Semi-automated hollow fiber system for viral transduction

Abstract

The system for introducing vectors includes a filter module defining an intracapillary space and an extracapillary space separated from the intracapillary space by a porous membrane. The system also includes a pair of intracapillary ports fluidly coupled to opposite ends of the intracapillary space, each port receiving a transduction medium, cells, and vectors. The system also includes a pair of extracapillary ports fluidly coupled to opposite ends of the extracapillary space, each port fluidly coupled to a source of extracapillary medium and a waste container.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 037,377, filed June 10, 2020. The disclosure of that prior application is considered part of the disclosure of this application and is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to semi-automated methods and systems for viral transduction using hollow fiber filter modules. [Background technology]

[0003] Cell therapy exploits the natural transduction process, using viral particles modified for safety and functionality as delivery vehicles (vectors) to introduce therapeutic genes into a patient's cells. Viral vector transduction is currently the most frequently used method in the manufacture of cell therapies to introduce therapeutic genetic material.

[0004] Current manufacturing transduction processes are labor-intensive and inefficient in their use of viral vectors, contributing to the high cost of manufacturing cell therapies and the long time required to produce these therapies. Thus, the current state-of-the-art in manufacturing transduction processes has significant limitations. Summary of the Invention [Means for solving the problem]

[0005] One aspect of the present disclosure provides a system for introducing vectors into cells. The system includes a filter module defining an intracapillary space and an extracapillary space separated from the intracapillary space by a porous membrane. The system also includes a pair of intracapillary ports fluidly coupled to opposite ends of the intracapillary space, each port receiving a transduction medium, cells, and vectors. The system also includes a pair of extracapillary ports fluidly coupled to opposite ends of the extracapillary space, each port fluidly coupled to a source of extracapillary medium and a waste container.

[0006] This aspect of the present disclosure may include one or more of the following optional features: In some examples, the system includes a collection vessel in fluid communication with at least one of the intracapillary ports. In some implementations, the system includes an intracapillary pump operable to supply respective flows of transduction medium, cells, and vectors to at least one of the intracapillary ports. Optionally, the intracapillary pump is operable in a first state to supply the cells and vectors to the intracapillary port during a first time period and in a second state to supply the transduction medium to the intracapillary port during a second time period.

[0007] In some examples, the system includes a waste container in communication with the extracapillary space via at least one extracapillary port. In some implementations, the system includes an extracapillary pump operable to supply a flow of extracapillary medium to each of the extracapillary ports. In some configurations, the system includes an extracapillary pump operable to supply a flow of waste fluid from the extracapillary ports to the waste container.

[0008] In some implementations, the porous membrane is cylindrical. In some instances, the porous membrane comprises pores that allow particles having a size less than about 50 kDa to pass through the pores from the intracapillary space. In some configurations, the intracapillary space defines a transduction zone.

[0009] Another aspect of the present disclosure provides a system for introducing a viral or non-viral vector into a cell. The system includes a hollow fiber defining an intracapillary space extending from a first end to a second end. The system also includes a casing surrounding one or more hollow fibers from the first end to the second end to define an extracapillary space between the hollow fiber and the casing, the casing including a first port in fluid communication with the intracapillary space adjacent the first end and a second port in fluid communication with the intracapillary space adjacent the second end. The system also includes a transduction medium source in fluid communication with the intracapillary space via each of the first and second ports. The system further includes a cell source containing cells and in fluid communication with the intracapillary space via each of the first and second ports. The system also includes a virus source containing a viral or non-viral vector and in fluid communication with the intracapillary space via each of the first and second ports.

[0010] This aspect of the present disclosure may include any one or more of the following features: In some examples, the system includes a collection vessel in fluid communication with the intracapillary space via at least one of a first port and a second port; In some implementations, the system includes an intracapillary pump including an inlet in fluid communication with each of a transduction medium source, a cell source, and a virus source; In some examples, the intracapillary pump includes a first outlet in fluid communication with the intracapillary space via the first port and a second outlet in fluid communication with the intracapillary space via the second port.

[0011] In some configurations, the casing includes a third port in communication with the extra-capillary space, and the system further includes a waste container in communication with the extra-capillary space via the third port. In some examples, the system includes an extra-capillary medium source in fluid communication with the extra-capillary space via the third port. In some configurations, the third port is disposed adjacent to a first end of the intra-capillary space, and the system further includes a fourth port in fluid communication with the extra-capillary space and disposed adjacent to a second end of the intra-capillary space. In some examples, the waste container and the extra-capillary medium source are each in communication with the extra-capillary space via the third port and the fourth port, respectively.

[0012] In some configurations, the hollow fiber comprises a plurality of hollow fibers. In some implementations, the hollow fiber comprises pores that allow particles having a size less than about 50 kDa to pass through the pores from the intracapillary space.

[0013] Yet another aspect of the present disclosure provides a method for introducing a viral or non-viral vector into cells using a hollow fiber defining an intracapillary space extending from a first end to a second end and an extracapillary space surrounding the intracapillary space from the first end to the second end, the method including loading the viral or non-viral vector into the intracapillary space of the hollow fiber and loading cells into the intracapillary space of the hollow fiber.

[0014] This aspect of the present disclosure may include any one or more of the following features: In some examples, loading the intracapillary space with a viral or non-viral vector includes loading the viral or non-viral vector from at least one of a first end and a second end of the intracapillary space; In some implementations, loading the intracapillary space with a viral or non-viral vector includes loading the viral or non-viral vector from each of the first end and the second end of the intracapillary space; In some configurations, loading the intracapillary space with cells includes loading the cells from at least one of the first end and the second end of the intracapillary space.

[0015] In some examples, loading the intracapillary space with cells includes loading the cells from each of the first and second ends of the intracapillary space. Optionally, the method may further include transducing the cells within the intracapillary space of the hollow fiber and recovering the transduced cells from the intracapillary space of the hollow fiber. In some examples, recovering the transduced cells from the intracapillary space includes loading a flushing fluid into the extracapillary space of the hollow fiber. In some implementations, recovering the transduced cells from the intracapillary space includes loading a flushing fluid into the intracapillary space from one of the first end or the second end.

[0016] In some examples, the method includes collecting waste material from the extracapillary space. In some implementations, the cells and the viral or non-viral vector are loaded simultaneously. In some configurations, the cells and the viral or non-viral vector are loaded separately. In some implementations, the cells are loaded before the viral or non-viral vector. In some configurations, the viral or non-viral vector is loaded before the cells.

[0017] In some instances, the cells are 1 x 10 3 From 1×10 10The cells are loaded at a concentration ranging between 1000 and 1000 cells / ml. In some implementations, loading the cells includes loading the cells at a rate that is a function of the size of the interior surface area of ​​the hollow fiber. In some configurations, the viral or non-viral vector is loaded as a viral particle. In some examples, the viral or non-viral vector is loaded as a nucleic acid vector.

[0018] In some examples, the method comprises a flow rate of about 5-100 μl / min / cm per square centimeter of interior surface area of ​​the hollow fiber. 2 In some examples, the loading rate per square centimeter of inner surface area of ​​the hollow fiber is about 5-20 μl / min / cm. 2 In some embodiments, the vector is derived from a lentivirus, retrovirus, adenovirus, adeno-associated virus, or hybrid virus. In some examples, the vector is a retrovirus. In some embodiments, the vector is a lentivirus. In some examples, the vector comprises a nanoparticle, a liposome, a lipid particle, carbon, a non-reactive metal, gelatin, and / or a polyamine nanosphere.

[0019] In some implementations, the cells and viral vectors are loaded into the intracapillary space at a multiplicity of infection (MOI) ranging from about 0.25 to about 4.0. In some examples, the cells and viral vectors are loaded into the intracapillary space at an MOI of about 2.5. In some configurations, the cells are B cells, T cells, NK cells, monocytes, progenitor cells, or cell lines.

[0020] Another aspect of the disclosure provides a population of cells produced by the method according to the above paragraph. Another aspect of the disclosure provides a pharmaceutical composition comprising cells produced by the method according to the above paragraph.

[0021] Another aspect of the present disclosure provides a method of manufacturing a cell therapy product comprising one or more transduced cells, the method including: (i) providing a system comprising a hollow fiber defining an intracapillary space extending from a first end to a second end for transducing cells, (ii) loading the intracapillary space with a population of cells and a viral or non-viral vector to effect transduction of one or more cells in the intracapillary space, and (iii) recovering a population of cells comprising the one or more transduced cells from the intracapillary space.

