Semi-automated hollow fiber system for viral transduction

Abstract

This invention provides a semi-automated method and system for introducing viral traits. [Solution] The system for introducing a vector includes a filter module that defines 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 that are fluid-coupled to both ends of the intracapillary space, each receiving a transduction medium, cells, and a vector. The system also includes a pair of extracapillary ports that are fluid-coupled to both ends of the extracapillary space, and connected 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 on Jun. 10, 2020. The disclosure of this prior application is considered a part of the disclosure of this application and is hereby incorporated by reference in its entirety into this specification.

[0002] The present disclosure relates to a semi - automated method and system for viral transduction using a hollow fiber filter module.

Background Art

[0003] Cell therapy uses virus particles modified for safety and functionality as a delivery medium (vector) to introduce therapeutic genes into a patient's cells, utilizing the natural transduction process. Viral vector transduction is currently the most frequently used method in the production of cell therapy for introducing therapeutic gene materials.

[0004] Current transduction processes for production are labor - intensive and inefficient in the use of viral vectors, increasing the manufacturing cost of cell therapy and the time required for the output of these therapies. Therefore, there are significant limitations in the current state - of - the - art technology for production transduction processes.

Summary of the Invention

Means for Solving the Problems

[0005] One aspect of the present disclosure provides a system for introducing a vector into a cell. This system ​​​​​​​​defines an intracapillary space and an extracapillary space separated from the intracapillary space by a porous membrane and includes a filter module. The system also fluidly couples to both ends of the intracapillary space and includes a pair of intracapillary ports, each of which receives a transduction medium, cells, and a vector . The system also couples to both ends of the extracapillary space and includes a pair of extracapillary ports fluidly connected to a source of extracapillary medium and a waste receptacle .

[0006] This aspect of the disclosure may include any one or more of the following features. In some examples , the system includes a collection container fluidly coupled to at least one of the intracapillary ports. In some implementations, the system includes an intracapillary pump operable to supply a flow 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 cells and vectors to the intracapillary port during a first period and in a second state to supply transduction medium to the intracapillary port during a second 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 port to the waste container .

[0008] In some implementations, the porous membrane is cylindrical. In some examples, the porous membrane is about 5 Pores that allow particles with a size of less than 0 kDa to pass through the capillary space. It is equipped with the following features. In some configurations, the capillary space defines the transduction zone.

[0009] Another aspect of this disclosure is a systolic method for introducing a virus or nonviral vector into cells. The system provides a capillary space extending from the first end to the second end. This system includes hollow fibers. The system also includes a casing, which extends from the first end At the second end, one or more hollow fibers are surrounded, and the hollow fibers and the cable A capillary space is defined between the sing and the capillary, and the first end is fluidly connected to the adjacent capillary space. The first port and the second port, which is fluidly connected to the capillary space adjacent to the second end, This includes the casing. This system also includes the first port and the second port respectively This also includes a transduction medium supply source that is fluidly connected to the capillary space via this. It includes and is fluidly connected to the capillary space via the first port and the second port, respectively. The system further includes a cell source. This system also includes a virus or non-viral vector. The will is fluidly connected to the capillary space through the first port and the second port, respectively. This also includes the source of the supply.

[0010] This aspect of the Disclosure may include one or more of the following features. In some examples, This system allows the capillary space to be accessed via at least one of the first port and the second port. It includes a fluid-connected recovery container. In some implementations, the system is a transduction medium. Inside the capillary tube, which includes an inlet fluidly connected to the respective supply source, cell supply source, and virus supply source. It is equipped with a pump. In some examples, the in-capillary pump is in the capillary via a first port A first outlet is fluid-connected to the space, and a second port is fluid-connected to the capillary space. Includes two exits.

[0011] In some configurations, the casing includes a third port that communicates with the space outside the capillary tube. The system further includes a waste container that communicates with the space outside the capillary tube via a third port. In some examples, this system fluidly connects a capillary to the space outside the capillary via a third port. Includes an extratubular culture medium supply source. In some configurations, the third port is at the end of the first capillary space. Located adjacent to the end, this system fluidly connects to the space outside the capillary and to the second space inside the capillary. It further comprises a fourth port located adjacent to the end of the terminal. In some examples, waste container Each of the container and the extracapillary culture medium supply source is connected via the third port and the fourth port, respectively. It is in communication with the space outside the capillary.

[0012] In some configurations, a hollow fiber contains multiple hollow fibers. In this implementation, the hollow fiber consists of particles with a size of less than approximately 50 kDa that form capillaries. It includes a hole that allows passage from the internal space through the hole.

[0013] A further aspect of this disclosure is a capillary space extending from a first end to a second end, and hair Hollows that define the extracapillary space surrounding the internal space of the capillary from the first end to the second end. This provides a method for introducing viral or nonviral vectors into cells using a viver. This method involves filling the capillary space of a hollow fiber with a virus or non-viral vector. This includes filling and filling the capillary space of the hollow fiber with cells.

[0014] This aspect of the Disclosure may include one or more of the following features. In some examples, Filling the capillary space with a virus or non-viral vector is a virus or A nonviral vector is injected from at least one of the first and second ends of the capillary space. This includes filling. In some implementations, viruses or non-viral vectors are capillary. Filling the space inside the tube means introducing viruses or non-viral vectors into the first space inside the capillary tube. This includes filling from the terminal and the second terminal, respectively. In some configurations, the cells are hairs. Filling the space inside the capillary means that cells are less likely to be found at the first and second ends of the space inside the capillary. This includes filling from a single unit.

[0015] In some cases, filling the capillary space with cells is the first step in filling the capillary space with cells. This includes filling from the end of the first and second ends, respectively. Optionally, this method may involve hollowing out the first end. Transduction into cells within the capillary space of fiber, and within the capillary space of hollow fiber This may further include recovering transduced cells from between. In some examples, capillaries Recovering transduced cells from the internal space involves flushing the fluid through the capillaries of hollow fibers. This includes filling the space outside the tube. In some implementations, the trait is introduced from the capillary space into the capillary tube. To recover the cells, flushing fluid is used to drain the capillary from either the first or second end. This includes filling the internal space.

[0016] In some cases, this method involves recovering waste from the space outside the capillary tube. In that implementation, cells and viruses or non-viral vectors are loaded simultaneously. In some configurations, cells and viruses or non-viral vectors are packed separately. In some implementations, cells are loaded prior to the viral or nonviral vector. In some configurations, the viral or non-viral vector is injected into the cell prior to its arrival. It will be filled.

[0017] In some examples, the cells are 1 × 10 3 From 1 x 10 10 Concentration in the range of cells / ml It is filled with. In some implementations, the cells are filled inside the hollow fiber. This involves filling cells at a rate that is a function of the size of the lateral surface area. In some configurations, Viruses or non-viral vectors are packed as viral particles. In some examples... The viral or non-viral vector is packed as a nucleic acid vector.

[0018] In some examples, this method measures the inner surface area of ​​the hollow fiber in one square centimeter. Approximately 5-100 μl / min / cm² 2 At this flow rate, cells and viruses or non-viral vectors This includes filling the tar. In some examples, the inner surface area of ​​the hollow fiber is 1 square meter The filling rate per centimeter is approximately 5-20 μl / min / cm 2 There are several. In its implementation, the vectors include lentiviruses, retroviruses, adenoviruses, and adenoviruses. It originates from an associated virus or a hybrid virus. In some cases, the vector It is a retrovirus. In some implementations, the vector is a lentivirus. In some cases, the vector is a non-reactive nanoparticle, liposome, lipid particle, carbon. It contains metals, gelatin, and / or polyamine nanospheres.

[0019] In some implementations, cells and viral vectors are in the range of approximately 0.25 to approximately 4.0. The capillary space is filled with the surrounding multiplicity of infection (MOI). In some cases, cells and The illus vector fills the capillary space with an MOI of approximately 2.5. In some configurations... Cells include B cells, T cells, NK cells, monocytes, progenitor cells, or cell lines.

[0020] Another aspect of this disclosure provides a population of cells generated by the method described in the preceding paragraph. Another aspect of this disclosure provides a pharmaceutical composition comprising cells produced by the method described in the preceding paragraph. To provide.

[0021] Another aspect of this disclosure provides a method for producing a cell therapy product comprising one or more transduced cells. Provided. This method involves (i) transducing cells, extending from the first end to the second end (ii) ) A population of cells and a virus or non-viral vector are packed into the capillary space, (iii) Inducing transduction of one or more cells in the internal space, and (iii) In the capillary space This includes recovering a population of cells containing one or more transduced cells.

[0022] This aspect of the present disclosure may include one or more of the following features: several implementation forms In this state, the cell population consists of αβ T cells, γδ T cells, NK cells, HSCs, and macrophages. Selected from dendritic cells and iPSCs. In some configurations, viruses or non-viruses. The ruth vector contains a recombinant receptor. In some configurations, the recombinant receptor is a chimeric anti- It is a primary receptor (CAR).

[0023] In some cases, transduced cells contain recombinant receptors on their cell surface. In its morphological form, the chimeric antigen receptor is CD44, CD19, CD20, CD22, CD23 CD30, CD89, CD123, CS-1, ROR1, Mesothelin, c-Met, P SMA, Her2, GD-2, CEA, MAGE A3 TCR, EGFR, HER2 / One or more of the following: ERBB2 / neu, EPCAM, EphA2, CEA, and BCMA It contains an extracellular ligand-binding domain that targets a selected tumor antigen.

[0024] In some configurations, this method includes the step of isolating transduced cells. In this configuration, the method involves the step of growing the cells recovered in a bioreactor. Furthermore, in some implementations, this method involves storing the recovered cells in a suitable cryopreservation medium. The system further includes the step of cryopreserving. In some implementations, the system A first port is fluidly connected to the capillary space adjacent to the first end, and a second port is adjacent to the second end It includes a capillary space and a second port that is fluid-connected to it.

