Systems and methods for large scale expansion and activation of immune cells

Abstract

Three-dimensional (3D) bioreactors for large scale expansion of immune cells and methods of using the same are disclosed. The 3D bioreactor comprises: at least one packed bed chamber comprising 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 configured to flow through the packed bed chamber having at least one porous coated scaffold; and at least one population of immune cells suspended in a fluid medium wherein the at least one porous scaffold coated with the ECM creates a fixed niche with low shear force that mimics the natural growth environment of the immune cells and allows large scale expansion of the population of immune cells flowing through the coated porous scaffold.

Description

Technical Field

[0001] The present invention relates to systems and methods for large-scale cultivation and / or activation of immune cells. More specifically, the present invention relates to large-scale cultivation and / or activation of immune cells in a packed bed bioreactor. Background Art

[0002] The cultivation of mammalian cells is complex in principle, due to their high sensitivity, relatively slow proliferation, complex differentiation process and absolutely sterile basic conditions. Large-scale cell culture is always a challenging process. Even small-scale cell growth requires attention and specific knowledge, because serious problems are often encountered. For example, during the cell culture preparation or testing process, the cell culture is subjected to stress or damage, and therefore, any analysis based on cultured cells may show results that are at least partially the consequences of such damage. In addition, applying the conclusions drawn from the results obtained on damaged cells to in vivo situations or using cells for immunotherapy may lead to fatal consequences. In addition, damage or stress conditions are not reproducible between single cell cultures and may affect the growth of cells. These challenges become even more critical when large-scale cell culture is carried out. Various attempts have been made in recent decades. U.S. Patent Application No. 2006 / 0194320 describes a device and method for culturing cells and / or tissues that simulates the cell structure and immune function of immunoactive tissues, however, the system and method are limited to a volume of 4 ml. Other patents and patent applications related to the field of the present invention are US 8911995 and US Patent No. 10472612.

[0003] In addition, the use of bioreactors for cell and tissue culture is well known. https: / / www.minerva-kg.de / libraryonline / upload / files / file_6400.pdf A detailed overview of bioreactor design, prototyping, and process control for reproducible three-dimensional tissue culture is provided in .

[0004] Cultivating immune cells in vitro is a further challenge. In recent years, the human immune system has been used as the basis for therapeutic technologies that can recognize and kill tumor cells and has been a core target of anticancer immunotherapy. Therefore, there is increasing interest in improving the effectiveness and accessibility of this technology so that it can be widely used in adoptive cell therapy (ACT) such as chimeric antigen receptor T (CAR-T) cells, tumor infiltrating lymphocytes (TIL), dendritic cells (DC), natural killer (NK) cells, etc. However, in order to implement this technology, the current available systems for culturing immune cells cannot meet the needs because they either damage the cells, have low efficiency, or have limited ability to scale up, and some require very high costs (see: Ganeeva I. et al. 2022 "Recent Advances in the Development of Bioreactors for Manufacturing of Adoptive Cell Immunotherapies", Bioengineering 2022, 9, 808. https: / / doi.org / 10.3390 / bioengineering9120808 ). In this article, the main culture methods for culturing immune cells known in the art so far and their advantages and disadvantages are reviewed. In more detail, a comparison is made between a stirred flask, a G-Rex flask, a rocking motion bioreactor, a stirred tank bioreactor, a hollow fiber bioreactor, and a CliniMACS Prodigy. Therefore, there is an urgent need for a scalable, cost-effective, and GMP-compliant bioreactor for culturing immune cells.

[0005] The present invention aims to provide systems and methods for large-scale culture and / or activation of immune cell populations. Summary of the invention

[0006] In one main aspect, the present invention relates to large-scale systems and methods for culturing and / or activating immune cells. The challenges that need to be addressed for efficient large-scale growth of immune cells are to avoid or minimize high shear stresses that may damage the cells to obtain high-scale homogenous systems, and to create conditions that allow for interaction between cells and activators and transfection agents, thereby creating physical niches and conditions that allow for high cell-cell interactions.

[0007] The present invention discloses, in one aspect, a method and system for growing immune cells, which consists of a porous stationary phase located in a flowing media. The stationary phase can be located in a bioreactor, or can be located in a separate chamber functionally connected to the bioreactor. The porous elements, as described in detail below, are located in a basket of a packed bed bioreactor and are immovable and do not move with the liquid flow around them and through them. The stationary phase can be implemented in various forms and is intended to create an environment with low flow and low shear.

[0008] As used herein, the terms "porous stationary phase", "porous scaffold", "porous element" and "porous coated scaffold" all refer to the same meaning and can be used interchangeably in the following description. In some embodiments, the porous stationary phase is coated with an extracellular matrix (ECM), and it can be further coated with an immune system cell activator to activate and expand immune cells. The combination of all components creates a niche for immune system cells that mimics their natural environment in tissues and lymph nodes.

[0009] As used herein, the terms "activator" and "immune cell activator" all refer to the same meaning and can be used interchangeably in the following description. In some embodiments, "activators" and "immune cell activators" are antigen presenting cells loaded with antigens presented on the cell surface. In another embodiment, the activator is an antibody against an activating receptor on the surface of an immune cell. In yet another embodiment, the activator is an antigen conjugated to a molecule that can be presented to an activating receptor on the surface of an immune cell.

[0010] The term "niche" as used herein refers to a stationary phase having pores, such as but not limited to scaffolds, beads and supports that allow liquids and particles to flow through. The particles can be cells, or other components, synthetic or natural.

[0011] The niche created mimics the lymph nodes / tissue in terms of the microenvironment in which immune cells naturally grow, allowing for optimal cell growth. Additionally, because the niche created mimics the natural environment of the cell, it allows for activation of the cell such that only certain cells respond to the activation, thereby allowing for some selection of desired cells.

[0012] Furthermore, the created niches allow for large-scale growth of immune cells while also maintaining relatively low shear forces to minimize cell damage.

[0013] The terms "media" and "medium" are intended to be synonymous and may be used interchangeably hereinafter.

[0014] The terms "packed bed chamber," "packed bed basket," "basket," "grow basket" are all intended to be synonymous and may be used interchangeably hereinafter.

[0015] Reference herein to "growth" of cells or cell populations is intended to be synonymous with expansion of the cell population, culturing of the cell population, with or without activation of the cells.

[0016] As used herein, the terms "immune cell", "immune cell population" and "lymphocyte" all refer to the same meaning and may be used interchangeably in the following description.

[0017] In certain embodiments, lymphocytes are expanded without substantial differentiation.In various embodiments, the expansion is described on a 2D substrate, on a 3D substrate, or on a 2D substrate followed by a 3D substrate.

[0018] In some embodiments, the lymphocytes are cultured 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 a generally sterile device (bioreactor) in which the cells are maintained under controlled conditions, as will be described below with reference to Figure 1 described.

[0019] Reference herein to "activation" of an immune cell or population of immune cells is intended to be synonymous with exposure of the immune cell to an antigen that results in a change in cell morphology and triggers an immune response detected by rapid proliferation and secretion of a wide variety of cytokines and chemokines.

[0020] Therefore, in one main aspect, the present invention relates to a three-dimensional (3D) bioreactor for large-scale expansion of immune cells, comprising: a) at least one packed bed chamber including 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 fluid culture medium, the fluid culture medium being configured to flow through the packed bed chamber having at least one porous coated scaffold; and d) at least one immune cell population suspended in the fluid culture medium; wherein the at least one porous scaffold coated with the ECM is configured to create a fixed niche with low shear forces that mimics the natural growth environment of the immune cells and allows large-scale expansion of the immune cell population flowing through the coated porous scaffold.

[0021] At least one porous support may be further coated with or connected with at least one immune cell activator. In some optional embodiments, the immune cell activator is an antigen presenting cell (APC) loaded with antigen or not loaded with antigen or directly with one of the antigens conjugated to the coated porous support. In the case where APC is not loaded with antigen, antigen can be presented at a later stage as needed to activate immune cells.

[0022] In a further option, after exposing the immune cell population to at least one porous coated scaffold conjugated to an immune cell activator, the expanded immune cells can be further activated within the packed bed chamber.