[0022] This aspect of the disclosure may include any one or more of the following features: In some implementations, the population of cells is selected from αβ T cells, γδ T cells, NK cells, HSCs, macrophages, dendritic cells, and iPSCs. In some configurations, the viral or non-viral vector comprises a recombinant receptor. In some configurations, the recombinant receptor is a chimeric antigen receptor (CAR).

[0023] In some examples, the transduced cells comprise a recombinant receptor on the surface of the cell. In some implementations, the chimeric antigen receptor comprises an extracellular ligand-binding domain that targets a tumor antigen selected from one or more of CD44, CD19, CD20, CD22, CD23, CD30, CD89, CD123, CS-1, ROR1, mesothelin, c-Met, PSMA, Her2, GD-2, CEA, MAGE A3 TCR, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, CEA, and BCMA.

[0024] In some configurations, the method includes isolating the transduced cells. In some configurations, the method further includes expanding the recovered cells in a bioreactor. In some implementations, the method further includes cryopreserving the recovered cells in a suitable cryopreservation medium. In some implementations, the system includes a first port in fluid communication with the intracapillary space adjacent the first end and a second port in fluid communication with the intracapillary space adjacent the second end.

[0025] In some examples, loading the intracapillary space with a virus or a non-viral vector comprises loading the virus or a non-viral vector from at least one of a first end and a second end of the intracapillary space. In some implementations, loading the intracapillary space with a virus or a non-viral vector comprises loading the virus or a non-viral vector from each of the first end and the second end of the intracapillary space. In some configurations, loading the intracapillary space with cells comprises loading the cells from at least one of the first end and the second end of the intracapillary space. In some implementations, loading the intracapillary space with cells comprises loading the cells from each of the first end and the second end of the intracapillary space.

[0026] Various aspects of the present disclosure are described in detail in the following sections. The use of the sections is not intended to limit the disclosure. Each section may be applicable to any aspect of the present disclosure. In this application, the use of "or" means "and / or" unless stated otherwise. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. [Brief explanation of the drawings]

[0027] [Figure 1A] 1 illustrates a hollow fiber system including a hollow fiber according to the present disclosure. [Figure 1B] 1B shows a horizontal cross section of a hollow fiber along line 1B-1B in FIG. 1A, where the hollow fiber is loaded with cells and viral or non-viral vectors. [Figure 1C] 1C shows a vertical cross section of a hollow fiber along line 1C-1C in FIG. 1A, where the hollow fiber is loaded with cells and viral or non-viral vectors. [Figure 1D] 1 is a schematic diagram of an example hollow fiber filter module including a plurality of hollow fibers according to the present disclosure. [Figure 2A]1 shows a hollow fiber system with hollow fibers showing fluid flow direction during cell and viral vector loading. [Figure 2B] 1 shows a horizontal cross section of a hollow fiber showing the direction of fluid flow during cell and viral or non-viral vector loading. [Figure 2C] 1 shows a vertical cross section of a hollow fiber showing the direction of fluid flow during cell and viral or non-viral vector loading. [Figure 3A] 1 shows a hollow fiber system including hollow fibers that indicate fluid flow direction during the delivery of viral or non-viral vectors into target or host cells. [Figure 3B] 1 shows a horizontal cross section of a hollow fiber showing the direction of fluid flow during the transfer of viral or non-viral vectors into target or host cells. [Figure 3C] 1 shows a vertical cross section of a hollow fiber showing the direction of fluid flow during the transfer of viral or non-viral vectors into target or host cells. [Figure 4A] 1 shows a hollow fiber system including hollow fibers showing fluid flow direction during cell harvesting. [Figure 4B] 1 shows a horizontal cross section of a hollow fiber with cells and viruses showing the fluid flow direction during cell harvesting. [Figure 4C] 1 shows a vertical cross section of a hollow fiber showing the fluid flow direction during cell collection. [Figure 5] Retroviral-transduced T cells under different transduction conditions are shown. [Figure 6] Figure 1 shows the survival rate of T cells after transduction under different conditions. [Figure 7] Retroviral-transduced NK cells under different transduction conditions are shown. [Figure 8] 1 shows lentiviral-transduced T cells under different transduction conditions. [Figure 9] 1 shows the technical layout of a semi-automated hollow fiber system for cell therapy transduction. DETAILED DESCRIPTION OF THE INVENTION

[0028] Current state-of-the-art Transduction is the process by which viruses infect target or host cells. Viruses have naturally evolved to undergo the transduction process and transfer genetic material into target cells very efficiently. For transduction to occur, viral particles must come into physical contact with the target cell, first binding to and penetrating the target cell, and finally transferring genetic material into the target cell. Binding occurs through specific protein-protein interactions with the correct proteins required by both the virus and the target cell.

[0029] Cell therapy exploits the natural transduction process, using viral particles modified for safety and functionality as delivery vehicles (vectors) to introduce therapeutic genes into patient cells. Viral vector transduction is currently the most frequently used method in the manufacture of cell therapies to introduce therapeutic genetic material into cells.

[0030] Current industry approaches to viral transduction include static transduction systems, the use of chemical enhancers, and spinoculation, each of which is further described below.

[0031] Viral transduction under static conditions is the most common method by which viral transduction is currently performed. In standard static transduction methods, most transduction is performed in standard culture flasks or bags under static culture conditions. In this technique, viral vectors are suspended in medium that can be approximately 100–1000 times deeper than the diameter of a single cell. Transduction using standard static methods faces various issues that result in inefficient transduction of cells. For example, static methods result in small vector particles that remain in suspension and cannot reach target cells. This occurs, at least in part, because large cells quickly settle to the floor of the culture vessel. The net result of using static culture methods for transduction is that only a small fraction of vector particles can reach cells by diffusion alone. As a result, transduction efficiency is low, and large amounts of viral vector are required to achieve significant cell transduction. This is because viral vector binding to target cells depends on receptor / ligand expression and physical contact. Therefore, the transduction rate is proportional to the local concentration of virus for a given cell. The need for large quantities of viral vectors to achieve sufficient transduction rates is costly and can create inefficiencies throughout the cell therapy manufacturing process.

[0032] Another standard method for transducing cells involves the use of chemical enhancers, which result in increased binding rates of vectors to cells. However, the use of methods relying on chemical enhancers is also costly, and the removal of chemical enhancers creates additional hurdles in the manufacturing process.

[0033] Another standard method for cell transduction is the use of spinoculation. Spinoculation refers to the inoculation of cells using centrifugal force. Spinoculation reduces the volume occupied by the cells. This technique has been shown to have various negative aspects, such as cell damage and difficulty in scaling up, and is generally less effective for small vectors.

[0034] Yet another method for improving the transduction efficiency of viruses, particularly retroviruses, is the use of cell adhesion substances that bind to retroviruses, such as fibronectin or the fibronectin fragment CH-296 [Retronectin® (recombinant human fibronectin fragment) or retronectin]. This method requires adding a solution containing a retroviral vector to a retronectin-coated vessel, followed by incubation for a period of time to allow only the viral vector to bind to retronectin, and then removing the supernatant containing inhibitors of viral infection before adding target cells. Coating the vessel surface with retronectin is time-consuming, making this method somewhat expensive. Furthermore, this method is difficult to scale up when large numbers of cells need to be transduced.

[0035] Cell transduction using hollow fiber systems The present disclosure relates to highly efficient methods of transducing cells using hollow fiber systems, e.g., tangential fluid flow methods, that allow for automated or semi-automated, high-efficiency cell transduction applicable to both lentiviral, retroviral, and other viral and non-viral vectors. The methods described herein provide an approach to hollow fiber transduction that circumvents the limitations of current state-of-the-art methods.

[0036] 1A illustrates a hollow fiber system 100 including one or more hollow fibers incorporated within a custom-designed pump / valve-based configuration. In some embodiments of the present disclosure, hollow fiber system 100 includes an intracapillary medium reservoir 104, a cell reservoir 108, a virus reservoir 112, an extracapillary medium reservoir 116, a waste reservoir 120, a collection reservoir 124, an intracapillary pump 128, an extracapillary pump 132, and a filter module 134 including one or more hollow fibers 136. As described in further detail below, filter module 134 provides a convenient means for introducing various materials into hollow fiber system 100, including retroviral materials.

