Multi-functional cell culture flask and its use in t-cell therapies

Abstract

A bioprocess vessel is provided that allows for T-cell activation and expansion in the same vessel without the use of beads for activation. A T-cell activator coating is employed on at least one interior wall of the bioprocess vessel. T-cells, or cells containing T-cells, are dispensed into the bioprocess vessel having the T-cell activator coating and incubated such that the T-cell activator coating and the cells are in contact with each other. Once activation is complete, expansion begins in the same bioprocess vessel by addition of expansion growth factors. Media exchange may be performed during the expansion phase. Cells can expand up to 2000-fold in the same vessel that activation occurred, all without the use of activation beads.

Description

MULTI-FUNCTIONAL CELL CULTURE FLASK AND ITS USE IN T-CELLTHERAPIESCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 667,421 filed on July 3, 2024, the content of which is relied upon and incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure relates to the field of bioprocess, and more specifically, to a bioprocess vessel for activating and expanding T-cells in a single vessel, including engineered T-cell immunotherapies from patient-derived donor cells, such as chimeric antigen receptor-modified T-cells and other engineered T-cell receptor therapy cells.BACKGROUND

[0003] Bioprocess includes the processes, devices, and components associated with the production of therapeutic products of interest from cultured cells. One use of a bioprocess is for engineered cell immunotherapies that use a patient’s own T-cells to create a tailored therapy against cancer cells. In particular, chimeric antigen receptor modified T-cells (CAR-T cells) and engineered T-cell receptor (TCR-T cells) immunotherapies are used as a treatment of some hematological malignancies and have a potential to treat other cancer types. The production of engineered cell therapies is complex and extremely expensive, on the order of several hundreds of thousands of dollars for a single treatment. Engineered cell therapies start by extracting a patient’s own blood and purifying it to obtain T-cells having certain markers. The extracted T- cells are then engineered to produce certain proteins on the outside of the T-cell in the form of a membrane receptor, which helps recognize and assist in destroying targeted malignant cells. In CAR-T cell therapies, which target certain types of blood cancers, a Chimeric Antigen Receptor (CAR) gene is incorporated into the activated T-cells. In TCR-T cell therapies, which target certain types of solid tumors, specific cancer-antigen specific T cell receptors (TCRs) can be incorporated into the activated T-cells. Once the engineered T-cells have been created (CAR-T cells or TCR-T cells), they are then activated by exposing the T-cells to certain types of antibodies. These activated engineered T-cells must then be multiplied to a therapeutic level,a phase called expansion. The cells are then harvested and then administered back to the patient as a therapeutic.

[0004] Current processes for engineered cell immunotherapies are problematic because they use beads for activation (micro or nano beads that act as an artificial antigen-presenting cell), and / or the activation step is carried out in a different vessel than the expansion step . Beads are problematic for immunotherapies because beads can be internalized by cells, creating silent contaminants. Using multiple vessels during the process creates an increased risk of contamination in a very expensive process for each vessel used. The present disclosure solves these problems.SUMMARY

[0005] According to one aspect of the present disclosure, a bioprocess vessel is provided that has a vessel body, at least one access port, and a T-cell activator coating on at least one interior wall of the vessel body. The vessel body has a top side, a bottom side, and sidewalls. The bioprocess vessel is configured to activate T-cells when cells comprising T-cells are contacted with the coating and to expand T-cells or cells comprising T-cells when expansion growth factors are added to the bioprocess vessel.

[0006] The bioprocess vessel may also have at least one cell culturing layer between the top side and bottom side of the vessel body. The bioprocess vessel may have a closure or connector for the at least one access port, and the closure or connector includes a porous membrane having pores sized to allow liquid to flow through the pores but sized to prevent T- cells from passing through the pores. The at least one interior wall of the vessel body that has the T-cell activator coating may be a sidewall. The T-cell activators of the T-cell activator coating may include anti-CD3 antibodies, anti-CD28 antibodies, or a combination thereof.

[0007] The T-cell activator coating may have a loading concentration of T-cell activators from 0.1 pg / cm2to 50 pg / cm2. T-cells that are activated and expanded in the bioprocess vessel may expand from 50 fold to 2000 fold over the number of activated T-cells in the bioprocess vessel. T-cells that are activated and expanded in the bioprocess vessel may expand at least 2-fold over the number of activated T-cells in the bioprocess vessel. At least 85% of T-cells in the bioprocess vessel may be viable T-cells three days after initiation of activation in the bioprocess vessel. At least 85% of T-cells in the bioprocess vessel may be viable T-cells three days after initiation of expansion in the bioprocess vessel.

[0008] The bioprocess vessel may be a sterilized bioprocess vessel, and the sterilization achieved with high-energy irradiation. The high-energy irradiation may be gamma irradiation, e-beam irradiation, or x-ray irradiation at a dosage between 1 kGy and 50 kGy.

[0009] According to another aspect of the present disclosure a method for activating and expanding T-cells is provided. The method steps include (a) providing a bioprocess vessel from the preceding paragraph, (b) providing an aqueous solution to the bioprocess vessel that is suitable for T-cell activation, (c) providing T-cells or cells comprising T-cells to the bioprocess vessel, (d) positioning the bioprocess vessel such that the T-cell activator coating on the bioprocess vessel is in contact with the T-cells in the aqueous solution to activate the T- cells to a preselected level of activation, (e) providing T-cell expansion growth factors to the bioprocess vessel once the preselected level of activation is achieved, and (f) culturing the T- cells or cells comprising T-cells that were activated by the T-cell activator coating until they have expanded at least 2-fold over the number of cells present after the desired level of activation is achieved. At least 70% of the T-cells may be viable three days after initiation of activation during step (d). At least 70% of the T-cells may be viable three days after culturing the T-cells with the provided T-cell expansion growth factors. The T-cells or cells comprising T-cells may be cultured until they have expanded at least 100-fold over the number of cells present after the desired level of activation is achieved. The T-cell activator coating may be on a sidewall of the bioprocess vessel. The method may include the step of positioning the bioprocess vessel on its bottom side for step (f) when the T-cell activator coating is on a sidewall of the bioprocess vessel.

[0010] The T-cells or cells comprising T-cells provided to the bioprocess vessel may be engineered T-cells. The engineered T-cells may include a gene for a chimeric antigen receptor or for a T-cell receptor. The T-cells or cells comprising T-cells provided to the bioprocess vessel may be patient-derived T-cells or cells comprising T-cells. The T-cell activator may include an antigen to an aP-T-cell receptor on the T-cells. The antigen may include anti-CD3 or anti-CD28, or a combination thereof. The bioreactor vessel may be a sterilized bioreactor vessel, irradiated with gamma irradiation, e-beam irradiation, or x-ray irradiation at a dosage between 1 kGy and 50 kGy.

