Systems and methods for bio-manufacturing

Abstract

A sterile bio-processing system for cell culturing, the system comprising at least two closed sterile elements, configured to contain fluid, wherein: each of the elements comprises at least one sterile tube, configured for fluid communication; and all of said sterile tubes are made of the same material, adapted to be welded one to another, to allow fluid communication between the different sterile elements of the system; and thereby enable the system to be a closed sterile system.

Description

SYSTEMS AND METHODS FOR BIO-MANUFACTURINGBACKGROUND OF THE INVENTION

[0001] Bio-Manufacturing of commercial scale living medicine must meet the highest regulatory requirements. Today’s bio-manufacturing processes include automatic devices combined with manual processes, carried out in a laminar flow hood to provide an aseptic work area, allowing the containment of infectious splashes or aerosols generated by many microbiological procedures. Bacterial, mycoplasma and other microorganism contaminations are one of the main concerns in culturing cells intended for cell therapies.

[0002] Accordingly, there is a long felt need to culture cells, in an entirely closed system, with minimal manual steps, especially for use in Cell and Gene Therapies (CGT).SUMMARY OF THE INVENTION

[0003] According to some embodiments of the invention a new sterile bio-processing system is provided configured for cell culturing. The system comprises at least two closed sterile elements, configured to contain fluid, wherein:• each of the elements comprises at least one sterile tube, configured for fluid communication; and• all of said sterile tubes are made of the same material, adapted to be welded one to another, to allow fluid communication between the different sterile elements of the system; and thereby enable the system to be a closed sterile system.

[0004] According to some embodiments, the sterile tubes are at least partially flexible.

[0005] According to some embodiments, the material of sterile tubes comprises PolyVinyl Chloride (PVC), or Thermo-Plastic Elastomer (TPE).

[0006] According to some embodiments, each of the sterile tubes is initially provided with a sterile fluid connection, at its proximal end, to its respective element and is sealed, at its distal end.

[0007] According to some embodiments, the sterile elements are: rigid vessels, flexible vessels, at least partially flexible vessels, and any combination thereof.

[0008] According to some embodiments, the system’ s elements comprise at least two of:• at least one culturing vessel; for a non-limiting example, a G-Rex® culturing vessel;• at least one container, configured to accommodate tissue and / or cells;• at least one bottle, configured to accommodate tissue and / or cells;• at least one syringe;• at least one filtering bag;• at least one filtering device;• at least one waste bag;• at least one fluid bag; according to some embodiments, the fluid is liquid media provided for the cell culture;• at least one manual or automatic pipette;• at least one harvesting element; for a non-limiting example, GatheRex®;• at least one washing element; for a non-limiting example, Lovo®; and• any combination thereof.

[0009] According to some embodiments, the cells comprise at least one of: tumor infiltrating lymphocytes (TILs), T cells, CAR-T, engineered T-cell receptor (TCR) cells, Natural Killer (NK) cells, CAR-NK, Natural killer T (NKT) cells, and CAR-NKT cells and any combination thereof.

[0010] According to some embodiments, the closed sterile system is configured to avoid the use of a laminar flow hood and / or any open process.

[0011] According to some embodiments a new method is provided for closing a sterile bioprocessing system for cell culturing, according to any one of the above mentioned embodiments; the method comprising:• sterile cutting two of the system’ s sterile tubes, providing each with a cut-edge; and• sterile welding cut-edges of both sterile tubes one to another, configured to allow sterile fluid communication between their respective elements.

[0012] According to some embodiments, the step of cutting is provided simultaneously.

[0013] According to some embodiments, the method further comprising sterile cutting and sterile sealing of at least one sterile tube, configured to aseptically disconnect its fluid communication.

[0014] According to some embodiments, the method further comprising the sterile cutting and the sterile welding any one of the system’s communication tubes more than one time, to allow fluid communication between its respective element, sequentially, with several different elements of the system, via their respective sterile tube.

[0015] According to some embodiments, the steps of sterile cutting and sterile welding are provided via a sterile welding machine.

[0016] According to some embodiments, the method further comprising avoiding the use of a laminar flow hood and / or any open process.

[0017] According to some embodiments, a new closed sterile bottle is provided, configured for cell bio-processing, wherein:• the bottle comprises at least one sterile tube, configured for fluid communication; and• said sterile tube is made of Poly -Vinyl Chloride (PVC) or Thermo-Plastic Elastomer (TPE), adapted to be welded to any other PVC or TPE tube, respectively.

[0018] According to some embodiments, a new closed sterile syringe is provided, configured for cell bio-processing, wherein:• the syringe comprises at least one sterile tube

[0200] , configured for fluid communication; and• said sterile tube is made of Poly -Vinyl Chloride (PVC) or Thermo-Plastic Elastomer (TPE), adapted to be welded to any other PVC or TPE tube, respectively.

[0019] According to some embodiments, a new method is provided for culturing cells, comprising seeding cells in the sterile bio-processing system, according to any one of the above mentioned embodiments.