[0037] The intracapillary medium container 104 contains intracapillary medium or transduction medium 106 and is connected to the intracapillary pump 128 via a transduction medium conduit 140. The cell container 108 contains cells 110 and is connected to the intracapillary pump 128 via a cell conduit 144. The cells may include B cells, T cells, NK (natural killer) cells, monocytes, or other lymphoid cells or progenitor cells.

[0038] The virus reservoir 112 contains virus or vector particles 114 and is connected to the intracapillary pump 128 via a virus conduit 148. The vector 114 can include a virus particle. In other examples, the virus can include a nucleic acid vector. In some examples, the virus is derived from a lentivirus, retrovirus, adenovirus, adeno-associated virus, or hybrid virus. In some embodiments, the virus can include a retrovirus or lentivirus. In some examples, non-viral vector(s) are used instead of a virus. Here, the non-viral vector can include liposomes, lipid particles, carbon, non-reactive metals, gelatin, polyamine nanospheres, and / or inorganic nanoparticles. Further examples of non-viral vectors include, for example, spheroplasts, erythrocyte ghosts, colloidal metals, inorganic nanoparticles, DEAE-dextran plasmids, or combinations thereof, among others. In some embodiments, the inorganic nanoparticles are calcium phosphate nanoparticles.

[0039] Although the present disclosure shows all three reservoirs 104, 108, 112 connected independently to the intracapillary pump 128 by conduits 140, 144, 148, respectively, two or more of the reservoirs 104, 108, 112 may share a common conduit. For example, all three reservoirs 104, 108, 112 may be connected to the intracapillary pump 128 via a single conduit. In another embodiment, the cell reservoir 108 and the virus reservoir 112 may be connected to the intracapillary pump 128 via a common conduit independent of the transduction medium conduit 140.

[0040] The intracapillary pump 128 receives one or more of the intracapillary medium 106, the cells 110, and the vector particles 114 and delivers them at a desired rate to the filter module 134. In the example shown, the intracapillary pump 128 includes a first outlet 152A and a second outlet 152B in fluid communication with the filter module 134. The first outlet 152A is fluidly coupled to the filter module 134 via a first intracapillary conduit 156A, and the second outlet 152B is fluidly coupled to the filter module 134 via a second intracapillary conduit 156B. As shown, filter module 134 is connected to first intra-capillary conduit 156A via first intra-capillary port 160A located at a first end of filter module 134 and to second intra-capillary conduit 156B via second intra-capillary port 160B located at an opposite second end of filter module 134. Intra-capillary ports 160A and 160B may include valves operable to selectively regulate the passage of fluid / media to filter module 134.

[0041] 1A , the extracapillary medium container 116 contains an extracapillary or collection medium 118, and the waste container 120 is configured to receive a fluid waste 122 from the filter module 134. The extracapillary pump 132 is configured to provide fluid flow between the filter module 134 and each of the extracapillary medium container 116 and the waste container 120. Here, the extracapillary pump 132 is connected to the extracapillary medium container 116 via an extracapillary medium conduit 176 and to the waste container 120 via a waste conduit 180. The extra-capillary pump 132 includes two or more pump ports 172A, 172B, each connected to the filter module 134 via a respective extra-capillary port 164A, 164B, which may include valves configured to regulate the flow of the extra-capillary medium 118 and waste material 122 into and out of the filter module 134. The first extra-capillary port 164A connects the filter module 134 to a first extra-capillary pump port 172A of the extra-capillary pump 132 via a first extra-capillary conduit 168A. The second extra-capillary port 164B connects the filter module 134 to a second extra-capillary pump port 172B of the extra-capillary pump 132 via a second extra-capillary conduit 168B.

[0042] Each of the intracapillary pump 128 and the extracapillary pump 132 may include any type of pump operable to provide fluid flow between the various vessels 104, 108, 112, 116, 120 and the filter module 134. While the illustrated example shows each pump 128, 132 embodied as a single pump, other embodiments of the system 100 may include multiple intracapillary pumps 128 and / or multiple extracapillary pumps 132, each operable to provide fluid to or from one or more of the vessels 104, 108, 112, 116, 120. The pumps 128, 132 may be embodied as manual pumps, such as syringes, or as powered pumps, such as metering pumps. Optionally, flow from each vessel 104, 108, 112, 116, 120 to each pump 128, 132 may be regulated by one or more valves implemented in the conduits 140, 144, 148, 176, 180 or vessels 104, 108, 112, 116, 120. In other examples, each conduit 140, 144, 148, 176, 180 may be individually connected to an independent pump 128, 132, such that flow from each vessel 104, 108, 112, 116, 120 is regulated directly by operation of the respective pump 128, 132.

[0043] FIG. 1B shows a horizontal cross-section of a simplified example of a hollow fiber 136. The horizontal cross-section is a cross-section of the hollow fiber 136 taken along line 1B-1B shown in FIG. 1A. The hollow fiber 136 can be enclosed within a casing 137 to form an example of a filter module 134. As shown, the space within the hollow fiber 136 defines an intracapillary space 138, and the space outside the hollow fiber 136 defines an extracapillary space 139. For example, the extracapillary space 139 is the space between the hollow fiber 136 and the casing 137. While the example shown shows a single hollow fiber 136 defining the intracapillary space 138, it should be understood that there can be multiple hollow fibers 136 arranged in parallel, such as in the example shown in FIG. 1D, that cooperatively define the intracapillary space 138. An example of a filter module 134 is Repligen's MicroKros hollow fiber, or the like. Continuing to refer to FIG. 1A, the first and second intra-capillary ports 160A, 160B are fluidly coupled to the intra-capillary space 138 at opposite ends of the hollow fiber 136, while the first and second extra-capillary ports 164A, 164B are fluidly coupled to the extra-capillary space 139 at opposite ends of the casing 137.

[0044] 1C shows a vertical cross-section of a hollow fiber 136 of the present disclosure. The vertical cross-section is a cross-section of the hollow fiber 136 along line 1C-1C shown in FIG. 1A. The vertical cross-section also shows the hollow fiber 136 disposed within a casing 137. The hollow fiber 136 includes a membrane having multiple pores that define a filter passage between an intra-capillary space 138 and an extra-capillary space 139. As described above and shown in FIG. 1D, multiple hollow fibers 136 may be implemented in a filter module 134, where all of the hollow fibers 136 are housed within the casing 137. Here, each hollow fiber 136 defines a separate portion of the intra-capillary space 138.

[0045] In one embodiment, the hollow fiber 136 is cylindrical and has a diameter of 500 μm. In some embodiments, the hollow fiber is cylindrical in shape. In some examples, the hollow fiber diameter is greater than about 80 μm, 100 μm, 150 μm, or 200 μm. The hollow fiber diameter is about 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, or about 1,000 μm.

[0046] In some embodiments, hollow fiber 136 includes a membrane having a plurality of pore sizes. In one embodiment, the membrane pore size is 750 kDa. In some examples, the membrane pore size of hollow fiber 136 may be between about 50 and 100 kDa. In some examples, the membrane pore size of hollow fiber 136 is greater than about 50 kDa. In some embodiments, the membrane pore size of hollow fiber 136 is between about 100 kDa and about 200 kDa. In some examples, the membrane pore size of hollow fiber 136 is about 300 kDa, 400 kDa, 500 kDa, 30 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1 μm.

[0047] In some embodiments, the hollow fiber membranes include polysulfone (PS), modified polyethersulfone (mPES), mixed cellulose esters (ME), polyethersulfone (PES), or a mixture thereof. In some examples, the hollow fiber membranes include ceramic(s), metal(s), or a mixture thereof. Optionally, the hollow fiber membranes do not contain retronectin, fibronectin, and / or polybrene (i.e., are free of retronectin, fibronectin, and / or polybrene). In some embodiments, the introduction of vectors 114 into cells 110 can be increased by coating the membrane of hollow fiber 136 with a compound. In some embodiments, hollow fiber 136 is coated with retronectin. In some implementations, hollow fiber 136 is coated with fibronectin. In some configurations, the membrane of hollow fiber 136 is coated with polybrene. In some examples, hollow fiber is coated with a mixture of retronectin, fibronectin, and / or polybrene.

