TW202136502A - Devices and methods for isolating tumor infiltrating lymphocytes and uses thereof - Google Patents

Abstract

The present invention provides methods for isolating and cryopreserving tumor infiltrating lymphocytes (TILs) and producing therapeutic populations of TILs, including methods via use of a kit and a semi-automatic device for aseptic disaggregation, enrichment, and cryopreservation of a resected tumor prior to expansion of the TIL population. The present invention also provides methods for expansion, and/or stabilization of TILs, for instance UTILs, compositions involving the same and methods of treatment involving the same.

Description

Device and method for separating tumor infiltrating lymphocytes and its use

The present invention provides methods and devices for separating and freezing tumor infiltrating lymphocytes (TIL) from excised tumors via semi-automatic aseptic tissue processing of tumors and thereby generating a therapeutic population of TILs.

T cells are derived from hematopoietic stem cells that reside in the bone marrow but then migrate to and mature in the thymus. During the maturation process, T cells undergo a series of selection events, thereby generating a diverse T cell repertoire. These cells are then released into the peripheral circulation to perform their specific functions as part of the acquired immune system.

T cells are not a homogeneous cell population, but are composed of many lineages, the main types of which are defined by the performance of two other cell markers. T cells expressing CD4 are generally called helper cells (Th) and are believed to coordinate many functions of the immune system through cell-cell contact and the production of mediator molecules called interleukins. CD8 T cells are considered to have cytotoxicity (Tc) and are considered to perform direct killing of target cells. These activities are controlled by the T cell receptor/antigen/MHC interaction-therefore, after successfully identifying the peptide/MHC on the target cell, CD4 and CD8 cells act synergistically through interleukin production and cytotoxic activity to eliminate Target cells include infected viruses and tumor cells.

T cells do not recognize complete proteins (antigens), but respond to short protein fragments that are presented on the surface of target cells by a specific protein called major histocompatibility complex (MHC). During the maturation process, T cells exhibit antigen-specific T cell receptors (TCR) on their cell surface, which recognize these short protein (peptide) antigens presented by MHC molecules. Therefore, only when the correct peptide is present on the surface of the target cell related to the correct MHC molecule, the T cell will activate its immune function. Therefore, the frequency of tumor-specific T cells is enriched in tumors, making them an ideal source of tumor-specific T cells (that is, tumor infiltrating lymphocytes (TIL)) (Andersen et al., Cancer Res. April 2012 1st; 72(7):1642-50. doi: 10.1158/0008-5472.CAN-11-2614. Epub 2012 February 6).

Of course, this description is highly simplified and represents a short general overview of T cell function. The acquired immune response does not work alone, but requires extensive interaction with a series of immune and non-immune cells to promote the efficient migration of T cells to the desired active site to ensure that the correct immune response is initiated and is no longer needed Then control and stop the immune response. Therefore, even in patients whose manufactured TIL initiates an immune response against tumors, it can be subsequently supported or weakened by the patient's own immune system and tumor environment.

Tumor-specific TILs are T cells isolated from tumors of patients with metastatic cancer. In most cancer patients, circulating tumor-specific T cells can hardly be detected in the blood. However, certain cancers such as skin melanoma appear to be immunogenic because of their ability to induce a significant number of T cells with anti-tumor activity during the natural process of tumor growth, especially in the tumor area (Muul et al., J Immunol. 1987 February 1;138(3):989-95). Tumor-reactive T cells that are "selected as T cells specific to tumors" can be isolated from tumor materials and expanded to a high number in vitro. Reports have shown that these cells contain anti-tumor reactivity, which can cause tumor destruction and clinical response when reinfused into patients (Dudley et al., Science. October 25, 2002; 298(5594): 850-4. Epub, September 19, 2002). In follow-up experiments, the importance of T cell characteristics is confirmed and the benefits of "young" fast-growing cells "young TIL" are confirmed, so that cells are "not specifically selected" at all. Notably, this produces about 50% of the TIL or the excellent response rate among the TIL selected by CD8 (Besser et al., Anticancer Res. January 2009; 29(1):145-54; Dudley et al., Clin Cancer Res. 2010 December 15; 16(24):6122-31. doi: 10.1158/1078-0432. CCR-10-1297. Epub 2010 July 28).

Andersen et al. (Cancer Res. 2012/4/1; 72(7):1642-50. doi: 10.1158/0008-5472.CAN-11-2614. Epub 2012/2/6) Compared with T cells in peripheral blood, tumors are enriched with melanoma-specific T cells (for known cancer antigens). This supports the following statement: when compared with early trials in melanoma patients using T cells isolated from peripheral blood and expanded in similar levels of IL2 or intravenous IL-2 alone, the isolated TIL population is the cause Enriched tumor-specific T cells with enhanced anti-tumor activity (LAK cells-Bordignon et al., Haematologica. December 1999; 84(12): 1110-49).

U.S. Patent No. 10,398,734 relates to a method for expanding TIL and generating a therapeutic population of TIL. The tumor of the '734 patent is transported as a bulk tumor, and the TIL inside the bulk tumor quickly becomes hypoxic and gradually deteriorates over time. The tumor of the '734 patent is also processed into fragments with degraded internal cell populations. In addition, the TIL used for manufacturing will only be the TIL amplified from the tissue fragments and will not be any TIL retained inside. Therefore, the resulting cell population may not reflect the complete diversity of the tumor environment.

The collection of TIL requires aseptic disaggregation of the solid tissue as the main tumor before culturing and expanding the TIL population. The conditions during the disaggregation of solid tissues and the time it takes to collect cells have a substantial impact on the survival rate and recovery rate of the final cellularized material. The solid tissue-derived cell suspension obtained using conventional methods usually includes a wide variety of different cell types, disaggregation media, tissue fragments, and/or fluids. This may require the use of selective targeting and/or isolation of cell types, for example before the manufacture of regenerative drugs, adoptive cell therapy, ATMP, diagnostic in vitro studies and/or scientific research.

Currently, selection or enrichment techniques generally use one of the following: size, shape, density, adhesion, and strong protein-protein interaction (ie, antibody-antigen interaction). For example, in some cases, the selection can be by providing a growth support environment and by controlling the culture conditions or interacting with more complex cell markers that are semi-permanently or permanently coupled to a magnetic or non-magnetic solid or semi-solid substrate. Role in progress.

For enrichment, separation or selection, any sorting technique can be used, such as affinity chromatography or any other antibody-dependent separation technique known in the art. Any ligand-dependent separation technique known in the art can be used in combination with positive and negative separation techniques that depend on the physical characteristics of cells. A particularly effective sorting technique is magnetic cell sorting. Methods of magnetically separating cells are commercially available from, for example, Thermo Fisher, Miltenyi Biotech, Stemcell Technologies, Cellpro Seattle, Advanced Magnetics, Boston Scientific or Quad Technologies. For example, monoclonal antibodies can be directly coupled to magnetic polystyrene particles (such as Dynal M 450 or similar magnetic particles) and used for, for example, cell separation. Dynabeads technology is not based on a tube column, but these magnetic beads and attached cells have liquid phase kinetics in the sample tube, and the cells are separated by placing the tube on a magnetic scaffold.

Enriching, sorting and/or detecting cells from a sample includes the use of monoclonal antibodies and colloidal superparamagnetic particles with organic coatings such as polysaccharides (e.g., magnetic activated cell sorting (MACS) technology (Miltenyi Biotec, Bergisch Gladbach) , Germany)). Particles (such as nanobeads or microbeads) can be directly combined with monoclonal antibodies or used in combination with anti-immunoglobulin, avidin or anti-hapten-specific microbeads, or coated with selective binding Characteristics of other mammalian molecules.

Magnetic particle selection techniques (such as those described above) separate cells positively or negatively by incubating cells with magnetic nanoparticles coated with antibodies or other parts of specific surface markers. This allows the cells expressing this marker to attach to the magnetic nanoparticles. After that, the cell solution is placed in a solid or flexible container in a strong magnetic field. In this step, cells are attached to the nanoparticles (expressing markers) and remain on the column, while other cells (not expressing markers) flow through. Using this method, cells can be separated positively or negatively with respect to one or more specific markers.

In the case of positive selection, after removing the column from the magnetic field, the cells attached to the magnetic column and exhibiting one or more relevant markers are washed out into different containers.

In the case of negative selection, the antibody or selective part used is directed against one or more unrelated surface markers known to be present on the cell. After the cell/magnetic nanoparticle solution is applied to the tube column, the cells expressing these antigens are bound to the tube column and the passing part is collected because it contains relevant cells. Since these cells are not coupled to one or more selective antibodies or partial labels of the nanoparticle, they are "unchanged." Known manual or semi-automated solid tissue processing steps are labor intensive and require knowledge of the technology.

In addition, when the material is used for therapeutic purposes, the treatment of environmental conditions that need to be strictly regulated during cell culture operations, such as as part of depolymerization, enzymatic digestion, and transfer to a storage device, or tissue treatment before it, or for decomposing Cultivation conditions for poly/cellularization and the rate of live tissue production. Usually, this process will require multiple pieces of laboratory and tissue processing equipment, as well as personnel with scientific and technical skills and knowledge. The critical stage is located in a hazard blockade or a sterile environment of one or more tissue processing facilities in order to safely perform the same activities and It also minimizes the risk of pollution.

The survival rate and recovery rate of the desired product from the tissue can be affected by the conditions during tissue collection, disaggregation, and cell collection. The present invention arises from the need to provide improved tissue processing, including equipment/devices that perform such processing to achieve the unmet needs described above.

The citation or identification of any documents in this application is not an admission that such documents can be used as the prior art of the present invention.

The present invention relates to a method for isolating a therapeutic population of tumor infiltrating lymphocytes (TIL), which may include: (a) Removal of the tumor from the individual; (b) Store the excised tumor in a single-use sterile kit, which contains: Depolymerization module for receiving and processing materials containing solid mammalian tissues; Optional enrichment module for filtering depolymerized solid tissue materials and separating undepolymerized tissues and filtrate; and A stabilization module for further processing and/or storage of depolymerized product materials as appropriate, Wherein each of the modules includes one or more flexible containers connected by one or more pipes adapted to enable tissue material to flow therebetween; and Each of the modules includes one or more ports to allow aseptic input of culture medium and/or reagents into one or more flexible containers; (c) Aseptically disaggregate and remove the tumor in the disaggregation module, thereby generating disaggregated tumors; (d) Perform the first expansion by culturing the disaggregated tumor in a cell culture medium containing IL-2 to produce a first population of UTIL; (e) Perform a second expansion by culturing the first population of UTIL together with additional IL-2, OKT-3 and antigen-presenting cells (APC) to produce a second population of TIL; (f) Collect and/or cryopreserve the second population of UTIL. In some embodiments, step a) is optional.

The present invention relates to a method for isolating a therapeutic population of cryopreserved tumor infiltrating lymphocytes (TIL), which may include: (a) Removal of the tumor from the individual; (b) Store the excised tumor in a single-use sterile kit, which contains: Depolymerization module for receiving and processing materials containing solid mammalian tissues; Optional enrichment module for filtering depolymerized solid tissue materials and separating undepolymerized tissues and filtrate; and A stabilization module for further processing and/or storage of depolymerized product materials as appropriate, Wherein each of the modules includes one or more flexible containers connected by one or more pipes adapted to enable tissue material to flow therebetween; and Each of the modules includes one or more ports to allow aseptic input of culture medium and/or reagents into one or more flexible containers; (c) Aseptically disaggregate and resect the tumor in the disaggregation module, thereby producing a disaggregated tumor. If the resected tumor can be stored at low temperature with minimal cell damage, it will be fully disaggregated; (d) Store the depolymerized tumor at low temperature in the stabilization module; (e) Perform the first expansion by culturing the disaggregated tumor in a cell culture medium containing IL-2 to produce a first population of UTIL; (f) Perform a second expansion by culturing the first population of UTIL together with additional IL-2, OKT-3 and antigen-presenting cells (APC) to produce a second population of TIL; (g) Collect and/or cryopreserve the second population of UTIL. In some embodiments, step a) is optional.

Depolymerization can include material understanding, enzymatic depolymerization, or physical and enzymatic depolymerization. In an advantageous embodiment, the depolymerized tumor is cellularized or purified.

In the present invention, the group of interconnected containers with specific individual functions maintains a sterile closed system to treat, optionally enrich but stabilize depolymerized and cellularized tumors. Basically, the present invention provides a rapid pre-sterilization environment to minimize the time required during the treatment of tumor resection and the risk of contamination or operator exposure.

Compared with standard non-closed tissue processing, the aseptic kit allows closed solid tissue processing, eliminating the risk of contamination of the final cellular product, especially when the process is performed in the tissue acquisition/acquisition site and needs to be stored before the final cell processing. Its ultimate utility. In addition, the safety of the operator is increased due to the reduction in direct contact with biologically hazardous materials that may contain infectious organisms, such as viruses. The kit also enables all or a portion of the final processed cellular material to be stabilized for delivery or storage before processing to achieve its final utility.

The present invention will enable the resected tumor to be treated during the resection or subsequently as needed without affecting the acquisition procedure or survival rate of the cellularized tumor.

In some embodiments, optional enrichment via a form of physical purification can be used to reduce impurities such as reagents that are no longer needed; cell debris; undepolymerized tumor tissue and fat. For this purpose, the sterile kit may have an enrichment module that can be selected according to the situation before stabilization. Individual cells or aggregates of small cell numbers can be enriched by excluding particles and fluids smaller than 5 μm or incompletely disaggregated materials of about 200 μm or larger in total to be stabilized after disaggregation, but this will depend on the tissue and The efficiency of disaggregation varies and depends on the final utility of the tissue or disaggregated tumor, and various embodiments in the form of tissue-specific kits can be used.

In another embodiment, a single cell suspension is provided after step (c).

In another embodiment, the first population of UTIL requires about 1 to 20 million UTILs.

In another embodiment, step (e) may further comprise the growth of UTIL from the excised tumor starting material followed by the rapid expansion of step (f).

In another embodiment, step (e) can be performed for about two weeks and step (f) can be performed for about two weeks.

In another embodiment, the additional step (h) involves suspending the second population of UTIL. Suspension can be in buffered saline, human serum albumin and/or dimethylsulfide (DMSO).

The present invention may also include a therapeutic population of cryopreserved UTIL obtained by any of the methods disclosed herein. The therapeutic population may contain about 5×10 ⁹ to 5×10 ¹⁰ T cells.

The present invention also covers cryopreservation bags for the therapeutic population disclosed herein. The cryopreservation bag can be used for intravenous infusion.

The present invention also encompasses a method of treating cancer, which may include administering the therapeutic population disclosed herein or the cryopreservation bag disclosed herein. The present invention also encompasses the therapeutic populations, pharmaceutical compositions or cryopreservation bags disclosed herein, which are used to treat cancer. The cancer can be bladder cancer, breast cancer, cancer caused by human papilloma virus, cervical cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), lung cancer, melanoma, ovarian cancer, non-small cell lung cancer ( NSCLC), renal cell carcinoma or renal cell carcinoma.

In another embodiment, one or more of the flexible containers of the sterile kit contains an elastically deformable material.

In another embodiment, one or more of the flexible containers of the disaggregation module of the sterile kit includes one or more sealable openings. One or more flexible containers of the depolymerization module and/or stabilization module may also include a heat-sealable melting port.

In another embodiment, one or more of the flexible containers of the sterile kit includes internal rounded edges.

In another embodiment, one or more of the flexible containers of the disaggregation module of the sterile kit includes a disaggregated surface adapted to mechanically squeeze and shear the solid tumor therein.

In another embodiment, one or more of the flexible containers of the enrichment module of the sterile kit includes a filter that retains the retentate of the cellularized and disaggregated solid tumor.

In another embodiment, one or more of the flexible containers of the stabilization module of the sterile kit contains a medium formulation for storing living cells in a solution form or in a cryopreserved state.

In another embodiment, the sterile kit further includes a digital, electronic or electromagnetic tag identifier. The tag identifier can be related to a specific program that defines a type of depolymerization and/or enrichment and/or stabilization process in which one or more types of media are used, including those suitable for controlled rate freezing The freezing solution selected according to the situation.

In another embodiment, the same flexible container may form part of one or more of the de-aggregation module, the stabilization module, and the enrichment module selected as appropriate.

In another embodiment, the disaggregation module of the sterile kit includes a first flexible container for receiving the tissue to be processed.

In another embodiment, the disaggregation module of the sterile kit includes a second flexible container that contains a culture medium for disaggregation.

In another embodiment, the optional enrichment module of the sterile kit includes a first flexible container and a third flexible container for receiving the enriched filtrate.

In another embodiment, both the depolymerization module and the stabilization module of the sterile kit include a second flexible container and the second flexible container includes a digestion medium and a stabilization medium.

In another embodiment, the stabilization module of the sterile kit includes a fourth flexible container, which includes a stabilized culture medium.

In another embodiment, the stabilization module of the sterile kit also includes a first flexible container and/or a third flexible container for storage and/or cryopreservation.

The present invention also provides a method for separating a therapeutic population of cryopreserved UTIL, which comprises: (a) Removal of the tumor from the individual; (b) Store the excised tumor in a single-use sterile kit, which contains: Depolymerization module for receiving and processing materials containing solid mammalian tissues; Optional enrichment module for filtering depolymerized solid tissue materials and separating undepolymerized tissues and filtrate; and A stabilization module for further processing and/or storage of depolymerized product materials as appropriate, Wherein each of the modules includes one or more flexible containers connected by one or more pipes adapted to enable tissue material to flow therebetween; and Each of the modules includes one or more ports to allow aseptic input of culture medium and/or reagents into one or more flexible containers; (c) Aseptically disaggregate and resect the tumor in the disaggregation module, thereby producing a disaggregated tumor. If the resected tumor can be stored at low temperature without cell damage, it will be fully disaggregated; (d) Store the depolymerized tumor at low temperature in the stabilization module; (e) Perform the first expansion by culturing the disaggregated tumor in a cell culture medium containing IL-2 to produce a first population of UTIL; (f) Perform a second expansion by culturing the first population of UTIL together with additional IL-2, OKT-3 and antigen-presenting cells (APC) to produce a second population of TIL; (g) Collect and/or cryopreserve the second population of UTIL. In some embodiments, step a) is optional.

In another embodiment, the automated device further includes a radio frequency identification tag reader for identifying the sterile kit, so that the sterile kit can be scanned during automated processing, such as in the automated device in the embodiment of the present invention. Recognition. Crucially, the label provides information about the conditions and steps required for automated processing, so simply by scanning the kit, any automated system used with the kit to process the organization can operate without further intervention or contamination. After the tissue sample has been placed in the disaggregation module, it can be sealed manually or automatically, for example before the start of the treatment.

The programmable processor of the automated device can also identify the sterile kit through the label and then perform the definition of the type of deaggregation, enrichment and stabilization process and the types of the respective media required for these processes, including suitable for controlled rates Freezing is a set program of freezing solution that exists depending on the situation. The programmable processor of the automation device is adapted to communicate with and control the following: a de-aggregation module; an enrichment module; and/or a stabilization module. In other words, the kit can therefore be read by an automated device that is used to perform a specific fully automated method to treat the tumor when inserted into such a device.

The programmable processor of the automated device can control the disaggregation module to enable the physical and/or biological decomposition of solid tissue materials. This decomposition can be physical or enzymatic decomposition of solid tissue materials. The enzymatic decomposition of solid tissue materials can be achieved by one or more medium enzyme solutions selected from the group consisting of collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase HI, pepsin and mixtures thereof.

In another embodiment, the programmable processor controls the depolymerization surface in the flexible container, which mechanically squeezes and shears the solid tissue. In some embodiments, the depolymerization surface is controlled by a mechanical piston.

In another embodiment, the programmable processor controls the stabilization module to enrich and deaggregate solid tissues in the cryopreservation container. This can be achieved using a programmable temperature setting, which is a condition determined by reading the label of the set inserted in the device.

In another embodiment, to perform different functions of the process, one or more of the additional components of the device and/or set are provided and can be used in any combination. This may include: a sensor capable of identifying whether the de-aggregation process has been completed in the de-aggregation module before transferring the de-aggregated solid tissue to the enrichment module that is optionally selected; measuring the de-aggregation module, enrichment module and/or A weight sensor that stabilizes the amount of culture medium required in one or more of the containers of the stabilization module, and controls the transfer of materials between the individual containers; controls the depolymerization module, the enrichment module, and/or stabilization A sensor for the temperature in the container of one or more of the modules; at least one bubble sensor that controls the transfer of the culture medium between the input and output ports of each container in the module; controls the input and output of the culture medium At least one pump for transfer between ports, as the case may be, a peristaltic pump; a pressure sensor that evaluates the pressure in the enrichment module; one or more valves that control the tangential flow filtration process in the enrichment module; and/ Or control the transfer of the culture medium between the input and output ports of each module or one or more clamps.

In another embodiment, the programmable processor of the automated device is adapted to maintain the optimal storage temperature range in the stabilization module until the container is removed; or to perform a controlled freezing step. This allows UTIL to be stored for a short period of time (a few minutes to a few days) or a longer period of time (a few days to a few years) before its final utility, depending on the type or stabilization process used with the stabilization module Certainly.

In another embodiment, the automated device further includes a user interface. The interface may include a display screen for displaying commands that guide the user to input parameters, confirm pre-programmed steps, warn of errors, or combinations thereof.

In another embodiment, the automated device is adapted to be movable and therefore may include dimensions that permit easy manipulation and/or assisted movement, such as wheels, tires, and/or handles.

The present invention also provides a semi-automatic sterile tissue processing method for separating therapeutic populations of cryopreserved UTIL, which includes the following steps: (a) Automatically determine the processing steps and related conditions of aseptic disaggregated tissue from the digital, electronic or electromagnetic tag identifiers related to the aseptic processing kit, where the aseptic kit includes: Depolymerization module for receiving and processing materials containing solid mammalian tissues; Optional enrichment module for filtering depolymerized solid tissue materials and separating undepolymerized tissues and filtrate; and A stabilization module for further processing and/or storage of depolymerized product materials as appropriate, Wherein each of the modules includes one or more flexible containers connected by one or more pipes adapted to enable tissue material to flow therebetween; and Each of the modules includes one or more ports to allow aseptic input of culture medium and/or reagents into one or more flexible containers; (b) Removal of the tumor from the individual; (c) Place the tumor in the flexible plastic container of the disaggregation module of the sterile kit; (d) Treat the tumor by communicating with and controlling the following automatic execution of one or more tissue processing steps: Disaggregation module; where the tumor is resected aseptically disaggregated to produce a disaggregated tumor, and if the resected tumor can be stored at a low temperature without cell damage, it will be fully disaggregated; Optional enrichment module, which filters the disaggregated tumor to remove the disaggregated solid tissue material and separate the undisaggregated tissue and filtrate; Stabilization module, in which the depolymerized tumor is stored at low temperature; (e) Perform the first expansion by culturing the disaggregated tumor in a cell culture medium containing IL-2 to produce a first population of UTIL; (f) Perform a second expansion by culturing the first population of UTIL together with additional IL-2, OKT-3 and antigen-presenting cells (APC) to produce a second population of TIL; (g) Collect and/or cryopreserve the second population of UTIL. In some embodiments, step b) is optional.

Flexible containers, such as bags, can be used to process tissue materials. The treatment may include separation or decomposition of tissues. For example, physical decomposition may be achieved by stirring, such as gentle agitation. Biological and/or enzymatic decomposition may include enzymatic digestion and/or extraction of components of tissue materials in the bag.

A flexible container (such as a bag) used to treat tissue may include one or more layers made of a sealable polymer with at least three flexible container edges that are sealed during manufacture and inserted into the tissue during use The open edge on the flexible container of the material. One or more connectors can be used to couple the flexible container to at least one element via a conduit. After the tissue is placed in the flexible container, the section of the flexible container close to the opening can be sealed or welded to form a sealed mouth. The sealing port may have a width of at least 3 mm and be arranged substantially parallel to the opening edge and spaced apart from the opening edge of the flexible container. In some cases, the sealing port may have a width greater than about 5 mm. For example, the bag can be sealed after the tissue is placed inside to have a sealing opening of at least 5 mm located close to the opening edge of the bag. The sealing mouth can be parallel to the opening edge and spaced apart from the opening edge of the bag.

The flexible container can be further fastened by a clamp that has a protrusion and is placed close to the sealing port and is further spaced apart from the opening edge of the flexible container than the sealing port.

In some cases, the seal and the flexible container are constructed so that the flexible container can withstand the 100 N force applied to the flexible container during use. In some cases, depending on the type of material used and/or the structure of the sealing port, it may be advantageous to use a clamp combined with such a sealing port. Therefore, during the use of a flexible container, such as a bag, the combination of the sealing port and the clamp may be able to withstand the 100 N force applied to the flexible container.

In some cases, the seal and the flexible container are constructed so that the flexible container can withstand the 75 N force applied to the flexible container during use. In some cases, depending on the type of material used and/or the structure of the sealing port, it may be advantageous to use a clamp combined with such a sealing port. Therefore, during the use of a flexible container, such as a bag, the combination of the sealing port and the clamp may be able to withstand the 75 N force applied to the flexible container.

The flexible container can be used to contain tissue during processing, such as the disaggregation of tissue material.

In some embodiments, a flexible container such as a bag can be used to depolymerize tissue material, filter depolymerized tissue material, and/or separate undisaggregated tissue and filtrate.

Flexible containers such as bags may be formed of elastically deformable materials. The material for flexible containers (such as bags) can be selected for one or more characteristics, including but not limited to airtightness (such as airtightness due to heat welding or the use of radio frequency energy), air permeability, and flexibility Properties (such as low-temperature flexibility (such as at -150°C or -195°C)), elasticity (such as low-temperature flexibility), chemical resistance, optical transparency, biocompatibility (such as cytotoxicity), hemolytic activity, resistance Leachability, low particle size, high transmission rate for specific gases (such as oxygen and/or carbon dioxide), and/or compliance with regulatory requirements.

Flexible containers, such as bags, may include indicators. The indicator can be used to identify the sample, the patient from which the sample is derived, and/or track the progress of a particular sample during treatment. In some cases, the indicator can be scanned by an automated or semi-automated system to track the progress of the sample.

Markings can be used on flexible containers, such as bags, to identify where the bag should be placed, handled, sealed, or any other action that can be taken with respect to the bag including tissue. Each bag may include multiple indicia that are sealed.

The open end of the bag can be sealed after the tissue is inserted into the bag. A sealing device (for example, a heating sealing machine) that operates under a predetermined pressure, a predetermined temperature, and a predetermined time range can be used to form any sealing port.

In some cases, a flexible container such as a bag can be used as a depolymerization container to serve as part of a depolymerization element that may also include a depolymerization device. In some embodiments, culture media and/or enzymes can be added to the bag inside the depolymerization element of the device. For example, the bag can be used with a device that mechanically squeezes tissue material placed in a flexible container.

In some embodiments, the tissue in a flexible container (such as a bag) can be sheared during disaggregation. In particular, the flexible container can be configured to cut tissue material.

The flexible container can be used in a semi-automated or automated process for aseptic depolymerization, stabilization and/or enrichment of mammalian cells or cell aggregates.

The kit for extracting the desired material from the tissue may include a disaggregation element, wherein at least some of the tissue is processed to form a treated fluid; an enrichment element (such as a filter), which is capable of enriching at least some of the treated fluid to form The desired material; the stabilization element, which can store a part of the desired material; and the indicator label positioned on at least one of the depolymerization element, the enrichment element, or the stabilization element, which can provide the source of the tissue and the state of the tissue regarding the process Or at least one of the identifiers.

The desired material can be a biological material or component of a specific size. For example, the desired material may be tumor infiltrating lymphocytes (TIL).

Different types of media can be used in various processes performed by depolymerization elements and stabilization elements. For example, cryopreservation medium can be provided in the kit and used in the stabilization element to control the freezing rate.

A kit for use in a device in which the depolymerization element can include a first flexible container and the stabilizing element can include a second flexible container.

The automated device for semi-automated aseptic depolymerization and/or enrichment and/or stabilization of cells or cell aggregates from mammalian solid tissues can include a programmable processor and a flexible container including the flexible container described herein. Set. The automated device may further include an indicator tag reader. For example, the indicator tag reader can be placed at any element (such as the de-aggregation, enrichment, or stabilization of the tissue material in the kit).

In some cases, the automated device may further include a radio frequency identification tag reader to identify the sample in the flexible container in the kit.

The automated device may include a programmable processor capable of identifying indicators positioned on components of the kit such as bags via indicator tags such as QR codes. After determining which sample is in the bag, the programmable processor then executes a program that defines the types of deaggregation, enrichment, and stabilization processes and the types of individual media required for these processes.

The kit for the automated device may include a depolymerized flexible container or bag. The programmable processor can control the depolymerization element and the depolymerization flexible container to enable physical and/or biological decomposition of solid tissue.

The programmable processor can control the components of the automated device, so that the depolymerized surface located close to the depolymerized flexible container can mechanically squeeze and shear the solid tissue in the depolymerized flexible container, depending on the situation. The surface is a mechanical piston.

The depolymerization element of the system can be controlled by the processor, so that the tissue in the depolymerization flexible container can realize the physical and enzymatic decomposition of the solid tissue. One or more medium enzyme solutions selected from collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase HI, pepsin or mixtures thereof can be provided to the depolymerization flexible container to help tissue enzymes break down.

The system may include a kit including a depolymerized flexible container and a stabilized flexible container and a programmable processor. The programmable processor may be adapted to control one or more of the following: depolymerization element; enrichment element; and stabilization element.

The programmable processor can control the stabilization element to store the enriched and depolymerized solid tissue in the stabilization container at low temperature. In some embodiments, the predetermined temperature can be programmed.

The automation device can include additional components in multiple combinations. The component may include a sensor capable of identifying whether the deaggregation process has been completed in the deaggregation module before transferring the deaggregated solid tissue to the enrichment element that may exist; measuring the deaggregation element, enrichment element and/or stabilization The amount of culture medium required in the container of one or more of the elements, and the weight sensor that controls the transfer of materials between the individual containers; controls the depolymerization element, the enrichment element and/or the stabilization element One or more sensors for the temperature in the container; at least one bubble sensor that controls the transfer of the culture medium between the input and output ports of each container in the element; controls the transfer of the culture medium between the input and output ports At least one pump, as the case may be, a peristaltic pump; a pressure sensor that evaluates the pressure in the enrichment element; one or more valves that control the tangential flow filtration process in the enrichment element; and/or control the media in each element Transfer one or more fixtures between input and output ports.

The automated device may include a programmable processor that is adapted to maintain the optimal storage temperature range in the stabilization module until the container is removed. In one embodiment, the programmable processor can perform the controlled freezing step.

In some cases, the automated device may include a user interface. The interface of the automation device may include a display screen for displaying commands that guide the user to input parameters, confirm pre-programmed steps, warn of errors, or combinations thereof.