[0020] According to some embodiments, the cells comprise at least one of: tumor infiltrating lymphocytes (TILs), T cells, CAR-T, engineered T-cell receptor (TCR) cells,Natural Killer (NK) cells, CAR-NK, Natural killer T (NKT) cells, and CAR-NKT cells and any combination thereof.

[0021] According to some embodiments, the method further comprising avoiding the use of a laminar flow hood and / or any open process.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0023] Fig. 1 schematically illustrates a bio-processing closed system, according to some embodiments of the invention;

[0024] Fig. 2 demonstrates a bio-processing closed culturing vessel, according to some embodiments of the invention;

[0025] Fig. 3 demonstrates a bio-processing closed filtering bag, according to some embodiments of the invention;

[0026] Fig. 4A demonstrates a welded connection of two fluid tubes, according to some embodiments of the invention;

[0027] Fig. 4B demonstrates a sealed and cut fluid tube, according to some embodiments of the invention;

[0028] Fig. 5 schematically illustrates a bio-processing closed bottle, according to some embodiments of the invention;

[0029] Figs. 6A and 6B schematically illustrate and demonstrate a bio-processing closed syringe, according to some embodiments of the invention; and

[0030] Fig. 7 schematically illustrates TILs manufacturing flow diagram.

[0031] Fig. 8 schematically illustrates the process of TIL therapy, according to one embodiment, including TIL manufacturing and administration.

[0032] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0033] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0034] As used herein, in one embodiment the term “about” refers to ± 10 %. In another embodiment, the term “about” refers to ± 9 %. In another embodiment, the term “about” refers to ± 9 %. In another embodiment, the term “about” refers to ± 8 %. In another embodiment, the term “about” refers to ± 7 %. In another embodiment, the term “about” refers to ± 6 %. In another embodiment, the term “about” refers to ± 5 %. In another embodiment, the term “about” refers to ± 4 %. In another embodiment, the term “about” refers to ± 3 %. In another embodiment, the term “about” refers to ± 2 %. In another embodiment, the term “about” refers to ± 1 %.

[0035] The present invention provides a sterile bio-processing system for cell culturing. According to some embodiments, the sterile bio-processing system is a closed system. According to some embodiments, the sterile bio-processing system described herein allows production of TILs with a reduced risk of contamination and without including non-sterile processes (such as open manipulation) or the use of a biological safety cabinet. Accordingly, the system and related methods described herein allow culturing TILs, suitable for therapeutic use, with less human intervention and exposure to the environment.

[0036] The term “closed system” as used herein may encompass a system which is closed to the outside environment. In some embodiments, after a tumor biopsy is added to the closed system, the system is not opened to the outside environment until the cells are ready to be used in a medicinal product. At the end of the process the cell therapy product is ready for infusion into the patient.

[0037] According to some embodiments, the terms “sterile” and “aseptic” are interchangeable and refer to an element and / or a system that is free of unintended microorganisms. According to some embodiments, element and / or the system is not open to the environment and therefore does not allow outside contamination, i.e., does not allow microorganisms to enter the system.

[0038] According to some embodiments, the term “non-sterile” means that the sterility cannot be assured, for example, following exposure to the environment, which could potentially lead to the presence of microorganisms.

[0039] According to some embodiments, the phrases “laminar flow cabinet”, “tissue culture hood”, “laminar flow hood”, and “biological safety cabinet”, are interchangeably used to refer to a partially enclosed bench work surface designed to prevent contamination of biological samples, semiconductor wafers, or any particle sensitive materials.

[0040] According to some embodiments, the phrase “open process”, or “open manipulation” as used herein, may encompass processes or steps performed outside of the closed system described herein. In some embodiments, such processes could expose the cell culture to the outside environment.

[0041] Very few instruments are designed specifically for Cell and Gene Therapy (CGT) processes. Repurposing bioprocessing equipment rather than tailor-made technologies is required to deliver advanced therapies to patients in the real world. The patient at the end of the process can then receive the treatment at a healthcare facility via medical devices.

[0042] According to some embodiments of the invention, new configurations are required to overcome a technological gap between the bioprocessing world, which tends to favor Thermoplastic Elastomers (TPE) tubing (such as C-Flex and Tygon) as standard, in contrast to the vastly used PVC standard in the medical device industry.

[0043] Tube welding is a key method in aseptic work required for the closed system manufacturing approach. TPE and PVC welding are two different technologies with their exclusive instrumentation.

[0044] On one hand, sterile bottles with transfer caps and pre-assembled syringes with tubing and stopcock for liquid management are commercially available only with TPEtubes. On the other hand, the culture vessel (G-Rex) and downstream instrumentation (like LOVO) are available only with PVC tubing.

[0045] Accordingly, incorporation of PVC tubing for bioprocess products is a new solution.

[0046] According to some embodiments of the invention, and as demonstrated for example in Fig. 1, a new sterile bio-processing system

[0100] is provided, configured for culturing cells; the system comprises at least two closed and sterile elements [110,121,122,141,142] that are configured to contain fluid, wherein:• each of the sterile elements comprises at least one sterile tube

[0200] , configured for fluid communication; and• all of said sterile tubes are made of the same material, adapted to be welded one to another

[0210] , as demonstrated in Fig. 4A for tubes [200A,200B], to allow fluid communication between the different sterile elements of the system; and thereby enable the system to be a closed sterile system.