[0011] The method may include the step of performing aqueous solution additions, aqueous solution exchanges, or a combination thereof during the culturing of the T-cells or cells comprising T-cells.

[0012] In yet another aspect of the present disclosure, a bioprocess vessel is provided that has a vessel body, a T-cell activator coating on at least one interior wall of the vessel body, and at least one access port and a closure or connector for the at least one access port comprising a porous membrane with pores sized to allow liquid to flow through the pores and sized to prevent T-cells from flowing through the pores. The vessel body has a top side, a bottom side, and sidewalls. The bioprocess vessel is configured to activate T-cells when T-cells or cells comprising T-cells are contacted with the coating and to expand T-cells or cells comprising T- cells when T-cell expansion growth factors are added to the bioprocess vessel.

[0013] The bioprocess vessel may expand the number of T-cells or cells comprising cells to a number of cells that is least 2-fold more than a number of cells at the end of activation. At least 70% of the number of T-cells at the end of activation may be viable and at least 70% of the number of T-cells at the end of expansion may be viable. The T-cell activator coating may include an antigen to an aP-T-cell receptor on the T-cells. The bioprocess vessel may be a sterilized bioprocess vessel, and the sterilization is gamma irradiation, e-beam irradiation, or x-ray irradiation at a dosage in a range from 1 kGy to 50 kGy. the T-cell activator coating comprises a loading concentration of T-cell activators from 0.1 pg / cm2to 50 pg / cm2.

[0014] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0015] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework to understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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 to scale, and certain features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0017] FIG. 1 depicts an exemplary bioreactor vessel with five layers and a single port with necked access and a cap closure. The bioreactor vessel has a T-cell activator coating on the interior side of the end sidewall.

[0018] FIG. 2 depicts an exemplary bioreactor vessel with five layers and a single port with necked access and a cap closure, during T-cell activation. The bioreactor vessel is positioned such that the aqueous solution containing the cells is in contact with the T-cell activator coating.

[0019] FIG. 3 depicts an exemplary bioreactor vessel with five layers and a single port with necked access and a cap closure, during T-cell expansion. The bioreactor vessel is positioned such that the aqueous solution containing the activated T-cells is distributed across the vessel and the layers.

[0020] FIG. 4 is a graph showing the change in the number of cells during T-cell activation and T-cell expansion in a multi-layer flask having three layers (bar graph), along with the change in fold expansion during T-cell activation and T-cell expansion (line graph).

[0021] FIG. 5 is a graph showing the change in the number of cells during T-cell activation and T-cell expansion in a T-175 Flask (bar graph), along with the change in fold expansion during T-cell activation and T-cell expansion (line graph).DETAILED DESCRIPTION

[0022] The various aspects and embodiments will now be fully described herein. These aspects and embodiments may, however, be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so the disclosure will be thorough and complete, and will fully convey the scope of the present subject matter to those skilled in the art. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0023] Modifications of the disclosure will occur to those skilled in the art and to those who make or use the disclosure. Therefore, it is understood that the embodiments shown in the drawings and described above are merely for illustrative purposes and not intended to limit the scope of the disclosure, which is defined by the following claims, as interpreted according to the principles of patent law, including the doctrine of equivalents.A. Definitions

[0024] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, particular methods and materials are now described.

[0025] As used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “a component” includes embodiments having two or more such components unless the context clearly indicates otherwise.

[0026] Unless otherwise stated, the use of individual numerical values is stated as approximations as though the values were preceded by the word “about” or “approximately.” Similarly, the numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about” or “approximately.” In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms “about” and “approximately” when referring to a numerical value shall have their plain and ordinary meanings to a person of ordinary skill in the art to which the disclosed subject matter is most closely related or the art relevant to the range or element at issue. The amount of broadening from the strict numerical boundary depends upon many factors. For example, some of the factors which may be considered include the criticality of the element and / or the effect a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those of skill in the art. As used herein, the use of differing amounts of significant digits for different numerical values is not meant to limit how the use of the words “about” or “approximately” will serve to broaden a particular numerical value or range. Thus, as a general matter, “about” or “approximately” broaden the numerical value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values plus the broadening of the range afforded by the use of the term “about” or “approximately.” Consequently, recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate valuefalling within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0027] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and / or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0028] As used herein, “have,” “having,” “include,” “including,” “comprise,” “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to.”

[0029] “Optional” or “optionally” means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component, or circumstance occurs and instances where it does not.

[0030] In this document, relational terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0031] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.B. Introduction

[0032] Primary T-cells isolated from patients and then engineered cannot grow in vitro without first being activated by T-cell activators. In vitro, engineered T-cell activation occurs either by artificial antigen-presenting cells (“aAPCs”) with a coating comprising T-cell activators or by surface mediated activation with a coating comprising T-cell activators. With aAPC activation, micrometer or nanometer sized beads are coated with T-cell activators such as anti-CD3 or anti-CD28 antibodies. Although bead-based activation has high activationefficiency, beads present safety concerns for use with therapeutics because during activation, beads can be internalized by the engineered T-cells and become difficult if not impossible to remove from the final products. With surface-mediated activation, T-cell activators such as anti-CD3 or anti-CD28 antibodies are coated onto a surface area of a surface and then that surface is contacted with the engineered T-cells. However, due to the cost of coating such surfaces and the small number of engineered T-cells to be activated in the process of patient derived immunotherapies, this necessitates the use of small vessels for activation and then transfer of activated T-cells into a larger vessel for proliferation (expansion of the number of engineered T-cells to create the therapeutic product). The transfer of activated T-cells can cause loss of the valuable engineered T-cells that have been activated and can introduce contamination, which destroys the entire batch. The present disclosure provides a solution that allows activation of T-cells and multi-layer expansion of the T-cells in a single vessel, without the use of beads.

[0033] Additional features and advantages will be set forth in the detailed description which follows and will be apparent to those skilled in the art from the description or recognized by practicing the embodiments as described in the following description, together with the claims and appended drawings.C. Bioprocess Vessel

[0034] According to aspects of the present disclosure, a bioprocess vessel for activating and expanding T-cells comprises a top side, a bottom side, sidewalls, at least one port, and a closure or connector for the at least one port. If more than one port exists, there may be a closure or connector for each port. The bioprocess vessel also comprises a coating with T-cell activators in the coating on at least one sidewall, the top side, or the bottom side of the vessel.