[0023] In another option, the expanded immune cells can be further activated within the packed bed chamber after exposure to a suspended soluble immune cell activating agent and further expanded with at least one porous ECM coated scaffold.

[0024] In some embodiments of the invention, the expanded and / or activated immune cell population is harvested or reactivated after exposing the APCs attached to at least one coated porous scaffold to an antigen to generate additional activation signals to the immune cell population.

[0025] Additionally, in some further embodiments, the immune cell population is harvested or reactivated by transferring the expanded immune cells to a different bioreactor comprising at least one porous scaffold coated with a different or similar immune cell activating agent.

[0026] The porous scaffold may be a single porous scaffold matrix extending within the interior space of the packed bed chamber, or it may be a plurality of mini porous scaffolds or micro porous scaffolds filling the packed bed chamber.

[0027] In some further optional embodiments of the present invention, the immune cell population is genetically modified by using a genetic modification agent incorporated into the bioreactor culture medium.

[0028] The present invention further relates to 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 culture medium from at least one container, the fluid culture medium being configured to flow through the packed bed chamber comprising at least one porous coated scaffold; and d) suspending at least one immune cell population in the circulating fluid culture medium; wherein the at least one porous scaffold coated with the ECM is configured to create a fixed niche with low shear forces that mimics the natural growth environment of the immune cells and allows large-scale expansion of the immune cell population flowing through the at least one porous scaffold.

[0029] The method may further include the step of coating at least one porous scaffold with at least one immune cell activator after the step of coating the scaffold with the ECM, and the step of exposing the immune cell population to at least one activator after suspending the at least one immune cell population in the circulating fluid culture medium to expand and activate the immune cell population within the packed bed chamber.

[0030] The method may further include the step of genetically modifying the immune cells within the 3D bioreactor using a genetic modification agent incorporated into the fluid culture medium.

[0031] In some optional embodiments, the method further comprises the step of harvesting the immune cells or a portion of the cells and further expanding and reactivating the immune cell population in the same bioreactor or in a different bioreactor.

[0032] Additionally or alternatively, the above method may further comprise the step of harvesting the immune cells, followed by the step of genetically modifying the cells outside the system, and then re-seeding the genetically modified immune cells into the same bioreactor or a different bioreactor.

[0033] In addition, in a further aspect, the present invention is intended to provide a three-dimensional (3D) bioreactor for large-scale expansion and activation of immune cell populations, comprising: a) at least one packed bed chamber comprising at least one porous antigen presenting cell mimetic scaffold (Antigen Presenting Cell Mimetic 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 fluid culture medium, the fluid culture medium being configured to flow through the coated porous APC-MS; and d) at least one immune cell population suspended in the fluid culture medium; wherein the at least one APC-MS creates a fixed microenvironment with low shear forces that mimics the natural growth environment of the immune cell population and allows large-scale expansion and / or activation of the immune cell population flowing therethrough.

[0034] The immune cell population may be reactivated after exposing the coated porous APC-MS to an antigen to generate additional activation signals to the immune cell population.

[0035] In some optional embodiments, the immune cell population is reactivated by transferring the cells to a different bioreactor comprising at least one coated multiporous APC-MS with a different or similar antigen.

[0036] According to an embodiment of the present invention, the at least one porous APC-MS may be made of a single unit expanded within the inner space of a packed bed chamber, or it may be a plurality of micro-porous APC-MS / micro-porous APC-MS filling a packed bed chamber.

[0037] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, the patent specification including definitions shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention is described herein by way of example only with reference to the accompanying drawings. Where specific reference is now made in detail to the drawings, it is emphasized that the details are shown by way of example and are presented only for the purpose of illustrative discussion of embodiments of the invention and in order to provide a useful and understandable description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a basic understanding of the invention, and the description in conjunction with the drawings makes it apparent to those skilled in the art how several forms of the invention may be implemented in practice.

[0039] In the attached picture:

[0040] Figure 1 is a schematic diagram of an optional packed bed bioreactor for growing, activating and harvesting immune cell populations according to embodiments of the present invention.

[0041] Figure 2 is a flow chart describing at a high level the sequence of major steps in preparing and using a packed bed bioreactor to activate, expand and harvest immune cells.

[0042] Figures 3A-3D yes Figure 1 Schematic partial elevation view of a packed bed bioreactor 100 at different stages of the growth process of an immune cell population, wherein Figure 3A An initial stage is shown in which the packed bed basket contains uncoated stents; Figure 3B A packed bed basket containing a coated scaffold and filled with an ECM protein solution is shown; Figure 3C It shows Figure 3B The coated stent is further connected to a packed bed basket of at least one type of immune cell activator; and Figure 3D A packed bed bioreactor is shown in which a coated scaffold is connected to one or more activating agents and immune cells flow in a culture medium.

[0043] Figure 4 Figure 2 is a flow cytometric plot of T cell activation and proliferation after 7 days of growth in a packed bed bioreactor, where activation and proliferation of cells were measured at days 0, 5, and 7.

[0044] Figure 5A Flow cytometric plots of activation and proliferation of MAIT cells grown in a packed bed bioreactor for 10 days, where activation and proliferation of cells were measured on days 0, 5, 7, and 10.

[0045] Figure 5B is a flow cytometric graph of MAIT cell growth after cells were transferred from the first bioreactor to the second bioreactor on day 10 and grown for an additional 7 days in the second bioreactor.

[0046] Figure 6 Schematic representation of the cell distribution inside and outside of the bioreactor packed bed for three different immune cell types: Jurkat, PBMC, and MAIT cells.

[0047] Figure 7 is a schematic diagram of packed bed bioreactors of different sizes, illustrating the high scalability of the system of the present invention. DETAILED DESCRIPTION

[0048] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited to the details set forth in the following description or exemplified by the examples in its application. The present invention can have other embodiments or practice or carry out in various ways. In addition, it should be understood that the wording and terminology adopted herein are for the purpose of description and should not be regarded as restrictive.

[0049] The present invention aims to provide systems and methods for large-scale expansion and activation of various immune cell populations.

[0050] In one main aspect, the present invention provides a 3D bioreactor for large-scale expansion of immune cells, comprising: a) at least one packed bed chamber comprising 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 fluid culture medium, the fluid culture medium being configured to flow through the packed bed chamber having at least one porous coated scaffold; and d) at least one immune cell population suspended in the fluid culture medium; wherein the at least one porous scaffold coated with the ECM is configured to create a fixed niche with low shear forces that mimics the natural growth environment of immune cells and allows large-scale expansion of the immune cell population flowing through the coated porous scaffold.

[0051] In a further aspect, the present invention provides 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 culture medium from at least one container, the fluid culture medium being configured to flow through the packed bed chamber comprising at least one porous coated scaffold; and d) suspending at least one immune cell population in the circulating fluid culture medium; wherein the at least one porous scaffold coated with the ECM is configured to create a fixed niche with low shear forces that mimics the natural growth environment of the immune cells and allows large-scale expansion of the immune cell population flowing through the at least one porous scaffold.

[0052] In addition, in a further aspect, the present invention provides a 3D bioreactor for large-scale expansion and activation of immune cell populations, comprising: a) at least one packed bed chamber including 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 culture medium, the fluid culture medium being configured to flow through the coated porous APC-MS; and d) at least one immune cell population suspended in the fluid culture medium; wherein the at least one APC-MS creates a fixed microenvironment with low shear forces that mimics the natural growth environment of the immune cell population and allows large-scale expansion and / or activation of the immune cell population flowing therethrough.

[0053] The main aspects of the present invention and optional modes of practicing the present invention will be better understood through the following detailed description of various exemplary non-limiting drawings and embodiments described below. Referring now to the drawings:

[0054] Figure 1 is a schematic cross-sectional view of an optional packed bed bioreactor 100 for growth, activation, and harvesting of immune cell populations according to an embodiment of the present invention.

