Packed bed reactor for engineered t cell production

Abstract

The present invention provides a bioreactor and a bioreactor system for mass production of engineered T cells. The bioreactor has a matrix comprising a single type of mesh or multiple types of meshes, and the matrix can be packed in the bioreactor in a stacked or coiled configuration. The bioreactor enables the conjugation, activation, and transduction of patient-derived T cells, followed by expansion of the resulting engineered T cells, all in a single bioreactor. The bioreactor system includes the bioreactor and also has a cell culture conditioning vessel with conditioned cell culture medium, and the bioreactor system can also include a cell culture medium feed vessel with fresh cell culture medium. The configuration of the bioreactor and the bioreactor system addresses the problem of efficient large-scale and commercial production of engineered T cells, reducing the time, cost, and contamination risk associated with current engineered T cell production technologies.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 601,910, filed November 22, 2023, pursuant to 35 USC §119, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to cell culture systems for manufacturing engineered T cells, including therapeutic cell lines such as highly efficient chimeric antigen receptor-modified T cells (CAR-T cells) and engineered T cell receptor cells (TCR-T cells). Background Technology

[0004] Over the past decade, immunotherapy has emerged as a promising new treatment option for cancer patients. In 2017, the Food and Drug Administration (FDA) made history by approving two chimeric antigen receptor-modified T-cell (CAR-T cell) therapies for hematologic malignancies. Currently, engineered T-cell receptor cells (TCR-T cells) targeting certain solid tumors are also being tested. Both types of engineered T-cell therapies utilize genetically modified T cells in the laboratory to enable them to more effectively target and destroy cancer cells.

[0005] Currently, over 300 CAR-T clinical trials are underway worldwide. Many of these trials have shown very encouraging results. Despite the promising nature of engineered T-cell technology, treatment costs for patients remain high. The estimated cost of a single treatment is approximately $350,000, excluding hospitalization and other related expenses. The primary reason for the high cost of these life-saving treatments is the inefficiency and costly manufacturing processes of current engineered T-cell therapies. This disclosure addresses the inefficiency of current engineered T-cell manufacturing processes by providing a novel fixed-bed bioreactor system that makes engineered T-cell manufacturing more efficient and cost-effective. Summary of the Invention

[0006] According to some aspects of this disclosure, a bioreactor is provided, comprising an inlet and an outlet, and a containment container including a matrix. The matrix includes a mesh substrate having single fibers or bundles of fibers configured for binding, activating, transducing, or culturing T cells. The bioreactor can perform one or more of binding, activating, transducing, and culturing T cells, and can perform all of these actions. In some embodiments, these steps are all performed in the same bioreactor.

[0007] According to some aspects of this disclosure, the matrix includes a mesh substrate filled in a stacked layer, and in other aspects, the matrix includes a mesh substrate filled in a roll. In some aspects, the matrix includes at least two types of mesh substrates, wherein a first mesh substrate is stacked with a second mesh substrate. In some aspects, different types of mesh substrates are filled together in a stacked manner, while in other aspects, different types of mesh substrates are filled together in a roll manner.

[0008] According to some aspects of this disclosure, the mesh substrate is composed of a single fiber, and the single fiber has a fiber thickness of about 5 μm to about 200 μm. In other aspects, the mesh substrate is composed of each braided fiber bundle forming the mesh, and each individual fiber bundle has a thickness of about 5 μm to about 200 μm.

[0009] Depending on the use of at least two different types of mesh substrates, each individual fiber of the first mesh substrate has a thickness of about 5 μm to about 30 μm, and each individual fiber of the second mesh substrate has a thickness of about 50 μm to about 200 μm. In some embodiments, each individual fiber of the first mesh substrate has a thickness of about 5 μm to about 20 μm, and each individual fiber of the second mesh substrate has a thickness of about 100 μm to about 160 μm.

[0010] According to some aspects of this disclosure, the bioreactor also includes a central support member. In some embodiments, a mesh substrate is attached to the central support member.

[0011] According to some aspects of this disclosure, the mesh substrate further includes a coating that binds to and activates CD3+ T cells. In some embodiments, the coating comprises an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD19 antibody, or a combination thereof. In some embodiments, when this type of coating is applied to the mesh substrate, the mesh substrate reactivates the T cells during the proliferation of engineered T cells.

[0012] According to some aspects of this disclosure, the mesh substrate also includes a coating that enhances the efficiency of viral vector transduction into T cells. In some embodiments, the coating comprises a recombinant fibronectin fragment. The recombinant fibronectin fragment includes, for example, a 63 kDa recombinant fragment from human fibronectin, including commercially available options such as RetroNectin from Taka Bio USA, Inc. ® .

[0013] According to some aspects of this disclosure, a bioreactor system is provided, comprising a bioreactor and a cell culture medium conditioning vessel. The bioreactor includes a containment vessel, an inlet, an outlet, and a substrate. The substrate includes a first mesh substrate for binding CD3+ T cells. In some aspects, the bioreactor system includes a pump between the bioreactor and the cell culture medium conditioning vessel. In some aspects, the bioreactor system includes a culture medium refeed vessel.

[0014] In some aspects, the matrix further includes a second mesh substrate for binding CD3+ T cells. When the matrix has at least two types of mesh substrates, in some embodiments, the first and second mesh substrates are filled as alternating layers between the first and second meshes. In some aspects, the thickness of a single fiber of the first mesh substrate is from about 5 μm to about 20 μm. In some aspects, the thickness of a single fiber of the first mesh substrate is from about 5 μm to about 30 μm, and the thickness of a single fiber of the second mesh substrate is from about 50 μm to about 200 μm. In some embodiments of these aspects, the first and second meshes have a coating comprising an antibody selected from anti-CD3, anti-CD28, and anti-CD-19, or combinations thereof. In some embodiments, the first mesh substrate has a coating for binding CD3+ T cells, and the second mesh substrate has a coating different from the coating on the first mesh for binding CD3+ T cells.

[0015] According to some aspects of this disclosure, a method for producing engineered T cells is provided, comprising the steps of: (a) providing a bioreactor according to claim 1; (b) providing CD3+ T cells to the bioreactor; (c) binding the CD3+ T cells to a mesh substrate; (d) activating the CD3+ T cells in the bioreactor; (e) transducing the activated CD3+ T cells in the bioreactor; (f) culturing the transduced engineered T cells in the bioreactor; and (g) harvesting the cultured engineered T cells from the bioreactor. The method for producing engineered T cells yields viable engineered T cells from at least 70% of the engineered T cells produced by the bioreactor. In some aspects, the step of providing CD3+ T cells to the bioreactor includes providing blood containing CD3+ T cells. In some embodiments, the blood is patient-derived blood.

[0016] Additional features and advantages will be set forth in the detailed description below, and will be partly apparent to those skilled in the art from the description or to be recognized by practice of the embodiments described herein, including the detailed description below, the claims and the drawings.

[0017] It should be understood that the foregoing general description and the following detailed description are merely exemplary and intended to provide an overview or framework for understanding the nature and characteristics of the claims. The accompanying drawings are included to provide further understanding and are incorporated in and form a part of this specification. The drawings illustrate one or more embodiments and, together with the description, serve to explain the principles and operation of the various embodiments. Attached Figure Description

[0018] The following is a description of the figures in the accompanying drawings, given purely by way of non-limiting example. The figures are not necessarily drawn to scale, and for clarity and simplicity, some features and views may be shown enlarged or schematically.