[0035] In some embodiments, there is at least one cell culturing layer between the top and bottom sides of the vessel. In some embodiments, there is a plurality of layers for culturing cells between the top and bottom sides of the vessel. Any number of layers may be present in a bioprocess vessel, for example, any number of cell culturing layers between 1 layer and 100 layers. In one embodiment, the bioprocess vessel comprises between 1 layer and 90 layers, between 1 layer and 80 layers, between 1 layer and 70 layers, between 1 layer and 60 layers, between 1 layer and 50 layers, between 1 layer and 40 layers, between 1 layer and 30 layers, between 1 layer and 20 layers, between 1 layer and 10 layers, between 5 layers and 30 layers,between 5 layers and 20 layers, or any number or range between 1 layer and 100 layers. In some embodiments, more than 100 cell culturing layers may be present. Cells may be cultured on either the top side of the layer, the bottom side of the layer, or both sides of the layer. Cells may also be cultured in the vessel on the interior portion of the bottom side of the vessel, the interior portion of the top side vessel, the interior portion of one or more of the sidewalls of the vessel, or any combination thereof.

[0036] Referring now to FIG. 1 , an exemplary bioprocess vessel 10 is shown, according to aspects of the disclosure. Bioprocess vessel 10 comprises a top side 15, a bottom side 20, a sidewall 15a at one end of the vessel that connects top side 15 to bottom side 20, a sidewall 15b that connects bottom side 20 to the section of vessel 10 that comprises a port 35, two sidewalls that extend the length of the vessel between the top and bottom sides (not shown), four cell culturing layers (30a, 30 / ?. 30c, 3 ( / ) between the top and bottom sides of the vessel, and closure 40 in the form of a cap. Bioprocess vessel 10 also comprises a coating 50 on sidewall 15a. The inside surface of the bottom side is also considered a cell culturing layer, such that the total number of cell culturing layers in the bioprocess vessel 10 is five layers.

[0037] The port(s) may be on one or more sidewalls of the bioprocess vessel, the top side of bioprocess vessel, or a combination thereof. The port(s) may also be on the bottom of the vessel in some embodiments. In some embodiments, there are at least two ports on the bioprocess vessel and liquid flow into the bioprocess vessel through one port and liquid flows out of the bioprocess vessel through the second port. In some embodiments, there is a single port on the bioprocess vessel where liquid can flow into the vessel and out of the vessel through the port. Ports may be flush on the bioprocess vessel, or they may be necked access ports. A necked access port is a port that extends outwardly from the vessel such that a tube or other connector, or a cap or other closure, may be secured over at least part of the neck portion of the necked access port.

[0038] Closures or connectors may be used for the port(s) and / or the necked access port(s). If a closure is employed, the closure may be any type of closure known to those of ordinary skill in the art, including but not limited to threaded caps, unthreaded caps, stoppers, and plugs. In one embodiment, the closure is a cap. In one specific embodiment, the closure is a threaded cap with matching threads on the port to secure the cap to the port. In some embodiments, at least one closure is removably attached to a port. In some embodiments, atleast one closure is permanently affixed to a port. In some embodiments, at least one closure is removably attached to a port.

[0039] If a connector is employed, the connector may be any type of connector that may be employed in creating a closed system, including but not limited to, tubing, pipes, and other hollow devices (devices with a fluid channel) that can fluidly connect the port to other vessels or to a cap or other sealing device.

[0040] In some embodiments, the closure is gas permeable, such that gas may flow into or out of the port but liquids may not. In one embodiment, the closure may be a vented cap that has a membrane with pores sized to allow gas to pass through but not liquid. The membrane may be a hydrophobic membrane insert made from material that will allow gas transport into the bioprocess vessel interior but prevent liquid from escaping. Examples of such membrane material include polytetrafluoethylene and polyvinylidenefluoride (PVDF), but any material known to those of ordinary skill in the art may be used. If a connector is employed, the connector may be gas-permeable or non-gas permeable. In such embodiments, the material of the connector itself is either gas-permeable or non-gas permeable.

[0041] In some embodiments, the closure or connector comprises a porous membrane or mesh sized with pores large enough to allow liquids and gasses to pass through the membrane but small enough to prevent T-cells from passing through the membrane. Pore sizes from about 0.1 pm to about 3 pm ensure a pore size small enough to prevent T-cells from passing through the membrane or mesh and large enough to allow liquid (and gas) to pass through, although other sizes below 0. 1 pm may still work to allow liquid through, and other sizes slightly larger than 3 pm (e.g., 4-7 pm) may prevent certain types of T-cells from passing through. The porous membrane or mesh spans across the closure or connector such that any fluid or cells that go through the port will encounter the porous membrane or mesh. With this type of closure or connector, the cell culture media may be exchanged without needing to remove the T-cells from the bioprocess vessel.

[0042] In one embodiment, a connector comprising a porous membrane or mesh sized with pores large enough to allow liquids to pass through the membrane but small enough to prevent T-cells from passing through the membrane (e.g., has pore sizes from about 0.1 pm to about 5 pm) has threads on the internal side of the connector that match threads on the outside of the port or necked access port of the bioprocess vessel. In some embodiments, the connectoralso has threads on the outside of the connector such that a cap or another connector can be threadedly connected to the outside of the connector with the porous membrane or mesh.

[0043] In embodiments where the bioprocess vessel has at least one port with this type of closure or connector, a closed system for media exchange may be employed, where liquid but not T-cells can exit the bioprocess vessel through a first port with the porous membrane or mesh and where new liquid can be added into the bioprocess vessel through a second port. In some embodiments, the second port also includes a porous membrane or mesh with pores sized small enough to prevent T-cells from passing through the pores but large enough to allow liquid to pass through.

[0044] In some embodiments, the bioprocess vessel may be part of a closed system that allows media, T-cell activators, T-cell expansion growth factors, or other nutrients to be added to the bioprocess vessel, and that allows the removal of spent, cultured fluid out of the bioprocess vessel. A closed system is sealed off from unfiltered environmental air while the system is in use. Closed systems may have a sealed media vessel that operably connects (such as fluidly connects) to an inlet port on the bioprocess vessel, and a sealed waste vessel that operably connects to an outlet port on the bioprocess vessel.

[0045] In such a system, fluid entering the bioprocess vessel may go through the inlet port, while fluid leaving the bioprocess vessel may exit through the outlet port. A porous membrane or mesh filter may be attached or connected to the outlet port, sized to prevent T- cells from exiting the vessel but to allow liquid to exit through the outlet port. Another such porous membrane or mesh filter may be attached or connected to the inlet port. The connectors between the vessels may have valves that allow for the addition of materials not in the media, and for controlling whether fluid flows to or from the vessel. The connectors may connect to pumps or other devices that aid in drawing fluid into or out from the bioprocess vessel.