[0047] According to some embodiments of the invention, and as demonstrated for example in Fig. 5, a new closed sterile bottle

[0140] is provided, configured for cell bio-processing; wherein:• the bottle comprises at least one sterile tube

[0200] , configured for fluid communication; and• said sterile tube is made of a material, adapted to be welded any one of the system’ s

[0100] sterile communication tubes

[0200] , to allow fluid communication between the bottle and anyone of the sterile elements of the system

[0100] ; and thereby enable the system to be a sterile closed system.

[0048] According to some embodiments of the invention, and as demonstrated for example in Figs. 6A and 6B, a new closed sterile syringe

[0180] is provided, configured for cell bioprocessing; wherein:• the syringe comprises at least one sterile tube

[0200] , configured for fluid communication; and• said sterile tube is made of a material, adapted to be welded any one of the system’ s

[0100] sterile communication tubes

[0200] , to allow fluid communication betweenthe syringe and anyone of the sterile elements of the system

[0100] ; and thereby enable the system to be a closed sterile system.

[0049] According to some embodiments, and as demonstrated in Fig. 1, the system’s elements comprise at least two of:• at least one culturing vessel [121,122]; for a non-limiting example, a G-rex® culturing vessel / s;• at least one container [110,141,142], configured to accommodate tissue and / or cells;• at least one bottle [140,141,142], configured to accommodate tissue and / or cells;• at least one syringe

[0180] ;• at least one filtering bag

[0130] ;• at least one filtering device;• at least one waste bag;• at least one fluid bag [151,152]; according to some embodiments, the fluid is liquid media provided for the cells’ culture;• at least one manual and / or automatic pipette;• at least one harvesting element

[0160] ; for a non-limiting example, GatheRex® device;• at least one washing element

[0170] ; for a non-limiting example, Lovo® device; and• any combination thereof.

[0050] According to some embodiments, and as demonstrated in Fig. 3, for a non-limiting example of a filtering bag

[0130] , the sterile tube

[0200] is in direct communication with the element’ s accommodated fluid.

[0051] According to some embodiments, and as demonstrated in Fig. 5, for a non-limiting example of a bottle

[0140] , the sterile tube

[0200] is in non-direct communication with the element’s accommodated fluid. According to such embodiments, the element

[0140] further comprises at least one of: a sterile connector

[0410] ; a sterile cap

[0420] ; a sterile sealer

[0430] ;• a sterile tube

[0400] that can be made of a different material; a material that is not adapted to be welded to the system’s closing sterile tubes

[0200] ; and• any combination thereof; for allowing the sterile fluid communication.

[0052] According to some embodiments, the cell culturing comprises cell expansion. According to some embodiments, the cells comprise at least one of: tumor infiltrating lymphocytes (TILs), T cells, CAR-T, engineered T-cell receptor (TCR) T cells, Natural Killer (NK) cells, CAR-NK, Natural killer T (NKT) cells, and CAR-NKT cells and any combination thereof. According to some embodiments, the cultured cells comprise TILs. According to some embodiments, the cultured cells comprise T cells. According to some embodiments, the cultured cells comprise CAR-T cells. According to some embodiments, the cultured cells comprise Natural Killer (NK) cells. According to some embodiments, the cultured cells comprise T-cell receptor (TCR) cells.

[0053] TILs are white blood cells that leave the bloodstream and migrate towards a tumor and try to attack it. According to embodiments, TILs comprise T cells. According to embodiments, TILs comprise B cells. According to embodiments, TILs comprise natural killer (NK) cells. According to embodiments, TILs comprise macrophages. According to embodiments, TILs comprise neutrophils. According to embodiments, TILs comprise dendritic cells. According to embodiments, TILs comprise mast cells. According to embodiments, TILs comprise eosinophils. According to embodiments, TILs comprise basophils. According to embodiments, TILs comprise plasma cells. According to embodiments, TILs comprise mature dendritic cells. According to embodiments, TILs comprise antigen presenting cells (APCs).

[0054] According to embodiments, TILs comprise CD45+ cells. According to embodiments, TILs comprise CD4+ cells. According to embodiments, TILs comprise CD8+ cells. According to embodiments, TILs comprise CD 163+ cells. According to embodiments, TILs comprise CD20+ cells. According to embodiments, TILs comprise CD3+ cells. According to embodiments, TILs comprise CD 138+ cells. According to embodiments, TILs comprise CD 163+ cells. According to embodiments, TILs comprise CD56+ cells. According to embodiments, TILs comprise FoxP3+ cells. According toembodiments, TILs comprise DC-LAMP+ cells. According to embodiments, TILs comprise CD28+ cells. According to embodiments, TILs comprise CD69+ cells.

[0055] According to embodiments, TILs comprise a combination of different types of lymphocytes. A skilled artisan would appreciate that different combinations of lymphocytes have different capacities for killing a tumor. According to embodiments, disclosed herein are specific combinations of lymphocytes particularly potent for killing a tumor.