[0055] In the depicted embodiment, a growth and vibration chamber 116 (hereinafter referred to as a "basket") is loaded with at least one porous support 10. The terms "support" and "support" may be used interchangeably and both refer to a structure configured as described in reference to FIG. Figure 2The basket 116 is a porous element coated as described in detail and creates a fixed niche within the basket that reduces shear forces and simulates the natural environment of the immune cell population. The basket wall 1161 is preferably separated from the bioreactor inner wall 40 and can move up and down. The bioreactor 100 is a liquid culture medium via an inlet tube 120, which is configured and operable to deliver various culture media into the bioreactor and then optionally autoclaved. In other embodiments, after sterilization, the liquid is replaced with a growth medium that fills the basket 116 and its contents. The basket 116 actually divides the fluid within the bioreactor 100 into three main parts: an upper part 122, which mainly contains fresh culture medium inserted via the inlet tube 120, a middle part 124, which contains culture medium within the basket 116 surrounding the coated scaffold, and a lower part 126, which mainly contains culture medium flowing through the basket and is generally lacking in nutrients consumed by the immune cells in the middle part (where their flow rate is reduced as they flow through at least one porous coated scaffold, interact with other cells, and / or become activated) and is enriched in compounds and debris secreted by the immune cells compared to the fresh culture medium at the upper part 122. In the embodiment described herein, the culture medium at the upper part 122 is stirred by an impeller 119, which produces movement of the fluid as shown by arrows 109. In still further embodiments, various parameters such as temperature, pH, dissolved oxygen concentration, etc. are set at the beginning of the process as a system setup procedure (see below). Figure 2 The process is described in detail below and is adjusted to suit the suspension conditions. In yet further embodiments, a slow initial stirring rate of the culture medium is used to promote cell adhesion to the coated scaffold, and then the stirring rate can be increased. If desired, cells can be harvested from the culture medium for final product manufacture or for further expansion of immune cells, as described in detail below ( Figure 2). In some embodiments, the rotation of the impeller 119 creates a negative pressure in the flow conduit 150, which pulls the culture medium with cells from the lower portion 126 through the flow conduit 150 and then into the upper portion 122 through the impeller 119 port, thereby circulating the culture medium and immune cells uniformly in a continuous loop in the direction according to arrow 109. In still further embodiments of the present invention, adjustments to the culture medium can be made by monitoring various parameters via electrodes 106 to control various parameters of the culture medium. In some optional embodiments, a ring sparger (not visible) is located inside the impeller aeration chamber 11 for oxygenating the culture medium flowing through the impeller 119 port via gas added from an external port 103 and a sparger line 7, and the external port 103 can be maintained inside the housing 5. In some other optional embodiments, gas can be added through the inlet 120. Alternatively, the injected gas can be confined in the distal chamber and absorbed by the nutrient medium that flushes the system. In some optional embodiments, a water jacket 117 covers the culture medium area within the bioreactor 100, with ports for moving jacket water in 13 and out 14. A removal tube 110 is disposed along the bioreactor and has an opening in the lower culture medium portion 126 to allow, if desired, harvesting of immune cells from the culture medium below the basket 116. The removal tube 110 can also be used to remove debris and refresh the culture medium after partial removal of spent culture medium and addition of fresh culture medium.

[0056] In some embodiments, a continuous stirred tank bioreactor can be used, wherein the culture medium is continuously fed into the bioreactor and the product is continuously extracted to maintain a time-constant steady state in the bioreactor. A stirred tank bioreactor with a fiber bed basket is available from, for example, New Brunswick Scientific Co., Edison, NJ). Other bioreactors that can be used are, for example, but not limited to, fixed bed bioreactors, perfusion bioreactors with multi-active foams, radial flow perfusion bioreactors including tubular poly-L-lactic acid (PLLA) porous supports, and any other bioreactors known in the art suitable for the purposes of the present invention. "Fixed bed bioreactor" refers to a bioreactor in which the cell growth substrate is generally not raised from the bottom of the incubation container in the presence of a growth medium. For example, the substrate can have sufficient density to prevent it from being raised and / or it can be filled by mechanical pressure to prevent it from being raised. The substrate can be a single body or multiple bodies. Typically, the substrate remains substantially in place during the standard stirring rate of the bioreactor. In some embodiments, a plurality of carriers are loosely filled, for example, forming a loosely packed bed immersed in a nutrient medium.

[0057] In addition, in certain embodiments, a perfusion bioreactor is used, wherein the perfusion chamber comprises a 3D substrate. In certain embodiments, the 3D substrate is in the form of a porous support 10. The porous support can be made of a single large unit that surrounds the entire volume or most of the volume of the packed bed chamber. Alternatively, the porous support used can be a plurality of microparticles. In some further optional embodiments, the porous support can be, for example, a macro carrier, a microcarrier, or a mixture thereof. Non-limiting examples of commercially available carriers include alginate-based (GEM, Global Cell Solutions), dextran-based ( GE Healthcare), collagen-based Percell Biolytica) and polystyrene-based (SoloHill Engineering) microcarriers.

[0058] In certain embodiments, T cells are cultured in a bioreactor having an "APC-MS", where the term "APC-MS" as used herein refers to a scaffold attached to or associated with a lymphocyte activation moiety (in a more specific embodiment, it can be any scaffold mentioned herein), and a more specific embodiment thereof is a fiber support or mesoporous silica micro-rod attached to or coated with an activation moiety.

[0059] Unless otherwise indicated, the term packed bed bioreactor refers to a bioreactor in which the cell growth substrate does not normally rise from the bottom of the incubation vessel in the presence of a growth medium. For example, the substrate may have sufficient density to prevent it from being lifted and / or it may be filled by mechanical pressure to prevent it from being lifted. The substrate may be a single body or multiple bodies. Typically, the substrate remains substantially in place during perfusion at the standard perfusion rate of the bioreactor. In certain embodiments, this definition does not exclude that the substrate may be lifted at an abnormally fast perfusion rate (e.g., greater than 200rpm).

[0060] In other embodiments, the biocontainer is used to expand cells, and in further embodiments, it is suitable for suspension culture. In various embodiments, the biocontainer is used and / or suitable for batch culture, fed-batch culture or continuous culture.

[0061] Figure 2 is a flow chart describing at a high level the sequence of major steps in the preparation and use of a packed bed bioreactor system for expanding, activating and harvesting immune cell populations according to embodiments of the present invention.

[0062] The system setup step 310 includes assembly of the system, filling the bioreactor basket with the required amount of porous scaffold, connecting the required electrodes for monitoring and controlling culture parameters (e.g., pH, dissolved oxygen, temperature) during use of the system, connecting the necessary tubing for fresh nutrient supply and byproduct removal, sealing and performing integrity testing, and sterilizing the system by steam sterilization in an autoclave. After sterilization, the system is connected to the bioreactor control station and the electrodes are calibrated.

[0063] The porous scaffold, also denoted as "carrier" hereinafter, can be made of natural or synthetic materials and can have various sizes. Some non-limiting examples of commercially available carriers include alginate-based (GEM, Global Cell Solutions), dextran-based ( GE Healthcare), collagen-based Percell Biolytica) and polystyrene-based carriers (SoloHill Engineering). Alternatively or additionally, the scaffold may include a fibrous material, optionally an adhesive fibrous material, which may be, for example, a woven fiber matrix, a nonwoven fiber matrix, or one of the two. A non-limiting example of a fiber scaffold is a carrier comprising a polyester mesh, such as New Brunswick Scientific Co. TM Plate, available from Eppendorf TM AG, Germany, and includes a polypropylene support; and a microporous carrier, such as BioNOC TM II carrier, commercially available from CESCO BioProducts (Atlanta, GA) and made of PET (polyethylene terephthalate). In certain embodiments, the fiber matrix mentioned includes polyester, polypropylene, polyalkylene, polyfluorochloroethylene, polyvinyl chloride, polystyrene or polysulfone. In more specific embodiments, the fiber matrix is ​​selected from polyester and polypropylene.

[0064] In order 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 on the disc make it possible to coat the scaffold with different materials by simply immersing the scaffold in a solution containing ECM proteins to generate electrostatic interactions. A detailed description of the effects of the extracellular matrix on immune cells is provided in Sutherland TE et al., "The extracellular matrix and the immune system: A mutually dependent relationship", Science. 2023 Feb. 17; 379(6633); PMID: 36795835. It should be clear that other scaffolds can be used and described A disk is a non-limiting example. Additionally, coating of the scaffold may be performed with natural ECM components or synthetic ECM components.

