Method and device for fabricating car t cells

Abstract

To provide a method for fabricating a population of T cells which express a chimeric antigen receptor.SOLUTION: A method includes the steps of: proving a fluid sample including a population of T cells; labeling the population of T cells by magnetic beads; extracting the population of T cells from the fluid sample by allowing the fluid sample to flow to a magnetic separator device; performing transduction to the population of T cells in order to express a chimeric antigen receptor (CAR) by bringing a vector having a CAR construct in contact with the population of T cells; and collecting the population of T cells by allowing a solution to flow to the magnetic separator device or another magnetic separator device.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Provisional Application No. 63 / 575,518, filed April 5, 2024, the entire contents of which are incorporated herein by reference.

[0002] BACKGROUND OF THE INVENTION The present invention relates to the generation of CAR T cells, and more particularly to methods and devices for generating the same.

[0003] Immunotherapy involving T cells engineered to express chimeric antigen receptors (CARs) has become an important treatment option for patients with B-cell lymphoma, acute lymphoblastic leukemia, or multiple myeloma. However, current methods for producing CAR T cells are complex and time-consuming, especially for autologous tissue procedures where cell samples are transported between the treatment site and a cell production facility. There is a need for methods and devices to reduce the cost and time required for CAR T cell production, especially for autologous tissue procedures.

[0004] SUMMARY OF THE INVENTION The present invention provides a method that meets this need. The method includes the features of the invention for generating a population of T cells that expresses a chimeric antigen receptor (CAR), the method comprising the steps of providing a fluid sample containing a population of T cells; labeling the population of T cells with magnetic beads; extracting the population of T cells from the fluid sample by flowing the fluid sample through a magnetic separation device; transducing the population of T cells to express a chimeric antigen receptor (CAR) by contacting the population of T cells with a vector having a chimeric antigen receptor construct; and collecting the population of T cells by flowing the solution through the magnetic separation device or another magnetic separation device. The method may further include activating the population of T cells prior to transducing the population of T cells.

[0005] According to another aspect of the present invention, a method for producing a population of T cells expressing a chimeric antigen receptor (CAR) includes the steps of: providing a fluid sample containing a population of T cells; labeling the population of T cells with magnetic beads; extracting the population of T cells from the fluid sample by flowing the fluid sample through a first conduit through a first magnetic separation device; transducing the population of T cells to express a chimeric antigen receptor (CAR) by contacting the population of T cells with a population of lentivirus in a solution contained in an incubation vessel; and collecting the population of T cells by flowing the solution through a second conduit through a second magnetic separation device, wherein the first conduit, the incubation vessel, and the second conduit are fluidly connected by a network of fluid lines. The first conduit, the incubation vessel, the second conduit, and the network of fluid lines are components of a closed fluid system. The method may further include activating the population of T cells prior to transducing the population of T cells.

[0006] According to yet another aspect of the present invention, a method for producing a population of T cells expressing a chimeric antigen receptor (CAR) includes the steps of providing a fluid sample containing a population of T cells; labeling the population of T cells with magnetic beads; extracting the population of T cells from the fluid sample by flowing the fluid sample through a conduit passing through a magnetic separation device; transducing the population of T cells to express a chimeric antigen receptor (CAR) by contacting the population of T cells with a population of lentivirus in a solution contained in an incubation vessel; and collecting the population of T cells by flowing the solution through an acoustic separation device, wherein the conduit, incubation vessel, and acoustic separation device are fluidly connected by a network of fluid lines. The conduit, incubation vessel, acoustic separation device, and network of fluid lines are components of a closed fluid system. The method may further include activating the population of T cells prior to transducing the population of T cells.

[0007] According to another aspect of the present invention, a method for producing a population of T cells expressing a chimeric antigen receptor (CAR) includes the steps of providing a fluid sample containing a population of T cells and other cells; labeling the other cells with magnetic beads; removing the other cells from the fluid sample by flowing the fluid sample through a conduit that passes through a magnetic separation device; transducing the population of T cells to express a chimeric antigen receptor (CAR) by contacting the population of T cells with a population of lentivirus in a solution contained in an incubation vessel; and collecting the population of T cells by flowing the solution through an acoustic separation device, wherein the conduit, incubation vessel, and acoustic separation device are fluidly connected by a network of fluid lines. The conduit, incubation vessel, acoustic separation device, and network of fluid lines are components of a closed fluid system. The method may further include activating the population of T cells prior to transducing the population of T cells.

[0008] According to yet another aspect of the present invention, there is provided a method for producing a population of T cells expressing a chimeric antigen receptor (CAR), comprising the steps of: providing a fluid sample containing a population of T cells; labeling the population of T cells with magnetic beads; extracting the population of T cells from the fluid sample by flowing the fluid sample through a first conduit past a first magnetic separation device; transducing the population of T cells to express a chimeric antigen receptor (CAR) by contacting the population of T cells with a population of lentivirus and removing the magnetic beads from the surface of the T cells in a solution contained in an incubation vessel; removing the magnetic beads from the solution by flowing the solution through a second conduit past a second magnetic separation device; and collecting the population of T cells by flowing the solution through an acoustic separation device, wherein the first conduit, the incubation vessel, the second conduit, and the acoustic separation device are fluidly connected by a network of fluid lines. The first conduit, the incubation vessel, the second conduit, the acoustic separation device, and the network of fluid lines are components of a closed fluidic system. The method may further comprise the step of activating the population of T cells prior to transducing the population of T cells. [Brief explanation of the drawings]

[0009] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings.

[0010] FIG. 1 is a block diagram illustrating an integrated system and its components that can be used to generate CAR T cells according to a first embodiment of the present invention.

[0011] FIG. 2 is a front view of an integrated system that can be used to generate CAR T cells from whole blood or leukopheresis products according to a first embodiment of the present invention.

[0012] FIG. 3 is a cross-sectional view corresponding to a column-free magnetic separation device for sorting magnetically labeled cells flowing through a conduit when the holder and conduit are removed from the magnetic assembly.

[0013] FIG. 4 is a cross-sectional view of the magnetic separation device of FIG. 3 when the conduit is pressed against the tip of the magnetic assembly by the holder during the magnetic separation process.

[0014] Figure 5 is a perspective view of the integrated system of Figure 2 using a closed fluidic assembly in the form of an integrated tubing set for manufacturing CAR T cells according to a first embodiment of the present invention.

[0015] FIG. 6 is a fluid circuit diagram corresponding to the integrated system of FIG.

[0016] FIG. 7 is a block diagram illustrating another integrated system and its components that can be used to generate CAR T cells according to a second embodiment of the present invention.

[0017] 8A-8E are various views of an acoustic separation device that can be used to sort cells according to physical properties.

[0018] FIG. 9 is a front view of an integrated system that can be used to generate CAR T cells from whole blood or leukopheresis products according to the first, second, and third embodiments of the present invention.

[0019] Figure 10 is a perspective view of the integrated system of Figure 9 using a closed fluidic assembly in the form of an integrated tubing set for manufacturing CAR T cells according to a second embodiment of the present invention.

[0020] FIG. 11 is a fluid circuit diagram corresponding to the integrated system of FIG.

[0021] FIG. 12 is a block diagram illustrating an integrated system and its components that can be used to generate CAR T cells according to a third embodiment of the present invention.

[0022] Figure 13 is a perspective view of the integrated system of Figure 9 using a closed fluidic assembly in the form of an integrated tubing set for manufacturing CAR T cells according to a third embodiment of the present invention.

[0023] FIG. 14 is a fluid circuit diagram corresponding to the integrated system of FIG.

[0024] FIG. 15 is a block diagram illustrating an integrated system and its components that can be used to generate CAR T cells according to a fourth embodiment of the present invention.

[0025] Figure 16 is a perspective view of the integrated system of Figure 9 using a closed fluidic assembly in the form of an integrated tubing set for manufacturing CAR T cells according to a fourth embodiment of the present invention.

[0026] FIG. 17 is a fluid circuit diagram corresponding to the integrated system of FIG.

[0027] 18A and 18B are dot plots showing T cell purity before and after the magnetic isolation step from leukopaque samples.

[0028] FIG. 19 is a dot plot showing T cell activation after 24 hours of incubation with CD3 / CD28 magnetic beads.

[0029] Figures 20A and 20B are dot plots of transduced T cells after magnetic collection.

[0030] For purposes of clarity and conciseness, like elements and components have the same names and numbering throughout the figures, but they are not necessarily drawn to scale.

[0031] Reference is made to specific features of the invention (including method steps) in the above summary and detailed description, and in the following claims, as well as in the accompanying drawings. It is to be understood that the disclosure of the invention herein includes all possible combinations of such specific features. For example, if a specific feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other specific aspects and embodiments of the invention, and in the invention generally.

[0032] When reference is made herein to a method including two or more defined steps, the defined steps may be performed in any order or simultaneously, unless the context excludes this possibility, and the method may include one or more other steps performed before any defined step, between two defined steps, or after all defined steps, unless the context excludes this possibility.

[0033] As used herein, the term "biological matter" includes cells, bacteria, viruses, molecules, particles containing RNA and DNA, cellular masses, bacterial masses, molecular masses and particulate masses.

[0034] The term "biological sample" as used herein includes blood, body fluids, tissue extracted from any part of the body, bone marrow, hair, nails, bones, teeth, liquids and solids from bodily waste, or surface swabs from any part of the body. A "fluid sample" or "sample fluid" or "liquid sample" or "sample solution" can include a biological sample in its original liquid form, biological matter that has been dissolved or dispersed in a buffer solution or dissociated from its original non-liquid form, and a biological sample that has been dispersed in a buffer solution. A buffer solution is a liquid in which biological matter can be dissolved or dispersed without introducing contaminants or undesirable biological matter. Biological matter and biological samples can be obtained from humans or animals. Biological matter can also be obtained from plants and the environment, including air, water, and soil. A fluid sample can contain various types of magnetic or optical labels or one or more chemical reagents that can be added during various process steps.

[0035] The term "sample flow rate" or "flow rate" is used herein to refer to the volumetric amount of fluid flowing through a cross section of a channel, conduit, fluidic component, fluid path, or fluidic line in a unit of time.

[0036] In the art of cell sorting and enrichment, a target population of biological objects is referred to as the "specific" object of interest, while the isolated but undesired biological objects are referred to as "non-specific." The term "purity" describes the frequency of the desired target or specific biological object and is quantified by the number of target biological objects divided by the total number of biological objects expressed as a percentage. The term "recovery fraction" or "recovery rate" refers to the efficiency of biological object sorting and is quantified by the number of target biological objects recovered after sorting divided by the number of target biological objects present in the initial sample expressed as a percentage.

[0037] The term "at least" followed by a number is used herein to indicate the beginning of a range beginning with that number, which may be a range with an upper limit or an open limit, depending on the variable being defined. For example, "at least 1" means 1 or more than 1. The term "up to" followed by a number is used herein to indicate the end of a range ending with that number, which may be a range with a lower limit of 1 or 0 or an open limit, depending on the variable being defined. For example, "up to 4" means 4 or less than 4, and "up to 40%" means 40% or less than 40%. When a range is given herein as "a first number to a second number" or "a first number minus a second number," this means a range with a lower limit of the first number and an upper limit of the second number. For example, "25 nm to 100 nm" means a range with a lower limit of 25 nm and an upper limit of 100 nm.

[0038] With reference to the orientation of the illustrated drawings, directional terms such as "front," "back," "top," and "bottom" may be used. Spatially relative terms such as "below," "under," "lower," "lower," "upper," and "above" may be used herein to describe the relationship of one element to another as illustrated in the drawings. Because articles and elements may be positioned in several different orientations, these terms are for illustrative purposes and are in no way limiting of the invention unless the context excludes that possibility.

[0039] When reference is made herein to a material AB composed of elements A and B, material AB may be an alloy, a compound, or a combination thereof, unless the context precludes that possibility.

[0040] Provided herein are production devices and methods for producing CAR T cells.

[0041] Embodiment 1: Positive Magnetic Selection and Positive Magnetic Purification

[0042] This embodiment uses a first magnetic separation device to isolate or extract magnetically labeled target cells from an initial sample for genetic modification, which may be performed in a cell incubator, and then uses a second magnetic separation device to purify the genetically modified target cells.

[0043] 1 is a block diagram illustrating an integrated system 100 and its components that can be used to generate CAR T cells. The integrated system 100, with control electronics, a user interface, hardware, software, and firmware (not shown), includes a first container or bag 102 for holding a sample containing target cells for processing, a first magnetic separation device 104, a cell incubation chamber 106, a second magnetic separation device 108, a second container or bag 112 containing a buffer or solution for eluting the target cells from the first magnetic separation device 104, an incubation component bank 114, a third container or bag 116 containing a buffer or solution for eluting the target cells from the second magnetic separation device 108, a fourth container or bag 110 for receiving the final cell product, and multiple fluid lines 118-130 that provide fluid interconnections between the individual components in the integrated system 100.

[0044] A first container or bag 102 holds a fluid sample containing target cells 132 (e.g., T cells) that have been magnetically labeled. The fluid sample may contain whole blood, leukopak, PBMCs, and / or other leukopheresis products containing target cells that have been magnetically labeled. The target cells may be magnetically labeled during an incubation step that takes place in the same first container or bag 102.

[0045] A first magnetic separation device 104, connected to a first container or bag 102 via a fluid line 118, is used to extract the magnetically labeled target cells from the fluid sample.

[0046] A second container or bag 112, connected to the inlet of the first magnetic separation device 104 through fluid line 120, contains a buffer or solution for eluting the magnetically labeled target cells remaining in the first magnetic separation device 104 after the passage of the fluid sample. The buffer or solution in the second container or bag 112 may contain a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0047] The cell incubation chamber 106 can be used for cell genome engineering, cell modification, cell transduction, or cell gene transfer. The cell incubation chamber 106 contains a cell container 134 connected to the outlet of the first magnetic separation device 104 through a fluid line 122. The cell container 134 contains magnetically labeled target cells, a buffer or solution for eluting the cells from the first magnetic separation device 104, and, optionally, one or more buffers or solutions from an incubation component bank 114 connected to the cell container 134 through a fluid line 124. One or more gas lines (e.g., CO2) (not shown) can be connected to the cell incubation chamber 106 to provide an environment with a desired gas composition (e.g., 5.0%±0.1% CO2). The cell incubation chamber 106 can also have a heating and cooling mechanism (not shown) capable of maintaining a constant temperature within the chamber (e.g., 37.0±0.1°C). Additionally, the cell incubation chamber 106 may also include an air circulation mechanism, such as a fan, for circulating air or other gas mixture therein to make the temperature more uniform throughout the chamber 106. The cell container 134 and the contents therein may be heated or cooled by convection through the surrounding environment in the cell incubation chamber 106. The cell container 134 may also have porous or permeable portions, such as membranes and vents, that allow gas molecules in the surrounding environment to diffuse through the flow-through, thereby exposing the contents of the cell container 134 to the air or other gas mixture inside the cell incubation chamber 106.

[0048] The incubation component tank 114 may contain one or more of the following items: culture media, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer.

[0049] The cell incubation chamber 106 may further include means, such as, but not limited to, cell electroporation, mechanical cell deformation, temperature, ultrasound, and optics, that can induce target cell membranes to open, thereby allowing genetic material to enter the target cells from the surrounding medium in the cell container 134. Genetic material may also be introduced into the target cells using droplet-based injection of genetic material or penetration of the target cell membrane.

[0050] The cell incubation chamber 106 can provide an environment for the transduction process. For example, lentivirus or other viruses can be used to transfer genomic material into target T cells to induce chimeric antigen receptor (CAR) proliferation on the cell surface.

[0051] A second magnetic separation device 108, connected to the cell container 134 in the cell incubation chamber 106 through fluid line 126, is used to extract target cells that still retain the magnetic label on their surface from the mixture of solutions and / or media used in the cell container 134 after genetic modification by transduction or gene transfer.

