Devices and methods for isolation, activation, transduction and expansion of t cells

Abstract

To provide devices and methods for the isolation, activation, transduction and expansion of T cells.SOLUTION: Provided are devices and method for the isolation, activation, transduction, and expansion of T cells. A three-dimensional (3D) bioreactor containing an antibody coating may be used. Such a 3D bioreactor can be used for the isolation of T cells from peripheral blood mononuclear cells, including attaching the T cells to the 3D bioreactor surface for activation and transduction with a lentiviral vector to generate CAR T cells. The CAR T cells can then be expanded in a separate downstream bioreactor, providing a scalable, automated system.SELECTED DRAWING: Figure 7A

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with federal support under FDA Contract No. 75F40119C10158. The federal government has certain rights in this invention.

[0002] Field

[0002] The present invention relates to an apparatus and method for the isolation, activation, transduction, and expansion of T cells. A three-dimensional (3D) bioreactor containing an antibody coating can be used. Such a 3D bioreactor can be used for the isolation of T cells from peripheral blood mononuclear cells, including attaching the T cells to the 3D bioreactor surface for activation and transduction with a lentiviral vector to generate CAR T cells. The CAR T cells can then be expanded in another downstream bioreactor, providing a scalable, automated system. [Background technology]

[0003] background Chimeric antigen receptor (CAR) T-cell therapy is considered a groundbreaking approach to curing cancer by relying on a patient's own immune cells. Five CAR T-cell-based therapies have received FDA approval in the past four years. Numerous clinical trials are underway. However, consistent, scalable, and cost-effective manufacturing remains an ongoing goal for CAR T-cell-based therapies. Currently, the relatively high cost of CAR T-cell-based therapies limits the number of patients who could potentially benefit from this novel cancer treatment, and the biotechnology industry remains committed to pursuing new platforms and protocols for generating CAR T cells.

[0004]

[0004] U.S. Patent No. 10,988,724, entitled "Three-Dimensional Bioreactors For Cell Expansion And Related Applications," describes a three-dimensional bioreactor consisting of interconnected voids having multiple pore openings in such voids.

[0005]

[0005] U.S. Patent No. 11,149,244, entitled "Three-Dimensional Bioreactor For T-Cell Activation And Expansion For Immunotherapy," describes a method for expanding T cells that relies on a 3D bioreactor containing multiple voids with surface areas for cell growth. A polydopamine coating is applied to the bioreactor surface, and then a tetrameric protein is attached to the polydopamine coating, and one or more biotinylated antibodies are immobilized on the tetrameric protein. T cells bearing T cell surface receptors that bind to and are activated by the biotinylated antibodies are then flowed into the 3D bioreactor. The T cells are then exposed to a perfusion medium containing signaling molecules to promote T cell proliferation.

[0006]

[0006] U.S. Patent No. 11,447,731, entitled "Three-Dimensional Bioreactors," describes three-dimensional bioreactors for cell growth similarly constructed with interconnected voids having multiple pore openings in the voids, the bioreactors being coated with substituted or unsubstituted poly(p-xylylene).

[0007]

[0007] U.S. Patent Application Publication No. 20210317396, entitled "Three-Dimensional Bioreactor For Viral Vector Production," describes a three-dimensional bioreactor having a plurality of interconnected voids and a plurality of pore openings in the voids, including seeding the bioreactor with viral vector-producing cells and flowing a perfusion medium through the bioreactor to promote proliferation of the viral vector cells.

[0008]

[0008] U.S. Patent Application Publication No. 20230139619, entitled "Devices For Cell Separation," describes devices for cell separation that include a plurality of non-random solid geometric structures and, optionally, a plurality of non-random solid interconnecting elements between such structures. The surfaces of such devices can be coated and functionalized to allow selective ligand or cell binding, and methods are provided for separating one or more target cells from a plurality of cells. Summary of the Invention [Problem to be solved by the invention]

[0009]

[0009] Nevertheless, there remains a need for improved bioreactor designs to provide both methods and devices for improving cell proliferation, particularly T cell proliferation. More specifically, there is a need for newer, scalable perfusion-based bioreactor systems that facilitate integrated protocols for T cell isolation, activation, transduction, and proliferation. [Means for solving the problem]

[0010] overview A method for isolating, activating, transducing and expanding T cells, comprising: a. i. a plurality of voids having a diameter D, including a void surface area for coating, and a plurality of pore openings of said voids having a diameter d, wherein 90% or more of said voids have a selected void volume (V) that does not vary by more than + / - 10.0%, and 90% or more of said pore openings of said voids have a value of d that does not vary by more than + / - 10.0%; or ii. A plurality of solid geometric structures having an exterior surface for coating, wherein 90% or more of said solid geometric structures have a volume (V) that does not vary by more than + / - 10.0%. providing a 3D bioreactor comprising: b. coating the 3D bioreactor with an antibody; c. Binding T cells to the antibody; d. activating the T cells; e. transducing the antibody-bound T cells with a transduction reagent; f. removing the transduced T cells from the 3D bioreactor and transferring them to a T cell expansion bioreactor, where the transduced T cells undergo expansion; A method comprising:

[0011] 1. A device for isolating, activating, transducing and expanding T cells, comprising: a. i. a plurality of voids having a diameter D, including a void surface area for coating, and a plurality of pore openings of said voids having a diameter d, wherein 90% or more of said voids have a selected void volume (V) that does not vary by more than + / - 10.0%, and 90% or more of said pore openings of said voids have a value of d that does not vary by more than + / - 10.0%; or ii. A plurality of solid geometric structures having an exterior surface for coating, wherein 90% or more of said solid geometric structures have a volume (V) that does not vary by more than + / - 10.0%. a 3D bioreactor comprising: b. a T cell expansion bioreactor connected to said 3D bioreactor; An apparatus comprising: [Brief explanation of the drawings]

[0012] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A]

[0012] A portion of a 3D bioreactor herein is shown having multiple non-random voids and non-random pore openings. [Figure 1B]

[0013] Non-random porosity is shown in cross section. [Figure 2A]

[0014] 1 illustrates a portion of a 3D bioreactor herein having non-random solid geometric structures and optional non-random solid interconnecting elements. [Figure 2B]

[0015] Also shown is a portion of a 3D bioreactor herein having non-random solid geometric structures and optional non-random solid interconnecting elements. [Figure 2C]

[0016]

[0023] Yet another example of a portion of the 3D bioreactor herein is provided having non-random solid geometric structures and optional non-random solid interconnecting elements. [Figure 3]

[0017] 10 shows a portion of a 3D bioreactor herein illustrating an exemplary use of non-random spheres and non-random interconnected elements. [Figure 4]

[0018] 1 shows a bioreactor in either a first or second configuration disposed within a housing between an inlet and an outlet for fluid flow. [Figure 5]

[0019] 1A and 2B show portions of a 3D bioreactor surface herein within a non-random void space as shown in FIG. 1A or on a surface of a non-random geometry as shown in FIG. 2A, which includes an antibody coating promoted on immobilized particles. [Figure 6A]

[0020] 1A or 2A placed within a housing for the inflow of a mixed population of T cells, B cells, monocytes, and NK cells. [Figure 6B]

[0021] The arrangement of bound / activated T cells within the 3D bioreactor 34 is shown. [Figure 6C]

[0022] An integrated bioreactor system is shown in which activated T cells are transduced to form CAR T cells, which are then dissociated from the antibody and flowed from the antibody-coated 3D reactor into a separate expansion reactor. [Figure 7A]

[0023] 1 shows a negative 3D bioreactor combined with a gas permeable fast growth reactor. [Figure 7B]

[0024] A preferred method of using the device of FIG. 7A for the purification, activation, transduction, and expansion of T cells is shown. [Figure 8A]

[0025] Figure 1 shows cell proliferation using the 3D bioreactor compared to using Dynabeads™ over the indicated time periods. [Figure 8B]

[0026] The average cell size over the indicated time periods is shown. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description

[0027] The present invention relates to a bioreactor system that provides a scalable automated system. That is, a first 3D bioreactor can be an antibody-coated "positive" or "negative" 3D bioreactor utilized for the isolation or purification, activation, and transduction of T cells. The first 3D bioreactor is then connected to a second bioreactor, a non-adherent T cell expansion reactor, that provides the proliferation (i.e., expansion and proliferation) of T cells to provide the T cell output.

[0014]

[0028] A first type of 3D reactor 10 for antibody coating is shown in FIG. 1A. The 3D bioreactor, shown in cutaway view, includes a continuously interconnected void surface region 12 comprising a plurality of interconnected, non-random voids 14, which are preferably spherical with an internal concave curve. By referring to non-random, it is understood that the 3D bioreactor allows for the specification of a targeted or selected number of voids that result in an actual repeating void size and / or geometry with a desired tolerance. The 3D bioreactor also includes interconnected, non-random pore openings 16 of the voids, which may also have a desired tolerance. The 3D bioreactor also ultimately defines a layer of non-random voids (see arrow "L"), and multiple layers of the 3D bioreactor may then allow for the specification of such non-random voids in columns (see arrow "C"). It is also useful to note here that such 3D bioreactors, as described, may be referred to as "negative" bioreactors because they rely on a plurality of interconnected, non-random void spaces and interconnected, non-random pore openings.

[0015]

[0029] The 3D bioreactor of FIG. 1A is preferably such that the plurality of voids 14 have a diameter D (the longest distance between any two points on the interior void surface) and the plurality of pore openings have a diameter d (the longest distance between any two points on the pore openings), where D>d. See FIG. 1B (pore openings not shown). Additionally, greater than 90.0% of the voids, or even greater than 95.0% of such voids, or even 99.0% to 100% of such voids, have a void volume (V) that varies to a tolerance of no more than + / −10.0%, or + / −5.0%, or + / −2.5%, or + / −1.0%, or + / −0.5%, or + / −0.1%.

[0016]

[0030] Furthermore, 90.0% or more of the pore openings of the voids, or even 95.0% or more of the pore openings, or even 99.0% to 100% of the pore openings exhibit a value of d, the tolerance of which does not vary by more than + / - 10.0%, or + / - 5.0%, or + / - 2.5%, or + / - 1.0%, or + / - 0.5%, or + / - 0.1%. The diameter (D) of the voids preferably has a value in the range of 0.09 mm to 100.0 mm, including all individual values ​​and increments therein. For example, the diameter of the voids may be in the range of 0.2 mm to 50.0 mm, or 0.2 mm to 50.0 mm, or 0.4 mm to 50.0 mm, or 0.4 mm to 25.0 mm. The diameter (d) of the pore openings preferably has a value in the range of 0.01 mm to 10.0 mm, including all individual values ​​and increments therein. For example, the diameter (d) of the pore opening can range from 0.05 mm to 2.0 mm, or from 0.1 mm to 2.0 mm.