[0065] In this way, for example, different 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, in order to simulate the structure of lymph nodes, the scaffold can be coated with type III collagen, which is the main building block of the reticular fibers as the main component of lymph node ECM. In addition, different organs have different ECM protein combinations and therefore different combinations or concentrations can be used for coating. In different embodiments, adhesion molecules can be conjugated to the protein of the scaffold or the scaffold coating to promote stronger interaction between immune cells and the scaffold coated with ECM. For example, E-selectin, P-selectin, ICAM1, ICAM2 and VCAM1 are known in the art to be promoters of interaction between leukocytes and ECM, and help guide leukocytes to inflamed tissues. Based on this, coating the scaffold with these molecules can increase the interaction between leukocytes and the scaffold coated with ECM. Optionally, ECM coating (step 312) occurs after system set up (step 310), and is followed by an activation coating step of the scaffold (step 314). In various embodiments, (depending on the product and its activating ligand) the activation coating step of the porous scaffold (step 314) occurs after ECM coating (step 312).

[0066] Step 314 describes the part of the stent coated with immune cell activation ligand. Part 314 can be performed before or after 312. Immune cells need to be activated by antigen presentation that usually occurs in lymph nodes or infected tissues. By coating the stent with ECM components and providing antigen presentation in a low shear environment, the niche created simulates the natural environment in which activation occurs. In one embodiment, after coating the ECM with serum ECM proteins, adherent or semi-adherent cells can be attached to the fibers that constitute the stent. Since some immune cells need to interact with antigens presented by antigen presenting cells (APC) in order to be activated, the APC loaded with antigens is inoculated during step 314 after the coating step 312. APC adheres to the stent and can present antigens to different immune cells, such as T cells, B cells and all subgroups thereof. In different embodiments, APC can be inoculated into a bioreactor and adhere to the stent in a non-antigen presentation form, and then after cell adhesion, the antigen can be incorporated into the culture medium that fills the bioreactor in order to present to immune cells such as T cells and B cells. A more specific example of this embodiment is to use monocytes to activate mucosal-associated invariant T cells (MAIT). In this example, PBMCs are inoculated into a bioreactor after coating the scaffold with fetal bovine serum albumin, monocytes adhere to the scaffold and the remaining cells are suspended in the culture medium. After the initial cell inoculation, 5-OP-RU (5-(2-oxopropylamino)-6-D-ribitol aminouracil) (antigen for activation of MAIT) is incorporated into the culture medium so that it is presented by monocytes and specifically activates MAIT cells. In a different embodiment of the present invention, the activation of immune cells can be regulated by antigens conjugated to a scaffold without APC. Antigens can be antibodies to specific activation receptors on immune cells, proteins recognized by activation receptors such as TCR. For example, the scaffold is coated with monoclonal anti-CD3 (OKT3) antibodies and anti-CD28 (CD28.2) antibodies to provide a co-stimulatory signal that binds to the T cell receptor. After the incubation period, a blocking step is performed, followed by a washing step. After the washing steps, coating of the scaffolds with fetal bovine serum albumin was performed. When the culture medium was changed and PBMCs were seeded into the bioreactor, the cells were activated through interaction with the ECM and antibody-coated scaffolds.

[0067] Another example is the use of MR1 monomers, tetramers and other forms loaded or not loaded with 5-OP-RU to activate MAIT cells. When MR1 not loaded with 5-OP-RU is used, the incorporation of 5-OP-RU occurs after performing blocking and washing steps.

[0068] Step 316 describes the part of the inoculation of the cells including the target cells for activation and amplification. In this step, immune cells from different sources can be used for inoculation of target cells, i.e., PBMCs collected from blood apheresis, PBMCs separated from specific organs or tumors. Immune cells can use fresh or frozen. In this step, immune cells are inoculated into a bioreactor. Using different environmental parameters, such as but not limited to stirring speed, pH, dissolved oxygen, temperature, etc., cells can adhere or remain suspended in a bioreactor. Adherent cells can enter the packed bed chamber, interact with the ECM proteins on the coated support, and create a microstructure (cellularorganized microstructure) of cell organization inside the packed bed. According to this embodiment, APC can be inoculated into a bioreactor and adhere to the support in a non-antigen presentation mode, and create an environment simulating lymph nodes until activation occurs. A more specific example of this embodiment is to use monocytes to activate mucosal associated constant T cells (MAIT). In such an example, after the scaffold is coated with fetal bovine serum albumin, PBMCs are seeded in a bioreactor, monocytes adhere to the scaffold and the remaining cells are suspended in the culture medium. Non-adherent cells can remain suspended in the bioreactor (in the mobile phase) and migrate into or out of the packed bed chamber to generate cell-to-cell interactions with adherent cells.

[0069] After the immune cells are inoculated in step 316, it is necessary to decide whether to perform gene editing on the expanded cell population (step 318). If gene editing is not performed, go to step 326.

[0070] Step 326 describes the expansion phase of the target cells, wherein environmental parameters such as stirring speed, pH, dissolved oxygen, temperature, etc. are controlled. In this step, the bioreactor is continuously heated and a preset gas mixture is provided to the system to maintain the desired conditions within a predetermined desired range. Once the cells are inoculated and activation occurs, whether by APCs with loaded ligands or by a scaffold coated with an activator (antibody), an infection state is simulated in the bioreactor and a series of mechanisms are triggered, which leads to clonal expansion of antigen-specific immune cells, during which antigen-specific immune cells are greatly expanded and up to 90% of the total immune cells may become antigen-specific. In order to support the rapid expansion of immune cells, sufficient nutrients are supplied and inhibitory metabolites are removed without disturbing the local microenvironment. In certain embodiments, the bioreactor can be operated in batch mode, fed batch mode and / or perfusion mode. In another embodiment of the present invention, a perfusion system (TFF, ATF, BioSep, etc.) can be connected to the bioreactor. Using a perfusion system, the culture medium from the bioreactor can be replaced without extracting cells from the system. The perfusion system allows the removal of conditioned medium from the system, the separation of suspended cells from the conditioned medium and the simultaneous supply of fresh medium to the bioreactor by command from a level electrode, by weight, according to a preset flow rate or manually. In this example, the conditioned medium is sampled daily to measure the number of suspended cells and the concentration of essential substrates in the medium, and the amount of fresh medium to be supplied is calculated based on the amount of cells and the concentration of nutrients.

[0071] In step 328, the culture medium is discharged from the system for harvesting target cells together with the immune cells and the washing step is preferably continued outside the bioreactor system. In this embodiment, the culture medium replacement step can be performed using an intermittent centrifuge or a continuous flow centrifuge (kSep and unifuge, etc.). After the culture medium is replaced, the cells or a portion of the cells can be returned to the bioreactor in step 332 to continue to expand with fresh growth medium. Alternatively, the harvested cells can be transferred to the downstream processing of the immune cells in step 330 to manufacture the final product.

[0072] If the target cell is gene edited, then in step 322, immune cells are genetically modified to add or delete specific traits. Use modifications such as adding new target receptors or activating receptors, or deleting specific receptors for self-recognition or deleting unwanted target receptors to transform immune cells for better treatment of different indications. In this step (322), gene modifiers can be inserted into bioreactors by non-viral agents such as liposomes (lipofectamine, etc.), polymers (PEI, etc.) or electroporation processes as described below, or by viral vectors such as retroviruses (Retrovirus), slow viruses (Lentivirus), adenoviruses (Adenovirus), adeno-associated viruses (Adeno-Associated virus) or other. These gene modifiers can be loaded with the following RNA or DNA constructs, which can use methods such as CRISPR / Cas9, transposons (Sleeping Beauty, PiggyBac) and DNA binding domains (zinc finger domains, etc.) to add new data to the genome or delete. In these embodiments, a genetically modified agent, such as a viral vector, is incorporated into the bioreactor culture medium after scaffold coating (312, 314), cell seeding (316) activation, and during cell expansion (326). At this stage, agitation and other environmental parameters such as pH and temperature may be altered to prepare the cells for better penetration by the genetically modified agent. After incorporation, an incubation period is performed under appropriate conditions to allow the genetically modified agent to penetrate into the cells.