[0048] Viral transduction process using hollow fiber systems As described in more detail below, introducing a viral or non-viral vector into cells using a hollow fiber system 100 according to the present disclosure generally involves three steps: 1) loading cells and a viral or non-viral vector into the intracapillary space 138, 2) introducing the viral or non-viral vector into cells within the intracapillary space 138, and 3) retrieving the cells and the viral or non-viral vector from the intracapillary space 138. The fluid flow direction at each step can be adjusted.

[0049] In some instances, the harvested cells, including the transduced immune cells, are transferred to a suitable bioreactor or culture vessel for expansion. The transduced cells are then grown in a suitable culture medium for 3-20 days, after which they are washed, suspended in a final formulation buffer, and cryopreserved as a formulation suitable for later therapeutic use.

[0050] In some examples, once harvested, the transduced cells are separated from non-transduced cells and vectors using any suitable means in the art, such as affinity isolation of the transduced cells from the vector and non-transduced cells using an antibody that binds to a chimeric antigen receptor (CAR) expressed on the cells of the transduced cells, or by using flow cytometry. Other suitable means in the art that can be used include, but are not limited to, size exclusion separation or some other method, such as the use of columns, membranes, etc., to separate cells from vectors. Once the cells are isolated, separated, or removed after the harvesting step, they may be expanded and then cryopreserved, or cryopreserved after the harvesting step, after which the cryopreserved cells can be used for later therapeutic use.

[0051] Fluid flow direction during cell and viral or non-viral vector loading 2A-2C illustrate the configuration and fluid flow direction of hollow fiber system 100 during the cell and vector loading process. The directions of the arrows in conduits 140, 144, 148, 156A, 156B, 168A, 168B, 176, and 180; intracapillary space 138; hollow fiber 136; and extracapillary space 139 indicate the fluid flow direction during the loading process. As shown in FIG. 2A, during the cell and vector loading process, intracapillary pump 128 receives a flow of cells 110 from cell container 108 and a flow of vectors 114 from virus container 112, but does not receive intracapillary medium 106 from intracapillary medium container 104. Thus, each container 104, 108, and 112 may be fluidly coupled to intracapillary pump 128, but the flow from each container may be selectively controlled (e.g., turned on and off) by one or more valves.

[0052] 2A , the intracapillary pump 128 supplies the cells 110 and vectors 114 to the intracapillary space 138 via first and second intracapillary conduits 156A, 156B, respectively. As previously described, the intracapillary conduits 156A, 156B may be connected to the intracapillary space 138 via intracapillary ports 160A, 160B located at opposite ends of the hollow fiber filter module 134. Thus, the cells 110 and vectors 114 are introduced into the intracapillary space 138 of the hollow fiber 136 from opposite ends of the hollow fiber 136 via the intracapillary conduits 156A, 156B, creating a backflow of the cells 110 and vectors 114 within the intracapillary space 138. As cells 110 and vectors 114 flow from opposite ends of intracapillary space 138, the counterflow of cells 110 and vectors 114 meets and / or intermingles in a common region within intracapillary space 138, defining a transduction zone. Thus, during the transduction step described below with respect to Figures 3A-3C, cells 110 can be transduced within a localized region of intracapillary space 138 based on the counterflow.

[0053] During the loading step, the cells 110 and the vectors 114 may be simultaneously loaded into the intracapillary space 138. In other examples, the cells 110 may be loaded into the intracapillary space 138 prior to the loading of the vectors 114. Conversely, in some examples, the vectors 114 may be loaded into the intracapillary space 138 before the cells 110. In another example, the cells 110 and the vectors 114 may be loaded into the intracapillary space 138 intermittently and alternatively through both ports 160A and 160B such that a layer of the cells 110 and the vectors 114 is loaded into the intracapillary space 138. Optionally, the cells 110 and the vectors 114 may be loaded through one of the ports 160A and 160B while the other port is closed.

[0054] In some embodiments, the cells are 1 x 10 3 From 1×10 10 The hollow fibers are loaded at a concentration ranging between about 1 x 10 cells / ml. In some embodiments, the cells are 6 From 1×10 9 The hollow fibers are loaded at a concentration of about 1 x 10 cells / ml. In some embodiments, the cells are 6 , 1×10 7 cells / ml, 2×10 7 cells / ml, 3×10 7 cells / ml, 4×10 7 cells / ml, 5×10 7 cells / ml, 6×10 7 cells / ml, 7×10 7 cells / ml, 8×10 7 cells / ml, 9×10 7 cells / ml or 1 x 10 8 The hollow fibers are loaded at a concentration of cells / ml.

[0055] In some embodiments, the viral particles are 1 x 10 6 IU virus / ml to 1 × 10 9 In some embodiments, the virus is loaded at a concentration ranging between about 1×10 IU virus / ml. 7 IU virus / ml, 2 × 10 7IU virus / ml, 3 × 10 7 IU virus / ml, 4 × 10 7 IU virus / ml, 5 × 10 7 IU virus / ml, 6 × 10 7 IU virus / ml, 7 × 10 7 IU virus / ml, 8 × 10 7 IU virus / ml, 9 × 10 7 IU virus / ml, 1 × 10 8 IU virus / ml, or 1 × 10 9 It is loaded at a concentration of IU virus / ml.

[0056] In some embodiments, the flow rate for loading viral or non-viral vectors is a function of the amount of inner surface area of ​​the membrane of the hollow fiber 136. In some embodiments, the flow rate per square centimeter of inner surface area of ​​the membrane of the hollow fiber 136 is 0.25 ml / min / cm 2 to 100 ml / min / cm 2 In some embodiments, the constant flow rate for loading cells into the hollow fiber ranges from 0.25 ml / min / cm 2 to 100 ml / min / cm 2 For example, in some implementations, the constant flow rate is between about 0.25 ml / min / cm 2 , 0.5ml / min, 1ml / min / cm 2 ,5ml / min / cm 2 , 10ml / min / cm 2 , 15ml / min / cm 2 ,20ml / min / cm 2 ,25ml / min / cm 2 ,30ml / min / cm 2 ,35ml / min / cm 2 ,40ml / min / cm 2 ,45ml / min / cm 2 ,50ml / min / cm 2 ,55ml / min / cm 2 ,60ml / min / cm 2 ,65ml / min / cm 2 ,70ml / min / cm 2 ,75ml / min / cm 2,80ml / min / cm 2 ,85ml / min / cm 2 ,90ml / min / cm 2 ,95ml / min / cm 2 or 100 ml / min / cm 2 is.

[0057] In some embodiments, the cells and viral particles are loaded into the hollow fiber capillary space at a multiplicity of infection (MOI) of about 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0. Thus, in some embodiments, the cells and viral particles are loaded at an MOI of about 0.25. In some embodiments, the cells and viral particles are loaded at an MOI of about 0.5. In some embodiments, the cells and viral particles are loaded at an MOI of about 1.0. In some embodiments, the cells and viral particles are loaded at an MOI of about 1.5. In some embodiments, the cells and viral particles are loaded at an MOI of about 2.0. In some embodiments, the cells and viral particles are loaded at an MOI of about 2.5. In some embodiments, the cells and viral particles are loaded at an MOI of about 3.0. In some embodiments, the cells and viral particles are loaded at an MOI of about 3.5. In some embodiments, the cells and viral particles are loaded at an MOI of about 4.0.

[0058] As the cells 110 and vectors 114 fill the intracapillary space 138, the hollow fibers 136 retain the cells 110 and vectors 114, concentrating them within the intracapillary space 138 of the hollow fibers 136. As a result, the cells 110 and vectors 114 are concentrated in the intracapillary space 138 (e.g., on the inner surface of the membrane of the hollow fibers 136). Waste or fluid 122 from the cells 110 and vectors 114 passes through the pores of the hollow fibers 136 from the intracapillary space 138 to the extracapillary space 139. As shown in FIG. 2B , the waste 122 flows in both directions through the extracapillary space 139 to extracapillary ports 164A, 164B located at either end of the filter module 134. Here, the counterflow of waste 122 towards extra-capillary ports 164A, 164B results in a cross-flow of the outgoing waste 122 stream against the incoming stream of cells 110 and vectors 114. The waste 122 travels from extra-capillary ports 164A, 164B through extra-capillary conduits 168A, 168B to extra-capillary pump ports 172A, 172B of extra-capillary pump 132, after which it is expelled by pump 132 through waste conduit 180 into waste container 120.