[0017]

[0031] The 3D bioreactor herein may also be constructed in a second configuration, preferably comprising a plurality of non-random solid geometric structures and, optionally, a plurality of interconnecting elements. Thus, this second configuration may be understood as a "positive" 3D bioreactor. The solid geometric structures may preferably comprise spheres, ellipses, and / or polygons, thereby presenting an exterior surface for the production of CAR T cells herein. As described, such solid geometric structures may optionally be connected by a plurality of solid interconnecting elements, which may assume various geometries, including rod-like or cylindrical, ellipses, and / or polygons. Such solid interconnecting structures may also provide an exterior surface for the production of CAR T cells. Neither the solid geometric structures nor the solid interconnecting elements themselves necessarily need be completely solid, but may contain a partially hollow interior for the placement of nutrients and / or other reagents to improve the performance of such a 3D bioreactor for the production of CAR T cells.

[0018]

[0032] 2A and 2B illustrate a second configuration of the 3D bioreactor 18 herein, in which the non-random solid geometric structures and optional non-random solid interconnecting elements preferably include spheres 20 and interconnecting rods 22. As shown in FIG. 2C, the 3D bioreactor 18 preferably has a diameter Φ ranging from 2.0 mm to 10,000 mm and a height H ranging from 1.0 mm to 5,000 mm. Preferably, the 3D bioreactor 18 has a Φ / H ratio ranging greater than 1:1. FIG. 3 illustrates a portion of the 3D bioreactor 18 with exemplary use of spheres and optional use of interconnecting rod elements. The non-random solid geometries herein preferably have a diameter D' (the longest distance between two points on the outer surface of the solid geometric structure and through the interior of the structure) ranging from 2.0 μm to 25.0 mm, including all values ​​and increments therein. Thus, D' may have a value in the range of 200 μm to 25.0 mm, or 5.0 μm to 10.0 mm, or 5.0 μm to 6.0 mm, or 1.0 mm to 25.0 mm. The solid geometric interconnection element (ICE) preferably has a diameter D'' in the range of 1.0 μm to 12.5 mm, including all values ​​and increments therein. Thus, D'' may preferably have a value of 1.0 μm to 3.0 mm. The length of the solid interconnection element (ICE L ) preferably has a value between 0.1 μm and 25.0 mm, including all values ​​and increments therein. L may have a value between 100.0 μm and 5.0 mm, or between 100.0 μm and 3.0 mm. The diameter of the solid interconnect structure (eg, rod) is preferably smaller than half the value of the diameter of the solid geometric shape (eg, sphere).

[0019]

[0033] Similar to the first configuration of the 3D bioreactor, the second configuration can also be characterized by its overall non-random nature, i.e., with respect to solid geometries (e.g., spheres 20), 90% or more of such solid geometries, or even 95.0% or more of such solid geometries, or even 99.0% to 100% of such solid geometries, have defined volumes with a tolerance such that the volumes do not vary by more than + / −10.0%, or + / −5.0%, or + / −2.5%, or + / −1.0%, or + / −0.5%, or + / −0.1%. Similarly, with respect to the optional use of solid interconnection elements (e.g., rods 20), 90% or more of such solid interconnection elements, or even 95.0% or more of such solid interconnection elements, or even 99.0% to 100% of such solid interconnection elements, will have defined volumes with a tolerance such that the volumes do not vary by more than + / -10.0%, or + / -5.0%, or + / -2.5%, or + / -1.0%, or + / -0.5%, or + / -0.1%.

[0020]

[0034] The 3D bioreactor device in the first or second configuration is preferably fabricated from a biocompatible or bioinert polymeric material, such as polystyrene, polycarbonate, acrylonitrile-butadiene-styrene (ABS), polylactic acid (PLA), or polycaprolactone (PCL), which are used in FDM (fused deposition modeling) 3D printing technology. References to biocompatible or bioinert should be understood to mean materials that are non-toxic to cultured cells. Additionally, the polymeric material for the device in the first or second configuration is preferably selected from polymers that are not susceptible to hydrolysis during cell culture, such that the amount of hydrolysis does not exceed 5.0 wt.%, more preferably does not exceed 2.5 wt.%, and most preferably does not exceed 1.0 wt.% of the polymeric material present. The device in the first or second configuration may also be fabricated from a biocompatible photosensitive material (e.g., Pro3Dure, Somos WaterShed XC 11122, etc.) used in SLA (stereolithography) and DLP (digital light processing) 3D printing technology. Additionally, the device in the first or second configuration may be formed of an interpenetrating polymer network (IPN), which refers to a polymer containing two or more networks that are at least partially entangled on a polymer scale, but are not covalently bonded to each other.

[0021]

[0035] The 3D bioreactor devices of the first or second configuration herein are also preferably formed from materials exhibiting a Shore D hardness of at least 10, or in the range of 10-95, more preferably in the range of 45-95. In this regard, it is also noteworthy that the devices herein preferably do not use hydrogel-type structures, which can be understood as hydrophilic polymer structures that contain some degree of cross-linking and absorb a significant amount of water (e.g., 10-40% by weight). It is also noteworthy that the devices herein preferably do not use collagen, alginate, fibrin, or other polymers that may be easily digested and remodeled by cells.