[0052] A third container or bag 116, connected to the inlet of the second magnetic separation device 108 through fluid line 128, contains a buffer or solution for eluting the magnetically labeled target cells remaining in the second magnetic separation device 108 after the fluid sample has passed through it. The buffer or solution in the third container or bag 116 may contain a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof.

[0053] A fourth container or bag 110, connected to the outlet of the second magnetic separation device 108 through a fluid line 130, contains genetically modified target cells (e.g., CAR T cells) in a solution that can be administered intravenously to a patient.

[0054] The network of fluid lines 118-130, the containers or bags 102, 112, 116, 110, the cell container 134 and the conduits passing through the first and second magnetic separation devices 104 and 108 may be constructed, interconnected and supplied as an integrated disposable set that can be sterilized and sealed from the surrounding environment.

[0055] Continuing with reference to FIG. 1, the process begins by providing a first container or bag 102 containing a first fluid sample containing magnetically labeled target cells 132 (e.g., T cells). In the case of a previously frozen sample, the first fluid sample can be prepared by first thawing the frozen sample and then extracting the target cells and other biological matter, if any, from the thawed sample fluid by centrifugation. The resulting target cells and other biological matter, if any, are resuspended in a buffer solution and filtered using a mesh size between 15 and 100 μm. A reagent containing a magnetic label is added to the filtered buffer solution containing the target cells, thereby forming the first fluid sample. Alternatively, the target cells can be magnetically labeled through an indirect process by first adding a reagent containing an intermediate link attached to the target cells before adding a reagent containing a magnetic label attached to the intermediate link.

[0056] For the first magnetic separation step, a first fluid sample is flowed through fluid line 118 into the inlet of first magnetic separation device 104. As the first fluid sample flows through first magnetic separation device 104, the magnetically labeled target cells 132 are retained in first magnetic separation device 104 by the magnetic field, while the removed first fluid sample exits first magnetic separation device 104 into a waste container or bag (not shown). After the first fluid sample has completely passed through first magnetic separation device 104, the magnetic field acting on the magnetically labeled target cells 132 is reduced or eliminated, and a first eluent, such as a buffer solution in second container or bag 112, flows through fluid line 120 into first magnetic separation device 104, eluting the magnetically labeled target cells 132 into cell container 134 via fluid line 122. Additionally, one or more solutions from incubation component tank 114, such as, but not limited to, culture medium, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer, are injected into cell container 134 through fluid line 124 to modify the genetic makeup of magnetically labeled target cells 132. Magnetically labeled target cells 132 (e.g., T cells) can be transduced to express a CAR by contacting the target cells with vectors bearing a CAR construct, such as viral vectors, e.g., lentiviruses and retroviruses, and non-viral vectors, e.g., plasmids, lipid nanoparticles, and mRNA.

[0057] The transduction or gene transfer process is carried out in the cell incubation chamber 106 while the magnetically labeled target cells 132 are immersed in a transduction or gene transfer medium containing one or more solutions from the first eluent and incubation component tank 114. The temperature and gas composition within the cell incubation chamber 106 can be controlled during the transduction or gene transfer process. For example, the temperature and gas composition within the cell incubation chamber 106 can be maintained at 37.0±0.1°C and 5.0%±0.1% CO2, respectively, for a period ranging from 10 minutes to 72 hours while the genetic makeup of the magnetically labeled target cells 132 is modified in the cell container 134.

[0058] After the transduction or gene transfer step is completed, a buffer to facilitate the subsequent magnetic separation step can be added from the incubation component tank 114 to the transduction or gene transfer medium in the cell container 134. The mixture of the transduction or gene transfer medium and the buffer to facilitate the magnetic separation step, if any, forms a second fluid sample containing magnetically labeled target cells with a modified genetic structure.

[0059] A second fluid sample is then flowed through fluid line 126 into the inlet of second magnetic separation device 108, where a second magnetic sorting step extracts magnetically labeled target cells having the modified genetic structure. As the second fluid sample flows through second magnetic separation device 108, the magnetically labeled target cells are retained therein by the magnetic field, while the removed second fluid sample exits second magnetic separation device 108 into a waste container or bag (not shown). After the second fluid sample has completely passed through second magnetic separation device 108, the magnetic field acting on the magnetically labeled target cells is reduced or eliminated, and a second eluent, such as a buffer solution in third container or bag 116, flows through fluid line 128 into second magnetic separation device 108, eluting the magnetically labeled target cells via fluid line 130 into fourth container or bag 110. After completion of the second magnetic separation step, the fourth container or bag 110 containing the magnetically labeled target cells with the modified genetic structure can be disconnected from the rest of the integrated system 100 without exposing its contents to the atmosphere. The contents in the fourth container or bag 110 can be administered intravenously to a patient.

[0060] 2 is a front view of an integrated system 100 that can be used to generate CAR T cells from whole blood or T-cell-containing samples (e.g., leukopheresis products) according to the steps described above. System 100 utilizes a closed fluidic assembly to provide a closed processing system that can be deployed in facilities that lack strict sterile or clean environments, such as medical centers, thereby alleviating many of the common logistical issues encountered in autologous tissue procedures.

[0061] 2 shows an automated production system 100 including a shell or housing structure 140, first, second, and third magnetic separation modules 142A-142C present in the housing structure 140, an incubation module 144, a sample rack 146 mounted on top of the housing structure 140, and first and second sample bag holders 164, 166. Fluid lines and connected sample bags, which may be supplied as an integrated disposable tubing set, and a computer used to control the system 100 are omitted from this view to present an unobstructive view of the magnetic separation modules 142A-142C.

[0062] Incubation module 144 includes an incubation chamber 106 located on top of a rocker base 147. Each of magnetic separation modules 142A-142C includes a first pinch valve 148A, 148B, 148C, a second pinch valve 150A, 150B, 150C, a third pinch valve 152A, 152B, 152C, a fourth pinch valve 154A, 154B, 154C, a first air detector 156A, 156B, 156C, a second air detector 158A, 158B, 158C, a peristaltic pump 160A, 160B, 160C, an occlusion sensor 162A, 162B, 162C, and a magnetic separation device 104, 105, 108.

[0063] System 100 may also include additional pinch valves 168, 170 located on one side of housing structure 140 and additional pinch valves 172, 174 located on the other side of housing structure 140. Electromechanical components 104-108, 147, 148A / B / C-162A / B / C, 168-174 may be controlled or automated by a computer or microprocessor (not shown).

[0064] Each of the magnetic separation devices 104, 105, 108 may have the same or substantially similar structure as magnetic separation device 188, the cross-sectional view of which is shown in Figure 3. Magnetic separation device 188 includes a magnetic assembly 190 for generating a magnetic field, a conduit 192 made of a soft and / or flexible material for conducting a fluid sample for sorting, and a holder 194 for supporting conduit 192. This figure shows conduit 192 and holder 194 removed from magnetic assembly 190. However, during the magnetic sorting process, conduit 192 is positioned in close proximity to magnetic assembly 190, thereby exposing conduit 192 to the magnetic field generated by magnetic assembly 190.

[0065] The magnetic assembly 190 for generating a magnetic field to attract magnetically labeled biological objects in the conduit 192 includes a magnetic flux source including first and second permanent magnets 193 and 195, a central flux guide 196 for guiding the magnetic flux from the magnetic flux source and forming the magnetic field, and first and second lateral flux guides 198 and 200 disposed on either side of the central flux guide 196 for guiding the magnetic flux from the magnetic flux source and forming the magnetic field in the gap between the flux guides 196-200.

[0066] The central flux guide 196 has a central tip 201 having a tapered shape and a central base 203 that is physically and / or magnetically coupled at a first pole (e.g., north pole) to the first and second permanent magnets 193 and 195. The central tip 201 may have a smaller cross-section, which may be defined herein as the cross-sectional area perpendicular to the flow of magnetic flux, than the central base 203, thereby concentrating magnetic flux from the central base 203 to the central tip 201. The first lateral flux guide 198 has a first lateral tip 202 and a first lateral base 204 that is physically and / or magnetically coupled at a second pole (e.g., south pole) to the first permanent magnet 193. The first lateral tip 202 may have a smaller cross-sectional area than the first lateral base 204, thereby concentrating magnetic flux from the first lateral base 204 at the first lateral tip 202. The second lateral flux guide 200 has a second lateral tip 206 and a second lateral base 208 that is physically and / or magnetically coupled at a second pole (e.g., south pole) to the second permanent magnet 195. The second lateral tip 206 may have a smaller cross-sectional area than the second lateral base 208, thereby concentrating magnetic flux from the second lateral base 208 to the second lateral tip 206. Thus, each of the tips 201, 202, and 206 may have a higher magnetic flux density than the corresponding base 203, 204, or 208. The first and second lateral flux guides 198 and 200 are parallel at their bases 204 and 208 and may bend or twist inward toward the central tip 201 at their tips 202 and 206, which may be pointed toward each other. The ends of the first and second lateral tips 202 and 206 may each have a chisel angle profile with an upwardly or outwardly sloping surface away from the central flux guide 196. The central tip 201 may be located below the first and second lateral tips 202 and 206. The conduit 192 may be operatively nested in a gap or recessed space defined by the tip of the central tip 201 and the slopes of the first and second lateral tips 202 and 206 during the magnetic separation process, thereby exposing the conduit 192 to the magnetic field generated by the magnetic assembly 190.

[0067] The first permanent magnet 193 may be disposed between the central base 203 and the first lateral base 204, and the second permanent magnet 195 may be disposed between the central base 203 and the second lateral base 208. The first and second permanent magnets 193 and 195 have opposite magnetization directions that may be oriented substantially perpendicular to the central flux guide 196.

[0068] The central base 203 is magnetically coupled to the first and second permanent magnets 193 and 195 at their first poles (e.g., north poles), while the first and second lateral bases 204 and 208 are magnetically coupled to the first and second permanent magnets 193 and 195, respectively, at their second poles (e.g., south poles), thereby causing the first and second lateral tips 202 and 206 (second polarity) and the central tip 201 (first polarity) to have opposite magnetic polarities, creating a strong magnetic field in or near the gap between the ends 201, 202 and 206, causing magnetically labeled biological matter to accumulate on the conduit wall.

[0069] 3 , holder 194 may have a first surface 210 facing conduit 192 and a second surface 212 opposite first surface 210. First surface 210 may have a ridge structure 214 protruding from first surface 210, which acts as a mechanical press to force conduit 192 into a gap or recessed space defined by the tip of central tip 201 and the slopes of first and second lateral tips 202 and 206 during the magnetic separation process. Additionally, ridge structure 214 of holder 194 may be made of a magnetic material that "floats" or conducts magnetic flux like an upper flux guide. In addition to acting like a mechanical press to press the conduit 192 against the tips 201, 202 and 206, the ridge structure 214 made of magnetic material may magnetically interact with the tips 201, 202 and 206 to further enhance the magnetic field between them, thereby increasing the magnetic sorting efficiency.

[0070] FIG. 4 is a cross-sectional view of the magnetic separation device 188 as the conduit 192 is squeezed between the ridge structure 214 of the holder 194 and the tips 201, 202, and 206 of the three magnetic flux guides 196-200 during the magnetic separation process. The holder 194 may further push the deformed or distorted conduit 192 into the gap between the central tip 201 and the first side tip 202 and the gap between the central tip 201 and the second side tip 206, where the magnetic field may be strongest. By forcing the conduit 192 against the tip of the central tip 201 and the slopes of the first and second side tips 202 and 206, more of the fluid sample flowing through the conduit 192 may be exposed to a stronger magnetic field. During the magnetic separation process, magnetically labeled biological objects 216 may accumulate at the bottom of the conduit 192 near the central tip 201, where the magnetic field gradient may be greatest.

[0071] In embodiments in which the ridge structure 214 is made of a soft magnetic material, the ridge structure 214 may act like an upper magnetic flux guide when positioned proximate to the tips 201, 202, and 206 during the magnetic separation process. The magnetic ridge structure 214 may conduct magnetic flux from the first and second lateral flux guides 198 and 200 and therefore may have the same magnetic polarity (second polarity) as the first and second lateral tips 202 and 206, thereby further enhancing the magnetic field between the ridge structure 214 and the central tip 201.

[0072] Each of the flux guides 196-200 may be made of a soft magnetic material or a material with a relatively high magnetic permeability, including any one of iron (Fe), cobalt (Co), nickel (Ni), or any combination thereof. For example, without limitation, any of the flux guides 196-200 may be made of iron. The conduit 192 may be made of any suitable flexible and / or pliable material that can be bent or deformed, such as, but not limited to, rubber, plastic, or any suitable polymeric material. The holder 194 may be made of any suitable non-magnetic material, such as, but not limited to, aluminum, glass, a non-ferrous metal or alloy, plastic, or any suitable polymeric material. In some embodiments, the ridge structure 214 of the holder 194 that contacts the conduit 192 may be made of a soft magnetic material, such as, but not limited to, any of the soft magnetic materials described above for the flux guides 196-200.

[0073] The operation of magnetic separation device 188, including the recovery of magnetically labeled target cells, is described in more detail in U.S. Patent Application Publication No. 18 / 795,047, which is incorporated herein by reference in its entirety. Other magnetic flux sources and column-free magnetic separation devices, such as those disclosed in U.S. Patent Application Publication No. 18 / 072,362, which is incorporated herein by reference, may also be used to separate magnetically labeled target cells from a fluid sample.

[0074] Referring back to FIG. 2 , the incubation module 144 includes a cell incubation chamber 106 located on a rocker base 147. The incubation chamber 106 has a heater that can maintain a uniform temperature within the chamber 106 between room temperature and 50°C. The CO2 concentration within the chamber 106 can be varied between 0 and 10% to provide an optimal environment for the cells. The rocker base 147 can move the chamber 106 at a speed of 2 to 4 rpm. The rocking motion of the chamber 106 caused by the rocker base 147 can facilitate uniform mixing of the fluid contents within the cell container 134. The rocker base 147 can remain stationary during cell activation, transduction, or expansion steps. After the incubation step, the rocker base 147 rocks the cell container 134 within the chamber 106, allowing the fluid in the cell container 134 to wash the cells off the surface of the cell container 134. The rocker base 147 can also be used to facilitate the draining of the cell container 134 by tilting the incubation chamber 106 to a fixed position to allow the end of the outlet tubing to reach the bottom of the fluid in the cell container 134.

[0075] System 100 has a modular design that includes three magnetic separation modules 142A-142C and one incubation module 144. Modules 142A-142C, 144 may be fluidly connected in series, parallel, or a combination thereof by a closed fluidic assembly that includes all fluid lines, including sample bags, cell containers, and conduits that pass through the magnetic separation device.

[0076] FIG. 5 is a perspective view of a system 100 for manufacturing CAR T cells that uses a closed fluidic assembly in the form of an integrated tubing set. For reasons of readability, the electromechanical components shown in FIG. 2 are not reproduced in the drawing to clearly show the routing of the fluid lines. The perspective view also shows pinch valves 152B, 154A, and 154B, which were previously obstructed by the cell incubation chamber 106 in the front view of FIG. 2, as well as pinch valves 168 and 170 located on one side of the housing structure 140. The lid and a portion of the sidewall of the incubation chamber 106 are further removed to expose a cell container 134 in the form of a common cell culture flask within the chamber 106. The cap of the cell culture flask 134 contains an air filter that vents to the environment within the incubation chamber 106 while maintaining sterility within the flask 134. FIG. 6 is a fluidic schematic corresponding to the system 100 shown in FIG. 5.

[0077] 2, 5, and 6, the fluidic assembly of system 100 is configured to use parallel, fluidly coupled first and third magnetic separation devices 104, 105 for extracting magnetically labeled target cells (e.g., T cells) from a first fluid sample in a first magnetic separation step, and a second magnetic separation device 108 for a subsequent second magnetic separation step. The use of two magnetic separation devices 104, 105 for the first magnetic separation step enables system 100 to increase sorting throughput and accommodate larger volumes of first fluid samples, such as whole blood.