[0059] Fluid flow direction during viral or non-viral vector delivery Once the cells 110 and vectors 114 have filled the intracapillary space 138, the hollow fiber system 100 is configured to introduce intracapillary medium 106 into the intracapillary space to facilitate transduction. The intracapillary fluid filling step promotes interaction between the cells 110 and the vectors 114 in the intracapillary space 138 (e.g., on the inner surface of the membrane of the hollow fiber 136), which results in binding of the vectors 114 to the cells 110 and the resulting entry of the vector particles 114 into the cells 110. Figures 3A-3C illustrate the configuration and fluid flow direction of the hollow fiber system 100 during the transduction process. The direction of the arrows indicates the fluid flow direction for the respective materials 122, 140 during the transduction process. 3A, during the transduction process, the cell reservoir 108 and the virus reservoir 112 are not in fluid communication with the intracapillary pump 128, but the intracapillary medium reservoir 104 is in fluid communication with the intracapillary pump 128. Thus, the intracapillary pump 128 receives a flow of intracapillary medium 106, but not cells 110 or vectors 114.

[0060] 3A, the intracapillary pump 128 supplies intracapillary medium to the intracapillary space 138 via the first and second intracapillary conduits 156A, 156B, respectively, to initiate vector transduction. Thus, like the cells 110 and vectors 114, the intracapillary medium 106 may fill the intracapillary space 138 from both ends of the hollow fiber 136. In one embodiment, the transduction time is approximately 90 minutes.

[0061] The intracapillary medium 106 may be supplied to the intracapillary space 138 using a continuous, constant fluid flow at a low rate to prevent virus from diffusing away from the cells. In some embodiments, the constant flow rate for introducing viral or non-viral vectors into cells is between 10 μl / min and 5 ml / min. In some embodiments, the constant flow rate for transducing vectors into cells is between 10 μl / min and 5 ml / min. For example, in some embodiments, the constant flow rate is about 10 μl / min, 25 μl / min, 50 μl / min, 100 μl / min, 250 μl / min, 500 μl / min, 750 μl / min, 1 ml / min, 2 ml / min, 3 ml / min, 4 ml / min, or 5 ml / min.

[0062] In some embodiments, the cells and viral or non-viral vector are exposed to fluid flow for about 5 minutes to approximately several days. In some embodiments, the cells and virus are exposed to fluid flow for 5 minutes to approximately 18 hours. In some embodiments, the cells and virus are exposed to fluid flow for 60 minutes to approximately 120 minutes. In some embodiments, the cells and virus are exposed to fluid flow for approximately 90 minutes. In some embodiments, the cells are further cultured in the hollow fiber system for several weeks after transduction.

[0063] During the transduction process, fluid enters the intracapillary space of the filter module 134 through ports 160A, 160B, passes from the intracapillary space 138 through the pores of the hollow fibers 136, and flows out into the extracapillary space 139. Waste or fluid 122 from the transduction process passes through the pores of the hollow fibers 136 from the intracapillary space 138 to the extracapillary space 139. As shown in FIG. 3B , the waste 122 flows in both directions through the extracapillary space 139 to extracapillary ports 164A, 164B located at opposite ends of the filter module 134. Here, the counterflow of waste 122 toward the extracapillary ports 164A, 164B results in a crossflow of the outgoing stream of waste 122 against the incoming stream of intracapillary medium 140. The extra-capillary pump 132 receives the waste 122 from the extra-capillary ports 164A, 164B via the extra-capillary conduits 168A, 168B and then expels the waste 122 via the waste conduit 180 to the waste container 120.

[0064] Fluid flow direction during cell and viral or non-viral vector recovery After the transduction process shown in Figures 3A-3C, the system 100 is configured to recover the transduced cells 126 from the intracapillary space 138. Figures 4A-4C show the configuration and fluid flow direction for the hollow fiber system 100 during the cell recovery process. The direction of the arrows indicates the fluid flow direction during the cell recovery process. As shown in Figure 4A, during the recovery process, the extracapillary pump 132 provides a flow of extracapillary medium 118 from the extracapillary medium container 116 to the extracapillary space 139 via extracapillary ports 164A and 164B, respectively. As shown in Figures 4B and 4C, the extracapillary medium 118 passes from the extracapillary space 139 to the intracapillary space 138, displacing the transduced cells 126 from the intracapillary space 138. For example, extracapillary medium 118 is introduced into the extracapillary space of filter module 134 via each of extracapillary ports 164A, 164B to maximize the migration of transduced cells 126 from the inner surface of the membrane of hollow fiber 136 into intracapillary space 138.

[0065] Continuing with FIG. 4A , the intracapillary pump 128 may also provide a flow of intracapillary medium 106 (or other flushing fluid) from the intracapillary medium container 104 to the intracapillary space 138 to flush out the transduced cells 126 released from the intracapillary space. However, unlike during the transduction process ( FIGS. 3A-3C ), in which the intracapillary medium 106 is provided from both ends of the hollow fiber 136 via both intracapillary ports 160A and 160B, during the recovery process, the intracapillary medium 106 is provided via only one intracapillary port 160A to initiate unidirectional flow through the intracapillary space 138. The repeated unidirectional fluid flow through the intracapillary space allows the transduced cells 126 to be recovered from the intracapillary space 138 via the other intracapillary port 160B into the recovery container 124.

[0066] In some examples, the transduced cells 126 are recovered from the intracapillary space 138 into complete culture medium and then transferred directly to a suitable bioreactor or culture vessel. The transduced cells are then grown in a product-dependent culture buffer for a growth period (e.g., 3-20 days). Once grown, the cells are washed, suspended in a final formulation buffer, and then frozen for therapeutic use. In other examples, the transduced cells 126 may be recovered from the intracapillary space 138 into a final formulation buffer. Here, the transduced cells 126 are introduced into a membrane-, column-, or other-based process for size selection of target cells and removal of unwanted virus. The selected target cells are then frozen for later therapeutic use.

[0067] Retroviral and lentiviral transduction using a semi-automated hollow fiber system Example 1. Retroviral transduction of T cells without retronectin using a semi-automated hollow fiber system This example illustrates a study demonstrating retroviral transduction of T cells without RetroNectin using a semi-automated hollow fiber system. This example compares the transduction rates achieved under six different conditions: a) untransduced (UTD) static bag only, b) static bag without RetroNectin (RN) coating, cells and virus co-incubated for 90 minutes, c) static bag with RetroNectin coating, 90 minutes incubation, d) static bag without RetroNectin coating, cells and virus co-incubated overnight, e) static bag with RetroNectin coating, cells and virus co-incubated overnight (standard process), and f) semi-automated hollow fiber system without RetroNectin, cells and virus co-incubated for 90 minutes. Comparative transduction rates for all six conditions are shown in Figure 5.

[0068] In this example, 3-fold dilutions of retrovirus were prepared to determine the optimal infection range. CD4 / CD8 isolated T cells were thawed and activated for 48 hours. In static control conditions, 7 million pre-activated T cells at a concentration of 1 million cells / mL were placed in culture bags. These pre-activated cells were then transduced with virus (MOI 2.5) either overnight or for 90 minutes. A retronectin control was prepared, in which cell bags were coated with retronectin at 10 μg / mL overnight. Retronectin-coated bags were pre-incubated with retrovirus for 2 hours.

[0069] In the semi-automated hollow fiber system 100, cells and virus were loaded into the filter module 134 at an MOI of 2.5 and transduced for 90 minutes. Retronectin was not used in the hollow fiber system 100. At the end of the 90-minute transduction process, the cells and virus were harvested from the filter module 134 and subsequently washed to remove the virus before seeding the cells onto GREX-6M. Cells from static bags transduced overnight underwent a similar process the following day. All cells were grown for 5 days post-transduction before being harvested for flow analysis.

[0070] The data showed that RetroNectin-coated bags exhibited higher transduction rates compared to bags without RetroNectin coating when cells were transduced for similar time intervals. For example, as shown in Figure 5, static bags with RetroNectin coating incubated for 90 minutes exhibited higher transduction rates compared to static bags without RetroNectin coating incubated for similar time intervals. Similarly, as shown in Figure 5, static bags with RetroNectin coating incubated overnight exhibited higher transduction rates compared to static bags without RetroNectin coating incubated overnight. As can be clearly seen in Figure 5, the semi-automated hollow fiber system 100 without RetroNectin coating incubated for 90 minutes exhibited a transduction rate nearly identical to that of static bags with RetroNectin coating incubated overnight (i.e., the standard process).