[0019] Figure 1A A perspective view of a three-dimensional model of a mesh substrate according to an embodiment is shown.

[0020] Figure 1B According to the embodiments Figure 1A A two-dimensional plan view of the substrate.

[0021] Figure 1C According to the embodiments Figure 1A A cross-sectional view of the substrate.

[0022] Figure 1D According to the embodiments Figure 1A The cross-sectional view of the substrate in the figure indicates the height h1 of the substrate and the widths w1 and w2 between the peaks and valleys.

[0023] Figure 2 This is a schematic diagram showing a cross-sectional view of a surface-functionalized substrate that facilitates the bonding of T cells to a substrate according to an embodiment.

[0024] Figure 3 This is a perspective view of an exemplary bioreactor having a matrix according to an embodiment, the matrix having stacked substrates.

[0025] Figure 4 This is an exploded view of an exemplary bioreactor with a matrix according to an embodiment, the matrix having stacked substrates.

[0026] Figure 5 This is an exploded view of a substrate having a wound base material according to an embodiment.

[0027] Figure 6 This is a cross-sectional view of a bioreactor with a matrix according to an embodiment, the matrix having a wound substrate and supporting members.

[0028] Figure 7 This is a schematic diagram of a bioreactor system for engineered T-cell production according to an embodiment.

[0029] Figure 8 This is a schematic diagram of a bioreactor system for engineered T-cell production according to an embodiment. Detailed Implementation

[0030] Various aspects and embodiments will now be fully described herein. However, these aspects and embodiments may be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided to make this disclosure thorough and complete and to fully convey the scope of the subject matter to those skilled in the art. All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety.

[0031] Modifications to this disclosure will be apparent to those skilled in the art, as well as to those who make or use this disclosure. Therefore, it should be understood that the embodiments shown in the accompanying drawings and described above are for illustrative purposes only and are not intended to limit the scope of this disclosure, which is defined by the appended claims as interpreted in accordance with the principles of patent law, including the doctrine of equivalents.

[0032] A. Definition

[0033] Unless otherwise defined, all terms and phrases used herein include their meanings as they have been acquired in the art, unless explicitly stated or clearly apparent from the context in which they are used. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, specific methods and materials are described hereafter.

[0034] As used herein, the terms “the,” “an,” or “a” mean “at least one,” and should not be limited to “only one” unless explicitly indicated otherwise. Thus, for example, unless the context otherwise clearly indicates, references to “component” include embodiments having two or more such components.

[0035] Unless otherwise stated, the use of individual numerical values ​​is described as approximations, as if the value were preceded by the words “about” or “approximately”. Similarly, unless otherwise explicitly stated, numerical values ​​within the various ranges specified in this application are described as approximations, as if the minimum and maximum values ​​within the stated ranges were preceded by the words “about” or “approximately”. In this way, deviations above and below the stated ranges can achieve substantially the same results as values ​​within the ranges. As used herein, when referring to numerical values, the terms “about” and “approximately” should have their common and ordinary meaning to those skilled in the art most closely related to the disclosed subject matter or to the scope or element discussed. The degree of deviation from strict numerical boundaries depends on a number of factors. Some factors that may be considered include, for example, the criticality of the element and / or the impact of a given amount of deviation on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. As used herein, the use of different numbers of significant figures for different numerical values ​​does not imply how the use of the words “about” or “approximately” will be used to broaden a particular numerical value or range. Therefore, as a general rule, “about” or “approximately” broadens the numerical value. Furthermore, the disclosure of ranges is intended to be as a continuous range, including each value between the minimum and maximum values ​​plus a range broadened by the use of the terms "about" or "approximately". Therefore, the description of numerical ranges herein is intended only as a shorthand method of individually referring to each individual value falling within said range, and each individual value is incorporated into this specification as if it were described separately herein.

[0036] As used herein, when used in a list of two or more items, the term "and / or" means that any one of the listed items may be used alone, or any combination of two or more of the listed items may be used. For example, if a composition is described as containing components A, B, and / or C, the composition may contain only A; only B; only C; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0037] As used in this article, words such as “have,” “having,” “include,” “including,” “comprise,” and “comprising” are used in their open-ended sense and usually mean “including but not limited to.”

[0038] "Optional" or "optionally" means that the element, component, or condition described below may or may not occur, and therefore the description includes both the occurrence and non-occurrence of the element, component, or condition.

[0039] In this document, relational terms such as first and second, top and bottom are used only to distinguish one entity or action from another, and do not necessarily require or imply any actual such relationship or order between these entities or actions.

[0040] Unless otherwise stated, all scientific and technical terms used herein have their common meaning in the art. The definitions provided herein are for ease of understanding of certain terms used frequently herein and are not intended to limit the scope of this disclosure.

[0041] B. Introduction

[0042] This disclosure provides a more efficient and cost-effective manufacturing process for the mass production of engineered T cells than currently available methods. Engineered T cell manufacturing typically involves four steps: (a) CD3+ T cell isolation, (b) viral transduction of T cell genes (e.g., using CAR or TCR genes), (c) T cell activation, and (d) engineered T cell culture (proliferation). CD3+ T cells are T cells with CD3+ surface markers on their outer surface. The CD3+ T cell complex is a lymphocyte that helps target and destroy foreign substances harmful to living organisms. For use in engineered T cell manufacturing, CD3+ T cells are first extracted from a patient by drawing blood and then purified to isolate the CD3+ T cells. This involves step (a) in the manufacturing process. To become engineered T cells, the CD3+ T cells are then genetically engineered to incorporate genes such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), so that those receptors can be prepared on the surface of the engineered T cells. This process is accomplished by transducing activated cells with a viral vector containing the desired gene to be expressed against a specific type of cancer (e.g., CAR, TCR), which involves step (b) in the manufacturing process. The engineered T cells are then activated with anti-CD3 and anti-CD28 monoclonal antibodies. This involves step (c) in the manufacturing process. The activated engineered T cells can then recognize and bind to specific types of cancer cells, and they can also proliferate. Finally, to produce a therapeutic dose of engineered T cells, the engineered T cells must be proliferated (involving step (d)) in the manufacturing process for a single treatment, in which the cells are reinfused into the patient's bloodstream to provide the original CD3+ T cells.

[0043] Current technologies for engineered T-cell production are inefficient, unable to produce large quantities of engineered T-cells, resulting in extremely high treatment costs. For example, the latest FDA-approved CAR-T cell therapy costs over $450,000 per treatment. This disclosure addresses this inefficiency by providing a novel packed-bed reactor system that allows each of four defined manufacturing steps to be performed in a single device. The packed-bed reactor system of this disclosure also provides a fixed bed with a large surface area and uniform flow, enabling efficient T-cell separation. It also possesses unique surface functions to increase antibody adsorption and maintain antibody conformation and activity, thereby effectively activating T-cells. In conventional engineered T-cell production systems, T-cell activation is performed using bead- or particle-based activation. These beads and particles can be internalized into cells and may ultimately enter the drug product, posing a safety concern. This invention uses a mesh instead of beads and particles for T-cell activation, avoiding the internalization problems experienced with bead and particle activation. Furthermore, this disclosure improves T-cell activation by providing improved surface curvature in the packed-bed reactor system. The system provides uniform flow to the fixed-bed reactor. The invention disclosed herein also eliminates the problems of contamination and operator error that plague current manufacturing processes. It also provides a perfusion system that enables better expansion of T cells and facilitates collection at the end of the culture process.

