step, by using the hydrophilic end groups of Fibra―Cel (registered trademark) disks, it is possible to coat scaffolds with various materials. This is done by simply immersing the scaffold in a solution containing ECM proteins to create electrostatic interactions. A detailed explanation of the effect of the extracellular matrix on immune cells is provided in "The Extracellular Matrix and the Immune System: Interdependence" by Sutherland T.E et al., Science, February 17, 2023, Volume 379 (Issue 6633), PMID: 36795835. Note that other scaffolds can also be used, and the described Fibra―Cel (registered trademark) disk is a non-limiting example. Furthermore, the coating of the scaffold can be performed using natural ECM components or synthetic ECM components.

[0059] In this way, various proteins such as, but not limited to, albumin, fibronectin, fibrin, fibrinogen, collagen, hyaluronic acid, elastin, laminin, selectin, etc. can be used to coat the scaffold and create an environment similar to the natural environment. For example, to mimic the structure of lymph nodes, type III collagen, which is a major component of reticular fibers, the main component of the ECM of lymph nodes, can be coated on the scaffold. Furthermore, since different organs have different combinations of ECM proteins, different combinations and concentrations can be used for coating. In another embodiment, by binding adhesion molecules to the scaffold or the protein-coated scaffold, a strong interaction between immune cells and the ECM-coated scaffold can be promoted. For example, E-selectin, P-selectin, ICAM1, ICAM2, and VCAM1 are known in the art to promote the interaction between leukocytes and the ECM and help induce leukocytes to inflamed tissues. Therefore, coating the scaffold with these molecules may increase the interaction between leukocytes and the ECM-coated scaffold. Alternatively, the ECM coating (step 312) is performed after the system setup (step 310), followed by the scaffold activation coating step (step 314). In different embodiments (depending on the product and its activation ligand), the activation coating step (step 314) of the porous scaffold is performed after the ECM coating (step 312).

[0060] Step 314 describes the portion where the scaffold is coated with the immune cell activation ligand. Step 314 can be performed before or after 312. Immune cells usually need to be activated by antigen presentation occurring in lymph nodes or infected tissues. By coating the scaffold with ECM components and performing antigen presentation in a low shear environment, the formed niche mimics the natural environment where activation occurs. In one embodiment, after ECM coating with serum ECM proteins, adherent or semi - adherent cells can attach to the fibers constituting the scaffold. Since some immune cells require interaction with antigens presented by antigen - presenting cells (APCs) to be activated, APCs loaded with antigens are seeded in step 314 after the coating step 312. The APCs can attach to the scaffold and present antigens to various immune cells such as T cells, B cells, and all their sub - populations. In another embodiment, APCs can be seeded into the bioreactor and attached to the scaffold without performing antigen presentation, and after the cells have attached, the antigen can be added to the medium filling the bioreactor to be presented to immune cells such as T cells and B cells. A more specific example of this embodiment is the use of monocytes to activate mucosal - associated invariant T cells (MAIT). In this example, after the scaffold is coated with fetal bovine serum proteins, PBMCs are seeded into the bioreactor, monocytes attach to the scaffold, and the remaining cells are suspended in the medium. After the initial cell seeding, 5 - OP - RU (5 - (2 - oxopropylideneamino) - 6 - D - ribitylaminouracil), which is the activation antigen of MAIT, is added to the medium and presented by monocytes to specifically activate MAIT cells. In another embodiment of the present invention, the activation of immune cells may be regulated by an antigen bound to a scaffold without APC. The antigen may be, for example, an antibody against a specific activation receptor on immune cells, a protein recognized by an activation receptor such as TCR, etc. For example, the scaffold is coated with both monoclonal anti-CD3 (OKT3) antibody and anti-CD28 (CD28.2) antibody to provide a co-stimulatory signal involved in the T cell receptor. After the incubation period, a blocking step is performed, followed by a washing step. After the washing step, the scaffold is coated with fetal bovine serum protein. When the medium is exchanged and PBMC is seeded into the bioreactor, the cells are activated by the interaction with the ECM and the antibody-coated scaffold.

[0061] As another example, for activating MAIT cells, there is the use of MR1 monomers, tetramers and other forms with or without 5-OP-RU loaded. When MR1 without 5-OP-RU is used, the addition of 5-OP-RU is performed after the blocking and washing steps.

[0062] Step 316 describes the seeding of cells including target cells for activation and proliferation. In this step, for seeding the target cells, various immune cell sources can be used, namely PBMC collected from blood component collection, PBMC isolated from specific organs or tumors, etc. The immune cells can be used in fresh or frozen state. In this step, the immune cells are seeded into the bioreactor. Using different environmental parameters such as, but not limited to, stirring speed, pH, dissolved oxygen, temperature, etc., the cells can either adhere or remain suspended in the bioreactor. The adherent cells enter the packed bed chamber and can interact with the ECM proteins on the coated scaffold to form an organized microstructure of cells within the packed bed. According to this embodiment, APC is seeded into the bioreactor, adheres to the scaffold in a form without antigen presentation, and can create an environment mimicking a lymph node until activation occurs. A more specific example of this embodiment is the use of monocytes to activate mucosal-associated invariant T cells (MAIT). In such an example, after the scaffold is coated with fetal bovine serum protein, PBMCs are seeded into the bioreactor, monocytes adhere to the scaffold, and the remaining cells are suspended in the medium. Non-adherent cells remain suspended within the bioreactor in the mobile phase and can enter and exit the packed bed chamber to create cell-cell interactions with the adherent cells.

[0063] After seeding the immune cells in step 316, a determination is made as to whether gene editing is to be performed on the expanded cell population (step 318). If gene editing is not performed, the process proceeds to step 326.