[0073] In addition, in some optional embodiments, the activated immune cells can be harvested from the bioreactor according to step (328) and aseptically transferred to an electroporation or other 2D flask device to allow the genetic modification agent to penetrate 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 for a wash step or in a 2D flask for a wash step.

[0074] After the incubation period, the washing step 324 is started. During the washing step (324), the culture medium in the bioreactor is replaced several times to extract the gene modifier from the system. In one embodiment of the present invention, a perfusion system (TFF, ATF and BioSep, etc.) is connected to the bioreactor. Using the perfusion system, the culture medium from the bioreactor can be replaced without extracting cells from the system. The perfusion system transfers out the old culture medium and simultaneously injects the new culture medium into the bioreactor by instructions from a liquid level electrode, weight or manually. This process can be performed for several chamber volumes until no gene modifier is found in the extracted culture medium. Alternatively, the culture medium with cells and gene modifiers is discharged or harvested from the system in step 328 after the expansion of the cells (step 326), and the washing step is continued outside the bioreactor. In this embodiment, the culture medium replacement step can be completed by using an intermittent centrifuge or a continuous flow centrifuge (e.g., kSep, Unifuge). After the culture medium is replaced, the cells can be re-inoculated into the starting bioreactor or a new bioreactor (332) for further expansion.

[0075] After reaching the amplification duration or reaching the desired final product or the immune cell concentration required for inoculating the cells to a larger scale bioreactor, a cell harvesting step (328, 340) is performed. In one embodiment, the harvesting step is performed by simply discharging the culture medium from the system with or without stirring. In different embodiments, the packed bed basket is connected to a harvesting system as described in detail in WO 2012 / 140519 by the same applicant, which is incorporated herein by reference in its entirety, and then during the discharge step, the slow vibration of the support basket is performed to release the captured suspended immune cells from the created packed bed support niche without causing physical damage to the cells. In addition, in all embodiments of the harvesting step, a cycle of refilling and discharging the culture medium can be performed to collect all cells from the system. The extracted cells are discharged into a sterile collection element for further processing in each cycle.

[0076] In a further embodiment of the invention, at the end of the harvesting step (328, 340), the cells or a portion of the harvested cells are subjected to further downstream processing (330, 342) to produce the final product. This step includes concentration and washing steps performed using different systems such as continuous flow centrifuges, filters, acoustic filtration devices, etc. The concentration and washing steps are followed by specific cell collection steps using a variety of separation methods and or final formulation of the product before the final product is packaged in its final packaging (vials or freezer bags).

[0077] In addition, in different embodiments of the present invention, after the harvesting step, the harvested immune cells or a portion of the harvested cells are used for further expansion (332). Further expansion is accomplished by performing step 316 in an existing bioreactor or transferring the cells to a larger scale bioreactor, such as a 1.5L bioreactor with a 30g scaffold, a 3.5L bioreactor with a 100g scaffold, or a 10L bioreactor with a 375g scaffold. In addition, the purpose of the new bioreactor can be to expand only using the ECM coated scaffold (step 334), or to perform another activation step to reactivate the cells. Reactivation can be accomplished, for example, with the same activator in a new bioreactor (step 336), or it can be performed in a new bioreactor with a new activator (step 338). In these two optional embodiments, the new bioreactor is prepared in advance according to steps 310 to 314 above.

[0078] In various optional embodiments, reactivation of the target cells can be performed in the first bioreactor or in 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 they are already in the bioreactor.

[0079] Figures 3A-3D is a schematic partial elevation view of a packed bed bioreactor at different stages of the immune cell culture process. Figure 3A A set-up phase of the system is shown in which the packed bed basket 116 of the bioreactor 100 contains only uncoated porous scaffolds 10 .

[0080] In more detail, the top and bottom boundaries of the packed bed chamber 116 are formed by perforated discs 132 each having a plurality of holes 1321 of a predetermined diameter. The perforated discs 132 are also hereinafter referred to as “discs”, “grids”, “upper grids”, “lower grids”, “middle grids”, which may be used interchangeably and refer to the upper wall, middle wall or lower wall of the basket 416. In some optional embodiments, as described above with reference to Figure 2 As described in detail, the porous scaffold is inserted into the basket 116 and can occupy part or most of the volume of the basket. At this stage, the bioreactor 100 can be free of liquid. The basket 116 is preferably connected to one or more vibration rods 136 configured to allow vertical movement of the basket 116. Vibration of the basket 116 can be used to Figure 2The target cells are harvested during or at the end of the expansion process. The basket 116 has a wall 1161 separated from the wall 40 of the bioreactor 100 to allow the basket to move up and down while maintaining the flow direction of the culture medium with the target cells only through the upper and lower trays 132. The basket 116 is located above the bottom of the bioreactor in the bioreactor 100, so that the upper part 122 and the lower part 126 define the middle part 124 including the packed bed basket 116.

[0081] Figure 3B A packed bed basket 116 containing a coated scaffold 10' and filled with a liquid culture medium 50 is shown. In order to create an environment suitable for simulating the natural environment of an immune cell population, as described above Figure 2 As described in detail in the embodiments of the present invention, the porous scaffold should first be coated with an ECM coating. After the ECM coating, the porous scaffold 10' can be further coated or connected to at least one type of antibody 1022 or antibody presenting cell (APC) 1033, such as Figure 3C After the porous scaffold is prepared, immune cells 1044 can be seeded in a bioreactor, such as Figure 3D As shown. Figure 3D As shown, immune cells are seeded with the flow of culture medium within all portions of the bioreactor and can be found in the upper portion 122, lower portion 126, and middle portion 124 within the basket 116 because the porous coated scaffold creates a niche with low shear forces that mimics the natural environment of immune cells, thereby allowing for optimal expansion of immune cells 1044. Additionally, upon exposure to antigens and / or APCs, immune cells 1044 are activated as previously described.

[0082] In certain embodiments, immune cells 1044 are incubated on an APC-mimicking scaffold (APC-MS) in a bioreactor 100. In further embodiments, the immune cells are subsequently harvested from the porous coated scaffold for incorporation into a pharmaceutical composition.

[0083] In further optional embodiments, for example in the case of a packed bed or solid scaffold, immune cells are inoculated under gentle agitation to promote uniform distribution. In the case of microcarriers, APC-MS and T cells are gently suspended and gently mixed. In either case, after inoculation, perfusion and agitation are stopped for at least a period of time to allow interaction between T cells and APC-MS and subsequent activation.

[0084] Figure 4 Figure 2 is a flow cytometric plot of T cell activation and proliferation after 7 days of growth in a packed bed bioreactor, where activation and proliferation of cells were measured at days 0, 5, and 7. The flow cytometer CytoFLEX includes 3 lasers (405 nm, 488 nm, 638 nm) and 13 channels for fluorescence detection.TM Analysis of cell population distribution.

[0085] PBMC cells were seeded onto a plate containing fixed antibodies (both anti-CD3 and anti-CD28 antibodies) that provide signals for T cell activation. The carrier is placed in a bioreactor packed bed. On days 0, 5, and 7, the activation of T cells was assessed by measuring the expression of CD69 (an inducible cell surface marker expressed after TCR activation) and CD25 (α-chain of the IL-2 receptor), which are activation markers generally associated with T cell activation. The results obtained demonstrated successful stimulation of T lymphocyte activation and proliferation, as indicated by an increase in the CD3 marker from 44% on day 0 to 91% on day 7. These results specifically show that T lymphocytes proliferate over a 7-day period. CD69 and CD25 were upregulated from 4% and 8% on day 0 to 42% and 90% on day 7, indicating the activation of T cells. In addition, after the harvesting step on day 7, no changes in cell markers were detected in the collected samples of all cells, indicating that there were no other cell populations in the bioreactor. In addition, no changes in the levels of activation markers were detected, indicating that the harvesting of cells did not affect the cell state.

[0086] CD69 is an inducible cell surface marker expressed upon activation via TCR or IL-2 receptor (CD25). It plays a role in the proliferation and survival of activated T lymphocytes.