[0078] The first, second, third, and fourth containers or bags 102, 112, 116, 110 are in the form of sample bags. Both sample bags 102 and 112 are fluidly connected to the inlets of the first and third magnetic separation devices 104, 105. In addition to sample bag 116, another sample bag 220, which may contain cryopreservation solution, storage medium, PBS-based solution, saline-based solution, human serum, glucose, or any combination thereof, is also fluidly connected to the inlet of second magnetic separation device 108. The incubation component bank 114 includes at least two sample bags 114A, 114B that are fluidly connected to the cell container 134 through the magnetic separation devices 104, 105. Each of sample bags 114A, 114B may contain culture medium, a vector for cell transduction or gene transfer, a lentivirus for transduction, a buffer or solution for non-viral gene transfer, or a cell activation reagent. Prior to the transduction or gene transfer step, the target T cells in the cell container 134 may be activated by flowing a cell activation reagent (e.g., a solution of CD3 / CD28 magnetic beads) into the cell container 134 from one of the sample bags 114A, 114B.

[0079] The fluidic assembly of system 100 further includes a sample bag 222 fluidly connected to the outlets of the first and third magnetic separation devices 104, 105 to collect waste from the devices 104, 105, and another sample bag 224 fluidly connected to the outlet of the second magnetic separation device 108 to collect waste from the second magnetic separation device 108. The sample bag 222 for collecting waste from the first and third magnetic separation devices 104, 105 can be placed in the first bag holder 164. The sample bags 110, 224 for collecting the final cell product and waste, respectively, from the second magnetic separation device 108 can be placed in the second bag holder 166. All other sample bags 102, 112, 114A, 114B, 116, 220 that provide materials for the manufacturing process can be suspended in the sample rack 146. The fluidic assembly of system 100 can include additional sample bags for various purposes. Each of the sample bags 102, 110, 112, 114A, 114B, 116, 220-224 may have an inlet port connected to a Luer connector and an outlet port connected to a drip chamber. Additionally, fluid flow through the inlet and outlet ports of each sample bag may be controlled or regulated by one or more manual pinch clamps attached thereto.

[0080] The fluidic assembly of system 100 may also include syringes 226, 228, 230. Syringes 226, 228, which may be fluidly connected to the inlet of cell container 134, may be used to extract a sample of magnetically separated target cells after the initial magnetic sorting step, or may inject culture medium, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer, or cell activation reagents into cell container 134. Syringe 230, which may be fluidly connected to the outlet of cell container 134, may be used to extract a sample of genetically modified target cells after the transduction or gene transfer step is completed.

[0081] Continuing to refer to FIG. 6, the fluid circuitry corresponding to system 100 includes sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, and cell container 134, all interconnected by a network of fluid lines passing through electromechanical components such as pinch valves 148A-148C, 150A-150C, 152A-152C, 154A-154C, 168-172, air detectors 156A-156C, 158A-158C, occlusion sensors 162A-162C, peristaltic pumps 160A-160C, and magnetic separation devices 104, 105, 108. The network of fluid lines, including the sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, and conduits passing through the magnetic separation devices 104, 105, 108, can be constructed, interconnected, sterilized, and supplied as an integrated disposable set. The electromechanical components of system 100 are external to the tubing set and therefore do not come into contact with the fluids in the tubing set.

[0082] A first container or sample bag 102, which may contain target cells for processing, is fluidly connected to the inlet of a conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through a first pinch valve 148A, a first air detector 156A, a second air detector 158A, a peristaltic pump 160A and an occlusion sensor 162A, and is also fluidly connected to the inlet of a conduit passing through the third magnetic separation device 105 by one or more fluid lines passing through a first pinch valve 148B, a first air detector 156B, a second air detector 158B, a peristaltic pump 160B and an occlusion sensor 162B. A second container or sample bag 112, which may contain a buffer solution or culture medium, is fluidly connected to the inlet of a conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through a second pinch valve 150A, a second air detector 158A, a peristaltic pump 160A and an occlusion sensor 162A, and is also fluidly connected to the inlet of a conduit passing through the third magnetic separation device 105 through one or more fluid lines passing through a second pinch valve 150B, a second air detector 158B, a peristaltic pump 160B and an occlusion sensor 162B.

[0083] The inlet of the cell container 134 is fluidly connected to the outlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through the third pinch valve 152A, and also fluidly connected to the outlet of the conduit passing through the third magnetic separation device 105 by one or more fluid lines passing through the third pinch valve 152B. Syringes 226, 228 may be fluidly connected to the fluid lines between the inlet of the cell container 134 and the third pinch valves 152A, 152B. A sample bag 222, which may store waste liquid such as sample fluid after target cell extraction, is fluidly connected to the outlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through the fourth pinch valve 154A, and also fluidly connected to the outlet of the conduit passing through the third magnetic separation device 105 by one or more fluid lines passing through the fourth pinch valve 154B.

[0084] The outlet of the cell container 134 is fluidly connected to the inlet of a conduit passing through the second magnetic separation device 108 by one or more fluid lines that pass through a first pinch valve 148C, a first air detector 156C, a second air detector 158C, a peristaltic pump 160C, and an occlusion sensor 162C. A syringe 230 may be fluidly connected to the fluid line connected to the outlet of the cell container 134. A third container or sample bag 116 is fluidly connected to the inlet of a conduit passing through the second magnetic separation device 108 by one or more fluid lines that pass through a pinch valve 172, a second pinch valve 150C, a second air detector 158C, a peristaltic pump 160C, and an occlusion sensor 162C. The sample bag 220 is fluidly connected to the inlet of a conduit passing through the second magnetic separation device 108 by one or more fluid lines passing through the pinch valve 174, the second pinch valve 150C, the second air detector 158C, the peristaltic pump 160C and the occlusion sensor 162C.

[0085] The outlet of the conduit passing through the second magnetic separation device 108 is fluidly connected to a fourth container or sample bag 110 by one or more fluid lines passing through a third pinch valve 152C, and is also fluidly connected to a sample bag 224 by one or more fluid lines passing through a fourth pinch valve 154C, which may be used to receive waste fluid.

[0086] The sample bag 114A of the incubation component bank 114 is fluidly connected to the inlet of the cell container 134 by one or more fluid lines passing through pinch valve 168, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104 and third pinch valve 152A, and by one or more fluid lines passing through pinch valve 168, second pinch valve 150B, second air detector 158B, peristaltic pump 160B, occlusion sensor 162B, third magnetic separation device 105 and third pinch valve 152B. Similarly, sample bag 114B of incubation component bank 114 is fluidly connected to the inlet of cell container 134 by one or more fluid lines passing through pinch valve 170, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104 and third pinch valve 152A, and by one or more fluid lines passing through pinch valve 170, second pinch valve 150B, second air detector 158B, peristaltic pump 160B, occlusion sensor 162B, third magnetic separation device 105 and third pinch valve 152B.

[0087] Embodiment 2: Positive magnetic selection and acoustic purification

[0088] This embodiment uses a magnetic separation device to isolate or extract magnetically labeled target cells for genetic modification, which can be done in a cell incubator, and then uses an acoustic separation device to purify the genetically modified target cells.

[0089] 7 is a block diagram illustrating an integrated system 300 and its components that can be used to generate CAR T cells. The integrated system 300, with control electronics, user interface, hardware, software, and firmware (not shown), includes a first container or bag 102, a magnetic separation device 104, a cell incubation chamber 106, an acoustic separation device 302, a second container or bag 112 containing a buffer or solution for eluting target cells from the magnetic separation device 104, an incubation component bank 114, a third container or bag 116 containing a buffer or solution for operating the acoustic separation device 302, a fourth container or bag 110 for the final cell product, and multiple fluid lines 118-124 and 304-308 that provide fluidic interconnections between the individual components in the integrated system 300. Integrated system 300 is similar to integrated system 100 shown in FIG. 1, except that second magnetic separation device 108 and its connected fluid lines 126-130 are replaced by acoustic separation device 302 and its connected fluid lines 304-308.

[0090] A first container or bag 102 holds a fluid sample containing target cells 132 (e.g., T cells) that have been magnetically labeled. The fluid sample may contain whole blood, leukopaques, PBMCs, and / or other leukopheresis products containing the target cells 132 that have been magnetically labeled. The target cells 132 may be magnetically labeled during an incubation step that takes place in the same first container or bag 102.

[0091] Magnetically labeled target cells 132 are extracted from the fluid sample using a magnetic separation device 104 connected to the first container or bag 102 through a fluid line 118. The magnetic separation device 104 may have the same or substantially similar structure as the magnetic separation device 188, the cross-sectional views of which are shown in Figures 3 and 4 and described above.

[0092] A second container or bag 112, connected to the inlet of the magnetic separation device 104 through a fluid line 120, contains a buffer or solution for eluting the magnetically labeled target cells 132 remaining in the magnetic separation device 104 after the passage of the fluid sample. The buffer or solution in the second container or bag 112 may contain a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0093] The cell incubation chamber 106 can be used for cell genome engineering, cell modification, cell transduction, or cell gene transfer. The cell incubation chamber 106 contains a cell container 134 connected to the outlet of the magnetic separation device 104 through a fluid line 122. The cell container 134 contains magnetically labeled target cells 132, a buffer or solution for eluting the cells from the magnetic separation device 104, and, optionally, one or more buffers or solutions from an incubation component bank 114 connected to the cell container 134 through a fluid line 124. One or more gas lines (e.g., CO) (not shown) can be connected to the cell incubation chamber 106 to provide an environment with a desired gas composition (e.g., 5.0%±0.1% CO). The cell incubation chamber 106 can also have a heating and cooling mechanism (not shown) capable of maintaining a constant temperature within the chamber (e.g., 37.0±0.1°C). Additionally, the cell incubation chamber 106 may also include an air circulation mechanism, such as a fan, for circulating air or other gas mixture therein to make the temperature more uniform throughout the chamber 106. The cell container 134 and the contents therein may be heated or cooled by convection through the surrounding environment in the cell incubation chamber 106. The cell container 134 may also have porous or permeable portions, such as membranes and vents, that allow gas molecules in the surrounding environment to diffuse through the flow-through, thereby exposing the contents of the cell container 134 to the air or other gas mixture inside the cell incubation chamber 106.

[0094] The incubation component tank 114 may contain one or more of the following items: culture media, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer.

[0095] The cell incubation chamber 106 may further include means, such as, but not limited to, cell electroporation, mechanical cell deformation, temperature, ultrasound, and optics, that can induce target cell membranes to open, thereby allowing genetic material to enter the target cells from the surrounding medium in the cell container 134. Genetic material may also be introduced into the target cells using droplet-based injection of genetic material or penetration of the target cell membrane.

[0096] The cell incubation chamber 106 can provide an environment for the transduction process. For example, lentivirus or other viruses can be used to transfer genomic material into target cells to induce chimeric antigen receptor (CAR) expression on the cell surface.

[0097] An acoustic separator 302, connected to the cell container 134 in the cell incubation chamber 106 through a fluid line 304, is used to extract the target cells from the mixture of solutions and / or media used in the cell container 134 after genetic modification by transduction or gene transfer. The acoustic separator 302 may have the same or substantially similar structure as the acoustic separator 428 shown in Figures 8A-8E.

[0098] 8A and 8B-8D are top and corresponding cross-sectional views, respectively, of an acoustic isolation device 428 assembled from a microfluidic chip 400. The microfluidic chip 400 includes a planar substrate 402 having a first planar surface 404 and a second planar surface 406 opposite the first planar surface 404, and a lid 408 attached to the planar substrate 402 at the first planar surface 404. The planar substrate 402 includes a network of channels 411 recessed from the first planar surface 404 and substantially covered by the lid 408. The network of channels 411 includes a separation channel 410 having an upstream end and a downstream end, a side inlet port 412 for introducing a first fluid into the separation channel 410, a pair of side inlet channels 414 connecting the side inlet port 412 to the separation channel 410 at or near its upstream end, a central inlet port 416 for introducing a second fluid into the separation channel 410, a central inlet channel 418 connecting the central inlet port 416 to the separation channel 410 at or near its upstream end, a side outlet port 420 for extracting a third fluid from the separation channel 410, a pair of side outlet channels 422 connecting the side outlet port 420 to the separation channel 410 at or near its downstream end, a central outlet port 424 for extracting a fourth fluid from the separation channel 410, and a central outlet channel 426 connecting the central outlet port 424 to the separation channel 410 at or near its downstream end.

[0099] The side and central inlet ports 412, 416 in Figure 8B and the central outlet port 424 in Figure 8D open to the exterior of the microfluidic chip 400 at the second planar surface 406 of the planar substrate 402. However, any of the ports 412, 416, 420, 424 may alternatively open to the exterior of the microfluidic chip 400 through the lid 408, which may include through-holes that are aligned with the respective port locations in the planar substrate 402.

[0100] Referring back to FIG. 8A , the separation channel 410 may have a linear shape with a nominal width W between its two sidewalls. W may be approximately in the range of 100 μm to 1 mm. One or more of the side inlet channel 414, the central inlet channel 418, the side outlet channel 422, and the central outlet channel 426 may be narrower than the nominal width of the separation channel 410. Referring to FIG. 8C , the separation channel 410 may have a nominal depth D, as measured from the first planar surface 404. D may be approximately in the range of 50 μm to 500 μm. One or more of the side inlet channel 414, the central inlet channel 418, the side outlet channel 422, and the central outlet channel 426 may be shallower than the nominal depth of the separation channel 410.

[0101] The pair of side inlet channels 414 connect to the separation channel 410 at two of its side walls near or at the upstream end. Thus, a first fluid flowing through the pair of side inlet channels 414 enters the separation channel 410 as two streams flowing adjacent to the two side walls of the separation channel 410. The central inlet channel 418 connects to the separation channel 410 at or near its center. A second fluid flowing through the central inlet channel 418 enters the separation channel 410 at the center and is sandwiched between the two streams of the first fluid at or near the upstream end of the separation channel 410.

[0102] The pair of side outlet channels 422 connect to the separation channel 410 at its two side walls at or near its downstream end. Thus, fluid flowing adjacent to the two side walls at or near the downstream end of the separation channel 410 is diverted by the pair of side outlet channels 422 to become a third fluid and exits the microfluidic chip 400 through the side outlet port 420. The remaining fluid at or near the center of the separation channel 410 that is not diverted by the pair of side outlet channels 422 becomes a fourth fluid and proceeds to flow through the central outlet channel 426 and exit the microfluidic chip 400 through the central outlet port 424.

[0103] FIG. 8E shows fluid paths through the microfluidic chip 400 according to the port locations described above. First and second fluids are introduced into the microfluidic chip 400 through side and central inlet ports 412 and 416, respectively. Third and fourth fluids are extracted from the microfluidic chip 400 through side and central inlet ports 420 and 424, respectively. The first fluid introduced through the side inlet port 412 may be a fluid sample containing particles or biological objects with different sizes or acoustic contrasts for separation by acoustic radiation pressure, while the second fluid introduced through the central inlet port 416 may be a buffer fluid containing no particles or biological objects. After passing through the separation channel 410, the third fluid extracted from the side outlet port 420 may contain particles or biological objects with relatively smaller sizes or smaller acoustic contrasts, while the fourth fluid extracted from the central outlet port 424 may contain particles or biological objects with relatively larger sizes or higher acoustic contrasts.

[0104] The microfluidic chip 400 is part of an acoustic separation device 428 and further includes a piezoelectric transducer 430 attached to the microfluidic chip 400, as shown in FIG. 8E. The piezoelectric transducer 430 may be attached to the second planar surface 406 of the planar substrate 402 opposite the separation channel 410. Alternatively, the piezoelectric transducer 430 may be replaced by a group of two or more piezoelectric transducers arranged along the separation channel 410 and operating at the same frequency. Alternatively, the piezoelectric transducer 430 or group of piezoelectric transducers may be attached to the microfluidic chip 400 on the outside of the lid 408. Alternatively, one or more piezoelectric transducers may be attached independently to each of the second planar surface 406 of the planar substrate 402 and the exterior of the lid 408.