[0022]

[0036] Furthermore, the 3D bioreactor devices herein in the first or second configuration are preferably fabricated from materials having a tensile modulus of at least 0.01 GPa. More preferably, the tensile modulus has a value ranging from 0.01 GPa to 20.0 GPa in increments of 0.01 GPa. Even more preferably, the tensile modulus of the materials of the devices herein ranges from 0.01 GPa to 10.0 GPa or from 1.0 GPa to 10 GPa. For example, with respect to the aforementioned polymeric materials suitable for fabricating the devices herein, polystyrene exhibits a tensile modulus of approximately 3.0 GPa, polycarbonate approximately 2.6 GPa, ABS approximately 2.3 GPa, PLA approximately 3.5 GPa, and PCL approximately 1.2 GPa.

[0023]

[0037] The 3D bioreactor devices herein, in either the first or second configuration, having such preferred regular geometric features and / or surface areas, are preferably fabricated by additive manufacturing techniques, such as fused deposition modeling (FDM), selective laser sintering (SLS), stereolithography (SLA), digital light processing (DLP) 3D printing techniques, according to computer-generated designs, such as those made available by the SolidWorks™ computer-aided design (CAD) program.

[0024]

[0038] The 3D bioreactor device in the first or second configuration can then be configured to be a fixed bed, with inlets and outlets to allow for the inflow and outflow of fluids. Referring to Figure 4, the 3D bioreactor device in either the first or second configuration described above can be placed within a housing 24, which can then be positioned between an inlet 26 and an outlet 28, where the inflow and outflow of fluids containing cells for separation can be provided.

[0025]

[0039] Regarding the optional use of a surface coating on the 3D bioreactor device in either the first or second configuration, preferably, such a coating is capable of providing affinity-based cell capture. Accordingly, the coating may preferably comprise substituted or unsubstituted poly(p-xylylene) from the polymerization of parylene monomers, β-casein, or polydopamine (PDA). Such a coating may preferably be present at a thickness ranging from 200 Angstroms to 100.0 μm.

[0026]

[0040] Thus, the coating procedure preferably relies on the use of parylene monomers, such as [2.2]paracyclophane, which can be preferably functionalized with the specified R, R, R, and R groups according to the following general reaction scheme: In the following scheme, it should be understood that initiation of polymerization is triggered remotely by ring opening in the low-pressure gas phase at high temperature (approximately 550°C) prior to deposition on the 3D device, which is preferably maintained at a relatively lower temperature (e.g., ≦100°C). [ka]

[0027]

[0041] In the above, where one of the R groups for each repeat unit "m" and / or repeat unit "n" is chlorine and the other R group is hydrogen, the above represents the polymerization of Parylene C, which is a USP Class VI and ISO-10993-6 certified biocompatible material. The values ​​of the specified crosslinking repeat units "m" and "n" are such that the molecular weight is relatively high, e.g., about 500,000. Therefore, the use of parylene monomers and subsequent polymeric coatings is believed to be such that devices of the first or second configuration described herein above can be coated with an impermeable film. The film can preferably have a thickness between 200 angstroms and 100.0 μm. It will be understood that R1, R2, R3, and R4 can be selected from hydrogen, halogen (-Cl or -Br), and other functional groups such as amines (-NH2), aliphatic aldehydes (-CHO), carboxylic acid functional groups (-COOH), hydroxyl (-OH), or carboxylate functional groups such as -C(O)CF3. Alternatively, a first layer of impermeable Parylene C can be first coated, followed by a coating of a different Parylene (e.g., R1, R2, R3, and R4 can be selected from amine (-NH2) and / or aldehyde (-CHO) functional groups). Thus, polymeric coatings for the devices herein in the first and / or second configurations can be provided, which coatings include multiple layers, each layer having its own specific and distinct chemical composition (i.e., the identity of at least one of R1, R2, R3, and R4 differs between at least two of the layers).

[0028]

[0042] One preferred method for coating the surface of the device herein with functionalized poly(p-xylylene) applies when one or more of the R1, R2, R3, and / or R4 groups described above contain a carboxylic acid ester functionality. In such cases, N-hydroxysuccinimide (NHS) can be used to form an ester bond. NH2-mPEG (methoxy-terminated oligoethylene glycol) or NH2-PEG-biotin can then be covalently attached to the device surface via an amine-NHS ester reaction to form an amide bond. Avidin, NeutrAvidin™ (deglycosylated native avidin protein), or streptavidin can then be conjugated to the biotin. Because avidin / deglycosylated native avidin protein / streptavidin has four binding sites, the remaining three sites are then available to bind biotinylated antibodies, such as anti-CD3 and anti-CD28, to capture T cells via surface receptors specific for these antibodies.