The automated device as described herein can be adapted to be movable.

The automatic tissue processing method may include automatically determining the conditions and related conditions for the processing steps from the digital, electronic or electromagnetic label indicators related to the components of the set. During use, the tissue sample can be placed in a flexible container with at least one open edge of the kit. After positioning the tissue in the flexible container, the opening edge can be sealed. During use, the tissue can be processed by automatically performing one or more tissue processing steps by conveying information related to the indicator and controlling the condition of approaching the flexible container and/or the position of the flexible container. In addition, the addition of materials to the set can be controlled based on information related to the indicator. At least some of the treated tissue can be filtered so that filtered fluid is produced. At least some of the filtered fluid can be provided to a cryopreservation flexible container to stabilize the desired materials present in the filtered fluid.

The processing as described herein may include stirring, extracting, and enzymatically digesting at least a portion of the tissue sample in the flexible container. In some cases, this treatment of the tissue can result in the extraction of the desired material from the tissue sample. For example, tumor infiltrating lymphocytes (TIL) can be extracted from a tissue sample.

Flexible containers (such as bags) used in the methods described herein may include heat-sealable materials.

The use of cryopreservation kits for tissue processing and extraction of self-organized materials can cause separation of the desired materials. In particular, materials such as tumor infiltrating lymphocytes (TIL) may be the desired materials.

In some cases, the cryopreservation kit and/or its components described herein can be used in an automated and/or semi-automated process for the depolymerization, enrichment and/or stabilization of cells or cell aggregates at a time. . In some embodiments, in some embodiments, bags used in cryopreservation kits, such as collection bags, can be used in multiple processes. For example, the collection bag can be repeatedly sealed at different locations to create separate compartments for processing tissue samples (such as biopsy samples and/or solid tissue).

The flexible container used in the present invention described herein, such as bags including collection bags and cryopreservation bags, may include at least a portion made of a predetermined material, such as thermoplastic, polyolefin polymer, ethylene vinyl acetate Ester (EVA), blends (such as copolymers such as vinyl acetate and polyolefin polymer blends (ie, OriGen Biomedical EVO film)), materials including EVA and/or co-extruded layers of sealable plastics . The collection bag, such as the tissue collection bag of the present invention, may include a bag made of a predetermined material for receiving tissue, such as ethylene vinyl acetate (EVA) and/or a material including EVA. The material for the bag can be selected for specific characteristics. In one embodiment, the bag including the collection bag may be substantially made of a blend of vinyl acetate and polyolefin polymers. For example, the relevant properties of materials that can be selected for cryopreservation kit components (such as collection bags and/or associated catheters) can be related to heat sealing.

The material for the bag can be selected for specific characteristics and/or a series of characteristics, such as tightness (such as heat sealability), air permeability, flexibility (such as low temperature flexibility), elasticity (such as low temperature elasticity), chemical resistance Properties, optical transparency, biocompatibility (such as cytotoxicity), hemolytic activity, resistance to leaching, with low particles.

In some embodiments, the material may be selected for the specific characteristics of the co-extruded material used to form at least one layer of the bag. The layer can be constructed so that when constructed, the inner layer of the bag is relatively biocompatible, that is, the material on the inner surface of the bag is stable and does not leach into the contents of the bag.

For example, the relevant properties of materials that can be selected for kit components such as collection bags, cryopreservation bags, and/or associated catheters can be related to sealing, such as heat sealing.

The bags (such as collection bags and/or cryopreservation bags) and any associated catheters can be substantially clear, transparent, translucent, any desired color, or combination thereof. Tissue collection bags and/or catheters can generally be manufactured in a manner similar to the manufacturing of closed and/or sealed blood and/or cryopreservation bags and associated catheters. The catheter in the present invention can be constructed of any desired material, including but not limited to polyvinyl chloride (PVC). For example, PVC may be the desired material because PVC facilitates welding and/or sealing.

In some embodiments, at least one end of the collection bag may be open for receiving tissue. In particular, in one embodiment, for example, a tissue sample from a biopsy can be placed in the bag via the open end (e.g., the top end). In some cases, the biopsy sample may be cancer tissue from animals (eg, domestic animals such as dogs or cats) or humans.

After the tissue is positioned in the bag, the bag can be sealed, and can then be processed. The treatment may include agitation of the tissue in the bag, such as gentle agitation, extraction, and/or enzymatic digestion. Tissue processing and extraction of required materials (such as tumor infiltrating lymphocytes (TIL)) can be in a closed system. Advantageous or preferred embodiments may include indicators that identify the patient from which the tissue is collected and/or markings showing where the collection bag can be clamped, sealed, acted upon, and/or attached in place in the instrument.

In some embodiments, the bag may be formed of a sealable material. For example, the bag may be formed of materials including, but not limited to, polymers such as aliphatic or semi-aromatic polyamides (for example, nylon), ethylene-vinyl acetate (EVA) and blends thereof Synthetic polymer, thermoplastic polyurethane (TPU), polyethylene (PE), vinyl acetate and polyolefin polymer blends and/or polymer combinations. Portions of the bag can be sealed and/or welded using energy, such as heat, radio frequency energy, high frequency (HF) energy, dielectric energy, and/or any other method known in the art.

The collection bag can be used as a treatment and/or depolymerization bag. The collection bag may have a width ranging from about 4 cm to about 12 cm and a width ranging from about 10 cm to about 30 cm. For example, the collection bag used for processing may have a width of about 7.8 cm and a length of about 20 cm. In particular, the bag may be heat-sealable, for example, using EVA polymer or blends thereof, vinyl acetate and polyolefin polymer blends, and/or one or more polyamides (nylon).

Indicators can include but are not limited to codes, letters, words, names, alphanumerics, numbers, images, barcodes, QR codes, tags, trackers (such as smart tracker tags or Bluetooth trackers) and/ Or any indicator known in the art. In some embodiments, the indicator may be printed, etched, and/or adhered to the surface of the components of the kit. The indicator can also be positioned on the bag using an adhesive, for example, a sticker or tracker can be placed on one bag and/or multiple bags. The collection bag and/or cryopreservation kit may include multiple indicators, such as numeric codes and/or QR codes.

Indicators (such as QR codes, tags such as smart tags and/or trackers) can be used to identify the samples in the bag and send instructions to the processor of the device so that the device will be de-aggregated according to the cryopreservation kit. The type of enrichment and/or stabilization process runs a specific program. Different types of media can be used in these processes, such as enzyme media that allows a controlled freezing rate, tumor digestion media, and/or cryopreservation media. In some embodiments, the cryopreservation kit and/or its components may include indicators that can be read by an automated device. The device can then perform a specific fully automated method for processing tissue when inserted into such a device. The invention is particularly suitable for sample processing, especially automated processing. In some cases, the cryopreservation kit and/or its components described herein can be used in an automated and/or semi-automated process for the depolymerization, enrichment and/or stabilization of cells or cell aggregates at a time. . In some embodiments, in some embodiments, bags used in cryopreservation kits, such as collection bags, can be used in multiple processes. For example, the collection bag can be repeatedly sealed at different locations to create separate compartments for processing tissue samples (such as biopsy samples and/or solid tissue).

In addition, markers can be placed in various locations on the bag, such as a tissue collection bag, to indicate where the bag can be sealed, clamped, and/or attached to an object. In some embodiments, markings where the display bag can be clamped, sealed, and/or attached to an object (such as an instrument) can be placed on the bag before use. For example, one or more markings can be positioned on the bag during manufacturing.

The positioner can be used to ensure that the tissue material in the bag can be properly handled during use, for example close to the positioning of the instrument. In some systems, the positioner can facilitate the use of the bag described in this article in an automated system. In particular, the positioner can be used to move the bag through an automated system.

The use of indicators such as QR codes may allow tracking of the process steps of a specific sample, making it possible to track the sample in a given process.

The present invention relates to and provides therapeutic cell populations, as discussed in the following numbered paragraphs:

Therefore, one objective of the present invention is that the present invention does not cover any previously known products, processes for manufacturing the products, or methods for using the products, so that the applicant reserves the right and hereby discloses any previously known products, processes or methods Disclaimer. In addition, it should be noted that the present invention is not intended to cover any product, process, or such that does not comply with the written description and implementation requirements of USPTO (35 USC §112, first paragraph) or EPO (EPC Article 83) within the scope of the present invention. The manufacture of the product or the method of using the product allows the applicant to reserve the right and hereby disclose any disclaimer of any previously described product, the manufacturing process of the product, or the method of using the product. It may be advantageous for the practice of the present invention to comply with EPC Article 53(c) and EPC Rules 28(b) and (c). We expressly reserve all rights to expressly waive any embodiment of the subject matter of any one or more granted patents of the applicant in the pedigree of this application or in any other pedigree or any third party in any previous application. Nothing in this article should be taken as a promise.

It should be noted that in the present invention and especially in the scope of patent application and/or paragraphs, terms such as "comprises/comprised/comprising" and similar terms may have the meaning assigned to them in the U.S. Patent Law; for example, they may Means "includes/included/including" and similar terms; and terms such as "consisting essentially of/consists essentially of" have the meaning assigned to them in the U.S. Patent Law, for example, It is allowed not to explicitly list the elements, but exclude the elements found in the prior art or affecting the basic or novel features of the present invention.

These and other embodiments are disclosed in or are obvious from the following embodiments and are covered by the following embodiments.

Related applications and incorporated by reference

This application claims from the U.S. patent application serial number 62/951,559 filed on December 20, 2019, the U.S. patent application serial number 62/982,470 filed on February 27, 2020, and the U.S. filed on July 2, 2020 Priority of patent application No. 29/740,293 and U.S. Patent Application Serial No. 63/047,431 filed on July 2, 2020, the contents of these documents are incorporated herein by reference in their entirety.

Refer to the UK patent application serial number GB1700621.4 filed on January 13, 2017, the European patent application EP18701791.8 filed on January 12, 2018, and the international patent application serial number PCT filed on January 12, 2018. /GB2018/050088, which was published as PCT Publication No. WO 2018/130845 on July 19, 2018, European Patent Publication: EP3568459, and U.S. Patent Application Serial No. 62/951,559 filed on December 20, 2019 , Which is incorporated herein by reference.

Refer to the UK patent application serial number GB1902763.0 filed on March 1, 2019, the UK patent application serial number GB1904249.8 filed on March 27, 2019, and the international patent application filed on February 28, 2020 Serial number PCT/EP2020/000053, which was published as WO 2020/177920 on September 10, 2020.

The aforementioned application, Biomarker Predictive of Tumour Infiltrating Lymphocyte Therapy and the Uses Thereof, WO2019145711A1 PCT/GB2019/050188, Tumor Infiltrating Lymphocyte Therapy and Uses Thereof USA, PCT/GB2020/051790 and US application serial number 62/878,001, Receptors Providing Targeted Costimulation for Adoptive Cell Therapy WO 2020/152451, U.S. Application Serial No. 62/951,770 and GB1900858.0, Cells Expressing Recombinant Growth Factor Receptors WO 2017/103596A1, U.S. Application Serial No. 16/061,435 and European Patent Publication EP3390436, and Chimeric Growth Factor Receptors WO2019243835A1 PCT/GB2019/051745, and all documents cited in or during its examination ("application cited documents"), and all documents cited or referenced in the application cited documents, and all documents cited in this article All documents cited or referenced ("Citations in this article"), and all documents cited or referenced in this article, and any product mentioned in this article or in any article incorporated by reference into this article The manufacturer's instructions, descriptions, product specifications, and product introductions are hereby incorporated by reference, and can be used in the implementation of the present invention. More specifically, all references are incorporated by citation, and the degree of incorporation is as if each individual document was specifically and individually instructed to be incorporated by citation.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by those familiar with the present invention.

The term "anti-CD3 antibody" refers to antibodies or variants thereof, such as monoclonal antibodies and includes human, humanized, chimeric, murine or mammalian antibodies, which are directed against CD3 in the T cell antigen receptor of mature human T cells Receptor. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 pure lines, also known as T3 and CD3.ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

When indicating the "anti-tumor effective amount", "tumor suppressive effective amount" or "therapeutic amount", the exact amount of the composition of the present invention to be administered can be determined by the physician considering the age, weight, tumor size, degree of infection or metastasis of the patient (individual) And individual differences in symptoms. General provisions may comprise the herein described tumor infiltrating lymphocytes (TIL e.g. two cytotoxic lymphocytes or of genetically modified) The pharmaceutical composition may be administered in the following doses: 10 ⁴ to 10 ¹¹ cells / kg body weight (e.g. 10 ⁵ to 10 ⁶ , 10 ⁵ to 10 ¹⁰ , 10 ⁵ to 10 ¹¹ , 10 ⁶ to 10 ¹⁰ , 10 ⁶ to 10 ¹¹ , 10 ⁷ to 10 ¹¹ , 10 ⁷ to 10 ¹⁰ , 10 ⁸ to 10 ¹¹ , 10 ⁸ to 10 ¹⁰ , 10 ⁹ to 10 ¹¹ or 10 ⁹ to 10 ¹⁰ cells/kg body weight), including all integer values within their range. The composition of tumor infiltrating lymphocytes (including genetically modified cytotoxic lymphocytes in some cases) can also be administered multiple times at the same dose. Tumor infiltrating lymphocytes (including genetics in some cases) can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Those who are familiar with medical technology can easily determine the best dose and treatment plan for a specific patient by monitoring the patient's disease symptoms and adjusting the treatment accordingly.

As used in this article, the terms "co-administration", "co-administering", "administered in combination with", and "administering in combination with" , "Simultaneous" and "concurrent" encompass the administration of two or more active pharmaceutical ingredients to an individual (in a preferred embodiment of the present invention, for example, a combination of at least one potassium channel agonist and a plurality of TILs), So that two active pharmaceutical ingredients and/or their metabolites exist in the individual at the same time. Co-administration includes simultaneous administration in separate compositions, administration in separate compositions at different times, or administration in the form of a composition in which two or more active pharmaceutical ingredients are present. It is preferable to administer in separate compositions at the same time and in the form of a composition in which two reagents are present.

As used herein, "cellularized/cellularization" refers to the process of disaggregation, which breaks down solid tissue, usually multicellular material composed of multiple cell lineages/types, into a small number of cells, including but not limited to one cell but can be It is a very small number of multiple cells of various lineages or cell types, that is, cell aggregates or cell aggregates.

As used herein, "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used with the methods of the present invention. The closed system includes, for example, but not limited to, closed G-Rex containers or cell culture bags. Once the tumor segment is added to the closed system, the system is not open to the external environment until the TIL is ready to be administered to the patient. In an advantageous embodiment, the closed system is the system disclosed in PCT Publication No. WO 2018/130845.

As used herein, "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreservation of cells. Such media may include media containing 2% to 10% DMSO. Exemplary media include CryoStor CS10, HypoThermosol, Bloodstor BS-55, and combinations thereof.

The term "cryopreserved TIL" herein means that primary, bulk, or amplified TIL (REP TIL) is processed and stored in the range of about -190°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the examples. For the sake of clarity, "Cryogenic TIL" can be distinguished from frozen tissue samples that can be used as a source of primary TIL.

As used herein, "depletion" refers to the process of negative selection that separates desired cells from undesired cells, which are labeled with a marker-binding fragment coupled to a solid phase.

As used herein, “disaggregation (disaggregation/disaggregate)” refers to the transformation of solid tissue into single cells or aggregates of small numbers of cells, where the diameter of a single cell as a spheroid is 5 μm, 6 μm, 7 μm, 8 μm , 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm or more, of which this is more often between 7 and 20 μm .

The term "effective amount" or "therapeutically effective amount" refers to the amount of a compound or combination of compounds as described herein, which is sufficient to achieve the intended application, including but not limited to disease treatment. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the individual to be treated and the disease condition (for example, the weight, age, and sex of the individual), the severity of the disease condition, or the mode of administration. The term also applies to the dose that will induce a specific response in the target cell (such as reduction of platelet adhesion and/or cell migration). The specific dosage will vary depending on the following: the specific compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered and the physical delivery system in which the compound is carried.

As used herein, "engineering" refers to the addition of nucleic acid materials or factors, which change the function of tissue-derived cells from its original function to a new or improved function with its ultimate utility.

As used herein, "enzyme medium" refers to a medium having enzymatic activity, such as collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase HI, pepsin, or a mixture thereof.

As used herein, "filtrate" refers to the material that passes through a filter, mesh, or membrane.

As used herein, "flexible container" refers to a flexible packaging system in multiple forms with one or more different types of films. Each membrane type is selected to provide specific characteristics to preserve the physical, chemical, and functional characteristics of the sterile fluid, solid tissue-derived cell material, and container integrity, depending on the process steps.

The "frozen solution" or "cryogenic storage solution" also called cryoprotectant in the art is a solution containing cryoprotective additives. These are generally permeable non-toxic compounds that change the physical stress that cells are exposed to during freezing to minimize freezing damage (that is, due to ice formation) and are most often one of or below a certain% vol/vol More: dimethylsulfoxide (DMSO); ethylene glycol; glycerol; 2-methyl-2,4-pentanediol (MPD); propylene glycol; sucrose; and trehalose.

The term "hematological malignancy" refers to mammalian cancers and tumors of hematopoietic and lymphoid tissues, including but not limited to blood, bone marrow, lymph nodes, and lymphatic system tissues. Hematological malignancies are also called "liquid tumors." Hematological malignancies include but are not limited to acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) ), acute monocytic leukemia (AMoL), Hodgkin's lymphoma and non-Hodgkin's lymphoma. The term "malignant B cell hematological disease" refers to a malignant hematological disease that affects B cells.

The term "IL-2" (also referred to as "IL2" herein) refers to the T cell growth factor called interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conserved amino acids Substitutions, glycoforms, biological analogues and their variants. IL-2 is described in, for example, Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 covers human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN), which can be purchased from multiple suppliers in 22 million IU/single use vials ), and the recombinant IL-2 form commercially supplied by CellGenix, Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Co., Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b), and from Other commercial equivalents of other suppliers. Aldeskinin (anti-Alanine-1, Serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses PEGylated forms of IL-2 as described herein, including the PEGylated IL2 prodrug NKTR-214 available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and PEGylated IL-2 suitable for the present invention are described in US Patent Application Publication No. US 2014/0328791 A1 and International Patent Application Publication No. WO 2012/065086 A1. Alternative forms of binding IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4902,502. Suitable IL-2 formulations for use in the present invention are described in U.S. Patent No. 6,706,289.

The term "IL-4" (also referred to herein as "IL4") refers to a cytokine called interleukin 4, which is produced by Th2 T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. After activation by IL-4, Th2 T cells then produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II performance, and induces class conversion from B cells to IgE and IgG1 performance. Recombinant human IL-4 suitable for use in the present invention can be purchased from multiple suppliers, including ProSpec-Tany TechnoGene Co., Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Waltham, Mass., USA (Human IL-15 recombinant protein, catalog number Gibco CTP0043).

The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine called interleukin 7, which can be obtained from stromal and epithelial cells and dendritic cells . Fry and Mackall, Blood 2002, 99, 3892-904 IL-7 can stimulate the production of T cells. IL-7 binds to IL-7 receptor, a heterodimer composed of IL-7 receptor α and common γ chain receptor, which belongs to a series that is important for the production of T cells in the thymus and the survival of the surrounding area. Signal. Recombinant human IL-7 suitable for use in the present invention can be purchased from multiple suppliers, including ProSpec-Tany TechnoGene Co., Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Waltham, Mass., USA (Human IL-15 recombinant protein, catalog number Gibco PHC0071).

The term "IL-12" (also referred to herein as "IL12") refers to a T cell growth factor called interleukin-12. Interleukin (IL)-12 is a secreted heterodimeric interleukin that contains two disulfide-linked glycosylated protein subunits, which are named p35 and p40 for their approximate molecular weights. IL-12 is mainly produced by antigen-presenting cells and drives cell-mediated immunity by binding to double-chain receptor complexes expressed on the surface of T cells or natural killer (NK) cells. The IL-12 receptor β-1 (IL-12Rpi) chain binds to the p40 subunit of IL-12, providing the main interaction between IL-12 and its receptor. However, the binding of IL-12p35, which is the second receptor chain IL-12RP2, confers intracellular signaling. IL-12 signalling that coincides with antigen presentation is believed to cause T cell differentiation against the T helper 1 (Thl) phenotype, which is characterized by interferon gamma (IFNy) production. It is believed that Th1 cells promote immunity against some intracellular pathogens, produce complement-fixing antibody isotypes, and contribute to tumor immune surveillance. Therefore, IL-12 is considered to be a significant component of the host's defense and immune mechanism. IL-12 is a part of the IL-12 family of cytokines, which also includes IL-23, IL-27, IL-35, and IL-39.

The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor called interleukin-15 and includes all forms of IL-15, including human and mammalian forms, conserved amino acids Substitutions, glycoforms, biological analogues and their variants. IL-15 is described in, for example, Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 and IL-2 share β and γ signaling receptor subunits. Recombinant human IL-15 is a single non-glycosylated polypeptide chain containing 114 amino acids (and N-terminal methionine) and a molecular weight of 12.8 kDa. Recombinant human IL-15 can be purchased from multiple suppliers, including ProSpec-Tany TechnoGene Co., Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Waltham, Mass., USA (human IL -15 recombinant protein, catalog number 34-8159-82).

The term "IL-18" (also referred to herein as "IL18") refers to a T cell growth factor called interleukin-15. Interleukin-18 (IL-18) is a pro-inflammatory cytokine belonging to the IL-1 family of cytokines due to its structure, receptor family and signal transduction pathway. Related cytokines include IL-36, IL-37, and IL-38.

The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein called interleukin-21 and includes all forms of IL-21, including human and mammalian forms, and conservative Amino acid substitutions, glycoforms, biological analogs and variants thereof. IL-21 is described in, for example, Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is mainly produced by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain with 132 amino acids and a molecular weight of 15.4 kDa. Recombinant human IL-21 can be purchased from multiple suppliers, including ProSpec-Tany TechnoGene Co., Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Waltham, Mass., USA (human IL -21 recombinant protein, catalog number 14-8219-80).

The term "liquid tumor" refers to an abnormal cell mass that is fluid in nature. Liquid tumor cancers include but are not limited to leukemia, myeloma and lymphoma, and other hematological malignancies. TIL obtained from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MIL).

As used herein, the "magnetic" in "magnetic particles" refers to all subtypes of magnetic particles, which can be prepared by methods well known to those skilled in the art, especially ferromagnetic particles, superparamagnetic particles, and paramagnetic particles. "Ferromagnetic" materials have strong magnetic susceptibility and can maintain magnetic properties when the magnetic field is removed. "Paramagnetic" materials only have weak magnetic susceptibility and quickly lose their weak magnetic properties when the magnetic field is removed. "Superparamagnetic" materials have high magnetic susceptibility, that is, when placed in a magnetic field, they become strong magnetic, but like paramagnetic materials, they lose their magnetism quickly.

As used herein, "marker" refers to a cell antigen, which is specifically expressed by a certain cell type. Preferably, the marker is a cell surface marker, so that the enrichment, separation and/or detection of living cells can be performed.

As used herein, "label binding fragment" refers to any part of the desired target molecule (ie antigen) that preferentially binds to the cell. The term part includes, for example, antibodies or antibody fragments. As used herein, the term "antibody" refers to multiple or monoclonal antibodies, which can be produced by methods well known to those skilled in the art. The antibody can be of any species, such as murine, rat, sheep, human. For therapeutic purposes, if non-human antigen-binding fragments are to be used, these fragments can be humanized by any method known in the art. The antibody can also be a modified antibody (e.g., oligomer, reduced, oxidized, and labeled antibody). The term "antibody" includes complete molecules and antibody fragments, such as Fab, Fab', F(ab')2, Fv, and single chain antibodies. In addition, the term "label-binding fragment" includes any part other than the antibody or antibody fragment that preferentially binds to the desired target molecule of the cell. Suitable parts include, but are not limited to, oligonucleotides called aptamers (Hermann and Pantel, 2000: Science 289: 820-825), carbohydrates, lectins, or any other antigen binding proteins (such as receptors) that bind to the desired target molecule. Body-ligand interaction).

"Media" means various solutions known in cell culture, cell treatment, and stabilization techniques for reducing cell death, including but not limited to one or more of the following media: organ preservation solution, selective dissolution solution, PBS, DMEM, HBSS, DPBS, RPMI, Iscove's medium, X-VIVO™, Lactated Ringer's solution, Ringer's acetate, physiological saline, PLASMAYTE™ solution , Crystal solution and IV fluid, colloidal solution and IV fluid, 5% dextrose aqueous solution (D5W), Hartmann's Solution (Hartmann's Solution). The culture medium can be a standard cell culture medium, such as the medium mentioned above, or for example for primary human cell culture (for example for endothelial cells, hepatocytes or keratinocytes) or stem cells (for example, dendritic cell maturation, hematopoietic expansion, keratinocytes) Cells, mesenchymal stem cells or T cell expansion) special medium. The culture medium may have supplements or reagents well known in the art, such as albumin and transporters, amino acids and vitamins, antibiotics, attachment factors, growth factors and cytokines, hormones, metabolic inhibitors or augmenters. Solvent. Various media are commercially available from, for example, ThermoFisher Scientific or Sigma-Aldrich.

As used herein, the term "microenvironment" can refer to the overall solid or hematological tumor microenvironment or to an individual subset of cells within the microenvironment. As used herein, the tumor microenvironment refers to the following complex mixture: "cells, soluble factors, signal transduction molecules, extracellular matrix and promotion of neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, promote therapeutic resistance Sex, and provide the mechanical signal of the niche that is dominantly transferred and thrive", as described in Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors exhibit antigens that should be recognized by T cells, it is rare for the immune system to clear tumors due to the immunosuppression of the microenvironment.

As used herein, the term "negative separation" refers to the active separation of cells bound by a label-binding fragment coupled to a solid phase, and these cells are not the desired cell population.

As used herein, "unlabeled" or "unaltered" refers to cells that are not bound by a label-binding fragment coupled to the solid phase. The unlabeled, unaltered cell part contains the desired target cell.

As used herein, "non-target cell" refers to a cell specifically bound by a marker-binding fragment coupled to a solid phase for removing unwanted cell types.

"OKT-3" (also referred to as "OKT3" in this article) refers to a monoclonal antibody to the CD3 receptor in the T cell antigen receptor of mature T cells or its biological analogs or variants, including humans and humans Chem, chimeric or murine antibodies, and include commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, San Diego, Calif., USA) and muromonab Or its variants, conservative amino acid substitutions, glycoforms or biological analogues.

As used herein, "particle" refers to a solid phase, such as colloidal particles, microspheres, nanoparticles, or beads. Methods of producing such particles are well known in the art. The particles can be magnetic particles or have other selective properties. The particles may be in the form of a solution or suspension or they may be in a lyophilized state before being used in the present invention. The lyophilized particles are then reconstituted in a suitable buffer before being brought into contact with the sample to be processed in accordance with the present invention.

The terms "peripheral blood mononuclear cells" and "PBMC" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMC is a type of antigen presenting cell.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents And inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic composition of the present invention is considered. Additional active pharmaceutical ingredients such as other drugs can also be incorporated into the described compositions and methods.

The term "cell population" (including TIL) herein means a plurality of cells sharing a common trait. Generally speaking, the number of groups is generally in the range of 1×10 ⁶ to 1×10 ¹² , and different TIL groups contain different numbers.

As used herein, "positive separation" refers to the active separation of cells bound by a label-binding fragment coupled to a solid phase, and these cells are the desired cell population.

As used herein, "negative separation" refers to the active separation of cells bound by a label-binding fragment coupled to a solid phase, and these cells are not the desired cell population.

As used herein, "purity" refers to the percentage of one or more target groups from the original solid tissue.

"Rapid expansion" means that the number of antigen-specific TIL increases by at least about 3 times (or 4 times, 5 times, 6 times, 7 times, 8 times or 9 times) over a week period, more preferably at least about 10 times over a week period. Times (or 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 800 times or 90 times), preferably at least about 100 times (or 200 times, 300 times, 400 times, 500 times) after a week , 600 times, 700 times, 800 times or 900 times), or at least about 1000 times or 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times or 9000 times during a period of one week). Several rapid amplification schemes are summarized below.

"One or more regenerative drugs", "one or more adoptive cell therapies" or "one or more advanced therapy pharmaceutical products" are used interchangeably herein and refer to the treatment of one or more mammals by any of the following Purpose of cell material: the function of part or all of the cell material; the supporting function of part or all of the cell material, which aims to improve the health of mammals after administration. Therapeutic cells may be used directly or may require further processing, expansion, and/or engineering to provide these effects.

As used herein, "sample" refers to a sample containing cells in any ratio. Preferably, these cells are living cells. In some cases, these cells can also be fixed or frozen cells that can be used for subsequent nucleic acid or protein extraction. The sample can be from an animal, especially a mammal, such as a mouse, rat or human. Any compressible solid tissue containing cells can be used. The present invention is mainly illustrated by separating hematopoietic and cancer cells from solid tumor tissues. However, the present invention relates to a method for separating a series of cells from any mammalian solid tissue.

As used herein, "solid phase" refers to the binding of a label to another substrate (eg particles, fluorophores, haptens such as biotin, polymers, or larger surfaces such as petri dishes and microtiter plates) Coupling of fragments (eg antibodies). In some cases, coupling allows direct immobilization of the antigen-binding fragment, for example, if the antigen-binding fragment is coupled to a larger surface of a petri dish. In other cases, this coupling causes indirect immobilization, such as direct or indirect (via biotin, for example) coupling to an antigen-binding fragment of a magnetic bead, and immobilization if the bead is held in a magnetic field. In other cases, the coupling of the antigen-binding fragment to other molecules does not cause direct or indirect fixation, but allows the enrichment, separation, separation and detection of cells according to the present invention, for example, if the label-binding fragment is coupled to a chemical or physical part, It then allows the identification of labeled and unlabeled cells, for example, via flow cytometry methods such as FACS sorting or fluorescence microscopy.

As used herein, "solid tissue" refers to one or more pieces of animal-derived mammalian solid tissue. The three-dimensional, that is, the length, width, and thickness of the geometric body is greater than the size of a unit based on multiple individual cells, and usually contains Connective material, such as collagen or a similar matrix that constitutes tissue structure, whereby the solid tissue cannot flow through a tube or be collected by a syringe or similar small tube or container, and that is, it has a size of 500 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 10 cm, 20 cm, 30 cm or larger sizes.

"Solid tumor" refers to a mass of extravagant tissue that does not usually contain a pavement or liquid area. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to malignant, neoplastic or cancerous solid tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The tissue structure of a solid tumor includes interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells that are scattered among the cancer cells and can provide a supporting microenvironment. In some embodiments, the cancer line is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma [HNSCC]) glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, three Negative breast cancer and non-small cell lung cancer. The tissue structure of a solid tumor includes interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells that are scattered among the cancer cells and can provide a supporting microenvironment.