[0064] Step 326 describes the growth phase of the target cells where environmental parameters such as, for example, agitation speed, pH, dissolved oxygen, temperature, etc. are controlled. In this step, the bioreactor is constantly heated and a pre-set gas mixture is supplied to the system to maintain the desired conditions within a predetermined desired range. When activation occurs by cells seeded, APCs loaded with ligands, or scaffolds coated with activators (antibodies), an infectious state is simulated within the bioreactor and a series of mechanisms are triggered. This results in clonal expansion of antigen-specific immune cells, during which the antigen-specific immune cells proliferate massively and up to 90% of the total immune cells can become antigen-specific. To support the rapid growth of immune cells, appropriate nutrient supply and removal of inhibitory metabolites are carried out without disturbing the local microenvironment. In certain embodiments, the bioreactor may operate in batch mode, fed-batch mode, and / or perfusion mode. In another embodiment of the present invention, a perfusion system (TFF, ATF, BioSep, etc.) may be connected to the bioreactor. By using a perfusion system, the medium can be exchanged from the bioreactor without extracting cells from the system. The perfusion system can remove the conditioned medium from the system, separate the suspended cells from the conditioned medium, and at the same time, supply fresh medium to the bioreactor based on level electrodes, weight, pre-set flow rates, or manual instructions. In this example, the conditioned medium is sampled daily, the number of suspended cells and the concentration of essential substrates in the medium are measured based on cell mass and nutrient concentration, and the amount of fresh medium to be supplied is calculated.

[0065] In step 328, to harvest the target cells, the medium is drained from the system together with the immune cells, and the washing step preferably continues outside the bioreactor system. In this embodiment, the medium exchange step can be performed using a batch centrifuge or a continuous flow centrifuge (kSep, unifuge, etc.). After the medium exchange, in step 332, the cells or some of the cells can be returned to the bioreactor to continue growth in fresh growth medium. Alternatively, the harvested cells can be transferred in step 330 to the downstream processing of the immune cells for final product manufacturing.

[0066] When gene editing is performed on target cells, in step 322, immune cells are genetically modified to add or delete specific properties. Modifications such as the addition of new targets or activation receptors, the deletion of specific receptors for self-recognition, and the deletion of unnecessary target receptors are used to transform immune cells for better treatment of various indications. In this step (322), gene modifiers may be inserted into the bioreactor by non-viral factors such as liposomes (e.g., Lipofectamine), polymers (e.g., PEI), etc., or by the electroporation process described later, or by viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or others. These gene modifiers can carry RNA or DNA constructs that can add or delete new data to the genome using methods such as CRISPR / Cas9, transposomes (Sleeping Beauty, PiggyBac), and DNA binding domains (e.g., zinc finger domains). In these embodiments, gene modifiers such as viral vectors are added to the bioreactor medium after the activation of the scaffold coating (312, 314) and cell seeding (316) and during cell growth (326). At this stage, stirring and other environmental parameters such as pH and temperature may be adjusted to prepare the gene modifier to penetrate the cells better. After addition, an incubation period is provided under appropriate conditions to allow the gene modifier to penetrate the cells.

[0067] Furthermore, in some optional embodiments, the activated immune cells are harvested from the bioreactor according to step (328) and transferred aseptically to an electroporation or other 2D flask device to allow the penetration of the gene modifier into the cells by electroporation or viral and non-viral methods in step 322. In this embodiment, after the incubation period, the immune cells can be re-seeded into the bioreactor to perform a washing step, or the washing step can be performed in a 2D flask.

[0068] After the incubation period, the washing step 324 is initiated. During the washing step 324, the medium in the bioreactor is exchanged several times to extract the gene modifier from the system. In one embodiment of the present invention, a perfusion system (such as TFF, ATF, BioSep, etc.) is connected to the bioreactor. By using the perfusion system, the medium in the bioreactor can be exchanged without extracting the cells from the system. The perfusion system discharges the old medium, and at the same time, new medium is injected into the bioreactor according to the level electrode, weight, or manual instruction. This process may be carried out for several chamber volumes until no gene modifier is found in the extracted medium. Alternatively, after cell growth (step 326), the medium with cells and the gene modifier is discharged or harvested from the system in step 328, and the washing step continues outside the bioreactor. In this embodiment, the medium exchange step can be performed using a batch centrifuge or a continuous flow centrifuge (e.g., kSep, Unifuge). After the medium exchange, the cells may be reseeded into the starting bioreactor or a new bioreactor (332) for further growth.

[0069] Once the proliferation duration is reached or the concentration of immune cells required for the desired final product or for seeding the cells into a larger bioreactor is reached, the cell harvesting steps (328, 340) are carried out. In one embodiment, the harvesting step is carried out by simply draining the medium from the system, with or without agitation. In another embodiment, after a packed bed basket is connected to a harvesting system described in detail in WO2012 / 140519 by the same applicant, which is incorporated herein by reference in its entirety, a slow vibration of the scaffold basket is carried out during the draining step to release the suspended immune cells captured from the niches of the packed bed scaffold formed without causing physical damage to the cells. Furthermore, in all embodiments of the harvesting step, a cycle of refilling and draining the medium may be carried out to collect all the cells from the system. In each cycle, the extracted cells are drained into a sterile collection element for further processing.

[0070] In a further embodiment of the invention, at the end of the harvesting steps (328, 340), the cells or a portion of the harvested cells are further downstream processed (330, 342) for final product manufacture. This step includes concentration and washing steps carried out using different systems such as continuous flow centrifuges, filters, acoustic filtration devices, etc. After the concentration and washing steps, a specific cell collection step using various separation methods and / or final formulation of the product is carried out, followed by filling of the final product into its final packaging (vials or cryobags).

[0071] Furthermore, in another embodiment of the present invention, after the harvesting step, the harvested immune cells or a partial portion of the harvested cells are used for further expansion (332). The further expansion is performed by executing step 316 in an existing bioreactor or by transferring the cells to a larger-scale bioreactor, such as a 1.5 L bioreactor equipped with a 30 g scaffold, a 3.5 L bioreactor equipped with a 100 g scaffold, or a 10 L bioreactor equipped with a 375 g scaffold. Furthermore, the purpose of the new bioreactor can only be utilized to grow using an ECM-coated scaffold (step 334) or to perform another activation step to reactivate the cells. The reactivation can be performed, for example, using the same activator (step 336) in the new bioreactor or using a new activator in the new bioreactor (step 338). In any of these embodiments, the new bioreactor is pre-prepared according to the aforementioned steps 310 - 314.