[0071] Furthermore, there was no significant difference in the viability of cells recovered from the bag (i.e., static control) and the hollow fiber after transduction, as shown in Figure 6. Similarly, there was no significant difference in cell expansion or proliferation between cells transduced in the bag and the hollow fiber system.

[0072] Example 2. Retroviral transduction of NK cells without retronectin using a semi-automated hollow fiber system This example presents a proof-of-concept study demonstrating retroviral transduction of NK cells using a hollow fiber system. This example compares the transduction rates achieved under two different conditions: a) static plate without retronectin coating, incubated for 90 minutes; b) semi-automated hollow fiber system 100 without retronectin coating, incubated for 90 minutes. The comparative transduction rates for these two conditions are shown in Figure 7.

[0073] In this example, retrovirus was prepared to an optimal infection range. Fresh umbilical cord blood NK cells were isolated and activated for 6 days before transduction. In static control conditions, 5 million pre-activated NK cells at a concentration of 1 million cells / mL were transduced with virus (MOI 2) for 90 minutes. In a semi-automated hollow fiber system, cells and virus were loaded into hollow fibers at an MOI of 2 and transduced for 90 minutes. At the end of the 90-minute transduction process, cells and virus were harvested from the hollow fiber system 100 and subsequently washed to remove virus before plating the cells onto tissue culture plates. Transduced static cells underwent a similar process. All cells were grown for 9 days after transduction before being harvested for flow analysis.

[0074] The data, shown in Figure 7, demonstrated that the hollow fiber system exhibited a higher transduction rate of NK cells compared to the static plate control.

[0075] Example 3. Lentiviral transduction using a semi-automated hollow fiber system This example shows a proof-of-concept study demonstrating lentiviral transduction using a semi-automated hollow fiber system. This example compares the transduction rates achieved under four different conditions: a) untransduced bags (i.e., static bags only), b) static bags incubated for 90 minutes, c) static bags incubated overnight, and d) semi-automated hollow fiber incubated for 90 minutes. The comparative transduction rates for all four conditions are shown in Figure 8.

[0076] In this example, a lentiviral vector carrying a ZsGreen reporter was used. CD4 / CD8 isolated T cells were thawed and activated for 48 hours. A single vial of cell and virus mixture (multiplicity of infection (MOI) of 1) was prepared and then aliquoted into separate vials to ensure equal MOIs.

[0077] In static control conditions, 7 million pre-activated T cells at a concentration of 1 million cells / mL were placed in the cell bag. These pre-activated cells were then transduced with virus at an MOI of 1 either overnight or for 90 minutes.

[0078] In a semi-automated hollow fiber system, the cell / virus mixture was loaded into the hollow fiber and transduced for 90 minutes. At the end of the 90-minute transduction process, the cells and virus were harvested from the hollow fiber and subsequently washed to remove the virus before seeding the cells onto GREX-6M. Cells transduced overnight underwent the same process the following day. All cells were grown for 5 days after transduction before being harvested for flow analysis.

[0079] As shown in Figure 8, static bags transduced overnight demonstrated higher transduction rates compared to static bags transduced for 90 minutes. As can be clearly seen in Figure 8, the semi-automated hollow fiber system 100 incubated for only 90 minutes demonstrated approximately 1.4-fold higher transduction rates compared to the static bags incubated overnight. Furthermore, there was no significant difference in viability between cells recovered from bags and hollow fibers after transduction. Similarly, there was no significant difference in proliferation between bags and hollow fibers after transduction.

[0080] Semi-automated hollow fiber system for cell therapy transduction 9 shows a schematic layout of another example hollow fiber system 200 for cell therapy transduction. The layout features input materials 206, 210, 214, 218, output materials 222, 226, and hollow fiber 236.

[0081] The input materials include transduction medium 206, cells 210, vectors 214, and recovery / harvesting medium 218. Each container 204, 208, 212, 216 for the input materials is also connected to a bubble sensor 284 and a valve 260A-D that controls the flow of the input material 206, 210, 214 to the hollow fiber 236. The bubble sensors 284A-D detect the presence of bubbles in the input materials 206, 210, 214, 218 and help ensure that the hollow fiber 236 receives the input material 206, 210, 214, 218 without bubbles.

[0082] The output materials include harvested cells 226 and waste material 222. Each output material container 220, 224 is also connected to one or more ports 164A, 164B that control the flow of fluid / media from the hollow fiber 236 to the output material container 220, 224.

[0083] Hollow fiber 236 is connected to several pumps 228A, 228B, 232 via valves 260E-260G, 264A-264D and pressure sensor 288. These pumps 228A, 228B, 232 control the rate of fluid flow into the intra- and extra-capillary spaces of hollow fiber 236 during the cell and virus loading, transduction, and harvesting processes performed using hollow fiber 236 in a manner similar to that described above with respect to hollow fiber 236.

[0084] The systems and methods disclosed herein significantly increase the efficiency of viral or non-viral vector transduction into cells by using a hollow fiber system to increase contact between the vector and target cells. In this way, a large number of cells are exposed to a sufficient concentration of vector to allow efficient cell transduction. This reduces the time to transduce cells while minimizing vector waste. Thus, the present disclosure provides systems and methods that not only reduce the total amount of vector used to achieve high cell transduction, but also significantly shorten transduction time. Thus, in one aspect, the systems and methods described herein achieve efficient cell transduction at a lower cost compared to conventional transduction systems. Additional advantages of the systems and methods disclosed herein include increased amounts of transduced cells, less virus consumed during the transduction process, shorter processing times, and reduced manufacturing costs. This ultimately benefits patients, at least because the methods allow for faster processing times and more effective therapeutics.

[0085] The method described herein uses a hollow fiber system that allows tangential fluid flow from one side of the hollow fiber to the other side of the hollow fiber. The hollow fiber system comprises one or more hollow fibers. The hollow fiber comprises a porous cylindrical surface that allows tangential fluid flow across the membrane. The tangential fluid flow brings the vector into contact / approach with the cells, which contributes to increased viral transduction efficiency.

[0086] The porous cylindrical surface of hollow fibers allows fluids and small molecules to pass through, while simultaneously blocking cells and larger molecules. Thus, in some embodiments described herein, hollow fibers contain pore sizes that selectively allow certain molecules to pass through the hollow fiber while simultaneously retaining cells and other large molecules. Furthermore, the hollow fibers described herein can be tailored to have pores between 50 kDa and 1 μm to further enhance the desired flow characteristics for achieving highly efficient transduction of viral or non-viral vectors into target or host cells. The pores of the hollow fiber can also be tailored based on the size of the viral or non-viral vector being used. In some embodiments, the pore size of the hollow fiber is one-quarter the size of the viral or non-viral particle. In some embodiments, the pore size of the hollow fiber is one-third the size of the viral or non-viral particle. In some embodiments, the pore size of the hollow fiber is one-half the size of the viral or non-viral particle. An additional parameter of the hollow fiber that can be adjusted to further optimize the efficiency of transduction of viral or non-viral vectors into target or host cells is the diameter of the hollow fiber itself.

[0087] Use of transduced cells Cells transduced with viral or non-viral vectors using the methods described herein allow the cells to be used for any purpose the modified cells can have. The modified cells maintain high viability (e.g., greater than 70%, 75%, 80%, 85%, or 90%, or up to 98%) and can be used for a variety of applications, such as for cell therapy, e.g., adoptive cell therapy applications.

[0088] In some embodiments, the viability and proliferation of transduced cells using the hollow fiber system is the same as the viability and proliferation of transduced cells using static conditions overnight.

[0089] Adoptive Cell Therapy The methods described herein can be used, inter alia, to genetically engineer cells for use in a variety of therapeutic methods, including, for example, for use in adoptive cell therapy applications.

[0090] Adoptive cell therapy ("ACT") refers to the infusion of autologous or allogeneic cells into a patient to treat disease. Various cell types, such as B cells, T cells, NK cells, monocytes, progenitor cells, or cell lines, can be used in ACT-based therapy. Progenitor cells can be isolated directly from the patient or a non-patient donor. Progenitor cells include, for example, adult stem cells and pluripotent cells, such as iPSCs, derived from the patient or a non-patient donor. In some embodiments, ACT uses the transplantation of genetically modified hematopoietic stem cells ("HSCs").