[0046] The bioprocess vessels of the present disclosure comprise a coating that assists in activating T-cells. The T-cells being activated by the coating may be naturally derived T- cells or engineered T-cells (such as those used in CAR-T and TCR-T therapies). The coating composition includes at least one T-cell activator and an aqueous solution suitable for the T- cell activator(s). Aqueous solutions that are suitable for the T-cell activators include phosphate buffered saline (“PBS”), Dulbecco’s Phosphate-Buffered Saline (“DPBS”), carbonate buffers,Tris buffers, acetate buffers, and any other buffer or media used with T-cell activators known to those of ordinary skill in the art.

[0047] A T-cell activator is a substance that stimulates T-cells to initiate targeted intracellular signals that increase a desired result (for example, overexpression of certain receptors on the outside of an engineered T-cell). Common examples of T-cell activators include but are not limited to antigens for aP-T-cell receptors (e.g., anti-CD3 monoclonal antibodies, anti-CD28 monoclonal antibodies, anti-CD19 monoclonal antibodies, and other like antigens), phytohemagglutinin (PHA) mitogen, certain cytokines such as IL-2 (interleukin- 2), and combinations thereof. Any T-cell activator known to those skilled in the field of T-cells can be provided as part of the coating for the bioprocess vessel of the present disclosure. In one embodiment, the T-cell activator is an antigen for a aP-T-cell receptor. In one specific embodiment, the T-cell activator is an antigen and is an anti-CD3 monoclonal antibody or an anti-CD28 monoclonal antibody, or a combination thereof. In some embodiments, the coating comprises at least one T-cell activator, and one or more different T-cell activators are added to the cell culture during T-cell activation.

[0048] To create T-cell signaling that extends long enough to lead to productive responses, the T-cell activators are surface-bound to at least one interior wall of the bioprocess vessel to mimic cellular interactions. Any known method of coating the activator to the surface of the walls of the bioprocess vessel may be used, as understood by those of ordinary skill in the art, such as adsorption. With adsorption, the T-cell activators in a T-cell activator coating will adhere to the surface of the bioreactor vessel where the coating is placed, and at least some of the adhered T-cell activators remain when excess coating is removed.

[0049] The coating with T-cell activators may be on at least one sidewall, the top side, or the bottom side of the bioprocess vessel, or on a combination thereof. In embodiments where the sidewalls have a smaller surface area than the surface area of the top or bottom sides of the vessel, the coating is preferably on one of the sidewalls (or a combination thereof) to save on coating costs and quantities. In embodiments where one or more sidewalls have a different surface area than other side walls, the coating is preferably on one of the smaller sized sidewalls (or a combination of smaller sized sidewalls) in the vessel.

[0050] The T-cell activator coating has a loading concentration, which is the amount of T-cell activators that are loaded per cm2of surface area of the bioreactor vessel that is coated.In some embodiments, the loading concentration of the T-cell activator coating on the bioreactor vessel may be from 0.1 pg / cm2to 50 pg / cm2. In some embodiments, the loading concentration of the T-cell activator coating on the bioreactor vessel is greater than 50 pg / cm2. In one embodiment, the loading concentration of the T-cell activator coating is from 0.1 pg / cm2to 40 pg / cm2, from 0. 1 pg / cm2to 30 pg / cm2, from 0. 1 pg / cm2to 20 pg / cm2, from 0.1 pg / cm2to 10 pg / cm2, from 0.1 pg / cm2to 5 pg / cm2, from 5 pg / cm2to 50 pg / cm2, from 10 pg / cm2to 50 pg / cm2, from 20 pg / cm2to 50 pg / cm2, from 30 pg / cm2to 50 pg / cm2, from 40 pg / cm2to 50 pg / cm2, or at any value or range from 0. 1 pg / cm2to 50 pg / cm2.

[0051] Once the coating is applied to an interior wall of the bioprocess vessel, the coating may then be incubated from about 5 minutes to about 48 hours, from about 5 minutes to 36 hours, from about 5 minutes to 24 hours, from about 5 minutes to about 20 hours, from about 5 minutes to about 16 hours, from about 5 minutes to about 12 hours, from about 5 minutes to about 8 hours, or from about 5 minutes to about 4 hours, from about 30 minutes to about 24 hours, from about 1 hour to about 24 hours, from about 2 hours to about 24 hours, from about 4 hours to about 24 hours, from about 8 hours to about 24 hours, from about 12 hours to about 24 hours, or from about 16 hours to about 24 hours, or any value or range between 5 minutes and 48 hours. The temperature at which the coating is incubated may be greater than 0 °C and less than 45 °C. In one embodiment, the temperature at which the coating is incubated is greater than 0 °C and less than 37 °C, greater than 0 °C and less than 25 °C, greater than 0 °C and less than 18 °C, greater than 0 °C and less than 12 °C, or than 0 °C and less than 8 °C, greater than 2 °C and less than 25 °C, or greater than 2 °C and less than 12 °C, or at any value or range between 0 °C and 45 °C.

[0052] After the T-cell activator coating has incubated on the interior wall surface of the bioprocess vessel, any excess coating may be removed (e.g. by draining or aspiration) and the coated interior wall may be rinsed with an aqueous solution such as PBS, DPBS or other aqueous solution for T-cell activators to eliminate unbound T-cell activators known to those of ordinary skill in the art.

[0053] In some embodiments, the bioprocess vessel may range in size from holding volumes of liquid from about 25 mb to about 50 liters (or any range or value between these two numbers), but smaller or larger sizes are also contemplated. In one embodiment, the bioprocess vessel may hold volumes of liquid of about 25 mb, about 50 mb, about 75 mb,about 100 mL, about 125 mb, about 250 mb, about 500 mb, about 750 mb, about one liter, about two liters, about three liters, about five liters, about 10 liters, about 20 liters, about 25 liters, about 35 liters, or about 50 liters. In another embodiment, the bioprocess vessel may hold volumes of liquid from 25 mL and 40 liters, from 25 mL to 30 liters, from 25 mL to 20 liters, from 25 mL to 15 liters, from 25 mL to 10 liters, from 25 mL to 5 liters, from 25 mL to 1 liter, or at any value or range from 25 mL to 40 liters.

[0054] In some embodiments, the bioprocess vessel is made from an injection molded polymer, for example polypropylene, polyethylene, polystyrene, polycarbonate, polyvinylchloride, polycarbonate, or any other appropriate polymer known to those of ordinary skill in the art. In one embodiment, the polymer is optically transparent and non-cytotoxic. Since the materials are made from lightweight polymers and the bioprocess vessel is presterilized during manufacture, the bioprocess vessel itself is disposable and there is no need for the end user to sterilize components of the system prior to use. In some embodiments, where the bioprocess vessel comprises cell culturing layers, the cell culturing layers are injection molded along with the outer body (the top side, bottom side, sidewalls, and port(s)) of the bioprocess vessel.