[0056] According to embodiments, TILs or TILs are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen, e.g., any of the cancer antigens described herein. According to embodiments, a TCRs comprises antigenic specificity for a melanoma antigen. According to embodiments, the antigen comprises gplOO or MART-1. According to embodiments, TILs or TILs are modified to express a chimeric antigen receptor (CAR) having antigenic specificity for a cancer antigen, e.g., any of the cancer antigens described herein. According to embodiments, TILs or TILs are modified to express a cell growth factor that promotes the growth and activation of TILs or TILs. According to embodiments, a growth factors comprise T-cell growth factors, IL-2, IL-7, IL-12, IL-15, or IL-18. According to embodiments, modified TILs express the T-cell growth factor at high levels. T-cell growth factor coding sequences, are readily available in the art, as are promoters, the operable linkage of which to a T-cell growth factor coding sequence promote high-level expression. Suitable methods of modification are known in the art. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Methods for modifying TILs are known in the art. For example, the TIL may be transduced to express a TCR having antigenic specificity for a cancer antigen using transduction techniques described in Morgan et al., Science 314(5796): 126-9 (2006) and Johnson et al. Blood 114:535-46 (2009).

[0057] According to some embodiments, the sterile tubes are at least partially flexible.

[0058] According to some embodiment, the material of sterile tubes comprises one of: Poly-Vinyl Chloride (PVC), Thermo-Plastic Elastomer (TPE).

[0059] According to some embodiments, the sterile elements are: rigid vessels, flexible vessels, at least partially flexible vessels, and any combination thereof.

[0060] According to some embodiment, the fluid is mostly liquid and wherein the liquid motion between the different elements of the system is provided, either by direct or indirect communication, via at least one of: a pump, a syringe, use of the gravitation force, a manual or automatic pipette, a siphon, use of the communicating vessels law, and any combination thereof.

[0061] According to some embodiments, and as demonstrated in Fig. 2, for a nonlimiting example of a culturing vessel

[0120] , each of the sterile tubes

[0200] is initially provided with a sterile fluid connection, at its proximal end

[0201] , to its respective element

[0120] and is sealed, at its distal end

[0202] .

[0062] According to some embodiments, and as demonstrated in Figs. 2 and 5, for a nonlimiting example of a culturing vessel

[0120] , at least one element of the system can be in non-direct communication with a non- sterile tube or any other non- sterile element or non- sterile environment (only environment is shown); for example, for gas communication. A material that is not adapted to be welded to the system’s closing sterile tubes

[0200] . According to such embodiments, the fluid communication is via a sterilizing filter

[0310] , and optionally a sterile tube

[0400] , such that the system remains sterilized. According to such embodiments, the non-sterile tube (not shown) can be made of a different material,

[0063] According to some embodiments of the invention, a method is provided for closing a sterile bio-processing system for cell culturing, according to any one of the above mentioned embodiments; the method comprising:• sterile cutting two of the system’s sterile tubes providing each with a cut-edge [203A,203B]; and• sterile welding

[0210] the cut-edges [203A,203B] of both sterile tubes [200A,200B] one to another, as demonstrated in Fig. 4A, configured to allow fluid communication between their respective elements, and accordingly to avoid any non-sterile connection with the system’s elements, including avoiding the need to use a laminar flow hood.

[0064] According to some embodiments, the method further comprising sterile sealing of a section of one of the sterile tubes [200C], along its length, and then cutting

[0220] that sterile tube [200C], as demonstrated in Fig. 4B, configured for sterile disconnecting its fluid communication, wherein said cutting and disconnection is such that:• only one sterile element remains closed; or• both previously connected sterile elements remain closed.

[0065] According to some embodiments, the sealed and cut sterile tube may be a communication tube of previously two sterile tubes [200A,200B], which were previously welded, as shown in Fig 4A, for fluid communication between their respective sterile elements (elements not shown in Fig. 4A). According to such embodiments, after the sealing, the cutting can be provided such that:• both the separated sterile tubes remain sealed, and therefore their respective elements remain closed and sterile; or• only one of the two separated (originally sterile) tubes remains sealed, and therefore only its respective element remains sterile, as the other element (originally sterile) is now not required for the sterile system.

[0066] According to some embodiments, the method further comprises the sterile cutting and the sterile welding of any one of the system’ s sterile tubes more than one time, to allow fluid communication between its respective element, sequentially, with several different elements of the system, via their respective sterile tubes, i.e., a repeated welding and connection of a specific sterile tube, and its respective element, each time to a different element of the system.

[0067] According to some embodiments, the steps of sterile cutting and sterile welding are provided via a sterile welding machine; for a non-limiting example, CompoDock® sterile tube connection system, designed for sterile medical PVC tube connections.

[0068] According to some embodiments of the invention, a method is provided for using the bio-processing closed sterile system

[0100] , as shown in Fig. 1, according to any one of the above mentioned embodiments.

[0069] According to some embodiments, for example for the TIL manufacturing process, all procedures are performed using closed sterile manipulations, while the G-Rex’s [121,122] are placed in the incubators.