[0025] In some cases, this involves filling the capillary space with a virus or non-viral vector. This involves a small amount of the virus or non-viral vector at the first and second ends of the capillary space. This includes filling with at least one. In some implementations, viruses or non-viruses Filling the capillary space with a virus vector allows a virus or non-viral vector to penetrate the hair. This includes filling from the first and second ends of the space inside the tubule, respectively. In the configuration, filling the capillary space with cells is done by placing the cells at the first end of the capillary space and This includes filling from at least one of the second ends. In some implementations, the cells are filled from at least one end. Filling the capillary space with cells is done at the first and second ends of the capillary space. This includes filling them.

[0026] Various aspects of this disclosure are described in detail in the following sections. This does not limit the scope of this disclosure. Each section may be applicable to any aspect of this disclosure. In this application, unless otherwise stated, the use of "or" means "and / or". To taste. When used herein, the singular forms "a," "an," and "the" are used in context. Unless otherwise explicitly indicated, it includes both singular and plural referents. [Brief explanation of the drawing]

[0027] [Figure 1A] This disclosure shows a hollow fiber system including hollow fibers. [Figure 1B] Figure 1A shows a horizontal cross-section of a hollow fiber along line 1B-1B, where the hollow fiber is filled with cells and viruses or non-viral vectors. [Figure 1C] Figure 1A shows a vertical cross-section of a hollow fiber along line 1C-1C, where the hollow fiber is filled with cells and viruses or non-viral vectors. [Figure 1D] This is a schematic diagram of an example of a hollow fiber filter module containing multiple hollow fibers according to the present disclosure. [Figure 2A] This shows a hollow fiber system including hollow fibers that indicate the direction of fluid flow during cell and viral vector loading. [Figure 2B] This shows a horizontal cross-section of a hollow fiber indicating the direction of fluid flow during cell and virus or non-viral vector loading. [Figure 2C]This shows a perpendicular cross-section of a hollow fiber indicating the direction of fluid flow during cell and virus or non-viral vector loading. [Figure 3A] This shows a hollow fiber system containing hollow fibers that indicate the direction of fluid flow during the introduction of a viral or nonviral vector into a target or host cell. [Figure 3B] A horizontal cross-section of a hollow fiber is shown, indicating the direction of fluid flow during the introduction of a viral or nonviral vector into a target or host cell. [Figure 3C] This shows a perpendicular cross-section of a hollow fiber indicating the direction of fluid flow during the introduction of a viral or nonviral vector into a target or host cell. [Figure 4A] This shows a hollow fiber system including hollow fibers that indicate the direction of fluid flow during cell harvesting. [Figure 4B] This image shows a horizontal cross-section of a hollow fiber containing cells and viruses, illustrating the direction of fluid flow during cell retrieval. [Figure 4C] This shows a vertical cross-section of a hollow fiber indicating the direction of fluid flow during cell harvesting. [Figure 5] This shows retrovirally transduced T cells under different transduction conditions. [Figure 6] This shows the survival rates of T cells after transduction under different conditions. [Figure 7] This shows retrovirally transduced NK cells under different transduction conditions. [Figure 8] This shows lentiviral transduction T cells under different transduction conditions. [Figure 9] This document describes the technical configuration of a semi-automated hollow fiber system for cell therapy transduction. [Modes for carrying out the invention]

[0028] Current cutting-edge technology Transduction is the process by which a virus infects a target cell or host cell. The process naturally leads to the efficient introduction of genetic material into target cells. This is how it has evolved. For transduction to occur, the virus particle must physically come into contact with the target cell. First, it must bind to the target cell, invade it, and finally introduce genetic material into the target cell. It must be. The binding must be with the correct protein required by both the virus and the target cell. It arises from protein-protein interactions.

[0029] Cell therapy uses a delivery medium (vector) to introduce therapeutic genes into patient cells. Using modified virus particles for safety and functionality, the natural transduction process To utilize. Viral vector transduction is currently used to introduce therapeutic genetic material into cells. This is the most frequently used method for manufacturing cell therapies.

[0030] Current industry approaches to viral transduction include static transduction systems. These include the use of chemical enhancers and spinoculation. Each of the approaches in this field will be explained further below.

[0031] Viral transduction under static conditions is the most effective method of viral transduction currently being performed. This is a common method. In standard static transduction methods, most transduction is performed under static culture conditions. This is done in a standard culture flask or bag. In this method, the viral vector is They are then suspended in a culture medium that can be approximately 100 to 1000 times deeper than the diameter of a single cell. Transduction using standard static methods presents various problems that lead to inefficient cell transduction. We face a problem. For example, if we use a static method, some remains in the suspension and cannot reach the target cells. This means that tiny vector particles exist. This is at least partially related to large cells. This occurs because it immediately settles at the bottom of the culture vessel. Final transduction using static culture method The result is that diffusion alone can only reach a very small fraction of the vector particles into the cell. As a result, the transduction efficiency is low, and to achieve transduction of a considerable number of cells, the virus The amount of the viral vector needs to be increased. This is because the binding of the viral vector to the target cells is This is because it is determined by the expression of receptors / ligands and physical contact. Therefore, traits The transduction rate is proportional to the local concentration of the virus in the given cells. Achieving this requires a large number of viral vectors, which is costly and also affects cells. This can create inefficiencies throughout the entire therapeutic manufacturing process.

[0032] Another standard method of transduction of cells is to increase the binding rate of the vector to the cell. This involves the use of chemical enhancers. However, the use of methods that rely on chemical enhancers Furthermore, the removal of chemical enhancers is costly and creates additional barriers in the manufacturing process. vinegar.

[0033] Another standard method for transduction of cells is the use of spinocuration. Spinocuration refers to the inoculation of cells using centrifugal force. The goal is to reduce the volume occupied by the cells. This method has drawbacks, such as potential cell damage and difficulties in scaling up. It has been shown that there are various negative aspects, and generally speaking, small vectors are not very... It has no effect.

[0034] Another method for improving the transduction efficiency of viruses, especially retroviruses, is , cell adhesion substances that bind to retroviruses, such as fibronectin or fibronectin Kucin fragment CH-296 [Retronectin (Registered) (Recombinant Human Fibronectin) This method involves the use of retronectin (fragment) or retronectin. The solution containing the viral vector is added to a container coated with retronectin, and then This allows only the viral vector to bind to retronectin for a certain period of time. After batting and removing the supernatant containing inhibitors against viral infection, the target cells are added. This requires coating the container surface with retronectin, which takes time. This method is somewhat costly. Furthermore, this method involves gene transfer to a large number of cells. Scaling up is difficult when it is necessary to do so.

[0035] Cell transduction using a hollow fiber system This disclosure covers lentiviruses, retroviruses, and other viruses, as well as nonviruses. Enables automated or semi-automated, highly efficient cell transduction applicable to both vectors. A highly efficient method for transducing cells using a hollow fiber system. For example, relating to tangential fluid flow methods. The methods described herein overcome the limitations of current state-of-the-art methods. This provides an approach to avoiding hollow fiber transduction.

[0036] Figure 1A shows one or more of the components incorporated within a custom-designed pump / valve base configuration. A hollow fiber system 100 including hollow fibers is shown. Some of the disclosures In this embodiment, the hollow fiber system 100 comprises a capillary culture medium container 104 and a cell volume The container 108, the virus container 112, the extracapillary culture medium container 116, and the waste container 120, A collection container 124, an in-capillary pump 128, an out-capillary pump 132, and one or more holograms. —Includes a filter module 134 containing fiber 136. Further details are provided below. As described, the filter module 134 uses a hollow fiber system with various materials. It provides a convenient means for introducing over 100 retroviral materials.

[0037] The capillary culture medium container 104 contains the capillary culture medium or the transduction medium 106, and transduction The cell container 108 is connected to the capillary pump 128 via the culture medium conduit 140. It contains 110 and is connected to the capillary pump 128 via the cell conduit 144. This includes B cells, T cells, NK (natural killer) cells, monocytes, and other lymphoid cells. For example, progenitor cells can be cited.

[0038] The virus container 112 contains a virus or vector particle 114 and a virus conduit 1 It is connected to the capillary pump 128 via 48. The vector 114 is a virus particle. One example is a child. Another example is a nucleic acid vector, which can be cited as a virus. Yes, it is possible. In some examples, viruses include lentiviruses, retroviruses, and adenoviruses. It originates from Rus, adeno-associated viruses, or hybrid viruses. Several implementation forms Examples of viruses in this context include retroviruses and lentiviruses. In some cases, non-viral vectors are used instead of viruses. Here, non-viral vectors include liposomes, lipid particles, carbon, non-reactive metals, and ze. Examples include latin, polyamine nanospheres, and / or inorganic nanoparticles. Further examples of viral vectors include, for example, spheroplasts and red blood cells. Ghost, colloidal metal, inorganic nanoparticles, DEAE dextran plasmid, or the These are some examples of combinations. In some embodiments, the inorganic nanoparticles are calcium phosphate. These are um nanoparticles.

[0039] This disclosure describes how the capillary pump 12 is independently operated by conduits 140, 144, and 148, respectively. This shows all three containers 104, 108, and 112 connected to 8, but container 104, Two or more of 108 and 112 may share a common conduit. For example, all three containers 1 04, 108, and 112 may be connected to the capillary pump 128 via a single conduit. In another embodiment, the cell container 108 and the virus container 112 are connected to the transduction medium conduit 140. It may be connected to the capillary pump 128 via a common conduit, independently of the others.

[0040] The capillary pump 128 pumps capillary culture medium 106, cells 110, and vector particles 114. It receives one or more of these and supplies them to the filter module 134 at the desired rate. In the example shown, the capillary pump 128 is fluidly connected to the filter module 134. It includes a first outlet 152A and a second outlet 152B. The first outlet 152A is a first capillary The filter module 134 is fluidly coupled via the internal conduit 156A, and the second outlet 152 B is fluidly coupled to the filter module 134 via the second capillary conduit 156B. It is. As shown, the filter module 134 is the filter module 134 The first capillary conduit 15 is accessed via the first capillary port 160A located at the first end. 6A and the second capillary located at the opposite second end of the filter module 134 It is connected to the second capillary conduit 156B via the internal port 160B. 60A and 160B selectively control the passage of fluid / culture medium to the filter module 134. It may include a valve that can be operated to control the operation.