[0072] In another optional embodiment, the reactivation of the target cells can be performed in the first bioreactor or a new bioreactor by adding soluble activators such as transact or anti-CD3 and anti-CD28 antibodies, or by adding soluble antigens that can be presented by APCs if the APCs are already placed in the bioreactor.

[0073] Figures 3A - 3D are schematic partial front views of a packed-bed bioreactor at different stages of the immune cell culture process. Figure 3A shows the setup stage of a system in which only the uncoated porous scaffold 10 is included in the packed-bed basket 116 of the bioreactor 100.

[0074] More specifically, the upper and lower boundaries of the packed bed chamber 116 are each constituted by a perforated disk 132 having a plurality of holes 1321 of a predetermined diameter. The perforated disk 132 is hereinafter also referred to as "disk", "grid", "upper grid", "lower grid", "intermediate grid", and these are all used interchangeably and can refer to the upper, middle, or lower walls of the basket 416. As described in detail with reference to FIGS. 2(1) and 2(2) above, in some optional embodiments, a porous scaffold may be inserted into the basket 116 and may occupy a partial or most of the volume of the basket. At this stage, the bioreactor 100 may not contain liquid. The basket 116 is preferably connected to one or more vibrating rods 136 configured to enable vertical movement of the basket 116. The vibration of the basket 116 may be used to harvest target cells during or at the end of the growth process, as described in FIGS. 2(1) and 2(2). The basket 116 has a wall 1161 independent of the wall 40 of the bioreactor 100, allowing movement above and below the basket while maintaining the flow direction of the medium containing target cells only through the upper and lower disks 132. Since the basket 116 is located above the bottom of the bioreactor within the bioreactor 100, the upper section 122 and the lower section 126 delimit the intermediate section 124 containing the packed bed basket 116.

[0075] Figure 3B shows a packed bed basket 116 containing a coated scaffold 10' and a liquid medium 50. To create an environment suitable for mimicking the natural environment of an immune cell population, the porous scaffold must first be coated with an ECM coating as described in detail in FIGS. 2(1), 2(2) and the examples of the present invention above. After the ECM coating is performed, the porous scaffold 10' may be further coated or bound with at least one antibody 1022 or an antibody presenting cell (APC) 1033 as shown in FIG. 3C. After the preparation of the porous scaffold is complete, as shown in FIG. 3D, immune cells 1044 can be seeded into the bioreactor. As shown in FIG. 3D, the immune cells are seeded with the flow of the medium within all sections of the bioreactor and can be found in the upper section 122, the lower section 126, and the middle section 124 within the basket 116. Thus, the porous coated scaffold forms a low shear stress niche that mimics the natural environment of the immune cells, allowing for optimal growth of the immune cells 1044. Further, when exposed to an antigen and / or APC, the immune cells 1044 are activated as previously described.

[0076] In certain embodiments, the immune cells 1044 are incubated in a bioreactor 100 on an APC mimicking scaffold (APC-MS). In further embodiments, the immune cells are harvested from the porous coated scaffold and then incorporated into a pharmaceutical composition.

[0077] In any further optional embodiments, the immune cells are gently agitated and seeded, for example, in the case of a packed bed or a solid state scaffold, to promote an even distribution. In the case of microcarriers, the APC-MS and T cells are gently suspended and gently mixed. In either case, after seeding, perfusion and agitation are stopped for a period of time to allow interaction between the T cells and the APC-MS and to promote subsequent activation.

[0078] Figures 4(1) to 4(4) are flow cytometry diagrams of the activation and proliferation of T cells after 7 days of growth in a packed-bed bioreactor, where the activation and proliferation of the cells were measured on day 0, day 5, and day 7. The distribution of the cell population was analyzed by a flow cytometer CytoFLEX including three lasers (405 nm, 488 nm, 638 nm) and 13 fluorescence detection channels. TM It was analyzed by

[0079] PMBC cells were seeded into a packed bed of a bioreactor containing a Fibra-Cel® carrier with immobilized antibodies (anti-CD3 and anti-CD28 antibodies) that provide activation signals for T cells. The activation of T cells was evaluated on day 0, day 5, and day 7 by measuring the expression of CD69 (an inducible cell surface marker expressed upon activation via the TCR) and CD25 (the α-chain of the IL-2 receptor), which are activation markers commonly associated with the activation of T cells. The results obtained showed successful stimulation of the activation and proliferation of T lymphocytes by the increase in the CD3 marker from -44% on day 0 to -91% on day 7. These results indicated, in particular, that T lymphocytes had proliferated over the 7-day period. The upregulation of CD69 and CD25 from -4%, -8% on day 0 to -42%, 90% on day 7 indicated the activation of T cells. Furthermore, the fact that no change was observed in the cell markers in the sample of all cells collected after the harvest step on day 7 indicated that no other cell populations were present in the bioreactor. Moreover, the fact that no change was observed in the levels of the activation markers suggested that the cell harvest had no effect on the cell state.

[0080] CD69 is an inducible cell surface marker expressed upon activation via the TCR or the IL-2 receptor (CD25). It plays a role in the proliferation and survival of activated T lymphocytes.