[0055] In some embodiments, the top side, bottom side, and sidewalls of the bioprocess vessel are permanently affixed to one another by, for example, ultrasonic welding along a weld line, laser welding, or adhesives. In embodiments where the bioprocess vessel comprises cell culturing layers, the cell culturing layers may also be permanently affixed to at least one sidewall of the bioprocess vessel. In some embodiments, the cell culturing layers may be permanently affixed to one, two, three, or four sidewalls of the bioprocess vessel. Permanently affixing the top side, bottom side, sidewalls, and cell culturing layers (if present) creates a completely and permanently integral unit. In embodiments having one or more necked access ports and caps, the caps are put into place and the unit is effectively sealed for shipment.

[0056] The integral unit may be sterilized. As most cell culture procedures are carried out under aseptic conditions by practicing sterile techniques, pre-sterilization of the bioprocess vessel provides a culture chamber that can be maintained in a sterile, closed environment. It is advantageous to have the cell culture process carried out in a system where the culture chamber is functionally closed to the external environment, with the sterile integrity maintained from the time the vessel is manufactured until it has been disposed of. One method of presterilization includes gamma irradiation. Other sterilization methods known to those skilled inthe art that could be used include ethylene oxide, electron beam irradiation, and x-ray irradiation. Doses of irradiation for sterilization may be between 1 kGy and 50 kGy. In some embodiments, sterilization is carried out before a T-cell activator coating is applied to an interior surface of the bioreactor vessel. In some embodiments, sterilization is carried out after a T-cell activator coating is applied to an interior surface of the bioreactor vessel.

[0057] T-cells that are activated and then expanded in a bioprocess vessel of the present disclosure may achieve expansions of the activated T-cells from 1.1 fold to 2000 fold in a single bioprocess vessel, or at any value or range therebetween. Expansions may be higher when fewer inoculating cells are used. In some embodiments, T-cells that are activated and expanded in a bioprocess vessel of the present disclosure may achieve expansions of the activated T-cells from 1.1 fold to 1500 fold, from 1.1 fold to 1000 fold, from 2 fold to 1000 fold, from 50 fold to 1000 fold, from 100 fold to 1000 fold, from 200 fold to 1000 fold, from 300 fold to 1000 fold, from 400 fold to 1000 fold, from 500 fold to 1000 fold, from 600 fold to 1000 fold, from 700 fold to 1000 fold, 1.1 fold to 900 fold, from 2 fold to 800 fold, from 2 fold to 700 fold, from 2 fold to 600 fold, from 2 fold to 500 fold, from 2 fold to 400 fold, or from 2 fold to 300 fold.

[0058] In some embodiments, the activated and expanded T-cells may achieve expansions of the activated T-cells from 2 fold to 10 fold after 2 days of expansion, or in any range or value therebetween. In some embodiments, the activated and expanded T-cells may achieve expansions of the activated T-cells from 15 fold to 80 fold after 5 days of expansion, or in any range or value therebetween. In some embodiments, the activated and expanded T- cells may achieve expansions of the activated T-cells from 15 fold to 80 fold after 5 days of expansion, or in any range or value therebetween. In some embodiments, the activated and expanded T-cells may achieve expansions of the activated T-cells from 50 fold to 200 fold after 10 days of expansion, or in any range or value therebetween. In some embodiments, T-cells that have been activating for at least 2 days have less than a 2-fold expansion over the number of inoculated cells for activation.

[0059] The bioprocess vessels of the present disclosure allow for the engineering, activation (and expansion) of engineered T-cells. Currently available non-bead, non-spinner flasks for activating and expanding T-cells cannot successfully activate and expand T-cells in the same vessel at any feasible level for commercial use, particularly when the expense of coatings containing biological agents are considered. The bioprocess vessel of the presentdisclosure solves this problem by providing a commercially feasible way to activate and expand T-cells in a single vessel, decreasing the need for multiple vessels along the cost of coating used. Methods of activating and expanding engineered T-cells with the bioprocess vessels of the present disclosure will now be described.D. Methods of Use

[0060] A method of activating and expanding T-cells includes the steps of (1) providing one of the bioprocess vessels of the present disclosure, (2) providing an aqueous solution into the cell culture chamber of the bioprocess vessel that is suitable for T-cell activation and expansion, (3) providing T-cells or cells comprising T-cells to the cell culture chamber of the bioprocess vessel (4) activating the T-cells by positioning the bioreactor vessel so the interior wall(s) with the T-cell activator coating are in continuous contact with the aqueous solution and T-cells, (6) providing T-cell expansion growth factors to the bioprocess vessel after the previous step, and (7) culturing the activated T-cells with the provided T-cell expansion growth factors until the T-cells have expanded (proliferated) to a desired level. Any of the embodiments for the bioprocess vessel described above will work for activating and expanding T-cells, including engineered T-cells.

[0061] The aqueous solution provided to the cell culture chamber of the bioreactor vessel may be any solution that allows activation of T-cells and / or expansion of T-cells to occur, such as Dulbecco’s Phosphate Buffered Saline (“DPBS”), Roswell Park Memorial Institute (“RPMI”) 1640 medium (Gibco®), HyClone® RPMI 1640 medium (Cytiva®), Iscov’s Modified Dulbecc’s Medium (“IMDM”), OpTmizer® CTS® T Cell Expansion Serum -Free Medium (Gibco), CTS® AIM-V® medium (Gibco®), X-VIVO® 15 (Lonza®), StemSpan® Serum-Free Expansion Medium or StemSpan® SFEM II medium (StemSpan® Technologies Canada, Inc.), and other like-media, including serum containing and serum-free variations, and other supplemented variations (e.g., with added L-glutamine, glucose, etc.). In some embodiments, the aqueous solution may further comprise additives or additional T-cell activators. The additives may be nutrients or other materials that aid in cell growth. The additional T-cell activators may be different from the T-cell activators used in the coating. Which additives and additional T-cell activators are added depend on the specific T-cell being activated and proliferated, and are known to those of ordinary skill in the art. In one nonlimiting example, the T-cell activator coating used on the bioprocess vessel may include anti-CD3 antibodies, and anti-CD28 antibodies may be added to the aqueous solution provided to the cell culture chamber.