[0041] Continuing with Figure 1A, the extracapillary culture medium container 116 is outside the capillary or the recovered culture medium 118 The waste container 120 contains the fluid waste 122 from the filter module 134. It is configured to take. The capillary external pump 132 is connected to the filter module 134 and the capillary To supply fluid flow between the outer culture medium container 116 and the waste container 120, respectively. This is how it is constructed. Here, the extracapillary pump 132 is connected to the extracapillary culture medium conduit 176. It is connected to the external culture medium container 116 and to the waste container 120 via the waste conduit 180. The capillary pump 132 has its respective capillary port 164A, 164 Two or more pump ports 172A connected to filter module 134 via B, Including 172B, the extracapillary ports 164A and 164B are extracapillary culture medium 118 and waste Flow from 122 to filter module 134 and out of filter module 134 It may include a valve configured to regulate the flow. First capillary outer port 164A The filter module 134 is pumped through the first capillary outer conduit 168A to the capillary outer pump. Connect to the first capillary external pump port 172A of 132. Second capillary external port 164 B pumps the filter module 134 through the second capillary outer conduit 168B to the capillary outer pump. Connect to the second capillary external pump port 172B of P132.

[0042] Each of the capillary pumps 128 and 132 is used in various containers 104, 1 A fluid flow is supplied between 08, 112, 116, 120 and filter module 134. It may include any type of pump capable of operating in that manner. The example shown is a single pump The images show the individual pumps 128 and 132 that are embodied as parts, but other parts of system 100 are also shown. The application method is to apply each to one or more of containers 104, 108, 112, 116, and 120 or Multiple capillary pumps 128 and / or capable of supplying fluid from them Multiple extracapillary pumps 132 may be included. Pumps 128 and 132 are syringes, etc. It may be implemented as a manual pump or as a powered pump such as a metering pump. (Optional) The flow from each container 104, 108, 112, 116, and 120 to each pump 128 and 132 These are conduits 140, 144, 148, 176, 180 or containers 104, 108, 112 , or may be regulated by one or more valves implemented in 116, 120. In other examples Each conduit 140, 144, 148, 176, 180 is connected to an independent pump 128, 132 They may be connected separately, thereby each container 104, 108, 112, 116, 120 The flow is directly regulated by the operation of pumps 128 and 132.

[0043] Figure 1B shows a simplified horizontal cross-section of hollow fiber 136. The horizontal cross-section is, This is a cross-section of the hollow fiber 136 along the line 1B-1B shown in Figure 1A. Fiber 136 is enclosed within casing 137, and an example of filter module 134 It can be formed. As shown, the space within the hollow fiber 136 is the space inside the capillary The space 138 is defined, and the space outside the hollow fiber 136 defines the space outside the capillary tube 139. For example, the space outside the capillary tube 139 is the space between the hollow fiber 136 and the casing 137. The example shown is a single hollow fiber 136 defining the capillary space 138. This shows that, naturally, in the example shown in Figure 1D, they are arranged in parallel. Even if there are multiple hollow fibers 136 that cooperatively define the capillary space 138, One example of filter module 134 is Repligen's MicroKros holo. —Fiber, or the same. Continue to refer to Figure 1A, the first and second hairs. The capillary ports 160A and 160B are located at both ends of the hollow fiber 136 within the capillary space 13 While fluid coupling with 8, the first and second capillary outer ports 164A and 164B are casing The ring 137 is fluidly coupled with the space outside the capillary tube 139 at both ends.

[0044] Figure 1C shows a vertical cross-section of the hollow fiber 136 of this disclosure. The vertical cross-section is shown in Figure 1A. This is a cross-section of the hollow fiber 136 along the indicated line 1C-1C. The perpendicular cross-section is also, The hollow fiber 136, which is placed inside the casing 137, is also shown. 6 is a plurality of elements that define the filter passage between the capillary space 138 and the capillary space 139. It includes a membrane having pores. As described above and shown in Figure 1D, it contains multiple hollow fibers 136 may be implemented in filter module 134, in which case all hollow The fiber 136 is housed within the casing 137. Here, each hollow fiber 1 36 defines a separate portion of the 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. The diameter of the hollow fiber is approximately 80 μm, 100 μm, 150 μm, and 200 μm. They are large. The hollow fiber diameters are approximately 250 μm, 300 μm, 350 μm, and 400 μm. , 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750 μm, 800μm, 850μm, 900μm, 950μm, or approximately 1,000μm ru.

[0046] In some embodiments, the hollow fiber 136 comprises a membrane having multiple pore sizes. In one embodiment, the pore size of the membrane is 750 kD. In some examples, hollow fibers are used. The pore size of the 136 membrane may be between approximately 50 and 100 kDa. In some examples, The pore size of the membrane of hollow fiber 136 is greater than approximately 50 kDa. In some embodiments, The pore size of the membrane of hollow fiber 136 is approximately 100 kDa to approximately 200 kDa. In some examples, the pore size of the Hollow Fiber 136 membrane is approximately 300 kDa, 400 kDa. a, 500kDa, 30nm, 40nm, 50nm, 100nm, 200nm, 300n m, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, also It is 1 μm.

[0047] In some embodiments, the hollow fiber membrane is polysulfone (PS), modified Polyethersulfone (mPES), cellulose mixed ester (ME), polyethers Examples include rhuzhon (PES) or mixtures thereof. In some examples, hollow rhuzhon (PES) Examples of Iber films include ceramics (multiple), metals (multiple), or mixtures thereof. It can be. Optionally, the hollow fiber membrane contains retronectin, fibronectin, and / or or does not contain polyblens (i.e., retronectin, fibronectin, and / or (Polyblen is absent). In some embodiments, vector 114 is introduced into cells 110. This can be increased by coating the film of the hollow fiber 136 with a compound. This can be done. In some embodiments, the hollow fiber 136 is made of retronectin. It is coated. In some implementations, the hollow fiber 136 is fibronect. It is coated with chin. In some configurations, the film of hollow fiber 136 is poly It is coated with Blenn. In some cases, hollow fibers are coated with retronectin. It is coated with a mixture of fibronectin and / or polybrene.

[0048] Viral phenotypic introduction process using hollow fiber systems As will be explained in more detail below, the hollow fiber system 100 according to this disclosure The introduction of a viral or nonviral vector into the cells used generally involves the following three steps: Includes: 1) Filling the capillary space 138 with cells and viruses or non-viral vectors. 2) the transmission of a virus or non-viral vector to a cell within the capillary space 138 Introduction, and 3) rotation of cells and viruses or non-viral vectors from the capillary space 138 To contain. The direction of fluid flow can be adjusted at each step.

[0049] In some cases, the harvested cells, including transduced immune cells, were suitable for proliferation. The transduced cells are then transferred to an ioreactor or culture vessel. Next, the transduced cells are placed in a suitable culture medium. After being allowed to grow for 3 to 20 days, they are washed and suspended in the final formulation buffer, and then... It is cryopreserved as a formulation suitable for therapeutic use.

[0050] In some cases, once recovered, the transduced cells are subjected to appropriate means in the art. By using this, for example, chimeric antigen receptors expressed on the cells of transduced cells Vectors using antibodies that bind to the body (CAR) and forms from untransduced cells Transduced cells are obtained by affinity isolation or by flow cytometry. It is isolated from cells and vectors that are not present. Other suitable in the art that can be used Possible methods include size exclusion separation or several other methods, such as separating vectors into fine particles. The use of columns, membranes, etc., for separating cells is possible, but is not limited to these. After the steps are taken, once the cells are isolated, separated, or removed, they are grown and then cryopreserved. They may be stored, or frozen after the recovery step, and thereafter the frozen stored particles The cells can be used for later therapeutic use.

[0051] Flow direction of fluids during cell and virus or non-viral vector loading Figures 2A-2C show the hollow fiber system 100 during the cell and vector loading process. The configuration and fluid flow direction are shown. Conduits 140, 144, 148, 156A, 156B, 168A, 168B, 176, and 180; Capillary space 138; Hollow fiber 13 6; Furthermore, the direction of the arrows in the capillary space 139 indicates the direction of fluid flow during the filling process. As shown in Figure 2A, during the cell and vector loading process, the capillary pump 128 This involves the flow of cells 110 from cell container 108 and vector 11 from virus container 112. It receives the flow from 4, but does not receive the capillary medium 106 from the capillary medium container 104. Therefore, each container 104, 108, and 112 is fluidly coupled to the capillary pump 128. It may be, but the flow from each container is selectively controlled by one or more valves (for example) (It may be turned on and off.)

[0052] Continuing with Figure 2A, the capillary pump 128 is connected to the first and second capillary conduits 1 Cells 110 and vector 114 are transported through capillary space 13 via 56A and 156B, respectively. It supplies to 8. As mentioned above, the capillary conduits 156A and 156B are hollow fiber Through the capillary ports 160A and 160B located at both ends of the filter module 134 The cells 110 and vector 114 may be connected to the capillary space 138. The hollow fiber 136 is carried out from both ends via the capillary conduits 156A and 156B. The fiber 136 is introduced into the capillary space 138, and cells 11 are introduced into the capillary space 138. Creates a backflow of 0 and vector 114. Cell 110 and vector 114 enter the capillary space 1 Because it flows from both ends of 38, the backflow of cells 110 and vector 114 occurs in the capillary space 138 They collide and / or mix in a common area within the same region, defining the trait introduction zone. Therefore, during the transduction step shown below with respect to Figures 3A-3C, cell 110 reverse Transduction can be performed within a localized region of the capillary space 138 based on the flow.

[0053] During the filling step, the cells 110 and the vector 114 may be supplied to the intracapillary space 138 simultaneously. Alternatively, the cells 110 may be supplied to the intracapillary space 138 prior to the supply of the vector 114. Conversely, in some examples, the vector 114 may be supplied to the intracapillary space 138 prior to the cells 110. In another example, the cells 110 and the vector 114 may be intermittently and alternatively supplied to the intracapillary space 138 through both ports 160A and 160B such that the layered cells 110 and the vector 114 are supplied into the intracapillary space 138. Optionally, the cells 110 and the vector 114 may be filled through the other of the ports 160A and 160B while one of the ports 160A and 160B is in a closed state.