[0081] Figures 5A(1) to 5A(4) are flow cytometry diagrams of the activation and proliferation of MAIT cells grown in a packed bed bioreactor for 10 days. The activation and proliferation of the cells were measured on day 0, day 5, day 7, and day 10. The distribution of the cell population was analyzed by the flow cytometer CytoFLEX TM and analyzed by

[0082] The mononuclear cells of IVB were seeded into a packed bed bioreactor containing an ECM-coated Fibra-Cel® carrier that mimics the natural environment and promotes the adhesion of APCs. The activation of MAIT cells was induced by 5-OP-RU antigen and IL-15. The activation and proliferation of MAIT cells were evaluated at several time points on day 0, day 5, day 7, and day 10. The population of MAIT cells was detected by the expression of CD3, Vα7.2, and CD161 markers. The results showed an increase in the proportion of MAIT cells starting from 22.6% on day 0 to a maximum of 96.26% on day 10. Furthermore, the expression of the activation markers CD69 and CD25 increased from day 0 to day 7 and decreased by day 10.

[0083] Figures 5B(1) to 5B(4) are flow cytometry diagrams showing the growth of MAIT cells grown for an additional 7 days in a second bioreactor after transferring the cells from the first bioreactor to the second bioreactor on day 10. Cells that reached their maximum growth capacity on day 10 in the first bioreactor were harvested, and then approximately 30% of these cells were seeded into a second bioreactor with a similar design to the first bioreactor. The cells were grown for an additional 7 days. The flow cytometry results showed that the proportion of MAIT cells was similar for most of the period but decreased from over 90% on day 14 to 82% on day 17. The upregulation of the CD69 marker expression from 30% to 87% on day 17 indicates that MAIT cells maintained the activation signal, while CD25 decreased gradually as expected.

[0084] Figure 6 is a graph showing the cell distribution inside and outside the bioreactor packed bed of three types of immune cell types (Jurkat, PBMC, and MAIT cells) on different growth days over two days. It is shown as a percentage.

[0085] The characteristics of the Fibra-Cel® disk and the packed bed structure enable the formation of a niche within the bioreactor system that mimics the natural environment of the cells, allowing the cells to remain inside for a certain period with low shear stress. Figure 6 shows the distribution of cells inside and outside the bioreactor packed bed. These values were calculated based on the cell numbers before and after harvesting of the packed bed, which was performed with several washing steps and vibration of the packed bed. The results indicate that at least 40% of the viable cells are inside the packed bed for all three specified types of immune cell types at different packed bed harvesting time points during the growth period.

[0086] Since a significant decrease in cell concentration was observed several hours after cell seeding, it can be assumed that the following cell distribution model is maintained throughout the cell growth period.

[0087] Figure 7 is a schematic diagram of packed-bed bioreactors of different sizes, showing the high scalability of the system of the present invention. According to any embodiment of the present invention, the initial seeding of the immune cell population is carried out in a mini-packed-bed bioreactor 400 having a mini-basket 416, and the total maximum volume of the mini bioreactor 400 is defined by its container dimensions 490. After cell growth in bioreactor 400, the cells or parts of the cells may be reseeded into a larger-sized bioreactor 500 having a packed-bed basket 516 larger than basket 416, which allows for more porous scaffolds and greater cell growth compared to bioreactor 400, and the total volume of bioreactor 500 is large and determined by its container dimensions 590. The same process is carried out for a larger bioreactor 600 having a basket 616 and a container 690, and then continues until reaching the largest bioreactor 700 having the largest basket 716 and container dimensions 790, thereby enabling multiple-fold expansion of immune cells. According to the foregoing methods and examples, it can be understood that the conditions for the growth and activation of the immune cell population in each bioreactor may be the same as or different from the previous growth session in a smaller bioreactor. At the end of the process, large-scale expansion of immune cells activated by the same or different activating agents is obtained.

Example

[0088] Reference is now made to the following examples, which describe specific embodiments in a non-limiting manner together with the above description.

Example

[0089] Growth of Jurkat cells in a packed-bed bioreactor.

[0090] After a 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® disks was assembled, it was autoclaved by steam sterilization at a temperature of 122.5 °C and a pressure 1 bar higher than atmospheric pressure for 30 minutes. Subsequently, the MiniBio reactor was connected to an Applikon MiniBio control station.

[0091] The Fibra-Cel® disks were supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) and 0.1% of 50 mg / ml gentamicin in RPMI-1640 medium and pre-incubated at 37 °C for approximately 24 hours. During incubation, serum proteins interacted electrostatically with the hydrophilic end groups of the Fibra-Cel® disks, creating an extracellular matrix (ECM) coating on the Fibra-Cel® that mimicked the natural environment of the cells.

[0092] 81.6×10 6 cells were thawed in RPMI-1640 medium supplemented with 10% HI-FBS and 0.1% of 50 mg / ml gentamicin, and the thawed cells were diluted to a target concentration of 0.24×10 6 cells / ml at seeding. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37 °C, dissolved oxygen (DO) 80%, pH 7.4, and agitation speed 150 rpm, and the total volume in the bioreactor reached 340 ml.

[0093] On day 3 of the culture, 25% of the medium was refreshed (i.e., 25% fresh medium was added). On day 4 of the culture, the cells were harvested from the packed-bed bioreactor for very short-term large-scale growth. The total cell number reached 830×10 6 cells, showing a 10.4-fold growth (results are detailed in Table 1). The distribution of cells inside and outside the packed bed is shown in Figure 6.

Example

[0094] Activation, proliferation, and harvesting of peripheral blood mononuclear cells (PBMCs) in a packed-bed bioreactor.

[0095] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and separated by filtration and density gradient medium Lymphoprep TM (Ficoll). Red blood cells (erythrocytes) were removed by RBC X1 lysis buffer. The isolated population was cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) cryopreservation solution.

[0096] After assembling a 0.5 L packed-bed MiniBio bioreactor containing 2.5 grams of Fibra-Cel® disks, autoclaving by steam sterilization was performed at a temperature of 122.5 °C and a pressure 1 bar higher than atmospheric pressure for 30 minutes. Subsequently, the MiniBio bioreactor was connected to an Applikon MiniBio control station.

[0097] Both Fibra-Cel® disks were coated with monoclonal anti-CD3 (OKT3) antibody and anti-CD28 (CD28.2) antibody, which provide co-stimulatory signals involved in the T cell receptor. The amount of each activator was calculated based on 0.21 μg / cm diluted in PBS 2 . The final solution was incubated at 100 rpm for 3 hours at room temperature (RT) and 1.5 hours at 37 °C.