[0070] According to some embodiments of the invention, a method for culturing cells using the bio-processing closed sterile system

[0100] , as described herein, is provided.

[0071] According to some embodiments of the invention, a method for cell expansion using the bio-processing closed sterile system

[0100] , as described herein, is provided. According to some embodiments, the method for culturing of cells comprises seeding cells in a bio-processing closed sterile system

[0100] described herein.

[0072] According to some embodiments of the invention, and as demonstrated for example in Fig. 8, a process of TIL therapy

[0800] is provided, comprising: obtaining a biopsy of a fresh resected tumor

[0802] from a patient

[0801] ; processing the tumor specimen into fragments

[0803] ; placing the fragments in the bio-processing closed sterile system; expanding cells in a pre-Rapid Expansion Protocol (pre-REP)

[0804] ; harvesting cells from pre-REP culture; optionally cryopreserving cells

[0805] ; expanding cells in a Rapid Expansion Protocol (REP)

[0806] ; harvesting cells from REP culture; formulating harvested cells into a product

[0807] ; preparing a final medicinal TIL product for quality control testing

[0808] ; and administering TILs to the patient

[0809] .

[0073] According to some embodiments, the method for culturing cells comprises: (a) placing fragments processed from a resected tumor from a patient into the bio-processing closed sterile system described herein; (b) expanding cells in a pre-Rapid Expansion Protocol (pre-REP); (c) harvesting cells from pre-REP culture; (d) expanding cells in a Rapid Expansion Protocol (REP); and (e) harvesting cells from REP culture to obtain TILs.

[0074] According to some embodiments, the method for culturing cells further comprises: cryopreserving cells. According to some embodiments, cryopreserving cells is after the step of expanding cells in a pre-Rapid Expansion Protocol.

[0075] According to some embodiments, the method for culturing cells further comprises: filtering cells. According to some embodiments, filtering cells is after the step of expanding cells in a pre-Rapid Expansion Protocol.

[0076] According to some embodiments, the method for culturing cells further comprises: supplementing the culture medium with IL-2.

[0077] According to some embodiments, the cultured cells comprise at least one of: tumor infiltrating lymphocytes (TILs), T cells, CAR-T, T-cell receptor (TCR) cells, and Natural Killer (NK) cells. According to some embodiments, the cultured cells comprise TILs. According to some embodiments, the cultured cells comprise T cells. According to some embodiments, the cultured cells comprise CAR-T cells. According to some embodiments, the cultured cells comprise Natural Killer (NK) cells. According to some embodiments, the cultured cells comprise T-cell receptor (TCR) cells.

[0078] According to some embodiments, TILs comprise at least one of: T cells, B cells, natural killer (NK) cells, dendritic cells, plasma cells, antigen presenting cells (APCs), CD4+, CD8+, CD163+, CD20+, CD3+, CD138+, CD163+, CD56+, CD28+, CD69+, FoxP3+, DC-LAMP+ cells, and any combination thereof.

[0079] According to some embodiments, a step of seeding cells is provided. Seeding of tumor fragments on Pre- Rapid Expansion Protocol (REP), i.e., day 0. Tumor fragments, suspended in culture medium, are collected into a custom-made 500ml collection bottle

[0141] with a transfer cap with PVC tubing

[0200] in the biological hood. The bottle then is connected to a port of the culturing vessel (G-Rex®)

[0121] by welding tubes

[0200] , to allow the passing of the tumor fragments from the bottle to the G-Rex

[0121] in a closed system.

[0080] According to embodiments, TILs are extracted from tissue. According to embodiments, TILs are extracted from a resected tumor. According to embodiments, TILs are extracted from a tumor fragment. According to embodiments, TILs are extracted from a non-ablated, non-treated tissue. According to embodiments, TILs are extracted from a biopsy. According to embodiments, the biopsy comprises tumor stroma. According to embodiments, the biopsy comprises a tumor. According to embodiments, the biopsy comprises an ablated tissue. According to embodiments, the biopsy comprises an ablated tumor. According to embodiments, the biopsy comprises a resected tumor. According to embodiments, the biopsy comprises an area surrounding a tumor. According to embodiments, the biopsy comprises necrotic tissue. According to embodiments, the biopsy comprises any combination of a tumor, a tumor stroma, or a tissue surrounding a tumor.According to embodiments, the biopsy comprises tumor tissue devoid of normal tissue and necrotic areas.

[0081] According to some embodiments, the tumor is selected from melanoma, nonHodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colorectal cancer, kidney cancer, renal cell carcinoma, uterine cancer, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, testicular cancer, gastric cancer, esophageal cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), or any combination thereof.

[0082] According to some embodiments, a step of adding media to the culturing vessel to the is provided. IL-2 addition can be provided at Pre-REP days: 5+1, 9+1 and 12+1. According to some embodiments, the required volume of IL-2 is aseptically extracted from the lOOmL bag

[0151] , using a custom-made sampling syringe

[0180] . According to some embodiments, the IL-2 is then added to the G-Rex by welding tube

[0200] of the syringe

[0180] to a port the G-Rex

[0121] , via its port

[0200] .