[0054] In some embodiments, the cells are filled into the hollow fiber at a concentration ranging between 1×10 3 to 1×10 10 cells / ml. In some embodiments, the cells are filled into the hollow fiber at a concentration of about 1×10 to 1×10 6 to 1×10 9 cells / ml. In some embodiments, the cells are about 1×10 , 1×10 6 cells / ml, 2×10 7 cells / ml, 3×1 7 [[ID=3�]]0 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 1×10 8 cells / ml and filled into the hollow fiber. <00 \ 00604>

[0055]

[0055] In some embodiments, the virus particles are 1 × 10 6 IU virus / ml to 1x1 0 9 It is filled with a concentration in the range of IU virus / ml. In some embodiments, Russ is approximately 1 x 10 7 IU virus / ml, 2 x 10 7 IU virus / ml, 3 x 10 7 IU virus / ml, 4 x 10 7 IU virus / ml, 5 x 10 7 IU virus / ml, 6×10 7 IU virus / ml, 7 x 10 7 IU virus / ml, 8 x 10 7 IU virus S / ml, 9 x 10 7 IU virus / ml, 1 x 10⁶ 8 IU virus / ml, or 1x 10 9 It is filled at a concentration of IU virus / ml.

[0056] In some embodiments, the flow rate for filling the viral or nonviral vector is , is a function of the size of the inner surface area of ​​the film of the hollow fiber 136. Several embodiments So, the flow rate per square centimeter of the inner surface area of ​​the hollow fiber 136 membrane is ,0.25ml / min / cm 2 From 100 ml / min / cm 2 It spans a range of several actual In the application method, the constant flow rate for filling the hollow fiber with cells is 0.25 ml / min. / cm 2 From 100 ml / min / cm 2 It is between. For example, in some implementations, The constant flow rate is approximately 0.25 ml / min / cm³. 2 , 0.5ml / min, 1ml / min / cm 2 , 5m l / 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 , 60 ml / 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 That is the case.

[0057] In some embodiments, the cells and virus particles are approximately 0.25, 0.5, 1.0, 1 Hollow with infection multiplicity (MOI) of 0.5, 2.0, 2.5, 3.0, 3.5, or 4.0 It fills the space inside the fiber capillary. Therefore, in some embodiments, cells and The virus particles are packed with an MOI of approximately 0.25. In some embodiments, cells and The virus particles are packed with an MOI of approximately 0.5. In some embodiments, cells and The Virus particles are packed with an MOI of approximately 1.0. In some embodiments, cells and Wi The Rus particles are packed with an MOI of approximately 1.5. In some embodiments, cells and viruses The particles are packed with an MOI of approximately 2.0. In some embodiments, cells and viruses The particles are packed with an MOI of approximately 2.5. In some embodiments, cells and virus particles are used. The child is filled with an MOI of approximately 3.0. In some embodiments, cells and virus particles are used. It is filled with an MOI of approximately 3.5. In some embodiments, the cells and virus particles are It is filled with an MOI of approximately 4.0.

[0058] When cells 110 and vector 114 are filled into the capillary space 138, hollow phi Bar 136 holds cells 110 and vector 114, and hollow fiber 136 capillaries The cells 110 and vector 114 are concentrated in the space 138 inside the capillary. It is concentrated in space 138 (for example, on the inner surface of the membrane of hollow fiber 136). Cell 1 Waste or fluid 122 from 10 and vector 114 enters the pores of hollow fiber 136 It passes through to the capillary space 138 and then to the capillary space 139. As shown in Figure 2B. The waste 122 flows in both directions through the capillary space 139 to the filter module 1 It leads to the capillary external ports 164A and 164B located at both ends of 34. Here, the capillary external ports The opposite flow of waste 122 toward 164A and 164B is directed toward cells 110 and vectors. This results in a cross-flow of waste 122 into the inflow of 114. Waste 122 is transported from capillary outer ports 164A, 164B to capillary outer conduits 168A, 168 The signal was transmitted via B to the extracapillary pump ports 172A and 172B of the extracapillary pump 132. Afterward, the waste is discharged to the waste container 120 via the waste conduit 180 by the pump 132.

[0059] Flow direction of the fluid during the introduction of a viral or non-viral vector When the cells 110 and vector 114 fill the capillary space 138, hollow fibers System 100 introduces the capillary culture medium 106 into the capillary space to promote transduction. It is configured to allow fluid to enter. The capillary fluid filling step involves the capillary space 138 (for example, Interaction between cells 110 and vector 114 on the inner surface of the membrane of hollow fiber 136 This promotes the action, which involves the binding of vector 114 to cell 110, and the resulting microorganisms. This leads to the entry of vector particles 114 into cells 110. Figures 3A-3C show the transduction process. The structure of the hollow fiber system 100 inside and the direction of fluid flow are shown. The direction of the arrows indicates: The diagram shows the fluid flow direction for each material 122 and 140 during the transduction process. As shown in 3A, during the transduction process, the cell container 108 and the virus container 112 Although the capillary pump 128 is not fluidly connected to the capillary culture medium container 104, the capillary pump It is fluidly connected to pump 128. Therefore, the capillary pump 128 controls the capillary culture medium 10 It receives the flow of 6, but neither cell 110 nor vector 114 receives it.

[0060] Continuing with Figure 3A, the capillary pump 128 is connected to the first and second capillary conduits 1 The capillary culture medium is supplied to the capillary space 138 via 56A and 156B respectively. Initiate vector introduction. Therefore, like cell 110 and vector 114, in capillary The culture medium 106 may be filled into the capillary space 138 from both ends of the hollow fiber 136. In one embodiment, the trait introduction time is approximately 90 minutes.

[0061] In capillary culture medium 106 is administered at a low flow rate to prevent the virus from detaching from the cells and spreading. The fluid may be supplied to the capillary space 138 using a continuous, constant flow of fluid. In the embodiment, a constant flow rate for introducing a virus or non-viral vector into a cell is The flow rate is between 10 μl / min and 5 ml / min. In some embodiments, the vector is introduced into the cells. The appropriate flow rate for transduction is between 10 μl / min and 5 ml / min. For example, In several embodiments, the constant flow rates are approximately 10 μl / min, 25 μl / min, 50 μl / min, 100μl / min, 250μl / min, 500μl / min, 750μl / min, 1ml / min, 2m The flow rates are l / min, 3 ml / min, 4 ml / min, or 5 ml / min.

[0062] In some embodiments, cells and viruses or nonviral vectors are used for about 5 minutes. They are exposed to a fluid flow for approximately several days. In some embodiments, cells and viruses The cells are exposed to a fluid flow for a period of 5 minutes to approximately 18 hours. In some embodiments, the cells The virus is then exposed to the fluid flow for 60 to approximately 120 minutes. Several implementations Morphologically, cells and viruses are exposed to a fluid flow for approximately 90 minutes. Several implementations In this state, the cells are cultured for several more weeks in a hollow fiber system after transduction. It can be done.

[0063] During the transduction process, the fluid is passed through ports 160A and 160B to the filter module. It enters the capillary space of L134, and the pore of the hollow fiber 136 emerges from the capillary space 138. It passes through and flows out into the space outside the capillary tube 139. Waste or fluid 12 from the transduction process. 2 passes through the pores of the hollow fiber 136 and moves from the capillary space 138 to the capillary space 13 This leads to 9. As shown in Figure 3B, the waste 122 passes through both the capillary space 139. Outer capillary ports 164A, 1, are located at both ends of the filter module 134, flowing in the direction of the flow. Proceed to 64B. Here, the waste 122 heading towards the capillary outer ports 164A and 164B is opposed. The flow is a ratio of the outflow flow of waste 122 to the inflow flow of capillary culture medium 140. This results in a loss of flow. The capillary pump 132 is connected to the capillary ports 164A, 164B or After receiving the waste 122 via the capillary outer conduits 168A and 168B, the waste conduit 1 The waste 122 is discharged into the waste container 120 via 80.

[0064] Fluid flow direction during the recovery of cells and viruses or non-viral vectors After the transduction process shown in Figures 3A-3C, system 100 enters the capillary space 13 The system is configured to recover transduced cells 126 from cell 8. Figures 4A-4C show the cell recovery process. The configuration and fluid flow direction of the hollow fiber system 100 in the cess are shown. The direction indicates the direction of fluid flow during the cell retrieval process. As shown in Figure 4A, the retrieval process is as follows: During the process, the extracapillary pump 132 operates the extracapillary ports 164A and 164B respectively. The flow of extracapillary culture medium 118 from the extracapillary culture medium container 116 to the extracapillary space 139 is supplied via this. As shown in Figures 4B and 4C, the extracapillary culture medium 118 is supplied to the extracapillary space 139. The transduced cells 126 pass through the capillary space 138 and move out of the capillary space 138. For example, extracapillary culture medium 118 is used in extracapillary ports 164A and 164B, respectively. It is introduced into the extracapillary space of the filter module 134 via and hollow fiber 1 Maximize the movement of transduced cells 126 from the inner surface of membrane 36 into the capillary space 138. .

[0065] Continuing to refer to Figure 4A, the capillary pump 128 also releases from the capillary space. To wash away the transduced cells 126, the capillary culture medium container 104 is removed from the capillary space 13. You may supply a flow of capillary culture medium 106 (or other flushing fluid) to 8. Then, the intracapillary culture medium 106 is hollowed out through both intracapillary ports 160A and 160B. Unlike the transduction process supplied from both ends of Iber 136 (Figures 3A-3C), During the harvesting process, the capillary culture medium 106 is supplied through only one capillary port 160A. Then, a unidirectional flow is initiated through the capillary space 138. The returning unidirectional fluid flow is directed through the capillary space via the other capillary port 160B. This makes it possible to recover the transduced cells 126 from 138 into the recovery container 124.

[0066] In some cases, transduced cells 126 are transferred from the capillary space 138 to the complete culture medium. After collection, the transduced cells are directly transferred to a suitable bioreactor or culture vessel. Next, the cells are grown in a product-dependent culture buffer for a growth period (e.g., 3-20 days). Once the cells have been grown, they are washed and suspended in the final formulation buffer before being treated. They are frozen for therapeutic use. In other examples, transduced cells 126 are placed in the capillary space 138 They may be recovered into the final formulation buffer. Here, the transduced cells 126 are the target cells A base for selecting the size and removing unnecessary viruses, such as a membrane, column, or other material. The cells are then introduced into the process. Next, the selected target cells are used for later therapeutic use. It will be frozen.