[0105] The piezoelectric transducer 430 or group of piezoelectric transducers may receive power in the form of an oscillating voltage having a frequency ranging from 100 kHz to 100 MHz to generate acoustic pressure waves in the separation channel 410 between its two sidewalls when liquid is present. When the channel width W is an integer multiple of half the wavelength of the acoustic pressure waves, acoustic standing waves may form in the separation channel 410, which may depend on the excitation frequency of the power applied to the piezoelectric transducer 430 and the compressibility and density of the liquid in the separation channel 410. When W is equal to half the wavelength of the acoustic waves generated by the piezoelectric transducer 430 or group of piezoelectric transducers, a standing wave is formed between the two sidewalls of the separation channel 410, with a single acoustic pressure node located along the center of the separation channel 410. Objects with relatively larger size and / or higher acoustic contrast may move toward the pressure node at a faster velocity, thereby enabling separation of objects by size or acoustic contrast.

[0106] 8A-8E, the operation of acoustic separation device 428 under single pressure node conditions will now be described. After completion of the transduction or gene transfer process, a fluid sample from cell container 134 containing genetically modified target cells 432, small debris or undesired biological matter 434 (i.e., non-target biological matter), if present, and the transduction or gene transfer medium is introduced into side inlet port 412 via fluid line 304, while a buffer fluid is introduced into central inlet port 416 from third container or bag 116 via fluid line 306. The target cells and undesired biological matter 432 and 434 may have sufficiently different physical sizes and / or acoustic contrasts, such as mass densities and compressibilities, to allow them to be separated by acoustic radiation pressure. For example, the target cells 432 may have a higher acoustic contrast, such as a larger physical size and / or a higher mass density and / or a lower compressibility, thereby allowing the acoustic radiation pressure to push the target cells 432 toward a pressure node located along the center of the separation channel 410 when operating under a single node.

[0107] A fluid sample containing target cells 432 and non-target biological matter 434 is introduced into the separation channel 410 at or near its upstream end via a pair of side inlet channels 414 as two laminar flows 436 and 438 flowing adjacent to the side walls. The two laminar flows 436 and 438 of the fluid sample in the separation channel 410, which may behave like laminar flows, are separated by a central flow 440 of buffer solution from the central inlet port 416. The central flow 440 of buffer solution may behave like a laminar flow and act as a sheath fluid to slow or prevent migration of non-target biological matter 434 toward a pressure node located along the center of the separation channel 410. As the fluid sample travels downstream in the separation channel 410, acoustic radiation pressure pushes the target cells 432 into the central flow 440, toward the pressure node located along the center of the separation channel 410, while the non-target biological matter 434 remains mostly in the two laminar flows 436 and 438 near the side walls. At the downstream end of separation channel 410, target cells 432, carried by the buffer solution, exit acoustic separator 428 and pass through central exit port 424 toward fourth container or bag 110. The removed fluid sample, which may contain non-target biological matter 434, flows near the side walls as laminar flows 436 and 438 and is diverted through a pair of side exit channels 422 to side exit port 420.

[0108] 7, a third container or bag 116, connected to the inlet of acoustic separation device 302 through fluid line 306, contains a buffer or solution that can act as a sheath fluid during acoustic separation of genetically modified target cells in acoustic separation device 302. The buffer or solution can also be used as a storage fluid for storing the genetically modified target cells in the fourth container for bag 110. The buffer or solution in third container or bag 116 can contain a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof.

[0109] A fourth container or bag 110, connected to the outlet of the acoustic isolation device 302 through a fluid line 308, contains the genetically modified target cells in a buffer or solution that can be administered intravenously to a patient.

[0110] The network of fluid lines 118-124, 304-308, the containers or bags 102, 110, 112, 116, the cell container 134 and the fluid lines passing through the magnetic separation device 104 and the acoustic separation device 302 may be constructed, interconnected and supplied as an integrated disposable tubing set that may be sterilized and sealed from the surrounding environment.

[0111] Continuing with reference to FIG. 7, the process begins by providing a first container or bag 102 containing a first fluid sample containing magnetically labeled target cells 132. In the case of a previously frozen sample, the first fluid sample may be prepared by first thawing the frozen sample and then extracting the target cells and other biological matter, if any, from the thawed sample fluid by centrifugation. The resulting target cells and other biological matter, if any, are resuspended in a buffer solution and filtered using a mesh size between 15 and 100 μm. A reagent containing a magnetic label is added to the filtered buffer solution containing the target cells, thereby forming the first fluid sample. Alternatively, the target cells may be magnetically labeled through an indirect process by first adding a reagent containing an intermediate link attached to the target cells before adding a reagent containing a magnetic label attached to the intermediate link.

[0112] For the magnetic separation process, a first fluid sample is introduced into the inlet of the magnetic separation device 104 via fluid line 118. As the first fluid sample flows through the magnetic separation device 104, the magnetically labeled target cells 132 are retained in the magnetic separation device 104 by the magnetic field, while the removed first fluid sample exits the magnetic separation device 104 into a waste container or bag (not shown). After the first fluid sample has completely passed through the magnetic separation device 104, the magnetic field acting on the magnetically labeled target cells 132 is reduced or eliminated, and a first eluent, such as a buffer solution in a second container or bag 112, flows into the magnetic separation device 104 via fluid line 120 and elutes the magnetically labeled target cells 132 into a cell container 134 via fluid line 122. Additionally, one or more solutions from incubation component tank 114, such as, but not limited to, culture medium, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer, are injected into cell container 134 through fluid line 124 to modify the genetic makeup of magnetically labeled target cells 132. Magnetically labeled target cells 132 (e.g., T cells) can be transduced to express a CAR by contacting the target cells with vectors bearing a CAR construct, such as viral vectors, e.g., lentiviruses and retroviruses, and non-viral vectors, e.g., plasmids, lipid nanoparticles, and mRNA.

[0113] The transduction or gene transfer process is carried out in the cell incubation chamber 106 while the magnetically labeled target cells 132 are immersed in a transduction or gene transfer medium containing one or more solutions from the first eluent and incubation component tank 114. The temperature and gas composition within the cell incubation chamber 106 can be controlled during the transduction or gene transfer process. For example, the temperature and gas composition within the cell incubation chamber 106 can be maintained at 37.0±0.1°C and 5.0%±0.1% CO2, respectively, for a period ranging from 10 minutes to 72 hours while the genetic makeup of the magnetically labeled target cells 132 is modified in the cell container 134.

[0114] After the transduction or gene transfer step is completed, a buffer to facilitate the subsequent acoustic sorting step can be added from the incubation component tank 114 to the transduction or gene transfer medium in the cell container 134. The mixture of the transduction or gene transfer medium and the buffer to facilitate the acoustic sorting step, if any, forms a second fluid sample containing the genetically modified target cells.

[0115] A second fluid sample is then introduced into the inlet of the acoustic separation device 302 via fluid line 304, and the genetically-modified target cells are extracted by an acoustic sorting process. Using the acoustic separation device 428 shown in FIGS. 8A-8E as an example, the second fluid sample containing the genetically-modified target cells is introduced into the side inlet port 412 via fluid line 304, while buffer solution is introduced into the central inlet port 416 from the third container or bag 116 via fluid line 306. The second fluid sample containing the genetically-modified target cells 432 and, if present, non-target biological matter 434, is introduced into the separation channel 410 at or near its upstream end via the pair of side inlet channels 414 as two laminar flows 436 and 438 flowing adjacent to the side walls. The two laminar flows 436 and 438 of the second fluid sample in the separation channel 410 are separated by a central flow 440 of buffer solution from the central inlet port 416. The central flow 440 of buffer solution behaves like a laminar flow and may act as a sheath fluid to slow or prevent the migration of non-target biological matter 434 toward a pressure node located along the center of the separation channel 410. As the second fluid sample proceeds downstream in the separation channel 410, acoustic radiation pressure pushes the genetically modified target cells 432 into the central flow 440 toward the pressure node located along the center of the separation channel 410, while the non-target biological matter 434 remains mostly in the two laminar flows 436 and 438 near the side walls. At the downstream end of the separation channel 410, the genetically modified target cells 432, carried by the buffer solution, exit the acoustic separation device 428 through the central exit port 424 via fluid line 308 and toward the fourth container or bag 110. The removed second fluid sample, which may contain non-target biological matter 434 , flows near the sidewalls in laminar flows 436 and 438 and is diverted to side outlet port 420 through a pair of side outlet channels 422 .

[0116] After the acoustic sorting process is complete, the fourth container or bag 110 containing the genetically modified target cells can be disconnected from the rest of the integrated system 300 without exposing its contents to the atmosphere. The contents in the third container or bag 300 can be administered intravenously to a patient.

[0117] 9 is a front view of an integrated system 300 that can be used to generate CAR T cells from whole blood or T-cell-containing samples according to the process described above. Utilizing a closed fluidic assembly, system 300 can be a closed processing system that can be deployed in facilities that lack strict sterile or clean environments, such as medical treatment facilities, thereby alleviating many of the common logistical issues encountered in autologous tissue procedures.

[0118] 9 shows an automated production system 300 including a shell or housing structure 140, first and second magnetic separation modules 142A, 142B and an acoustic separation module 310 present in the housing structure 140, an incubation module 144, a sample rack 146 mounted on top of the housing structure 140, and first and second sample bag holders 164, 166. Fluid lines and connected sample bags, which may be supplied as an integrated disposable tubing set, and a computer used to control the system 300 are omitted from this view to present an unobstructive view of the magnetic separation modules 142A, 142B and the acoustic separation module 310. The production system 300 is similar to the system 100 shown in FIG. 2, except for the replacement of the magnetic separation module 142C with the acoustic separation module 310.

[0119] Incubation module 144 includes an incubation chamber 106 located on top of a rocker base 147. Each of magnetic separation modules 142A, 142B includes a first pinch valve 148A, 148B, a second pinch valve 150A, 150B, a third pinch valve 152A, 152B, a fourth pinch valve 154A, 154B, a first air detector 156A, 156B, a second air detector 158A, 158B, a peristaltic pump 160A, 160B, an occlusion sensor 162A, 162B, and a magnetic separation device 104, 105. The acoustic isolation module 310 includes first and second peristaltic pumps 312, 314, first and second occlusion sensors 316, 318, first and second air detectors 320, 322, an acoustic isolation device 302, and first, second, and third pinch valves 324-328.

[0120] System 300 may also include additional pinch valves 168, 170 disposed on one side of housing structure 140 and additional pinch valves 172, 174 disposed on the other side of housing structure 140. Electromechanical components 104-106, 147, 148A / B-162A / B, 168-174, 312-328 may be controlled or automated by a computer or microprocessor (not shown).

[0121] Each of the magnetic separation devices 104, 105 may have the same or substantially similar structure as the magnetic separation device 188 shown in Figures 3 and 4 and described above. Other magnetic flux sources and column-free magnetic separation devices, such as those disclosed in U.S. Patent Application No. 18 / 072,362, which is incorporated herein by reference, may also be used to separate magnetically labeled target cells from a fluid sample.

[0122] Acoustic isolator 302 may have the same or substantially similar structure as acoustic isolator 428 shown in Figures 8A-8E and described above.

[0123] The incubation module 144 includes a cell incubation chamber 106 located on a rocker base 147. The incubation chamber 106 has a heater that can maintain a uniform temperature within the chamber 106 between room temperature and 50°C. The CO2 concentration within the chamber 106 can be varied between 0 and 10% to provide an optimal environment for the cells. The rocker base 147 can move the chamber 106 at a speed of 2 to 4 rpm. The rocking motion of the chamber 106 caused by the rocker base 147 can facilitate uniform mixing of the fluid contents within the cell container 134. The rocker base 147 can remain stationary during cell activation, transduction, or expansion steps. After the incubation step, the rocker base 147 rocks the cell container 134 within the chamber 106, allowing the fluid in the cell container 134 to wash the cells off the surface of the cell container 134. The rocker base 147 can also be used to facilitate the draining of the cell container 134 by tilting the incubation chamber 106 to a fixed position to allow the end of the outlet tubing to reach the bottom of the fluid in the cell container 134.

[0124] System 300 has a modular design that includes two magnetic separation modules 142A, 142B, one acoustic separation module 310, and one incubation module 144. Modules 142A, 142B, 144, and 310 can be fluidly connected in series, parallel, or a combination thereof by a closed fluidic assembly that includes sample bags, cell containers, acoustic separation devices, and all fluid lines, including conduits that pass through the magnetic separation devices.

[0125] FIG. 10 is a perspective view of a system 300 for manufacturing CAR T cells that uses a closed fluidic assembly in the form of an integrated tubing set. For reasons of readability, the electromechanical components shown in FIG. 9 are not shown in this drawing to clearly show the routing of the fluid lines. The perspective view also shows pinch valves 152B, 154A, 154B, 328, which were previously obstructed by the cell incubation chamber 106 in the front view of FIG. 9, as well as pinch valves 172, 174 located on the side of the housing structure 140. The lid and a portion of one sidewall of the incubation chamber 106 are further removed to expose a cell container 134 in the form of a common cell culture flask within the chamber 106. The cap of the cell culture flask 134 contains an air filter that vents to the environment within the incubation chamber 106 while maintaining sterility within the flask 134. The second bag holder 166 is also omitted from the drawing to show the sample bags 110, 224 within the holder 166. FIG. 11 is a fluid circuit diagram corresponding to the system 300 shown in FIG.

[0126] 9-11, the fluidic assembly of system 300 is configured to use first and second fluidic separation devices 104, 105 that are fluidly coupled in parallel to extract magnetically labeled target cells (e.g., T cells) from a first fluid sample in a magnetic separation step, and acoustic separation device 302 for a subsequent acoustic separation step. The use of two magnetic separation devices 104, 105 for the magnetic separation step increases the sorting throughput of system 300, allowing it to accommodate larger volumes of first fluid samples, such as whole blood.

[0127] The first, second, third, and fourth containers or bags 102, 112, 116, 110 are in the form of sample bags. Both sample bags 102 and 112 are fluidly connected to the inlets of the first and second magnetic separation devices 104, 105. In addition to sample bag 116, another sample bag 220, which may contain cryopreservation solution, preservation medium, PBS-based solution, saline-based solution, human serum, glucose, or any combination thereof, is also fluidly connected to the inlet of acoustic separation device 302. Incubation component bank 114 includes at least two sample bags 114A, 114B that are fluidly connected to cell container 134 through magnetic separation devices 104, 105. Each of sample bags 114A, 114B may contain culture medium, a vector for cell transduction or gene transfer, a lentivirus for transduction, a buffer or solution for non-viral gene transfer, or a cell activation reagent. Prior to the transduction or gene transfer step, the target T cells in the cell container 134 may be activated by flowing a cell activation reagent (e.g., a solution containing CD3 / CD28 magnetic beads) into the cell container 134 from one of the sample bags 114A, 114B.

[0128] The fluidic assembly of system 300 further includes a sample bag 222 fluidly connected to the outlets of the first and second magnetic separation devices 104, 105 to collect waste from the first and second magnetic separation devices 104, 105, and another sample bag 224 fluidly connected to the outlet of acoustic separation device 302 to collect waste from acoustic separation device 302. Sample bag 222 for collecting waste from the first and second magnetic separation devices 104, 105 can be placed in first bag holder 164. Sample bags 110, 224 for collecting the final cell product and waste, respectively, from acoustic separation device 302 can be placed in second bag holder 166. All other sample bags 102, 112, 114A, 114B, 116, 220 that provide materials for the manufacturing process can be suspended in sample rack 146. The fluidic assembly of system 300 can include additional sample bags for various purposes. Each of the sample bags 102, 110, 112, 114A, 114B, 116, 220-224 may have an inlet port connected to a Luer connector and an outlet port connected to a drip chamber. Additionally, fluid flow through the inlet and outlet ports of each sample bag may be controlled or regulated by one or more manual pinch clamps attached thereto.