[0044] Additional features and advantages will be set forth in the following detailed description and will be apparent to those skilled in the art from the description or will be recognized by practice of the embodiments described below, as well as in the claims and drawings.

[0045] C. Substrate used in reactors

[0046] The bioreactor used in the system includes a substrate that captures CD3+ T cells for further processing and facilitates the steps of activating CD3+ T cells, transducing activated T cells into engineered T cells, and culturing (proliferating) engineered T cells. The substrate can be made of monofilaments or multifilaments of polymeric materials compatible with cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. In one specific embodiment, the material used for the substrate includes polyethylene terephthalate (PET). In some embodiments, the substrate is a polymer-based material and can be formed as a molded polymer sheet; a polymer sheet having openings punched through its thickness; multiple filaments fused into a mesh layer; or a 3D-printed substrate.

[0047] Embodiments of this disclosure include a substrate with a defined and ordered structure. This contrasts with substrates (i.e., nonwoven substrates with randomly ordered fibers) typically used in cell culture bioreactors, which have indefined or disordered structures. The defined and ordered structure ensures consistency and predictability of cell culture results. Furthermore, the substrate has an open, porous structure that prevents cell retention and allows cells to flow uniformly through the packed bed. This configuration enables improved CD3+ T cell activation, genetic engineering of CD3+ T cells with genes of selected receptors to enable nutrient delivery through said receptors, genetically engineered T cell growth, and cell harvesting. The substrate has multiple pores or openings. In some embodiments, the substrate may be arranged in a thin sheet-like configuration having a first side and a second side separated by a relatively small thickness, such that the sheet thickness is small relative to the width and / or length of the first and second sides of the substrate. The size and geometry of the substrate material allow T cells to adhere to antibodies on the substrate material or be captured by the substrate material, while also allowing sufficient fluid to flow around the substrate material and through the openings. The physical structure of the substrate produces a high surface-to-volume ratio for culturing T cells. According to various embodiments, the substrate can be arranged or filled in a bioreactor in some of the ways discussed in this disclosure to achieve uniform inoculation and improve activation, growth and harvesting.

[0048] In some embodiments, the substrate may include a mesh. The mesh substrate may have a pattern or weave, including, for example, knitting, warp knitting, or woven fabric. In some embodiments, the mesh may be woven. Examples of weave types include plain weave, plain Dutch weave, twill weave, twill Dutch weave, and five-needle knit. In some embodiments, the mesh may alternatively be extruded, oriented, expanded, or tubular. In other embodiments, the mesh may be knitted, welded, expanded, wintered, photochemically etched, coiled, or electroformed.

[0049] For embodiments including a mesh substrate, the mesh substrate may include mesh size, linear fiber density, and open area size. Mesh size (also called mesh count) is the number of openings per linear inch measured from the center of one fiber to the center of an adjacent fiber. An open area is the space between parallel fibers adjacent to each other. Linear fiber density is the fiber density per unit length of the mesh. When a single type of mesh is used, the mesh substrate comprises multiple fibers having an individual fiber thickness between about 5 μm and about 200 μm. In one embodiment, a single type of mesh is used, and the mesh substrate comprises multiple fibers having an individual fiber thickness between 5 μm and 50 μm.

[0050] Two or more different types of meshes can be used as substrates in a matrix, which is a hybrid mesh matrix. The types of meshes used in the hybrid mesh matrix can have different materials, different surface functions, different fiber diameters or opening sizes, different mesh sizes, different linear fiber densities, different fiber strand or bundle curvatures, or any combination thereof. In some embodiments, the matrix comprises two mesh types, wherein a first mesh substrate is laminated onto a second mesh substrate, and wherein the first mesh substrate is different from the second mesh substrate. In one embodiment, the first mesh substrate has a different single fiber thickness and / or opening size (distance between two parallel fibers in the mesh) than the second mesh substrate. The size difference of the single fibers of the two mesh substrates allows T cell binding and activation to occur while providing sufficient space for cells and culture medium to pass through the substrate.

[0051] Figure 1A and 1B A three-dimensional (3D) perspective view and a two-dimensional (2D) plan view of an exemplary single mesh substrate 100 according to one or more embodiments of the present disclosure are shown respectively. The mesh substrate 100 is a woven mesh layer made of a first plurality of fibers 102 extending in a first direction and a second plurality of fibers 104 extending in a second direction. The woven fibers of the mesh substrate 100 form a plurality of openings 106, which may be defined by one or more widths or diameters (e.g., D1, D2). The size and shape of the openings may vary based on the weave type (e.g., the number, shape, and size of filaments; the angle between intersecting filaments, etc.). The woven mesh can be characterized as a two-dimensional sheet or layer on a macroscopic scale. However, close observation of the woven mesh reveals that a three-dimensional structure is formed due to the undulations of the intersecting fibers of the mesh. Therefore, as... Figure 1C As shown, the thickness T of the woven mesh 100 can be greater than the thickness of a single fiber (e.g., t1). As used herein, the thickness T is the maximum thickness between the first side 108 and the second side 110 of the woven mesh. Without wishing to be bound by theory, it is believed that the three-dimensional structure of the substrate 100 is advantageous because it provides a large surface area for culturing T cells, and the structural rigidity of the mesh can provide a consistent and predictable cell culture substrate structure, enabling uniform fluid flow.

[0052] exist Figure 1BIn this design, opening 106 has distances D1 and D2, where distance D1 is defined as the distance between opposite fibers 102, and distance D2 is defined as the distance between opposite fibers 104. D1 and D2 may be equal or unequal, depending on the weave geometry. When D1 and D2 are unequal, the larger one may be referred to as the primary distance, and the smaller one as the secondary distance. In some embodiments, the distance spanning the opening may refer to the widest portion of the opening. Unless otherwise stated, opening distance as used herein refers to the distance between parallel fibers on opposite sides of the opening.

[0053] A given fiber in multiple fibers 102 has a thickness t1, and a given fiber in multiple fibers 104 has a thickness t2. For example... Figure 1C The fibers shown have a circular cross-section or other three-dimensional cross-section, and thicknesses t1 and t2 are the maximum diameter or thickness of the fiber cross-section. According to some embodiments, multiple fibers 102 all have the same thickness t1, and multiple fibers 104 all have the same thickness t2. Alternatively, t1 and t2 may be equal. However, in one or more embodiments, t1 and t2 are not equal, for example, when multiple fibers 102 and multiple fibers 104 are different. Furthermore, multiple fibers 102 and multiple fibers 104 may each contain two or more fibers of different thicknesses (e.g., t1, t2, t2, t3, t4, t2, t3, t4, t3 ... 1a t 1b etc., and t 2a t 2b (etc.). According to the embodiment, the thicknesses t1 and t2 are related to the size of the cells cultured thereon, such that the fibers approximate a curved surface from the cell's perspective, which allows for better cell attachment and growth compared to some other solutions with larger fiber sizes (e.g., much larger than the cell diameter). Due to the three-dimensional properties of the woven mesh, such as... Figure 1A-1C As shown, the 2D surface area of ​​fibers that can be used for cell attachment and proliferation exceeds the attachment surface area on an equivalent planar 2D surface.