[0067] Retrovirus and lentiviral phenotypes using a semi-automated hollow fiber system introduction Example 1. A semi-automated hollow fiber system without retronectin. Retroviral transduction of T cells This example uses retronectin with a semi-automated hollow fiber system. This example shows a test demonstrating retroviral transduction of T cells. This example involves the following six different... Compare the transduction rates achieved under the following conditions: a) Static batch without transduction (UTD) b) Static bags without retronectin (RN) coating, cells and viruses c) Static bag with retronectin coating, 90 d) Static bag without retronectin coating, cells and wi e) Incubate the cells overnight in a static bag coated with retronectin. and the virus is co-incubated overnight (standard process), and f) retronectin is used No semi-automated hollow fiber system, cells and viruses co-incubate for 90 minutes. Figure 5 shows the comparative trait introduction rates for all six conditions.

[0068] In this embodiment, a 3-fold dilution of the retrovirus was prepared to determine the optimal infection range. CD4 / CD8 isolated T cells were thawed and activated for 48 hours under static control conditions. 7 million pre-activated T cells at a concentration of 1 million cells / mL were placed in a culture bag. Next, these pre-activated cells were exposed to the virus (MOI 2.5) overnight or for nine days. Transduction was performed for 0 minutes. A retronectin control was prepared and the cell bag was treated with 10 μg / The retronectin-coated bags were coated overnight with retronectin in mL. It was pre-incubated with the retrovirus for 2 hours.

[0069] In the semi-automated hollow fiber system 100, cells and viruses are separated by an MOI of 2. The filter module 134 was filled with 5, and transduction was performed for 90 minutes. Hollow fiber s Stem 100 did not use retronectin. End of 90-minute transduction process. Next, the cells and viruses are collected from the filter module 134 and then washed. After removing the ruth, the cells were seeded in GREX-6M. Cells in static bags transduced overnight. The same process was carried out the following day. After growing all the cells for 5 days after transduction, It was collected for flow analysis.

[0070] The data shows that when cells are transduced at similar time intervals, retronectin-coated cells The bag showed a higher transduction rate compared to bags without retronectin coating. This was shown. For example, as shown in Figure 5, retronectin cultures incubated for 90 minutes Static bags with coating, without retronectin coating, underwent similar time intervals. It showed a higher transduction rate compared to incubated static bags. Similarly, as shown in Figure 5, Therefore, static bags with retronectin coating that have been incubated overnight are retronectin Higher phenotypic development compared to static bags incubated overnight without nectin coating. The inclusion rate was shown. As can be clearly seen in Figure 5, retrone that had been incubated for 90 minutes. The semi-automated hollow fiber system 100 without kuchin coating is incubated overnight. Static bags with retronectin coating (i.e., standard process) It showed a trait introduction rate that was almost the same as the quality introduction rate.

[0071] Furthermore, as shown in Figure 6, the cells recovered from the bag after transduction (i.e., static pairs) There was no significant difference in the viability of cells recovered from (Irradiated) and hollow fibers. Similarly, Cells transduced in a globule and cells transduced in a hollow fiber system There was no significant difference in cell enlargement or proliferation between the two groups.

[0072] Example 2. N without retronectin using a semi-automated hollow fiber system Retroviral transduction of K cells This example demonstrates retroviral phenotypic induction of NK cells using a hollow fiber system. This demonstrates a proof-of-concept test. This embodiment was achieved under the following two different conditions. To compare the transduction rates: a) Static plate without retronectin coating, 90 minutes Inter-incubation, b) Semi-automated hollow fiber without retronectin coating Stem 100, incubated for 90 minutes. The transduction rates for comparison under these two conditions were... This is shown in Figure 7.

[0073] In this example, the retrovirus was prepared to the optimal infection range. Fresh umbilical cord blood NK cells It was isolated and activated for 6 days before transduction. Under static control conditions, 1 million cells / Using a virus (MOI 2) in 5 million pre-activated NK cells at a mL concentration Transduction was performed for 90 minutes. Cells and viruses were transduced in a semi-automated hollow fiber system. The substance was packed into hollow fibers at an MOI of 2 and transduced for 90 minutes. At the end of the process, the cells and viruses are recovered from the hollow fiber system 100, and After washing and removing the virus, the cells were seeded onto tissue culture plates. Transduction was performed. The static cells underwent a similar process. After all cells were grown for 9 days following transduction, They were collected for flow analysis.

[0074] As shown in Figure 7, the data shows that the hollow fiber system performed better compared to the static plate control. This demonstrated a high transduction rate into NK cells.

[0075] Example 3. Lentiviral transduction using a semi-automated hollow fiber system This example demonstrates lentiviral transduction using a semi-automated hollow fiber system. This presents a proof-of-concept test demonstrating this. This embodiment was achieved under the following four different conditions: Compare the transduction rates: a) bags that are not transduced (i.e., static bags only), b a) Static bags incubated for 90 minutes, a) Static bags incubated overnight, and d) Semi-automated hollow fiber incubated for 90 minutes. All four conditions Figure 8 shows the comparative transduction rates for each.

[0076] In this embodiment, a lentiviral vector having a ZsGreen reporter is used. The isolated CD4 / CD8 T cells were thawed and activated for 48 hours. The cells and viruses were mixed. After preparing a single vial of the compound [Multiple Infections (MOI) 1], ensure equal MOI. To do this, it was divided equally into separate vials.

[0077] Under static control conditions, 7 million pre-activated T cells at a concentration of 1 million cells / mL were used. The cells were placed in cell bags. Next, these pre-activated cells were subjected to a wiping reaction at MOI 1. Transduction was performed using Russ either overnight or for 90 minutes.

[0078] In a semi-automated hollow fiber system, the cell / virus mixture is placed in the hollow fiber. The cells were filled and transduced for 90 minutes. At the end of the 90-minute transduction process, the cells and The virus was recovered from the hollow fiber, then washed to remove it, and then the cells were removed. The cells were seeded in GREX-6M. Cells transduced overnight were subjected to the same process the following day. All cells were grown for 5 days after transduction and then harvested for flow analysis.

[0079] As shown in Figure 8, static bags transfected overnight are compared to static bags transfected for 90 minutes. It showed a higher trait introduction rate compared to [another method]. As is clearly visible in Figure 8, the ink was used for only 90 minutes. The incubated semi-automated hollow fiber system 100 was incubated overnight. It showed approximately 1.4 times higher transduction compared to the bag transduction. Furthermore, after transduction, the bag There was no significant difference in the survival rate of cells recovered from the bag and cells recovered from the hollow fiber. Similarly, there was no significant difference in proliferation between the bags and hollow fibers after transduction. .

[0080] Semi-automated hollow fiber system for cell therapy transduction Figure 9 shows a schematic of another example of the Hollow Fiber System 200 for cell therapy transduction. The arrangement is as follows: Input materials 206, 210, 214, 218; Output materials 222, 22 The material is characterized by 6 and hollow fiber 236.

[0081] The input materials are transduction medium 206, cells 210, vector 214, and recovery / recovery medium. Includes 218. Each container for the input material, 204, 208, 212, and 216, is equipped with a foam sensor. Control the flow of materials 206, 210, and 214 to be fed into 284 and hollow fiber 236. It is also connected to valves 260A~260D. The foam sensors 284A~D are connected to the input material 20 The presence of bubbles in 6, 210, 214, and 218 is detected, and it is confirmed that the hollow fiber 236 is not a bubble. Helps you receive input materials 206, 210, 214, and 218 that are not present.

[0082] The output material includes recovered cells 226 and waste 222. Each output material is in a container 220. 224 is a fluid / culture medium from hollow fiber 236 to production material container 220, 224 It is also connected to one or more ports 164A, 164B that control the flow.

[0083] Hollow fiber 236 is used for valves 260E~260G, 264A~264D and pressure Several pumps 228A, 228B, and 232 are connected via sensor 288. These pumps 228A, 228B, and 232 are described above with respect to hollow fiber 236. Cell and virus loading is performed using hollow fiber 236 in a similar manner to the above. The capillary cavity of hollow fiber 236 during the process, transduction process, and recovery process. This controls the velocity of fluid flow between the tubes and into the space outside the capillaries.

[0084] The systems and methods disclosed herein use a hollow fiber system to perform a vector By increasing contact between the ter and target cells, the virus or non-virus enters the cells. This significantly increases the efficiency of introducing the ruth vector. In this way, a large number of cells can be efficiently introduced into the cells. The subject is exposed to a sufficient concentration of the vector to enable transduction. To minimize waste while shortening the time required to transduce cells. Therefore, this disclosure , not only reduce the total amount of vectors used to achieve high transduction of cells, but also , provide a system and method that also significantly shorten the transduction time. Thus, in one aspect , the systems and methods described herein achieve efficient cell transduction at a low cost compared to conventional transduction systems. Further advantages of the systems and methods disclosed herein include an increase in the amount of transduced cells, a decrease in the virus consumed during the transduction process , a reduction in processing time, and a reduction in manufacturing costs. This is because at least the method makes the processing time faster and creates more effective therapeutic agents, thus bringing benefits to patients.