[0087] Figure 5A Figure 2 is a flow cytometric plot of activation and proliferation of MAIT cells grown in a packed bed bioreactor for 10 days, where activation and proliferation of cells were measured on days 0, 5, 7, and 10. TM Analysis of cell population distribution.

[0088] IVB monocytes were seeded onto ECM-coated plates that mimicked the natural environment and promoted the attachment of APCs. The MAIT cells were cultured in a packed bed bioreactor of a carrier. MAIT cell activation was induced by 5-OP-RU antigen and IL-15. The activation and proliferation of MAIT cells were evaluated at several time points (days 0, 5, 7 and 10). The MAIT cell population was detected by the expression of CD3, Vα7.2, CD161 markers. The results showed that the proportion of MAIT cells increased, starting from 22.6% on day 0 and as high as 96.26% on day 10. In addition, the expression of activation markers CD69 and CD25 increased from day 0 to day 7 and decreased by day 10.

[0089] Figure 5BFigure 1 is a flow cytometric plot of MAIT cell growth after cells were transferred from the first bioreactor to the second bioreactor on day 10 and grown for an additional 7 days in the second bioreactor. Cells that reached maximum growth capacity by day 10 in the first bioreactor were harvested, and approximately 30% of these cells were then inoculated into the second bioreactor, which was designed similarly to the first bioreactor. The cells were grown for an additional 7 days. Flow cytometric results demonstrated that the MAIT percentages were similar for most of the time, but showed a decrease from >90% on day 14 to 82% on day 17. CD69 marker expression was upregulated from 30% to 87% on day 17, indicating that MAIT cells maintained their activation signal, while CD25 gradually decreased as expected.

[0090] Figure 6 Figure 1 is a graphic representation of the cell distribution inside and outside the bioreactor packed bed for three different immune cell types: Jurkat, PBMC and MAIT cells, at two different growth days. Shown as percentages.

[0091] The tray properties and packed bed structure allow the creation of niches inside the bioreactor system that mimic the natural environment of the cells, where the cells can reside for a period of time with low internal shear stress. Figure 6 The distribution of cells inside and outside the bioreactor packed bed is shown. These values ​​were calculated based on cell counts before and after packed bed harvest performed by several washing steps accompanied by packed bed vibration. The results show that at different packed bed harvest time points throughout the growth period, for all three given immune cell types, at least 40% of all viable cells were inside the packed bed.

[0092] Since a significant decrease in cell concentration was observed a few hours after cell seeding, it can be inferred that the following cell distribution pattern was maintained throughout the cell expansion period.

[0093] Figure 7Schematic diagram of packed bed bioreactors of different sizes, showing the high scalability of the system of the present invention. According to an optional embodiment of the present invention, the first inoculation of the immune cell population can be carried out in a micro packed bed bioreactor 400 with a micro basket 416, wherein the total maximum volume of the micro bioreactor 400 is defined by its container size 490. After the cells are expanded in the bioreactor 400, the cells or a portion of the cells can be re-inoculated in a larger size bioreactor 500 with a packed bed basket 516 that is larger than the basket 416 and allows a larger number of porous scaffolds and higher cell expansion relative to the bioreactor 400, because the total volume of the bioreactor 500 is higher and is determined by its container size 590. The same process can be carried out for a larger bioreactor 600 with a basket 616 and a container 690, and so on until a maximum bioreactor 700 with a container size 790 that allows multiple growth of immune cells is reached, with a maximum basket 716. Based on the methods and examples provided above, it can be understood that in each bioreactor, the conditions for the growth and activation of the immune cell population can be similar to the previous growth stage in the smaller bioreactor, or can be different. At the end of this process, a massive growth of immune cells activated by similar or different activators is obtained.

[0094] Example

[0095] Reference is now made to the following examples, which together with the above descriptions illustrate certain embodiments in a non-limiting fashion.

[0096] Example 1

[0097] Expansion of Jurkat cells in a packed bed bioreactor.

[0098] Assemble a 0.5 L packed bed MiniBio reactor containing 2.5 g The trays were then sterilized by steam sterilization in an autoclave at a temperature of 122.5° C. and a pressure of 1 bar above atmospheric pressure for 30 minutes. Afterwards, the MiniBio reactor was connected to an Applikon MiniBio control station.

[0099] Will The plates were pre-incubated with RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) and 0.1% 50 mg / ml gentamicin at 37°C for approximately 24 hours. During the incubation period, serum proteins were The hydrophilic end groups on the disk interact electrostatically and An extracellular matrix (ECM) coating is created on the surface of the cells that mimics their natural environment.

[0100] 81.6×10 6Thaw the cells in RPMI-1640 supplemented with 10% HI-FBS and 0.1% 50 mg / ml gentamicin. Dilute the thawed cells to 0.24 × 10 6 The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature of 37°C, 80% dissolved oxygen (DO), pH 7.4, and stirring at 150 rpm, so that the total volume inside the bioreactor reached 340 ml.

[0101] On the third day of culture, the medium was renewed by 25% (meaning 25% of fresh medium was added). On the fourth day of culture, cells were harvested from the packed bed bioreactor due to the large proliferation in a very short period of time. The total cell number reached 830×10 6 cells, which means a 10.4-fold expansion (see Table 1 for details). The distribution of cells inside and outside the packed bed is as follows Figure 6 shown.

[0102] Example 2

[0103] Activation, expansion and harvesting of peripheral blood mononuclear cells (PBMCs) in packed bed bioreactors.

[0104] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and cultured by filtration and density gradient medium Lymphoprep TM (Ficoll) separation. Red blood cells (erythrocytes) depleted by RBC X1 lysis buffer. The separated population was cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0105] Assemble a 0.5 L packed bed MiniBio reactor containing 2.5 g The trays were then sterilized by steam sterilization in an autoclave at a temperature of 122.5° C. and a pressure of 1 bar above atmospheric pressure for 30 minutes. Afterwards, the MiniBio reactor was connected to an Applikon MiniBio control station.

[0106] Coating with monoclonal anti-CD3 (OKT3) and anti-CD28 (CD28.2) antibodies disk, both of which provide co-stimulatory signals that bind to T cell receptors. Diluted in PBS to 0.21 μg / cm 2 The amount of each activator was calculated. The final solution was incubated at 100 rpm for 3 hours at room temperature (RT) and at 37°C for 1.5 hours.

[0107] After incubation with the activator, the blocking procedure was followed by an additional incubation with 1% BSA solution for 1 hour at RT and 100 rpm. Then, the cells were drained and washed with PBS at 150 rpm for 10 minutes.

[0108] The packed bed bioreactor was then prepared and equilibrated at 37°C for approximately 24 hours with growth medium consisting of RPMI-1640 supplemented with 10% HI-FBS, 1% sodium pyruvate (100 mM) and 0.1% 50 mg / ml gentamicin. During this incubation period, serum proteins and The hydrophilic end groups on the disk interact electrostatically and Create an ECM coating that mimics the natural environment of cells.

[0109] 262×10 6 The cells were thawed into RPMI-1640 supplemented with 10% HI-FBS, 1% sodium pyruvate (100 mM), IL-2 (100 U / L) and 0.1% 50 mg / ml gentamicin. The thawed cells were diluted to 0.97 × 10 6 The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature of 37°C, 80% DO, pH 7.4 and agitation of 100 rpm, so that the total volume inside the bioreactor reached 270 ml. After inoculation, 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 inoculation.

[0110] During the growth phase, the growth medium and the 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 Bioanalyzer), and flow cytometry (CytoFLEX TM ) to measure cell population distribution.

[0111] Medium renewals of 5.5%, 32%, and 20% were performed on days 3, 5, and 6, respectively. On day 7 of culture, cells were harvested from the packed bed bioreactor because maximum growth capacity had been reached. The total cell count reached 1073 × 10 6 91% of the cells were CD3 positive, which means an 8.5-fold expansion. The proportions of different cell populations in the culture and their changes over time are shown in Figure 2. Figure 4 The cell distribution inside and outside the packed bed is shown in Figure 6 shown.

[0112] Example 3

[0113] Cultivation of mucosa-associated invariant T cells (MAIT) in packed bed bioreactors: activation, expansion, harvest, re-expansion, and second harvest.