[0054] In some embodiments, when a single mesh is used as a substrate, the thickness (t1 or t2) of a single fiber can range from about 5 μm to about 200 μm; about 5 μm to about 160 μm; about 5 μm to about 125 μm; about 5 μm to about 100 μm; about 5 μm to about 50 μm; about 5 μm to about 30 μm; or about 5 μm to about 15 μm. These thicknesses and thickness ranges are exemplary. Other thicknesses or ranges can be used, but preferably, the fiber thickness produces a curvature close to the cell size that T cells will bind to. In some other embodiments, when a mixed mesh is used as a substrate, the first mesh substrate may include a first plurality of fibers, the thickness of which a single fiber (t1 or t2) is... 1a or t 2aThe thickness of the fiber is between approximately 5 μm and approximately 30 μm. In one specific embodiment, the first mesh substrate comprises a first plurality of fibers, the thickness of which is between approximately 5 μm and approximately 20 μm or between approximately 5 μm and approximately 15 μm. In some embodiments using a hybrid mesh, the second mesh substrate comprises a second plurality of fibers, the thickness of which is between approximately 5 μm and approximately 30 μm. 1b (or t2) is between about 50 μm and about 200 μm. In one specific embodiment, the second mesh substrate includes a second plurality of fibers, the thickness of each fiber being between about 100 μm and about 160 μm.

[0055] The mesh openings (having a distance D1 or D2) can range from about 15 μm to about 400 μm. In some embodiments, the mesh openings can be between about 30 μm and about 200 μm, between about 30 μm and about 100 μm, between about 20 μm and about 75 μm, between about 50 μm and about 150 μm, or any other range between 15 μm and 400 μm. When a hybrid mesh is used, the mesh opening size in the first mesh can differ from the mesh opening size in the second mesh. For example, the first mesh substrate can have mesh openings between about 30 μm and 100 μm, while the second mesh substrate can have mesh openings between about 150 μm and 350 μm. In some embodiments, when a hybrid mesh is used, the first mesh will have smaller mesh openings, while the second mesh will have larger mesh openings than the first mesh.

[0056] The ranges of fiber thickness and opening size provided are examples of some embodiments and are not intended to limit the possible characteristic dimensions of the mesh according to all embodiments. The combination of fiber thickness and opening size is selected based on the desired size of the surface area for T cell binding and activation and the expected proliferation rate relative to a unit volume of reactor space. For example, when a smaller thickness is used for a single fiber (e.g., 10 μm for a single type of mesh), the opening size can be smaller (e.g., between 25 μm and 50 μm). As another example, when a larger thickness is used for a single fiber (e.g., 100 μm), the opening size can be larger (e.g., between 150 μm and 300 μm).

[0057] In embodiments where the mesh substrate is composed of fiber bundles, the thickness of each bundle can be as described above for a single fiber. The dimensions of the individual fibers in each bundle can be different, as long as the total thickness of the bundle remains the same as the total thickness used for the individual fibers.

[0058] A given single fiber in the mesh also has a curvature k along its length (in embodiments with fiber bundles, the fiber bundles have curvature). The amount of curvature k at a point in the fiber is influenced by the height h between the peaks and valleys of the fiber in the mesh substrate 100, the width w1 between two adjacent peaks of the fiber in the mesh substrate 100, and the width w2 between two adjacent valleys of the fiber in the mesh substrate 100, as shown below. Figure 1D A schematic cross-sectional view of the mesh is shown. According to some embodiments, multiple fibers 102 all have the same width w1 and height h1, and multiple fibers 104 all have the same width w2 and height h2. Furthermore, w1 and w2 can be equal, or h1 and h2 can be equal, or w1 and w2 can be equal and h1 and h2 can be equal. However, in one or more embodiments, w1 and w2 or h1 and h2 are not equal, for example, when the multiple fibers 102 are different from the multiple fibers 104. Additionally, due to the different heights and widths of the peaks and valleys between fibers (e.g., w... 1a w 1b And w 2a w 2b And h 1a h 1b And h 2a h 2b (etc.), multiple fibers 102 and multiple fibers 104 may each contain two or more fibers with different curvatures.

[0059] In one specific embodiment, width w1 is substantially the same as width w2, and falls between 20 μm and 800 μm, between 20 μm and 700 μm, between 20 μm and 600 μm, between 20 μm and 500 μm, between 20 μm and 400 μm, between 20 μm and 300 μm, between 20 μm and 200 μm, or between 20 μm and 100 μm. In another specific embodiment, width w1 is substantially the same as width w2, and falls between 200 μm and 800 μm, or between 400 μm and 640 μm. In yet another specific embodiment, the width w1 is approximately the same as the width w2, and is between 20 μm and 90 μm, between 20 μm and 80 μm, between 20 μm and 70 μm, between 20 μm and 60 μm, between 20 μm and 50 μm, or between 20 μm and 40 μm.

[0060] In one specific embodiment, the height h1 is between 7 μm and 400 μm, between 7 μm and 300 μm, between 7 μm and 200 μm, between 7 μm and 100 μm, between 7 μm and 50 μm, or between 7 μm and 20 μm. In another specific embodiment, the height h1 is between 60 μm and 400 μm, between 60 μm and 350 μm, between 100 μm and 350 μm, or between 120 μm and 320 μm.

[0061] In some embodiments, the surface of the substrate is functionalized to help improve the interaction between the substrate and antibodies, cells, or both. Such modification can be achieved by chemically treating the polymeric material of the mesh substrate, or by grafting antibodies or other proteins or protein complexes that promote T-cell binding to the fiber surface. Alternatively, the mesh substrate can be coated with a thin layer of antibodies that promote T-cell binding, including, for example, anti-CD3, anti-CD28, concanavalin A, or combinations thereof. Alternatively, the surface of the fibers of the mesh substrate can be endowed with T-cell adhesion properties through treatment processes using various types of plasma, process gases, and / or chemicals known in industry. Figure 2 An embodiment of a surface-functionalized mesh substrate is shown in a cross-sectional view of mesh substrate 100, wherein multiple fibers 102 are generally perpendicular to multiple fibers 104 and have been surface-functionalized with antibody 114, which facilitates the recruitment and binding of T cells 112.

[0062] D. Engineered T-cell reactor

[0063] The engineered T-cell reactor disclosed herein is a bioreactor comprising a containment container, an inlet, an outlet, and a matrix having a substrate. In some embodiments, the matrix comprises a stack of laminated substrates. In some embodiments, the matrix comprises a wound substrate. The matrix is ​​contained within the containment container. The inlet provides liquid feed into the containment container. The outlet processes the liquid from the containment container.