[0129] The fluidic assembly of system 300 may also include syringes 226, 228, 230. Syringes 226, 228, which may be fluidly connected to the inlet of cell container 134, may be used to extract a sample of magnetically separated target cells after the magnetic sorting step, or may inject culture medium, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer, or cell activation reagents into cell container 134. Syringe 230, which may be fluidly connected to the outlet of cell container 134, may be used to extract a sample of genetically modified target cells after the transduction or gene transfer step is completed.

[0130] Continuing to refer to FIG. 11, the fluid circuit corresponding to system 300 includes sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, and acoustic separator 302, all interconnected by a network of fluid lines passing through electromechanical components such as pinch valves 148A / B-154A / B, 168-172, 324-328, air detectors 156A / B, 158A / B, 320, 322, occlusion sensors 162A, 162B, 316, 318, peristaltic pumps 160A, 160B, 312, 314, and magnetic separators 104, 105. The network of fluid lines, including the sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, acoustic separator 302, and conduits passing through magnetic separators 104, 105, can be constructed, interconnected, sterilized, and supplied as an integrated disposable set. The electromechanical components of system 300 are external to the tubing set and therefore do not come into contact with the fluids in the tubing set.

[0131] A first container or sample bag 102, which may contain target cells for processing, is fluidly connected to the inlet of a conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through a first pinch valve 148A, a first air detector 156A, a second air detector 158A, a peristaltic pump 160A and an occlusion sensor 162A, and is also fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 by one or more fluid lines passing through a first pinch valve 148B, a first air detector 156B, a second air detector 158B, a peristaltic pump 160B and an occlusion sensor 162B. A second container or sample bag 112, which may contain a buffer solution or culture medium, is fluidly connected to the inlet of a conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through a second pinch valve 150A, a second air detector 158A, a peristaltic pump 160A and an occlusion sensor 162A, and is also fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 through one or more fluid lines passing through a second pinch valve 150B, a second air detector 158B, a peristaltic pump 160B and an occlusion sensor 162B.

[0132] The inlet of the cell container 134 is fluidly connected to the outlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through the third pinch valve 152A, and also fluidly connected to the outlet of the conduit passing through the second magnetic separation device 105 by one or more fluid lines passing through the third pinch valve 152B. Syringes 226, 228 may be fluidly connected to the fluid lines between the inlet of the cell container 134 and the third pinch valves 152A, 152B. A sample bag 222, which may store waste liquid such as sample fluid after target cell extraction, is fluidly connected to the outlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through the fourth pinch valve 154A, and also fluidly connected to the outlet of the conduit passing through the second magnetic separation device 105 by one or more fluid lines passing through the fourth pinch valve 154B.

[0133] The outlet of the cell container 134 is fluidly connected to the inlet of the acoustic isolation device 302 (e.g., side inlet port 412 in FIG. 8A ) by one or more fluid lines that pass through a first peristaltic pump 312, a first occlusion sensor 316, and a first air detector 320. A syringe 230 may be fluidly connected to the fluid line connected to the outlet of the cell container 134. A third container or sample bag 116 is fluidly connected to the inlet of the acoustic isolation device 302 (e.g., central inlet port 416 in FIG. 8A ) by one or more fluid lines that pass through a pinch valve 172, a second peristaltic pump 314, a second occlusion sensor 318, and a first pinch valve 324. The sample bag 220 is fluidly connected to the inlet of the acoustic isolation device 302 (e.g., central inlet port 416 in FIG. 8A ) by one or more fluid lines that pass through the pinch valve 174, the second peristaltic pump 314, the second occlusion sensor 318, and the first pinch valve 324.

[0134] A fourth container or sample bag 110 is fluidly connected to the outlet of the acoustic isolation device 302 (e.g., central outlet port 424 in FIG. 8A ) by one or more fluid lines that pass through a second pinch valve 326. A sample bag 224, which may be used to receive waste fluid, is fluidly connected to the outlet of the acoustic isolation device 302 (e.g., side outlet port 420 in FIG. 8A ) by one or more fluid lines that pass through a second air detector 322 and a third pinch valve 328.

[0135] The sample bag 114A of the incubation component bank 114 is fluidly connected to the inlet of the cell container 134 by one or more fluid lines passing through pinch valve 168, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104 and third pinch valve 152A, and by one or more fluid lines passing through pinch valve 168, second pinch valve 150B, second air detector 158B, peristaltic pump 160B, occlusion sensor 162B, second magnetic separation device 105 and third pinch valve 152B. Similarly, sample bag 114B of incubation component bank 114 is fluidly connected to the inlet of cell container 134 by one or more fluid lines passing through pinch valve 170, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104 and third pinch valve 152A, and by one or more fluid lines passing through pinch valve 170, second pinch valve 150B, second air detector 158B, peristaltic pump 160B, occlusion sensor 162B, second magnetic separation device 105 and third pinch valve 152B.

[0136] Embodiment 3: Negative magnetic selection and acoustic purification

[0137] This embodiment uses a magnetic separation device to isolate or extract non-magnetically labeled cells from non-target cells that have been magnetically labeled for genetic modification, which can be done in a cell incubator, and then uses an acoustic separation device to purify the genetically modified target cells.

[0138] 12 is a block diagram illustrating an integrated system 500 and its components that may be used to generate CAR T cells. The integrated system 500, with control electronics, user interface, hardware, software, and firmware (not shown), includes a first container or bag 102, a magnetic separation device 104, a cell incubation chamber 106, an acoustic separation device 302, a second container or bag 112 containing a buffer or solution for eluting magnetically labeled non-target cells from the magnetic separation device 104, an incubation component bank 114, a third container or bag 116 containing a buffer or solution for operating the acoustic separation device 302, a fourth container or bag 110 for the final cell product, and multiple fluid lines 118, 120, 124, 304-308, 502 that provide fluidic interconnections between the individual components in the integrated system 500. Components 102-106, 110-120, 124, 134, 302-308 of integrated system 500 are similar to components 110-120, 124, 134, 302-308, respectively, of integrated system 300, as shown in FIG. 4 and described above, except that fluid line 122 is replaced by fluid line 502 to provide a negative selection step (i.e., non-target cells are magnetically labeled).

[0139] A first container or bag 102 holds a fluid sample containing non-target cells 504 with attached magnetic labels and target cells 506 without magnetic labels. The fluid sample may contain whole blood, leukopaque, PBMCs, and / or other cell suspensions with attached magnetic labels to the non-target cells 504. The magnetic labels may be attached to the non-target cells 504 during an incubation step that takes place in the same sample container or bag 102.

[0140] Magnetically labeled non-target cells 504 are extracted from the fluid sample using a magnetic separation device 104 connected to the first container or bag 102 through a fluid line 118. The magnetic separation device 104 may have the same or substantially similar structure as the magnetic separation device 188, the cross-sectional views of which are shown in Figures 3 and 4 and described above.

[0141] A second container or bag 112, connected to the inlet of the magnetic separation device 104 through fluid line 120, contains a buffer or solution for eluting the magnetically labeled non-target cells 504 remaining in the magnetic separation device 104 after the passage of the fluid sample. The buffer or solution in the second container or bag 112 may contain a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0142] The cell incubation chamber 106 can be used for cell genome engineering, cell modification, cell transduction, or cell gene transfer. The cell incubation chamber 106 contains a cell container 134 connected to the outlet of the magnetic separation device 104 through a fluid line 502. The cell container 134 contains non-magnetic target cells 506 and one or more buffers or solutions from an incubation component bank 114 connected to the cell container 134 through a fluid line 124. One or more gas lines (e.g., CO2) (not shown) can be connected to the cell incubation chamber 106 to provide an environment with a desired gas composition (e.g., 5.0%±0.1% CO2). The cell incubation chamber 106 can also have a heating and cooling mechanism (not shown) that can maintain a constant temperature within the chamber (e.g., 37.0±0.1°C). Additionally, the cell incubation chamber 106 may also include an air circulation mechanism, such as a fan, for circulating air or other gas mixture therein to make the temperature more uniform throughout the chamber 106. The cell container 134 and the contents therein may be heated or cooled by convection through the surrounding environment in the cell incubation chamber 106. The cell container 134 may also have porous or permeable portions, such as membranes and vents, that allow gas molecules in the surrounding environment to diffuse through the flow-through, thereby exposing the contents of the cell container 134 to the air or other gas mixture inside the cell incubation chamber 106.

[0143] The incubation component tank 114 may contain one or more of the following items: culture media, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer.

[0144] The cell incubation chamber 106 may further include means, such as, but not limited to, cell electroporation, mechanical cell deformation, temperature, ultrasound, and optics, that can induce target cell membranes to open, thereby allowing genetic material to enter the target cells from the surrounding medium in the cell container 134. Genetic material may also be introduced into the target cells using droplet-based injection of genetic material or penetration of the target cell membrane.

[0145] The cell incubation chamber 106 can provide an environment for the transduction process. For example, lentivirus or other viruses can be used to transfer genomic material into target cells to induce chimeric antigen receptor (CAR) expression on the cell surface.

[0146] An acoustic separator 302, connected to the cell container 134 in the cell incubation chamber 106 through a fluid line 304, is used to extract the target cells from the mixture of solutions and / or media used in the cell container 134 after genetic modification by transduction or gene transfer. The acoustic separator 302 may have the same or substantially similar structure as the acoustic separator 428 shown in Figures 8A-8E and described above.

[0147] A third container or bag 116, connected to the inlet of acoustic separation device 302 through fluid line 306, contains a buffer or solution that can act as a sheath fluid during acoustic separation of the genetically modified target cells in acoustic separation device 302. The buffer or solution can also be used as a storage fluid for storing the genetically modified target cells in the fourth container for bag 110. The buffer or solution in third container or bag 116 can contain a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof.

[0148] A fourth container or bag 110, connected to the outlet of the acoustic isolation device 302 through a fluid line 308, contains the genetically modified target cells in a buffer or solution that can be administered intravenously to a patient.

[0149] The network of fluid lines 118, 120, 124, 304-308, 502, the containers or bags 102, 110, 112, 116, the cell container 134 and the fluid lines passing through the magnetic separation device 104 and the acoustic separation device 302 may be constructed, interconnected and supplied as an integrated disposable tubing set that may be sterilized and sealed from the surrounding environment.

[0150] 12, the process begins by providing a first container or bag 102 containing therein a first fluid sample containing magnetically labeled non-target cells 504 and non-magnetic target cells 506. The first fluid sample is flowed through fluid line 118 into the inlet of magnetic separation device 104 for the magnetic separation process.

[0151] As the first fluid sample flows through the magnetic separation device 104, the magnetically labeled non-target cells 504 are retained in the magnetic separation device 104 by the magnetic field, while the non-magnetic target cells 506 retained by the removed fluid sample (i.e., without the magnetically labeled non-target cells 504) exit the magnetic separation device 104 via fluid line 502 and enter the cell container 134. One or more solutions from the incubation component tank 114, such as, but not limited to, culture medium, a vector for cell transduction or gene transfer, a lentivirus for transduction, a buffer or solution for non-viral gene transfer, etc., are injected into the cell container 134 through fluid line 124 to modify the genetic makeup of the non-magnetic target cells 506. The non-magnetic target cells 506 (e.g., T cells) can be transduced to express a CAR by contacting the target cells with a vector bearing a CAR construct, for example, viral vectors, such as lentiviruses and retroviruses, and non-viral vectors, such as plasmids, lipid nanoparticles, and mRNA.

[0152] The transduction or gene transfer process is carried out in the cell incubation chamber 106 while the target cells 506 are immersed in a transduction or gene transfer medium containing one or more solutions from the first eluent and incubation component tank 114. The temperature and gas composition within the cell incubation chamber 106 can be controlled during the transduction or gene transfer process. For example, the temperature and gas composition within the cell incubation chamber 3c can be maintained at 37.0±0.1°C and 5.0%±0.1% CO2, respectively, for a period ranging from 10 minutes to 72 hours while the genetic structure of the target cells 506 is modified in the cell container 134.

[0153] After the transduction or gene transfer step is completed, a buffer to facilitate the subsequent acoustic sorting step can be added from the incubation component tank 114 to the transduction or gene transfer medium in the cell container 134. The mixture of the transduction or gene transfer medium and the buffer to facilitate the acoustic sorting step, if any, forms a second fluid sample containing the genetically modified target cells.

[0154] A second fluid sample is then introduced into the inlet of the acoustic separation device 302 via fluid line 304, and the genetically-modified target cells are extracted by an acoustic sorting process. Using the acoustic separation device 428 shown in FIGS. 8A-85E as an example, a second fluid sample containing genetically-modified target cells is introduced into the side inlet port 412 via fluid line 304, while a buffer solution is introduced into the central inlet port 416 from the third container or bag 116 via fluid line 306. The second fluid sample containing genetically-modified target cells 432 and, if present, non-target biological matter 434, is introduced into the separation channel 410 at or near its upstream end via the pair of side inlet channels 414 as two laminar flows 436 and 438 flowing adjacent to the side walls. The two laminar flows 436 and 438 of the second fluid sample in the separation channel 410 are separated by a central flow 440 of buffer solution from the central inlet port 416. The central flow 440 of buffer solution behaves like a laminar flow and may act as a sheath fluid to slow or prevent the migration of non-target biological matter 434 toward a pressure node located along the center of the separation channel 410. As the second fluid sample proceeds downstream in the separation channel 410, acoustic radiation pressure pushes the genetically modified target cells 432 into the central flow 440 toward the pressure node located along the center of the separation channel 410, while the non-target biological matter 434 remains mostly in the two laminar flows 436 and 438 near the side walls. At the downstream end of the separation channel 410, the genetically modified target cells 432, carried by the buffer solution, exit the acoustic separation device 428 through the central exit port 424 via fluid line 308 and toward the fourth container or bag 110. The removed second fluid sample, which may contain non-target biological matter 434 , flows near the sidewalls in laminar flows 436 and 438 and is diverted to side outlet port 420 through a pair of side outlet channels 422 .

[0155] After the acoustic sorting process is complete, the fourth container or bag 110 containing the genetically modified target cells can be disconnected from the rest of the integrated system 500 without exposing its contents to the atmosphere. The contents in the fourth container or bag 110 can be administered intravenously to a patient.

[0156] 9 is a front view of an integrated system 500 that can be used to generate CAR T cells from whole blood or T-cell-containing samples according to the steps described above. Utilizing a closed fluidic assembly, system 500 can be a closed processing system that can be deployed in facilities that lack strict sterile or clean environments, such as medical treatment facilities, thereby alleviating many of the common logistical issues encountered in autologous tissue procedures.

[0157] 9 shows an automated production system 500 including a shell or housing structure 140, first and second magnetic separation modules 142A, 142B and acoustic separation module 310 present in housing structure 140, an incubation module 144, a sample rack 146 mounted on top of housing structure 140, and first and second sample bag holders 164, 166. Fluid lines and connected sample bags, which may be supplied as an integrated disposable tubing set, and a computer used to control system 500 are omitted from this view to present an unobstructive view of magnetic separation modules 142A, 142B and acoustic separation module 310. Production system 500 is similar to system 300, except for the routing of fluid lines to accommodate negative selection of the magnetic separation step, as described below.

[0158] Incubation module 144 includes an incubation chamber 106 located on top of a rocker base 147. Each of magnetic separation modules 142A, 142B includes a first pinch valve 148A, 148B, a second pinch valve 150A, 150B, a third pinch valve 152A, 152B, a fourth pinch valve 154A, 154B, a first air detector 156A, 156B, a second air detector 158A, 158B, a peristaltic pump 160A, 160B, an occlusion sensor 162A, 162B, and a magnetic separation device 104, 105. The acoustic isolation module 310 includes first and second peristaltic pumps 312, 314, first and second occlusion sensors 316, 318, first and second air detectors 320, 322, an acoustic isolation device 302, and first, second, and third pinch valves 324-328.