[0091] One category of ACT therapy, hematopoietic stem cell ("HSC") transplantation, involves the infusion of autologous or allogeneic stem cells to restore hematopoietic function in patients with damaged or deficient bone marrow or immune systems. It also allows for the introduction of genetically modified HSCs, for example, to treat congenital genetic diseases. In a typical HSC transplant, HSCs are obtained from bone marrow, peripheral blood, or umbilical cord blood.

[0092] In some embodiments, cells obtained from peripheral blood are genetically engineered for use in ACT. Peripheral blood is used for autologous transplantation because it contains a higher content of stem and progenitor cells compared to bone marrow or umbilical cord blood. Furthermore, HSCs obtained from peripheral blood have shown faster engraftment after transplantation. Because HSCs are present in low concentrations in peripheral blood, donors are typically treated with mobilizing factors, such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), which affect the adhesion of HSCs to the bone marrow environment and release them into the peripheral blood.

[0093] In some embodiments, the methods described herein are used to genetically modify T cells for T cell immunotherapy-based ACT procedures. T cell immunotherapy is another category of ACT procedures, involving the infusion of autologous or allogeneic T lymphocytes that have been selected and / or engineered ex vivo to target a specific antigen, such as a tumor-associated antigen. T lymphocytes are typically obtained from a donor's peripheral blood by leukapheresis. In some T cell immunotherapies, donor-derived T lymphocytes, such as tumor-infiltrating lymphocytes ("TILs"), are expanded in culture and selected for antigen specificity without altering their native specificity. In other T cell immunotherapy methods, donor-derived T lymphocytes are engineered ex vivo, usually by transduction with a viral expression vector, to express a chimeric antigen receptor ("CAR") of a predetermined specificity. CARs typically comprise an extracellular domain, such as a binding domain from an scFv, that confers specificity for a desired antigen, a transmembrane domain, and one or more intracellular domains that elicit T cell effector function, such as an intracellular domain from CD3ζ or FcRγ, and optionally one or more costimulatory domains derived, for example, from CD28 and / or 4-1BB. In yet other T cell immunotherapies, T lymphocytes obtained from a donor are engineered ex vivo, typically by transduction with a viral expression vector, to express a T cell receptor ("TCR") that confers the desired specificity for an antigen presented in the context of a particular HLA allele.

[0094] In some embodiments, the methods described herein are used to genetically modify hematopoietic stem cells (HSCs). In some embodiments, the HSCs undergo additional treatment to expand the population of HSCs or are manipulated by recombinant methods described herein to introduce heterologous genes or additional functions into the allogeneic HSCs prior to transplantation into the recipient subject. In certain embodiments, the additional treatment results in maturation of the HSCs.

[0095] HSCs obtained from either autologous or allogeneic donors can undergo additional processing before transplantation into a recipient subject. In some embodiments, the HSCs are processed to expand the population of HSCs, for example, by culturing one or more HSCs in an appropriate medium.

[0096] In some embodiments, either autologous or allogeneic HSCs are recombinantly engineered to introduce heterologous genes using the methods disclosed herein. Such genetic engineering can be used to correct genetic defects and / or introduce additional functionality into HSCs prior to transplantation. In some embodiments, functional wild-type genes are introduced into HSCs to correct genetic defects, such as congenital hematopoietic disorders (e.g., beta-thalassemia, Fanconi anemia, hemophilia, sickle cell anemia, etc.), primary immunodeficiencies (e.g., adenosine deaminase deficiency, X-linked severe combined immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, Janus kinase 3 deficiency, purine nucleoside phosphorylase (PNP) deficiency, leukocyte adhesion deficiency type 1, etc.), and congenital metabolic diseases (e.g., mucopolysaccharidosis (MPS) types I, II, III, and VII, Gaucher disease, and X-linked adrenoleukodystrophy, etc.). In certain embodiments, HSCs are genetically engineered by genome editing using a recombinase system, such as a CRISPR / Cas9 system or Cre / Lox recombinase. For example, recombinase systems can be used to remove genes or correct gene defects. In various embodiments, other methods for altering HSC function include the introduction of antisense nucleic acids, ribozymes, and RNAi, among others.

[0097] In some embodiments, progenitor cells or cell lines are modified by introducing a viral or non-viral vector into the progenitor cells or cell lines. Any suitable progenitor cells or cell lines can be used in accordance with the methods and systems described herein. Non-limiting examples of suitable progenitor cells include, for example, cells isolated directly from a patient or a non-patient donor. Progenitor cells include, for example, adult stem cells and pluripotent cells, such as iPSCs, derived from a patient or a non-patient donor. Various cell lines can also be used with the methods and systems described herein, including, for example, mammalian cell lines of human or non-human origin.

[0098] Other features, objects, and advantages of the present disclosure will become apparent in the following examples. It should be understood, however, that the examples, while illustrating embodiments of the present disclosure, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the examples.

[0099] definition Adoptive Cell Therapy: As used herein, the terms "adoptive cell therapy," "adoptive cell transfer," or "ACT" refer to the transplantation of cells into a patient in need of such transplantation. The cells may be obtained and expanded from the patient in need thereof, or may be obtained from a donor other than the patient. In some embodiments, the cells are immune cells such as lymphocytes. Various cell types can be used in ACT, for example, T cells, CD8+ cells, CD4+ cells, NK cells, delta-gamma T cells, regulatory T cells, and peripheral blood mononuclear cells. In some embodiments, the cells are genetically modified to introduce a chimeric antigen receptor (CAR).

[0100] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0101] Approximately or about: As used herein, when applied to one or more values ​​of interest, the term "approximately" or "about" refers not only to the stated value but also to values ​​similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction of (above or below) the stated reference value, unless otherwise specified or clear from the context (except in cases where such number exceeds 100% of possible values).

[0102] Chimeric Antigen Receptor (CAR): As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that can confer antigen specificity to cells (e.g., immune cells, e.g., NK cells, iPSC-derived NK cells (iNK cells), T cells, e.g., naive T cells, central memory T cells, effector memory T cells, gamma delta T cells, regulatory T cells, or combinations thereof) transduced using the methods described herein. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In various embodiments, the CARs described herein may comprise one or more of an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, optionally one or more costimulatory domains, and an intracellular signaling domain.

[0103] Cryopreservation: As used herein, the term "cryopreservation" generally refers to freezing biological material (e.g., a population of cells or transduced cells) to a temperature low enough to stop chemical reactions that might otherwise damage the material, thereby protecting the material. Cryopreserved cells maintain viability in the frozen state for extended periods of time, such as 1, 5, 10 years or more in the frozen state. Once thawed, cryopreserved cells can be expanded for both in vitro and in vivo applications.

[0104] Host cell or target cell: As used herein, the terms "host cell" or "target cell" include cells that have not been transfected, infected, or transduced. In some embodiments, the terms "host cell" or "target cell" include those that have been transfected, infected, or transduced with a recombinant vector or polynucleotide of the present disclosure. Host cells may include packaging cells, producer cells, and cells infected with a viral vector. In certain embodiments, host cells infected with a viral vector of the present disclosure are suitable for administration to a subject in need of treatment. In some embodiments, the target cell is a stem or progenitor cell. In certain embodiments, the target cell is a somatic cell, e.g., an adult stem cell, progenitor cell, or differentiated cell. In preferred embodiments, the target cell is a hematopoietic cell, e.g., a hematopoietic stem or progenitor cell. In some embodiments, the target cell includes a B cell, a T cell, a NK cell, a monocyte, or a progenitor cell. In some embodiments, the target cell is a mammalian cell, an insect cell, a bacterial cell, or a fungal cell.

[0105] mammalian cell lines In some embodiments, a "host cell" or "target cell" includes a cell line. A variety of cell lines are known in the art and are suitable for use with the present disclosure. Suitable cell lines include, for example, mammalian cell lines of human or non-human origin.

[0106] Any mammalian cell or cell type capable of cell culture and expression of a polypeptide can be utilized as a host or target cell in accordance with the present disclosure. Non-limiting examples of mammalian cells that can be used in accordance with the present disclosure include human embryonic kidney 293 cells (HEK293), HeLa cells; BALB / c mouse myeloma line (NSO / 1, ECACC number: 85110503); human retinoblastoma cells (PER.C6 (CruCell, Leiden, The Netherlands)); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4139 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W136, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma line (Hep G2). In some embodiments, a suitable mammalian cell is not an endosomal acidification-deficient cell.