[0064] In some embodiments, the engineered T-cell reactor of this disclosure can capture and activate CD3+ T cells using a mesh substrate of a bioreactor. In one embodiment, this is achieved by attaching a monoclonal antibody (through coating, covalent linking, or other techniques) to the mesh substrate, the monoclonal antibody binding to the CD3 protein complex on the surface of the CD3+ T cells (cell attachment) and simultaneously activating the CD3+ T cells. In some embodiments, the activated CD3+ T cells in the bioreactor are then transduced. Transduction of activated T cells can be achieved by providing the bioreactor with various viral vectors containing genes for proteins or protein complexes that are desired to be expressed by the activated T cells. Examples of such genes include, but are not limited to, CAR genes or TCR genes. The transduced T cells in the bioreactor are considered engineered T cells and can now target cancer cells in a patient's body. In some embodiments, the engineered T cells are proliferated in the bioreactor using a cell culture medium for T cell growth. Once the desired level of engineered T cells is reached, the cells can be harvested from the bioreactor.

[0065] It should be understood that the steps of purification, activation, transduction, and amplification can all be performed in the bioreactor of this disclosure, but are not required to be. In some embodiments, only some steps may be performed in the bioreactor. For example, in some embodiments, purification of T cells from blood is performed separately from the bioreactor, but the activation, transduction, and amplification steps are performed within the bioreactor. As another example, purification, activation, and amplification may occur within the bioreactor, but transduction is performed separately. In yet another example, transduction is performed after purification, separately from the bioreactor, and then activation and amplification are performed in the bioreactor. These examples are merely exemplary combinations of steps that can be performed in a bioreactor, and other combinations of steps may be performed, as those skilled in the art will understand. For example, only activation and transduction may be performed in the bioreactor.

[0066] Over time, some activated T cells may dissociate from the substrate and float or circulate within the bioreactor, or even be recycled through the bioreactor while other steps (e.g., proliferation) are still in progress. Dissociated T cells may undergo at least some deactivation. In some embodiments, deactivated T cells are reactivated to their activated state upon rebinding to the substrate in the bioreactor. Conventional bioreactor systems for engineered T cell production cannot reactivate T cells because activation materials (e.g., beads and particles) must be removed after the activation step to prevent contamination of the drug product (proliferated engineered T cells). This is a significant advantage of this disclosure.

[0067] Typically, the flow direction of fluids in a bioreactor can be configured in different directions, including flow approximately perpendicular to the substrate layer or flow approximately parallel to the substrate layer. Figure 3 In one exemplary embodiment of the bioreactor 300 with a containment container 302 shown, the overall flow direction is from inlet 310 to outlet 312, and in this example, the first and second sides of the substrate layer 308 in the stacked configuration are perpendicular to the overall flow direction. In contrast, Figure 4 The example shown is an embodiment with stacked substrates, wherein the bioreactor 320 includes a stack of receiving container 322 and substrates 328 layered within a cell culture chamber 324, the substrates having a first side and a second side parallel to the overall flow direction, the overall flow direction corresponding to the direction indicated by the flow lines from the inlet 330 and the outlet 332. Therefore, the substrates of the embodiments of this disclosure can be used in any configuration. Figure 3 As shown, matrix 306 includes stacked substrates 308 and an internal space defined by cell culture chambers 304. In each bioreactor 300 and 320, the mesh substrates 308, 328 are sized and shaped to fill the internal space defined by cell culture chambers 304, 324, such that the culture space in each containment vessel is filled with a cell growth surface, thereby maximizing efficiency, in terms of cells per unit volume. Although Figure 4 Multiple inlets 330 and multiple outlets 332 are shown, but it is conceivable that the bioreactor 320 could be fed by a single inlet and have a single outlet. Furthermore, although... Figure 3-4 This is an example of a bioreactor with stacked substrates, but the flow direction configuration is also applicable to bioreactors with wound substrates.

[0068] According to various embodiments described herein, a distribution plate can be used to help distribute culture media, cells, or nutrients across the entire cross-section of a packed bed (a stacked or wound mesh substrate), thereby improving the uniformity of fluid flow through the packed bed. When the containment container has multiple inlets, multiple inlets indicate how the distribution plate can be configured with multiple holes across the cross-section of the packed bed to produce a more uniform flow.

[0069] Factors such as fiber thickness, opening size, and weave type / pattern will determine the surface area available for T cell attachment and engineered T cell growth in a bioreactor. Furthermore, when the matrix used in the reactor includes other arrangements of stacked, wound, or overlapping substrates, the matrix packing density will affect the surface area of ​​the packed bed matrix. Packing density can vary with the packing thickness of the substrate material (e.g., the space required for the substrate layers). For example, if the substrate stack has a certain height, each layer of the stack can be considered to have a certain packing thickness, which is determined by dividing the total height of the stack by the number of layers in the stack. Packing thickness will vary based on fiber thickness and weave pattern, but it can also vary based on the arrangement of adjacent layers in the stack. For example, due to the three-dimensional nature of the woven layers, adjacent layers can be interlocked or overlapped to some extent based on their arrangement. In the first arrangement, adjacent layers can be tightly fitted together, but in the second arrangement, adjacent layers may not overlap, for example, when the lowest point of the upper layer is in direct contact with the highest point of the lower layer. For certain applications, it may be necessary to provide a matrix with a lower layer fill density (e.g., when higher permeability is preferred) or a higher fill density (e.g., when maximizing substrate surface area is preferred). According to one or more embodiments, the fill thickness may be about 20 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μm to about 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400 μm; or about 200 μm to about 300 μm. In another embodiment, the fill thickness may be about 20 μm to about 200 μm, about 20 μm to about 150 μm, about 20 μm to about 100 μm, or about 20 μm to about 50 μm.

[0070] The aforementioned structural factors determine the surface area of ​​the bioreactor substrate, whether it is a single-layer substrate or a substrate with multiple layers. For example, in one specific embodiment, a single-layer woven mesh substrate with a circular shape of 6 cm in diameter can have a surface area of ​​approximately 68 cm². 2 The effective surface area. As used herein, “effective surface area” is the total surface area of ​​fibers in a portion of the substrate material that can be used for cell attachment and growth. Unless otherwise stated, reference to “surface area” refers to this effective surface area. According to one or more embodiments, the effective surface area of ​​a single woven mesh substrate layer with a diameter of 6 cm can be approximately 50 cm². 2 Approximately 90 cm 2 Approximately 53 cm 2 Approximately 81 cm 2 Approximately 68 cm 2 Approximately 75 cm 2 or approximately 81 cm 2These effective surface area ranges are provided as examples only, and some embodiments may have different effective surface areas.

[0071] Stacked substrate bioreactor

[0072] According to one or more embodiments, a bioreactor is provided in which a substrate is used within a culture chamber within a containment vessel of the bioreactor. In one embodiment, the substrate comprises a stack of mesh substrate layers. The stack of mesh substrate layers may comprise a single type of mesh, or may comprise two or more types of mesh. Figure 3 An example of a bioreactor 300 is shown, comprising a containment container 302, within which a cell culture chamber 304 is located. Within the cell culture chamber 304 is a substrate 306 comprising a stack of substrate layers 308. The substrate layers 308 are stacked such that a first or second side of one substrate layer faces the first or second side of an adjacent substrate layer. The bioreactor 300 has an inlet 310 at one end for introducing culture medium, cells, and / or nutrients into the culture chamber 304, and an outlet 312 at the opposite end for removing culture medium, cells, or cell products from the culture chamber 304. By allowing the substrate layers to be stacked in this manner, the system can be easily scaled up without negatively impacting cell attachment and proliferation, because the stacked substrate structure is well-defined and fluid flow is efficient. While bioreactors are generally described as having an inlet and an outlet, some embodiments may use one or both inlets and outlets to allow culture medium, cells, or other contents to flow into and out of the culture chamber. For example, using… Figure 3 In the illustrated embodiment, inlet 310 can be used to allow culture medium or cells to flow into culture chamber 304 during cell seeding, activation, perfusion, or culture phases, but can also be used to remove one or more of culture medium, cells, or cell products through inlet 310 during harvest phases. Therefore, the terms "inlet" and "outlet" are not intended to limit the function of these openings.