[0062] Cells (along with media or other liquid sustenance for the cells) are provided to a bioprocess vessel having the T-cell activator coating to start T-cell activation. The cells provided to the vessel for T-cell activation may be T-cells themselves or may be a cell type that contains T-cells. Cell types that contain T-cells include cells such as peripheral blood mononuclear cells (“PBMCs”) (e.g., PBMCs from human or animal origin) and other T- lymphocyte containing cells known to those of ordinary skill in the art. In some embodiments, the cells provided to the bioprocess vessel for T-cell activation are non-engineered T-cells that have been derived from a patient or other living being that produces T-cells and / or cells that produce T-cells. Non-limiting examples of such non-engineered T-cells include patient derived CD3+ T-cells, CD28+ cells, or other patient derived T-cells. In some embodiments, the cells provided to the vessel for T-cell activation are engineered T-cells, which originate patient- derived (or other living being-derived) T-cells but have then been genetically modified. In some embodiments, the T-cell is an engineered T-cell. Engineered T-cells include T-cells that have been transduced with a viral vector that carries desired genes, such as CAR or TCR. However, any T-cell that has been genetically altered is an engineered T-cell and may be used. In some embodiments, the cells provided to the bioprocess vessel for T-cell activation may be a cell type that comprises a T-cell. In one specific embodiment, the cell type comprising T- cells is a PBMC.

[0063] To activate the T-cells, the T-cells are combined with the aqueous liquid and the mixture is contacted with the coating on the bioreactor vessel comprising T-cell activators. The vessel may be tilted or stood on an end or side to maximize contact between the T-cells and the T-cell activators of the coating. One such example of this is shown in FIG. 2, where bioreactor vessel 10 is positioned on its end such that sidewall 25a with T-cell activator coating 50 is horizontal while bottom side 20 and the additional cell culturing layers 30a, 30 / ?. 30c, and 30<7 are vertical. Cap 40 is removed and aqueous solution 60 for T-cell activation along with T-cells 65 (naturally occuring or engineered) are added to the vessel through port 35. The cap 40 may then be replaced. The T-cells 65 contact the T-cell activators in the coating 50 and become activated T-cells. However, it should be understood that if the coating is on a different interior wall of the bioreactor vessel, for activation of T-cells, the vessel will be positionedsuch that the wall having the coating will be positioned horizontally with the coating at the bottom of the vessel in that position.

[0064] The activation of T-cells may occur with the vessel in a static position. In some embodiments, the length of time for T-cell activation may be from about 5 minutes to about five days. In one embodiment, the length of time for T-cell activation may be from about 5 minutes to about 4 days, from about 5 minutes to about 3 days, from about 5 minutes to about 2 days, from about 5 minutes to about 1 day, from about 1 day to about 5 days, from about 2 days to about 5 days, from about 3 days to about 5 days, from about 4 days to about 5 days, from about 1 day to about 4 days, from about 2 days to about 4 days, or any value or range between about 5 minutes to about 5 days. In some embodiments, the activation of T-cells may occur at a temperature greater than 0 °C and less than 45 °C. In one embodiment, the temperature at which the activation of T-cells is carried out is greater than 0 °C and less than 37 °C, greater than 0 °C and less than 25 °C, greater than 0 °C and less than 18 °C, greater than 0 °C and less than 12 °C, or than 0 °C and less than 8 °C, greater than 2 °C and less than 25 °C, or greater than 2 °C and less than 12 °C, or at any value or range between 0 °C and 45 °C. In one specific embodiment, the temperature for T-cell activation is between 25 °C and 40 °C.

[0065] Once the T-cells have been activated, the T-cells are then expanded (proliferated). The activated T-cells may be expanded at any time, but preferably, expansion begins after (1) the T-cells have increased in diameter (due to activation), or (2) the number of cells in the bioprocess vessel exceeds the number of cells provided to the bioprocess vessel, or (3) the T-cells in the bioprocess vessel being activated begin expressing CD25+ or CD95+ (or both) receptors, or (4) a combination thereof. The average T-cell size and the number of cells in the bioprocess vessel can be determined by an automatic cell counter such as a ViCELL cell counter (Beckman Coulter). Expression of CD25+ or CD95+ can be determined using flow cytometry with a fluorophore on anti-CD25 antibodies for CD25+ expression or anti-CD29 antibodies for CD95+ expression that bind to T-cells expressing these receptors.

[0066] Expansion begins when expansion growth factors are added into the bioprocess vessel containing the activated T-cells. Expansion growth factors are specific materials that signal the T-cell to start the T-cell’s growth phase (as opposed to the T-cell’s activation phase). Specific cellular materials indue the growth of T-cells, such as certain cytokines. Expansion growth factors for T-cell expansion include interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin- 15 (IL-15), and interleukin-3 (IL-3), among other T-cell expansion growth factorsknown to those of ordinary skill in the art. An additional amount of aqueous solution for expanding the T-cells (with or without the expansion growth factors) may be added to the bioreactor vessel after the activation step is completed to provide starting nutrients and volume for cell expansion.

[0067] After the activation step, the bioreactor vessel may then be positioned to distribute activated T-cells across the bioprocess vessel and on any cell culturing layers that are contacting the aqueous solution. In embodiments where the cell culturing layers are present and are approximately parallel to the bottom side of the bioreactor vessel, cell expansion is preferably performed with both the bottom side of the vessel and the cell culturing layers being positioned to be horizontal to the surface the vessel is sitting on.

[0068] One example of expanding the activated T-cells inside the bioreactor vessel is shown in FIG. 3. Additional aqueous solution 60 has been added into bioreactor vessel 10 through port 35 (cap 40 is removed during the addition of the liquid and then replaced). As the T-cell activator coating 50 is on the end of the vessel on sidewall 25a, the vessel has been repositioned from its configuration in FIG. 2 during T-cell activation to a flat position where bottom side 20 and cell culturing layers 30a-30<7 are horizontal for the expansion stage. T-cells 65 are activated and distributed across the bottom side 20 and the layers 30a-30<7 of the vessel for the expansion stage.

[0069] Additional aqueous solution may be added to the bioreactor vessel during the expansion stage as needed. Likewise, aqueous solution may be removed and replaced with fresh aqueous solution through any ports as needed during the expansion stage. The exchange of aqueous solution may be needed in some embodiments to ensure the cells have enough nutrients and oxygen for growth during expansion. In some embodiments, the exchange of aqueous solution may involve removing some or all the aqueous liquid with cells, spinning the cells down in a centrifuge, then resuspending them in fresh aqueous liquid, and adding the resuspension (and any additional amounts of aqueous solution if needed) back into the bioprocess vessel. In some embodiments, some or all of the aqueous solution may be removed out of the vessel through a porous membrane that has pores sized large enough to allow liquid to flow in and out but sized small enough to keep the T-cells in the vessel, and then fresh aqueous solution may be added through the same port or a different port. In some embodiments, the exchange of aqueous solution occurs in a closed system (the cells and other contents are not exposed to unfiltered air).

[0070] The expansion process may occur over an extended period of time. In some embodiments, the expansion of activated T-cells may be a length of time between about 1 day and about 30 days, between about 1 day and about 25 days, between about 1 day and about 20 days, between about 1 day and about 15 days, between about 1 day and about 10 days, between about 5 days and about 30 days, between about 10 days and about 30 days, between about 15 days and about 30 days, between about 20 days and about 30 days, between about 3 days and about 21 days, or at any value or range between about 1 day and 30 days.