[0098] After incubation with the activator, the Fibra-Cel® disks were followed by a blocking treatment and further incubated at 100 rpm for 1 hour at RT with 1% BSA solution. Subsequently, the Fibra-Cel® disks were drained and washed with PBS at 150 rpm for 10 minutes.

[0099] Subsequently, the packed-bed bioreactor was prepared for culturing and equilibrated at 37°C for approximately 24 hours in a growth medium supplemented with 10% HI-FBS, 1% sodium pyruvate (100 mM), and 0.1% 50 mg / ml gentamicin in RPMI-1640 medium. During this incubation, serum proteins interact electrostatically with the hydrophilic end groups of the Fibra-Cel® disks, creating an ECM coating on Fibra-Cel® that mimics the natural environment of the cells.

[0100] 262×10 6 cells were thawed in RPMI-1640 medium supplemented with 10% HI-FBS, 1% sodium pyruvate (100 mM), IL-2 (100 U / L), and 0.1% 50 mg / ml gentamicin, and the thawed cells were diluted to a target concentration of 0.97×10 6 cells / ml at seeding. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm, and the total volume in the bioreactor reached 270 ml. At seeding, the cells were distributed between the packed bed and the "external" environment within the bioreactor, and a decrease in cell concentration was observed 3 hours after seeding.

[0101] During the growth period, the growth medium and cell suspension (in the "external" environment) were sampled daily to measure pH, cell concentration (by Vi-Cell), cell metabolic activity based on nutrient consumption (by Cedex bio analyzer), and the distribution of the cell population by flow cytometer (CytoFLEX TM ).

[0102] Medium replacements were performed at 5.5%, 32%, and 20% on days 3, 5, and 6, respectively. On day 7 of the culture, since the maximum growth capacity was reached, the cells were harvested from the packed-bed bioreactor. The total cell number was 1073×10 6It reached individual cells, 91% were CD3 positive, and showed 8.5-fold growth. The ratios of different cell populations in the culture and their variations over time are shown in FIGS. 4(1) to 4(4). The distribution of cells inside and outside the packed bed is shown in FIG. 6.

Example

[0103] Culture of Mucosal Associated Invariant T cells (MAIT) in a packed bed bioreactor: activation, proliferation, harvesting, regrowth and secondary harvesting.

[0104] Peripheral blood mononuclear cells were isolated from human placental venous blood (IVB) and separated by filtration and density gradient medium Lymphoprep TM (Ficoll). Red blood cells (erythrocytes) were removed with RBC X1 lysis buffer. The isolated population was cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) cryopreservation solution.

[0105] After assembling a 0.5 L packed bed MiniBio reactor containing 2.5 grams of Fibra-Cel® disks, autoclaving by steam sterilization was performed at a temperature of 122.5 °C and a pressure 1 bar higher than atmospheric pressure for 30 minutes. Then, the MiniBio reactor was connected to an Applikon MiniBio control station.

[0106] Fibra-Cel® disks were supplemented with 10% HI-FBS in RPMI-1640 medium and incubated at 37 °C for about 24 hours. During incubation, serum proteins interacted electrostatically with the hydrophilic end groups of Fibra-Cel® disks, creating an ECM coating on Fibra-Cel® that mimics the natural environment of cells.

[0107] 300±20×10 6 cells were thawed in 4Cell® Nutri-T GMP medium supplemented with 1% L-glutamine 200 mM and 0.1% of 50 mg / ml gentamicin, and the thawed cells were adjusted to a target concentration of 1×10 6It was diluted to cells / ml. The prepared cell suspension was seeded into a bioreactor system set at the following conditions: temperature 37°C, DO 80%, pH 7.4, and stirring speed 100 rpm, and reached a final volume of 300 ml. At the time of seeding, the cells were distributed between the packed bed and the "external" environment within the bioreactor, and a decrease in cell concentration was observed 3 hours after seeding.

[0108] Three hours after seeding, MAIT cells were activated via the T cell receptor (TCR). The activation pathway via the TCR requires co-stimulatory signals including recognition of riboflavin metabolites derived from microorganisms presented on the MHC class I-like molecule MR1 and co-stimulation by CD28, TLR agonists, bacterial products, or cytokines.

[0109] The initial cell population isolated from human placental venous blood IVB contains various antigen-presenting cells (APCs) such as dendritic cells, monocytes, and B cells, which can activate MAIT cells via MR1. The microbial-derived riboflavin intermediate 5-OP-RU was added to the growth medium at a concentration of 250 nM, presented on MR1 by APCs, and recognized by the TCR of MAIT cells. Furthermore, IL-15 at a concentration of 50 ng / ml was also added to the growth medium to stimulate MAIT cells (via the IL-15R expressed on MAIT cells) to produce IFN-γ and release granzyme B and perforin.

[0110] During the growth period, the growth medium and cell suspension were sampled daily to measure the pH, cell concentration (by Vi-Cell), the metabolic activity of the cells based on nutrient consumption (by Cedex TM bio analyzer), and the distribution of the cell population by flow cytometer (CytoFLEX TM ).

[0111] The cells were cultured in a packed-bed bioreactor for 10 days, and the medium was renewed at 5%, 5.2%, and 100% on the 3rd, 5th, and 7th days, respectively. On the 10th day of culture, since the maximum growth capacity was reached, the cells were harvested from the packed-bed bioreactor. The total cell number reached 1089×10 6 cells, with 94% being MAIT cells (Vα7.2 positive, CD161 high), showing a 43.5-fold proliferation. The ratios of different cell populations in the culture and their variations over time are shown in Figures 5A(1) to 5A(4).