[0073] In one or more embodiments, the flow resistance and bulk density of the packed bed can be controlled by interleaving layers of substrate of different types, sizes, and / or geometries. Specifically, mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the fluid flow resistance of the packed bed configuration. By interleaving different types, sizes, and / or geometries of meshes, the flow resistance of one or more specific sections of the bioreactor can be controlled or altered. This results in more uniform liquid infusion in the packed bed. In one example, the bioreactor matrix may include a first layer of mesh of a first type beneath a second layer of mesh of a second type. This layering pattern continues until the desired matrix height or density is achieved. When more than one type of mesh is used, the meshes are distinct from each other. As another example, the bioreactor matrix may include two layers of mesh of the first type, followed by five layers of mesh of the second type, and then three layers of mesh of the third type. This layering pattern continues until the desired matrix height or density is achieved.

[0074] In some embodiments, the mesh substrate (having a single type or multiple types of mesh substrate) can be rotated interlayer to achieve different fluid flow resistance and different substrate accessibility patterns than those achieved with a single layering direction. For example, the matrix may include a first layer of a first type of mesh, followed by a second layer of a first type of mesh rotated 45 degrees relative to the first layer, then a third layer of a first type of mesh rotated 45 degrees relative to the second layer, and then a fourth layer of a first type of mesh rotated 45 degrees relative to the third layer. This pattern continues until the matrix reaches the desired fill height or density. In another example, a first layer of a first type of mesh is stacked on top of a second layer of a second type of mesh rotated 30 degrees relative to the first layer. This layering pattern is then repeated until the matrix reaches the desired fill height or density. Interlayer rotation also prevents nesting, a phenomenon where at least a portion of a mesh substrate layer extends downwards into a second mesh layer, leading to uneven fluid flow and uneven T-cell binding sites.

[0075] The examples of layering and rotation described above are merely illustrative and for purposes of explanation, and are not intended to limit possible combinations. In reality, various combinations of different types of meshes or adjacent meshes in different orientations can yield different distributions of volumetric density and flow resistance on the cell growth surface. For example, packed bed columns with different volumetric cell density regions or substrate accessibility regions (e.g., a series of regions producing low / high / low / high density or substrate accessibility patterns) can be assembled by interlacing meshes of different sizes, types, and / or geometries.

[0076] Wound substrate reactor

[0077] According to one or more embodiments, a bioreactor is provided in which a substrate is used in a culture chamber within a containment vessel of the bioreactor. In one embodiment, the substrate is filled in a roll form. The substrate may be filled in a cylindrical roll form within the containment vessel of the bioreactor (see...). Figure 5 and 6 In such embodiments, the scalability of the packed bed bioreactor can be achieved by increasing the overall length and height of the mesh substrate. The amount of mesh used in a cylindrical roll configuration can vary based on the desired packing density of the packed bed. For example, the cylindrical rolls can be densely packed as tight rolls or loosely packed as loose rolls. The packing density is typically determined by the required surface area of ​​the substrate for a given application or scale. In one embodiment, the required mesh length can be calculated from the diameter of the packed bed bioreactor using the following formula:

[0078] Equation 1

[0079] Where L is the total length of the mesh required to fill the bioreactor, R is the inner radius of the packed bed culture chamber, and r is the inner support component of the mesh winding ( Figure 6 The radius of the support member 366 in the structure is given, and t is the thickness of a layer of mesh. In this configuration, the bioreactor can be expanded by increasing the diameter or width of the cylindrical roll of the packed bed (i.e., Figure 5 (W in the middle) and / or increase the height of the filled bed cylindrical drum (i.e., Figure 5 This is achieved by using H in the formula, thereby providing a larger substrate surface area for inoculation and growth of adherent cells.

[0080] When the matrix comprises more than one type of mesh, the meshes can be stacked in the desired number of mesh layers before forming a cylindrical roll configuration. For example, if two types of meshes are required, one type of mesh can be stacked on top of different types of meshes, and the stacked layers can then be rolled into a matrix with a cylindrical roll configuration. Similarly, if a single type of mesh is required, but layers of meshes with different orientations between layers are present, one type of mesh can be stacked on top of meshes of the same type but with different orientations, and the stacked layers can then be rolled into a matrix with a cylindrical roll. These exemplary configurations reduce or eliminate any nesting effects caused by adjacent meshes of the same type. These are merely examples and are for illustrative purposes only, and are not intended to limit possible combinations. In practice, various combinations of meshes of different sizes can yield different distributions of volumetric density and flow resistance on the cell growth surface.

[0081] Figure 5An embodiment of the matrix is ​​shown, wherein the substrate is formed as a cylindrical roll 350. For example, a sheet of matrix material including a mesh substrate 352 is wound into a cylinder around a central longitudinal axis y. The cylindrical roll 350 has a width W along a dimension perpendicular to the central longitudinal axis y and a height H along a direction perpendicular to the central longitudinal axis y. In one or more preferred embodiments, the matrix including the cylindrical roll 350 is designed within a containment container such that the central longitudinal axis y is parallel to the overall flow direction F of fluid through the bioreactor or culture chamber containing the cylindrical roll. Figure 6 A cross-section of a bioreactor 360 is shown, the bioreactor having a containment container 362 that holds a substrate 364 arranged in this cylindrical roll configuration. Similar to... Figure 5 The cylindrical roll 350 and the substrate 364 have a central longitudinal axis, which is located in... Figure 6 The bioreactor 360 also includes a central support member 366 around which the substrate 364 is positioned. According to some embodiments, the central support member 366 may be provided purely for physical support and / or alignment of the substrate 364, but may also provide other functions. For example, the central support member 366 may be provided with one or more openings for supplying media to the substrate 364 along the length H of the substrate. In other embodiments, the central support member 366 may include one or more attachment points for securing one or more portions of the substrate 364 to the inner portion of a cylindrical reel. These attachment points may be hooks, loops, posts, clamps, or other components for attaching the substrate to the central support member 366.