"Thawed cryopreserved TIL" herein means a TIL population that has been previously cryopreserved and then processed to return to room temperature or higher temperature (including but not limited to cell culture temperature or temperature at which TIL can be administered to patients).

The terms "treatment", "treating", "treat" and similar terms refer to obtaining the desired pharmacological and/or physiological effects. The effect may be preventive in terms of completely or partially preventing the disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects attributable to the disease. As used herein, "treatment" encompasses any treatment of diseases in mammals, especially humans, and includes: (a) preventing the occurrence of the disease in individuals who may be susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease , That is, curb its development or progress; and (c) alleviate the disease, that is, promote the regression of the disease and/or alleviate one or more symptoms of the disease. "Treatment" is also intended to encompass the delivery of an agent in order to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, such as in the case of a vaccine.

Herein "tumor infiltrating lymphocytes" or "TIL" means the population of cells initially obtained as white blood cells that have left the individual's bloodstream and migrated into the tumor. TIL includes, but is not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), Thi and Thi 7 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TIL includes primary TIL and secondary TIL. "Primary TILs" are those TILs obtained from patient tissue samples as outlined herein (sometimes referred to as "fresh collections"), and "secondary TILs" are any population of TIL cells that have been expanded or proliferated as discussed herein , Including but not limited to body TIL and amplified TIL ("REP TIL" or "REP TIL"). The population of TIL cells may include genetically modified TIL. TIL can generally be defined biochemically, using cell surface markers, or based on its ability to infiltrate tumors and achieve treatment. TIL can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD62L, CD95, PD-1 and CD25. Additionally and alternatively, TIL can be functionally defined in terms of its ability to infiltrate solid tumors when reintroduced into a patient. TIL can be further characterized by performance-for example, if TIL is produced in response to TCR conjugation, for example, interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, it can be considered as potent or functional, or better, by flow cytometry, after TCR-induced stimulation, CD137, CD107a, INF-γ, TNF-α and IL- 2. Intracellular staining, individual cells can be effective.

As used herein, "retentate" refers to materials that do not pass through filters, nets, or membranes.

As used herein, "ultimate utility" refers to the manufacture or direct use of regenerative drugs, adoptive cell therapy, ATMP, diagnostic in vitro research or scientific research.

The present invention relates to tumor infiltrating lymphocytes (TIL), specifically unmodified TIL (UTIL), which can be isolated from tumors of patients with metastatic cancer, and relates to autologous TIL produced from the same cancer patient and returned to the same cancer patient . The present invention also relates to a method for isolating a therapeutic population of cryopreserved TIL or UTIL, and to a TIL obtained or obtainable by using a device including a single-use sterile kit to treat and resect the tumor by the method described herein And UTIL.

In general, TIL is initially obtained from a patient tumor sample ("primary TIL") and then amplified for further manipulation as described herein, cryopreservation as outlined herein, re-stimulation, and optionally assessed as TIL health The phenotype of the indicators and a larger population of metabolic parameters.

Patient tumor samples can be obtained using methods known in the art, generally through surgical resection, needle biopsy or other methods used to obtain samples containing a mixture of tumor and TIL cells. In general, tumor samples can be from any solid tumor, including primary tumors, aggressive tumors, or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. Solid tumors can be of any cancer type, including but not limited to breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, stomach cancer, and skin cancer (including but not limited to squamous cell carcinoma). Shape cell carcinoma, basal cell carcinoma and melanoma). In some embodiments, TIL is obtained from malignant melanoma tumors, as these tumors have been reported to have particularly high TIL content.

Generation generally involves a two-stage process. In stage 1, the initial tumor material is dissected, placed in a sterile kit with a disaggregation module, enzyme digested and/or fragmented, and the tumor in the disaggregation module is homogenized to obtain a single cell suspension. Although homogenized cells can be further purified in a separate enrichment module in a sterile kit to remove components, such as reagents that are no longer needed; cell debris; undisaggregated tissues, cells can be stored directly at low temperature to stabilize the starting material It is used for TIL manufacturing and storage in the stabilization module of the sterile kit until stage 2 is required. Stage 2 generally involves the growth of TIL from the excised tumor starting material (2 weeks), followed by a rapid expansion process of TIL cells (rapid expansion program "REP"-2 weeks). The final product was washed and collected, then suspended in buffered saline, 8.5% HAS, and 10% DMSO, and stored at low temperature to form a solid sterile product, which was thawed in a single dose form before infusion without further modification.

There are three separate elements for treatment that may contribute to therapeutic activity. The core element is TIL, that is, tumor-derived T cells, which can target and eliminate tumor cells by a variety of methods in which T cells are used as part of their normal functions. These methods include direct methods (ie, perforin-mediated cytotoxicity) and indirect methods (ie, cytokine production). Although mouse models indicate that the production of interferon gamma is essential for effective therapy, it is not clear which of these methods is most important for the anti-tumor effect in vivo. Two other elements that contribute to therapy are pretreatment chemotherapy and high-dose intravenous IL-2. It is believed that these two elements work by supporting the transplantation of T cells in patients after infusion: initially via modulating chemotherapy, which removes competing and modulating immune cells; and then the IL-2 component that supports T cell survival.

The structure of the cell therapy product is produced by directly growing TIL from the enzymatically digested tumor mass with the help of growth support cell culture medium and T cell support growth factor interleukin-2 (IL-2). This allows tumor-specific T cells to selectively survive and grow from a mixture of tumor cells, while T cells that do not recognize tumor antigens will not be stimulated and will be selectively lost. Products include products based on autologous T cells, where T cells have been derived from the patient's own cancer tissue and rapidly expanded to form a pure T cell population and T cells as defined by CD3 surface markers.

In short, TIL, especially UTIL, can be produced using tumor biopsy as starting material in a two-stage process: stage 1 (usually carried out in 2 to 3 hours) using dissection, enzymatic digestion, and homogenization using kits and semi-automatic devices The initial collection and processing of tumor materials to produce a single cell suspension, which can be directly stored at low temperature using the set’s stabilization module to stabilize the starting material for subsequent manufacturing; and stage 2, which can be within a few days or It happened a few years later. Stage 2 can be carried out over 4 weeks, which can start with the thawing of the product of stage 1, and grow TIL from the tumor starting material (about 2 weeks), followed by a rapid expansion process of TIL cells (about 2 weeks) to increase cell mass and Therefore, the continuous process of increasing the dosage. The TIL, especially UTIL, is concentrated and washed before being formulated into a liquid suspension of cells. The sterile medicine can be stored at low temperature in the bag, which will be thawed in a single dose form before intravenous infusion without further modification.

In one embodiment, the bag of the present invention is a collection bag and/or a cryopreservation bag. The bag and any associated conduits can be substantially clear, transparent, translucent, any desired color, or combination thereof. Tissue collection bags and/or catheters can generally be manufactured in a manner similar to the manufacturing of closed and/or sealed blood and/or cryopreservation bags and associated catheters. The catheter in the present invention can be constructed of any desired material, including but not limited to polyvinyl chloride (PVC). For example, PVC may be the desired material because PVC facilitates welding and/or sealing.

The collection bag, such as the tissue collection bag of the present invention, may include at least a part of a bag for receiving tissue made of a predetermined material, such as polyolefin polymer, ethylene vinyl acetate (EVA), copolymer (such as acetic acid) A blend of vinyl ester and polyolefin polymers (that is, OriGen Biomedical EVO film) and/or materials including EVA. The material for the bag can be selected for specific characteristics and/or a series of characteristics, such as tightness (such as heat sealability), air permeability, flexibility (such as low temperature flexibility), elasticity (such as low temperature elasticity), chemical resistance Properties, optical transparency, biocompatibility (such as cytotoxicity), hemolytic activity, resistance to leaching, with low particles.

The seal can be formed with energy (e.g. heat) during use to create a weld zone. The sealing port formed during use may have a width in the range of about 2.5 mm to about 7.5 mm. Generally speaking, the sealing port 140 is formed after placing the tissue material in the bag 140 and may have a width of about 5 mm. The seal peel test (ie ASTM F88/F88M) and/or burst test (ie ASTM F1140/F1140M or ASTM F2051/F2054M) can be used to test the strength of the seal.

In some embodiments, the bag or flexible container can withstand a force of 100 Newtons during use when properly sealed and when positioned in a device for handling and/or processing, when further secured with a clamp. The bag or flexible container embodiment may be constructed to withstand a force of 75 Newtons during use when properly sealed and when positioned within a device for handling and/or processing, when further secured with a clamp.

When forming a sealing port or a fusion port on a flexible container (such as a bag, for example, a collection bag and/or a cryopreservation bag), the sealing device can be used at a predetermined temperature, pressure, and amount of time depending on the material used for the bag Apply heat and/or pressure. For example, some heat sealers may need to apply heat and pressure for about eight seconds. After 8 seconds, the heat of the device can be turned off, however, pressure can be applied for an additional 2 to 3 seconds.

In some embodiments, the bag may have a length in the range of about 10 cm to about 50 cm. In particular, the bag used in the present invention described herein may have a length in the range of about 15 cm to about 30 cm. For example, the bag may have a length in the range of about 18 cm to about 22 cm.

Some conduits can be weldable. The weldable conduit may be made of a polymer material such as polyvinyl chloride (PVC).

Can be used at locations along the catheter including, but not limited to, valves without needle valves. In some embodiments, the bag may have a length in the range of about 10 cm to about 40 cm. In particular, the bag used in the present invention described herein may have a length in the range of about 15 cm to about 30 cm. For example, the bag may have a length in the range of about 18 cm to about 22 cm.

Cryopreservation bags may need to be suitable for cryopreservation using cryoprotectants such as dimethylsulfoxide ("DMSO"). In some embodiments, the cryopreservation bag can be constructed so that the bag can contain a volume of material in the range of about 5 ml to about 45 ml. In detail, the cryopreservation bag may include a volume of material contained in the range of about 10 ml to about 35 ml. For example, some embodiments include cryopreservation bags that can contain a volume of material to be stored in the range of about 15 ml to about 30 ml. The cryopreservation bag may have a size such that a desired predetermined volume is achieved. In some embodiments, the cryopreservation bag may have a width in the range of about 4 cm to about 11 cm and a length in the range of about 10 cm to about 18 cm. For example, the cryopreservation bag may have a width in the range of about 5.8 cm to about 9.8 cm and a length in the range of about 12 cm to about 16 cm. In detail, an embodiment of the cryopreservation bag may have a width of about 7.8 cm and a length of about 14 cm.

Before use, the cryopreservation kit and/or its specific components can be sterilized. The material used to form the bag may be heat-sealable. The material used for the bag may include, but is not limited to, polymers such as EVA, polyamide (e.g. nylon), and combinations thereof. The open bag can be used for processing and/or deaggregation after the bag is closed using a sealing port and/or clamp.

The filter may be an inline filter, a blood filter (such as a blood administration filter), a biological filter, and/or a line agglutinate removal filter. The filter can be configured to remove material larger than a predetermined size from the treated tissue to form the desired material. For example, clumps of tissue can be separated from depolymerized tissue using a filter. In particular, the tissue composition that enters the catheter after filtration may have a component with an average size of less than about 200 µm, so that the desired material is formed. For example, the desired material may include tumor infiltrating lymphocytes (TIL) with an average size of less than about 170 µm.

The filter can be selected so that the treated tissue composition entering from the catheter can be enriched so that after the filter, the desired material flowing into the catheter in the direction of the stabilizing element has a size in the range of about 15 µm to about 500 µm The components. In some embodiments, the filter may be configured such that the tissue composition entering the catheter in the direction of the stabilizing element after filtration has a component having a size in the range of about 50 µm to about 300 µm. For example, in one embodiment, the filter may be configured so that the tissue composition entering the catheter after filtration has a component having a size in the range of about 150 µm to about 200 µm.

In some embodiments, the filter of the enrichment element can remove material outside the predetermined size range of about 5 µm to about 200 µm from the treated tissue to form the desired material. For example, the desired material may include TIL with an average size in the range of about 5 µm to about 200 µm. The valve can be placed at a predetermined distance from the collection bag. For example, the needleless valve can be positioned approximately 20 cm from the collection bag. Valves such as needleless valves can be used to add material to the collection bag. For example, the enzyme culture medium can be inserted into the needle-free valve in order to add the culture medium to the collection bag. The material to be provided via the valve includes, for example, tumor digestion medium and/or cryoprotectant or cryopreservation medium, such as DMSO and/or its solution, such as 55% DMSO and 5% polydextrose cryopreservation medium (for example, BloodStor 55-5).

The syringe can be used to provide tumor digestion medium and 55% DMSO solution, such as 55% DMSO and 5% polydextrose cryopreservation medium via needle-free valves 290 and 292, respectively. During processing, the material can be selectively provided to the cryopreservation kit at a predetermined time. In addition, the clamp can be used to control the flow of provided materials such as tumor digestion medium and/or cryoprotective agents, such as DMSO solution can be provided to devices such as collection bags, filters and/or cryopreservation bags at a predetermined time.

In some embodiments, after such a valve, there may be a predetermined amount of conduit to allow room for additional components to be welded to the cryopreservation kit. For example, after some valves, at least ten (10) cm catheter can be positioned before the next element. The conduit 199 may be sealable and/or weldable. For example, the material used for the catheter may include, but is not limited to, polyvinyl chloride (PVC) and/or other materials known in the art. In some embodiments, the catheter can be sized to fit the connector. For example, the catheter may have an inner diameter in the range of about 1.5 mm to about 4.5 mm and an outer diameter in the range of about 2.1 mm to about 6.1 mm. For example, an embodiment of a cryopreservation kit may include a catheter having an inner diameter in the range of about 2.9 mm to about 3.1 mm and an outer diameter in the range of about 4.0 mm to about 4.2 mm. The length of the catheter used in the cryopreservation kit 191 can vary, wherein the length of the individual catheter elements is in the range of about 1 cm to about 30 cm.

The clamp can be used to prevent and/or prevent enzymatic media and/or digested tissue from moving into the filter. For example, clamps can be used to prevent and/or prevent enzymatic media and/or digested tissue from moving into the filter before the desired filtration step. Another clamp 198 prevents and/or prevents the cryoprotectant from undesirably moving into the filter.

In a particular embodiment, two or more bags may be coupled together to ensure that the depolymerization product material can be properly stored.

In some embodiments, the present invention may include an automated device for semi-automated aseptic depolymerization, enrichment, and/or stabilization of cells and/or cell aggregates from tissues, such as solid mammalian tissues. The automation device used with the present invention may include a programmable processor and a cryopreservation kit. In some embodiments, the cryopreservation kit may be single-use. The present invention further relates to semi-automatic sterile tissue processing methods.

In some embodiments, bags such as collection bags may be used in the collection kit. A bag with an open end allows adding samples, such as tissue samples. The connector in the collection kit can couple the bag to the catheter. The catheter material can be sealable and/or weldable. For example, the catheter can be sealed using energy (such as heat, radio frequency, etc.). The catheter material can be made of PVA.

In some embodiments, the catheter can be coupled to a valve to allow the addition of one or more media enzyme solutions, including but not limited to collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase HI, pepsin, or Its mixture. For example, the valve may be a needleless valve. The catheter used in the cryopreservation kit may include a catheter having an outer diameter in the range of about 3.0 mm to about 5.0 mm, wherein the inner diameter of the catheter is in a range of about 2.0 mm to about 4 mm. In particular, the catheter may have an outer diameter of 4.1 +/- 0.1 mm and an inner diameter of about 3.0 +/- 0.1 mm. The length of the catheter depends on the configuration of the collection set. For example, one embodiment of the collection kit may include a catheter with a length ranging from about 10 cm to about 20 cm.

In some embodiments of the collection kit, the prototype may include one or more clamps to prevent and/or prevent tissue and/or enzyme culture from moving. In particular, the enzyme culture medium and/or tissue can be prevented from moving into the filter before the filtration step.

There are three separate elements for treatment that may contribute to therapeutic activity. The core element is TIL, such as UTIL, which has the potential to eliminate tumor cells through multiple mechanisms that T cells use as part of their normal functions.

These mechanisms include: direct cytotoxicity, by [a] releasing cytotoxins (such as perforin, granzyme, and granulysin), which enter target cells by tight junctions and induce cell death; and by [b] Cell surface interactions between T cells and targets, such as binding FAS ligand-mediated cytotoxicity to induce apoptosis; and indirect methods (such as cytokine production), which can recruit and stimulate secondary effector cells to participate And induce tumor cell death.

TIL, especially UTIL, is an autologous product; therefore, each batch manufactured provides a single dose for the designated patient. There is no mixing of sub-lots or batches. The medicine is a small amount of aseptically prepared T cells (5× 10 ⁹ to 5×10 ¹⁰ ) batches.

Compared with US Patent No. 10,398,734 (the "'734 Patent"), there are several advantages in the present invention. The first step in the '734 patent transforms the tumor itself into fragments of TIL cultured therefrom. In contrast, the present invention releases TIL from a tumor, the tumor is stored and depolymerized in a sterile kit under aseptic conditions after resection, a cell suspension is prepared therefrom, and the resulting TIL is stored by cryopreservation. The present invention provides a diverse population of TILs representing the diversity that exists within tumors. And because it is a homogeneous suspension, the TIL expanded in culture will retain this diversity, which provides the greatest opportunity to solve the diverse population of cancer cells present in the tumor.

In contrast, the manufacturing process of the '734 patent starts with fragments of tissue that have undergone internal cell population degradation during shipment and any other delays before starting processing. In addition, the TIL used for manufacturing will only be TIL amplified from tissue fragments, and not any TIL retained in the interior, so that the resulting cell population may not reflect the complete diversity of the tumor environment.

Another difference is that the process of entering the closed manufacturing process occurs much earlier in the process of the present invention than in the process of the '734 patent and the probability of contamination is lower. Specifically, the destruction of tumor tissue in this application occurs in a closed processing system, rather than the '734 patent describes a deep fragmentation process that takes place in the form of an open operation in a biological safety cabinet.

Because the starting material of the present invention is stored under sterile conditions in a sterile kit, the complete manufacturing process for cryopreserved tumor cell suspensions can be arranged and performed with high capacity and efficiency. In contrast, since the '734 patent starts with unfrozen tissue, the fragmentation and "growth out" steps are performed on a stand-by basis, which has a lower capacity utilization efficiency. In the '734 patent, the removal of this intermediate freezing step shortens the manufacturing process as a whole, but it means that the entire process is performed on a standby basis. This means that manufacturing downtime has a significant impact on the manufacturing facilities of the '734 patent because there cannot be any Delays, and planning the manufacturing downtime requires all products in the process to be completed and new operations need to be stopped.

The advantage of the process of this application is that the tissue in the form of tumor resection can be collected before TIL therapy is needed, transported, processed, cryopreserved, and stored in a sterile kit until and if required to manufacture, so it can be used to treat patients with early disease Of patients, while they have alternative therapies, they are collected and stored. Therefore, there is little or no impact on the time or geographic location of tumor collection and subsequent manufacturing. In the '734 patent, this is not possible, and the complete manufacture of the drug must occur before the cells can be frozen and preserved.

As mentioned above, these are very different culture processes that will produce different cell populations for the starting REP culture, as reflected by the very different number of cells required to inoculate the REP culture, between 1 million and 20 million ( The present invention) is relative to 25 million to 200 million (the '734 patent). In the present invention, during the initial TIL expansion, the culture is inoculated with a cell suspension (that is, cells grown from depolymerized and cryopreserved cells, which will be a mixture of resident and emerging T cells) (As opposed to overgrowth from fragments (ie, newborn cells)); this means that REP is not only seeded with newborn T cells. In addition, the present invention can utilize both solid and flexible closed containers, wherein the flexible container can achieve a higher volume based on the derived tumor suspension (instead of multiple fragments as defined in the '734 patent). Good environment].

Use standard surgical practices to surgically remove metastatic tumor material in the operating room. Before depolymerization, the extra material (ie, non-tumor material as defined macroscopically) is removed and the tumor material is transferred into a sterile bag.

The following may involve the acceptance test of tumor starting materials. First, the source tissue was confirmed as tumor material. Second, assess the microbial load of a representative sample of depolymerized tissue and where the current antibiotic susceptibility is defined (manufacturing can be done at antibiotic risk), but the final material must be negative for microbial growth. Third, the number and survival rate of TIL and tumor cells can be evaluated by flow cytometry.

The method of the present invention includes the following steps: aseptically disaggregating tumors resected from an individual to produce disaggregated tumors, wherein if the resected tumors can be stored at low temperature without cell damage, they are fully disaggregated. In an advantageous embodiment, the programmable processor of the semi-automatic device can control the depolymerization, so that the surface of the depolymerized flexible container can mechanically squeeze and shear the solid tissue (see, for example, PCT Publication No. WO 2018/ 130845). The de-agglomerated surface can be controlled, for example, by a mechanical piston.

For enzymatic digestion, an enzyme mixture of DNase 1 and collagenase (type IV) was used to produce a cell suspension (containing both T cells and tumor cells) from the resected metastatic tumor. The combination of repeated mechanical compression exposes an additional surface for the enzyme to obtain, and the enzyme reaction accelerates the process of solid tissue becoming a cell suspension before cryopreservation, which is optional. In one embodiment, after the depolymerization step is completed, the DMSO-based cryoprotectant is added just before the controlled rate refrigeration cycle. In some embodiments, the enzymatic decomposition of solid tissues can be achieved by selecting and providing one or more medium enzyme solutions, such as collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase H1, pepsin or the like. Any mixture. The enzymatic digestion of the resected metastatic tumor can be carried out in the depolymerization flexible container of a semi-automatic device.

For example, in another embodiment of the method of the present invention, when the depolymerization process is supplemented with enzymatic digestion, the medium formulation used for enzymatic digestion must be supplemented with enzymes that help protein breakdown and make cell-to-cell boundary breakdown. .

Various liquid formulations known in cell culture or cell processing technology can be used as liquid formulations for cell depolymerization and enzymatic digestion of solid tissues, including but not limited to one or more of the following media: organ preservation solution, selective Dissolving solution, PBS, DMEM, HBSS, DPBS, RPMI, Ebbe's medium, XVIVO™, AIM-V™, lactated Ringer's solution, Ringer's acetate solution, physiological saline, PLASMAYTE™ solution, crystal solution and IV Fluid, colloidal solution and IV fluid, 5% dextrose aqueous solution (D5W), Hartmann's solution DMEM, HBSS, DPBS, RPMI, AIM-V™, Escher medium, XVIVO™, each may be supplemented with additional cells as appropriate Supporting factors such as fetal bovine serum, human serum or serum substitutes or other nutrients or cytokines to help cell recovery and survival or specific cell depletion. The culture medium can be a standard cell culture medium, such as the medium mentioned above, or for example for primary human cell culture (for example for endothelial cells, hepatocytes or keratinocytes) or stem cells (for example, dendritic cell maturation, hematopoietic expansion, keratinocytes) Cells, mesenchymal stem cells or T cells) special medium. The culture medium may have supplements or reagents well known in the art, such as albumin and transporters, amino acids and vitamins, one or more metal ions, antibiotics, linking factors, delinking factors, surfactants, growth factors and cells Interleukins, hormones or solubilizers. Various media are commercially available from, for example, ThermoFisher, Lonza or Sigma-Aldrich or similar media manufacturers and suppliers.

The liquid formulation required for enzymatic digestion must have sufficient calcium ions present in at least 0.1 mM up to 50 mM, optimally in the range of 2 to 7 mM, and ideally 5 mM.

The solid tissue to be digested can be washed with a liquid formulation containing chelating agents EGTA and EDTA after depolymerization to remove adhesion factors and inhibitory proteins, followed by washing and removing EDTA and EGTA, and then enzymatic digestion.

The liquid formulation required for enzyme digestion preferably has the least chelating agents EGTA and EDTA, which can severely inhibit enzyme activity by removing calcium ions required for enzyme stability and activity. In addition, β-mercaptoethanol, cysteine and 8-hydroxyquinoline-5-sulfonate are other known inhibitory substances.

Use dissection, enzyme digestion and homogenization to process tumor materials to produce TIL, especially UTIL single cell suspension, which can be directly stored at low temperature to stabilize the starting material for subsequent processing, which is used for cell suspension through TIL, especially UTIL The first amplification in IL-2 to obtain the first population of TIL, especially UTIL.

These methods also include the step of cryopreservation to disaggregate tumors, such as cell suspensions. The cryopreservation of the disaggregated tumor is performed on the same day as the step of aseptically disaggregating the tumor resected from the individual to generate a disaggregated tumor. If the resected tumor can be stored at low temperature without cell damage, it will be fully disaggregated. For example, cryopreservation is 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90 minutes after the step of disaggregating tumors, or 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 hours. Depolymerization of tumors, as the cryopreservation of the single cell suspension obtained by enzymatic depolymerization in the depolymerization module of a semi-automatic device, by cooling and/or maintaining the suspension at a temperature between 8°C and at least -80°C or lower To proceed. Depolymerization can be as fast as 5 minutes, but most often 45 minutes to 1 hour, and cryogenic storage can be as fast as 60 minutes or up to 150 minutes. In one embodiment, the methods include storing cryopreserved disaggregated tumors. As described in the preferred embodiment, the device includes at least one cell container for cryopreservation, wherein the container is a flexible container made of an elastic deformable material. In this embodiment of the device, the final container is directly transferred to the freezer -20 to -190°C or lower, optimally located in a controlled rate freezing equipment associated with the device or supplied separately (limited by Planer Products or Asymptote, for example) In company manufacturing), the temperature of the freezing chamber and one or more flexible storage containers used to contain the enriched and depolymerized solid tissue container is controlled by the following: inject cold air (usually nitrogen, such as Planer products) ; Or by removing heat from one or more controlled cooling surfaces. Both methods make it possible to control the freezing process of one or more specific cells to be frozen at the required rate accurately based on the desired survival rate of the freezing solution and product with an error of less than 1°C or better 0.1°C. This cryopreservation process must consider the ice crystal growth temperature, which is ideally as close as possible to the melting temperature of the frozen solution. Then the crystals grow in the aqueous solution, the water is removed from the system as ice, and the concentration of the remaining unfrozen solution increases. As the temperature decreases, more ice is formed, reducing the remaining unfrozen portion, and its concentration further increases. In aqueous solutions, there is a large temperature range in which ice coexists with concentrated aqueous solutions. Finally, the temperature is lowered, and the solution reaches the glass transition state. At this time, the frozen solution and the cells turn from the viscous solution to a solid-like state. Below this temperature, the cells will not undergo further biological changes and therefore stabilize in several years, possibly decades. Until needed.

Ice crystal growth and crystal growth involve the release of heat to the freezing solution and the cell microenvironment, and it is desirable to keep the cells and the freezing solution cool, even when the freezing fluid resists temperature changes during a phase change. Depends on whether the depolymerization includes enzymatic depolymerization and the optimal temperature for enzymatic digestion of a given enzyme, enzyme concentration and tissue type, the temperature at the beginning of cryopreservation includes but not limited to 40℃, 39℃, 38℃, 37℃ , 36℃, 35℃, 34℃, 33℃, 32℃, 31℃, 30℃, 29℃, 28℃, 27℃, 26℃, 25℃, 24℃, 23℃, 22℃, 21℃ and 20 °C, that is, the temperature in the range of mammalian body temperature to room temperature, and further includes lower refrigeration temperatures, such as but not limited to 10°C, 8°C, 6°C, 5°C, 4°C, 3°C, and 2°C. The target temperature of cryogenic cooling includes but is not limited to -60°C, -65°C, -70°C, -75°C, -80°C, -85°C, -90°C and the temperature in between, and as low as the liquid nitrogen vapor storage temperature (-195.79) ℃) lower temperature. In some embodiments, the methods and devices used in accordance with the present invention are designed or programmed to minimize the time from physiological temperature or digestion temperature to cryogenic storage temperature. In certain embodiments, the methods and devices for cryopreservation used in accordance with the present invention are advantageously designed and programmed so that as the culture medium crystallizes, heat is released to the environment including the cells, to the environment, to the environment around the environment, or to the environment. Cooling under conditions that are minimized or avoided. In some embodiments, the method is designed and/or the device is programmed for continuous cooling from the depolymerization temperature to the low temperature target temperature. Exemplary programmed cooling rates include, but are not limited to, -0.5°C/min, -1°C/min, -1.5°C/min, -2°C/min, or -2.5°C/min. The cooling rate is the program target and can be changed in the cooling cycle. The cooling rate can be varied, for example, ±0.1°C/min, ±0.2°C/min, ±0.3°C/min, ±0.4°C/min, or ±0.5°C/min. In an embodiment of the present invention, the cryogenic storage temperature is -80℃±10℃ and the device is programmed to reduce the temperature by 1℃/min or 1.5℃/min or 2℃/min or 1℃/min±0.5℃ /min or 1.5°C/min±0.5°C/min or 2°C/min±0.5°C/min.

In some embodiments, the methods of the present invention provide for obtaining young TIL, which can provide increased replication cycles when administered to an individual/patient and therefore can provide superiority to older TIL (ie, further prior to administration to the individual/patient). The additional therapeutic benefit of TIL) after more rounds of replication. The characteristics of young TIL have been described in the literature, for example, Donia et al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother 32:415-423 (2009); Robbins et al., J Immunol 2004; 173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou et al., J Immunother, 28:53-62 (2005); and Tran et al. , J Immunother, 31:742-751 (2008), all of which are incorporated herein by reference in their entirety.

The diverse antigen receptor systems of T and B lymphocytes are produced by somatic cell recombination of a limited but large number of gene segments. These gene segments: variable (V), diversity (D), junction (J) and constant (C), determine the binding specificity and downstream applications of immunoglobulin and T cell receptor (TCR). The present invention provides a method for generating TIL that exhibits and increases the diversity of the T cell repertoire. In some embodiments, the TIL obtained by the method of the present invention exhibits an increase in the diversity of the T cell repertoire. In some embodiments, the TIL obtained by the method of the present invention exhibits an increase in the diversity of the T cell repertoire compared to freshly collected TIL and/or TIL prepared using methods other than the provided methods. In some embodiments, the TIL obtained by the method of the present invention exhibits an increase in the diversity of the T cell repertoire compared to the freshly collected TIL and/or TIL. In some embodiments, the TIL obtained in the first expansion exhibits an increase in the diversity of the T cell repertoire. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin light chain in the immunoglobulin. In some embodiments, the diversity is in the T cell receptor. In some embodiments, the diversity is in one of T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, the expression of T cell receptor (TCR) alpha and/or beta is increased. In some embodiments, the expression of T cell receptor (TCR) alpha is increased. In some embodiments, the expression of T cell receptor (TCR) β is increased. In some embodiments, the performance of TCRab (ie, TCRα/β) is increased.