[0029] Coating of 3D bioreactors with antibodies

[0043] The 3D bioreactors described herein can be coated with antibodies. This can be achieved by directly coating the biocompatible or bioinert polymeric materials described above, which are used to form the 3D bioreactor and hydrophobic surface, with various antibodies, such as anti-CD2, anti-CD3, or anti-CD28, via hydrophobic-hydrophobic interactions. Alternatively, the 3D bioreactor can be first coated with, for example, a tetrameric protein, followed by a biotinylated antibody that binds to the tetrameric protein. Alternatively, the surface of the 3D bioreactor can be first coated with a surface coating containing substituted or unsubstituted poly(p-xylylene) from the polymerization of parylene monomers, β-casein, or polydopamine (PDA). Following such coating, the coating on the 3D bioreactor itself can be coated with one or more selected antibodies. For example, a polydopamine coating on the 3D bioreactor can itself be coated with a tetrameric protein and then coated with a biotinylated antibody. Alternatively, the 3D bioreactor herein can be first coated with poly(p-xylylene), followed by polydopamine, tetrameric protein, and biotinylated antibody.

[0030]

[0044] The 3D bioreactor herein may more preferably be coated with antibody-labeled particles on the surface of the 3D bioreactor, as disclosed in U.S. Patent Application No. 18 / 640,078. As described, the antibody coating can be directly disposed on the interior surface of the 3D bioreactor. The particles preferably include any biocompatible particles that can serve to immobilize antibodies. Thus, the particles may include silica (SiO2) particles and / or biocompatible polymer particles. Thus, antibody-labeled particles herein refer to particles having antibodies immobilized thereon, preferably by covalent bonding or, for example, by secondary interactions (e.g., hydrophobic-hydrophobic interactions). The particles preferably have a diameter of 1.0 μm or less. Thus, the particles preferably have a diameter in the range of 10 nm to 1000 nm (1.0 μm), including all individual values ​​and increments within that range.

[0031]

[0045] Therefore, particles can be preferably coated with biotin-binding molecules such as avidin, streptavidin, or a tetrameric protein (a protein having a quaternary structure) containing deglycosylated native avidin protein. The particles bearing the biotin-binding molecules can then be coated with biotinylated antibodies. More specifically, biotinylated antibodies such as anti-CD3, anti-CD19, anti-CD22, anti-CD25, and anti-CD28 can be immobilized on the particles coated with biotin-binding molecules, for example, via a biotin-avidin / streptavidin binding mechanism. Biotinylated antibodies refer to any one or more of the specified antibodies covalently bound to a biotin-binding molecule bound to the particle surface.

[0032]

[0046] The antibody-labeled particles described herein provide for relatively easy convection of fluid-suspended particles into the 3D bioreactor herein and subsequent attachment to the 3D bioreactor surface. Such attachment to the 3D bioreactor surface is believed to occur via hydrophobic-hydrophobic interactions. The immobilized antibody-labeled particles preferably provide an array of individual antibody-labeled particles on the 3D bioreactor surface. FIG. 5 illustrates a portion of a 3D bioreactor surface herein, where controlling the number of antibody-labeled particles 30 on the surface 32 of the 3D bioreactor allows for determining the average surface density of the antibody-labeled particles for a relatively uniform coating. The 3D bioreactor surface of FIG. 5 may be a portion of a 3D bioreactor surface within a non-random void 14 shown in FIG. 1A or on the surface of a non-random geometric structure 20 (e.g., spheres) or non-random interconnected element (e.g., rods) shown in FIG. 2A. The average surface density of antibody-labeled particles on the surface of the 3D bioreactor is preferably 3.9×10. 5 particles / cm 2 ~3.2×10 7 particles / cm 2 The average distance between antibody-labeled particles on the surface of the 3D bioreactor is preferably within the range of 1.0 μm to 9.0 μm.

[0033]

[0047] Therefore, it will be understood that the 3D bioreactor herein can be directly coated with an antibody. Alternatively, the 3D bioreactor herein can be first coated with poly(p-xylylene), or β-casein or polydopamine (PDA), and then coated with an antibody. The 3D bioreactor herein can also be preferably coated with antibody-labeled particles. Any one of these coated 3D bioreactors can be advantageously integrated with the cell growth device herein to provide a scalable automated system.

[0034]

[0048] Referring to Figure 6A, the antibody-coated 3D bioreactor 34 (shown in Figure 1A or Figure 2A) is positioned within a housing for fluid inflow and outflow so that it can receive a mixed population of T cells, B cells, monocytes, and NK cells. More specifically, the mixed population is 70-90% lymphocytes (T cells, B cells, and NK cells), 10-20% monocytes, and 1-2% dendritic cells. The frequency of cell types within the lymphocyte population includes 70-85% CD3+ T cells, 5-10% B cells, and 5-20% NK cells. CD3+ lymphocytes are composed of CD4+ and CD8+ T cells at an approximately 2:1 ratio. Cell surface receptors on T cells (e.g., CD3+ and CD28+ coactivator receptors) can bind to antibodies (e.g., anti-CD3 and anti-CD28) immobilized on the 3D bioreactor surface.