[0082] E. Bioreactor System

[0083] The bioreactor system disclosed herein is designed to produce large quantities of engineered T cells in fewer steps and less time than conventional systems. In one embodiment, the matrix provides at least one substrate having surface area for T cell binding, activation, transduction, and / or proliferation, possessing good mechanical strength and forming a highly uniform, multi-layered, interconnected fluid network when assembled in a packed bed or other bioreactor. In a particular embodiment, a mechanically stable, non-degradable woven mesh can be used as a substrate to support the generation of engineered T cells from CD3+ T cells. The matrix disclosed herein supports T cell attachment, activation, transduction, and / or proliferation in a high volumetric density. When CD3+ T cells are seeded directly from blood, the bioreactor system of this disclosure can purify large volumes of blood due to the large surface area available for binding in the bioreactor. For example, depending on the concentration of CD3+ T cells in the blood, even 1 L to 5 L of blood can be perfused through the bioreactor, and in some embodiments, the blood can be recycled back through the bioreactor for more efficient capture and seeding of the bioreactor. The matrix of this disclosure enables uniform cell seeding and efficient collection of cells or other products from the bioreactor. Furthermore, embodiments of this disclosure support cell culture to provide a uniform cell distribution during the seeding step and allow multiple steps of engineered T cell production to be performed in a single container, which is not possible with current systems used for engineered T cell production. Therefore, the matrix also eliminates diffusion limitations during bioreactor operation. Moreover, the matrix enables simple and efficient CAR-T cell production because it allows all steps of CAR-T production to be performed in a single container, minimizing cell loss due to contamination (a problem present in current processes that require cell transfer between production steps) and enabling efficient, large-scale production of CAR-T cells. The well-defined matrix of one or more embodiments enables complete cell recovery from the packed bed of a bioreactor and continuous cell collection.

[0084] Figure 7An exemplary bioreactor system 400 according to one or more embodiments is illustrated. System 400 includes a bioreactor 402 that contains a substrate and base material of one or more embodiments disclosed herein. Bioreactor 402 is fluidly connected to a culture medium conditioning vessel 404, and the system is capable of supplying cell culture medium 406 from the conditioning vessel 404 to the bioreactor 402. The culture medium conditioning vessel 404 may include sensors and control components present in typical bioreactors used in the bioprocessing industry for suspension batch culture, fed-batch culture, or perfusion culture. These sensors and control components include, but are not limited to, a DO oxygen sensor, a pH sensor, an oxygenator / gas bubbling unit, a temperature probe, and nutrient and alkali addition ports. The gas mixture supplied to the bubbling unit can be controlled by a gas flow controller for N2, O2, and CO2 gases. The culture medium conditioning vessel 404 may also include an impeller for culture medium mixing. All culture medium parameters measured by the sensors listed above can be controlled by a culture medium adjustment control unit 418, which communicates with the culture medium adjustment container 404 and is capable of measuring the conditions of the cell culture medium 406 and / or adjusting the conditions of the cell culture medium to the desired level. Figure 7 As shown, the culture medium conditioning container 404 is provided as a separate container from the bioreactor 402. This can have the advantage of separating culture medium conditioning from cell culture, and then supplying the conditioned medium to an area surrounding the substrate. However, in some embodiments, culture medium conditioning may be performed within the bioreactor container 402.

[0085] Culture medium 406 from culture medium conditioning container 404 is delivered to bioreactor 402 via inlet 408, which may also include an injection port for inoculating cell seed material and initiating cell culture. Bioreactor 402 may also include one or more outlets 410 through which the cell culture medium 406 exits the bioreactor 402. Additionally, cells or cell products may be discharged through outlet 410. To analyze the contents of the effluent from bioreactor 402, one or more sensors 412 may be provided in the pipeline. In some embodiments, bioreactor system 400 includes a flow control unit 414 for controlling the flow rate into bioreactor 402. For example, flow control unit 414 may receive signals from one or more sensors 412 (e.g., O2 sensors) and, based on these signals, regulate the flow rate into bioreactor 402 by sending a signal to a pump 416 (e.g., a peristaltic pump) upstream of inlet 408 of bioreactor 402. Thus, based on one or a combination of factors measured by sensor 412, pump 416 may control the flow rate into bioreactor 402 to obtain the desired cell culture conditions.

[0086] The culture medium perfusion rate is controlled by a signal processing unit 414, which collects and compares sensor signals from the culture medium conditioning vessel 404 and a sensor located at the bioreactor outlet 410. Due to the fluidity of the culture medium perfused through the bioreactor 402, nutrient, pH, and oxygen gradients are formed along the packed bed. The perfusion flow rate of the bioreactor can be automatically controlled by a flow control unit 414 operably connected to the peristaltic pump 416.

[0087] The bioreactor system disclosed herein may have other configurations and other settings. For example, in one embodiment, the bioreactor system includes a bioreactor, a substrate contained within the bioreactor, an outlet, an inlet, a culture medium conditioning vessel, and a culture medium refeed vessel. Figure 8 An example of this embodiment is depicted. The bioreactor system 400 has a bioreactor 402 having an inlet 408, an outlet 410, a culture medium conditioning vessel 404, a culture medium refeed vessel 405, and a substrate 450 contained within the bioreactor 402. Figure 8 In this embodiment, matrix 402 comprises a wound substrate. However, matrix 402 may alternatively comprise a stacked layer substrate as described above. CD3+ T cells isolated from the patient can enter the bioreactor through inlet 408. Cells can enter the bioreactor 402 directly through a port (not shown) on inlet 408, or as described above. Figure 8 As shown, it can be achieved by having an insertion device (e.g.) Figure 8 Cells are added to the inlet of the syringe 440 (shown) via a port (not shown) on the tubing. The port can be of any type known to those skilled in the art. Cell culture medium 406 can flow through the tubing that connects the culture medium conditioning container 404 to the bioreactor 402 via an inlet 408. Used culture medium flows out from an outlet 410 and can be returned to the culture medium conditioning container 404, or alternatively, can be discarded as culture medium waste. Figure 8(Seen as waste bin 411). Pump 416 can control the flow of cell culture medium 406 from culture medium conditioning container 404, as previously described. Flow control valves 415 (415a, 415b, 415c) can be used to control the flow to the containers in the bioreactor system. For example, flow control valve 415b can be used to control the flow between culture medium refeed container 405 and culture medium conditioning container 404. As another example, flow control valve 415c can be used to control the flow from the outlet of bioreactor 402 back to culture medium refeed container 404. As yet another example, flow control valve 415a can be used to control the flow from the outlet of bioreactor 402 to waste bin 411. Flow control valves can operate simultaneously, or some flow control valves can be used without others, and which flow control valves are actively operating can vary throughout the manufacturing process.

[0088] In the production of engineered T cells, CD3+ T cells already in the bioreactor can be dispersed throughout the cell culture chamber and bind to monoclonal antibodies (or other CD3+ T cell binders) already coated onto a substrate in the bioreactor. This binding of CD3+ T cells helps purify them when blood flows directly into the bioreactor. When activating antibodies such as anti-CD3, anti-CD28, or combinations thereof are used as the monoclonal antibodies binding to the substrate, the CD3+ T cells will bind to the monoclonal antibody and will also be activated by the antibody. It should be understood that other activating antibodies, such as anti-CD19 and other activating antibodies known to those skilled in the art, can also be used. If a non-activating binder is used instead as an adhesion agent between the CD3+ T cells and the substrate, an activator can be added after the CD3+ T cells have bound to the non-activating binder in a manner similar to the addition of CD3+ T cells, and the activator can then be dispensed into the bioreactor to activate the bound CD3+ T cells.