[0114] Blood mononuclear cells were isolated from human placental venous blood (IVB) and purified by filtration and density gradient medium Lymphoprep TM (Ficoll) separation. Red blood cells (erythrocytes) depletion by RBC X1 lysis buffer. The separated populations were cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0115] Assemble a 0.5 L packed bed MiniBio reactor containing 2.5 g The trays were then sterilized by steam sterilization in an autoclave at a temperature of 122.5° C. and a pressure of 1 bar above atmospheric pressure for 30 minutes. Afterwards, the MiniBio reactor was connected to an Applikon MiniBio control station.

[0116] Will The plate was incubated with RPMI-1640 supplemented with 10% HI-FBS at 37°C for approximately 24 hours. During the incubation period, serum proteins and The hydrophilic end groups on the disk interact electrostatically and Create an ECM coating that mimics the natural environment of cells.

[0117] 300±20×10 6 Thaw cells into medium supplemented with 1% L-glutamine 200 mM and 0.1% 50 mg / ml gentamicin. The thawed cells were diluted to 1×10 6 The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature of 37°C, 80% DO, pH 7.4 and agitation of 100 rpm, to a final volume of 300 ml. After inoculation, 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 inoculation.

[0118] Three hours after inoculation, MAIT cells are activated via their T cell receptor (TCR). The activation pathway through the TCR requires co-stimulatory signals, which include recognition of microbial-derived riboflavin metabolites presented on the MHC Class I-like molecule MR1, and co-stimulation by CD28, TLR agonists, bacterial products, or cytokines.

[0119] The initial cell population isolated from human placenta IVB includes various antigen presenting cells (APCs) such as dendritic cells, monocytes, and B cells that can activate MAIT cells via MR1. 5-OP-RU, a microbial-derived riboflavin intermediate, was added to the growth medium at a concentration of 250 nM to be presented to the MAIT cell TCR by the APCs on MR1. IL-15 at a concentration of 50 ng / ml was also added to the growth medium to stimulate MAIT cells (via IL-15R expressed on MAIT cells) to produce IFN-γ and release granzyme B and perforin.

[0120] During the growth phase, 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 TM Bioanalyzer) and flow cytometry (CytoFLEX TM ) to measure cell population distribution.

[0121] The cells were grown in the packed bed bioreactor for 10 days, with 5%, 5.2%, and 100% medium renewals on days 3, 5, and 7, respectively. On day 10 of culture, the cells were harvested from the packed bed bioreactor because the maximum growth capacity had been reached. The total cell count reached 1089 × 10 6 94% of the cells were MAIT cells (Vα7.2 positive, CD161 high), which means a 43.5-fold expansion. Figure 5A shown.

[0122] After harvesting the cells from the bioreactor on day 10 of culture, the cells were examined for further expansion. An additional 0.5 L packed bed MiniBio reactor was assembled and the 2.5 g The sterile system of the plate was pre-incubated with RPMI-1640 supplemented with 10% HI-FBS at 37°C for about 24 hours. The dish provides an ECM coating and creates a natural environment for the reseeded cells.

[0123] After ECM coating, the packed bed bioreactor was filled with a 1% L-glutamine 200 mM and 0.1% 50 mg / ml gentamicin supplemented with The growth medium consisting of Nutri-T GMP medium was pre-balanced. 295 × 10 6 The harvested cells were inoculated into growth medium and diluted to 1 × 10 6The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature of 37°C, 80% DO, pH 7.4 and agitation of 100 rpm, to a final volume of 300 ml. IL-15 at a concentration of 50 ng / ml was added to the growth medium to induce MAIT cell expansion.

[0124] During the growth phase, 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 Bioanalyzer), and flow cytometry (by CytoFLEX TM ) to measure cell population distribution.

[0125] The cells were grown in the packed bed bioreactor for an additional 7 days, with 26.5% and 100% medium renewals performed on the 12th and 14th days of culture, respectively. On the 17th day of culture, the cells were harvested from the packed bed bioreactor. The total cell number reached 490 × 10 6 88% of the total MAIT cells (Vα7.2 positive, CD161 high) were MAIT cells, which means a 1.5-fold expansion. The relative percentage of MAIT cells in the culture and its changes over time are shown in Figure 2. Figure 5B shown.

[0126] Table 1 below summarizes the initial and final total viable cells of three different immune cell types, Jurkat, PBMC and MAIT, their relative population shares and the expansion folds during the growth phase:

[0127] Table 1

[0128]

[0129] As shown in the table, all expansion fold results were above 1, indicating cell expansion in the second growth phase for all three given immune cell types as well as MAIT cells. In addition, Table 1 shows a shift in the balance of the cell population, which reached more than 94% of target cells at the end of the growth phase examined (T cells after 7 days of growth and MAIT cells after 10 days of growth). The second growth phase of MAIT showed a slight decrease in the percentage of MAIT cells (from 94% to 86%).

[0130] Example 4

[0131] Expansion, activation and harvesting of B cells derived from peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0132] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and cultured by filtration and density gradient medium Lymphoprep TM(Ficoll) separation. Red blood cells (erythrocytes) depletion by RBC X1 lysis buffer. The separated populations were cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0133] Assemble a 0.5 L packed bed MiniBio reactor containing 2.5 g The trays were then sterilized by steam sterilization in an autoclave at a temperature of 122.5° C. and a pressure of 1 bar above atmospheric pressure for 30 minutes. Afterwards, the MiniBio reactor was connected to the Applikon MiniBio control system.

[0134] Coating with anti-CD40 antibody The amount of activator was diluted in PBS to 1 μg / cm 2 The final solution was incubated at 100 rpm for 3 hours at room temperature and at 37°C for 1.5 hours. The incubation with the activator was followed by a blocking procedure with an additional incubation with 1% BSA solution at room temperature, 100 rpm for 1 hour. Then drained and washed with PBS at 150 rpm for 10 minutes.

[0135] Will The plates were pre-incubated with RPMI 1640 supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin at 37°C for approximately 24 hours. During the incubation period, serum proteins and The hydrophilic end groups on the disk interact electrostatically and Create an ECM coating that mimics the natural environment of cells.

[0136] 300×10 6 10 cells were thawed in RPMI 1640 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, recombinant human IL-4 (10 ng / ml) and IL-21 (10 ng / ml). The thawed cells were diluted to 1×10 at the time of plating. 6 The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature of 37°C, 80% DO, pH 7.4 and agitation of 100 rpm, so that the total volume inside the bioreactor reached 300 ml. After inoculation, the cells were distributed between the packed bed and the "external" environment around the packed bed basket in the bioreactor, and samples were taken every day to measure pH, cell concentration (by Vi-Cell), and cell metabolic activity.

[0137] During the growth phase, the growth medium and cell suspension (in the “external” environment) were analyzed based on nutrient consumption (by Cedex Bioanalyzer) and flow cytometry (CytoFLEX TM ) of the cell population.

[0138] A 50% medium renewal was performed on days 4 and 6. On day 8 of culture, cells were harvested from the packed bed bioreactor.

[0139] Example 5

[0140] Activation, expansion, and harvesting of iNKT cells derived from peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0141] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and cultured by filtration and density gradient medium Lymphoprep TM (Ficoll) separation. Red blood cells (erythrocytes) depletion by RBC X1 lysis buffer. The separated populations were cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0142] Assemble a 0.5 L packed bed MiniBio reactor containing 2.5 g The trays were then sterilized by steam sterilization in an autoclave at a temperature of 122.5° C. and a pressure of 1 bar above atmospheric pressure for 30 minutes. Afterwards, the MiniBio reactor was connected to the Applikon MiniBio control system.

[0143] Will The plates were pre-incubated with RPMI 1640 supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin at 37°C for approximately 24 hours. During the incubation period, serum proteins were The hydrophilic end groups on the disk interact electrostatically and Create an ECM coating that mimics the natural environment of cells.