[0107] Additionally, any number of commercially and non-commercially available hybridoma cell lines that express polypeptides or proteins can be utilized in accordance with the present disclosure. Those skilled in the art will understand that hybridoma cell lines may have different nutritional requirements and / or may require different culture conditions for optimal growth and polypeptide or protein expression, and will be able to modify conditions as needed.

[0108] non-mammalian cell lines Any non-mammalian cell or cell type capable of cell culture and polypeptide expression can be used as a host cell in accordance with the present disclosure. Non-limiting examples of non-mammalian host cells and cell lines that can be used in accordance with the present disclosure include yeast cells such as Pichia pastoris, Pichia methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Yarrowia lipolytica; insect cells such as Sodoptera frugiperda, Trichoplusis ni, Drosophila melangoster, and Manduca sexta; bacterial cells such as Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Bacillus lichenifonnis, Bacteroides fragilis, Clostridia perfringens, and Clostridia difficile; and amphibian cells and cell lines such as Xenopus laevis.

[0109] Functional equivalent or derivative: As used herein, the term "functional equivalent" or "functional derivative," in the context of a functional derivative of an amino acid sequence, refers to a molecule that retains a biological activity (either function or structure) substantially similar to that of the original sequence. Functional derivatives or equivalents may be naturally occurring or synthetically prepared. Exemplary functional derivatives include amino acid sequences with one or more amino acid substitutions, deletions, or additions, provided that the biological activity of the protein is preserved. It is desirable that the substituted amino acid have similar chemical and physical properties to the substituted amino acid. Desirable similar chemical and physical properties include similarities in charge, bulk, hydrophobicity, hydrophilicity, etc.

[0110] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than inside a multicellular organism.

[0111] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term may also be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0112] Non-viral vector: As used herein, the term "non-viral vector" includes, for example, nanoparticles, liposomes, lipid particles, carbon, non-reactive metal, gelatin and / or polyamine nanospheres.

[0113] Primary cells: The term "primary cells" refers to cells that are isolated directly from a subject and then expanded.

[0114] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked via peptide bonds. The term is used to refer to an amino acid chain of any length, but those of skill in the art will understand that the term is not limited to long chains and can refer to a minimal chain comprising two amino acids linked via a peptide bond. Polypeptides may be processed and / or modified, as known to those of skill in the art.

[0115] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as individual units. When a single polypeptide is a separate functional unit and does not require permanent or temporary physical association with other polypeptides to form the separate functional unit, the terms "polypeptide" and "protein" may be used interchangeably. When a separate functional unit is composed of multiple polypeptides that are physically associated with each other, the term "protein" refers to the multiple polypeptides that are physically associated and function together as a separate unit.

[0116] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, a subject is a human. A subject may be a patient, which refers to a human who visits a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0117] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, reach perfection and / or progress to perfection or achieve or avoid certain results. The term "substantially" is therefore used to express the potential lack of perfection inherent in many biological and chemical phenomena.

[0118] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0119] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a symptom(s) of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. Those skilled in the art will appreciate that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one dose.

[0120] Treating: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, or prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of, one or more symptoms or characteristics of a particular disease, injury, and / or condition. Treatment may also be administered to subjects who do not show signs of disease and / or who show only early signs of disease, with the intent of reducing the risk of developing pathology associated with the disease.

[0121] Vector: As used herein, the term "vector" refers to a combination of any carrier and any exogenous gene(s). Vectors may include, inter alia, non-viral vectors, viral vectors, and any combination thereof. For example, non-viral vectors include, inter alia, but are not limited to, liposomes, spheroplasts, erythrocyte ghosts, colloidal metals, calcium phosphate, DEAE-dextran plasmids, or combinations thereof. Viral vectors may include, inter alia, but are not limited to, retroviral vectors, lentiviral vectors, pseudotyped vectors, adenoviral vectors, adeno-associated viral vectors, hybrid viruses, and any combination thereof.

[0122] Transduction: As used herein, the term "transduction" refers to the process by which foreign DNA is introduced into another cell via a viral vector. Various viral vectors are known in the art, including, for example, retroviral vectors, lentiviral vectors, pseudotyped vectors, adenoviral vectors, adeno-associated viral vectors, and any combination thereof, among others.

[0123] Transfection: As used herein, the term "transfection" refers to the process of introducing nucleic acid into a cell by non-viral methods. In some embodiments, the methods described herein are suitable for transfecting a cell of interest.

[0124] The recitation herein of numerical ranges by endpoints includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0125] Various aspects of the present disclosure are described in detail in the following sections. The use of the sections is not intended to limit the disclosure. Each section may be applicable to any aspect of the present disclosure. In this application, the use of "or" means "and / or" unless stated otherwise. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

Claims

1. A system for introducing a vector into a cell, the system comprising: a filter module defining an intra-capillary space and an extra-capillary space separated from the intra-capillary space by a porous membrane; a pair of intracapillary ports fluidly coupled to opposite ends of the intracapillary space, each port receiving transduction medium, cells, and vectors; a pair of extracapillary ports coupled to opposite ends of the extracapillary space and fluidly connected to a source of extracapillary medium and a waste container; The system comprising:

2. The system of claim 1 , further comprising a collection vessel in fluid communication with at least one of the intra-capillary ports.

3. 3. The system of claim 1 or 2, further comprising an intracapillary pump operable to provide a respective flow of the transduction medium, the cells, and the vector to at least one of the intracapillary ports.

4. 4. The system of claim 3, wherein the intracapillary pump is operable in a first state to supply the cells and the vector to the intracapillary port for a first period of time and in a second state to supply the transduction medium to the intracapillary port for a second period of time.

5. The system of any one of claims 1 to 4, further comprising a waste container in communication with the extracapillary space via at least one extracapillary port.

6. The system of any one of claims 1 to 5, further comprising an extracapillary pump operable to provide a flow of the extracapillary medium to each of the extracapillary ports.

7. The system of any preceding claim, further comprising an extracapillary pump operable to provide a flow of waste fluid from the extracapillary port to the waste container.

8. The system according to any one of claims 1 to 7, wherein the porous membrane is cylindrical.

9. 9. The system of claim 1, wherein the porous membrane comprises pores that allow particles having a size of less than about 50 kDa to pass from the intracapillary space through the pores.

10. The system of any one of claims 1 to 9, wherein the intracapillary space defines a transduction zone.

11. 1. A system for introducing a viral or non-viral vector into a cell, the system comprising: a hollow fiber defining an intracapillary space extending from a first end to a second end; a casing surrounding the one or more hollow fibers from the first end to the second end to define an extra-capillary space between the hollow fibers and the casing, the casing comprising a first port in fluid communication with the intra-capillary space adjacent the first end and a second port in fluid communication with the intra-capillary space adjacent the second end; a transduction medium source in fluid communication with the intracapillary space via each of the first port and the second port; a cell source containing the cells and in fluid communication with the intracapillary space via each of the first port and the second port; a viral source comprising the viral or non-viral vector and in fluid communication with the intracapillary space via each of the first port and the second port; The system comprising:

12. The system of claim 11 , further comprising a collection vessel in fluid communication with the intracapillary space via at least one of the first port and the second port.

13. 13. The system of claim 11 or 12, further comprising an intracapillary pump including an inlet in fluid communication with each of the transduction medium source, the cell source, and the virus source.

14. 14. The system of claim 13, wherein the intracapillary pump includes a first outlet in fluid communication with the intracapillary space through the first port and a second outlet in fluid communication with the intracapillary space through the second port.

15. The system of any one of claims 11 to 14, wherein the casing includes a third port in communication with the extracapillary space, and the system further comprises a waste container in communication with the extracapillary space via the third port.

16. 16. The system of claim 15, further comprising an extracapillary medium source in fluid communication with the extracapillary space via the third port.

17. 17. The system of claim 16, wherein the third port is positioned adjacent to the first end of the intracapillary space, the system further comprising a fourth port in fluid communication with the extracapillary space and positioned adjacent to the second end of the intracapillary space.

18. 18. The system of claim 17, wherein the waste container and the extracapillary medium source are each in communication with the extracapillary space via the third port and the fourth port, respectively.

19. The system of any of claims 11 to 18, wherein the hollow fiber comprises a plurality of hollow fibers.

20. 20. The system of any of claims 11 to 19, wherein the hollow fiber comprises pores that allow particles having a size of less than about 50 kDa to pass through the pores from the intracapillary space.

Images