[0089] After activation, activated CD3+ T cells in the bioreactor can be transduced within the bioreactor. Transduction of activated T cells can be achieved using any transduction method known to those skilled in the art. For example, a lentivirus-derived viral vector containing the desired target gene (e.g., CAR, TCR, etc.) can be perfused into the bioreactor. The perfusion flow rate used for transduction can be from about 0.1 mL / min to 100 mL / min, depending on the size of the bioreactor and the efficiency of transduction. In one embodiment, the flow rate can be from about 0.1 mL / min to about 75 mL / min, from about 0.1 mL / min to about 50 mL / min, from about 0.1 mL / min to about 25 mL / min, or any other value or range between 0.1 mL / min and 100 mL / min. In some embodiments, the perfusion flow rate during the transduction phase can be set low to facilitate the transduction of the viral vector into CD3+ T cells. In one embodiment, the transduction perfusion flow rate can be from 0.1 mL / min to 0.5 mL / min. In some embodiments, the transduction perfusion flow rate is 0.1 mL / min to 1 mL / min, or 0.1 mL / min to 5 mL / min, or 0.1 mL / min to 10 mL / min. Furthermore, in some embodiments, the compound that improves transduction efficiency can be perfused into the bioreactor or can be coated onto a substrate. For example, compounds known to those skilled in the art for improving viral transfection and transduction, such as recombinant fibronectin fragments, including recombinant fibronectin fragments derived from humans, can be used. A commercially available example is RetroNectin. ® (Taka BioUSA, Inc.) A 63 kDa recombinant fibronectin polypeptide derived from human genes can be used to improve the viral transfection efficiency of T cells.

[0090] After transduction, as understood by those skilled in the art, the engineered T cells are proliferated in a bioreactor by adjusting the cell culture medium to a medium suitable for the growth of engineered T cells. The engineered T cells proliferate through perfusion until the desired cell level is reached. As previously described, cells that have lost their activation activity (common in conventional methods) can be reactivated during this phase by rebinding antibodies coated onto a substrate in the bioreactor. Therefore, the bioreactor and bioreactor system utilize the substrate of this disclosure to establish a reactivation mechanism during proliferation. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, or more of the engineered T cells are reactivated during the proliferation phase.

[0091] The embodiments of this disclosure achieve the harvesting of viable cultured cells, as measured by the trypan blue exclusion method. In one aspect of this disclosure, viable engineered T cells, as measured by the trypan blue exclusion method, can be harvested from a bioreactor system, comprising 70% to 100% viable engineered T cells, or about 80% to about 100% viable engineered T cells, or about 90% to about 100% viable engineered T cells. For example, as measured by the trypan blue exclusion method, at least 70% of the harvested engineered T cells are viable, at least 80% are viable, at least 80% are viable, at least 85% are viable, at least 90% are viable, at least 91% are viable, at least 92% are viable, at least 93% are viable, at least 94% are viable, at least 95% are viable, at least 96% are viable, at least 97% are viable, at least 98% are viable, or at least 99% are viable.

[0092] While this disclosure includes a limited number of embodiments, those skilled in the art who benefit from this disclosure will understand that other embodiments can be devised without departing from the scope of this disclosure.

Claims

1. A bioreactor comprising: Entrance and exit; as well as A container, the container comprising a substrate; The matrix includes a mesh substrate having single fibers or bundles of fibers configured for binding, activating, transducing, or culturing T cells.

2. The bioreactor of claim 1, wherein the mesh substrate is configured for binding, activating, transducing and culturing T cells in the same bioreactor.

3. The bioreactor of claim 1, wherein the substrate further comprises a mesh substrate filled in layers.

4. The bioreactor of claim 1, wherein the substrate further comprises a mesh substrate filled in a roll.

5. The bioreactor of claim 1, wherein the substrate further comprises at least two types of mesh substrates, and wherein the first type of mesh substrate is laminated with the second type of mesh substrate.

6. The bioreactor of claim 5, wherein the at least two types of mesh substrates are filled in a stacked manner.

7. The bioreactor of claim 5, wherein the at least two types of mesh substrates are filled in a roll manner.

8. The bioreactor of claim 1, wherein the individual fibers of the mesh substrate have a fiber thickness of about 5 μm to about 200 μm.

9. The bioreactor of claim 1, wherein the fiber bundles of the mesh substrate have a thickness of about 5 μm to about 200 μm.

10. The bioreactor of claim 5, wherein each individual fiber of the first mesh substrate has a thickness of about 5 μm to about 30 μm, and each individual fiber of the second mesh substrate has a thickness of about 50 μm to about 200 μm.

11. The bioreactor system of claim 5, wherein each individual fiber of the first mesh substrate has a thickness of about 5 μm to about 20 μm, and each individual fiber of the second mesh substrate has a thickness of about 100 μm to about 160 μm.

12. The bioreactor according to claim 7, further comprising a central support member.

13. The bioreactor of claim 12, wherein the mesh substrate is attached to the central support member.

14. The bioreactor of claim 1, wherein the mesh substrate further comprises a coating that binds to and activates CD3+ T cells.

15. The bioreactor of claim 14, wherein the coating comprises an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD19 antibody, or a combination thereof.

16. The bioreactor of claim 15, wherein the mesh substrate reactivates T cells during engineered T cell proliferation.

17. The bioreactor of claim 1, wherein the mesh substrate further comprises a coating that improves the efficiency of viral vector transduction into T cells.

18. The bioreactor of claim 17, wherein the coating comprises recombinant fibronectin fragments.

19. A bioreactor system comprising: A bioreactor, comprising a containment vessel, an inlet, an outlet, and a substrate; as well as Cell culture medium conditioning container; The matrix includes a first reticular substrate that binds CD3+ T cells.

20. The bioreactor system of claim 19, further comprising a pump between the bioreactor and the cell culture medium conditioning vessel.

21. The bioreactor system of claim 19, wherein the matrix comprises a second mesh substrate for binding CD3+ T cells.

22. The bioreactor system of claim 20, further comprising a culture medium refeeding vessel.

23. The bioreactor system of claim 19, wherein the first mesh substrate and the second mesh substrate are filled as alternating layers between the first mesh and the second mesh.

24. The bioreactor system of claim 19, wherein the first mesh substrate has a single fiber thickness of about 5 μm to about 20 μm.

25. The bioreactor system of claim 21, wherein the first mesh substrate has a mesh size of about 5 μm to about 30 μm, and wherein the second mesh substrate has a mesh size of about 50 μm to about 200 μm.

26. The bioreactor system of claim 19, wherein the first mesh substrate and the second mesh substrate have a coating comprising an antibody selected from anti-CD3, anti-CD28 and anti-CD19 or a combination thereof.

27. The bioreactor system of claim 21, wherein the first mesh substrate has a coating that binds CD3+ T cells, and wherein the second mesh substrate has a coating that is different from the coating that binds CD3+ T cells on the first mesh.

28. A method for producing engineered T cells, comprising the following steps: (a) Providing the bioreactor according to claim 1; (b) Providing CD3+ T cells to the bioreactor; (c) Binding the CD3+ T cells to the reticular substrate; (d) Activate CD3+ T cells in the bioreactor; (e) Transducing activated CD3+ T cells in the bioreactor; (f) Culturing transduced engineered T cells in the bioreactor; and (g) Harvesting cultured engineered T cells from the bioreactor; At least 70% of the engineered T cells from the bioreactor are viable.

29. The method of claim 28, wherein the step of providing CD3+ T cells to the bioreactor comprises providing blood containing CD3+ T cells in the blood.

30. The method of claim 29, wherein the blood is patient-derived blood.

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