The method of the present invention also includes the following steps: performing the first expansion by culturing the disaggregated tumor in a cell culture medium containing IL-2 to produce a first population of TIL, especially UTIL. The cells produced by the above steps are cultured in serum containing IL-2 under conditions that promote the growth of TIL compared to tumors and other cells. In some embodiments, tumor digests are grown in 2 mL wells in a medium containing inactivated human AB serum with 6000 IU/mL IL-2. This primary cell population is cultured for several days, usually 3 to 14 days, to produce a bulk TIL population, usually about 1×10 ⁸ bulk TIL cells. In some embodiments, the primary cell population is cultured for 7 to 14 days to produce a bulk TIL population, usually about 1×10 ⁸ bulk TIL cells. In some embodiments, the primary cell population is cultured for 10 to 14 days to produce a bulk TIL population, usually about 1×10 ⁸ bulk TIL cells. In some embodiments, the primary cell population is cultured for about 11 days to produce a bulk TIL population, usually about 1×10 ⁸ bulk TIL cells.

In a preferred embodiment, the amplification of TIL can use the initial bulk TIL amplification step as described below and herein, followed by the second amplification (including rapid amplification protocol (REP) step) as described below and herein And then stimulate the REP step) to proceed.

In an advantageous embodiment, the cryopreserved depolymerized tumor tissue is thawed and 1:9 resuspended in T cell culture medium (T cell culture medium manufactured by Immetacyte contract, supplemented with the following additives 10% FBS and 3000 IU/mL IL-2 ), then filtered through the line 100 to 270 μm filter and centrifuged in a 50 mL centrifuge tube, and then resuspended in 20 mL. Samples can be obtained for flow cytometry analysis to quantify a variety of HLA-A, B, C, CD58 ⁺ and DRAQ7¯ cells. In some embodiments, this can be used instead of manual (such as but not limited to hemocytometer) or alternative automated total viable cell counting device (such as but not limited to NucleoCounter™; Guava ^® ; automated blood analysis and counter; pipette-based cells Counter, such as but not limited to Scepter™) inoculation.

In one embodiment, the resuspended cryopreservation disaggregated tumor tissue is cultured in serum containing IL-2 under conditions that promote the growth of TIL compared to tumors and other cells. In some embodiments, the tumor digest is contained in inactivated human AB serum with 6000 IU/mL IL-2 (or in some cases, as outlined herein, in the presence of an artificial antigen presentation [aAPC] cell population) Incubate the medium in 2 mL wells. This primary cell population is cultured for several days, usually 10 to 14 days, to produce a bulk TIL population, usually about 1×10 ⁸ bulk TIL cells. In some embodiments, the growth medium contains IL-2 or a variant thereof during the first expansion. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution has a specific activity of ^{20 to 30×10 6 IU/mg for a 1 mg vial.} In some embodiments, the IL-2 stock solution has a specific activity of ^{20×10 6 IU/mg for a 1 mg vial.} In some embodiments, the IL-2 stock solution has a specific activity of ^{25×10 6 IU/mg for a 1 mg vial.} In some embodiments, the IL-2 stock solution has a specific activity of ^{30×10 6 IU/mg for a 1 mg vial.} In some embodiments, the final concentration of the IL-2 stock solution is 4-8×10 ⁶ IU/mg IL-2. In some embodiments, the final concentration of the IL-2 stock solution is 5-7×10 ⁶ IU/mg IL-2. In some embodiments, the final concentration of the IL-2 stock solution is 6×10 ⁶ IU/mg IL-2. In some embodiments, the first expansion medium comprises about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6000 IU/mL IL-2 or about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium contains about 6,000 IU/mL IL-2. In one embodiment, the cell culture medium further contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In one embodiment, the cell culture medium further contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU/mL IL-2. In one embodiment, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL , About 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL or about 8000 IU/mL IL- 2. In one embodiment, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL , 7000 to 8000 IU/mL or about 8000 IU/mL IL-2.

In some embodiments, the first expansion medium comprises about 500 IU/mL IL-12, about 400 IU/mL IL-12, about 300 IU/mL IL-12, about 200 IU/mL IL-12, about 180 IU/mL IL-12, about 160 IU/mL IL-12, about 140 IU/mL IL-12, about 120 IU/mL IL-12, or about 100 IU/mL IL-12. In some embodiments, the first expansion medium contains about 500 IU/mL IL-12 to about 100 IU/mL IL-12. In some embodiments, the first expansion medium contains about 400 IU/mL IL-12 to about 100 IU/mL IL-12. In some embodiments, the first expansion medium contains about 300 IU/mL IL-12 to about 100 IU/mL IL-12. In some embodiments, the first expansion medium contains about 200 IU/mL IL-12. In some embodiments, the cell culture medium contains about 180 IU/mL IL-12. In one embodiment, the cell culture medium further contains IL-12. In a preferred embodiment, the cell culture medium contains about 180 IU/mL IL-12.

In some embodiments, the first expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the first expansion medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium contains about 200 IU/mL IL-15. In some embodiments, the cell culture medium contains about 180 IU/mL IL-15. In one embodiment, the cell culture medium further contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU/mL IL-15.

In some embodiments, the first expansion medium comprises about 500 IU/mL IL-18, about 400 IU/mL IL-18, about 300 IU/mL IL-18, about 200 IU/mL IL-18, about 180 IU/mL IL-18, about 160 IU/mL IL-18, about 140 IU/mL IL-18, about 120 IU/mL IL-18, or about 100 IU/mL IL-18. In some embodiments, the first expansion medium contains about 500 IU/mL IL-18 to about 100 IU/mL IL-18. In some embodiments, the first expansion medium contains about 400 IU/mL IL-18 to about 100 IU/mL IL-18. In some embodiments, the first expansion medium contains about 300 IU/mL IL-18 to about 100 IU/mL IL-18. In some embodiments, the first expansion medium contains about 200 IU/mL IL-18. In some embodiments, the cell culture medium contains about 180 IU/mL IL-18. In one embodiment, the cell culture medium further contains IL-18. In a preferred embodiment, the cell culture medium contains about 180 IU/mL IL-18.

In some embodiments, the first expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL- twenty one. In some embodiments, the first expansion medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the first expansion medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In one embodiment, the cell culture medium further includes IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU/mL IL-21.

The medium also considers a combination of interleukins, such as but not limited to IL-2, IL-12, IL-15, IL-18 and IL-21. Also consider other cytokines, such as IL-23, IL-27, IL-35, IL-39, IL-18, IL-36, IL-37, IL-38, IFN-α, IFN-β, IFN- γ or a combination of IL-2, IL-12, IL-15, IL-18 and IL-21. Also consider antibodies, such as Th2 blocking reagents, such as but not limited to IL-4 (aIL4), anti-IL-4 (aIL4R), anti-IL-5R (aIL5R), anti-IL-5 (aIL5), anti-IL13R (aIL13R) Or anti-IL13 (aIL13).

In some embodiments, the first TIL amplification can be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days. Days or 14 days. In some embodiments, the first TIL expansion can be performed for 1 to 14 days. In some embodiments, the first TIL expansion can be performed for 2 to 14 days. In some embodiments, the first TIL expansion can be performed for 3 to 14 days. In some embodiments, the first TIL expansion can be performed for 4 to 14 days. In some embodiments, the first TIL expansion can be performed for 5 to 14 days. In some embodiments, the first TIL expansion can be performed for 6 to 14 days. In some embodiments, the first TIL expansion can be performed for 7 to 14 days. In some embodiments, the first TIL expansion can be performed for 8 to 14 days. In some embodiments, the first TIL expansion can be performed for 9 to 14 days. In some embodiments, the first TIL expansion can be performed for 10 to 14 days. In some embodiments, the first TIL expansion can be performed for 11 to 14 days. In some embodiments, the first TIL expansion can be performed for 12 to 14 days. In some embodiments, the first TIL expansion can be performed for 13 to 14 days. In some embodiments, the first TIL expansion can be performed for 14 days. In some embodiments, the first TIL expansion can be performed for 1 to 11 days. In some embodiments, the first TIL expansion can be performed for 2 to 11 days. In some embodiments, the first TIL expansion can be performed for 3 to 11 days. In some embodiments, the first TIL expansion can be performed for 4 to 11 days. In some embodiments, the first TIL expansion can be performed for 5 to 11 days. In some embodiments, the first TIL expansion can be performed for 6 to 11 days. In some embodiments, the first TIL expansion can be performed for 7 to 11 days. In some embodiments, the first TIL expansion can be performed for 8 to 11 days. In some embodiments, the first TIL expansion can be performed for 9 to 11 days. In some embodiments, the first TIL expansion can be performed for 10 to 11 days. In some embodiments, the first TIL expansion can be performed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the first amplification period. In some embodiments, IL-2, IL-7, IL-15, and/or IL-21 and any combination thereof may be included during the first amplification period. In some embodiments, the combination of IL-2, IL-15, and IL-21 is used as a combination during the first amplification period.

In some embodiments, the first amplification is performed in a closed system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is an example G-REX-10 or G-REX-100 or advantageously a device of WO 2018/130845. In some embodiments, the closed system bioreactor is a single bioreactor.

Advantageously, the TIL population obtained from the first expansion (referred to as the second TIL population) can undergo a second expansion (which may include the expansion sometimes referred to as REP). Similarly, where the genetically modified TIL will be used for therapy, the first TIL population (sometimes referred to as the bulk TIL population) or the second TIL population (which in some embodiments may include those referred to as the REP TIL population) The population) can be genetically modified before expansion or after the first expansion and before the second expansion for suitable treatment.

Bean virus is an effective gene transfer vehicle due to its ability to transduce dividing and non-dividing cells. Although the most well-studied legumevirus gene therapy vector is derived from type 1 human immunodeficiency virus (HIV), it has also been developed based on other primate and non-primate legumeviruses, including HIV-2, SIV, Feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), goat arthritis encephalitis virus (CAEV), visna virus (visna virus) and Jembrana disease virus (Jembrana disease virus, JDV) Gene therapy vector.

Replication-defective viral vectors are necessary to prevent patients from being infected with potentially lethal viruses. Legumevirus vectors have been developed to become safer and more effective. The latest third-generation vector removes all auxiliary toxic and pathogenic auxiliary genes while separating the remaining genes, which is essential for the performance of the transgene in the three plastids. See, for example, U.S. Patent Publication 2006/0024274.

The EIAV gene transfer vector has been proved to be effective in _{transducing proliferation and G 1 suppression cells in vitro.} Mitrophanous et al., 1999. Stable gene transfer to the nervous system using a non-primate lentiviral vector. Gene Ther. 6: 1808-1818; Olsen, JC, 1998, Gene transfer vectors derived from equine infectious anemia virus. Gene Ther. 5 : 1481-1487; Olsen, JC, 2001, EIAV, CAEV and Other Lentivirus Vector Systems, Somat Cell Mol Genet, Volume 26, Issue 1/6, 131-45.

Heemskerk, B. et al. 2008, Adoptive cell therapy for patients with melanoma, using tumor-infiltrating lymphocytes genetically engineered to secrete interleukin-2. Human gene therapy, 19(5), 496-510 describes genetically engineered to express IL -2 to extend the TIL survival period of TIL. The patient's TIL was transfected with a retroviral vector based on Moloney murine leukemia virus (MMLV) during the first amplification period, and then the second amplification was performed to obtain a sufficient number for treatment.

In short, the SBIL2 vector containing the MFG backbone derived from Moloney murine leukemia virus (MMLV) and the cDNA copy of the human IL-2 gene under the control of the 5'long terminal repeat (LTR) promoter is in Pseudotyped in the PG13 encapsulated cell line, which provides the gibbon ape leukemia virus (GaLV) envelope protein. A stable production clone (PG13SBIL2#3) containing three copies of integrated retroviral IL-2 DNA was produced. The clinical GMP grade SBIL2 retrovirus supernatant was produced by the National Gene Vector Laboratory of Indiana University (Indianapolis, IN). For TIL transduction, 6-well non-tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ) are coated with recombinant human fibrin fragments (Retronectin) (CH-296, 25 μg/ml phosphate buffered saline [PBS] solution, GMP Grade; Takara Bio, Otsu, Japan), blocked with PBS-2% human serum albumin (HSA), and pretreated with thawed SBIL2 virus supernatant (5 ml/well) _{at 32°C and 10% CO 2} Load for 4 hours. Add TIL at 3 ml/well at 37°C and 5% CO ₂ for 18-24 hours, transfer to the second set of SBIL2 load plates, and incubate for another 18-24 hours, after which TIL is collected and resuspended in fresh Medium.

Zhang, L. et al., 2015, Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma, Clinical Cancer Research 21(10), 2278-2288 TIL of IL-12 is selectively secreted. TIL is transduced with a MSGV1 γ-retroviral vector carrying a gene encoding single-chain IL-12 driven by the nuclear factor of activated T cell (NFAT) promoter.

MSGV-1 is derived from the MSGV vector that uses the long terminal repeat sequence of the murine stem cell virus and contains the extended gag region and Kozak sequence. The gene encoding human single-chain IL-12 was synthesized by the NFAT-responsive promoter in the order of IL-12 p40, linker G6S and IL-12 p35, and inserted into the MSGV-1 vector in the opposite direction of the 5'LTR. The production cell line based on high titer PG13 cells was produced and the retroviral supernatant was produced by NCI Surgery Branch Vector Production Facility (Bethesda, MD) under Good Manufacturing Practice (GMP) conditions. The vector supernatant was tested and passed all currently required US Food and Drug Administration regulations for the production of recombinant gamma-retroviral vectors for clinical applications.

The transduction procedure was performed by irradiating allogeneic PBMC feeder cells with 30 ng/ml anti-CD3 mAb Orthoclone OKT3 (Centocor Ortho Biotech, Raritan, NJ), 3000 IU/ml recombinant human IL-12 and 4 Gy to feed 200 cells per TIL The ratio of cells stimulates tumor infiltrating lymphocytes (TIL) to initiate. On day 4 and/or day 5, cells were collected for transduction using a non-tissue culture 6-well plate coated with recombinant human fibrin fragment (CH-296; Takara Bio, Otsu, Japan). The carrier supernatant was "spin-loaded" by centrifugation at 2000 g at 32°C for 2 hours on the coated pan. The retroviral vector supernatant was aspirated from the wells, and 2×10 ⁶ samples were added to each well. The stimulated TIL cells were then centrifuged at 1000 g for 10 minutes. The plates were incubated at 37°C overnight and the cells were collected the next day for the second transduction. The cells of the first 21 patients underwent two transductions. Patient 12 The cell undergoes only one transduction.

Jones, S. et al., 2009, Lentiviral vector design for optimal T cell receptor gene expression in the transduction of peripheral blood lymphocytes and tumor-infiltrating lymphocytes. Human gene therapy, 20(6), 630-640 described development for bean-shaped The promoter of the viral vector expresses genes in the transduced T lymphocytes and constructs effective anti-tumor T cells.

TIL was obtained from surgical samples. PBL was thawed from a frozen stock solution stored at -180°C and placed in AIM-V and interleukin-2 (IL-2; Cetus, Emeryville, CA) at 300 IU/ml in the culture. For OKT3 stimulation, cells are initially placed in a medium with 50 ng/ml of anti-CD3 antibody OKT3 (Ortho Biotech, Bridgewater, NJ), or placed in OKT3 medium at the initial change of the medium after transduction. ^{For the transduction of PBL or TIL, adjust 1×10 6} cells to a final volume of 1 ml with virus supernatant and polybrene (final concentration, 8 μg/ml) in a 24-well tissue culture treatment dish. The cells were transduced by centrifuging the plate at 1000× g , 32°C for 1.5 hours. The dish was placed in a humidified 5% CO ₂ incubator at 37°C overnight, and the medium was replaced the next day. Use OKT3 (50 ng/ml), IL-2 (5000 IU/ml) and irradiated allogeneic peripheral blood mononuclear cells (TIL:feeder cell ratio, 1:100) from three different donors for TIL The rapid amplification protocol (REP) as previously described. Six days after REP, TIL was transduced and returned to culture as described.

Beane, JD et al., 2015, Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma. Molecular therapy: 23(8), 1380-1390 described by encoding PD-1 Specific zinc finger nuclease (ZFN)-mediated gene editing of mRNA electroporation and PD-1 clinical-scale gene editing.

In order to generate a sufficient number of transduced T cells for adoptive cell transfer, REP was used to induce TIL proliferation. 46 In brief, 1×10 ⁷ TILs and 1×10 ⁹ allogeneic irradiated (5,000 rad) peripheral blood mononuclear cells (PBMC) were combined, and these cells were suspended in 30 ng/ml OKT3 400 ml T cell culture medium. Cells were cultured in G-Rex100 flasks at 37°C and 5% CO2. After five days, aspirate and replace 200 ml of medium. Seven days after the start of REP, TIL was collected and washed twice with Hyclone electroporation buffer (Hyclone Laboratories, Logan, UT). The cells were then counted and resuspended in electroporation buffer at a concentration of ^{1×10 8 /ml.} The cells were then transferred to the MaxCyte CL-2 treatment assembly and mixed with 120 μg/ml PD-1 ZFN mRNA (or GFP mRNA, TIL/GFP for GFP transfection). Electroporation was performed according to the MaxCyte protocol. After electroporation, the TIL from the processing assembly was transferred to a T-175 flask and placed in an incubator at 37°C for 20 minutes. After this incubation step, TIL was resuspended in AIM-V medium at a concentration of ^{1×10 6 /ml.} The cells were then placed in an incubator set at 30°C for overnight low-temperature incubation as previously described. The next day, TIL was transferred to a 37°C incubator and kept undisturbed until day 10 of REP (3 days after electroporation).

In some embodiments, the TIL obtained from the first amplification until the phenotype is stored for selection. In some embodiments, the TIL obtained from the first amplification is not stored and is directly subjected to the second amplification. Therefore, the method includes the following steps: by culturing TIL, especially the first population of UTIL, with additional IL-2, OKT-3, and antigen-presenting cells (APC) to perform a second expansion to produce a second population of TIL . In some embodiments, the TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from the first amplification to the second amplification is about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days after the cryopreserved depolymerized tumor tissue is thawed. Occurs at 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, in some embodiments, the transition from the first amplification to the second amplification occurs about 3 to 21 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs about 4 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs about 4 to 10 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs about 7 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs about 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the inoculation of the REP culture is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, after the cryopreserved depolymerized tumor tissue is thawed. Occurs at 17, 18, 19, 20, or 21 days.

In some embodiments, the transition from the first amplification to the second amplification is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after cryopreserved depolymerized tumor tissue is thawed. Occurs on days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the transition from the first amplification to the second amplification occurs 1 to 14 days after cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the first TIL expansion can be performed for 2 to 14 days. In some embodiments, the transition from the first amplification to the second amplification occurs 3 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 5 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 6 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 7 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 9 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 10 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 11 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 12 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 13 to 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 14 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 1 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 2 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 3 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 5 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 6 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 7 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 9 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 10 to 11 days after the cryopreserved depolymerized tumor tissue is thawed. In some embodiments, the transition from the first amplification to the second amplification occurs 11 days after the cryopreserved depolymerized tumor tissue is thawed.

In some embodiments, TIL is not stored after the first amplification and before the second amplification, and the TIL directly proceeds to the second amplification. In some embodiments, as described herein, the transformation occurs in a closed system. In some embodiments, the TIL from the first expansion, the second population of TIL, directly proceeds to the second expansion without the transition phase.

In some embodiments, the conversion from the first amplification to the second amplification is performed in a closed system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 or Xuri WAVE bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, after collection and initial bulk processing, the number of TIL cell populations is expanded. This further amplification is referred to herein as second amplification, and it may include an amplification process commonly referred to as a rapid amplification process in the art. The second expansion is generally achieved in a gas-permeable or gas exchange container using a medium containing a variety of components, including feeder cells, a source of cytokines, and anti-CD3 antibodies.

In some embodiments, the second expansion of TIL or the second expansion of TIL can be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL expansion can be performed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL expansion can be performed for about 7 days to about 14 days. In some embodiments, the second TIL expansion may be performed for about 8 days to about 14 days. In some embodiments, the second TIL expansion may be performed for about 9 days to about 14 days. In some embodiments, the second TIL expansion may be performed for about 10 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 11 days to about 14 days. In some embodiments, the second TIL expansion may be performed for about 12 to about 14 days. In some embodiments, the second TIL expansion may be performed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 14 days.

In one embodiment, the second amplification can be performed in a gas-permeable container using the method of the present invention. For example, TIL can use non-specific T cell receptors to stimulate interleukin-2 (IL-2) or interleukin-7 (IL-7) or interleukin-15 (IL-15), IL Rapid amplification in the presence of -12. Non-specific T cell receptor stimulators may include, for example, anti-CD3 antibodies, such as about 30 ng/ml OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, Calif. ) Or UHCT-1 (available from BioLegend, San Diego, Calif., USA). TIL can be amplified to induce further stimulation of TIL in vitro by including one or more cancer antigens (including its antigenic portion, such as one or more epitopes) during the second expansion period, the antigen may vary depending on the situation From carrier expression, such as human leukocyte antigen A2 (HLA-A2) binding peptide, for example 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217 (210M), depending on the situation, T cell growth factor, Such as the presence of 300 IU/mL IL-2 or IL-15. Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TIL can also be rapidly amplified by re-stimulation with the same antigen or antigens pulsed to the cancer on the antigen-presenting cell expressing HLA-A2. Alternatively, TIL may be further stimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of the second amplification. In some embodiments, the second expansion is performed in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In one embodiment, the cell culture medium further contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In one embodiment, the cell culture medium contains about 100 IU/mL, about 200 IU/mL, about 300 IU/mL, about 400 IU/mL, about 500 IU/mL, about 600 IU/mL, about 700 IU/mL , About 800 IU/mL, about 900 IU/mL, 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL or about 8000 IU/ mL IL-2. In one embodiment, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL , 7000 to 8000 IU/mL or 8000 IU/mL IL-2.

In one embodiment, the cell culture medium contains OKT3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL OKT3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL , About 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL OKT3 antibody. In one embodiment, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL OKT3 antibodies.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-7, IL-15, and/or IL-21 and any combination thereof may be included during the second amplification period. In some embodiments, the combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21 and any combination thereof may be included.

In some embodiments, the second expansion can be performed in a supplemented cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second expansion occurs in supplemented cell culture medium. In some embodiments, the supplemental cell culture medium includes IL-2, OKT-3, and antigen presenting feeder cells. In some embodiments, the second cell culture medium includes IL-2, OKT-3, and antigen-presenting cells (APC; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (ie, antigen-presenting cells).

In some embodiments, the second expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second expansion medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium contains about 200 IU/mL IL-15. In some embodiments, the cell culture medium contains about 180 IU/mL IL-15. In one embodiment, the cell culture medium further contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU/mL IL-15.

In some embodiments, the second expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL- twenty one. In some embodiments, the second expansion medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second expansion medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In one embodiment, the cell culture medium further includes IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APC) are PBMC. In one embodiment, the ratio of TIL to PBMC and/or antigen presenting cells in rapid expansion and/or second expansion is about 1:25, about 1:50, about 1:100, about 1:125, About 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, About 1:400 or about 1:500. In one embodiment, the ratio of TIL to PBMC in the rapid amplification and/or the second amplification is between 1:50 and 1:300. In one embodiment, the ratio of TIL to PBMC in the rapid amplification and/or the second amplification is between 1:100 and 1:200.

In one embodiment, the REP and/or second amplification is performed in a flask, wherein the body TIL is combined with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL -2 mixed in 150 ml medium. Media replacement (usually 2/3 media replacement with fresh media via aspiration) is performed until the cells are transferred to the replacement growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable containers, as discussed more fully below.

In some embodiments, the second amplification (which may include a process called the REP process) is shortened to 7 to 14 days, as discussed in the examples and figures. In some embodiments, the second expansion is shortened to 11 days.

In one embodiment, the REP and/or the second amplification can use the T-175 flask and breathable bag as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or a gas-permeable incubator (G-Rex flask). In some embodiments, the second amplification (including the amplification called rapid amplification) is performed in a T-175 flask, and about 1×10 ⁶ TIL suspended in 150 mL of medium can be added to each T- 175 in the flask. TIL can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/ml anti-CD3. T-175 flasks can be incubated in 5% CO ₂ at 37°C. Half of the medium can be replaced on the 5th day with 50/50 medium with 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined in a 3 L bag and 300 mL AIM V with 5% human AB serum and 3000 IU/mL IL-2 is added to 300 ml TIL suspension. Count the number of cells in each bag every day or every two days, and add fresh medium to keep the cell count between 0.5 and 2.0×10 ⁶ cells/ml.

In one embodiment, the second amplification can be performed in a 500 mL gas-permeable flask (G-Rex 100, available from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA) with a 100 cm gas-permeable silica bottom. ×10 ⁶ or 10×10 ⁶ TILs can be cultured with PBMC in 400 mL 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2 and 30 ng/ml anti-CD3 (OKT3). G-Rex 100 flasks can be incubated in 5% CO ₂ at 37°C. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491×g) for 10 minutes. The TIL pellets can be resuspended in 150 mL of fresh medium with 5% human AB serum and 3000 IU/mL IL-2 and added back to the original G-Rex 100 flask. When TIL is continuously expanded in G-Rex 100 flasks, the TIL in each G-Rex 100 can be suspended in 300 mL of medium in each flask on the 7th day, and the cell suspension can be divided into 3 for inoculation Three 100 mL aliquots of the G-Rex 100 flask. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL IL-2 can be added to each flask. The G-Rex 100 flasks can be incubated in 5% CO ₂ at 37° C. and after 4 days, 150 mL of AIM-V with 3000 IU/mL IL-2 can be added to each G-REX 100 flask. The cells can be collected on the 14th day of culture.

In one embodiment, the second expansion (including the expansion called REP) is performed in a flask, wherein the body TIL is combined with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 is mixed in 150 ml medium. In some embodiments, the medium replacement is performed until the cells are transferred to the replacement growth chamber. In some embodiments, 2/3 of the medium is replaced with fresh medium by aspiration. In some embodiments, the alternative growth chamber includes a G-REX flask and a gas-permeable container, as discussed more fully below.

In one embodiment, the second amplification (including the amplification called REP) is performed and further includes the step of selecting TIL for good tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058 A1 can be used to select TIL for superior tumor responsiveness.

Optionally, cell viability analysis can be performed after the second amplification (including the amplification called REP amplification) using standard analysis known in the art. For example, trypan blue exclusion analysis can be performed on samples of bulk TIL, which selectively labels dead cells and allows assessment of survival rate. In some embodiments, the Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, Mass.) can be used to count and determine the survival rate of TIL samples.

In some embodiments, the second amplification of TIL (including the amplification called REP) can use the T-175 flask and breathable bag as previously described (Tran KQ, Zhou J, Durflinger KH et al., 2008, J Immunother., 31:742-751, and Dudley ME, Wunderlich JR, Shelton TE et al., 2003, J Immunother., 26:332-342) or a gas-permeable G-Rex flask. In some embodiments, the second amplification is performed using a flask. In some embodiments, the second amplification is performed using a gas-permeable G-Rex flask. In some embodiments, the second amplification is performed in T-175 flasks, and about 1×10 ⁶ TILs are suspended in about 150 mL of medium and added to each T-175 flask. TIL and irradiated (50 Gy) allogeneic PBMC as ``feeding'' cells were cultured together at a ratio of 1:100 and the cells were supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3 CM and AIM-V Culture in a 1:1 mixture of medium (50/50 medium). The T-175 flask was incubated at 37°C in 5% CO ₂ . In some embodiments, half of the medium is replaced with 50/50 medium with 3000 IU/mL IL-2 on day 5. In some embodiments, on day 7, cells from 2 T-175 flasks were combined in a 3 L bag and 300 mL AIM-V with 5% human AB serum and 3000 IU/mL IL-2 was added to 300 mL TIL suspension. The number of cells in each bag can be counted every day or every two days, and fresh medium can be added to keep the cell count between about 0.5 and about 2.0×10 ⁶ cells/ml.

In some embodiments, the second amplification (including the amplification called REP) is performed in a 500 mL volumetric flask (G-Rex 100, Wilson Wolf) ^{with a 100 cm 2 gas-permeable silica bottom, about 5×10 6} or 10×10 ⁶ TILs were cultured in 400 mL 50/50 medium supplemented with 3000 IU/mL IL-2 and 30 ng/ml anti-CD3 with irradiated allogeneic PBMC at a ratio of 1:100. The G-Rex 100 flask was incubated at 37°C in 5% CO ₂ . In some embodiments, on day 5, 250 mL of supernatant was removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL IL-2 and added back to the original G-Rex 100 flask. In the example of continuous expansion of TIL in G-Rex 100 flasks, the TIL in each G-Rex 100 was suspended in 300 mL of medium in each flask on the 7th day, and the cell suspension was divided into three for inoculation Three 100 mL aliquots from the G-Rex 100 flask. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL IL-2 was added to each flask. G-Rex 100 flasks were incubated in 5% CO ₂ at 37° C. and after 4 days, 150 mL of AIM-V with 3000 IU/mL IL-2 was added to each G-REX 100 flask. The cells were collected on the 14th day of culture.

The diverse antigen receptor systems of T and B lymphocytes are produced by somatic cell recombination of a limited but large number of gene segments. These gene segments: variable (V), diversity (D), junction (J) and constant (C), determine the binding specificity and downstream applications of immunoglobulin and T cell receptor (TCR). The present invention provides a method for generating TIL that exhibits and increases the diversity of the T cell repertoire. In some embodiments, the TIL obtained by the method of the present invention exhibits an increase in the diversity of the T cell repertoire. In some embodiments, the TIL obtained in the second expansion exhibits an increase in the diversity of the T cell repertoire. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin light chain in the immunoglobulin. In some embodiments, the diversity is in the T cell receptor. In some embodiments, the diversity is in one of T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, the expression of T cell receptor (TCR) alpha and/or beta is increased. In some embodiments, the expression of T cell receptor (TCR) alpha is increased. In some embodiments, the expression of T cell receptor (TCR) β is increased. In some embodiments, the performance of TCRab (ie, TCRα/β) is increased.

In some embodiments, the second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APC).

In some embodiments, the second amplification is performed in a closed system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is a device such as G-REX-10 or G-REX-100 or advantageously WO 2018/130845. In some embodiments, the closed system bioreactor is a single bioreactor.

In one embodiment, the second expansion procedure described herein and the procedure referred to as REP) require overfeeding of cells during the REP TIL expansion period and/or during the second expansion period. In many embodiments, the feeder cell line is obtained from peripheral blood mononuclear cells (PBMC) of a standard whole blood unit of a healthy blood donor. PBMC is obtained using standard methods, such as Ficoll-Paque gradient separation.

Generally speaking, allogeneic PBMC are not activated by irradiation or heat treatment, and are used in the REP procedure, which provides an exemplary protocol for evaluating replication incompetence of irradiated allogeneic PBMC.