[0112] After harvesting the cells from the bioreactor on the 10th day of culture, further cell proliferation was investigated. An additional 0.5L packed-bed MiniBio reactor was assembled and prepared as before. This is a sterile system containing 2.5 grams of Fibra-Cel® disks, which were pre-incubated at 37°C for about 24 hours with RPMI-1640 supplemented with 10% HI-FBS to provide an ECM coating on the Fibra-Cel® disks and create a natural environment for the re-seeded cells.

[0113] After ECM coating, the packed-bed bioreactor was pre-equilibrated for culture using a growth medium composed of 4Cell® Nutri-T GMP medium supplemented with 1% L-glutamine 200 mM and 0.1% gentamicin 50 mg / ml. Of the harvested cells, 295×10 6 cells were seeded into the growth medium and diluted to a target concentration of approximately 1×10 6 cells / ml. The prepared cell suspension was seeded into a bioreactor system set at the following conditions: temperature 37°C, DO 80%, pH 7.4, and stirring speed 100 rpm, reaching a final volume of 300 ml. To induce the proliferation of MAIT cells, IL-15 was added to the growth medium at a concentration of 50 ng / ml.

[0114] During the growth period, the growth medium and cell suspension were sampled daily to measure pH, cell concentration (by Vi-Cell), cell metabolic activity based on nutrient consumption (by Cedex bio analyzer), and the distribution of the cell population by flow cytometer (CytoFLEX TM ).

[0115] The cells were further cultured in a packed-bed bioreactor for 7 days, and medium replacements of 26.5% and 100% were performed on days 12 and 14 of the culture, respectively. On day 17 of the culture, the cells were harvested from the packed-bed bioreactor. The total cell number reached 490×10 6 cells, with 88% being MAIT cells (Vα7.2 positive, CD161 high), showing a 1.5-fold increase. The relative proportion of MAIT cells in the culture and its variation over time are shown in Figures 5B(1) to 5B(4).

[0116] Table 1 below summarizes the initial and final total viable cell numbers, relative population proportions, and growth multiples during the growth period for three different immune cell types (Jurkat cells, PBMCs, and MAIT cells).

[0117]

Table 1

[0118] As shown in Table 1, all growth multiples exceed 1, and cell growth has been confirmed in all three predetermined immune cell types and even during the secondary growth period of MAIT cells. Furthermore, Table 1 shows changes in the balance of the cell population, with the proportion of target cells exceeding 94% at the end of the investigated growth period (T cells after 7 days of growth, MAIT cells after 10 days of growth). During the secondary growth period of MAIT cells, a slight decrease in the proportion of MAIT cells is shown (from 94% to 86%).

Example

[0119] Proliferation, activation, and harvesting of B cells derived from peripheral blood mononuclear cells (PBMCs) in a packed-bed bioreactor.

[0120] Peripheral blood mononuclear cells (PBMCs) are isolated from human peripheral blood and separated by filtration and density gradient medium Lymphoprep TM (Ficoll). Red blood cells (erythrocytes) are removed by RBC X1 lysis buffer. The isolated population is cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) cryopreservation solution.

[0121] After a 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® disks is assembled, autoclaving by steam sterilization is performed at a temperature of 122.5 °C and a pressure 1 bar higher than atmospheric pressure for 30 minutes. Subsequently, the MiniBio reactor is connected to an Applikon MiniBio control system.

[0122] Fibra-Cel® disks are coated with anti-CD40 antibody to provide an activation signal. The amount of activator is calculated based on 1 μg / cm diluted with PBS 2 . The final solution is incubated at 100 rpm for 3 hours at RT and 1.5 hours at 37 °C. After incubation with the activator, Fibra-Cel® disks are blocked and additionally incubated at 100 rpm for 1 hour at RT with 1% BSA solution. Subsequently, Fibra-Cel® disks are drained and washed with PBS at 150 rpm for 10 minutes.

[0123] Fibra-Cel® disks are pre-incubated at 37 °C for approximately 24 hours in RPMI1640 medium supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin. During this incubation, serum proteins interact electrostatically with the hydrophilic end groups of Fibra-Cel® disks to create an ECM coating on Fibra-Cel® that mimics the natural environment of cells.

[0124] 300×106 Individual cells are thawed in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, 100 IU / ml penicillin-streptomycin, human recombinant IL-4 (10 ng / ml) and IL-21 (10 ng / ml). The thawed cells are diluted to a target concentration of 1×10 6 cells / ml at the time of seeding. The prepared cell suspension is seeded into a bioreactor system set to the following conditions: temperature 37°C, DO 80%, pH 7.4, and stirring speed 100 rpm so that the total volume in the bioreactor reaches 300 ml. At the time of seeding, the cells are distributed between the "external" environment surrounding the packed bed and the packed bed basket in the bioreactor and sampled daily to measure pH, cell concentration (by Vi-Cell), and cell metabolic activity.

[0125] During the growth period, the growth medium and cell suspension (in the "external" environment) are based on nutrient consumption (by Cedex bio analyzer) and the distribution of the cell population by flow cytometer (CytoFLEX TM ).

[0126] The medium is renewed by 50% on the 4th and 6th days. On the 8th day of culture, the cells are harvested from the packed bed bioreactor.

Example

[0127] Activation, proliferation and harvesting of iNKT cells derived from peripheral blood mononuclear cells (PBMC) in a packed bed bioreactor.

[0128] Peripheral blood mononuclear cells (PBMC) are isolated from human peripheral blood and separated by filtration and density gradient medium Lymphoprep TM (Ficoll). Red blood cells (erythrocytes) are removed by RBC X1 lysis buffer. The isolated population is cryopreserved in HI-FBS and DMSO cryopreservation solution.

[0129] After a 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® disks is assembled, it is autoclaved by steam sterilization at a temperature of 122.5 °C and a pressure 1 bar higher than atmospheric pressure for 30 minutes. Thereafter, the MiniBio reactor is connected to an Applikon MiniBio control system.