[0159] System 500 may also include additional pinch valves 168, 170 disposed on one side of housing structure 140 and additional pinch valves 172, 174 disposed on the other side of housing structure 140. Electromechanical components 104-106, 147, 148A / B-162A / B, 168-174, 312-328 may be controlled or automated by a computer or microprocessor (not shown).

[0160] Each of the magnetic separation devices 104, 105 may have the same or substantially similar structure as the magnetic separation device 188 shown in Figures 3 and 4 and described above. Other magnetic flux sources and column-free magnetic separation devices, such as those disclosed in U.S. Patent Application No. 18 / 072,362, which is incorporated herein by reference, may also be used to separate magnetically labeled target cells from a fluid sample.

[0161] Acoustic isolator 302 may have the same or substantially similar structure as acoustic isolator 428 shown in Figures 8A-8E and described above.

[0162] The incubation module 144 includes a cell incubation chamber 106 located on a rocker base 147. The incubation chamber 106 has a heater that can maintain a uniform temperature within the chamber 106 between room temperature and 50°C. The CO2 concentration within the chamber 106 can be varied between 0 and 10% to provide an optimal environment for the cells. The rocker base 147 can move the chamber 106 at a speed of 2 to 4 rpm. The rocking motion of the chamber 106 caused by the rocker base 147 can facilitate uniform mixing of the fluid contents within the cell container 134. The rocker base 147 can remain stationary during cell activation, transduction, or expansion steps. After the incubation step, the rocker base 147 rocks the cell container 134 within the chamber 106, allowing the fluid in the cell container 134 to wash the cells off the surface of the cell container 134. The rocker base 147 can also be used to facilitate the draining of the cell container 134 by tilting the incubation chamber 106 to a fixed position to allow the end of the outlet tubing to reach the bottom of the fluid in the cell container 134.

[0163] System 500 has a modular design that includes two magnetic separation modules 142A, 142B, one acoustic separation module 310, and one incubation module 144. Modules 142A, 142B, 144, and 310 can be fluidly connected in series, parallel, or a combination thereof by a closed fluidic assembly that includes sample bags, cell containers, acoustic separation devices, and all fluid lines, including conduits that pass through the magnetic separation devices.

[0164] FIG. 13 is a perspective view of a system 500 for manufacturing CAR T cells that uses a closed fluidic assembly in the form of an integrated tubing set. For reasons of readability, the electromechanical components shown in FIG. 9 are not shown in this drawing to clearly show the routing of the fluid lines. The perspective view also shows pinch valves 152B, 154A, 154B, 328, which were previously obstructed by the cell incubation chamber 106 in the front view of FIG. 9, as well as pinch valves 172, 174 located on one side of the housing structure 140. The lid and a portion of the sidewall of the incubation chamber 106 are further removed to expose a cell container 134 in the form of a common cell culture flask within the chamber 106. The cap of the cell culture flask 134 contains an air filter that vents to the environment within the incubation chamber 106 while maintaining sterility within the flask 134. The second bag holder 166 is also omitted from the drawing to show the sample bags 110, 224 within the holder 166. FIG. 14 is a fluid circuit diagram corresponding to the system 500 shown in FIG.

[0165] 9, 13, and 14, the fluidic assembly of system 500 is configured to use parallel, fluidly coupled first and second magnetic separation devices 104, 105 for extracting magnetically labeled non-target cells from a first fluid sample in a magnetic separation step, and an acoustic separation device 302 for purifying the non-magnetic target cells. The use of two magnetic separation devices 104, 105 for the magnetic separation step increases the sorting throughput of system 500, allowing it to accommodate a larger volume of the first fluid sample.

[0166] The first, second, third, and fourth containers or bags 102, 112, 116, 110 are in the form of sample bags. Both sample bags 102 and 112 are fluidly connected to the inlets of the first and second magnetic separation devices 104, 105. In addition to sample bag 116, another sample bag 220, which may contain cryopreservation solution, preservation medium, PBS-based solution, saline-based solution, human serum, glucose, or any combination thereof, is also fluidly connected to the inlet of acoustic separation device 302. Incubation component bank 114 includes at least two sample bags 114A, 114B that are fluidly connected to cell container 134 through magnetic separation devices 104, 105. Each of sample bags 114A, 114B may contain culture medium, a vector for cell transduction or gene transfer, a lentivirus for transduction, a buffer or solution for non-viral gene transfer, or a cell activation reagent. Prior to the transduction or gene transfer step, the target T cells in the cell container 134 may be activated by flowing a cell activation reagent (e.g., a solution containing CD3 / CD28 magnetic beads) into the cell container 134 from one of the sample bags 114A, 114B.

[0167] The fluidic assembly of system 500 further includes a sample bag 222 fluidly connected to the outlets of the first and second magnetic separation devices 104, 105 to collect waste from the first and second magnetic separation devices 104, 105, and another sample bag 224 fluidly connected to the outlet of acoustic separation device 302 to collect waste from acoustic separation device 302. The sample bag 222 for collecting waste from the first and second magnetic separation devices 104, 105 can be placed in the first bag holder 164. The sample bags 110, 224 for collecting the final cell product and waste, respectively, from acoustic separation device 302 can be placed in the second bag holder 166. All other sample bags 102, 112, 114A, 114B, 116, 220 that provide materials for the manufacturing process can be suspended in the sample rack 146. The fluidic assembly of system 500 can include additional sample bags for various purposes. Each of the sample bags 102, 110, 112, 114A, 114B, 116, 220-224 may have an inlet port connected to a Luer connector and an outlet port connected to a drip chamber. Additionally, fluid flow through the inlet and outlet ports of each sample bag may be controlled or regulated by one or more manual pinch clamps attached thereto.

[0168] The fluidic assembly of system 500 may also include syringes 226, 228, 230. Syringes 226, 228, which may be fluidly connected to the inlet of cell container 134, may be used to extract a sample of magnetically separated target cells after the magnetic sorting step, or may inject culture medium, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer, or cell activation reagents into cell container 134. Syringe 230, which may be fluidly connected to the outlet of cell container 134, may be used to extract a sample of genetically modified target cells after the transduction or gene transfer step is completed.

[0169] Continuing to refer to FIG. 14, the fluid circuit corresponding to system 500 includes sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, and acoustic separator 302, all interconnected by a network of fluid lines passing through electromechanical components such as pinch valves 148A / B-154A / B, 168-172, 324-328, air detectors 156A / B, 158A / B, 320, 322, occlusion sensors 162A, 162B, 316, 318, peristaltic pumps 160A, 160B, 312, 314, and magnetic separators 104, 105. The network of fluid lines, including the sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, acoustic separator 302, and conduits passing through magnetic separators 104, 105, can be constructed, interconnected, sterilized, and supplied as an integrated disposable set. The electromechanical components of system 500 are external to the tubing set and therefore do not come into contact with the fluids in the tubing set.

[0170] A first container or sample bag 102, which may contain target cells for processing, is fluidly connected to the inlet of a conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through a first pinch valve 148A, a first air detector 156A, a second air detector 158A, a peristaltic pump 160A and an occlusion sensor 162A, and is also fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 by one or more fluid lines passing through a first pinch valve 148B, a first air detector 156B, a second air detector 158B, a peristaltic pump 160B and an occlusion sensor 162B. A second container or sample bag 112, which may contain a buffer solution or culture medium, is fluidly connected to the inlet of a conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through a second pinch valve 150A, a second air detector 158A, a peristaltic pump 160A and an occlusion sensor 162A, and is also fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 through one or more fluid lines passing through a second pinch valve 150B, a second air detector 158B, a peristaltic pump 160B and an occlusion sensor 162B.

[0171] The inlet of the cell container 134 is fluidly connected to the outlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through a fourth pinch valve 154A, and also fluidly connected to the outlet of the conduit passing through the second magnetic separation device 105 by one or more fluid lines passing through a fourth pinch valve 154B. Syringes 226, 228 may be fluidly connected to the fluid lines between the inlet of the cell container 134 and the fourth pinch valves 154A, 154B. A sample bag 222, which may store waste fluid containing magnetically labeled non-target cells, is fluidly connected to the outlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through a third pinch valve 152A, and also fluidly connected to the outlet of the conduit passing through the second magnetic separation device 105 by one or more fluid lines passing through a third pinch valve 152B.

[0172] The outlet of the cell container 134 is fluidly connected to the inlet of the acoustic isolation device 302 (e.g., side inlet port 412 in FIG. 8A ) by one or more fluid lines that pass through a first peristaltic pump 312, a first occlusion sensor 316, and a first air detector 320. A syringe 230 may be fluidly connected to the fluid line connected to the outlet of the cell container 134. A third container or sample bag 116 is fluidly connected to the inlet of the acoustic isolation device 302 (e.g., central inlet port 416 in FIG. 8A ) by one or more fluid lines that pass through a pinch valve 172, a second peristaltic pump 314, a second occlusion sensor 318, and a first pinch valve 324. The sample bag 220 is fluidly connected to the inlet of the acoustic isolation device 302 (e.g., central inlet port 416 in FIG. 8A ) by one or more fluid lines that pass through the pinch valve 174, the second peristaltic pump 314, the second occlusion sensor 318, and the first pinch valve 324.

[0173] A fourth container or sample bag 110 is fluidly connected to the outlet of the acoustic isolation device 302 (e.g., central outlet port 424 in FIG. 8A ) by one or more fluid lines that pass through a second pinch valve 326. A sample bag 224, which may be used to receive waste fluid, is fluidly connected to the outlet of the acoustic isolation device 302 (e.g., side outlet port 420 in FIG. 8A ) by one or more fluid lines that pass through a second air detector 322 and a third pinch valve 328.

[0174] The sample bag 114A of the incubation component bank 114 is fluidly connected to the inlet of the cell container 134 by one or more fluid lines passing through pinch valve 168, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104 and fourth pinch valve 154A, and by one or more fluid lines passing through pinch valve 168, second pinch valve 150B, second air detector 158B, peristaltic pump 160B, occlusion sensor 162B, second magnetic separation device 105 and fourth pinch valve 154B. Similarly, sample bag 114B of incubation component bank 114 is fluidly connected to the inlet of cell container 134 by one or more fluid lines passing through pinch valve 170, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104 and fourth pinch valve 154A, and by one or more fluid lines passing through pinch valve 170, second pinch valve 150B, second air detector 158B, peristaltic pump 160B, occlusion sensor 162B, second magnetic separation device 105 and fourth pinch valve 154B.

[0175] Embodiment 4: Positive magnetic selection, delabeling, sonic purification

[0176] This embodiment uses a first magnetic separation device to isolate or extract magnetically labeled target cells, a cell incubator to genetically modify the magnetically labeled target cells and, particularly when larger magnetic labels are used, to remove the magnetic label from the target cells, and an acoustic separation device to purify the genetically modified target cells.

[0177] Figure 15 is a block diagram illustrating an integrated system 600 and its components that can be used to generate CAR T cells. An integrated system 600 having control electronics, a user interface, hardware, software, and firmware (not shown) includes a first container or bag 102, a first magnetic separation device 104, a cell incubation chamber 106, a second magnetic separation device 105, an acoustic separation device 302, a second container or bag 112 containing a buffer or solution for eluting target cells from the first magnetic separation device 104, an incubation component bank 114, a third container or bag 116 containing a buffer or solution for operating the acoustic separation device 302, a fourth container or bag 110 for the final cell product, a fifth container or bag 602 containing a buffer or solution for eluting magnetic labels from the second magnetic separation device 105 and / or for priming the second magnetic separation device 105, and a plurality of fluid lines 118-126, 306, 308, 604, 606 providing fluidic interconnections between the individual components in the integrated system 600.

[0178] A first container or bag 102 holds a fluid sample containing target cells 132 (e.g., T cells) that have been magnetically labeled. The fluid sample may contain whole blood, leukopaques, PBMCs, and / or other leukopheresis products that contain magnetically labeled target cells 132. The magnetic labels may be attached to the target cells 132 during an incubation step that takes place in the same first container or bag 102.

[0179] A first magnetic separation device 104, connected to the first container or bag 102 through a fluid line 118, is used to extract the magnetically labeled target cells 132 from the fluid sample. The first magnetic separation device 104 may have the same or substantially similar structure as the magnetic separation device 188, the cross-sectional views of which are shown in Figures 3 and 4 and described above.

[0180] A second container or bag 112, connected to the inlet of the first magnetic separation device 104 through fluid line 120, contains a buffer or solution for eluting the magnetically labeled target cells 132 remaining in the first magnetic separation device 104 after the passage of the fluid sample. The buffer or solution in the second container or bag 112 may contain a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0181] The cell incubation chamber 106 can be used for cell genome engineering, cell modification, cell transduction, or cell gene transfer. The cell incubation chamber 106 contains a cell container 134 connected to the outlet of the first magnetic separation device 104 through a fluid line 122. The cell container 134 contains magnetically labeled target cells 132, a buffer or solution for eluting the cells from the first magnetic separation device 104, and, optionally, one or more buffers or solutions from an incubation component bank 114 connected to the cell container 134 through a fluid line 124. One or more gas lines (e.g., CO) (not shown) can be connected to the cell incubation chamber 106 to provide an environment with a desired gas composition (e.g., 5.0%±0.1% CO). The cell incubation chamber 106 can also have a heating and cooling mechanism (not shown) capable of maintaining a constant temperature within the chamber (e.g., 37.0±0.1°C). Additionally, the cell incubation chamber 106 may also include an air circulation mechanism, such as a fan, for circulating air or other gas mixture therein to make the temperature more uniform throughout the chamber 106. The cell container 134 and the contents therein may be heated or cooled by convection through the surrounding environment in the cell incubation chamber 106. The cell container 134 may also have porous or permeable portions, such as membranes and vents, that allow gas molecules in the surrounding environment to diffuse through the flow-through, thereby exposing the contents of the cell container 134 to the air or other gas mixture inside the cell incubation chamber 106.

[0182] The incubation component tank 114 may contain one or more of the following items: culture medium, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer, reagents for removing magnetic labels from target cells in the delabeling step.

[0183] The cell incubation chamber 106 may further include means, such as, but not limited to, cell electroporation, mechanical cell deformation, temperature, ultrasound, and optics, that can induce target cell membranes to open, thereby allowing genetic material to enter the target cells from the surrounding medium in the cell container 134. Genetic material may also be introduced into the target cells using droplet-based injection of genetic material or penetration of the target cell membrane.

[0184] The cell incubation chamber 106 can provide an environment for a transduction process. For example, lentivirus or other viruses can be used to transfer genomic material into target cells to induce chimeric antigen receptor (CAR) proliferation on the cell surface. Magnetic labels attached to target cells 132 can also be removed from the target cells in the cell container 134 before, during, or after a genetic modification process (e.g., transduction or gene transfer) by adding a delabeling reagent from the incubation component tank 114 to the cell container 134.

[0185] A second magnetic separation device 105, connected to a cell container 134 in the cell incubation chamber 106 through a fluid line 126, is used to remove free magnetic label removed from the genetically modified target cells in a solution and / or medium mixture in the cell container 134, which may include a buffer for eluting the genetically modified target cells from the first magnetic separation device 104, one or more reagents for transduction or gene transfer, and a reagent for delabeling the target cells 132. The second magnetic separation device 105 retains free magnetic label as the solution and / or medium mixture containing the delabeled genetically modified target cells flows through the second magnetic separation device 105 and into the inlet of the acoustic separation device 302 through a fluid line 604. The second magnetic separation device 105 may have the same or substantially similar structure as the magnetic separation device 188, the cross-sectional views of which are shown in FIGS. 3 and 4 and described above.

[0186] In an alternative scheme, the delabeled target cells may flow from the second magnetic separation device 105 into a container or bag before further flowing to the inlet of the acoustic separation device 302. The container or bag may act as a reservoir for temporarily storing fluid therein to allow the downstream second magnetic separation device 105 and the upstream acoustic separation device 302 to operate at different flow rates as needed.