In some embodiments, if the total number of live cells on the 14th day is less than the initial live cells placed in the culture on the 0th day of the REP and/or the 0th day of the second expansion (that is, the start day of the second expansion) Cell number, PBMC is deemed to be incompetent for replication and accepted for use in the TIL expansion procedure described herein.

In some embodiments, if cultured in the presence of OKT3 and IL-2, the total number of viable cells on day 7 and day 14 is compared to day 0 of REP and/or day 0 of second expansion ( That is, on the start day of the second expansion) the number of initial living cells placed in the culture did not increase, PBMC was deemed to be replication incompetent and accepted for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/ml OKT3 antibody and 3000 IU/ml IL-2.

In some embodiments, if cultured in the presence of OKT3 and IL-2, the total number of viable cells on day 7 and day 14 is compared to day 0 of REP and/or day 0 of second expansion ( That is, on the start day of the second expansion) the number of initial living cells placed in the culture did not increase, PBMC was deemed to be replication incompetent and accepted for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5 to 60 ng/ml OKT3 antibody and 1000 to 6000 IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 10 to 50 ng/ml OKT3 antibody and 2000 to 5000 IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/ml OKT3 antibody and 2000-4000 IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 25 to 35 ng/ml OKT3 antibody and 2500 to 3500 IU/ml IL-2.

In some embodiments, the antigen presenting feeder cell is PBMC. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TIL to antigen-presenting feeder cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, About 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:50 and 1:300. In one embodiment, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:100 and 1:200.

In one embodiment, the second expansion procedure described herein requires a ratio of ^{about 2.5×10 9} feeder cells: about 100×10 ^{6 TIL.} In another embodiment, the second expansion procedure described herein requires a ratio of ^{about 2.5×10 9} feeder cells: about 50×10 ^{6 TIL.} In yet another embodiment, the second expansion procedure described herein requires about 2.5×10 ⁹ feeder cells: about 25×10 ⁶ TIL.

In one embodiment, the second expansion procedure described herein requires overfeeding of cells during the second expansion. In many embodiments, the feeder cell line is obtained from peripheral blood mononuclear cells (PBMC) of a standard whole blood unit of a healthy blood donor. PBMC is obtained using standard methods, such as Ficoll-Paque gradient separation. In one example, artificial antigen presenting (aAPC) cells are used instead of PBMC.

Generally speaking, allogeneic PBMC are not activated by irradiation or heat treatment, and are used for TIL amplification procedures.

In one example, artificial antigen presenting cells are used in the second expansion instead of PBMC or in combination with PBMC.

The amplification methods described herein generally use a medium with a high dose of cytokines, especially IL-2, as known in the art.

Alternatively, it is also possible to use a combination of cytokines for rapid expansion and/or second expansion of TIL, wherein the combination of two or more of IL-2, IL-15 and IL-21 is as disclosed in the international The case No. WO 2015/189356 and W International Publication No. WO 2015/189357 are generally summarized, and these documents are expressly incorporated herein by reference in their entirety. Therefore, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, with the latter being specific in many embodiments. use. The use of a combination of cytokines is particularly advantageous for the production of lymphocytes, and especially T cells as described herein.

In some embodiments, the medium used in the amplification methods described herein (including the amplification method called REP) also includes anti-CD3 antibodies. The combination of anti-CD3 antibody and IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen in the case of full-length antibodies and Fab and F(ab')2 fragments, where the former is generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is incorporated by reference in its entirety. Into this article.

Those familiar with this technology should understand that there are a variety of anti-human CD3 antibodies suitable for use in the present invention, including anti-human CD3 multi-strain and monoclonal antibodies from various mammals, including but not limited to murines, humans, and primates. , Rat and canine antibodies. In a specific embodiment, the OKT3 anti-CD3 antibody (available from Ortho-McNeil, Raritan, N.J. or Miltenyi Biotech, Auburn, Calif.) is used.

After the second expansion step, the cells can be collected. In some embodiments, TIL is collected after one, two, three, four, or more amplification steps. In some embodiments, TIL is collected after two amplification steps.

The TIL can be collected in any suitable and sterile manner, including, for example, by centrifugation. The TIL collection method is well known in the art and any such known method can be used with the process of the present invention. In some embodiments, TIL is collected using an automated system.

The cell harvester and/or cell processing system can be purchased from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. Any cell-based collector can be used with the method of the invention. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, the cell collection line is via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump cell-containing solutions through membranes or filters (such as rotating membranes or rotating membranes) in a sterile and/or closed system environment. Filter), allowing continuous flow and cell processing to remove the supernatant or cell culture medium without pelleting. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed sterile system.

In some embodiments, collection is performed from a closed system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is a device such as G-REX-10 or G-REX-100 or advantageously WO 2018/130845. In some embodiments, the closed system bioreactor is a single bioreactor.

The cells are transferred to the container for administration to the patient. In some embodiments, once a therapeutically sufficient number of TIL is obtained using the amplification method described above, it is transferred to a container for administration to the patient.

In one embodiment, the TIL amplified using the APC of the present invention is administered to a patient in the form of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TIL in a sterile buffer. The TIL amplified using the PBMC of the present invention can be administered by any suitable method known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic.

In one embodiment, the TIL amplified using the method of the present invention is administered to the patient in the form of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TIL in a sterile buffer. The TIL amplified using the PBMC of the present invention can be administered by any suitable method known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs are administered, with an average of about 7.8×10 ¹⁰ TILs, especially if the cancer is melanoma. In one embodiment, about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs are administered. In some embodiments, about 3×10 ¹⁰ to about 12×10 ¹⁰ TILs are administered. In some embodiments, about 4×10 ¹⁰ to about 10×10 ¹⁰ TILs are administered. In some embodiments, about 5×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, about 6×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, about 7×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ . In some embodiments, the therapeutically effective dose is about 7.8×10 ¹⁰ TILs, especially if the cancer is melanoma. In some embodiments, the therapeutically effective dose is about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is about 3×10 ¹⁰ to about 12×10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is about 4×10 ¹⁰ to about 10×10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is about 5×10 ¹⁰ to about 8×10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is about 6×10 ¹⁰ to about 8×10 ^{10 TIL} . In some embodiments, the therapeutically effective dose is about 7×10 ¹⁰ to about 8×10 ¹⁰ TIL.

In some embodiments, the number of TIL provided in the pharmaceutical composition of the present invention is about 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ 2×10 ¹⁰ 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1× 10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2× 10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3× 10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In one embodiment, the number of TIL provided in the pharmaceutical composition of the present invention is within the following range: 1×10 ⁶ to 5×10 ⁶ , 5×10 ⁶ to 1×10 ⁷ , 1×10 ⁷ to 5 ×10 ⁷ , 5×10 ⁷ to 1×10 ⁸ , 1×10 ⁸ to 5×10 ⁸ , 5×10 ⁸ to 1×10 ⁹ , 1×10 ⁹ to 5×10 ⁹ , 5×10 ⁹ to 1 ×10 ¹⁰ , 1×10 ¹⁰ to 5×10 ¹⁰ , 5×10 ¹⁰ to 1×10 ¹¹ , 5×10 ¹¹ to 1×10 ¹² , 1×10 ¹² to 5×10 ¹² and 5×10 ¹² to 1 ×10 ¹³ .

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% of the pharmaceutical composition. %, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% , 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002 % Or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75% of the pharmaceutical composition , 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6 %, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75 %, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003% , 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is within the following range: about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30% of the pharmaceutical composition. %, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, About 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5 % To about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w /v or v/v.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is within the following range: about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5 of the pharmaceutical composition %, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, About 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g , 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention exceeds 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g , 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

The TIL provided in the pharmaceutical composition of the present invention is effective in a wide dosage range. The exact dosage will depend on the route of administration, the form of compound administration, the sex and age of the individual to be treated, the weight of the individual to be treated, and the preference and experience of the attending physician. The clinically determined dose of TIL can also be used when appropriate. The amount of the pharmaceutical composition administered using the method herein, such as the dosage of TIL, will depend on the human or mammal to be treated, the severity of the disease or condition, the rate of administration, the configuration of the active pharmaceutical ingredients, and the judgment of the prescribing physician Depends.

In some embodiments, TIL can be administered in a single dose. Such administration can be by injection, such as intravenous injection. In some embodiments, TIL can be administered in multiple doses. The administration can be once, twice, three times, four times, five times, six times, or more than six times per year. The administration can be once a month, once every two weeks, once a week, or once every other day. The investment of TIL may continue as needed.

In some embodiments, the effective dose of TIL is about 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some embodiments, the effective dose of TIL is in the following range: 1×10 ⁶ to 5×10 ⁶ , 5×10 ⁶ to 1×10 ⁷ , 1×10 ⁷ to 5×10 ⁷ , 5×10 ⁷ to 1×10 ⁸ , 1×10 ⁸ to 5×10 ⁸ , 5×10 ⁸ to 1×10 ⁹ , 1×10 ⁹ to 5×10 ⁹ , 5×10 ⁹ to 1×10 ¹⁰ , 1×10 ¹⁰ to 5×10 ¹⁰ , 5×10 ¹⁰ to 1×10 ¹¹ , 5×10 ¹¹ to 1×10 ¹² , 1×10 ¹² to 5×10 ¹² and 5×10 ¹² to 1×10 ¹³ .

In some embodiments, the effective dose of TIL is in the following range: about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg , About 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg /kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg To about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is in the following range: about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg To about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg , About 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of TIL can be administered by any of the recognized modes of administration of agents with similar utility, including intranasal and transdermal routes, by intraarterial injection, intravenous, intraperitoneal, parenteral, intramuscular, Subcutaneously, locally, by transplantation or by inhalation, administered in single or multiple doses.

The present invention also includes a kit suitable for using the TIL of the present invention, especially UTIL for diagnosis and prognostic analysis. The kit of the present invention includes buffers, cytokines, flasks, culture media, product containers, reagents and instructions.

The following presents a non-limiting multi-step example to set up TIL growth from a tumor, set up a rapid expansion process, confirm that the irradiated PBMC feeder cells do not expand and transfer the static culture to the WAVE bioreactor (see e.g. https:/ /www.gelifesciences.com/en/us/shop/cell-culture-and-fermentation/rocking-bioreactors/consumables-and-accessories/single-use-readytoprocess-wave-cellbag-bioreactors-p-00346#overview) and Blending and filling.

In step one (day 0), the cryopreserved depolymerized tumor tissue is thawed and resuspended in T cell culture medium supplemented with 10% FBS and 3000 IU/mL IL-2 1:9, and then goes through the pipeline 100 to 270 Filtered on a μm filter and centrifuged in a 50 mL centrifuge tube, then resuspended in 20 mL. Obtain samples for flow cytometry analysis SOP-to quantify a variety of HLA-A, B, C, CD58 ⁺ and DRAQ7¯ cells.

In step two, the cell suspension was then incubated in the cell vessel to ≥0.25 × 10 ⁶ to ≤0.75 × 10 ⁶ th HLA-A, B, C and DRAQ7¯ and CD58 ⁺ cells / ml supplemented with adding the antibacterial Inoculated into CM-T (T cell culture medium supplemented with 10% fetal bovine serum) and interleukin-2 (IL-2) 1000 IU/ml . T cells grow in CM-T over a period of 2 weeks. From day 5, remove half of the medium and use fresh medium supplemented with 10% fetal bovine serum, amphotericin B, gentamicin and IL-2 CM-T replacement. This is repeated every 2/3 days between the 5th day and the 10th day to ensure that the cells are maintained at ≤0.1×10 ⁶ to 2×10 ⁶ CD45 ⁺ CD3 ⁺ phospholipid binding protein -V ^-ve (Annexin-V ^-ve ) DRAQ7 ^-ve cells/ml. According to PH Eur 2.6.27, the microbiological examination of the supernatant of TIL culture (day 5 to day 7) confirmed that there was no microbial growth. Flow cytometric analysis of measurement (day 7 to day 10) the quantitative CD45 ⁺ CD3 ⁺ concentration of phospholipid binding proteins and -V ^¯ DRAQ7¯ of cells.

In step three, Ficoll (density 1.078 g/ml) was used to separate 4×10 ⁹ irradiated PBMCs (25-50 Gy) from multiple allogeneic donors (white blood cell layer from healthy blood supply). Flow cytometry analysis and quantification of CD45 ⁺ phospholipid binding protein-V and DRAQ7 cells. Microbial growth was determined by the microbiological inspection test of irradiated PBMC.

In step 4, quantify the amount of TIL available to start the rapid amplification process (day 12). Flow cytometry analysis and quantitative analysis of CD45 ⁺ CD3 ⁺ phospholipid binding protein-V and DRAQ7 cells.

In step 5, in a closed static cell culture bag, prepare a culture mixture of feeder cells (PBMC separated by irradiated ficoll) and growth supplements in 3 LT cell mixed medium, which contains: ≥3 to ≤5×10 ⁹ One irradiated PBMC-CD45 ⁺ phospholipid binding protein-V and DRAQ7 cells, 7-9% human AB serum, 2000 to 4000 IU/mL IL-2 and 20 to 40 ng/ml OKT-3 antibody.

In step 6, before adding TIL, a representative sample of the culture mixture of feeder cells (PBMC separated by irradiated ficoll) was obtained for use in the control flask.

In step 7, add TIL to the REP culture: ≥1 to ≤20×10 ⁶ tumor-derived TIL-CD45 ⁺ CD3 ⁺ phospholipid binding protein-V and DRAQ7 cells.

In step 8, the static culture was incubated in a dry incubator at 3.5% to 6% carbon dioxide at 35°C to 38.5°C for 6 days. ^{The number of CD45 +} phospholipid binding protein -V ^- and DRAQ7 ^- cells in the control flasks (collected in step 6) containing the REP mixture without TIL to ensure that the irradiated feeder cells are not expanded on the 14th and 18th days and Survival rate. Flow cytometry analysis and quantification of CD45 ⁺ phospholipid binding protein-V and DRAQ7 cells.

In step 9, the WAVE bioreactor bag is pre-conditioned with 1.7 L TCM supplemented with 7 to 9% human AB serum and 2000 to 4000 IU/mL IL-2 at 35 to 38.5°C and 3.5% to 6% carbon dioxide. 1 To 2 hours.

In step 10, TIL is transferred and amplified in the WAVE bioreactor system.

In step 11, connect a 1×TCM 10 L bag supplemented with 2000 to 4000 IU/mL IL-2.

In step 12 (day 19 to 22), adjust the perfusion rate between day 19 and day 22.

In step 13 (day 24), the filling is stopped, and the waste and feed are disconnected.

In step 14, the TIL is concentrated and washed.

In step 15, in an infusion bag with a total volume ranging from 125 to 270 mL, cells suspended in PBS containing 10% DMSO and 8.5% HSA were used to prepare the final drug formulation.

In step 16, a sample of the final product bag containing TIL is obtained for QC analysis and sample retention. The QC analysis of fresh medicines includes microbiological inspection testing and color and visible particle testing. Prepare retained samples for cell dose, survival rate, phenotype and efficacy; microbiological examination and endotoxin analysis.

In step 17, the final product container is labeled and overlaps the final product label.

In step 18, cryopreserve by freezing at a controlled rate of -1°C/min to -60°C and transfer to storage at ≤-130°C. The QC analysis of cryopreserved drugs includes qPCR mycoplasma test, T cell dose and survival rate test, endotoxin test as measured by the kinetic color LAL test, and evaluation of CD137 after co-cultivation with cell lines expressing anti-CD3 fragments ⁺ , IFN-γ ⁺ , TNFα ⁺ or CD107a ⁺ combination of CD2 ⁺ performance CD45 ⁺ DRAQ7 ^- performance test. Table 1-Overview of manufacturing using only static culture bags CTU-TIL number (gender) TIL from the first amplified tumor (×10 ⁷ ) Live CD3+ cells in the second expansion (×10 ⁷ ) Amplification factor* Final tissue live CD3+ (×10 ¹⁰ ) 1 (F) 2.1 1.5 690 1.0 3 (M) 3.8 2.0 1100 2.2 5 (M) 8.2 1.5 1281 2.0 Mean ± SD 6.0±2.2 1.7±0.23 1023±303 1.7±0.52 *-Equal to the TIL used in the final manufactured TIL/REP Table 2-Overview of manufacturing using perfusion bioreactor CTU-TIL number (gender) TIL from the first amplified tumor (×10 ⁷ ) Live CD3+ cells in the second expansion (×10 ⁷ ) Amplification factor* Final tissue live CD3+ (×10 ¹⁰ ) 12 (F) 5.4 2.0 1600 3.2 13 (M) 14.0 2.0 1010 2.02 14 (M) 5.8 2.0 2100 4.2 15(M) 5.1 2.0 3100 6.2 16 (M) 3.0 1.8 3000 5.4 19 (M) 7.6 2.0 3400 6.8 20 (M) 1.4 1.1 5409 5.95 21 (F) 1.4 1.4 3646 4.85 27(M) 5.1 2.0 1845 3.69 28 (F) 8.9 2.0 1590 3.18 32 (F) 34.0 2.0 1835 3.67 32 (F) N/A ** 2.0 1985 3.97 35 (M) 8.6 2.0 3125 6.25 36 (M) 3.2 1.6 2050 3.28 37 (F) 4.0 2.0 1265 2.53 38 (M) 0.55 0.32 3969 1.27 39 (M) 0.83 0.83 1398 1.16 40 (F) 1.4 0.71 7444 5.3 41 (M) 9.0 2.0 1555 3.11 42 (M) 9.8 2.0 1965 3.93 43 (F) 25.0 2.0 2310 4.62 47 (F) 2.67 2.0 1450 2.9 48 (F) 2.73 2.0 1865 3.73 51 (M) 4.1 2.0 1780 3.56 54 (M) 27.5 2.0 395 7.9 57 (M) 2.3 1.5 764 1.13 60 (F) 3.1 1.1 1486 1.56 63 (M) 0.84 0.89 5842 5.24 64 (M) 0.72 0.72 2993 2.14 67 (M) 0.38 0.37 7526 2.82 Mean ± SD 6.61±8.2 1.56±0.61 2650±1770 3.52±1.69 *-Equal to the TIL used in the final manufactured TIL/REP **-Patients treated with TIL from the original tumor source twice

The invention provides a depolymerization system or device. In some embodiments, the disaggregation device is in the form of a treadle device that disaggregates tissue into individual cells or cell aggregates. In some embodiments, the depolymerization device provides thermal control during the depolymerization process. In some embodiments, the present invention provides a cryopreservation system or device. In some embodiments, a device for depolymerization and cryopreservation and thermal control is provided. In another aspect, the present invention provides one or more flexible containers or a system containing a plurality of containers, the containers including one or more depolymerization/cryopreservation systems or devices adapted for use in the present invention Flexible container for depolymerization, cryopreservation or both depolymerization and cryopreservation. In some embodiments, one or more containers or a plurality of containers are interconnected and suitable for use in a closed system. The above aspects are indicated in the scope of the patent application attached to this article. In view of the following detailed description that provides an example of the present invention, more advantages and benefits of the present invention will become readily apparent to those skilled in the art.

In certain embodiments, the deaggregator includes one or more movable surfaces, such as plates and/or paddles, and is designed to apply compressive and shear forces to the tissue sample. In one embodiment, the digester includes a first surface and a second surface that can move relative to each other. In some embodiments, the surface is an opposing surface positioned to apply pressure to the sample. In an embodiment, at least one of the surfaces moves in a direction perpendicular to the direction of the surface in order to apply pressure to the sample. In one embodiment, the surfaces are aligned in parallel and are designed to move together and separate in a repetitive or cyclic manner, so that the sample is repeatedly compressed between the surfaces in a cyclic manner and then relaxed. In the embodiments of the present invention, the compression and relaxation of the sample generates shear forces in the sample.

In an embodiment, one of the first and second surfaces remains stationary when the other surface moves. In another embodiment, both the first and second surfaces move. In one embodiment, the tissue sample is contained in a flexible and/or elastic container, which contains the tissue sample and optionally a depolymerization fluid or solution. In some embodiments, the container adapts to changes in volume between the first and second surfaces as the surfaces move. In certain embodiments, the container is elastic and confines the tissue sample and depolymerization fluid to the opposing surface. In some embodiments, the container is flexible and the surrounding air pressure helps confine the tissue sample and depolymerization fluid to the opposing surface. In some embodiments, the air pressure is ambient pressure. In some embodiments, air pressure is applied in the enclosed chamber and the pressure is greater than the ambient pressure.

In certain embodiments, the deaggregation device includes two or more opposing surface groups arranged side by side. In some such embodiments, the surface groups share one surface (for example, a single plate, which remains stationary as appropriate), and the second surfaces of each group are positioned side by side and apply pressure against the stationary plate. The second surface can alternately apply pressure in a pedaling motion. In certain such embodiments, a flexible container is used, which confines the tissue sample and depolymerized fluid in the space between the stationary surface and the moving surface, while allowing the contents of the container to flow back and forth between the moving surfaces. In certain embodiments, the container is adapted to limit or prevent such back and forth movement of the contents. In one embodiment, the flow of contents from one side to the other is blocked across the sealed opening of the container. In another embodiment, a baffle across the container restricts the flow of contents from one side to the other.

The heald surface can be actuated by any suitable mechanism. This document discloses an example of the side rod system of the device 100 which is designed to alternately move the tread surface against the flexible container. There is a spring on the surface of the heddle. The spring is designed to press the surface of the heddle against the container while allowing the thickness of the container to change and the grain size in the container. In some embodiments, the spring is preloaded. Also disclosed herein is an example of the cam actuation design of the device 200 characterized by two tread surfaces. In the device 200, the pre-loaded spring presses the heddle surface against the flexible container and the cam mechanism cyclically raises one heddle surface, and then the other one moves away from the flexible container. In another embodiment, one or more rocker arms or rods are used to lift the heddle surface away from the container. In yet another embodiment, the heddle surface is raised and lowered hydraulically. In yet another embodiment, the heddle surface is raised and lowered pneumatically. Although in the 200 device, there are two heddle surfaces that alternately contact the disaggregation vessel, in some embodiments, the actuation mechanism allows all moving surfaces to apply pressure at the same time, including when the system is at rest. Such features can be used to empty the contents of the depolymerization container at the end of the depolymerization process. For example, instead of the heddle surface being in the middle position or one raised and one lowered, all the heddle surfaces are lowered relative to the disaggregation container, and their contents are squeezed out through the connected tube, filtered as appropriate, to the second level Receiving container, such as a cryopreservation container.

In the completely closed depolymerization and cryopreservation system exemplified in this article, it is characterized by automated depolymerization, followed by manual filtration and transfer to a cryopreservation container by a sealing system of a syringe and tube, and automated cryopreservation. Advantageously, the depolymerized tumor tissue is manually transferred from the depolymerization container to the cryopreservation container, and the depolymerization and cryopreservation steps are performed by the same automated device programmed to sequentially manage the two steps. In other embodiments, the disaggregation procedure is designed so that upon termination, the disaggregated tumor tissue is automatically moved from the disaggregation container to the cryopreservation container. In some embodiments, the peristaltic pump and valve in contact with the connecting tube control the flow of the contents. In some embodiments, the tread surface of the deaggregator is arranged to push or squeeze the deaggregated tumor solution from the deaggregation container into the cryopreservation container via the filter as appropriate, and the valve controls the flow of the contents. In such embodiments, as exemplified herein, the depolymerization and cryopreservation in a closed system and any transfer of materials are preferably controlled and performed by the same device.

Several depolymerization systems have been tested and optimized with respect to variables including force, digestion time and speed (RPM or cycles/minute). Determine the results and predictions of certain tissue types for the combination of force, time, and speed variables, including forces at most and above 60 N, digestion time at most and above 60 minutes, and speeds at most and above 240 RPM. In some embodiments of the present invention, the force is 20-200 N, or 30-120 N, or 30-90 N, or 40-60 N, or 10-20 N, or 20-30 N, or 30- 40 N, or 40-50 N, or 40-45 N, or 45-50 N, or 50-55 N, or 55-60 N, or 60-65 N, or 65-70 N, or 70-75 N , Or 75-80 N. A typical heddle foot has a surface area of ^{about 20 to 50 cm 2.} Based on the 30 cm ² tread surface, the tread pressure is 0.5-6.5 N/cm ² , or 1-4 N/cm ² , or 1-3 N/cm ² , or 1-2 N/cm ² , or 1.5- 2.5 N/cm ² , or 2-3 N/cm ² , or 2.5-3.5 N/cm ² , or 1.5 N/cm ² ±0.5 N/cm ² , or 2 N/cm ² ±0.5 N/cm ² , Or 2.5 N/cm ² ±0.5 N/cm ² , or 3 N/cm ² ±0.5 N/cm ² , or 4 N/cm ² ±0.5 N/cm ² , or 5 N/cm ² ±0.5 N/cm ² . The nominal pressure can be measured with a pressure sensor, and is preferably calibrated for the thickness of the depolymerization vessel. In some embodiments, the deaggregation device incorporates a pressure sensor. In certain embodiments of the present invention, the digestion time is 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less, or 5-120 minutes, or 15-100 Minutes, or 30-90 minutes, or 40-60 minutes, or 5-10 minutes, or 10-20 minutes, or 20-30 minutes, or 30-40 minutes, or 40-45 minutes, or 45-50 minutes, Or 50-60 minutes, or 60-65 minutes, or 65-70 minutes, or 40 minutes ± 5 minutes, or 45 minutes ± 5 minutes, or 50 minutes ± 5 minutes, or 55 minutes ± 5 minutes, or 60 minutes ± 5 minutes, or 65 minutes ± 5 minutes, or 70 minutes ± 5 minutes. In some embodiments, the depolymerization device operates at 60-360 RPM, or 120-340 RPM, or 180-300 RPM, or 210-270 RPM, 80-160 RPM, or 120-200 RPM, or 160-240 RPM , Or 200-280 RPM, or 240-320 RPM, or 280-360 RPM, or 60 RPM±20 RPM, or 80 RPM±20 RPM, or 100 RPM±20 RPM, or 120 RPM±20 RPM, or 140 RPM ±20 RPM, or 160 RPM±20 RPM, or 180 RPM±20 RPM, or 200 RPM±20 RPM, or 220 RPM±20 RPM, or 240 RPM±20 RPM, or 260 RPM±20 RPM, or 280 RPM± Operate at 20 RPM, or 300 RPM±20 RPM, or 320 RPM±20 RPM, or 340 RPM±20 RPM, or 360 RPM±20 RPM.

In certain embodiments, the material is understood to be continuous. In certain embodiments, the material is understood to be periodic or intermittent. For example, when an increase in temperature is observed in a depolymerized sample, it may be advantageous to temporarily slow down or interrupt the polymerization to reduce or prevent the temperature increase or allow the temperature to equilibrate to a set point. Without wishing to be bound by theory, the temperature increase can be through the physical manipulation of the sample by the disaggregation device, the heat transfer from the movable pedal mechanism of the device, the reduced physical contact during the disaggregation process, the heat transfer from the sample to the freezing unit, or other reasons happen. In certain embodiments, periodic or intermittent deaggregation may be beneficial to the deaggregation device. In the cam driving device as disclosed herein, the life expectancy of the cam mechanism can be increased by periodically reversing the direction of rotation of the cam from time to time, thus extending the life of the cam by distributing wear on both sides of the cam. In the embodiment of the present invention, the activity time period of physical gathering includes but is not limited to 15 to 30 seconds, 20 to 40 seconds, 30 to 60 seconds, 45 to 75 seconds, 60 to 90 seconds, at least 20 seconds, and at least 30 seconds. Seconds, at least 40 seconds, at least 1 minute, at least 1.5 minutes, or at least 2 minutes. The duration of inactivity can be but not limited to 1 second to 10 seconds, 10 seconds to 20 seconds, 20 seconds to 30 seconds, 30 seconds to 40 seconds, 40 seconds to 60 seconds, 5 seconds, 10 seconds, 20 seconds, 30 Seconds, 40 seconds, 60 seconds, 90 seconds, 120 seconds or duration in between. The duration of inactivity can be as short as the time necessary for the depolymerization device to reverse direction.

In some embodiments, the surfaces are opposing surfaces that are positioned to move laterally with respect to each other. In some such embodiments, the lateral movement includes linear lateral movement. In certain such embodiments, lateral movement includes orbital lateral movement. In a certain embodiment, there are both linear and orbital lateral movement.

In one embodiment, the opposite surface is flat. In an embodiment, at least one of the surfaces includes a convex area and is arranged to move in a rocking motion relative to the other surface. One aspect of convex surface and rocking motion provides creep-like motion.

According to the present invention, the movement of the surface is controlled. Such control includes controlling one or more aspects of the surface movement, including but not limited to speed, sample compression, system pressure, duration, and cycle frequency. In some embodiments, one or more aspects of plate movement are constant. In some embodiments, one or more states of plate movement depend on the state of disaggregation. In some embodiments, the state of disaggregation is defined by the time of the disaggregation process, such as one or more predefined stages, such as early, middle, late, or more accurately measured in hours, minutes, and seconds. period. In some embodiments, the state of disaggregation is defined by the size distribution of the tumor mass. For example, in one embodiment of the present invention, as the size of the tumor mass decreases, the pressure increases.

Examples of depolymerization devices and alternative solutions

Referring to FIG. 41, there is shown a heddle device 100 that disaggregates tissue into individual cells or cell aggregates in a closed and at least initially sterile sample container bag 10 that is substantially flat and relatively thin. The device includes a temperature control device that can be removably inserted into a temperature control device (such as a controlled temperature rate change freezer, thawing device, or warmer, for example, a commercially available freezer called Via Freeze™, or a temperature control device that provides a controlled rate of temperature change). Any other device is schematically shown in FIG. 41 and generally described herein as a housing 110 formed by an assembly of components in a freezer 40). In practice, the housing will include an unspecified cover. In use, the device and bag provide a closed system to disaggregate tissue (such as resected tumors, resected tumors or parts of needle biopsy, etc.), and then cryopreserve the resulting cell suspension for subsequent analysis without the need to disaggregate The sample is transferred from the bag 10.

The housing 110 has a chassis 112 to which a motor unit 114 including an electric motor and a gearbox is connected, which has an output speed of 10 to 300 rpm. The output shaft of the motor and gearbox 114 has a crank 116 driving a connecting rod 118, which in turn is pivotally connected to the pedal mechanism 120, which will move through a pedal cycle for each rotation of the crank 116, that is Heald cycle between 0.2 and 6 seconds. In more detail, the pedaling mechanism has a parallelogram four-bar linkage device, which includes two spaced apart pivots 122 and 124 rigidly mounted to the chassis 112, which are respectively pivotally mounted on two relatively parallel horizontal rods 126 and 128. Each of the horizontal rods has two parallel harness rods 130 and 132, which are pivotally connected to one on each side of the pivot shafts 122 and 124, together forming a parallelogram linkage. The connecting rod 118 is suitably pivotally held to the extension of the top horizontal rod so that the movement of the extension causes a cyclic up and down movement of the heddles 130 and 132 (in the orientation shown). Each heald rod 130 and 132 is connected with foot assemblies 134 and 136, which, by virtue of the above-mentioned cyclic movement, will move up and down in a sequential manner with the movement of the crank 116, that is, when one foot is up, the other will move up and down. Will go down, and vice versa.