[0085] The method described herein uses a hollow fiber system that allows for a tangential flow of fluid from one side of the hollow fiber to the other side of the hollow fiber. This hollow fiber system comprises one or more hollow fibers. The hollow fiber comprises a porous cylindrical surface that allows for a tangential flow of fluid across the membrane. The tangential flow of fluid brings the vector into contact / close proximity with the cells, which contributes to an increase in viral transduction efficiency . The porous cylindrical surface of the hollow fiber allows the flow of fluids and small molecules, but at the same time does not allow the flow of cells and large molecules. Thus, in some embodiments described herein , the hollow fiber comprises a pore size that selectively allows certain molecules to flow out through the hollow fiber while at the same time retaining the cells and other large molecules. Further, the

[0086] hollow fibers described herein are used to deliver viral or non-viral vectors to target or host cells . In some embodiments described herein, the hollow fiber selectively allows certain molecules to flow out through the hollow fiber while at the same time retaining the cells and other large molecules. comprises a pore size that allows the passage of certain molecules while retaining cells and other large molecules. Further, the hollow fibers described herein are used to deliver viral or non-viral vectors to target or host cells To further enhance the desired flow characteristics for achieving high-efficiency vector introduction, 50kD It can be adjusted to have pores between a and 1 μm. The pores of the hollow fiber are Furthermore, it can be adjusted based on the size of the virus or non-viral vector used. In some embodiments, the pore size of the hollow fiber is determined by whether it is viral or nonviral. It is one-quarter the size of the particle. In some embodiments, the pore size of the hollow fiber is It is one-third the size of a virus or non-viral particle. In some embodiments, The pore size of the low fiber is half the size of a virus or non-viral particle. To further optimize the efficiency of introducing viral or nonviral vectors into host cells. Further adjustable parameters for the hollow fiber include the direct action of the hollow fiber itself. It is the diameter.

[0087] Use of transduced cells The virus or non-viral vector introduced using the method described herein The cells may be used for any purpose that the modified cells may have. To enable modified cells to maintain a high survival rate (e.g., 70%, 75%, 80%). 85%, or over 90%, or up to 98%, for example, in applications such as adoptive cell therapy. It can be used for various purposes, such as cystic therapy.

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

[0089] Adoptive cell therapy The methods described herein include, in particular, those for use in adoptive cell therapy applications. It can be used to genetically modify cells for use in various therapeutic methods.

[0090] Adoptive cell therapy ("ACT") involves the use of autologous cells or allogeneic cells to treat a disease. This refers to the injection of B cells, T cells, NK cells, monocytes, progenitor cells, or cell lines into a patient. Various cell types such as these can be used in ACT-based therapies. Progenitor cells are used in patients They can be isolated directly from donors other than the person or patient. Examples of progenitor cells include: Examples include adult stem cells and pluripotent cells such as iPSCs derived from patients or non-patient donors. In some embodiments, ACT is used to modify genetically modified hematopoietic stem cells ("HSCs"). Use transplantation.

[0091] Hematopoietic stem cell (HSC) transplantation, one category of the ACT method, uses autologous stem cells. This includes the injection of allogeneic stem cells into patients with damaged or deficient bone marrow or immune systems. It restores blood function. Furthermore, it can be used for purposes such as treating congenital genetic diseases, including the introduction of genetically modified HSCs. This also makes it possible. In a typical HSC transplant, the HSC is obtained from bone marrow, peripheral blood, or umbilical cord blood. It is possible.

[0092] In some embodiments, cells obtained from peripheral blood are genetically modified for use in the ACT method. Peripheral blood has a higher content of stem cells and progenitor cells compared to bone marrow or umbilical cord blood. Therefore, it is used for autotransplantation. Furthermore, HSCs obtained from peripheral blood have a better chance of engraftment after transplantation. This indicates that it is faster. Because HSCs are present in peripheral blood at low concentrations, donors usually have bone Granulocyte colony punctures affect the adhesion of HSCs to the medulla environment, causing them to be released into the peripheral blood. are treated with mobilizing factors such as granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF).

[0093] In some embodiments, the methods described herein are used to genetically modify T cells for adoptive cell therapy (ACT) based T cell immunotherapy. T cell immunotherapy is another category of ACT methods, and is selected to target specific antigens, such as tumor-associated antigens, and involves the injection of autologous or allogeneic T lymphocytes that have been engineered ex vivo and / or in vitro. T lymphocytes are typically obtained from the peripheral blood of a donor by apheresis. In some T cell immunotherapies, T lymphocytes obtained from a donor, such as tumor infiltrating lymphocytes (“TILs”), are cultured and selected for antigen specificity without altering their native specificity. In other methods of T cell immunotherapy, T lymphocytes obtained from a donor are typically engineered ex vivo by transduction with a viral expression vector to express a chimeric antigen receptor (“CAR”) of a desired specificity. A CAR typically includes an extracellular domain, such as a binding domain derived from a scFv, that confers specificity for a desired antigen, a transmembrane domain, and one or more intracellular domains that induce T cell effector functions, such as the intracellular domain from CD3ζ or FcRγ, and optionally one or more costimulatory domains derived from, for example, CD28 and / or 4-1BB. In yet other T cell immunotherapies, T lymphocytes obtained from a donor are typically engineered ex vivo by transduction with a viral expression vector to express a T cell receptor (“TCR”) that confers a desired specificity for an antigen presented in the context of a particular HLA allele situation. ​​​​​​​​​​​​​​​​​

[0094] In some embodiments, the methods described herein genetically modify hematopoietic stem cells (HSCs). Used to modify. In some embodiments, the HSC is used to modify the recipient target. Prior to transplantation, the HSCs undergo additional treatment to expand the population, or as described herein. The recombination method described above manipulates heterologous genes or additional functions in allogeneic HSCs. To introduce. In certain embodiments, additional treatment leads to the maturation of HSCs.

[0095] HSCs obtained from either an autologous or allogeneic donor may not be suitable for the recipient. Additional processing can be performed before transplantation. In some embodiments, HSCs are, for example, By culturing one or more HSCs in a suitable medium, the HSC population can be expanded. It is processed for that purpose.

[0096] In some embodiments, either in-house or allogeneic HSCs are recombinant A heterologous gene is manipulated and introduced by a method disclosed herein. The procedure can be used to correct genetic defects and / or add to HSCs before transplantation. The function can be introduced. In some embodiments, a functional wild-type gene is introduced into HSCs. Genetic defects, such as congenital hematopoietic disorders (e.g., β-thalassemia, Fanconi anemia, blood (e.g., phobias, sickle cell anemia), primary immunodeficiency (e.g., adenosine deaminase deficiency) disease, X-linked severe combined immunodeficiency, chronic granulomatosis, Wiskott-Aldrich syndrome Janus kinase 3 deficiency, purine nucleoside phosphorylase (PNP) deficiency, leukemia (e.g., globular adhesion disorder type 1), and congenital metabolic disorders (e.g., mucopolysaccharidosis (MPS) type 1, Correction of (Type II, Type III, Type VII, Gaucher disease, X-linked adrenoleukodystrophy, etc.) In certain embodiments, HSCs are recombinase systems, such as CRISPR / Genetic manipulation by genome editing using Cas9 or Cre / Lox recombinase For example, a recombinase system is used to remove genes or repair gene defects. It can be corrected. Other ways to modify the functionality of HSC in various embodiments In particular, the introduction of antisense nucleic acids, ribozymes, and RNAi can be cited. .

[0097] In some embodiments, the progenitor cell or cell line is a virus or non-viral vector - is modified by introducing it into progenitor cells or cell lines. The methods described herein and Any suitable progenitor cell or cell line can be used according to the system. As a standard practice, suitable progenitor cells include, for example, those directly obtained from the patient or a non-patient donor. Examples of separated cells include progenitor cells derived from the patient or a non-patient donor. Examples include adult stem cells and pluripotent cells such as iPSCs. Various cell lines are also included in this specification. It can be used in conjunction with the methods and systems described herein, for example, human Alternatively, non-human mammalian cell lines may be used.

[0098] Other features, purposes, and advantages of this disclosure are evident in the following embodiments. However, The examples provided illustrate, but are not limiting, embodiments of the present disclosure. It should be understood that various changes and modifications within the scope of this disclosure are possible, as can be seen from the examples. This will become clear to those skilled in the art.

[0099] definition Adoptive cell therapy: When used herein, “adoptive cell therapy” and “adoptive cell transplantation” are also used. The term "ACT" refers to the transplantation of cells into patients who require cell transplantation. It may be obtained from patients who need it and propagated, or from donors other than patients. It may also be obtained. In some embodiments, the cells are immune cells such as lymphocytes. For example, T cells, CD8+ cells, CD4+ cells, NK cells, delta-gamma T cells. Various cell types, such as regulatory T cells and peripheral blood mononuclear cells, can be used in ACT. In some embodiments, cells are genetically modified to introduce chimeric antigen receptors (CARs). It will be modified.

[0100] Animals: As used herein, the term "animal" refers to any member of the animal kingdom. It refers to. In some embodiments, "animal" refers to a human being at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits). These include monkeys, dogs, cats, sheep, cows, primates, and / or pigs. In the application form, animals include mammals, birds, reptiles, amphibians, fish, insects, and / or worms. This includes, but is not limited to, the animals. In some embodiments, the animals are transgenic animals. This may be an object, a genetically modified animal, and / or a clone.

[0101] Approximately or about: When used herein, when applied to one or more intended values, The terms "approximately" or "about" refer to the reference value mentioned, not just the value mentioned. It also refers to similar values. In certain embodiments, the terms "approximately" or "about" are specified separately. Unless otherwise stated, or unless it is clear from the context that such a number is a possible value Except in cases where it exceeds 100%, the reference value (whether above or below it) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14% in either direction. 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, This refers to a range of values ​​that are within 1%.

[0102] Chimeric antigen receptor (CAR): When used herein, "chimeric antigen receptor" also The term "CAR" refers to cells transduced using the method described herein (for example) For example, immune cells, such as NK cells, iPSC-derived NK cells (iNK cells), T cells, etc. For example, naive T cells, central memory T cells, effector memory T cells, cancer Conferring antigen specificity to madelta T cells, regulatory T cells, or a combination thereof. This refers to a manipulated receptor that can perform this action. CAR stands for artificial T cell receptor, chimeric T cell receptor, or Also known as chimeric immune receptors. In various embodiments, the CAR described herein This includes an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, and one or more co-domains. It may include one or more stimulating domains and intracellular signaling domains.

[0103] Cryopreservation: As used herein, the term "cryopreservation" generally refers to the preservation of biological materials. (For example, a population of cells or transduced cells) that could otherwise damage the material To stop certain chemical reactions and thereby protect the material, it is necessary to freeze it at a sufficiently low temperature. This refers to the fact that cryopreserved cells can be stored in a cryopreserved state for 1, 5, 10 years or longer. These cells maintain their viability for a long period of time in a frozen state. Furthermore, it can be propagated for both in vitro and in vivo applications.