[0130] The Fibra-Cel® disks are supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin in RPMI1640 medium and pre-incubated at 37 °C for about 24 hours. During incubation, serum proteins interact electrostatically with the hydrophilic end groups of the Fibra-Cel® disks to create an ECM coating on the Fibra-Cel® that mimics the natural environment of the cells.

[0131] 600×10 6 cells are thawed in RPMI1640 medium supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 0.1 mM MEM non-essential amino acids, 5.5 μM β-mercaptoethanol, 100 IU / ml penicillin-streptomycin, and 100 IU / ml human recombinant IL-2. The thawed cells are diluted to a target concentration of 2×10 6 cells / ml at the time of seeding. The prepared cell suspension is seeded into a bioreactor system set to the following conditions: temperature 37 °C, DO 80%, pH 7.4, and agitation speed 100 rpm such that the total volume in the bioreactor reaches 300 ml. At the time of seeding, the cells are distributed between the packed bed and the "external" environment within the bioreactor. Three hours after seeding, the activation step is initiated and 100 ng / ml of α-galactosylceramide is added to the bioreactor medium to be presented on CD1d located on antigen-presenting cells (derived from the PBMC population) attached to the Fibra-Cel® disks.

[0132] During the growth period, the growth medium and the cell suspension (in the "external" environment) are sampled daily to measure the pH, cell concentration (by Vi-Cell), cell metabolic activity based on nutrient consumption (by Cedex bio analyzer), and the distribution of the cell population by a flow cytometer (CytoFLEX TM ).

[0133] The medium is renewed by 70% on the 3rd and 6th days. On the 7th day of culture, the cells are harvested from the packed bed bioreactor.

Example

[0134] Activation, proliferation, and harvesting of γδ T cells derived from peripheral blood mononuclear cells (PBMC) in a packed bed bioreactor.

[0135] Peripheral blood mononuclear cells (PBMC) are isolated from human peripheral blood and separated by filtration and density gradient medium Lymphoprep TM (Ficoll). Red blood cells (erythrocytes) are removed by RBC X1 lysis buffer. The isolated population is cryopreserved in HI-FBS and DMSO cryopreservation solution.

[0136] After assembling a 0.5 L packed bed MiniBio reactor containing 2.5 grams of Fibra-Cel® disks, autoclaving by steam sterilization is performed at a temperature of 122.5 °C and a pressure 1 bar higher than atmospheric pressure for 30 minutes. Then, the MiniBio reactor is connected to an Applikon MiniBio control station.

[0137] The Fibra-Cel® disks are supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin in RPMI1640 medium and pre-incubated at 37 °C for about 24 hours. During incubation, serum proteins interact electrostatically with the hydrophilic end groups of the Fibra-Cel® disks, creating an ECM coating on the Fibra-Cel® that mimics the natural environment of the cells.

[0138] 300×10 6 cells are thawed in RPMI 1640 medium supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 0.1 mM MEM non-essential amino acids, 50 μm β-mercaptoethanol, 100 IU / ml penicillin-streptomycin, and 300 IU / mL IL-2. The thawed cells are diluted to a target concentration of 1×10 6 cells / ml at the time of seeding. The prepared cell suspension is seeded into a bioreactor system set to the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm so that the total volume in the bioreactor reaches 300 ml. At the time of seeding, the cells are distributed between the packed bed and the "external" environment within the bioreactor. Three hours after seeding, the activation step is initiated and 5 μM zoledronic acid is added to the bioreactor medium.

[0139] During the growth period, the growth medium and cell suspension (in the "external" environment) are sampled daily to measure pH, cell concentration (by Vi-Cell), cell metabolic activity based on nutrient consumption (by Cedex bio analyzer), and the distribution of the cell population by flow cytometer (CytoFLEX TM ).

[0140] The medium is renewed by 50% on days 4, 7, 10, and 13. On day 14 of the culture, the cells are harvested from the packed bed bioreactor.

Example

[0141] Activation, proliferation, and harvesting of natural killer (NK) cells derived from peripheral blood mononuclear cells (PBMC) in a packed bed bioreactor.

[0142] Natural killer (NK) cells are isolated from human peripheral blood mononuclear cells (PBMCs) using RosetteSep (STEMCELL Technologies; usually >95% CD56+CD3-) and separated by filtration and density gradient medium Lymphoprep TM (Ficoll). Red blood cells (erythrocytes) are removed by RBC X1 lysis buffer. The isolated population is cryopreserved in HI-FBS and DMSO cryopreservation solution.

[0143] A 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® disks is assembled and then autoclaved by steam sterilization at a temperature of 122.5 °C and a pressure 1 bar higher than atmospheric pressure for 30 minutes. Thereafter, the MiniBio reactor is connected to an Applikon MiniBio control station.

[0144] Fibra-Cel® disks are supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin in RPMI1640 medium and pre-incubated at 37 °C for about 24 hours. During incubation, serum proteins interact electrostatically with the hydrophilic end groups of the Fibra-Cel® disks to create an extracellular matrix (ECM) coating on Fibra-Cel® that mimics the natural environment of the cells.

[0145] 900×10 6 Individual NK cells are thawed in RPMI1640 medium supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 0.1 mM MEM non-essential amino acids, 100 IU / ml penicillin-streptomycin. The thawed cells are seeded at a target concentration of 3×10 6It is diluted to cells / ml. The prepared cell suspension is seeded into a bioreactor system set to the following conditions: temperature 37°C, DO 80%, pH 7.4, and stirring speed 100 rpm so that the total volume in the bioreactor reaches 300 ml. At the time of seeding, the cells are distributed between the packed bed and the "external" environment in the bioreactor. For the pre-activation treatment, the medium is supplemented with human recombinant IL-12 (10 ng / mL), IL-18 (50 ng / mL), and IL-15 (50 ng / mL) and cultured for 16 ± 2 hours, and then, after a washing step, cultured in a growth medium supplemented with human recombinant IL-15 (1 ng / mL).