The foot assemblies 134 and 136 each include a flat bottom plate 138 and 140, and each plate is spring-mounted to the upper foot frame 142 and 144 by a spiral metal spring 146, respectively. In the configuration described above or an equivalent configuration (if used), the spring 146 is preloaded. In this case, for each foot, the combined preload is preferably 40 to 80 N, more preferably 30 to 70 N, and preferably about 60 N. The combined spring strain rate is 1 N to 5 N per millimeter of movement, preferably about 3 N per millimeter, and the expected foot movement is about 8 mm to 12 mm, preferably about 10 mm. In addition, the surface area of each foot is intended to be about 20 to 50 cm ² , preferably about 35 cm ² . This makes the imaginary pressure on the bag between zero (when the foot is lifted from the bag or is substantially empty, and up to about 6 N/cm ² (about 9 psi). The preferred imaginary pressure is about 2 N/cm ² (about 3 psi). However, given that the bag may or may not, at least at the beginning of the heddle process, contain homogeneous materials, there will be clumps of material in which the applied force will concentrate, and therefore the pressure is described as an "hypothetical" ideal situation, For example, when the heddling process is about to end, the minimum pressure difference resistance of the bag 10 applied is provided.

At the bottom of the chassis is the receiving area 148 of the flexible bag 10 and the adjacent receiving area 148 is the heat transfer plate 150. The area 148 is large enough to allow the sample processing bag 10 to be slid onto the plate 150 via the front part of the chassis (the front part is shown in FIG. 41). The board includes the upper surface 151 of the bag 10 on the upper surface, and the lower surface 152 exposed for external heating or cooling during use. The upper surface 151 is generally parallel to the bottom plates 138 and 140 of each foot, so that the bottom plate moves generally parallel to the surface 151. In other words, the flat bottom plate moves in a direction substantially perpendicular to the surface 151, which prevents significant side forces on the mechanism 120. The plate 150 is formed of metal, preferably aluminum or copper, or gold or silver, or alloys containing these metals. The thermal conductivity is preferably higher than 100 and more preferably higher than 200 W/m K, which is measured at 20°C. The thickness of the plate 150 material is about 3 mm or less, and provides a low thermal mass, and therefore provides a faster response to the contents of the bag 10 following temperature changes on the opposite side of the plate.

Also referring to FIGS. 42 and 43, the device operates by supplying current to the motor unit 114 to drive the crank 116 clockwise in this example as indicated by the arrow C. The crank causes the connecting rod 118 to operate the pedal mechanism 120 described above. It should be noted that the top and bottom of the stroke of the crank (where the maximum force is applied to the mechanism 120) coincide with the lowest positions of the respective foot assemblies 134 and 136. The foot assembly moves up and down in the directions of the arrows U and D to sequentially massage the sample bag 10 so that the contents of the bag 10 have the opportunity to move to a side away from the respective heddles. Since the potentially solid tissue sample in the bag can move away from the pedal foot, and because the bottom plates 138 and 140 of each foot are spring-loaded, which provide additional elastic movement to the foot, even when it is at the bottom of its stroke, it should be understood It is less likely that the organization will get stuck when the quality of the organization is higher. Sequential pedaling actions also reduce the probability of bag 10 breaking.

Figure 44 is a plan view of the device 100 described above, but in this view, the bag 10 is not in place. In particular, the relative side-by-side positions of the foot assemblies 134 and 136 can be seen, which are spaced apart and have a collective area viewed in plan view, which is approximately equal to the area of the bag 10 when laid flat, but the area of the bag 10 The area difference of about ±10% is effective.

FIG. 45 shows another plan view of the device 100', which is similar in construction to the device 100 described above, but in this alternative, the motor 113 of the motor unit 114 is transverse to its transmission by using a 90-degree transmission 115 The output shaft of 115 is configured so that the motor 113 does not protrude beyond the back wall 111 of the device 100'. Therefore, if necessary, the device 100' can be adapted to a smaller freezer volume.

During the depolymerization process described above, the force applied by the foot assemblies 134 and 136 is reacted by the heat transfer plate 150. This means that the sample bag 10 is pressed against the contact surface 151 of the plate 150 during processing, providing good surface contact between the sample bag 10 and the plate surface 151, and thus improving heat energy transfer.

Figures 46, 47 and 48 show different embodiments of the flexible sample bag 10 mentioned above. Slide the bag in use to an appropriate position in the receiving area 148 in the device 100 or 100' and place it under the two feet 134 and 136 mentioned. Therefore, the bag has a generally flat configuration with a thickness of approximately up to 12 mm, and has some additional compliance in order to fit the tissue sample therein. As can be seen from FIG. 46, a configuration of the bag 10 is shown, which is formed by two layers of plastic material sealed only at its periphery 14 to form a central cavity 12 and a port 16 for entering the cavity 12. The bag may be formed of EVA. In use, preferably, the port 16 or at least one of them is large enough, that is, about 10 mm in diameter or larger, to accept that it has been chopped into small pieces if necessary and transferred to the bag cavity by means of a syringe Sample of 12. However, it is also possible to include a so-called "zip-lock" port at the end of the bag opposite the port, so that a large tissue sample can be placed in the bag and then the bag can be resealed. The "zip lock" can be folded one or more times to form a seam, which is folded and held inside the elastic channel or by another clamp or multiple clamps (not shown) to reduce the probability of leakage. As an alternative, the bag 10 can be opened and tissue can be added. The bag can then be heat sealed with its contents in place. The bag 10 includes corner holes 18 for positioning the bag in the device in use and holding the bag in place during heald riding. Although the figure shows a bag 10 with one cavity 12, it will be possible to provide a bag with more than one cavity, such as two, three, four or five cavities, such as each of a plurality of cavities It is elongated and has an initially open, heat-sealable end, and a sealable port at the other end, which is used to introduce reagents such as depolymerase, and is used to take out after depolymerization is completed or substantially completed Disaggregate the sample.

FIG. 47 shows the bag 10 of FIG. 46, which is installed in the positioning frame 20 by means of the pegs 24 on the frame, and the pegs are fitted into the corner holes 18. The frame 20 is an alternative way of positioning and holding the bag 10 in place within the device 100/100'. The frame 20 includes positioning holes 22 that cooperate with the device for positioning and holding the bag in place during heddles. The frame has an internal open window 26 with a smooth rounded internal edge 23 to accommodate the cavity 12 and the pedal feet 134 and 136 in use. The frame 20 makes it easier to load and unload the bag 10 to and from the device 100/100'.

FIG. 48 shows an alternative frame 20 ′ having two substantially symmetrical halves each similar in construction to the frame 20. Each frame half additionally has a flexible shell 30 which is molded to the frame 20 ′ so that the two halves such as clam shells are combined to enclose the bag 10. If the bag 10 breaks internally during use, the top and bottom flexible shells serve as a protective wall (bund). This feature is especially suitable for infectious tissue samples.

Another alternative (not shown) can be a simple bag-in-bag configuration to contain the leakage. In yet another alternative, the bag may include a substrate with independent pores that are elastic (at least at room temperature), so that an aliquot of the sample can be removed without using the entire sample, for example, after freezing as described below. Alternatively, the sealable bag can further heat seal the components to allow the sample to separate.

The processing of the sample put into the bag 10 can mainly follow the steps described in WO2018/130845 in one example. In this configuration, the tissue to be contained in the sealed bag is suspended in an aqueous solution, which may contain digestive enzymes (such as collagenase and protease) to promote tissue decomposition, and is introduced into the bag through the port 16. Here, the bag is placed on the plate 150 and the temperature is raised to about 35° C. from, for example, an external heat source to promote the tissue digestion rate. An important difference presented here is that a single sample processing bag is used, and digestive enzymes can be introduced through one of the ports 16 in the bag before or during depolymerization. The heat transfer plate 150 can be used to introduce heat energy into the bag by heating the plate on its bottom surface to provide a desired temperature in the bag for enzyme action. The heat can conveniently come from an electric heating heating plate, or an electric heating element in or on the plate 150. The amount of disaggregation will depend on many parameters, such as the size, density and elasticity of the initial tissue sample, and therefore the time of disaggregation and the rate of walking will vary significantly. Too long or too violent stepping can cause the cell survival rate to decrease. Therefore, the speed of the motor unit and the disaggregation time period are important. One option to solve this problem is to control the processing time based on a look-up table including the time required to disaggregate similar samples and the output speed. Another option is to measure the instantaneous electric power or electrical energy required for the de-aggregation process over time, or to measure the force or stress applied to the plate 150 or another part of the mechanism, and stop after reaching a predetermined threshold. To indicate that the sample has fully depolymerized. As the power/force/stress decreases, the de-agglomeration is closer to completion. Another option is to measure the absorbance of light passing through the bag-the greater the absorbance, the closer the sample is to complete disaggregation. After the depolymerization is complete, the contents of the bag can be transferred, and the cells or other related components can be separated and placed back into a fresh bag for freezing in the device 100/100'. Alternatively, and preferably all of the depolymerized material can be left in the bag and the device for freezing. The cryoprotectant is introduced into the bag via port 16.

Another difference between the method of the present invention and the method described in WO2018/130845 is that after the cryoprotectant is introduced, the device with the depolymerized sample and the cryoprotectant in the bag is installed (or kept in the device), and the whole The device is installed in the freezer 40 as described above. The base of the freezer is cold, and therefore heat energy is drawn from the bag 10 via the heat transfer plate 150. In order to control ice formation and prevent the sample from being too cold when the bag is cooled, it can be massaged by the feet 134 and 136 in the manner described above, albeit at a slower rate than deaggregation, to control ice crystal growth and thus increase the cell survival rate after thawing. Electric energy can be supplied to the motor unit 114 via a wire conductor to maintain the movement of the mechanism 120 inside the freezer, such as the freezer 40 (FIG. 41).

Since the device can be removed from the freezer, it is easier to clean after use.

When needed, the frozen deagglomerated sample in the bag 10 can be further externally heated by the heating plate 150 and/or by partially immersing the device 100/100' in a warm water bath, keeping it at about 37°C and removing the cryogenic protection And quickly thaw in the device 100/100'. In each case, massage the bag during thawing. If the enzyme is still present, it can also be removed when needed, for example by means of filtration. Generally speaking, it will have little or no effect on cells during cryopreservation, because its effect is suspended at low temperatures. All process manipulation, heating, deaggregation, cooling, freezing and subsequent thawing of the sample occur in the same sealed flexible bag 10 and can be performed in a single device. This is not only time and space efficient, but also allows a single record to capture all things that happen to the sample during processing, such as temperature, duration, depolymerization speed, freezing scheme, and reduce the chance of error, such as the sample in the processor. Excessive time spent in an uncontrolled environment in between.

More specific examples of equipment and techniques for tissue sample processing and freezing are given below.

FIG. 49 shows an example of a bag 10 formed of a thermoplastic material such as EVA or PVG film and having an opening 11 for receiving a tissue sample T. FIG. The bag includes a conduit 13 connected to one or more ports 16 (FIG. 46). The conduit includes one or more shunts 17, compression valves 19, and standard Luer type connectors 15. The single catheter line shown is merely illustrative-the bag 10 may include additional parallel catheters connected via a plurality of ports 16.

Once the tissue T is inside the bag 10. The opening 11 can be sealed by the following: shown as closed and sealed in FIG. 50 and shown as open by a dotted line in the same figure, and/or by using the heat as shown in FIG. 51A The sealing machine 50 performs heat sealing to produce one or more heat-sealed closed strips (for example, a plurality of parallel strips) 8, and each method forms a sealed cavity 12 (FIGS. 46, 47, and 49).

Alternative or additional ways for sealing the bag 10 are shown in Figures 51B and 51C. As shown in FIG. 51C, after heat sealing at the sealing port 8, the bag 10 can be clamped in a two-part clamp 60, which includes a top rod 62 and a bottom rod 64 that are forced together by a pair of screws 66. FIG. 51B shows the clamp 60 in an exploded state, but in use, the screw 66 does not have to be completely removed from the remaining clamp before being inserted into the bag 10. The top rod 62 has a tapered groove 68 in which there is a complementary wedge-shaped formation 61 when clamped. The grooves and wedges concentrate the clamping force at the apex 65 of the wedge, and provide a clamping force at the apex that is higher than that of a flat clamping surface. For even more clamping force, the apex 65 has a small channel 67 at its peak, which, in use, is joined by a complementary ridge formation 69 in the top rod. In some embodiments, the force is sufficient to offset the need for heat sealing the port 8. In some embodiments, a heat-sealed port or other bag sealing mechanism is required, for example to provide for handling of the sample-containing bag outside the deagglomerator. In some embodiments, the clamping device ensures the integrity of the sealing port. The clamping force is further enhanced by the thickness and rigidity of the top and bottom rods that are not easily bent, and therefore the clamping force exerted by the screw 66 is maintained. Figure 51C shows the clamp 60 in a clamped state. The protrusion 63 is in contact with the parts of the heddling device 100/100' or 200 (as described below) to prevent the clamp during heddling, and thus clamp the movement of the bag 10. The outer periphery and height of the clamp 60 have a size and shape set to fit in the complementary portion of the sample receiving area 148 (or 248, Fig. 62 and below), and thus provide further positioning of the clamping bag 10 during heald riding. Although not illustrated, the clamp 60 can also incorporate additional frames 20, 20' as shown in FIGS. 47 and 48, and allow the clamp to be rigidly mounted to one end of the frame and one or more ports 16 (FIGS. 46 and 49 ) Supported at the other end of the frame.

Referring to FIG. 52, in use, once sealed, digestive enzyme E can be introduced into the cavity 12 through the catheter 13, for example, by using a syringe 5 connected to the shunt connector 17 to inject the enzyme into the bag. With the holding bag in an upright orientation, the air can then be removed from the cavity 12 by drawing out the plunger of the syringe 5, as shown in FIG. 53. The initial mixing of Enzyme E and Tissue T can be performed manually, as shown in Figure 54.

The bag 10 can then be loaded into the heddle device 100 for disaggregation, with or without the frame 20/20' and the protective cover 30, as illustrated in FIG. 55.

The depolymerization process is then carried out as described above. Once completed (which can take between several minutes and several hours, for example about 10 minutes to 7 hours, preferably 40 minutes to 1 hour), the bag device described above and an additional connection to the diverter 17 can be used, for example The sample aliquot bag 7 (shown in Figure 56) divides the depolymerized liquefied sample into aliquots. In that case, the syringe 5 is used to draw the liquefied sample out of the bag 10 in the direction of arrow F, the valves 19a and 19b are opened and the valve 19c adjacent to the sample aliquot bag 7 is closed. Once sufficient sample has been drawn into the syringe 5, valve 19b is closed, valve 19a remains open, and valve 19c is opened. The syringe is then used to force the liquid in the direction of arrow F in FIG. 57 into the sample aliquot bag 7. The tube 13 of the aliquot bag 7 can be heat-sealed by means of a clamping heat-sealing machine 55 and is shown in FIG. 58. This process can be repeated until enough aliquots are obtained or until there are no more samples left. The bag may already be partially zoned to make it easier to seal the compartments.

As described above, the sample bag 10 can be held in the heddle device 100 (FIG. 55) and then the heddle device may be loaded into the controlled rate temperature change device, in this case the freezer 40 as shown in FIG. 59 middle. The technology allows the hedging to continue during freezing to inhibit ice crystal formation, but in practice the bag 10 can be removed before freezing, and the freezer 40 then only cools the sample via the heat transfer plate during the hedging. In the alternative, the aliquot sample bag 7 can replace the entire sample bag 10. In another alternative, the freezer 40 can be used to gently cool the untreated or processed sample to about 4°C by installing the heddle device 100 on the top of the freezer 40, where the freezer lid is opened so The substrate 150 is allowed to cool, as shown in FIG. 60. In another alternative, it is possible to remove the base 150 and place it in the freezer with the freezer lid closed, as shown in FIG. 61. In yet another alternative (not shown), the bag 10 or 7 can be directly frozen in the freezer 40.

The present invention should not be regarded as limited by the embodiments described above, but can be varied within the scope of the appended patent application, as can be easily seen by those familiar with the art. For example, the heddle mechanism described above is preferable because it provides a fully pivoting mechanical interconnection, which is less likely to get stuck in cold conditions than a sliding surface, but the mechanism can be used for any purpose. Replacement of mechanically equivalent components for two or more foot pedals in sequence. The described flat feet can be replaced with roller feet, in which the pedaling movement is left and right instead of up and down. The speed of the described pedaling harness or its mechanical equivalent motion is preferably 2 or 3 pedaling harnesses per second for each foot to optimize the disaggregation and maximize the cell recovery rate, and is a stable pedaling harness, but for different cells Type, the heddle can be faster or slower or intermittent.

Since the device 100/100' is intended to be placed in a freezer and withstand extremely low temperatures (for example, -80°C or lower), it is preferable to use metal parts, especially those parts such as spring 146, because Polymeric parts become much more rigid at low temperatures. In addition, tightly fitting components (such as pistons and barrels) can become stuck or poorly fitted at extremely low temperatures, so a simple pivotable linkage of the mechanism 120 as described is preferable.

Figures 62, 63, and 64 show an alternative heddle device 200, which is similar in size and function to the device 100 described above. The device 200 has certain differences that are described in more detail below.

Referring to FIG. 62, the main difference between the device 100 and the device 200 is that the device 200 has a pedaling mechanism 220 that is different from the mechanism 120 of the device 100. The two heddles 234, 236 are driven by a 24 volt DC electric motor 213 (Fig. 63) in a cyclical alternative pedaling motion similar to the motion shown in FIGS. 42 and 43, which is a part of the electric motor unit 214, The electric motor unit has a rotary encoder that provides feedback to the controller 221 (Figure 63) for monitoring and controlling the speed of the pedaling movement. The motor drives the camshaft 224 via the toothed belt 222. The camshaft includes a pair of cams 230, 232 that are offset by 180 degrees. In this case, the respective contours are cycloid shapes to provide simple harmonic motion of the cam followers. Each cam is operable to move the cam followers including the associated elastic driven wheels 225, 227 riding on the cam profile, and the driven wheel shafts 221, 223 in a force-transmitting relationship with the spring follower brackets 226, 228 Assembly. Each bracket 226, 228 slides in the linear guide 229, and the respective feet 234, 236 are connected to the bracket. As the respective cams are rotated by the motor overcoming the thrust of the return spring 231, each assembly is then forced upward by each of the driven wheels as it rides the cam profile with its feet away from the pedaling state. . When the cam rotates further and the profile of the cam retreats, the spring 231 associated with each follower assembly forces the assembly and the foot downward with the pedaling force.

As a result, the pedaling force is limited to the spring rate of the associated follower assembly spring 231 instead of the power of the driving motor. 1. The force applied to the bag is limited by the spring during use. This is because the mechanism drives the foot upward and the spring pushes it back downward. This ensures:

a. The motor will not stall (regardless of the size or texture of the tumor);

b. The sample is not compressed by force and the bag will not crack;

c. The maximum pressure applied to the bag is lower than the pressure tested during the manufacture of the bag; and

d. As described below, the hinged bag receiving area 248 can accept the sample bag and any clamps used without pre-positioning the feet. In other words, when receiving the bag, the foot can be in any position because the hinged sample area 248 is closed against the foot, and if necessary, because the hinged area is closed against the foot, any sample can be compressed by the foot at that time.

63 and 64, the device 200 further includes a flexible sealing film 241 extending from the device housing 210 to the upper part of the two feet 234, 236, which provides flow resistance between the sole of the foot and the rest of the pedal mechanism 220 And dust-proof seal. If the compression bag breaks during use, it is configured to prevent contamination of the mechanism. Although the membrane 241 is preferred, the feet can slide in the seal, such as a lip seal that is installed to separate the mechanism 220 from the pocket area 248 and achieve a similar prevention of mechanism contamination when needed.

The device 200 further includes a heat transfer plate 250 that performs the same function as the heat transfer plate 150. However, this plate 250 is hinged to one side of the housing at the hinge 255 (Figure 64), making it easier to insert and remove the bag to be treaded (as shown in Figures 46, 47 and 48). The heat transfer plate 250 includes a temperature sensor 256 that allows the controller to monitor and record the temperature of the plate 250 and the bag receiving area 248 for quality control. The board 250 has a first surface 251 and a second surface 252, which have the same functions as the surfaces 151 and 152 described above.

The height of each foot can be adjusted relative to the heat transfer plate 250 of the device 200, and the indication of its movement is also monitored by the controller. Therefore, even though the rotary encoder can indicate that the motor is rotating, a mechanical failure such as a failure of the toothed belt 222 can still be detected by the controller, and suitable actions can be implemented, such as issuing an alarm.

The device 200 has the same external dimensions as the device 100, and the housing 210 of the device is intended to slide inside the controlled rate freezer 40, where the freezer cover is as described above and illustrated in FIG. 61.

For convenience, terms such as upper, lower, upper and lower and more descriptive terms such as feet, pedals and harnesses have been used to describe the invention shown in the drawings, but in practice, the devices shown may Oriented in any way so that these terms become, for example, the reverse or less descriptive in their new orientation. Therefore, the restriction on orientation should not be interpreted by such terms or synonymous terms.

The present invention provides a device (100/100') for disaggregating tissue samples into individual cells or cell aggregates in a closed flexible bag (10). The device includes a mechanical disaggregation mechanism (120) and a tissue sample bag receiving area (148), the device further comprises a heat transfer plate (150) for transferring heat energy to or from the area (148), the plate has a first plate surface (151) adjacent to the area (148) and a departure area (148) The opposite surface (152) exposed to external heat.

The cryopreservation of tumor tissues at the time of collection enables separation of manufacturing and tumor collection. This means that UTIL manufacturing can be used as a single manufacturing process planning and proceeding from the thawing of tumor digests to the final TIL collection washing, drug formulation, filling, labeling and cryopreservation.

The cryopreservation of the final product allows all release testing to be performed before adjusting chemotherapy and patient treatment to be separated from the final product manufacturing location.

Use flow cytometry to characterize and quantify the manufactured products. TILd is defined as a T cell that expresses the cell surface marker CD3, which has been derived from a metastatic tumor by pathological evaluation of a representative sample of the starting material. The survival rate is based on the percentage ^{of all CD3 +} cells that do not bind to the early cell death marker phospholipid binding protein-V and/or the survival rate dye DRAQ7 (equivalent to trypan blue or PI). Purity is defined as the percentage of live T cells (CD3 ⁺ , phospholipid binding protein -V ^-ve and DRAQ7 ^-ve ) in the living hematopoietic cell population (CD45 ⁺ , phospholipid binding protein -V ^-ve and DRAQ7 ^-ve ).

Before the rapid expansion protocol (REP), most of the cells were T cells expressing CD3. In research and clinical batches, a different distribution of CD3+CD8+ and CD3+CD4+ TIL was observed and it will contain a subset of tumor-reactive cells. Since TIL uses anti-CD3 amplification in REP, the final product contains almost only live CD3+ T cells (>94%).

Theoretically, the final product can still contain tumor cells, but this is due to the culture conditions that strongly and selectively promote T cell growth and T cell-mediated killing of tumor cells are extremely unlikely. The clinical data of hundreds of TIL infusions did not show the presence of tumor cells by cytology. In order to organize the data to finalize the specifications, tests have been incorporated to identify all non-hematopoietic living cell materials in the original IPC analysis and the frequency of cancer biomarkers will also be tested.

The TIL cell medicine is a suspension in approximately 125 to 270 ml buffer isotonic saline containing 8.5% human serum albumin and 10% DMSO. The number of cells present depends on the capacity of the individual TIL cells to be expanded in the culture, as well as the culture conditions and the reproducibility of manufacturing. Table 3-Exemplary drug composition Component Quantity (per infusion bag) Function Tumor-derived T cells 5×10 ⁹ to 5×10 ¹⁰ CD45 ⁺ , CD3 ⁺ , phospholipid binding protein ^- V-, DRAQ7 ^- cells Active ingredient 20% human serum albumin 8.5% HSA W/V Absorption inhibitor Phosphate buffered saline 125 to 270 ml Isotonic thinner DMSO 10% V/V Cryoprotectant

Referring to FIG. 1, the disaggregation module of the device is disclosed. In embodiments involving enzymatic digestion, the device may include a flexible container 1a for depolymerization and digestion. The open end 1b permits the transfer of solid tumor tissue material into the container 1a . The hanging hole 1c allows the container 1a to be hung and supported during transportation or use. In order to maintain the sterile condition of the device, the target thermal welding position 1d allows the container 1a to be sealed using a thermal welding device 13c or other similar methods. The container 1a may have rounded edges 1e on the inner surface of the container 1a to reduce loss, and the loss may appear as part of the transfer to the example illustrated in FIGS. 2A-C or 3A or 3B. The conduit 1f enables the medium 3a to be transferred to the container 1a via the sterile filter 2a . The sterile filter 2a contains spikes that allow the sealing port to be pierced in the subsequent module to facilitate the transfer of the culture medium 3a. The catheter 1g makes it possible to transfer the digestive enzyme 3b to the container 1a via the sterile filter 2b . The sterile filter 2b contains spikes that allow piercing of the seal to facilitate the transfer of digestive enzymes 3b into the container 1a. After the solid tumor tissue is depolymerized, especially involving enzymatic digestion, the depolymerized mixture is transferred out of the catheter via a filter unit 4a containing a sterile filter 4b for 1 h , and then enters the incubation stage. The filter unit 4a may be flexible to allow distortion without affecting the effectiveness of the filtering process. The filter 4b removes undisaggregated tissue. The catheter clamp 5a allows the culture medium 3a to enter the flexible container 1a via the sterile filter 2a . In the embodiment involving enzyme digestion, the catheter clamp 5b allows the enzyme 3b to enter the flexible container 1a via the sterile filter 2b . The catheter clamp 5c allows the contents of the flexible container 1a to enter through the filter unit 4a to identify one or more examples in FIGS. 2A-C or 3A or 3B.

According to Fig. 2A, the sterile filter 2c permits the introduction of the culture medium 3a and/or the freezing solution 3c required for cryopreservation of the depolymerized tumor tissue. The filter 4d can be required for extra size separation of cell/tissue agglutinates. The filter 4d is enclosed in the filter unit 4c , and the filter unit can be flexible to allow distortion without affecting the effectiveness of the filtering process. In one embodiment, the filter 4e may be required to retain the cells, but allow the culture medium and cell fragments to be washed away. The filter 4d is enclosed in the filter unit 4c in a similar manner. In one embodiment, the conduit clamp 5d is in place to prevent the material from the container 1a that has passed through the filter units 4a and 4c from returning to the container 1a . In one embodiment, the catheter clamp 5e is in place to allow waste from the container 1a that has passed through the filter units 4a , 4c, and 4e to enter the waste container 6a , but prevents the medium 3a or 3c from entering through the sterile filter 2c. Before the waste enters the waste container 6a through the catheter clamp 5e , the catheter clamp 5f prevents the material from the container 1a that has passed through the filter units 4a , 4c, and 4e from returning to the source of the culture medium 3a or 3c or transferring to FIG. 3A or FIG. 3B One of the examples described in. Once the waste is exhausted, the catheter clamps 5e and 5d are closed, and the catheter clamp 5f allows the culture medium 3a or 3c to transfer the cells in the filter 4e to one of the examples illustrated in FIG. 3A or FIG. 3B. The waste container 6a has hanging holes to support the waste container 6a during use and/or transportation.

Figure 2B illustrates the enrichment module of the device. The catheter clamp 5g allows the contents of the container 1a to enter the flexible container 7a of the enrichment module through the filter unit 4a . The catheter clamp 5h allows the contents of the container 7a to pass through the filter unit 8a , retaining and enriching cells, while allowing waste and debris to pass through the pressure controlled by the valve 8c before the enriched cells return to the container 7a through the open catheter clamp 5i The filter 8b enters the waste container 6a . The catheter clamp 5i allows the contents of the container 7a to pass through the filter unit 8a through the open catheter clamp 5h , retaining and enriching cells, while allowing waste and debris to pass through the pressure controlled by the valve 8c before the enriched cells return to the container 7a Filter 8b . After cell enrichment occurs, the catheter clamp 5h is closed and the catheter clamp 5j is opened to allow the contents of the container 7a to be transferred to one of the examples illustrated in Figure 3A or Figure 3B. The waste container 6a has hanging holes 6b to support the waste container 6a during use and/or transportation. The container 7a of the enrichment module has hanging holes 7b to support the container 7a during use and/or transportation. The container 7a may have rounded edges 7c on the inner surface of the container 7a to reduce wastage, which may appear as part of the transfer to the example illustrated in FIG. 3A or FIG. 3B. The duct 7d allows the container 7a to receive the contents of the container 1a via the filter unit 4a and the filter unit 8a. The duct 7e allows the contents of the container 7a to pass through the filter unit 8a , retaining and enriching the cells, while allowing waste and debris to pass through the filter by the pressure controlled by the valve 8c before the enriched cells return to the container 7a through the open duct clamp 5i The device 8b enters the waste container 6a . The conduit 7f allows the contents of the container 7a to pass through the filter unit 8a , retaining and enriching the cells, while allowing waste and debris to pass through the filter 8b through the filter 8b before the enriched cells are returned to the container 7a through the pressure controlled by the valve 8c In the container 6a .

Figure 2C illustrates another embodiment of the enrichment module. The catheter clamp 5g allows the contents of the container 1a to enter the flexible container 7a through the filter unit 4a . The catheter clamp 5h allows the contents of the container 7a to pass through the filter unit 9a , retaining and enriching cells, while allowing waste and debris to pass through the pressure controlled by the valve 9c before the enriched cells return to the container 7a through the open catheter clamp 5i The filter 9b enters the waste container 6a . The catheter clamp 5i allows the contents of the container 7a to pass through the filter unit 9a through the open catheter clamp 5h , retaining and enriching cells, while allowing waste and debris to pass through the pressure controlled by the valve 9c before the enriched cells return to the container 7a Filter 9b . After cell enrichment occurs, the catheter clamp 5h is closed and the catheter clamp 5j is opened to allow the contents of the container 7a to be transferred to one of the examples illustrated in Figure 3A or Figure 3B. The waste container 6a has hanging holes 6b to support the waste container 6a during use and/or transportation. The container 7a of the enrichment module has hanging holes 7b to support the container 7a during use and/or transportation. The container 7a may have rounded edges 7c on the inner surface of the container 7a to reduce wastage, which may appear as part of the transfer to the example illustrated in FIG. 3A or FIG. 3B. The duct 7d allows the container 7a to receive the contents of the container 1a via the filter unit 4a and the filter unit 9a. The duct 7e allows the contents of the container 7a to pass through the filter unit 9a , retaining and enriching the cells, while allowing waste and debris to pass through the filter by the pressure controlled by the valve 9c before the enriched cells return to the container 7a through the open duct clamp 5i The device 9b enters the waste container 6a . The conduit 7f allows the contents of the container 7a to pass through the filter unit 9a , retaining and enriching the cells, while allowing waste and debris to pass through the filter 9b through the filter 9b before the enriched cells are returned to the container 7a through the pressure controlled by the valve 9c In the container 6a . The filter unit 9a 7a help filter the contents of the container to return to the enriched cell by a pressure control valve 9c of the waste before the container 7a 9B medium and debris removed to the waste container via a filter 6a. The filter 9b can be wound into a coil to increase the distance that the waste must dissolve before reaching the waste container 6a to improve the purification of the cell culture medium and facilitate the transportation and storage of the improved filter 9b.