[0144] 600×10 6 10 cells were thawed into RPMI 1640 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 recombinant human IL-2. The thawed cells were diluted to 2 × 10 at the time of plating. 6The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature of 37°C, 80% DO, pH 7.4 and agitation of 100 rpm, so that the total volume inside the bioreactor reached 300 ml. After inoculation, the cells were distributed between the packed bed and the "external" environment within the bioreactor. Three hours after inoculation, 100 ng / ml α-galactosylceramide was spiked into the bioreactor medium to The activation step begins with presentation of the antigen on CD1d on antigen presenting cells (from the PBMC population) on the plate.

[0145] During the growth phase, the growth medium and the 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 Bioanalyzer), and flow cytometry (CytoFLEX TM ) to measure cell population distribution.

[0146] A 70% medium renewal was performed on days 3 and 6. On day 7 of culture, cells were harvested from the packed bed bioreactor.

[0147] Example 6

[0148] Activation, expansion and harvesting of γδ T cells derived from peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0149] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and cultured by filtration and density gradient medium Lymphoprep TM (Ficoll) separation. Red blood cells (erythrocytes) depletion by RBC X1 lysis buffer. The separated populations were cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0150] Assemble a 0.5 L packed bed MiniBio reactor containing 2.5 g The trays were then sterilized by steam sterilization in an autoclave at a temperature of 122.5° C. and a pressure of 1 bar above atmospheric pressure for 30 minutes. Afterwards, the MiniBio reactor was connected to an Applikon MiniBio control station.

[0151] Will The plates were pre-incubated with RPMI 1640 supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin at 37°C for approximately 24 hours. During the incubation period, serum proteins were The hydrophilic end groups on the disk interact electrostatically and Create an ECM coating that mimics the natural environment of cells.

[0152] 300×10 6 10 cells were thawed into RPMI 1640 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 were diluted to 1×10 at the time of plating. 6 The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature of 37°C, 80% DO, pH 7.4 and agitation of 100 rpm, so that the total volume inside the bioreactor reached 300 ml. After inoculation, the cells were distributed within the bioreactor between the packed bed and the "external" environment. The activation step was started 3 hours after inoculation by spiking 5 μM zoledronic acid into the bioreactor medium.

[0153] During the growth phase, the growth medium and the 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 Bioanalyzer), and flow cytometry (CytoFLEX TM ) to measure cell population distribution.

[0154] A 50% medium renewal was performed on days 4, 7, 10 and 13. On day 14 of culture, cells were harvested from the packed bed bioreactor.

[0155] Example 7

[0156] Activation, expansion and harvesting of natural killer (NK) cells derived from peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0157] Natural killer (NK) cells were isolated from human peripheral blood mononuclear cells (PBMCs) using RosetteSep (STEMCELL Technologies; routine ≥95% CD56+CD3-) and purified by filtration and density gradient medium Lymphoprep TM (Ficoll) separation. Red blood cells (erythrocytes) depletion by RBC X1 lysis buffer. The separated populations were then cryopreserved in HI-FBS and DMSO freezing solution.

[0158] Assemble a 0.5 L packed bed MiniBio reactor containing 2.5 g The trays were then sterilized by steam sterilization in an autoclave at a temperature of 122.5° C. and a pressure of 1 bar above atmospheric pressure for 30 minutes. Afterwards, the MiniBio reactor was connected to an Applikon MiniBio control station.

[0159] Will The plates were pre-incubated with RPMI 1640 supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin at 37°C for approximately 24 hours. During the incubation period, serum proteins were The hydrophilic end groups on the disk interact electrostatically and An extracellular matrix (ECM) coating is created on the surface of the cells that mimics their natural environment.

[0160] 900×10 6 NK cells were thawed into RPMI 1640 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, and 100 IU / ml penicillin-streptomycin. The thawed cells were diluted to 3 × 10 6 The target concentration of cells / ml was 10 cells / ml. The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature of 37°C, 80% DO, pH 7.4 and stirring at 100 rpm, so that the total volume inside the bioreactor reached 300 ml. After inoculation, the cells were distributed between the packed bed and the "external" environment within the bioreactor. For pre-activation, the culture medium was supplemented with recombinant human IL-12 (10 ng / mL), IL-18 (50 ng / mL) and IL-15 (50 ng / mL) and cultured for 16±2 hours, followed by a washing step and then cultured in growth medium supplemented with recombinant human IL-15 (1 ng / mL).

[0161] During the growth phase, the growth medium and the 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 Bioanalyzer), and flow cytometer CytoFLEX TM Measuring cell population distribution.

[0162] 30% medium renewal was performed on days 4 and 7. On day 8 of culture, cells were harvested from the packed bed bioreactor.

[0163] It should be understood that certain features of the present invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the present invention described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable sub-combination.

[0164] Although the present invention has been described in conjunction with specific embodiments of the present invention, many substitutes, modifications and variations will be apparent to those skilled in the art.Therefore, the present invention is intended to include substitutes, modifications and variations that fall within the spirit and broad scope of claims and specification sheets.All publications, patents and patent applications and GenBank accession numbers mentioned in this specification sheet are incorporated herein by reference in the specification sheet with their entirety, and their degree is specifically and individually indicated to be incorporated herein by reference as each single publication, patent or patent application or GenBank accession number.In addition, quoting or identification of any reference in the application should not be construed as admitting that this type of reference can be used as prior art of the present invention.

Claims

1. A three-dimensional (3D) bioreactor for large-scale expansion of immune cells, comprising: a. comprising at least one packed bed chamber comprising at least one porous support; b. at least one porous scaffold coated with one or more extracellular matrix proteins (ECM); c. at least one container comprising a fluid culture medium configured to flow through the packed bed chamber having at least one porous coated support; and d. at least one immune cell population suspended in said fluid culture medium; Wherein, the at least one porous scaffold coated with the ECM is configured to create a low shear immobilization niche that mimics the natural growth environment of immune cells and allows large-scale expansion of immune cell populations 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 connected with at least one immune cell activator.

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

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

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

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

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

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

9. The 3D bioreactor of any one of the preceding claims, wherein the immune cell population is genetically modified using a genetic modification agent incorporated into the bioreactor culture medium.

10. A method for large-scale expansion of immune cells in a three-dimensional (3D) bioreactor, comprising: a. inserting at least one porous support 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 culture medium from at least one container, the fluid culture medium being configured to flow through the packed bed chamber comprising at least one porous coated support; and d. suspending at least one immune cell population in a circulating fluid culture medium; Wherein, the at least one porous scaffold coated with the ECM is configured to create a low shear fixation niche that mimics the natural growth environment of immune cells and allows large-scale expansion of immune cell populations flowing through the at least one porous scaffold.

11. The method according to claim 10 further comprises the step of coating the at least one porous support with at least one immune cell activator after step 9(b) and the step of exposing the immune cell population to the at least one activator after step 9(d) to expand and activate the immune cell population within the packed bed chamber. 12 . The method according to claim 11 , further comprising the step of genetically modifying immune cells inside the 3D bioreactor using a genetic modification agent incorporated into the fluid culture medium.

13. The method according to claims 10-12, further comprising the step of harvesting the immune cells or a portion of said cells and further expanding and reactivating said immune cell population in the same bioreactor or in a different bioreactor.

14. The method of claim 13, further comprising the step of harvesting the immune cells, followed by the step of genetically modifying the cells outside the system, and then re-seeding the genetically modified immune cells into the same bioreactor or a different bioreactor.

15. A three-dimensional (3D) bioreactor for large-scale expansion and activation of immune cell populations, comprising: a. comprising at least one packed bed chamber of 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 comprising a fluid culture medium configured to flow through the coated porous APC-MS; and d. at least one immune cell population suspended in said fluid culture medium; Therein, the at least one APC-MS creates a fixed microenvironment with low shear forces that mimics the natural growth environment of the immune cell population and allows large-scale expansion and / or activation of the immune cell population flowing therethrough. 16 . The 3D bioreactor of claim 15 , wherein the immune cell population is reactivated after exposing the coated porous APC-MS to an antigen to generate additional activation signals to the immune cell population.

17. The 3D bioreactor of claims 15-16, wherein the immune cell population is reactivated by transferring the cells to a different bioreactor comprising at least one coated multiporous APC-MS with a different or similar antigen.

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

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