[0104] Host cell or target cell: When used herein, "host cell" or "target cell" The term "cells" includes cells that have not been transfected, infected, or transduced. In that embodiment, the terms “host cell” or “target cell” refer to the recombinant vectors of the herein. Includes those transfected, infected, or transduced with tar or polynucleotides. Hmm. Host cells contain packaging cells, producer cells, and viral vectors. Infected cells may be present. In certain embodiments, the viral vector of this disclosure Infected host cells are suitable for administration to subjects in need of treatment. Several implementations Morphologically, the target cells are stem cells or progenitor cells. In certain embodiments, the target cells These are somatic cells, such as adult stem cells, progenitor cells, or differentiated cells. In a preferred embodiment, In some embodiments, the target cells are hematopoietic cells, such as hematopoietic stem cells or progenitor cells. So, target cells include B cells, T cells, NK cells, monocytes, or progenitor cells. In some embodiments, the target cells are mammalian cells, insect cells, bacterial cells, or fungal cells. That is the case.

[0105] mammalian cell lines In some embodiments, “host cells” or “target cells” include cell lines. The cell line is known in the art and is suitable for use with respect to this disclosure. Examples of cell lines include human or non-human mammalian cell lines.

[0106] Any mammalian cell or cell type capable of cell culture and polypeptide expression, These can be used as host cells or target cells in accordance with this disclosure. An example of a non-limiting mammalian cell type that can be used is human fetal kidney 293 cells (HEK29). 3), HeLa cells; BALB / c mouse myeloma line (NSO / l, ECACC number: 85 110503); human retinoblast cells (PER.C6 (CruCell, Leiden, Th e Netherlands); monkey kidney C transformed by SV40 V1 strain (COS-7, ATCC CRL 1651); human fetal kidney strain (in suspension culture) Subcloned 293 or 293 cells for proliferation, Graham et al. al., J. Gen Virol., 36:59 (1977)); baby hamsters Tar renal 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 le kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (He La, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Farlow 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 (Mat her et al., Annals NY Acad. Sci., 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (He Examples include p G2). In some embodiments, suitable mammalian cells are endosaw These are not cells lacking acidification.

[0107] Furthermore, commercially and non-commercially available peptides or proteins express Any number of ibridomas cell lines can be used in accordance with this disclosure. A person skilled in the art will know Ibridomas cell lines may have different nutritional requirements, and / or optimal growth and Different culture conditions may be required for polypeptide or protein expression. You will be able to understand this and modify the conditions as needed.

[0108] non-mammalian cell lines Any non-mammalian cells or cell cultures capable of cell culture and polypeptide expression. Ip can be used as a host cell in accordance with this disclosure. Non-limiting examples of possible non-mammalian host cells and cell lines include Pichia in relation to yeast. pastoris, Pichia methanolica, Pichia angus ta, Schizosacccharomyces pombe, Saccharomy ces cerevisiae and Yarrowia lipolytica; insects Regarding Sodoptera frugiperda, Trichoplusis ni, Drosophila melangoster and Manduca sexta; Regarding bacteria, Escherichia coli and Salmonella typh imurium, Bacillus subtilis, Bacillus liche nifonnis, Bacteroides fragilis, Clostridia perfringens, Clostridia difficile; and amphibians Examples include cells and cell lines derived from Xenopus Laevis of the same species.

[0109] Functional equivalent or derivative: When used herein, “functional equivalent” or “functional The term "functional derivative" is used in the context of functional derivatives of amino acid sequences, meaning that the original sequence is a derivative of the original sequence. This refers to molecules that possess substantially similar biological activity (either functional or structural) to that of [another molecule]. Functional derivatives or equivalents may be natural derivatives or synthetically prepared. It may also be a functional derivative that preserves the biological activity of a protein. Under the condition that it exists, it has one or more amino acid substitutions, deletions, or additions. Examples include anoacid sequences. Substituted amino acids have the same chemistry and physical properties as substituted amino acids. This is desirable. Desired similar chemical and physical properties include electric charge, bulkiness, hydrophobicity, hydrophilicity, etc. The similarity is included.

[0110] In vitro: As used herein, the term “in vitro” refers to the in vitro of a multicellular organism. It occurs not in the body, but in artificial environments, such as in a test tube or reaction vessel, or in cell culture. It refers to an event or phenomenon.

[0111] In Vivo: As used herein, the term "in vivo" refers to human and non-human animals. This refers to events that occur within multicellular organisms such as objects. In the context of cell-based systems, this The term refers to events that occur within living cells (as opposed to, for example, in vitro systems). It is sometimes used for that purpose.

[0112] Nonviral vectors: When used herein, the term "nonviral vectors" is used in this specification. For example, nanoparticles, liposomes, lipid particles, carbon, non-reactive metals, gelatin and / or It contains polyamine nanospheres.

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

[0114] Polypeptide: As used herein, the term "polypeptide" means peptide bond This term refers to a continuous chain of amino acids linked together via a fusion chain. This term can refer to amino acid chains of any length. It is used to refer to, but to those skilled in the art, this term is not limited to long chains and pep It is understood that it can also refer to the smallest chain containing two amino acids linked by a tide bond. It will be done. As is known to those skilled in the art, polypeptides are processed and / or modified. It's okay if it's not allowed.

[0115] Protein: As used herein, the term "protein" refers to individual units and It refers to one or more polypeptides that function as a single polypeptide. and permanent or temporary physical interactions with other polypeptides to form separate functional units When no binding is required, the terms "polypeptide" and "protein" can be used interchangeably. It can be used. A separate functional unit is composed of multiple polypeptides that physically associate with one another. In this context, the term "protein" refers to a group of organisms that are physically bound together and function as separate units. This refers to multiple polypeptides capable of performing certain functions.

[0116] Subject: As used herein, the term "subject" means human or any non-human animal. Things (for example, mice, rats, rabbits, dogs, cats, cows, pigs, sheep, horses, or This refers to primates. Humans include prenatal and postnatal forms. In many embodiments, the subject is The subject is a human being. The subject may be a patient, and the patient is seeking a medical provider for the diagnosis or treatment of a disease. This refers to a person who visits. The term "subject" in this specification means "individual" or "patient". It is used interchangeably with "[...]. The subject may be suffering from a disease or disability or They are susceptible to these conditions, but may or may not exhibit symptoms of disease or disorder.

[0117] Substantially: As used herein, the term “substantially” refers to the characteristics covered by the subject matter. This refers to a qualitative state that represents all or nearly all range or degree of a property. Those skilled in the art in the field will know that biological and chemical phenomena are rarely, if any, completely. It does not reach and / or progress to completeness or does not achieve a certain result or It will be understood that this does not avoid. The term "substantially" is therefore used by many organisms. It is used to express the inherent lack of integrity in scientific and chemical phenomena.

[0118] Afflicted: An individual who is "afflicted" with a disease, disorder, and / or condition is an individual suffering from a disease, disorder Diagnosed with or exhibiting one or more symptoms of the condition and / or state.

[0119] Therapeutic effective dose: As used herein, the term “therapeutic effective dose” of a therapeutic agent means Administered to subjects who have or are susceptible to diseases, disorders and / or conditions. When a disease, disorder and / or condition has symptoms(s)(multiple) onset, treatment, diagnosis, prevention and This means a sufficient amount to cause / delay. A therapeutically effective dose is typically at least 1 Those skilled in the art will understand that it is administered according to a dosage plan that includes the number of doses.

[0120] To treat: When used herein, "to treat," "to treat," or "to treat" The term "and" refers to one or more symptoms or characteristics of a particular disease, injury, and / or condition. To partially or completely reduce, improve, alleviate, inhibit, or prevent their onset, or to delay their onset. To reduce the severity and / or decrease their incidence This refers to any method used to reduce the risk of developing disease-related symptoms. For the purpose of this, it is also applied to subjects who do not show signs of the disease and / or who only show early signs of the disease. You may do so.

[0121] Vector: As used herein, the term "vector" means any carrier and any This refers to a combination of foreign genes (multiple genes are possible). Vectors include, in particular, non-viral genes. This may include vectors, viral vectors, and any combination thereof. For example, non Viral vectors include, in particular, liposomes, spheroplasts, red blood cell ghosts, and Lloyd's metal, calcium phosphate, DEAE dextran plasmid, or combinations thereof This includes, but is not limited to, combinations of viral vectors, including retro. Viral vectors, lentiviral vectors, pseudotype vectors, adenoviruses Vectors, adeno-associated virus vectors, hybrid viruses, and any combination thereof This may include, but is not limited to, combinations.

[0122] Transduction: As used herein, the term "transduction" refers to the introduction of foreign DNA into a foreign DNA. This refers to the process by which viruses are introduced into other cells via a viral vector. The vectors are known in this field, for example, retroviral vectors, and moreover, retroviral vectors, etc. Viral vectors, pseudotype vectors, adenovirus vectors, adeno-associated virus This includes ruth vectors and any combination thereof.

[0123] Transfection: As used herein, “transfection” The term refers to the process of introducing nucleic acids into cells by nonviral methods. In terms of application, the method described herein is suitable for transfection of target cells. .

[0124] In this specification, the enumeration of numerical ranges by endpoints includes all numbers and that fall within that range. Includes decimals (for example, 1-5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4 and 5) (Included). All numbers and their decimals are presumed to be modified by the term "approximately". It should also be understood that this is the case.

[0125] Various aspects of this disclosure are described in detail in the following sections. This does not limit the scope of this disclosure. Each section may be applicable to any aspect of this disclosure. In this application, unless otherwise stated, the use of "or" means "and / or". To taste. When used herein, the singular forms "a," "an," and "the" are used in context. Unless otherwise explicitly indicated, it includes both singular and plural referents.

Claims

1. A system for introducing a vector into cells, wherein the system is The capillary space and the extracapillary space separated from the capillary space by a porous membrane are defined. The filter module that makes up the, The capillary space is fluidly connected to both ends, with each being a transduction medium, cells, and a vector, respectively. A pair of capillary ports that receive, It is connected to both ends of the space outside the capillary tube and is fluidly connected to the source of the culture medium outside the capillary tube and the waste container. A pair of capillary outer ports and The system comprising the above.