Figure 3A illustrates an example of a stabilization module. The catheter clamp 5k allows: via the filter unit 4a as illustrated in FIG. 1, or via the contents of the container 1a of the filter unit 4c as illustrated in FIG. 2A; or via the filter unit 8a as illustrated in FIG. 2B, Or transfer the contents of the container 7a of the filter unit 9a to the container 10a of the stabilization module as illustrated in FIG. 2C. The container 10a of the stabilization module has a hanging hole 10b to support the container 10a during use and/or transportation. The container 10a may have a rounded edge 10c on the inner surface of the container 7a to reduce loss, which may occur as part of the transfer away from the duct 10e or 10f. The duct 10e enables the contents of the container 10a to be drawn out via the connector 10h. The catheter 10f contains a flexible film to enable sterile spikes to be introduced through the sterile cover plate 10g , so that the contents of the container 10a can be drawn out.

Figure 3B illustrates another embodiment of the stabilization module. The catheter clamp 51 allows: the contents of the container 1a via the filter unit 4a as illustrated in FIG. 1, or via the filter unit 4c as illustrated in FIG. 2A; or via the filter unit 8a as illustrated in FIG. 2B, Or transfer the contents of the container 7a of the filter unit 9a to the container 11a of the stabilization module as illustrated in FIG. 2C. The container 11a of the stabilization module has a hanging hole 11b to support the container 10a during use and/or transportation. The container 10a may have a rounded edge 10c on the inner surface of the container 7a to reduce loss, which may occur as part of the transfer away from the duct 11f. The catheter clamp 5m allows the culture medium 3c to enter the flexible container 11a through the sterile filter 2c . The catheter clamp 5n allows the contents of the container 11a to enter one of the cryopreservation containers 12a depending on the open or closed state of the catheter clamps 5o , 5p , 5q , 5r , 5s, and 5t . The catheter clamps 5o , 5p , 5q , 5r , 5s, and 5t allow the contents of the container 11a to enter one of the cryopreservation containers 12a . The conduit 11d enables the container 11a to receive: the contents of the container 1a via the filter unit 4a as illustrated in FIG. 1 or via the filter unit 4c as illustrated in FIG. 2A; or via the filter as illustrated in FIG. 2B Unit 8a , or the contents of the container 7a through the filter unit 9a as illustrated in Figure 2C. The duct 11e allows the cryopreservation medium 3c to be transferred to the container 11a . The conduit 11f enables the contents of the container 11a to be transferred to the cryopreservation container 12a , where the final depolymerized UTIL product as a single cell suspension is stored for future use in a rapid expansion process. The cryopreservation container 12a has a fixing member 12b to allow aseptic transfer of TIL from the cryopreservation container 12a. The cryopreservation container 12a has a space 12c suitable for the volume of the UTIL cell suspension to be stored. The cryopreservation container 12a also has a target position 12d for welding the pipe 11f to the cryopreservation container 12a .

Figure 4 illustrates another example of the device and set. The peg 13a allows the medium 3a , 3b, and 3c to be suspended. The peg 13b is connected to a weight sensor for hanging the container 1a and may include one or more of the containers 7a , 10a, and/or 11a depending on the embodiment used. The weight sensor is used to define the judgment stage to control the automatic processing of materials. Heat-sealing device 13c may be used in solid tumor tissue after the removal of the container into the container 1a, 1a sealed at the target site. The depolymerization module 13d has an opening, which can be closed and locked to enable depolymerization and the temperature can be controlled between 0°C and 40°C (tolerable deviation of 1°C) to enable digestion, wherein digestive enzymes are used for depolymerization Solid tumor tissue. The disaggregation module 13d also has a built-in sensor to evaluate the solid tissue disaggregation level by measuring the difference in light distribution over time to identify changes, and thus to identify the completion of the disaggregation process, which can take several seconds to several hours. Occurs within the segment. The depolymerization module 13d may also include a depolymerization surface 13f , which directly contacts the container 1a and bears against the back of the housing of the depolymerization module 13d , which can be closed and locked during depolymerization and digestion using enzymes. The final blending module 13e has a cover that allows the temperature of the container 10a or 11a to be controlled depending on the embodiment used, which can control the temperature between 0°C and the ambient temperature (to a tolerance of 1°C). The catheter clamps 13g and 13j serve as input and output ports, which are placed in the catheter positioner 13i , and depending on the embodiment used, facilitate the transfer of the depolymerized tumor product between the containers 1a , 10a, or 11a . The peristaltic catheter pump 13h controls the transfer of the culture medium 3a or 3c between the catheter clamps 13g and 13j serving as input and output ports. The conduit valve 13k helps to control the pressure via the valves 8c and 9c in the enrichment module, as illustrated in Figures 2B and 2C. Depending on the embodiment used, the pegs 13l allow the waste container 6a and/or the cryopreservation container 12a to be hung. The embodiment may also include a conduit fusion splicer 13m required to connect the cryopreservation container 12a to the device as illustrated in FIG. 3B. The embodiment may also include a catheter cutter 13n that disconnects the cryopreservation container 12a from the device as illustrated in FIG. 3B. The controlled rate cooling module 13o can be cooled or maintained at any temperature between 8°C and at least -80°C to help the cryopreservation process.

The method of the present invention is exemplified according to the following procedure. It is clearly stated that, in addition to the basic characteristics of the method, the various optional steps listed in this article can be independently combined to achieve related technical advantages associated with the types of sampling and results to be achieved.

The semi-automatic aseptic tissue processing method includes: as appropriate, according to the set described in this article, automatically determine the aseptic disaggregated tissue processing step and one or more other tissue processing steps and related conditions from the digital label identifier on the aseptic processing set; Place the tissue sample in the flexible plastic container of the aseptic processing kit; and process the tissue sample by communicating with and controlling the following to automatically execute one or more tissue processing steps: disaggregation module; optional enrichment as appropriate Module; and stabilization module.

Basically, the process includes the open-ended bag (the first flexible container, which is part of the disaggregation module) that can receive biopsy/tissue samples (preferably for tumor removal) has been connected by one or more pipes to or can be manually The aseptic connector controlled by the operator is connected to

I. A single container with digestion medium (the second flexible container, which is part of the depolymerization module) and with or without a stabilizing solution (the same second flexible container is also part of the stabilization module)

II. One container with digestion solution (the second flexible container is part of the depolymerization module) and another container with the stabilization solution (the fourth flexible container is part of the stabilization module)

When adding a biopsy and sealing the open end pocket, the digestion medium can be added and processed through a pipe or a sterile connector (such as the pipe/port of the technical solution 1).

When the digestion is complete, the tissue is now a single or a few aggregated cell suspensions, and the cells may be filtered before step 4 as appropriate (the optional enrichment module used for filtration includes the first flexibility containing the sample Container and filter to the third container for receiving the enriched filtrate).

When the stabilization medium does not exist in the same flexible container, the container with the stabilization solution is completed by opening the connected pipe for addition or a sterile connection controlled by manual operation, and the connector should be opened to This enables the stabilizing solution to be added in both cases before the process continues.

Single or a few aggregated cell suspensions in the original flexible container or can be divided into multiple storage stabilization containers as appropriate, and then maintained in a stable state on the device and/or will undergo cryopreservation, and then removed for transportation , Storage and/or use for its ultimate utility. The stabilization module also includes the first or third container, as used in storage/freezing/storage.

In another non-limiting example of the process:

a) Place the tissue sample collected by a separate procedure (not part of the present invention) such as a biopsy or surgery for collecting the required tissue material in an initial flexible plastic container (see, for example, Figure 1, container 1a).

b) Before using one or more of the following examples of the present invention to depolymerize, transfer the culture medium (see, for example, Figure 1, culture medium 3a) to the depolymerization chamber, or in one example also add and collect the enzyme (see For example, Figure 1, enzyme 3b), a mechanism such as a weight sensor (see, for example, Figure 1, 13b as part of module 13d) evaluates the required amount of medium to be added, which is determined by: direct operator input or The weight of solid tissue.

c) Single use of flexible disaggregation container, solid tissue, culture medium and in one example, the enzyme is combined during the polymerization process of a minimum of seconds to several hours, of which the optimal time between 1 and 10 minutes is required to Decompose the solid tissue until there are no visual changes (see Figure 5 and Table 1). The disaggregation device is designed to use variable speed and time to compress the tissue depending on the time it takes to disaggregate and the feedback via the sensor in the disaggregation module (see Figure 1, 13d).

d) In an embodiment where an enzyme is present, this will need to be at an optimal temperature between 30 and 37°C, but can be as low as 0°C up to 40°C for at least 1 minute to several hours, but more preferably 15 to 45-minute incubation period.

e) Step c and step d) in the enzyme example can be repeated until the tissue stops changing, or the example has been depolymerized into a liquid cell suspension, no matter which one appears first, it can be depolymerized by the depolymerization module. Detector (see Figure 1, 13d) to monitor.

f) In one embodiment, the incompletely disaggregated tissue, related materials and impurities are removed so that the cell suspension can be enriched by delivering the disaggregated tissue and culture medium using one or more of the following embodiments:

i. Pass directly through one or more mechanical filters with pores of at least >0.1 μm to 1000 μm, but preferably between 50 μm and 250 μm, and more preferably 100 μm to 200 μm (illustrated in Figure 2A ).

ii. Density-based separation using centrifugation and/or sedimentation with or without a cell aligned density retention solution (such as Ficoll-paque GE Healthcare).

iii. Hydrodynamic filtration, in which fluid flow and flow obstructing materials enhance the analysis and classification of cells and impurities based on size and shape

iv. Field flow classification, in which the applied field (such as flow, electricity, gravity, centrifugation) acts in the vertical or opposite direction to the selected flow (such as tangential flow filtration, hollow fiber flow filtration, asymmetric flow filtration, Centrifugal flow filtration). In this case: the cells or impurities that respond the most to the force are driven to the wall, where the flow is the smallest and therefore the residence time is longer; while the cells or impurities that respond the least to the force remain stratified and dissociate quickly to the flow (Figure 2B and C) Tangential flow filtration as described in).

v. Acoustophoresis, in which one or more audio frequencies tuned or harmonized with a population of cells or impurities are used to drive the desired cells or impurities to the input stream in a tangential path.

g) In one embodiment, the depolymerized and enriched tissue product will be resuspended in fresh medium (Figure 2A, using medium 3a), such as:

i. Cell enrichment medium for independent targeted enrichment procedures as previously described

ii. Direct cell culture or cold storage medium (such as HypoThermosol ^® from BioLife Solutions.

h) In the example adopted in g), transfer the resuspended depolymerized solid tissue source product to one of the final product containers (illustrated in Figure 3A) of the example for storage for several hours to several days, and then Used for its ultimate utility.

i) In addition, after step f), the application example (illustrated in Figure 3B) will be applicable in the following situations: the depolymerized solid tissue-derived product is resuspended in a cryoprotectant (Figure 3B, medium 3c), that is, used Store depolymerized solid tissue-derived products in frozen solutions that last for several days to several years, such as CryoStor ^® Frozen Solution from BioLife Solution.

j) At this stage, the depolymerized solid tissue-derived product is resuspended in the freezing solution (Figure 4, module 13e) and transferred to one or more flexible cryogenic storage containers (illustrated in Figure 3A, container 12a) And in one embodiment of the device, there is a controlled rate freezing process (Figure 4, module 13o).

k) Thereafter, the bag can be separated from the device and sterile processing kit for independent storage or distribution.

In other embodiments, the disposable kit of the present invention can be used with an automatic device for semi-automatic aseptic processing of tissue samples. Figures 6 and 7 depict the disposable kit of the present invention.

Figure 6 depicts a semi-automatic aseptic tissue processing method using multiple flexible containers for different starting solutions as part of a module for the process of depolymerization and stabilization.

Process Step 1-The user can log in to the device and use the device to scan the label on the sterile kit to send the automatic processing steps to be used. The device processor identification tag is provided with information required to perform specific processing commands related to a specific set.

Process Step 2-The flexible bag containing the digestion medium (part of the depolymerization module) and the flexible bag containing the low temperature/stabilization solution (part of the stabilization module) are respectively suspended or fastened to the device.

Process Step 3-The biopsy or tissue sample for processing can be placed in the flexible container (part of the two modules) of the sterile kit through the open end.

Process Step 4-The open end (to add the sample during the initial processing) can then be closed using heat fusion to seal the flexible container containing the sample.

Process Step 5-The user can then interact with the user interface of the processor to confirm the existence of the tissue sample and enter any other tissue material specific information when needed.

Process step 6-Digestion medium and low temperature/stabilization solution flexible container is connected to the flexible container containing the sample, and then it can be placed in the device for automatic processing.

Process Step 7-The device circulates according to the set information to perform the depolymerization of the sample and the stabilization/low-temperature storage of the obtained cells.

Process step 8-When stabilizing/freezing, disconnect and discard the medium and low temperature/stabilization container of the set used. Disconnected tissue is processed into a single-cell or multi-cell solution in a flexible container, and then transferred to a storage or transportation container before its final use.

In another embodiment, FIG. 7 depicts that the flexible container containing the culture medium for the process can be shared between the modules of the aseptic processing kit and method.

Process Step 1-The user can log in to the device and use the device to scan the label on the sterile kit to send the automatic processing steps to be used.

Process Step 2-Hang or otherwise fasten the flexible bag (part of the depolymerization/stabilization module) containing both the culture medium and the low temperature/stabilization solution to the device.

Process Step 3-The biopsy or tissue sample for processing can be placed in another flexible container (part of the two modules) of the sterile kit through the open end.

Process Step 4-The open end can then be closed by heat welding to seal the flexible container containing the sample.

Process Step 5-The user can then interact with the user interface of the processor to

Confirm that the tissue sample exists and enter any tissue material-specific information if needed.

Process step 6-Digestion medium and low temperature/stabilization solution flexible container is connected to the flexible container containing the sample, and then it can be placed in the device for automatic processing.

Process Step 7-The device is cycled to enable the depolymerization of the sample and the stabilization of the resulting cells, optionally via cryopreservation.

Process step 8-When the freezing/stabilization is complete, the user disconnects and discards all the flexible containers used in the set. Disconnect the tissues processed into single or multi-cell solutions in the remaining flexible containers, and then transfer them to storage or transportation containers before their final use.

For example, in another embodiment of the method of the present invention, when the depolymerization process is supplemented with enzymatic digestion, the medium formulation used for enzymatic digestion must be supplemented with enzymes that help protein breakdown and make cell-to-cell boundary breakdown. , As described above.

Various liquid formulations known in cell culture or cell processing technology can be used as liquid formulations for cell depolymerization and enzymatic digestion of solid tissues, including but not limited to one or more of the following media: organ preservation solution, selective Dissolving solution, PBS, DM EM, HBSS, DPBS, PM I, Ebbe's medium, XVIVO™, AIM-V™, lactated Ringer's solution, Ringer's acetate solution, physiological saline, PLASMAYTE™ solution, crystal solution And IV fluid, colloidal solution and IV fluid, 5% dextrose aqueous solution (D5W), Hartmann's solution DM EM, HBSS, DPBS, RPMI, AIM-V™, Escher medium, XVIVO™, each can be supplemented according to the situation There are additional cell support factors, such as fetal bovine serum, human serum or serum substitutes or other nutrients or interleukins to help cell recovery and survival or specific cell depletion. The culture medium can be a standard cell culture medium, such as the medium mentioned above, or for example for primary human cell culture (for example for endothelial cells, hepatocytes or keratinocytes) or stem cells (for example, dendritic cell maturation, hematopoietic expansion, keratinocytes) Cells, mesenchymal stem cells or T cells) special medium. The culture medium may have supplements or reagents well known in the art, such as albumin and transporters, amino acids and vitamins, one or more metal ions, antibiotics, linking factors, delinking factors, surfactants, growth factors and cells Interleukins, hormones or solubilizers. Various media are commercially available from, for example, ThermoFisher, Lonza or Sigma-Aldrich or similar media manufacturers and suppliers.

The liquid formulation required for enzymatic digestion must have sufficient calcium ions present in at least 0.1 mM up to 50 mM, optimally in the range of 2 to 7 mM, and ideally 5 mM.

The solid tissue to be digested can be washed with a liquid formulation containing chelating agents EGTA and EDTA after depolymerization to remove adhesion factors and inhibitory proteins, followed by washing and removing EDTA and EGTA, and then enzymatic digestion.

The liquid formulation required for enzyme digestion preferably has the least chelating agents EGTA and EDTA, which can severely inhibit enzyme activity by removing calcium ions required for enzyme stability and activity. In addition, b-mercaptoethanol, cysteine and 8-hydroxyquinoline-5-sulfonate are other known inhibitory substances.

As described in the preferred embodiment, the final cell container for cryopreservation is a flexible container made of elastic deformable material. In this embodiment of the device, the final container is directly transferred to the freezer -20 to -190°C or lower, optimally located in a controlled rate freezing equipment associated with the device or supplied separately (limited by Planer Products or Asymptote, for example) In company manufacturing), the temperature of the freezing chamber and one or more flexible storage containers used to contain the enriched and depolymerized solid tissue container is controlled by the following: inject cold air (usually nitrogen, such as Planer products) ; Or by removing heat from one or more controlled cooling surfaces. Both methods make it possible to control the freezing process of one or more specific cells to be frozen at the required rate accurately based on the desired survival rate of the freezing solution and product with an error of less than 1°C or better 0.1°C. This cryopreservation process must consider the ice crystal growth temperature, which is ideally as close as possible to the melting temperature of the frozen solution. Then the crystals grow in the aqueous solution, the water is removed from the system as ice, and the concentration of the remaining unfrozen solution increases. As the temperature decreases, more ice is formed, reducing the remaining unfrozen portion, and its concentration further increases. In aqueous solutions, there is a large temperature range in which ice coexists with concentrated aqueous solutions. Finally, the temperature is lowered, and the solution reaches the glass transition state. At this time, the frozen solution and the cells turn from the viscous solution to a solid-like state. Below this temperature, the cells will not undergo further biological changes and therefore stabilize in several years, possibly decades. Until needed.

The depolymerized cell product obtained by the method of the present invention can be cultured and/or analyzed (characterized) according to all methods known to those skilled in the art.

The TIL that can be obtained by the method disclosed herein can be used in subsequent steps, such as research, diagnosis, tissue bank, biobank, pharmacology, or clinical application known to those skilled in the art. The TIL can then be cultured using a medium optimized for this application, such as a mixture of T cells that usually contain but are not limited to growth factors (such as IL-2, IL-7, IL-15, IL-21) or stimulation conditions Cellular Therapeutics, such as antibody-coated dishes or polystyrene beads. In the present invention, the separated cells are inoculated into a culture vessel and maintained using procedures standardized by those skilled in the art, such as humid atmosphere (1% to 20% usually 5% CO ₂ , 80% to 99% usually 95% Air), the temperature is between 1 to 40°C, usually 37°C, for several weeks, and supplements can be added, supplemented with 10% FBS and 3000 IU/mL IL-2.

The enriched TIL can be used as a therapy before and/or after cell culture, such as a pharmaceutical composition in cell therapy or disease prevention. The pharmaceutical composition can be used to treat and/or prevent diseases in mammals, especially humans, and may include administering a pharmaceutically effective amount of the pharmaceutical composition to the mammal.

In addition to being formulated as drugs for the treatment of various cancers, such TIL cultures can also be used to study cell function, tumor cell killing, cell signaling, biomarkers, cell pathways, nucleic acids, and can be used to identify donors, tissues, tissues, etc. Other cell or tissue related factors of cell or nucleic acid state.

The disease can be any disease, which can be treated and/or prevented by the presence of solid tissue-derived cells and/or by increasing the concentration of related cells in/at the relevant location (ie, tumor or disease site). The disease to be treated and/or prophylactically treated can be any disease, such as cancer or degenerative disease. The treatment can be the transplantation of enriched, engineered, or expanded cells or any combination of these cells, and administration to relevant parts of the body or systemic supply.

The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although the appropriate dose can be determined by clinical trials.

As described herein, the present invention provides a kit that allows receiving, processing, storing and/or separating materials such as tissues, especially mammalian tissues. In addition, the present invention provides kits of components, such as flexible containers, such as bags, filters, valves, brackets, clamps, connectors, and/or pipes, such as catheters. In detail, the bag may be coupled to one or more tubes or sections of the catheter, which are adapted to enable tissue material to flow between the various components of the cryopreservation kit.

The processing of tissue into cells using cryopreservation kits and/or collection bags may include automated and/or semi-automated devices and methods.

In addition, by using the bags, sets, devices, and processes described herein, combined with general skills in the art, other embodiments of the present invention can be easily identified. Those familiar with this technology will easily understand the known variations.

Design patent application serial number 29/740,293 provides a tissue collection bag suitable for tissue collection. The top of the tissue collection bag of the present invention is open for receiving tissue, for example, tissue biopsy, such as animal (for example, domestic animals, such as dogs or cats) or human cancer tissue. The tissue collection bag should be sealed with the tissue collected in it, and for the tissue so sealed in it for processing in it, for example, the treatment may include stirring and/or compression, such as gentle stirring and/or compression, and/or enzymatic digestion of the tissue in it . Advantageously, the tissue processing and the extraction of desired materials (such as tumor infiltrating lymphocytes (TIL)) can be in a closed system. Advantageous or preferred embodiments may include a mark indicating the patient from which the tissue is collected and/or a mark displayed in the instrument where the collection bag can be clamped or attached in place to apply agitation and/or the collection bag may be displayed, for example, by A sign of the location sealed by heat sealing (which can be part of the instrument used for processing). Advantageously, before applying the treatment, the collection bag is clamped or affixed to the instrument for treatment and/or sealing, such as heat sealing. In some descriptions, the catheter can be displayed with dotted lines or dots to show that the catheter is not necessarily regarded as part of the design of the present invention; but in some embodiments, it may be regarded as part of the design of the present invention. The dotted lines or dots should be interpreted as the presence or absence of the catheter and can be claimed as either or both, that is, in the entire drawing, the catheter may form part of the design of the present invention (and may not necessarily be the design of the present invention). Part). In addition, although some instructions do not show signs, they can indicate the patient who obtained the sample, the patient who obtained the sample and the position where the tissue collection bag can be clamped or attached to the instrument, and the patient who obtained the sample. And the location where the tissue collection bag can be clamped or attached to the instrument and the location where the tissue collection bag can be sealed, such as a heat-sealed location mark. It should be understood that the design of the present invention may include its variations, for example, The patient who obtained the sample and the mark of the position where the tissue collection bag can be heat-sealed, but no sign showing the position where the tissue collection bag can be clamped or attached to the instrument; and the design of the present invention may include a mark indicating that the tissue collection bag can be heat-sealed The location mark and/or the mark showing the location where the tissue collection bag can be clamped or attached to the instrument without the mark indicating the patient who obtained the sample (including the patient mark can be imprinted on the tissue collection bag while in use, and The mark for clamping or attaching or heat sealing may be on the tissue collection bag before use). The tissue collection bag including any associated catheters can generally be clear or transparent or translucent or any desired color. Tissue collection bags including any associated catheters can generally be manufactured in a manner similar to the following: closed or sealed, blood collection, tissue culture, biological treatment or cryopreservation bags and associated catheters. The associated conduit in the present invention can be constructed from any desired material and polyvinyl chloride (PVC) or a material including PVC as the desired material because it facilitates welding and/or sealing. The part of the tissue collection bag of the present invention for receiving tissue can be made from any desired material and ethylene vinyl acetate (EVA) or a material including EVA as the desired material because it facilitates heat sealing.

As shown in FIG. 11A, an embodiment of the set 2 for treating tissues, such as the disaggregation, enrichment, and/or stabilization of tissues. The tissue to be processed may include solid eukaryotes, especially mammalian tissues, such as tissues from samples and/or biopsies. The set 2 includes components such as bags 4 and 6, such as a collection bag 4 and a cryopreservation bag 6. The set as depicted in Figures 11A-D can be used in automatic or semi-automatic devices for processing.

In some embodiments, the set of components may include indicators, such as codes, letters, words, names, letters, numbers, images, barcodes, quick response (QR) codes, trackers (such as smart trackers and/ Or Bluetooth tracker), tags (such as radio frequency tags and/or other digitally identifiable identification tags), so that they can be scanned during automated and/or semi-automated processing, such as in the embodiment of the present invention in an automated device And recognition. For example, tags can provide information about conditions and/or steps that need to be processed automatically. For example, scanning kit components such as bags may allow automated systems used with the kit to process tissue without further intervention and/or contamination. In particular, the tissue sample has been placed in a collection bag for processing in the disaggregation element of the device. The collection bag can be sealed before the start of the treatment. In some embodiments, the collection bag can use energy, such as heat, radio frequency energy, high frequency (HF) energy, dielectric energy, and/or any other methods known in the art, before processing begins. Manual and/or automatic seal.

In some embodiments, a heat sealing machine with heating rods (for example, Van der Staehl MS-350, Uline H-190 Impulse sealing machine or similar sealing machines known in the art), the heating rods can be used to produce on the bag Seal the mouth.

In a particular embodiment, when a heat sealer is used, it may be advantageous to form a seal at a temperature below about 100°C and a pressure in the range of about 0.8 bar to about 2.8 bar. This high temperature and pressure can be applied for about eight seconds, after which the temperature can be lowered, but in some embodiments, the pressure is continuously applied for about 2 to 3 seconds. The values of temperature, pressure and time will vary based on the formulation of the material forming the bag and especially the material forming the seal. For example, another material may require a sealing machine to reach a temperature higher than about 210°F (98.9°C) for a minimum of about 3 seconds, after which the heating rod can be cooled for 5 seconds before the heating rod is removed.

The positioning of the material to be sealed can be critical to the strength of the seal formed. For example, incomplete seals, wrinkles, channels, and/or gaps in the material to be sealed can reduce the strength of the seal.

The seal peel test (ie ASTM F88/F88M) and/or burst test (ie ASTM F1140/F1140M or ASTM F2051/F2054M) can be used to test the strength of the seal.

In some embodiments, the bag or flexible container can withstand a force of 100 Newtons during use when properly sealed and when positioned in a device for handling and/or processing, when further secured with a clamp. The bag or flexible container embodiment may be constructed to withstand a force of 75 Newtons during use when properly sealed and when positioned within a device for handling and/or processing, when further secured with a clamp.

As shown in FIG. 11A, the set 2 includes a disaggregation element 4 in which a collection bag 5 can be processed, an enrichment element 8 in which a filter 9 can be positioned, and a stabilization element in which a cryopreservation bag 7 is used to store the desired material 6. In the components of the set 2, such as the collection bag 5, the tissue is processed. For example, the collection bag 5 can be used for the disaggregation of solid tissues derived from eukaryotic cells. The tissue can be processed in such a way that most of the resulting tissue can be a single cell and/or aggregates of small cell numbers after processing. In addition, in particular, the treatment can be carried out in a kit and/or especially in a collection bag.

The enrichment of the treated tissue can be performed at the enrichment element 8 in the filter 9. The filter 9 may be selected so that the filtered composition (ie, the desired material) entering the conduit 11 may have a predetermined size component. The filter 9 may be selected so that the desired material composition entering the catheter 11 may have components such as tumor infiltrating lymphocytes (TIL) with an average size of less than about 200 µm. In particular, in one embodiment, the desired material may include tumor infiltrating lymphocytes (TIL) with an average size of less than about 170 µm.

In some embodiments, the desired material may include tumor infiltrating lymphocytes (TIL) ranging from about 15 µm to about 500 µm. For example, in one embodiment, the filter 9 may be configured such that the tissue composition entering the catheter 11 has a component with an average size of less than about 200 µm. In particular, the desired material leaving the filter and entering the conduit 11 after filtration may have a component with an average size of less than about 170 µm.

In some embodiments, the filter 9 is configured such that the filtered composition entering the catheter 11 has a component having a size in the range of about 50 µm to about 300 µm. For example, in one embodiment, the filter 9 may be configured such that the tissue composition entering the catheter 11 has a component with an average size in the range of about 150 µm to about 200 µm.

As shown in FIG. 11A, the cryopreservation bag 7 in the stabilization element 6 of the system for treating tissue can be used to stabilize the tissue composition for storage and/or transportation.

Figure 11B depicts the set 2 with valves 12,13. The valve can be a needleless valve. The valves 12, 13 can be used to provide enzyme culture media, such as tumor digestion media, cryoprotectants, and/or cryopreservation media. In particular, the valve 12 can be used to provide enzyme culture medium to the catheter 10. The enzyme culture medium can be moved to the collection bag 4 to assist in processing the tissue placed in the bag 5.

The valve 13 can be used to provide a cryoprotectant such as a DMSO solution to the catheter 11 so that the DMSO solution can be moved to the cryopreservation bag 7. In some embodiments, a cryoprotectant such as a DMSO solution may be mixed with the filtered material entering the catheter 11 so that the combined composition of the DMSO solution and the filtered material enters the cryopreservation bag 7. The filtered material entering the catheter 11 may include components such as tumor infiltrating lymphocytes (TIL) having a predetermined average size. For example, in some embodiments, the average size of the components in the filtered composition may be less than about 200 µm.

In some embodiments, as shown in FIG. 11C, the set 2 includes a clamp 14 around the filter 9 to ensure that the material provided via the valves 12, 13 is prevented from flowing into the filter 9. The valve 13 can be used to provide a cryoprotective agent to the conduit 11 so that the cryoprotectant can be mixed with the filtered material entering the conduit 11 from the filter 9. For example, the clamp 14 may be positioned to prevent and/or prevent the cryoprotectant from flowing in the direction of the filter 9. In some embodiments, after the filtered solution starts to flow from the filter 9, the clamp 14 is released so that the combined composition of the cryoprotectant and the filtered material enters the cryopreservation bag 7 at the stabilization element 6. The filtered material entering the catheter 11 may include components such as tumor infiltrating lymphocytes (TIL) having a predetermined average size. For example, in some embodiments, the average size of the components in the filtered composition may be less than about 200 µm.