[0083] According to some embodiments, a step of sampling is provided. Sampling can be provided at pre-REP days: 9+1, 12+1 and REP-day 11+1. Sampling of cells or culture medium is performed using a custom-made syringe

[0180] , aseptically welded via tube

[0200] to the G-Rex [121,121].

[0084] According to some embodiments, a step of pre-REP harvest is provided. The pre- REP culture harvest includes two steps:• first medium depletion from the G-Rex, and• then the removal of tumor fragments from single cells.

[0085] According to some embodiments, medium depletion is performed by extracting the upper level of the culture medium, into a waste bag, for example using the GatheRex® pump.

[0086] According to some embodiments, the tumor fragments are then separated from the single cells, using a straining bag

[0130] , with a 40pm mash filter separating its two sides.According to some embodiments, the straining bag

[0130] is welded via tubes

[0200] to the G-Rex, then using the GatheRex® pump, the G-Rex® content is transferred into the bag

[0130] and then strained through the mash, allowing the passage of only single cells, without any tumor fragments. The single cells are then collected in a 3L bag “cells master mix bag” [HO].

[0087] According to some embodiments, a step of REP culture seeding is provided. For the REP culture seeding, feeder cells and culture medium are added to the “cells master mix bag”

[0110] , containing the single cells collected from the Pre-REP process. The feeder cells are pre-packed in bags with PVC tubes and are added to the “cells master mix bag” by welding both bags

[0110] and gravitation. Culture medium packed in a bag with PVC tube (arrives packed in bags or pre-packed in suitable bags by the manufacturer team) is added to the “cells master mix bag” by welding, via tubes

[0200] , both bags and measuring the accurate volume using a hook-weight According to some embodiments, the seeding of the REP culture is performed by welding via tubes

[0200] the “cells master mix bag” to a port of each G-Rex500cs and transferring an accurate volume of mix, using a hook-weight.

[0088] According to some embodiments, another step of medium addition is provided. Medium addition can be provided at REP day 5 / 6, 7+1, and 11+1. Culture medium supplemented with IL-2 is added, during the REP stage. The required volume of IL-2 is then aseptically extracted from the lOOmL bag into a custom-made sampling syringe

[0180] and later added to the culture medium bag (arrives packed in bags or pre-packed in suitable bags by the manufacturer team). The culture medium is added to the G-Rex®.

[0089] According to some embodiments, a step of Cell harvesting is provided. The REP culture harvest includes two steps:• first medium depletion, and• then harvest of the cells.

[0090] According to some embodiments, the upper level of the culture medium (that does not contain cells) is removed from each G-Rex500cs

[0122] , into a waste bag welded, via tubes

[0200] , to a port of the G-Rex500cs, using the GatheRex® pump. Then, the remaining medium containing cells is collected into a collection bag or bottle, welded via another tube

[0200] to another port of the G-Rex500cs using the GatheRex® pump.

[0091] According to some embodiments, a step of Final formulation is provided. The bag containing the cells collected from all G-Rex500 vessels is welded, via tubes

[0200] , to the LOVO® sterile kit

[0170] . The cells are washed, formulated, and packed by the LOVO® system working in a closed system.

[0092] According to some embodiments, any one of the above mentioned embodiments or a combination thereof, is configured to avoid or at least minimize the use of a laminar flow hood.

[0093] Examples: TILs Manufacturing Process and Process Controls

[0094] Aim: demonstrate the ability to culture TILs which meet all acceptance criteria of TILs to be used as a medicinal product, in the closed system described herein in detail.

[0095] Methods: TILs were manufactured in a continuous process, starting with a patient’ s tumor biopsy and ending with a product configured to be provided to the patient via an infusion, for an autologous treatment. All processing took place within a closed system

[0100] as disclosed in any one of the above mentioned embodiments.

[0096] Processing was initiated with a 14+2 days pre-Rapid Expansion Protocol (pre-REP) process, involving the isolation of TILs from a patient’s tumor and the establishment of a primary culture of 5-50xl06TILs. Then, in the REP process, TILs were further expanded for additional 14+2 days to produce >10 xlO9TILs for a subsequent re-infusion back to the patient. The manufacturing process

[0700] includes manufacturing steps [701-705], as illustrated in Error! Reference source not found. 7 and described in the following paragraphs.

[0097] pre-REP Culture Seeding (Pre-REP day 0)

[0701]

[0098] A tumor specimen was resected in the hospital, collected into sterile a tube containing SPS-1® (UW Solution) Static Preservation Solution with 50 pg / mL gentamicin and transferred to a manufacturing facility at 2-8°C, where it was kept refrigerated, until use. Within 48 hours, from resection, the tumor tissue processing started. The tumor tissue was first washed four (4) times, by filtering through a Steriflip filter tube using phosphate- buffered saline (PBS) supplemented, with 50 pg / ml gentamicin, for the first three washes,and 50 pg / ml gentamicin for the last wash. The tumor specimen was then dissected into fragments smaller or equal to 8 mm3, using a sharp scalpel. About 100-400 fragments were placed in up to two units of a G-RexlOOcs

[0121] (closed system; 100-200 fragments per unit) in 250 mL of PRIME-XV T cell chemically defined media (CDM) supplemented with 3,000 lU / mL IL-2. The G-RexlOOcs flasks were then incubated in a humidified incubator at 37°C in 5% CO2.