[0146] During the growth period, the growth medium and the cell suspension (in the "external" environment) are sampled daily to measure pH, cell concentration (by Vi-Cell), the metabolic activity of the cells based on nutrient consumption (by Cedex bio analyzer), and the distribution of the cell population by a flow cytometer (CytoFLEX TM )

[0147] The medium is renewed by 30% on the 4th and 7th days. On the 8th day of the culture, the cells are harvested from the packed bed bioreactor.

[0148] It can be seen that certain features of the present invention are described in the context of individual embodiments for clarity, but can also be provided in combination in a single embodiment. Similarly, various features of the present invention are described in the context of a single embodiment for brevity, but can also be provided separately or in any suitable sub-combination.

[0149] Although the present invention has been described in connection with specific embodiments of the invention, it will be recognized by those skilled in the art that many alternatives, modifications, and variations are apparent. Accordingly, the present invention is intended to embrace alternatives, modifications, and variations that fall within the spirit and broad scope of the claims and the description. All publications, patents, and patent applications, and GenBank accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application, or GenBank accession number were specifically and individually indicated to be incorporated by reference. Further, any citation or identification of a document in this application should not be construed as an admission that such document is available as prior art to the present invention.

Claims

1. A three-dimensional (3D) bioreactor for the large-scale expansion of immune cells, comprising: a. At least one packed bed chamber containing at least one porous scaffold; b. At least one porous scaffold coated with one or more extracellular matrix proteins (ECM); c. At least one container containing a flowing medium, the flowing medium being configured to flow through the packed bed chamber having at least one porous coated scaffold; d. At least one population of immune cells suspended in the flowing medium, wherein the at least one porous scaffold coated with the ECM is configured to form a stationary niche having a low shear force that mimics the natural growth environment of immune cells; A 3D bioreactor that enables large-scale expansion of the population of immune cells flowing through the coated porous scaffold.

2. The 3D bioreactor according to claim 1, wherein the at least one porous scaffold is further coated or linked with at least one immune cell activator.

3. The 3D bioreactor according to claim 2, wherein the immune cell activator is either an antigen-presenting cell (APC) loaded or not loaded with an antigen, or an antigen directly bound to the coated porous scaffold.

4. The 3D bioreactor according to any one of the preceding claims, wherein the expanded immune cells are further activated in the packed bed chamber by exposing the population of immune cells to the at least one porous coated scaffold bound to the immune cell activator.

5. The 3D bioreactor according to any one of the preceding claims, wherein the expanded immune cells are further activated in the packed bed chamber by exposure to a soluble immune cell activator in suspension and further expanded by at least one porous ECM-coated scaffold.

6. The 3D bioreactor according to any one of the preceding claims, wherein the population of immune cells is harvested or reactivated by exposing APCs attached to at least one coated porous scaffold to an antigen, thereby generating additional activation signals for the population of immune cells.

7. The 3D bioreactor according to any one of the preceding claims, wherein the immune cell population is harvested or reactivated by transferring the expanded immune cells to another bioreactor containing at least one porous scaffold coated with a different or similar immune cell activator.

8. The 3D bioreactor according to any one of the preceding claims, wherein the at least one porous scaffold is either a single porous scaffold matrix expanded within the internal space of the packed bed chamber or a plurality of mini or micro porous scaffolds filling the packed bed chamber.

9. The 3D bioreactor according to any one of the preceding claims, wherein the immune cell population is genetically modified using a gene modifier added to the bioreactor medium.

10. A method for large-scale expansion of immune cells in a three-dimensional (3D) bioreactor, comprising: a. inserting at least one porous scaffold into at least one packed bed chamber; b. coating the at least one porous scaffold with one or more extracellular matrix proteins (ECM); c. circulating a fluid medium from at least one container, the fluid medium being configured to flow through the packed bed chamber having the at least one porous coated scaffold; d. suspending at least one immune cell population in the circulated fluid medium, wherein the at least one porous scaffold coated with the ECM forms a fixed niche having a low shear force that mimics the natural growth environment of immune cells and is configured to enable large-scale expansion of the immune cell population flowing through the at least one porous scaffold.

11. After step b, coating the at least one porous scaffold with at least one immune cell activator to proliferate and activate the immune cell population within the packed bed chamber; After step d, further comprising exposing the immune cell population to the at least one activator. The method according to claim 10.

12. The method according to claim 11, further comprising genetically modifying immune cells within the 3D bioreactor using a gene modifier added to the fluid medium.

13. The method according to any one of claims 10 to 12, further comprising the step of harvesting immune cells or a part of cells, and further proliferating and reactivating the immune cell population in the same bioreactor or a different bioreactor.

14. The method according to claim 13, further comprising the step of performing genetic modification outside the system after the step of harvesting the immune cells, and then reseeding the genetically modified immune cells into the same bioreactor or a different bioreactor.

15. A three-dimensional (3D) bioreactor for large-scale proliferation and activation of an immune cell population, comprising: a. At least one packed bed chamber containing at least one porous antigen-presenting cell mimicking scaffold (APC-MS); b. At least one APC-MS coated with one or more extracellular matrix proteins (ECM); c. At least one container containing a flowing medium, the container being configured such that the flowing medium flows through the coated porous APC-MS; d. At least one immune cell population suspended in the flowing medium, wherein the at least one APC-MS forms a fixed microenvironment with low shear force that mimics the natural growth environment of the immune cell population, enabling large-scale proliferation and / or activation of the immune cell population flowing through the microenvironment.

16. The 3D bioreactor according to claim 15, wherein the immune cell population is reactivated by exposing the coated porous APC-MS to an antigen, so as to generate an additional activation signal for the immune cell population.

17. The 3D bioreactor according to claim 15 or 16, wherein the immune cell population is reactivated by transferring cells to different bioreactors containing at least one coated porous APC-MS having different or similar antigens.

18. The 3D bioreactor according to any one of claims 15 to 17, wherein the at least one porous APC-MS is a single unit expanded within the internal space of the packed bed chamber or a plurality of mini / micro porous APC-MS filling the packed bed chamber.

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