[0035]

[0049] Figure 6B shows bound / activated T cells in the 3D bioreactor 34. Next, as shown in Figure 6C, an integrated bioreactor system is shown in which the activated T cells can then be transduced to form CAR T cells, which can then be dissociated from the antibody and flowed from the antibody-coated 3D bioreactor to a separate expansion reactor that provides oxygen and nutrients for CAR T cell expansion. See also Figure 7. Accordingly, preferred expansion reactors include stirred tank bioreactors that rely on mechanical agitation, wave bioreactors that rely on rocking motion, and gas-permeable fast-growth bioreactors sold as G-Rex™ cell culture bioreactors. Additionally, CAR T cells flowing out of the 3D bioreactor are prevented from being recirculated back into the antibody-coated 3D bioreactor, avoiding restimulation of the CAR T cells, which could result in apoptosis (cell death). [Example]

[0036] Example Example 1 – Coating of 3D bioreactors with antibody-labeled silica particles

[0050] Silica particles having a diameter of 1.0 μm were coated with streptavidin and then coated with biotinylated anti-CD19, anti-CD22, and anti-CD25 antibodies as disclosed in U.S. Patent Application No. 18 / 640,078. The antibody-coated particles were suspended in 2.0 ml of phosphate buffer solution (PBS) and coated with a 100 cm internal surface area as shown and described in FIGS. 2A-2C and 3. 2 The antibody-coated silica particles were circulated through the 3D bioreactor with the PBS solution. Circulation was performed at 2°C to 8°C for approximately 16 hours. The flow rate was 0.1 mL / min. After coating, unbound particles were washed away by flowing 10 mL of PBS from the inlet to the outlet of the 3D bioreactor at a rate of 1 mL / min. A second 10 mL of PBS was flowed through the 3D bioreactor from the outlet to the inlet at the same flow rate. The unbound particles flowing out of the reactor were collected and counted to determine the number of antibody-coated particles remaining in the 3D bioreactor. Table 1 below shows that on average, at least 98% of the antibody-coated silica particles remained attached to the 3D bioreactor surface.

[0037] [Table 1]

[0038] Example 2 – Coating of 3D bioreactors with antibody-labeled silica particles

[0051] As disclosed in U.S. Patent Application No. 18 / 640,078, an internal surface area of ​​250 cm 2 is shown and described in FIG. 1A. 2 A 3D bioreactor with an internal liquid volume of approximately 13 mL was also coated with antibody-coated silica particles. 9Streptavidin-coated 1.0 μm silica particles (Bands Lab, Cat. #CS1001) were added to a 1.5 ml Eppendorf tube. The particles were washed twice with sterile PBS to remove preservatives such as sodium azide (NaN3). After washing, the particles were first coated with 5 μg of biotinylated sheep anti-mouse secondary antibody, followed by 15 μg / mL of mouse IgG anti-CD3 and 15 μg / mL of mouse IgG anti-CD28 antibodies. All coatings were performed at room temperature for 1 hour.

[0039]

[0052] The antibody-coated silica particles were suspended in 40 mL of PBS, which was circulated through the 3D bioreactor at a flow rate of 0.1 mL / min. Perfusion-based particle coating was performed at 2°C to 8°C for approximately 16 hours. After coating, 130 mL of PBS, equivalent to 10 times the internal void volume of the 3D bioreactor, was flowed through the 3D bioreactor by gravity to remove unbound particles. Typically, approximately 3 × 10 6 The particles are distributed over 250 cm of the 3D bioreactor. 2 The average distance between antibody-labeled particles (between two silica particles with a diameter of 1.0 μm) on the surface of the 3D bioreactor was 1.88 μm.

[0040] Example 3 - 3D bioreactor combined with gas permeable fast growth bioreactor as a scalable automated system

[0053] As shown in Figure 7A, the available surface area is 250 cm 2 The negative 3D bioreactor 10 disclosed herein, having a plurality of interconnected non-random voids and interconnected non-random pore openings, with an internal volume of approximately 13.0 mL, was combined with a gas-permeable rapid growth bioreactor 36 and a peristaltic perfusion pump 38. The 3D bioreactor surface was coated with 1.0 μm diameter silica particles functionalized with anti-CD3 and anti-CD28 antibodies. It is noteworthy that the 3D bioreactor 10 may also be a positive 3D bioreactor described herein. The gas-permeable rapid growth bioreactor was configured to accommodate 5×10 6activating cells, perhaps 300 × 10 6 ~400×10 6 The G-Rex™ 6M was capable of expanding up to 100 cells. During the initial loading of peripheral blood mononuclear cells (PBMCs), the medium volume within the G-Rex™ 6M is only approximately 5.0 mL. The PBMC suspension is perfused through the 3D bioreactor to promote interaction between the cells and the antibody coating and achieve T cell binding and activation. During the 48 hours of perfusion, the medium could be aspirated from the bottom of the G-Rex 6M and circulated back into the 3D bioreactor, from where it could be recycled back into the G-Rex 6M.

[0041]

[0054] After 48 hours, the G-Rex 6M was first filled with fresh medium (approximately 100 mL). The medium intake position was switched from the bottom of the gas-permeable rapid growth reactor 36 to the top of the reactor. Because the cells that flowed out of the 3D bioreactor 10 by gravity settled to the bottom of the reactor due to their higher density compared to the medium, the medium introduced from the top of the reactor was cell-free. In other words, this system prevented washed-out cells from re-entering the 3D bioreactor 10 and causing restimulation, which could lead to cell apoptosis. The cells that settled to the bottom of the reactor 36 received oxygen through the gas-permeable membrane underneath. The supernatant medium was then refreshed as needed to provide oxygen and additional nutrients to induce continued proliferation.