[0099] IL-2 Supplement (Pre-REP Days 5±1, 9±1, 12±1)

[0702]

[0100] On days pre-REP 5+1, 9+1, 12+1, the culture medium was supplemented with 3,000 lU / ml IL-2, withdrawn from sterile bag, and connected by welding a syringe

[0180] to the G-Rex

[0121] without opening the system (as demonstrated in Fig. 1).

[0101] Pre-REP Culture Harvest (Pre-REP Day 9±1 or 12±1 or 14±2) and REP culture seeding / REP Day 0

[0703]

[0102] On pre-REP Days 9+1 and 12+1, the possibility of early harvest was evaluated according to lactate concentration, cell number, viability, and identity. First, lactate concentration in each G-RexlOOcs

[0121] was measured. If lactate concentration was equal or greater than 2mM in one of the G-RexlOOcs vessels, then a cell count was performed, for each G-RexlOOcs. If the total cell number (in both G-RexlOOcs together) was equal or greater than 35xl06, with at least 70% viability, then a cell identity test using flow cytometry was performed. The pre-REP culture was harvested within 24 hours, if the percentage of CD45+cells in each of the G-RexlOOcs was at least 70%. On pre-REP Day 14+2, the cells were harvested regardless of any criteria.

[0103] The pre-REP culture harvest was performed using a closed system

[0100] . First, the upper level of the culture medium (approximately 150 mL) that does not contain cells was removed from the G-RexlOOcs

[0121] using the Gatherex pump. Then, the lower level of the medium, containing cells and tumor fragments was collected into a cell-collection bottle through a standard transfusion set with a 200pm mesh filter separating its two sides. The G-RexlOOcs content was transferred and strained through the mesh

[0130] , allowing the passage of strictly only cells without tumor fragments. The harvest process was repeated for the second G-RexlOOcs into the same cell collection bottle, in order for TILs from the twoG-RexlOOcs to be pooled. The pooled collection bottle was sampled, and the following tests were performed: cell count, viability, and identity. The acceptance criteria for proceeding to REP were a cell count of at least 7xl06, Viability of at least 70% and CD45+cells of at least 70%. At this point, the pre-REP TILs were directly seeded for rapid expansion (REP).

[0104] The cells were seeded using a closed system

[0100] having one (1) to four (4) G- Rex500cs vessels

[0122] at a cell density of 4-10xl06TILs per G-Rex500 in 625 mL medium. The medium was composed of PRIME-XV T Cell CDM supplemented with 30 ng / mL anti-CD3 antibody (OKT-3) and 3,000 lU / mL IL-2.

[0105] Medium addition (REP Day 5±1, 7±1, 11±1)

[0704]

[0106] On REP Day 5+1 , 625 ml of PRIME XV CDM culture media supplemented with 3,000 lU / ml IL-2 were added into each G-Rex500cs. On REP days 7+1 and 11+1, 1,875 ml of culture media supplemented with 3,000 lU / ml IL-2 were added into each G-Rex500cs for a total final volume of 5L. All media additions were performed in a closed system.

[0107] TILs Harvest and Final Formulation (REP Day 14±1)

[0705]

[0108] The upper level of the culture medium that did not contain cells (approximately 4.5L) was removed from each G-Rex500cs using the Gatherex pump. Then, the remaining medium containing cells was collected from all G-Rex500cs vessels and pooled into a single collection bag. Next, the collection bag was welded to the LOVO sterile kit. LOVO is an automated and fully closed cell processing device. The cells were washed and formulated by the LOVO system in Plasma-Lyte containing 4% (w / v) human serum albumin (HSA). The final product was packaged in a single 500mL bag at a volume of 100-500mL and a cell concentration of 100-200xl06cells / mL.

[0109] Results’.

[0110] TILs were prepared from tumors of 3 patients (termed ER03, ER05, and ER06) in the closed system in a R&D Lab.

[0111] Table 1 shows the engineering run data of the 3 TIL batches

[0112] Table 1 : engineering runs data:

[0113] Table 2 shows the test results of 3 TIL batches produced in a closed system located in a standard R&D lab. It demonstrates successful manufacturing of a sterile TILs product using the closed system. Safety tests included Bacterial endotoxins, Sterility, and Mycoplasma testing, which all confirmed that there were no contaminations of the culture.

[0114] Table 2: Release testing of TILs produced in an R&D lab

[0115] TILs were prepared from 3 qualification batches (termed 100-pp-QB201DP- IL001090, 100-PP-QB201DP-IL00052, and 100-PP-QB201DP-IL00058) in the closed system in clean rooms in a GMP facility.

[0116] Table 3 shows the qualification data of the 3 TIL.