[0187] A fifth container or bag 602, connected to the inlet of the second magnetic separation device 105 through fluid line 606, contains a buffer or solution for eluting free magnetic labels remaining in the second magnetic separation device 105 after the passage of the fluid sample. The buffer or solution in the fifth container or bag 602 may contain a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0188] Acoustic separation device 302, connected to the outlet of second magnetic separation device 105 through fluid line 604, is used to extract genetically modified target cells from the mixture of solution and / or medium discharged from second magnetic separation device 105. Acoustic separation device 302 may have the same or substantially similar structure as acoustic separation device 428 shown in Figures 8A-8E.

[0189] A third container or bag 116, connected to the inlet of acoustic separation device 302 through fluid line 306, contains a buffer or solution that can act as a sheath fluid during acoustic separation of the genetically modified target cells in acoustic separation device 302. The buffer or solution can also be used as a storage fluid for storing the genetically modified target cells in the fourth container for bag 110. The buffer or solution in third container or bag 116 can contain a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof.

[0190] A fourth container or bag 110, connected to the outlet of the acoustic isolation device 302 through a fluid line 308, contains the genetically modified target cells in a buffer or solution that can be administered intravenously to a patient.

[0191] The network of fluid lines 118-126, 306, 308, 604, 606, the fluid lines passing through the containers or bags 102, 110, 112, 116, 602, the cell container 134 and the first and second magnetic separation devices 104, 105 and the acoustic separation device 302 may be constructed, interconnected and supplied as an integrated disposable tubing set that may be sterilized and sealed from the surrounding environment.

[0192] Continuing with reference to FIG. 15, the process begins by providing a first container or bag 102 containing a first fluid sample containing magnetically labeled target cells 132. In the case of a previously frozen sample, the first fluid sample may be prepared by first thawing the frozen sample and then extracting the target cells and other biological matter, if any, from the thawed sample fluid by centrifugation. The resulting target cells and other biological matter, if any, are resuspended in a buffer solution and filtered using a mesh size between 15 and 100 μm. A reagent containing a magnetic label is added to the filtered buffer solution containing the target cells, thereby forming the first fluid sample. Alternatively, the target cells may be magnetically labeled through an indirect process by first adding a reagent containing an intermediate link attached to the target cells before adding a reagent containing a magnetic label attached to the intermediate link.

[0193] For the magnetic separation process, a first fluid sample is introduced into the inlet of the magnetic separation device 104 via fluid line 118. As the first fluid sample flows through the magnetic separation device 104, the magnetically labeled target cells 132 are retained in the magnetic separation device 104 by the magnetic field, while the removed first fluid sample exits the magnetic separation device 104 into a waste container or bag (not shown). After the first fluid sample has completely passed through the magnetic separation device 104, the magnetic field acting on the magnetically labeled target cells 132 is reduced or eliminated, and a first eluent, such as a buffer solution in a second container or bag 112, flows into the magnetic separation device 104 via fluid line 120 and elutes the magnetically labeled target cells 132 into a cell container 134 via fluid line 122. Additionally, one or more solutions from incubation component tank 114, such as, but not limited to, culture medium, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer, are injected into cell container 134 through fluid line 124 to modify the genetic makeup of magnetically labeled target cells 132. Magnetically labeled target cells 132 (e.g., T cells) can be transduced to express a CAR by contacting the target cells with vectors bearing a CAR construct, such as viral vectors, e.g., lentiviruses and retroviruses, and non-viral vectors, e.g., plasmids, lipid nanoparticles, and mRNA.

[0194] The transduction or gene transfer step is performed in the cell incubation chamber 106 while the magnetically labeled target cells 132 are immersed in a transduction or gene transfer medium containing one or more solutions from the first eluent and incubation component tank 114. The temperature and gas composition within the cell incubation chamber 106 can be controlled during the transduction or gene transfer step. For example, the temperature and gas composition within the cell incubation chamber 106 can be maintained at 37.0±0.1°C and 5.0%±0.1% CO2, respectively, for a period ranging from 10 minutes to 72 hours while the genetic makeup of the magnetically labeled target cells 132 is modified in the cell container 134. To remove the magnetic label from the target cells after the transduction or gene transfer step is completed, reagents can be injected from the incubation component tank 114 into the cell container 134. Alternatively, this delabeling step can be performed before or during the transduction or gene transfer step.

[0195] After the genetic modification and delabeling steps of the target cells are completed, a buffer to facilitate the subsequent acoustic sorting step can be added from the incubation component tank 114 to the transduction or gene transfer medium and delabeling reagent in the cell container 134. The mixture of the transduction or gene transfer medium, the delabeling reagent, and the buffer to facilitate the acoustic sorting step, if any, forms a second fluid sample containing the genetically modified target cells without their magnetic labels.

[0196] The second fluid sample is then flowed through fluid line 126 into the inlet of second magnetic separation device 105, where the free magnetic labels are extracted by a second magnetic sorting step. As the second fluid sample flows through second magnetic separation device 105, the free magnetic labels, including those removed from the genetically modified target cells, are retained in second magnetic separation device 105 by the magnetic field, while the genetically modified target cells carried by the removed second fluid sample (i.e., without free magnetic labels) exit second magnetic separation device 105 through fluid line 604 to acoustic separation device 302.

[0197] 8A-8E as an example, a cleared second fluid sample containing genetically-modified target cells 432 is introduced into the side inlet port 412 via fluid line 604, while buffer solution is introduced into the central inlet port 416 from the third container or bag 116 via fluid line 306. The cleared second fluid sample containing the genetically-modified target cells 432 and, if present, non-target biological matter / debris 434 is introduced into the separation channel 410 at or near its upstream end via the pair of side inlet channels 414 as two laminar flows 436 and 438 flowing adjacent to the side walls. The two laminar flows 436 and 438 of the cleared second fluid sample in the separation channel 410 are separated by a central flow 440 of buffer solution from the central inlet port 416. The central flow 440 of buffer solution behaves like a laminar flow and may act as a sheath fluid to slow or prevent the movement of non-target biological matter / debris 434 toward the pressure node located along the center of the separation channel 410. As the removed second fluid sample proceeds downstream in the separation channel 410, acoustic radiation pressure pushes the genetically modified target cells 432 into the central flow 440 toward the pressure node located along the center of the separation channel 410, while the non-target biological matter / debris 434 remains mostly in the two laminar flows 436 and 438 near the side walls. At the downstream end of the separation channel 410, the genetically modified target cells 432, carried by the buffer solution, exit the acoustic separation device 428 through the central exit port 424 via fluid line 308 and toward the fourth container or bag 110. The second fluid sample after further removal may contain non-target biological matter / debris 434 and flows near the side walls as laminar flows 436 and 438 and is diverted to the side outlet port 420 through a pair of side outlet channels 422.

[0198] After the acoustic sorting process is complete, the fourth container or bag 110 containing the genetically modified target cells can be disconnected from the rest of the integrated system 600 without exposing its contents to the atmosphere. The contents in the fourth container or bag 110 can be administered intravenously to a patient.

[0199] 9 is a front view of an integrated system 600 that can be used to generate CAR T cells from whole blood or T-cell-containing samples according to the steps described above. Utilizing a closed fluidic assembly, system 600 can be a closed processing system that can be deployed in facilities that lack strict sterile or clean environments, such as medical treatment facilities, thereby alleviating many of the common logistical issues encountered in autologous tissue procedures.

[0200] 9 shows an automated production system 600 including a shell or housing structure 140, first and second magnetic separation modules 142A, 142B and acoustic separation module 310 present in housing structure 140, an incubation module 144, a sample rack 146 mounted on top of housing structure 140, and first and second sample bag holders 164, 166. Fluid lines and connected sample bags, which may be supplied as an integrated disposable tubing set, and a computer used to control system 600 are omitted from this view to present an unobstructive view of magnetic separation modules 142A, 142B and acoustic separation module 310. Production system 600 is similar to systems 300 and 500, except for the routing of the fluid lines, as described below.

[0201] Incubation module 144 includes an incubation chamber 106 located on top of a rocker base 147. Each of magnetic separation modules 142A, 142B includes a first pinch valve 148A, 148B, a second pinch valve 150A, 150B, a third pinch valve 152A, 152B, a fourth pinch valve 154A, 154B, a first air detector 156A, 156B, a second air detector 158A, 158B, a peristaltic pump 160A, 160B, an occlusion sensor 162A, 162B, and a magnetic separation device 104, 105. The acoustic isolation module 310 includes first and second peristaltic pumps 312, 314, first and second occlusion sensors 316, 318, first and second air detectors 320, 322, an acoustic isolation device 302, and first, second, and third pinch valves 324-328.

[0202] System 600 may also include additional pinch valves 168, 170, 608 disposed on one side of housing structure 140 and additional pinch valves 172, 174 disposed on the other side of housing structure 140. Electromechanical components 104-106, 147, 148A / B-162A / B, 168-174, 312-328, 608 may be controlled or automated by a computer or microprocessor (not shown).

[0203] Each of the magnetic separation devices 104, 105 may have the same or substantially similar structure as the magnetic separation device 188 shown in Figures 3 and 4 and described above. Other magnetic flux sources and column-free magnetic separation devices, such as those disclosed in U.S. Patent Application No. 18 / 072,362, which is incorporated herein by reference, may also be used to separate magnetically labeled target cells from a fluid sample.

[0204] Acoustic isolator 302 may have the same or substantially similar structure as acoustic isolator 428 shown in Figures 8A-8E and described above.

[0205] The incubation module 144 includes a cell incubation chamber 106 located on a rocker base 147. The incubation chamber 106 has a heater that can maintain a uniform temperature within the chamber 106 between room temperature and 50°C. The CO2 concentration within the chamber 106 can be varied between 0 and 10% to provide an optimal environment for the cells. The rocker base 147 can move the chamber 106 at a speed of 2 to 4 rpm. The rocking motion of the chamber 106 caused by the rocker base 147 can facilitate uniform mixing of the fluid contents within the cell container 134. The rocker base 147 can remain stationary during cell activation, transduction, or expansion steps. After the incubation step, the rocker base 147 rocks the cell container 134 within the chamber 106, allowing the fluid in the cell container 134 to wash the cells off the surface of the cell container 134. The rocker base 147 can also be used to facilitate the draining of the cell container 134 by tilting the incubation chamber 106 to a fixed position to allow the end of the outlet tubing to reach the bottom of the fluid in the cell container 134.

[0206] System 600 has a modular design that includes two magnetic separation modules 142A, 142B, one acoustic separation module 310, and one incubation module 144. Modules 142A, 142B, 144, and 310 can be fluidly connected in series, parallel, or a combination thereof by a closed fluidic assembly that includes sample bags, cell containers, acoustic separation devices, and all fluid lines, including conduits that pass through the magnetic separation devices.

[0207] FIG. 16 is a perspective view of a system 600 for manufacturing CAR T cells that uses a closed fluidic assembly in the form of an integrated tubing set. For reasons of readability, the electromechanical components shown in FIG. 9 are not shown in this drawing to clearly show the routing of the fluid lines. The perspective view also shows pinch valves 152B, 154A, 154B, 328, which were previously obstructed by the cell incubation chamber 106 in the front view of FIG. 9, as well as pinch valves 172, 174 located on one side of the housing structure 140. The lid and a portion of the sidewall of the incubation chamber 106 are further removed to expose a cell container 134 in the form of a common cell culture flask within the chamber 106. The cap of the cell culture flask 134 contains an air filter that vents to the environment within the incubation chamber 106 while maintaining sterility within the flask 134. The second bag holder 166 is also omitted from the drawing to show the sample bags 110, 224 within the holder 166. FIG. 17 is a fluid circuit diagram corresponding to the system 600 shown in FIG.

[0208] 9, 16, and 17, in contrast to the fluidic assemblies of systems 100, 300, and 500, the fluidic assembly of system 600 is not configured to use both first and second magnetic separation devices 104, 105 to extract magnetically labeled non-target cells from a first fluid sample in a magnetic sorting process. The first magnetic separation device 104 is used to extract magnetically labeled target cells from the first fluid sample, while the second magnetic separation device 105 is used to remove free magnetic label after the magnetic label has been removed from the target cells. The acoustic separation device 302 is then used to purify the non-magnetic target cells.

[0209] The first, second, third, and fourth containers or bags 102, 112, 116, 110 are in the form of sample bags. Both sample bags 102 and 112 are fluidly connected to the inlet of the first magnetic separation device 104. In addition to sample bag 116, another sample bag 220, which may contain cryopreservation solution, storage medium, PBS-based solution, saline-based solution, human serum, glucose, or any combination thereof, is also fluidly connected to the inlet of the acoustic separation device 302. The incubation component bank 114 includes at least three sample bags 114A-114C that are fluidly connected to the cell container 134 through the magnetic separation device 104. Each of the sample bags 114A, 114B, 114C may contain culture medium, a vector for cell transduction or gene transfer, a lentivirus for transduction, a buffer or solution for non-viral gene transfer, a cell activation reagent, or a reagent for removing magnetic labels from target cells (i.e., a delabeling reagent). Prior to the transduction or gene transfer step, the target T cells in cell container 134 may be activated by flowing a cell activation reagent (e.g., a solution containing CD3 / CD28 magnetic beads) into cell container 134 from one of sample bags 114A-114C.

[0210] The fluidic assembly of system 600 further includes a sample bag 222 fluidly connected to the outlets of the first and second magnetic separation devices 104, 105 to collect waste from the first and second magnetic separation devices 104, 105, and another sample bag 224 fluidly connected to the outlet of acoustic separation device 302 to collect waste from acoustic separation device 302. The sample bag 222 for collecting waste from the first and second magnetic separation devices 104, 105 can be placed in the first bag holder 164. The sample bags 110, 224 for collecting the final cell product and waste, respectively, from acoustic separation device 302 can be placed in the second bag holder 166. All other sample bags 102, 112, 114A-114C, 116, 220 providing materials for the manufacturing process can be suspended in the sample rack 146. The fluidic assembly of system 600 can include additional sample bags for various purposes. Each of the sample bags 102, 110, 112, 114A-114C, 116, 220-224 may have an inlet port connected to a Luer connector and an outlet port connected to a drip chamber. Additionally, fluid flow through the inlet and outlet ports of each sample bag may be controlled or regulated by one or more manual pinch clamps attached thereto.

[0211] The fluidic assembly of system 600 may also include syringes 226, 228, 230. Syringes 226, 228, which may be fluidly connected to the inlet of cell container 134, may be used to extract a sample of magnetically separated target cells after the magnetic sorting step, or may inject culture medium, vectors for cell transduction or gene transfer, lentivirus for transduction, buffers or solutions for non-viral gene transfer, cell activation reagents, or delabeling reagents into cell container 134. Syringe 230, which may be fluidly connected to the outlet of cell container 134 or the outlet of second magnetic separation device 105, may be used to extract a sample of genetically modified target cells after the transduction or gene transfer step is completed.

[0212] Continuing to refer to FIG. 17, the fluid circuit corresponding to system 600 includes sample bags 102, 110, 112, 114A-114C, 116, 220-224, syringes 226-230, cell container 134, and acoustic separator 302, all interconnected by a network of fluid lines passing through electromechanical components such as pinch valves 148A / B-154A / B, 168-172, 324-328, air detectors 156A / B, 158A / B, 320, 322, occlusion sensors 162A, 162B, 316, 318, peristaltic pumps 160A, 160B, 312, 314, and magnetic separators 104, 105. The network of fluid lines, including the conduits passing through the sample bags 102, 110, 112, 114A-114C, 116, 220-224, syringes 226-230, cell container 134, acoustic separator 302, and magnetic separators 104, 105, can be constructed, interconnected, sterilized, and supplied as an integrated disposable set. The electromechanical components of system 600 are external to the tubing set and therefore do not come into contact with the fluids in the tubing set.