[0042]

[0055] More preferably, to the general procedure described above, the system shown in Figure 7B is first primed with 24 mL of medium before loading the PBMCs, pre-oxygenating the medium and eliminating air bubbles in the circuit. Approximately 13 x 10 PBMCs uniformly suspended in 2 mL of complete culture medium are used. 6PBMCs are loaded into the 3D bioreactor 10 at a rate of 0.2 mL / min at three-way stopcock D using a syringe driven by peristaltic pump 38. Once cell loading is complete, the pump begins driving oscillation media flow by first pushing the media upwards within the bioreactor at a rate of 0.1 mL / min for 10 minutes, and then pushing the media downwards within the bioreactor at the same rate of 0.1 mL / min for 10 minutes. The upward / downward oscillation flow cycle is repeated overnight, or for approximately 16 hours. The oscillation media flow moves the cells upward and downward within the 3D bioreactor 10, promoting interaction between the cells and the antibody-coated bioreactor surface and enhancing T cell activation.

[0043]

[0056] After overnight oscillatory flow, the bioreactor system was switched to a relatively low flow rate of 0.04 mL / min unidirectional circulation to mimic quiescent conditions and promote T cell activation, which resulted in some T cell size expansion and clustering.

[0044]

[0057] At 48 hours after T cell activation, the G-Rex 6M was first filled with 100 mL of medium and then flushed at a relatively high flow rate of 50 mL / min for 1 minute to disaggregate clusters formed by activated T cells. Next, perfusion at a flow rate of 1 mL / min was applied to continuously flush and remove dissociated cells from the 3D bioreactor 10 for transfer and collection of the T cells in the G-Rex 6M. The medium flowing back into the bioreactor was taken from near the top surface of the 100 mL of medium in the G-Rex 6M in this cell-free zone. Approximately 54 hours after T cell activation on day 2, the 3D bioreactor 10 was flushed again at 50 mL / min for 1 minute to dissociate any remaining cells. Approximately 65 hours after T cell activation on day 3, the 3D bioreactor 10 was flushed again. The 3D bioreactor was then disconnected. The disconnected bioreactor and tubing were flushed by pumping 130 mL of PBS at 50 mL / min. The remaining cells were collected and added to G-Rex 6M. The cells in G-Rex continued to divide and proliferate, achieving 5–7 population doublings.

[0045]

[0058] Anti-CD3-anti-CD28 coated Dynabeads™-based T cell activation and proliferation experiments were performed simultaneously as a control. Half of the PBMCs used in the bioreactor studies (i.e., 7.0 × 10 6 1000 cells / well) were used in the Dynabeads™ experiments. PBMCs were seeded into two wells of a 6-well plate and mixed with 2x Dynabeads™ in 6.5 mL of medium in each well. 65 hours after T cell activation, PBMCs were isolated from the Dynabeads™ and then transferred to G-Rex 6M for expansion.

[0046]

[0059] It will therefore be appreciated herein that the 3D bioreactor in combination with the expansion bioreactor may preferably be configured as a scalable automated system. As shown in Figure 7B, software control, such as LabVIEW™ software, can be used to program and automate the indicated steps of priming, loading of PBMCs, binding of T cells to the 3D bioreactor, activation and transduction to form CAR T cells, dissociation of CAR T cells and transfer of the dissociated cells to the expansion bioreactor (G-Rex expansion), and proliferation of CAR T cells in the expansion bioreactor followed by recovery of CAR T cells.

[0047]

[0060] Table 2 below shows the cell population profiles as a function of time during the above 3D bioreactor and Dynabeads™ experiments. Figure 8A shows the change in cell number in the 3D bioreactor over the indicated time periods relative to the use of Dynabeads™. Figure 8B shows the average cell size over the indicated time periods. The data show the proliferation of activated cells in both the 3D bioreactor and Dynabeads™ experiments.

[0048] [Table 2]

[0049]

[0061] Table 3 then summarizes flow cytometry analysis of cells recovered after 13 days of T cell activation and expansion in the 3D bioreactor and Dynabeads™ experiments. The data demonstrate the expansion of CD4+ and CD8+ T cells. PBMCs activated and expanded using Dynabeads™ showed a relatively higher fold increase than those in the 3D bioreactor system. In both experiments, recovered cells reached a T cell purity of over 92%, a significant increase from 46.2% in the original PBMCs. The CD4+ and CD8+ ratios were better in cells recovered from the 3D bioreactor, closer to the more desirable 1:1 ratio. The majority of monocytes were captured by the bioreactor or 6-well plate during T cell activation. Minimal B cells (<1%) and NK cells (<2%) were present in the cells recovered after T cell activation and expansion.

[0050] [Table 3]

[0051] Example 4 – Testing of a negative 3D bioreactor in combination with a G-Rex cell expansion device for the purification, activation, transduction and expansion of T cells

[0062] Next, we fabricated the above negative 3D bioreactor (surface area 250 cm) coated with 1 μm silica particles functionalized with antibodies. 2 The same perfusion system (with GFP-LVV) was tested for T cell transduction in addition to T cell activation in the same perfusion system connected to a G-Rex 6M. In this experiment, T cells were transduced using a lentiviral vector encoded to produce green fluorescent protein (GFP-LVV) at a concentration of 13.6E+06 TU / mL. 24 h after T cell activation, 0.5 mL of GFP-LVV was first diluted to 2 mL and then injected into the 3D bioreactor. The same oscillatory flow as above was used to enhance the interaction between the transducing lentiviral vector and T cells bound to the bioreactor surface.