[0071] T-cells or cellular materials may be harvested after expansion through the access ports by means of pipette, pouring, pumping, or other like way.

[0072] In some embodiments, using the methods described above and the bioprocess vessels of the present disclosure, at least 70% of T-cells are viable three days after initiation of activation of the T-cells. In one embodiment, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of T-cells are viable three days after initiation of activation of the T-cells. Automatic cell counters known in the industry, such as the Vi-CELL cell counter line of machines from Beckman-Coulter, can provide the viable cell density, the percentage of viable cells, and the average cell diameter values of a culture (including for T-cells). In some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of T-cells are viable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14, days, 15 days, or more, after initiation of expansion of the T-cells. In one embodiment, at least 80% of T-cells are viable 3 days after initiation of the expansion phase.

[0073] While the present disclosure includes a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the present disclosure.EXAMPLESExample 1

[0074] A Falcon® 525 cm2rectangular straight neck cell culture flask with three layers and a vented cap with a gas permeable membrane was employed for T-cell activation and expansion. The flask was positioned so it was standing upright on its short end (sidewall) such that the bottom of the flask and the layers were vertical, and the short end (sidewall) of theflask was horizontal while the port and cap were now at the highest point. Anti-CD3 antibodies (Clone 0KT3, ThermoFisher) at a concentration of 1 mg / mL were mixed with Dulbecco’s Phosphate-Buffered Saline (DPBS) to a concentration of 5 pg / mL and then 10 mb of the mixture was pipetted onto the interior surface of the short end (sidewall) of the flask, which had a surface area of 35.0 cm2. The loading concentration of the T-cell activator on the sidewall was 1.43 pg / cm2. The flask was then stored at 4 °C overnight to allow adsorption of the antibodies on the interior surface of the flask. Excess coating was then removed. The T-cell activator coating was then washed two times with 20 m DPBS, with each wash then discarded.

[0075] Next, primary human Peripheral Blood Mononuclear Cells (“hPBMC”) (STEMCELL Technologies™) were inoculated into 18 mL of OpTmizer™ CTS™ T-cell Expansion Media (Life Technologies A1048501) to a concentration of 0.97 x 106hPBMCs / mL. 10 mL of the inoculation solution was added to the coated flask along with 40 pL of Anti-CD28 antibody (Clone CD28.2, ThermoFisher) at 1 mg / mL concentration, and the coated flask was stood on the short end that had the coating for the activation stage. The flask was then left for 3 days in a humidified incubator at 37 °C and 5% carbon dioxide on its short end for activation of the T-cells to occur.

[0076] After the 3 -day activation stage, the cell density was analyzed with a cell counter (see Table 1, row 3BD (before dilution)) and then additional OpTmizer™ CTS™ T- cell Expansion Media was added to the flask until the activated T-cells were diluted down to 0.25xl06cells / mL (see Table 1, row 3 AD (after dilution)). The flask was positioned so that the bottom side of the flask and the layers were horizonal. The media with activated T-cells was distributed across the bottom of the flask and the layers. The activated T-cells were then expanded (proliferated) for 9 days with the bottom side of the flask set horizontal. The viable cell density (“VCD”), the percentage of viable cells, and average cell diameter were checked each day during activation and expansion with a ViCELL Cell Counter (Beckman-Coulter). The measurements shown in Table 1 columns 2-7 were taken before any dilution or media exchange. After the measurements were taken, the cells were allowed to continue to grow, were diluted, or were subjected to a partial media exchange, as described below in the last column (column 8) of Table 1 . The flask used did not have a closed media exchange system, so media exchange was performed by removing the cells and spent media through the port on the flask and then spinning down the cells in a centrifuge at 300g for 10 minutes followed by resuspension in 60 mL of fresh OpTmizer™ CTS™ T-cell Expansion Media.

[0077] Table 1

[0078] As shown in FIG. 4, the T-cell numbers stay relatively flat during the first 3 days during the activation stage. Then the cells expand over the next 9 days during the expansion phase until the cells reach approximately a plateau of over 100-fold expansion with over 1000 x 106total cells by the end of the 9 days of expansion.Example 2

[0079] A Coming® 175 cm2rectangular straight neck cell culture flask without any layers and a vented cap with a gas permeable membrane was employed for T-cell activation and expansion. The flask was positioned so it was standing upright on its short end (sidewall) such that the bottom of the flask was vertical, and the short end (sidewall) of the flask was horizontal with the port and cap at the highest point. The sidewall had a surface area of 60.5 cm2. Anti-CD3 antibodies (Clone OKT3, ThermoFisher) at a concentration of 1 mg / mL were mixed with DBPS to a concentration of 5 pg / mL and pipetted 5 mL of the mixture onto theinterior surface of the short end (sidewall) of the flask. The loading concentration of the T-cell activator was 0.51 pg / cm2. The flask was then stored at 4 °C overnight to allow adsorption of the antibodies on the interior surface of the flask. Excess coating was then removed. The T-cell activator coating was then washed two times with 10 mL DPBS, with each wash then discarded.

[0080] Next, T-cells from a hPBMC cell line (STEMCELL Technologies™) were inoculated into 18 mL of OpTmizer™ CTS™ T-cell Expansion Media (Life Technologies A1048501) to a concentration of 0.97 x 106hPBMCs / mL. 5 mL of the inoculation solution was added to the coated flask along with 20 pL of Anti-CD28 antibody (Clone CD28.2, ThermoFisher) at 1 mg / mL concentration, and the coated flask was stood on the short end that had the coating for the activation stage. The flask was then left for 3 days in a humidified incubator at 37 °C and 5% carbon dioxide on its short end for activation of the T-cells to occur.

[0081] After the 3 -day activation stage, the cell density was analyzed with a cell counter (see Table 2, row 3BD (before dilution)) and then additional OpTmizer™ CTS™ T- cell Expansion Media was added to the flask until the activated T-cells were diluted down to 0.25xl06cells / mL (see Table 2, row 3 AD (after dilution)). The flask was positioned so that the bottom side of the flask was horizonal. The activated T-cells were then expanded (proliferated) for 9 days with the bottom side of the flask set horizontal. The viable cell density (“VCD”), the percentage of viable cells, and average cell diameter were checked each day during activation and expansion with a ViCELL Cell Counter (Beckman-Coulter). The measurements shown in Table 2 columns 2-7 were taken before any dilution or media exchange. After the measurements were taken, the cells were allowed to continue to grow, were diluted, or were subjected to a partial media exchange, as described below in the last column (column 8) of Table 2. The flask used did not have a closed media exchange system, so media exchange was performed by removing the cells and spent media through the port on the flask and then spinning down the cells in a centrifuge at 300g for 10 minutes followed by resuspension in 30 mL of fresh OpTmizer™ CTS™ T-cell Expansion Media.