[0213] A first container or sample bag 102, which may contain target cells for processing, is fluidly connected by one or more fluid lines passing through a first pinch valve 148A, a first air detector 156A, a second air detector 158A, a peristaltic pump 160A, and an occlusion sensor 162A to an inlet of a conduit passing through the first magnetic separation device 104. A second container or sample bag 112, which may contain a buffer solution or culture medium, is fluidly connected by one or more fluid lines passing through a second pinch valve 150A, a second air detector 158A, a peristaltic pump 160A, and an occlusion sensor 162A to an inlet of a conduit passing through the second magnetic separation device 105, and also fluidly connected by one or more fluid lines passing through a second pinch valve 150B, a second air detector 158B, a peristaltic pump 160B, and an occlusion sensor 162B. In this arrangement shown, the first and second magnetic separation devices 104, 105 use the same buffer or culture medium solution stored in the second container or sample bag 112 instead of separate containers or sample bags 112, 602 as shown in Figure 15. However, the separate container or sample bag 602 may be fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 for eluting the free magnetic labels collected by the second magnetic separation device 105.

[0214] The inlet of the cell container 134 is fluidly connected to the outlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through the third pinch valve 152A. Syringes 226, 228 may be fluidly connected to the fluid line between the inlet of the cell container 134 and the third pinch valve 152A. A sample bag 222, which may store waste liquid containing non-target cells and / or free magnetic labels, is fluidly connected to the outlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines passing through the fourth pinch valve 154A, and is also fluidly connected to the outlet of the conduit passing through the second magnetic separation device 105 by one or more fluid lines passing through the fourth pinch valve 154B.

[0215] The outlet of the cell container 134 is fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 by one or more fluid lines passing through a first pinch valve 148B, a first air detector 156B, a second air detector 158B, a peristaltic pump 160B and an occlusion sensor 162B.

[0216] The outlet of the conduit passing through the second magnetic separation device 105 is fluidly connected to the inlet of the acoustic separation device 302 (e.g., side inlet port 412 in FIG. 8A ) by one or more fluid lines passing through the third pinch valve 152B, the first peristaltic pump 312, the first occlusion sensor 316, and the first air detector 320. The syringe 230 is fluidly connected to a fluid line connected to the outlet of the cell container 134 or the outlet of the conduit passing through the second magnetic separation device 105, as shown. The third container or sample bag 116 is fluidly connected to the inlet of the acoustic separation device 302 (e.g., central inlet port 416 in FIG. 8A ) by one or more fluid lines passing through the pinch valve 172, the second peristaltic pump 314, the second occlusion sensor 318, and the first pinch valve 324. The sample bag 220 is fluidly connected to the inlet of the acoustic isolation device 302 (e.g., central inlet port 416 in FIG. 8A ) by one or more fluid lines that pass through the pinch valve 174, the second peristaltic pump 314, the second occlusion sensor 318, and the first pinch valve 324.

[0217] A fourth container or sample bag 110 is fluidly connected to the outlet of the acoustic isolation device 302 (e.g., central outlet port 424 in FIG. 8A ) by one or more fluid lines that pass through a second pinch valve 326. A sample bag 224, which may be used to receive waste fluid, is fluidly connected to the outlet of the acoustic isolation device 302 (e.g., side outlet port 420 in FIG. 8A ) by one or more fluid lines that pass through a second air detector 322 and a third pinch valve 328.

[0218] Sample bag 114A of incubation component bank 114 is fluidly connected to the inlet of cell container 134 by one or more fluid lines that pass through pinch valve 168, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104, and third pinch valve 152A. Sample bag 114B of incubation component bank 114 is fluidly connected to the inlet of cell container 134 by one or more fluid lines that pass through pinch valve 170, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104, and third pinch valve 152A. Similarly, sample bag 114C of incubation component bank 114 is fluidly connected to the inlet of cell container 134 by one or more fluid lines that pass through pinch valve 608, second pinch valve 150A, second air detector 158A, peristaltic pump 160A, occlusion sensor 162A, first magnetic separation device 104 and third pinch valve 152A.

[0219] As will be appreciated by those skilled in the art, the steps described in embodiments 1-4 may be performed using other systems or equipment not described herein. For example, the initial magnetic isolation / extraction and / or final magnetic collection / purification of T cells may be performed using a column-based magnetic separation device; or the magnetic separation device, acoustic separation device, and cell container may not be fluidly connected or integrated into a system (i.e., separate devices).

[0220] Example

[0221] The following examples are offered to illustrate, but not to limit, the present invention.

[0222] The target cell recovery reported herein, when measured by a flow cytometer (CytoFlex, Beckman Coulter), is calculated from the number of events or cells for target cells in the fluid sample after magnetic or acoustic sorting divided by the number of events or cells for target cells in the initial fluid sample before any sorting. The target cell purity reported herein, when measured by a flow cytometer, is calculated from the number of events or cells for target cells divided by the number of all events or cells in the fluid sample.

[0223] Example 1: CAR T cells generated from fresh leukopacks using positive magnetic isolation and purification

[0224] This embodiment uses a system 100 with a closed fluidic assembly as shown in Figures 2, 5, and 6 and described above. System 100 includes three magnetic modules 142A-142C and an incubation module 144. The closed fluidic assembly includes sample bags 102, 110, 112, 114A, 114B, 116, 220-224, cell containers 134 in the form of cell culture flasks, conduits passing through magnetic separation devices 104, 105, 108, and fluid lines connecting these components.

[0225] The sample preparation process involves approximately 6 × 10 8 The leukopheresis sample, consisting of approximately 1 / 10 leukopak containing 3 × 10 cells, was incubated with CD4 / CD8 magnetic beads (Beijing T&L Biological Technology) to magnetically label T cells for 30 minutes at room temperature while rocking on a mixer. After incubation, the leukopheresis sample was diluted with buffer (MARS® MAG buffer, Applied Cells) to approximately 3 × 10 cells. 7The T cells are diluted to a cell concentration of 100 / ml and a total volume of about 20 ml to obtain a first fluid sample, which is then transferred to a first sample bag 102. It is worth noting that the magnetic separation device shown in Figures 3 and 4 can also be used to directly extract or isolate T cells from whole blood without leukopheresis or buffy coat extraction, as reported in U.S. Patent Application No. 18 / 795,047, the entire contents of which are incorporated herein by reference.

[0226] Isolation or extraction of T cells from a first fluid sample begins by flowing the first fluid sample from a first sample bag 102 through a pair of magnetic separation devices 104, 105 at a total flow rate of 1 ml / min. As the first fluid sample flows through the magnetic separation devices 104, 105, the magnetically labeled T cells are retained in the devices 104, 105 by the magnetic field, while the depleted first fluid sample exits the devices 104, 105 and is collected by the sample bag 222. After the first fluid sample has completely passed through the magnetic separation devices 104, 105, the magnetic field acting on the magnetically labeled T cells is removed and a first eluent comprising T cell culture medium (OptiVitro, ExcellBio) containing IL-2 (300 IU / ml), IL-7 (10 ng / ml) and IL-15 (10 ng / ml) (TL) is flowed from the second sample bag 112 through the magnetic separation devices 104, 105 to elute the magnetically labeled T cells into a cell culture flask 134 located inside the cell incubation chamber 106.

[0227] Figure 18A shows a dot plot of a first fluid sample comprising the initial leukopheresis sample prior to the magnetic isolation step, and the culture medium containing T cells extracted from the magnetic isolation step. The cytometry data indicate that the magnetic isolation step increases the purity of T cells as a percentage of total white blood cells from 60% to 96%, with 59% of the T cells recovered from the magnetic isolation step (i.e., recovery rate). The cytometry data in Figure 18B show that the CD4 + / CD8 + It is further shown that the ratio remains relatively constant after the magnetic isolation step.

[0228] After the magnetic isolation step, the process of activating the T cells in the cell culture flask 134 is initiated by flowing an activation reagent containing ActCel CD3 / CD28 magnetic beads (Beijing T&L Biological Technology) diluted in T cell culture medium from the sample bag 114A into the cell culture flask 134. After adding the activation reagent to the cell culture flask 134, the cell incubation chamber 106 is rocked on the rocker base 147 to uniformly mix the fluid mixture in the cell culture flask 134. The T cells are then incubated in the cell culture flask 134 with the CD3 / CD28 magnetic beads in the cell incubation chamber 106 for 24 hours at 37°C in a 5% CO environment.

[0229] Figure 19 is a dot plot showing the expression of activation markers CD25 and CD69 on T cells incubated with CD3 / CD28 magnetic beads for 24 hours. The plot shows that 80%, 74%, and 69% of T cells express the CD25, CD69, and CD25+CD69 (double positive) activation markers, respectively.

[0230] After the activation step, the process of transducing T cells in cell culture flask 134 is initiated by flowing a lentivirus solution containing CD19 CAR-T lentivirus (Jiangsu Hillgene BioPharma Co., Ltd.) suspended in T cell culture medium from sample bag 114B into cell culture flask 134 at a multiplicity of infection (MOI) of 5. After adding the lentivirus solution to cell culture flask 134, cell incubation chamber 106 is rocked on rocker base 147 to uniformly mix the fluid mixture in cell culture flask 134. T cells in cell culture flask 134 are then incubated with the lentivirus in cell incubation chamber 106 for 24 hours at 37°C in a 5% CO environment.

[0231] After the transduction step, the T cell collection / purification step is initiated by flowing a second fluid sample in cell culture flask 134 containing T cells and residual lentivirus through magnetic separation device 108 at a flow rate of 0.5 ml / min. As the second fluid sample flows through magnetic separation device 108, the magnetically labeled T cells are retained in device 108 by the magnetic field, while the depleted second fluid sample containing residual lentivirus exits device 108 and is collected by sample bag 224. After the second fluid sample has completely passed through magnetic separation device 108, the magnetic field acting on the magnetically labeled T cells is removed, and a second eluent containing a buffer (MARS® MAG buffer, Applied Cells) flows from third sample bag 116 into magnetic separation device 108, eluting the T cells into fourth sample bag 110 for subsequent analysis, including cytometry. The magnetic collection / purification step recovers 91% of the T cells from the second fluid sample after the activation and transduction steps. Alternatively, the cryopreservation fluid stored in the sample bag 220 may be used as a second eluent to preserve the T cells in the fourth sample bag 110 for cryopreservation.

[0232] Figures 20A and 20B are dot plots corresponding to the final T cell product after the transduction and magnetic collection / purification steps. Figure 20A shows that 65% of the T cells express the CAR. Furthermore, Figure 20B shows that 88% of the CAR T cells express the CCR7 marker (memory T cells), and more specifically, 54% of the CAR T cells are T stem cell memory (TSCM) cells. The percentage of TSCM cells generated by this method is significantly higher than traditional production methods that require T cell expansion.

[0233] While the present invention has been shown and described with reference to certain preferred embodiments, it is to be understood that those skilled in the art will no doubt devise certain changes and modifications thereto which will nevertheless encompass the true spirit and scope of the invention. Accordingly, the scope of the present invention should be determined not by the examples given, but by the appended claims and their legal equivalents.

[0234] Any claim element that does not expressly recite a "means" for performing a particular function or a "step" for performing a particular function, as identified in 35 U.S.C. § 112, ¶ 6, should not be construed as a "means" or "step" clause. In particular, the use of "step" in the claims herein is not intended to invoke the provisions of 35 U.S.C. § 112, ¶ 6.

Claims

1. 1. A method for generating a population of T cells expressing a chimeric antigen receptor (CAR), comprising: providing a fluid sample containing a population of T cells; labeling the population of T cells with magnetic beads; extracting a population of T cells from the fluid sample by passing the fluid sample through a magnetic separation device; transducing a population of T cells to express a chimeric antigen receptor (CAR) by contacting the population of T cells with a vector carrying a CAR construct; and collecting the population of T cells by passing the solution through a magnetic separator or another magnetic separator; A method comprising:

2. 10. The method of claim 1, further comprising activating the population of T cells prior to transducing the population of T cells.

3. 3. The method of claim 2, wherein the step of activating the population of T cells is carried out by contacting the population of T cells with CD3 / CD28 magnetic beads.

4. The method of claim 1 , wherein the vector is a lentivirus.

5. The method of claim 1 , wherein the vector is a retrovirus.

6. The method of claim 1 , wherein the fluid sample comprises whole blood.

7. The method of claim 1 , wherein the fluid sample comprises a leukopheresis product.

8. 1. A method for generating a population of T cells expressing a chimeric antigen receptor (CAR), comprising: providing a fluid sample containing a population of T cells; labeling the population of T cells with magnetic beads; extracting a population of T cells from the fluid sample by flowing the fluid sample through a first conduit that passes through a first magnetic separation device; transducing the population of T cells to express a chimeric antigen receptor (CAR) by contacting the population of T cells with a population of lentivirus in a solution contained in an incubation vessel; and collecting the population of T cells by flowing the solution through a second conduit that passes through a second magnetic separation device. Including, The method, wherein the first conduit, the incubation vessel and the second conduit are fluidly connected by a network of fluid lines.

9. 9. The method of claim 8, wherein the first conduit, the incubation vessel, the second conduit and the network of fluid lines are components of a closed fluid system.

10. 9. The method of claim 8, further comprising activating the population of T cells prior to transducing the population of T cells.

11. The method of claim 8 , wherein the fluid sample comprises whole blood.

12. The first and second magnetic separation devices each include: a permanent magnet having first and second poles; a central flux guide including a central tip portion having a tapered shape and a central base portion magnetically coupled to the first pole; a bottom flux guide magnetically coupled to the second pole; a first side flux guide including a first side base magnetically coupled to the second pole through a first side tip and a bottom flux guide; a second side flux guide including a second side base magnetically coupled to the second pole through a second side tip and bottom flux guide; Including, 9. The method of claim 8, wherein the first and second side flux guides are positioned on opposite sides of the central flux guide with the first and second side apices bent toward the central apice.

13. 13. The method of claim 12, wherein the first and second magnetic separation devices each further comprise a floating magnetic flux guide that presses the first or second conduit against the central tip and the first and second lateral tips.

14. 1. A method for generating a population of T cells expressing a chimeric antigen receptor (CAR), comprising: providing a fluid sample containing a population of T cells; labeling the population of T cells with magnetic beads; extracting a population of T cells from the fluid sample by flowing the fluid sample through a conduit that passes through a magnetic separation device; transducing the population of T cells to express a chimeric antigen receptor (CAR) by contacting the population of T cells with a population of lentivirus in a solution contained in an incubation vessel; collecting the population of T cells by flowing the solution through an acoustic separator; Including, The method wherein the conduit, the incubation vessel and the acoustic separation device are fluidly connected by a network of fluid lines.

15. 15. The method of claim 14, further comprising activating the population of T cells prior to transducing the population of T cells.

16. 15. The method of claim 14, wherein the conduit, incubation vessel, acoustic separator and network of fluid lines are components of a closed fluid system.

17. The acoustic isolation device a planar substrate having first and second planar surfaces; a network of channels recessed from the first planar surface; a lid attached to the first planar surface and covering the network of channels; one or more piezoelectric transducers attached to an outer surface of the lid opposite the inner surface of the lid facing the network of channels; 15. The method of claim 14, comprising:

18. The magnetic separation device a permanent magnet having first and second poles; a central flux guide including a central tip portion having a tapered shape and a central base portion magnetically coupled to the first pole; a bottom flux guide magnetically coupled to the second pole; a first side flux guide including a first side base magnetically coupled to the second pole through a first side tip and a bottom flux guide; a second side flux guide including a second side base magnetically coupled to the second pole through a second side tip and bottom flux guide; Including, 15. The method of claim 14, wherein the first and second side flux guides are positioned on opposite sides of the central flux guide with the first and second side apices bent toward the central apice.

19. 20. The method of claim 18, wherein the magnetic separation device further comprises floating magnetic flux guides that press the conduit against the central tip and the first and second side tips.

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