[0052]

[0063] Forty-eight hours after T cell activation or 24 hours after T cell transduction, cells in the 3D bioreactor system were released from the 3D bioreactor into a G-Rex expansion reactor. The process was the same as described above. The expanded T cells were harvested on day 7 after the experiment. Table 4 shows the viability and proliferation of PBMCs after activation and transduction. Cell viability is similar to the T cell activation study described above. Cell proliferation (or proliferation) appears to be slightly higher than in the previous T cell activation study.

[0053] [Table 4]

[0054]

[0064] Table 5 then shows flow cytometry analysis of cells before seeding into the 3D bioreactor and after recovery from the 3D bioreactor. The data show that by day 7, over 30% of T cells were transduced. The T cell population was also enriched from 50.3% to 94% after activation, transduction, and expansion. The majority of B cells and monocytes were removed by the bioreactor and culture process.

[0055] [Table 5] [Explanation of symbols]

[0056] 10. 3D Bioreactor 12 Void surface area 14 Non-random voids 16 Non-random pore openings 18. 3D Bioreactor 20 spheres 22 interconnecting rod 24 Housing 26 Entrance 28 Exit 30 Antibody labeled particles 32 Surface 34 3D Bioreactor 36 Gas-permeable fast growth bioreactor 38 Peristaltic Perfusion Pump

Claims

1. 1. A method for isolating, activating, transducing and expanding T cells, comprising: a. i. a plurality of voids having a diameter D, including a void surface area for coating, and a plurality of pore openings of said voids having a diameter d, wherein 90% or more of said voids have a selected void volume (V) that does not vary by more than + / - 10.0%, and 90% or more of said pore openings of said voids have a value of d that does not vary by more than + / - 10.0%; or ii. A plurality of solid geometric structures having an exterior surface for coating, wherein 90% or more of said solid geometric structures have a volume (V) that does not vary by more than + / - 10.0%. providing a 3D bioreactor comprising: b. Coating the 3D bioreactor with an antibody; c. Binding T cells to the antibody; d. activating the T cells; e. transducing the antibody-bound T cells with a transduction reagent; f. removing the transduced T cells from the 3D bioreactor and transferring them to a T cell expansion bioreactor, where the transduced T cells undergo expansion; A method comprising:

2. 10. The method of claim 1, wherein the binding of T cells to the antibody comprises perfusing peripheral blood mononuclear cells (PBMCs) through the 3D bioreactor.

3. 10. The method of claim 1, wherein the transduced T cells removed from the 3D bioreactor are prevented from being transferred back into the 3D bioreactor.

4. 10. The method of claim 1, wherein the 3D bioreactor comprises a biocompatible material.

5. 10. The method of claim 1, wherein the void surface area or outer geometric surface of the 3D bioreactor is first coated with substituted or unsubstituted poly(p-xylylene), β-casein or polydopamine prior to being coated with the antibody.

6. 10. The method of claim 1, wherein said coating said 3D reactor with an antibody comprises coating said 3D bioreactor with antibody-labeled particles.

7. The method of claim 6, wherein the antibody-labeled particles comprise particles having a particle diameter of 10 nm to 1.0 μm.

8. The method of claim 6 , wherein the antibody-labeled particles comprise silica particles.

9. The method of claim 6 , wherein the antibody-labeled particles comprise polymeric particles.

10. 7. The method of claim 6, wherein the antibody-labeled particles comprise particles coated with biotin-binding molecules, the biotin-binding molecules being coated with biotinylated antibodies.

11. The method of claim 10 , wherein the biotin-binding molecule comprises a tetrameric protein.

12. 12. The method of claim 11, wherein the tetrameric protein comprises avidin, streptavidin, or deglycosylated native avidin protein.

13. The method of claim 10, wherein the biotinylated antibody is selected from the group consisting of an anti-CD3 antibody, an anti-CD22 antibody, an anti-CD25 antibody, and an anti-CD28 antibody.

14. 10. The method of claim 1, wherein the T cells are transduced with a lentiviral vector.

15. 1. A device for isolating, activating, transducing and expanding T cells, comprising: a. i. a plurality of voids having a diameter D, including a void surface area for coating, and a plurality of pore openings of said voids having a diameter d, wherein 90% or more of said voids have a selected void volume (V) that does not vary by more than + / - 10.0%, and 90% or more of said pore openings of said voids have a value of d that does not vary by more than + / - 10.0%; or ii. A plurality of solid geometric structures having an exterior surface for coating, wherein 90% or more of said solid geometric structures have a volume (V) that does not vary by more than + / - 10.0%. a 3D bioreactor comprising: b. a T cell expansion bioreactor connected to said 3D bioreactor; An apparatus comprising:

16. 16. The device of claim 15, wherein T cells in said T cell expansion reactor are prevented from entering said 3D bioreactor.

17. 16. The apparatus of claim 15, wherein the 3D reactor comprises a biocompatible material.

18. 16. The device of claim 15, wherein the 3D bioreactor is coated with an antibody.

19. 16. The device of claim 15, wherein the void surface area or the outer surface of the geometric structure is coated with substituted or unsubstituted poly(p-xylylene), β-casein, or polydopamine.

20. 20. The device of claim 18, wherein the antibody coating comprises antibody-labeled particles.

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