[0082] Table 2

[0083] As shown in FIG. 5, the T-cell numbers stay relatively flat during the first 3 days during the activation stage. Then the cells expand over the next 9 days during the expansion phase until the cells reach approximately a plateau of over 190-fold expansion with over 875 x 106total cells by the end of the 9 days of expansion.

Claims

CLAIMSWhat Is Claimed Is:

1. A bioprocess vessel, comprising: a vessel body comprising a top side, a bottom side, and sidewalls; at least one access port; and a T-cell activator coating on at least one interior wall of the vessel body; wherein the bioprocess vessel is configured to activate T-cells when cells comprising T-cells are contacted with the coating and to expand T-cells or cells comprising T-cells when expansion growth factors are added to the bioprocess vessel.

2. The bioprocess vessel of claim 1, further comprising at least one cell culturing layer between the top side and bottom side of the vessel body.

3. The bioprocess vessel of any one of claims 1-2, wherein the bioprocess vessel comprises a closure or connector for the at least one access port, and the closure or connector comprises a porous membrane having pores sized to allow liquid to flow through the pores but sized to prevent T-cells from passing through the pores.

4. The bioprocess vessel of any one of claims 1-3, wherein the T-cell activator coating is on a sidewall.

5. The bioprocess vessel of any one of claims 1-4, wherein the T-cell activator coating comprises anti-CD3 antibodies, anti-CD28 antibodies, or a combination thereof.

6. The bioprocess vessel of any one of claims 1-5, wherein the T-cell activator coating comprises a loading concentration of T-cell activators from 0.1 pg / cm2to 50 pg / cm2.

7. The bioprocess vessel of any one of claims 1-6, wherein T-cells activated and expanded in the bioprocess vessel expand at least 2-fold over the number of T-cells in the bioprocess vessel at the end of activation.

8. The bioprocess vessel of any one of claims 1-6, wherein T-cells activated and expanded in the bioprocess vessel expand from 50 fold to 2000 fold over the number of T- cells in the bioprocess vessel at the end of activation.

9. The bioprocess vessel of any one of claims 1-6, wherein at least 85% of T-cells in the bioprocess vessel are viable three days after initiation of activation.

10. The bioprocess vessel of any one of claims 1-7, wherein at least 85% of T-cells in the bioprocess vessel are viable three days after initiation of expansion.

11. The bioprocess vessel of any one of claims 1-10, wherein the bioprocess vessel is a sterilized bioprocess vessel, and the sterilization is high-energy irradiation.

12. The bioprocess vessel of claim 11, wherein the high-energy irradiation is gamma irradiation, e-beam irradiation, or x-ray irradiation at a dosage between 1 kGy and 50 kGy.

13. A method for activating and expanding T-cells, comprising the steps of:(a) providing the bioprocess vessel of any of the preceding claims;(b) providing an aqueous solution to the bioprocess vessel that is suitable for T-cell activation;(c) providing T-cells or cells comprising T-cells to the bioprocess vessel;(d) positioning the bioprocess vessel such that the T-cell activator coating on the bioprocess vessel is in contact with the T-cells or cells comprising T-cells in the aqueous solution to activate the T-cells;(e) providing T-cell expansion growth factors to the bioprocess vessel after step (d); and(f) culturing the T-cells with the provided T-cell expansion growth factors until the T- cells have expanded at least 2-fold over the number of T-cells present at the end of activation.

14. The method of claim 13, wherein at least 70% of the T-cells are viable three days after initiation of activation during step (d).

15. The method of claim 14, wherein at least 70% of the T-cells are viable three days after culturing the T-cells with the provided T-cell expansion growth factors.

16. The method of claim 13, wherein the T-cells are cultured until they have expanded at least 100-fold over the number of cells present at the end of activation.

17. The method of any one of claims 13-16, wherein T-cell activator coating is on a sidewall of the bioprocess vessel.

18. The method of claim 17, further comprising the step of positioning the bioprocess vessel on its bottom side for step (f).

19. The method of any one of claims 13-18, wherein the T-cells or cells comprising T- cells provided to the bioprocess vessel are engineered T-cells.

20. The method of claim 19, wherein the engineered T-cells comprise a gene for a chimeric antigen receptor or for a T-cell receptor.

21. The method of any of claims 13-18, wherein the T-cells or cells comprising T-cells provided to the bioprocess vessel are patient-derived T-cells or patient derived cells comprising T-cells.

22. The method of any of claims 13-21, wherein the T-cell activator comprises an antigen to an aP-T-cell receptor on the T-cells.

23. The method of claim 22, wherein the antigen comprises anti-CD3 or anti-CD28, or a combination thereof.

24. The method of any of claims 13-23, further comprising the step of performing aqueous solution additions, aqueous solution exchanges, or a combination thereof during the culturing of the T-cells or cells comprising T-cells.

25. The method of any of claims 12-24, wherein the bioreactor vessel is a sterilized bioreactor vessel, irradiated with gamma irradiation, e-beam irradiation, or x-ray irradiation at a dosage between 1 kGy and 50 kGy.

26. A bioprocess vessel, comprising: a vessel body comprising a top side, a bottom side, and sidewalls; at least one access port and a closure or connector for the at least one access port comprising a porous membrane with pores sized to allow liquid to flow through the pores and sized to prevent T-cells from flowing through the pores; anda T-cell activator coating on at least one interior wall of the vessel body; wherein the bioprocess vessel is configured to activate T-cells when T-cells or cells comprising T-cells are contacted with the coating and to expand T-cells when T-cell expansion growth factors are added to the bioprocess vessel.

27. The bioprocess vessel of claim 26, wherein expansion in the bioprocess vessel achieves a number of T-cells that is least 2-fold more than a number of T-cells at the end of activation.

28. The bioprocess vessel of claim 27, wherein at least 70% of the number of T-cells at the end of activation are viable and wherein at least 70% of the number of T-cells at the end of expansion are viable.

29. The bioprocess vessel of any one of claims 26-28, wherein the T-cell activator coating comprises an antigen to an aP-T-cell receptor on the T-cells.

30. The bioprocess vessel of any one of claims 26-29, wherein the bioprocess vessel is a sterilized bioprocess vessel, and the sterilization is gamma irradiation, e-beam irradiation, or x-ray irradiation at a dosage in a range from 1 kGy to 50 kGy.

31. The bioprocess vessel of any one of claims 26-30, wherein the T-cell activator coating comprises a loading concentration of T-cell activators from 0.1 pg / cm2to 50 pg / cm2.

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