2. The claim further comprises a recovery container fluidly connected to at least one of the capillary ports. The system described in 1.

3. The transduction medium, the cells, and the vector are placed in at least one of the capillary ports. - Further comprising an in-capillary pump capable of supplying each of the flows, claim The system described in 1 or 2.

4. The capillary pump transports the cells and the vector into the capillary port during the first period. During the first state and second period of supplying to the port, the transduction medium is supplied to the capillary port. The system according to claim 3, which is operable in a second state of supplying to.

5. A waste container communicating with the space outside the capillary tube via at least one of the capillary tube outer ports. The system according to any one of claims 1 to 4, further comprising:

6. A device that can be operated to supply the flow of the extracapillary culture medium to each of the extracapillary ports. The system according to any one of claims 1 to 5, further comprising an extracapillary pump.

7. It is operable to supply a flow of waste fluid from the capillary external port to the waste container. The system according to any one of claims 1 to 6, further comprising an extracapillary pump.

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

9. The porous membrane allows particles having a size of less than approximately 50 kDa to pass through the pores from the capillary space. The system according to any one of claims 1 to 8, comprising the holes that allow passage.

10. The capillary space defines a transduction zone, as described in any one of claims 1 to 9. Stem.

11. A system for introducing a virus or non-viral vector into a cell, wherein the The stem is, A hollow fiber that defines the space inside the capillary extending from the first end to the second end, A casing, wherein one or more holograms are located from the first end to the second end. - Surrounding the fiber, a capillary space is formed between the hollow fiber and the casing. A first port is defined between the first end and the capillary space adjacent to it, and is fluidly connected to the capillary space. , comprising a second port that is fluidly connected to the adjacent capillary space at the second end, The casing and, Fluid flow is connected to the capillary space through the first port and the second port, respectively. The combined transduction medium supply source, The cells include the capillaries, which are connected via the first port and the second port, respectively. A cell supply source fluidly connected to the internal space of the tube, The virus or non-viral vector comprises the first port and the second port A virus supply source is fluidly connected to the capillary space via each of the to The system comprising the above.

12. The capillary space and The system according to claim 11, further comprising a fluid-connected recovery container.

13. Each of the transduction medium source, the cell source, and the virus source and fluid The sys according to claim 11 or 12, further comprising a capillary pump including a connected inlet. Tem.

14. The capillary pump is fluidly connected to the capillary space via the first port. The outlet 1 and the second outlet, which is fluidly connected to the capillary space via the second port The system according to claim 13, including.

15. The casing includes a third port communicating with the space outside the capillary tube, and the system The device further comprises a waste container that communicates with the space outside the capillary tube via the third port. A system described in any of the requirements 11 to 14.

16. The extracapillary culture medium supply source, which is fluidly connected to the space outside the capillary via the third port, The system according to claim 15, which includes the following:

17. The third port is located adjacent to the first end of the capillary space, and the The stem is fluidly connected to the space outside the capillary and adjacent to the second end of the space inside the capillary. The system according to claim 16, further comprising a fourth port arranged therein.

18. The waste container and the capillary culture medium supply source are each connected to the third port and the The fourth port is in communication with the space outside the capillary tube, as described in claim 17. system.

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

20. The hollow fiber contains particles having a size of less than approximately 50 kDa within the capillary space. The hole is provided that allows something to pass through from the hole, according to any one of claims 11 to 19. The system.

21. The capillary space extending from the first end to the second end and the space extending from the first end to the second end Using hollow fibers to define the extracapillary space surrounding the capillary space at the end, A method for introducing a RUSS or non-viral vector into cells, The virus or non-viral vector is filled into the capillary space of the hollow fiber. The steps to take, The steps include filling the capillary space of the hollow fiber with the cells and The method, including the method described above.

22. Filling the space inside the capillary with the aforementioned virus or nonviral vector is as described above. The virus or non-viral vector is placed at the first end and the second end of the capillary space. The method according to claim 21, comprising filling from at least one of the ends.

23. Filling the space inside the capillary with the aforementioned virus or nonviral vector is as described above. The virus or non-viral vector is placed at the first end and the second end of the capillary space. The method according to claim 21 or 22, comprising filling from each end.

24. Filling the capillary space with the cells is the process of filling the capillary space with the cells Claims 21 to 2 include filling from at least one of the first end and the second end. The method described in any of the three points.

25. Filling the capillary space with the cells is the process of filling the capillary space with the cells The following are claims 21 to 23, which include filling from each of the ends of the first and the second ends. Either method.

26. The steps include transduction into the cells within the capillary space of the hollow fiber, The steps include: recovering the transdermal cells from the capillary space of the hollow fiber; The method according to any one of claims 21 to 25, further comprising:

27. Recovering the transduced cells from the capillary space is done by using the flushing fluid. The method according to claim 26, comprising filling the space outside the capillary with low fiber.

28. Recovering the transduced cells from the capillary space is done by using the flushing fluid in front of This includes filling the capillary space from one of the first or second ends. The method according to claim 26 or 27.

29. Any of claims 21 to 28 further includes recovering waste from the space outside the capillary tube. The method used.

30. The cells and the virus or nonviral vector are packed together, claim 2. The method described in any of 1 to 29.

31. The cells and the virus or nonviral vector are packed separately, claim 2 The method described in any of 1 to 29.

32. The cells are loaded prior to the virus or nonviral vector, claim 2. The method described in any of 1 to 29.

33. Claim 2, the virus or nonviral vector is loaded prior to the cells. The method described in any of 1 to 29.

34. The aforementioned cells are 1 × 10 ３ from 1 x 10 １０ It is filled with a concentration in the range of cells / ml. The method according to any one of claims 21 to 33.

35. The amount of cells used to fill the hollow fiber is a function of the size of the inner surface area of ​​the hollow fiber. The method according to any one of claims 21 to 34, comprising filling cells at a rate.

36. Claim 2, the virus or non-viral vector is filled as a viral particle. The method described in any of 1 to 35.

37. Claim 2, the virus or non-viral vector is packed as a nucleic acid vector. The method described in any of 1 to 36.

38. Approximately 5-100μl / min / cm ２ Between these, the inner surface area of ​​the hollow fiber is 1 square centimeter The cells and the virus or non-viral vector are filled at a flow rate per inch meter. The method according to any one of claims 21 to 37, further comprising doing the following.

39. The filling of the hollow fiber at a flow rate per square centimeter of the inner surface area is as follows: Approximately 5-20μl / min / cm ２ The method according to any one of claims 21 to 38.

40. The vector is a lentivirus, retrovirus, adenovirus, adeno-associated virus. The person according to any one of claims 21 to 39, derived from Rus or a hybrid virus. Law.

41. The method according to claim 40, wherein the vector is a retrovirus.

42. The method according to claim 40, wherein the vector is a lentivirus.

43. The vectors include nanoparticles, liposomes, lipid particles, carbon, non-reactive metals, gelatin, and / or the method according to any one of claims 21 to 39, comprising polyamine nanospheres. 。

44. The cells and the viral vector have an infection multiplicity in the range of about 0.25 to about 4.

0. The method according to any one of claims 21 to 43, wherein the capillary space is filled with MOI.

45. The cells and the viral vector are packed into the capillary space at an MOI of approximately 2.

5. The method according to claim 44.

46. The cells are B cells, T cells, NK cells, monocytes, progenitor cells, or cell lines, claim The method described in any of items 21 to 45.

47. A population of cells produced by the method described in any one of claims 21 to 46.

48. A pharmaceutical composition comprising cells produced by the method described in any one of claims 21 to 46. 。

49. A method for producing a cell therapy product containing one or more transduced cells, (i) The capillary space extending from the first end to the second end for transduction into the cell To prepare a system equipped with a defining hollow fiber, (ii) Fill the capillary space with a population of cells and a virus or non-viral vector. This results in the transduction of one or more cells within the capillary space, (iii) Collect a population of cells containing one or more transduced cells from the space inside the capillary. thing and The method, including the method described above.

50. The aforementioned cell populations include αβ T cells, γδ T cells, NK cells, HSCs, and macrophages. The method according to claim 49, selected from dendritic cells and iPSCs.

51. The virus or nonviral vector comprises a recombinant receptor, claim 49 or 5 The method described in 0.

52. The method according to claim 51, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

53. The method according to claim 49, wherein the transduced cells contain a recombinant receptor on the surface of the cells.

54. The aforementioned chimeric antigen receptors are CD44, CD19, CD20, CD22, CD23, CD 30, CD89, CD123, CS-1, ROR1, Mesothelin, c-Met, PSMA , Her2, GD-2, CEA, MAGE A3 TCR, EGFR, HER2 / ERB Select one or more from B2 / neu, EPCAM, EphA2, CEA, and BCMA. The method according to claim 52, comprising an extracellular ligand-binding domain that targets a tumor antigen. Law.

55. The method described above further comprises the step of isolating the transduced cells, according to claims 49 to 54. One of the methods described above.

56. The above method includes the step of growing the recovered cells in a bioreactor. The method according to any one of claims 49 to 55, including the above.

57. The method includes the step of freezing the recovered cells in a suitable cryopreservation medium. The method according to any one of claims 49 to 56, further comprising:

58. The system has a first port that is fluidly connected to the first end and the adjacent capillary space. It includes a second port that is fluidly connected to the capillary space adjacent to the second end. The method according to any one of claims 49 to 57.

59. Filling the space inside the capillary with the aforementioned virus or nonviral vector is as described above. The virus or non-viral vector is placed at the first end and the second end of the capillary space. The method according to any one of claims 49 to 58, comprising filling from at least one end. 。

60. Filling the space inside the capillary with the aforementioned virus or nonviral vector is as described above. The virus or non-viral vector is placed at the first end and the second end of the capillary space. The method according to any one of claims 49 to 58, comprising filling from each end.

61. Filling the capillary space with the cells is the process of filling the capillary space with the cells Claims 49 to 6 include filling from at least one of the first end and the second end. The method described in any of the following ways.

62. Filling the capillary space with the cells is the process of filling the capillary space with the cells The following are claims 49 to 60, which include filling from each of the ends of the first and second ends. Either method.

Images