[0117] Table 3: qualification batches (QB) data:

[0118] Safety tests included Bacterial endotoxins, Sterility, and Mycoplasma testing, which all confirmed that there were no contaminations of the culture.

[0119] Table 4 shows the test results of 3 TIL batches produced in a closed system located in a clean room, in a GMP facility. It demonstrates successful manufacturing of a sterile TILs product using the closed system. Safety tests included Bacterial endotoxins, Sterility, and Mycoplasma testing, which all confirmed that there were no contaminations of the culture.

[0120] Table 4: Release testing of TILs produced in clean rooms in GMP facility:

[0121] Conclusion: The examples provided herein demonstrate successful preparation of TILs from tumors collected from 6 patients, 3 prepared in R&D labs (engineering runs) and 3 in cleanrooms (qualification batches). The test results meet all acceptance criteria of TILs to be used as a medicinal product. Advantageously, the closed system described herein allows production of TILs with a reduced risk of contamination and without including non- sterile processes or the use of a biological safety cabinet. Accordingly, the system and related methods described herein allow culturing TILs, suitable for therapeutic use, with less human intervention and at a reduced cost.

[0122] Different embodiments are disclosed herein. Features of certain embodiments may be combined with features of other embodiments; thus certain embodiments may be combinations of features of multiple embodiments.

[0123] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those ofordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMSWhat is claimed is:

1. A sterile bio-processing system for cell culturing, the system comprising at least two closed sterile elements, configured to contain fluid, wherein:• each of the elements comprises at least one sterile tube, configured for fluid communication; and• all of said sterile tubes are made of the same material, adapted to be welded one to another, to allow fluid communication between the different sterile elements of the system; and thereby enable the system to be a closed sterile system.

2. The system of claim 1, wherein the sterile tubes are at least partially flexible.

3. The system of claim 1, wherein the material of sterile tubes comprises Poly-Vinyl Chloride (PVC), or Thermo-Plastic Elastomer (TPE).

4. The system of claim 1, wherein each of the sterile tubes is initially provided with a sterile fluid connection, at its proximal end, to its respective element and is sealed, at its distal end.

5. The system of claim 1, wherein the sterile elements are selected from: rigid vessels, flexible vessels, at least partially flexible vessels, and any combination thereof.

6. The system of claim 1, wherein the system’s elements comprise at least two of:• at least one culturing vessel;• at least one container, configured to accommodate tissue and / or cells;• at least one bottle, configured to accommodate tissue and / or cells;• at least one syringe;• at least one filtering bag;• at least one filtering device;• at least one waste bag;• at least one fluid bag;• at least one manual or automatic pipette;• at least one harvesting element;• at least one washing element; and• any combination thereof.

7. The system of claim 1, wherein the cells comprise at least one of: tumor infiltrating lymphocytes (TILs), T cells, CAR-T, engineered T-cell receptor (TCR) cells, Natural Killer (NK) cells, CAR-NK, Natural killer T (NKT) cells, and CAR-NKT cells and any combination thereof.

8. The system of claim 1, wherein the closed sterile system is configured to avoid the use of a laminar flow hood and / or any open process.

9. A method for closing a sterile bio-processing system for cell culturing, according to claim 1; the method comprising:• sterile cutting of two of the system’s sterile tubes, providing each with a cutedge; and• sterile welding the cut-edges of both sterile tubes one to another, configured to allow sterile fluid communication between their respective elements.

10. The method of claim 9, wherein the step of cutting is provided simultaneously.

11. The method of claim 9, further comprising sterile cutting and sterile sealing of at least one sterile tube, configured to aseptically disconnect its fluid communication.

12. The method of claim 9, further comprising the sterile cutting and the sterile welding any one of the system’s communication tubes more than one time, to allow fluid communication between its respective element, sequentially, with several different elements of the system, via their respective sterile tube.

13. The method of claim 9, wherein the steps of sterile cutting and sterile welding are provided via a sterile welding machine.

14. The method of claim 9, further comprising avoiding the use of a laminar flow hood and / or any open process.

15. A closed sterile bottle, configured for cell bio-processing, the bottle comprises at least one sterile tube, configured for fluid communication; wherein said sterile tube is made of Poly-Vinyl Chloride (PVC) or Thermo-Plastic Elastomer (TPE), adapted to be welded to any other PVC or TPE tube, respectively.

16. A closed sterile syringe, configured for cell bio-processing, the syringe comprises at least one sterile tube, configured for fluid communication; wherein said sterile tube is made of Poly-Vinyl Chloride (PVC) or Thermo-Plastic Elastomer (TPE), adapted to be welded to any other PVC or TPE tube, respectively.

17. A method for culturing cells comprising seeding cells in the sterile bio-processing system according to claim 1.

18. The method of claim 17, wherein the cells comprise at least one of: tumor infiltrating lymphocytes (TILs), T cells, CAR-T, engineered T-cell receptor (TCR) cells, Natural Killer (NK) cells, CAR-NK, Natural killer T (NKT) cells, and CAR-NKT cells and any combination thereof.

19. The method of claim 17, further comprising avoiding the use of a laminar flow hood and / or any open process.

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