The embodiment of the kit 2 may include the port 16 on the cryopreservation bag 7 as shown in FIG. 11D. The port can be used to add and/or remove material from the cryopreservation bag 7. For example, the test sample can be removed from the cryopreservation bag.

Figure 12A shows a perspective view of an embodiment of a bag 22 for use in a set. The bag 22 may include a connector 24, an opening section 26, a sealing section 21 and a locator 23. The connector 24 can be used to couple the bag 22 to the conduit 25. The positioner 23 may be an opening in the bag 22.

The bags (such as collection bags and/or cryopreservation bags) and any associated catheters can be substantially clear, transparent, translucent, any desired color, or combination thereof. Bags (eg, collection bags and/or cryopreservation bags) and/or catheters can generally be manufactured in a manner similar to the manufacturing of closed and/or sealed blood and/or cryopreservation bags and associated catheters.

The bags used in the present invention described herein include collection bags and cryopreservation bags, and may include at least a portion made of predetermined materials such as thermoplastics, polyolefin polymers, ethylene vinyl acetate (EVA), blends Compounds (such as copolymers, such as blends of vinyl acetate and polyolefin polymers (ie, OriGen Biomedical EVO film)), materials including EVA and/or co-extruded layers of sealable plastics.

The material for the bag can be selected for specific characteristics and/or a series of characteristics, such as tightness (such as sealing due to heat welding or use of radio frequency energy), air permeability, flexibility (such as low temperature flexibility (such as At -150°C or -195°C)), elasticity (such as low-temperature elasticity), chemical resistance, optical transparency, biocompatibility (such as cytotoxicity), hemolytic activity, leaching resistance, low particulate, specific High transmission rate of gases (such as oxygen and/or carbon dioxide) and/or compliance with regulatory requirements. For example, when tested according to the tensile strength test method outlined in ASTM D-638, the material used for the bag can be selected to have a tensile strength greater than about 2500 psi (172 bar). In particular, when tested according to the tensile strength test method outlined in ASTM D-638, embodiments of flexible containers (such as bags) use materials having a tensile strength greater than about 2800 psi (193 bar).

In some embodiments, the material may be selected for the specific characteristics of the co-extruded material used to form at least one layer of the bag. The layer can be constructed so that when constructed, the inner layer of the bag is relatively biocompatible, that is, the material on the inner surface of the bag is stable and does not leach into the contents of the bag.

For example, the relevant properties of materials that can be selected for kit components such as collection bags, cryopreservation bags, and/or associated catheters can be related to sealing, such as heat sealing.

The seal peel test (ie ASTM F88/F88M) and/or burst test (ie ASTM F1140/F1140M or ASTM F2051/F2054M) can be used to test the strength of the seal.

In some embodiments, the bag or flexible container can withstand a force of 100 Newtons during use when properly sealed and when positioned in a device for handling and/or processing, when further secured with a clamp. The bag or flexible container embodiment may be constructed to withstand a force of 75 Newtons during use when properly sealed and when positioned within a device for handling and/or processing, when further secured with a clamp.

The bag, especially the size of the collection bag and/or storage bag, can be specific to the device used for processing and/or processing. The bag size should be adjusted based on the configuration and/or size of one or more devices used for processing. Particular attention should be paid to the placement and/or size of any components (such as ports, connectors, or the like) that extend beyond the boundary of the bag. Components such as ports can interfere with the operation of devices used for processing and/or processing. In addition, care should be taken to ensure that the thickness of the bag is consistent with the requirements of the machine, especially with regard to the sealing material such as the manufactured seal.

The catheter in the present invention can be constructed of any desired material, including but not limited to polyvinyl chloride (PVC). For example, PVC may be the desired material because PVC facilitates welding and/or sealing.

In some embodiments, as depicted in Figures 12A-12E, 13A-13E, 14, 20A-20E, 21A-21E, 22A-22D, 27A, 28, 33, and 34, at least one end of the collection bag can be opened with To the receiving organization. In particular, in one embodiment, for example, a tissue sample from a biopsy can be placed in the bag via the open end (e.g., the top end). In some cases, the biopsy sample may be cancer tissue from animals (eg, domestic animals such as dogs or cats) or humans.

As shown in Figure 12A, the bag 22 can be used as a tissue collection bag. For example, after the tissue is positioned in the bag, the bag can be sealed, and then can be processed. The treatment may include agitation of the tissue in the bag, such as gentle agitation, extraction, and/or enzymatic digestion. Tissue processing and extraction of desired materials from it (such as tumor infiltrating lymphocytes (TIL)) can be in a closed system. Advantageous or preferred embodiments may include an indicator indicating the patient from which the tissue is collected and/or a mark showing the location in the instrument where the collection bag can be clamped, sealed, acted upon by a device, and/or attached in place.

In some embodiments, the bag 22 may be formed of a sealable material. For example, the bag 22 may be formed of materials including, but not limited to, polymers such as aliphatic or semi-aromatic polyamides (for example, nylon), ethylene-vinyl acetate (EVA) and blends thereof. Compounds, blends of vinyl acetate and polyolefin polymers, thermoplastic polyurethane (TPU), polyethylene (PE) and/or synthetic polymers of polymer combinations. Portions of the bag can be sealed and/or welded using energy, such as heat, radio frequency energy, high frequency (HF) energy, dielectric energy, and/or any other method known in the art.

The collection bag can be used as a treatment and/or depolymerization bag. The collection bag may have a width ranging from about 4 cm to about 12 cm and a width ranging from about 10 cm to about 30 cm.

For example, the collection bag used for processing may have a width of about 7.8 cm and a length of about 20 cm. In particular, the bag can be heat-sealable, for example using EVA polymer and blends thereof, blends of vinyl acetate and polyolefin polymers, and/or one or more polyamides (nylon).

As depicted in Figure 12A, the bag 22 can be used as a tissue collection bag for sealing tissue therein for use in the treatment of the present invention.

Figure 12B shows a perspective view of an embodiment of the bag 22 used as a tissue collection bag. The tissue can be sealed in a bag and then processed. The bag 22 as shown in FIG. 12B may be marked with indicators 27, 28, such as a patient identifier that can identify a patient who has obtained or obtained a tissue sample or biopsy.

Indicators can include but are not limited to codes, letters, words, names, alphanumerics, numbers, images, barcodes, QR codes, tags, trackers (such as smart tracker tags or Bluetooth trackers) and/ Or any indicator known in the art. In some embodiments, the indicator may be printed, etched, and/or adhered to the surface of the components of the kit. For example, the indicator can be directly printed on the surface of at least one component of the set, as shown in FIG. 12B. The indicator can also be positioned on the bag using an adhesive, for example, a sticker or tracker can be placed on one bag and/or multiple bags. For example, as shown in FIG. 12B, the bag 22 includes a plurality of indicators 28 (digital code), 27 (QR code).

Figure 12C shows a perspective view of a bag used as a tissue collection bag. Tissue can be inserted into the bag 22 for processing. The indicator can be used to identify patients for whom a tissue sample and/or biopsy has been obtained or obtained. As shown in Figure 12C, the indicators 27, 28 include QR codes and identification numbers for tracking the sample, locating the sample, and/or tracking the status of the sample in the process. For example, in some embodiments, the indicator can be used to locate a sample at any given location in the laboratory. The indicator may be placed on the bag before and/or during use, for example, the patient indicator may be embossed on the bag when the bag is removed for use with the sample. In addition, the bag 22 may include indicia 29. Markers can be used to show where seals, clamps, and/or instruments should be located.

Indicators (such as QR codes, tags such as smart tags and/or trackers) can be used to identify the samples in the bag and send instructions to the processor of the device so that the device will be de-aggregated according to the cryopreservation kit. The type of enrichment and/or stabilization process runs a specific program. Different types of media can be used in these processes, such as enzyme media that allows a controlled freezing rate, tumor digestion media, and/or cryopreservation media. In some embodiments, the cryopreservation kit and/or its components may include indicators that can be read by an automated device. The device can then perform a specific fully automated method for processing tissue when inserted into such a device. The invention is particularly suitable for sample processing, especially automated processing.

In some cases, the cryopreservation kits and/or components thereof described herein may be single-use. The cryopreservation kit and/or its components can be used in automated and/or semi-automated processes for the disaggregation, enrichment and/or stabilization of cells or cell aggregates. In some embodiments, in some embodiments, bags used in cryopreservation kits, such as collection bags, can be used in multiple processes. For example, the collection bag can be repeatedly sealed at different locations to create separate compartments for processing tissue samples (such as biopsy samples and/or solid tissue).

In addition, markers can be placed at various positions on the bag (such as a tissue collection bag) to indicate where the bag can be sealed, clamped, and/or attached to an object. In some embodiments, markings where the display bag can be clamped, sealed, and/or attached to an object (such as an instrument) can be placed on the bag before use. For example, one or more markings can be positioned on the bag during manufacturing.

The seal can be formed with energy (e.g. heat) during use to create a weld zone. The sealing port formed during use may have a width in the range of about 2.5 mm to about 7.5 mm. Generally speaking, the sealing port 140 is formed after placing the tissue material in the bag 140 and may have a width of about 5 mm.

The seal peel test (ie ASTM F88/F88M) and/or burst test (ie ASTM F1140/F1140M or ASTM F2051/F2054M) can be used to test the strength of the seal.

In some embodiments, the bag or flexible container can withstand a force of 100 Newtons during use when properly sealed and when positioned in a device for handling and/or processing, when further secured with a clamp. The bag or flexible container embodiment may be constructed to withstand a force of 75 Newtons during use when properly sealed and when positioned within a device for handling and/or processing, when further secured with a clamp.

When forming a sealing port or a fusion port on a flexible container (such as a bag, for example, a collection bag and/or a cryopreservation bag), the sealing device can be used at a predetermined temperature, pressure, and amount of time depending on the material used for the bag Apply heat and/or pressure. For example, some heat sealers may need to apply heat and pressure for about eight seconds. After 8 seconds, the heat of the device can be turned off, however, pressure can be applied for an additional 2 to 3 seconds.

Figure 12D shows a perspective view of an embodiment of a tissue collection bag used to seal tissue therein for the treatment of the present invention. The indicators 27, 28 are positioned on the bag 22 so that the user can easily identify the patient during use. In addition, these indicators can be used to identify the material in the bag and to track the progress of the material in the bag during a specific treatment method. In some embodiments, the bag contains a volume of medium in the range of about 0.1 ml to about 25 ml and a volume of tissue in the range of about 0.1 ml to about 10 ml in the bag during processing. The ratio of the volume of medium in the bag to the volume of tissue during processing should be in the range of about 1.0 to about 2.5. In some embodiments, the ratio of medium volume to tissue volume is in the range of about 1.7 to about 2.3. Specifically, the ratio of the volume of the medium to the volume of the tissue is in the range of about 2.0 to about 2.2.

As shown in FIG. 12D, the marking 29 is positioned close to the open end 26 of the bag 22. As shown in FIG. During use, the marker 29 may be placed on the bag based on the method used to process the tissue sample and/or the biopsy sample. During use, for example, based on the processing method being used or to be used and/or the equipment to be used, the mark can be placed on the bag. In some embodiments, the indicia can be positioned on the bag during manufacturing. For example, the location of the mark regarding the sealing and/or clamping position may vary based on the treatment method and/or the volume of the tissue to be treated.

Figure 12E shows a perspective view of the tissue collection bag. The tissue can be sealed in the bag 22 for processing. The connector 24 can provide access to the bag. As shown, the connector 24 may be connected to other devices, such as filters, bags, etc., using a conduit 25. The port 20 can be used to obtain samples from the bag 22 and/or provide material from the bag 22 during use.

Figure 13A shows a front view of a bag for tissue collection. The tissue can be sealed in the bag during use. The bag 30 can be manufactured with a sealed edge 31. As shown in FIG. 13A, the sealing edge 31 may be located on three edges, and the fourth edge may include an open section 36.

The positioner 33 on the bag 30 can be used to position the bag. For example, one or more locators can be used to ensure that the bag can be properly handled during use, such as being positioned close to the instrument. In some systems, the positioner can facilitate the use of the bag described in this article in an automated system. In particular, the positioner can be used to move the bag through an automated system.

As shown in FIG. 13B, the bag 30 may have indicators 36, 37 for identifying samples, such as indicators for identifying patients who have obtained or obtained a tissue sample or biopsy. The use of an indicator 37 such as a QR code may allow tracking of the process steps of a particular sample, making it possible to track the sample in a given process.

Figure 13C shows the front view of the tissue collection bag. The tissue can be sealed in the bag and processed and/or processed in it. The bag 30 may have indicators 37, 38 for identifying the sample, such as an indicator for identifying a patient whose tissue sample or biopsy has been obtained or obtained. The use of an indicator 37 such as a QR code may allow tracking of the process steps of a particular sample, making it possible to track the sample in a given process. The positioner 33 can be used to position the bag 30 for processing. The connector 34 may allow tissue, processed tissue, etc. to be coupled to other devices via the catheter 35.

Figure 13D depicts a front view of a tissue collection bag with indicators 37, 38 to identify samples. The use of an indicator 37 such as a QR code may allow tracking of the process steps of a particular sample, making it possible to track the sample in a given process. The marking 39 and/or the positioner 33 can be used to control the positioning of the bag during processing and/or processing. A marker is placed close to the open end to indicate where to position, seal, and/or clamp the bag during use. The bag 30 can be manufactured with a sealed edge 31. As shown in FIG. 13D, the sealing edge 31 may be located on three edges, and the fourth edge may include an open section 36.

Figure 13E shows a front view of a tissue collection bag that can be sealed after the tissue is placed therein. The connector 34 and the port 32 can provide access to the bag. One or more ports may be positioned on the collection bag such that the ports allow the input of media and/or reagents and/or the extraction of samples from the bag.

As shown, the connector 34 may be coupled to other devices such as filters, bags, etc., using a conduit 35. Depending on the purpose, the marks and indicators can be placed on one or more sides of the bag. In particular, as shown in FIG. 13E, the positioner 33, marks 39 and/or indicators 37, 38 can be used to position the bag 30 for processing, such as applying agitation, for example by heat sealing (which can be used for processing) Part of the instrument) is sealed, and materials are added for processing and/or extraction. Advantageously, before applying the treatment, the collection bag is clamped or affixed to the instrument for treatment and/or sealing, such as heat sealing.

Figure 14 shows a rear view of the bag used for tissue collection. In particular, the bag 40 can be sealed and disposed of with the tissue placed therein. The sealing port may be located close to the open end 46 and substantially parallel thereto. As shown, the connector 44 may use a conduit 46 to connect to other devices, such as filters, bags, and the like. The bag 40 can be manufactured with a sealed edge 41. As shown in FIG. 14, the sealing edge 41 may be located on three edges, and the fourth edge may include an open section 46. The positioner 43 can be surrounded by the manufactured sealing edge 41.

Figure 15 depicts a side view of a bag 50 for tissue collection capable of sealing tissue therein and allowing the tissue to be processed during use of the bag. The bag 50 can be coupled to the conduit 54 by the connector 52.

Figure 16A shows a top view of an unsealed tissue collection bag. The bag 60 may include a sealed part 66 and an open part 64. The connector 62 is visible through the bag 60. After the tissue is placed in the open portion of the bag at the top of the bag 60, the bag can be sealed.

Figure 16B shows a bottom view of a tissue collection bag 60 with a sealed edge 66 for sealing the tissue therein for disposal. The connector 62 is visible on the bag 60.

Figure 17A shows a top view of a partially open bag. The bag 70 may include a sealed part 76 and an open part 74. The connector 72 is visible through the bag 70. After the tissue is placed in the open portion of the bag at the top of the bag 70, the bag can be sealed.

Figure 17B shows a bottom view of a tissue collection bag used to seal the tissue for disposal. The connector 72 is visible on the bag 70.

Figure 18A depicts a top view of a partially open bag. The tissue can be inserted through the open end 84 of the bag 80. The connector 82 is shown arranged at the bottom of the bag 80.

Figure 18B shows a top view of a fully open bag for collecting and/or processing tissue. The open end 84 of the bag 80 can receive tissue for processing, such as processing, separation, and/or separation. The sealing edge 86 can be created during manufacturing.

Figure 19A depicts a top view of a partially open bag 90 with a sealed edge 96 on the side of the bag. As shown, tissue can be inserted through the open end 94 of the bag 90. The connector 92 is shown arranged at the bottom of the bag 90.

Figure 19B shows a top view of a fully open bag for collecting and/or processing tissue with a sealed edge 96 on the side of the bag. The open end 94 of the bag 90 can receive tissue for processing, such as processing, separation, and/or separation. The connector 92 is shown installed at the bottom of the bag 94.

Figures 20A-20E show a front view of an embodiment of a tissue collection bag. As shown in Figure 20A, a bag 100 with a sealed edge 101 and an open end 102 can be connected to a device (not depicted) via a conduit 105 and/or a connector 104. For example, the connector 104 is disposed in the bag 100, and the y-connector 106 may be disposed along the catheter. Figure 20B shows another embodiment of a bag 100 that includes indicators 107, 108 to allow a user to identify a patient who has obtained or obtained a tissue sample or biopsy. In addition, an embodiment of a bag 100 including a mark 109 and indicators 107, 108 is depicted in FIG. 20C. The use of the positioner 103 may allow constant positioning of the bag, which allows constant treatment of the tissue in the bag. The indicators 107, 108 identify the sample with the sample and/or patient information. In some cases, the indicator can be used to identify and/or track samples, such as tissue samples and/or biopsy samples. FIG. 20D depicts a bag 100 with multiple indicators 107, 108 and indicia 109. FIG. The mark can show where the bag 100 should be sealed. For example, the mark 109 may indicate the location where the bag 100 should be sealed, clamped, and/or coupled to another device. The sealed mark can be positioned close to the opening edge of the bag, for example, such a mark can be positioned at a predetermined distance from the opening edge. In some embodiments, the sealed mark may be substantially parallel to the edge of the opening. As shown, the bag 100 can include a connector 104 and a conduit 105.

In one embodiment, as shown in FIG. 20E, the bag 100 includes a port 110 and a connector 104. The port may allow the addition of material and/or the removal of material from the sample. For example, during tissue treatment, samples can be taken multiple times throughout the treatment. In addition, the port 110 may allow the aseptic input of culture medium and/or reagents into the bag 100.

Figure 21A shows a front view of a bag 100 for collecting and/or processing tissue. The tissue can be placed in the bag 100 through the open end 102. The connector 104 can be used to couple the bag 100 with the catheter 105 and the clamp 112.

Figures 21B-21E show front views of additional embodiments of the bag 100. Figures 21B-11D show various configurations including indicators 107, 108 and/or mark 109. The bag may include indicators such as codes, letters, words, names, alphanumerics, numbers, images, barcodes, QR codes, tags, trackers (such as smart tracker tags or Bluetooth trackers) and/ Or any indicator known in the art. In some embodiments, the indicator may be printed, etched, and/or adhered to the surface of the components of the kit. The indicator can also be positioned on the bag using an adhesive, for example, a sticker or tracker can be placed on one bag and/or multiple bags. The collection bag and/or cryopreservation kit may include multiple indicators, such as numeric codes and/or QR codes.

Indicators (such as QR codes, tags such as smart tags and/or trackers) can be used to identify the samples in the bag and send instructions to the processor of the device so that the device will be de-aggregated according to the cryopreservation kit. The type of enrichment and/or stabilization process runs a specific program.

Figure 21E depicts a front view of another embodiment of a bag 100 for collecting, processing, processing, and/or separating materials. The tissue to be processed can be sealed in the bag 100. The tube 105 can couple the bag 100 to the clamp 112 via the connector 104. The port 114 may allow input and/or removal from the bag 100. For example, the port may allow for sampling and/or for aseptic input of culture medium and/or reagents into a flexible container (such as a bag of a cryopreservation kit).

FIG. 22A shows a front view of another embodiment of a tissue collection bag 120 having a sealing edge 121 for sealing tissue therein for processing. The bag 120 includes a positioner 123 and a connector 124 coupled to the catheter 125.

FIG. 22B shows a front view of a tissue collection bag 120 with a sealed edge 121 and an open end 122. FIG. The indicators 127, 128 can be positioned on the bag 120 so that they can be easily acquired by an automated system. The opening defining the positioner 123 may be surrounded by the sealing edge 121. The indicator can be used to identify patients for whom a tissue sample or biopsy has been obtained or obtained.

As shown in FIG. 22C, the bag 120 includes indicators 127, 128 and indicia 129. FIG. 22D depicts a collection bag 120 with a plurality of markings 129. FIG. The sealed mark can be positioned close to the opening edge of the bag. Such marks can be positioned at a predetermined distance from the edge of the opening. In some embodiments, the sealed mark may be substantially parallel to the edge of the opening.

FIG. 23 depicts a front view of the sealed bag 130 positioned so that the bottom of the bag 130 is shown at the top of the page, with the conduit 135 extending from the connector 134. The bag 130 includes an indicator 137 on the sealed portion 131 of the bag 130. The indicator on the sealing portion can be placed during and/or after the sealing of the bag 130. In general, the bag is sealed after the tissue is provided. The indicator 138 on the surface of the bag 130 may be a barcode. The positioner 133 can be positioned close to the connector 134.

The bags (such as collection bags and/or cryopreservation bags) and any associated catheters can be substantially clear, transparent, translucent, any desired color, or combination thereof. Tissue collection bags and/or catheters can generally be manufactured in a manner similar to the manufacturing of closed and/or sealed blood and/or cryopreservation bags and associated catheters. The catheter in the present invention can be constructed of any desired material, including but not limited to polyvinyl chloride (PVC). For example, PVC may be the desired material because PVC facilitates welding and/or sealing.

The collection bag, such as the tissue collection bag of the present invention, may include at least a part of a bag for receiving tissue made of a predetermined material, such as polyolefin polymer, ethylene vinyl acetate (EVA), copolymer (such as acetic acid) A blend of vinyl ester and polyolefin polymers (that is, OriGen Biomedical EVO film) and/or materials including EVA. The material for the bag can be selected for specific characteristics and/or a series of characteristics, such as tightness (such as heat sealability), air permeability, flexibility (such as low temperature flexibility), elasticity (such as low temperature elasticity), chemical resistance Properties, optical transparency, biocompatibility (such as cytotoxicity), hemolytic activity, resistance to leaching, with low particles.

As shown in FIG. 24, the bag 140 may include a plurality of marks 141, 142 that are placed such that if the area including the marks is sealed, the compartment 143 may be formed in the bag 140. The bag 140 has a pre-welded section 145 formed during bag manufacturing, which can be used to form a sample compartment during use. Figure 24 depicts an embodiment of a collection bag that can be formed so that it has multiple compartments. Each compartment can be formed in the bag by placing multiple sealing ports and/or fusion ports (for example, heat sealing). For example, after placing the tumor suspension in the collection bag, using energy such as heat, the closed open end can be welded, and additional marks 141 such as weld lines 142 can be welded to form a compartment.

The positioner 143 on the bag 140 ensures that the bag is properly positioned relative to the instrument (such as a sealing device such as an RF heat sealer and/or a syringe).

The seal can be formed with energy (e.g. heat) during use to create a weld zone. The sealing port formed during use may have a width in the range of about 2.5 mm to about 7.5 mm. Generally speaking, the sealing port 140 is formed after placing the tissue material in the bag 140 and may have a width of about 5 mm.

The seal peel test (ie ASTM F88/F88M) and/or burst test (ie ASTM F1140/F1140M or ASTM F2051/F2054M) can be used to test the strength of the seal.

In some embodiments, the bag or flexible container can withstand a force of 100 Newtons during use when properly sealed and when positioned in a device for handling and/or processing, when further secured with a clamp. The bag or flexible container embodiment may be constructed to withstand a force of 75 Newtons during use when properly sealed and when positioned within a device for handling and/or processing, when further secured with a clamp.

When forming a sealing port or a fusion port on a flexible container (such as a bag, for example, a collection bag and/or a cryopreservation bag), the sealing device can be used at a predetermined temperature, pressure, and amount of time depending on the material used for the bag Apply heat and/or pressure. For example, some heat sealers may need to apply heat and pressure for about eight seconds. After 8 seconds, the heat of the device can be turned off, however, pressure can be applied for an additional 2 to 3 seconds.

In some systems, the positioner can facilitate the use of the bag described in this article in an automated system. Therefore, the tissue that has been placed in the bag 140 can be divided into separate compartments 144, 146, 147. As shown, each compartment 144, 146, 147 includes ports 148, 149, 150, respectively. Each port allows direct access to the compartment. This may allow individual addition, storage, and/or testing of samples. For example, a sealed collection bag can help store and test TIL for the suitability and/or microbiological characteristics of complex samples. Because this type of test may require freezing smaller aliquots of digested material in a collection bag so that the smaller aliquots of digested material can be thawed separately. In some embodiments, the bag 140 as depicted in FIG. 24 can be used as a collection bag and/or cryopreservation bag.

Figure 25 shows a front view of an embodiment of the collection bag. In this embodiment, the length of the collection bag 152 is about 150 mm (that is, 15 cm) and the width is about 90 mm (that is, 9 cm). The bag 152 includes an opening that serves as a locator 160. One or more positioners can be used to control the orientation of the bag to ensure that the bag is properly positioned for processing and/or handling during use, such as close to the instrument positioning. In some systems, the positioner can facilitate the use of the bag described in this article in an automated system. In particular, the positioner can be used to move the bag through an automated system. The sealing port 156 is about 5 mm. The seal can be formed using energy (e.g. heat) during use to create a weld zone. The sealing port may have a width in the range of about 2.5 mm to about 7.5 mm. Generally speaking, the sealing port 156 is formed after placing the tissue material in the bag 152. As shown in FIG. 25, the bag 152 has a pre-welded section 158 formed during bag manufacturing.

As shown in Figure 26, the collection bag can be coupled to the conduit and valve. In some embodiments, the bag may have a length in the range of about 10 cm to about 50 cm. In particular, the bag used in the present invention described herein may have a length in the range of about 15 cm to about 30 cm. For example, the bag may have a length in the range of about 18 cm to about 22 cm. The bag 162 as shown in FIG. 26 has a length of about 20 cm. The collection bag used as described herein may have a width in the range of about 6.8 cm to about 8.8 cm. As shown in FIG. 26, the width of the collection bag 162 is about 7.8 cm. Can be used at locations along the catheter including, but not limited to, valves without needle valves. For example, the needleless valve 164 is positioned approximately 20 cm from the bag 162 coupled by the catheter 166. Before adding another element or component, the catheter 166 extends from the needleless valve 164 at least 10 cm.

As depicted in Figure 27A, the open bag 170 is coupled to the conduits 172, 174, 176 prior to use. The bag 170 may be constructed of a sealable material. In particular, the bag can be sealed using a heat sealer (such as a benchtop heat sealing device). Some of the conduits, such as conduit 174, may be non-weldable. Can be used at locations along the catheter including, but not limited to, valves without needle valves. For example, the needleless valve 178 is positioned at the ends of the catheters 174, 176.

In some embodiments, the bag may have a length in the range of about 10 cm to about 50 cm. In particular, the bag used in the present invention described herein may have a length in the range of about 15 cm to about 30 cm. For example, the bag may have a length in the range of about 18 cm to about 22 cm. The bag 170 as shown in Fig. 27A has a length of about 20 cm.

Figure 27B shows a front view of an embodiment of a collection bag that has been sealed, for example, after the material is deposited in the bag. The bag 180 is constructed of a sealable material. In particular, the bag can be sealed using a heat sealer (such as a benchtop heat sealing device). The sealing port can be positioned close to the opening edge of the bag. In some cases, the marking can be positioned at a predetermined distance from the opening edge. In some embodiments, the sealing port may be substantially parallel to the edge of the opening.

Some of the conduits, such as conduits 182, 184, 186, may be weldable. The weldable conduit may be made of a polymer material such as polyvinyl chloride (PVC).

Can be used at locations along the catheter including, but not limited to, valves without needle valves. For example, the needleless valve 188 is positioned at the ends of the catheters 184, 186. In some embodiments, the bag may have a length in the range of about 10 cm to about 40 cm. In particular, the bag used in the present invention described herein may have a length in the range of about 15 cm to about 30 cm. For example, the bag may have a length in the range of about 18 cm to about 22 cm. The bag 180 as shown in Figure 27A has a length of about 20 cm.

As shown in FIG. 28, the embodiment of the cryopreservation kit is shown face up and includes an open bag 190 and a cryopreservation bag 192. As shown, the cryopreservation bag 192 may include indicators 193, 194. Cryopreservation bags may need to be suitable for cryopreservation using cryoprotectants such as dimethylsulfoxide ("DMSO"). In some embodiments, the cryopreservation bag can be constructed so that the bag can contain a volume of material in the range of about 5 ml to about 45 ml. In detail, the cryopreservation bag may include a volume of material contained in the range of about 10 ml to about 35 ml. For example, some embodiments include cryopreservation bags that can contain a volume of material to be stored in the range of about 15 ml to about 30 ml. The cryopreservation bag 192 may have a size such that a desired predetermined volume is achieved. In some embodiments, the cryopreservation bag may have a width in the range of about 4 cm to about 11 cm and a length in the range of about 10 cm to about 18 cm. For example, the cryopreservation bag may have a width in the range of about 5.8 cm to about 9.8 cm and a length in the range of about 12 cm to about 16 cm. In particular, an embodiment of the cryopreservation bag as depicted in FIG. 28 may have a width of about 7.8 cm and a length of about 14 cm.

Before use, the cryopreservation kit and/or its specific components can be sterilized. For example, the bags 190, 192 can be sterilized. The materials used to form the bags 190, 192 may be heat-sealable. The material used for the bag may include, but is not limited to, polymers such as EVA, polyamide (e.g. nylon), and combinations thereof. The open bag 190 may be used for processing and/or depolymerization after the bag is closed using a sealing port and/or clamps (not shown).

The set 191 further includes valves 195, 196, clamps 197, 198, a tube 199, and a filter 200. The filter 200 may be a line filter, a blood filter (such as a blood administration filter), a biological filter, and/or a line agglutinate removal filter. The filter can be configured to remove material larger than a predetermined size from the treated tissue to form the desired material. For example, clumps of tissue can be separated from depolymerized tissue using a filter. In particular, the tissue composition that enters the catheter after filtration may have a component with an average size of less than about 200 µm, so that the desired material is formed. For example, the desired material may include tumor infiltrating lymphocytes (TIL) with an average size of less than about 170 µm.

The filter can be selected so that the treated tissue composition entering from the catheter can be enriched so that after the filter, the desired material flowing into the catheter in the direction of the stabilizing element has a size in the range of about 15 µm to about 500 µm The components. In some embodiments, the filter may be configured such that the tissue composition entering the catheter in the direction of the stabilizing element after filtration has a component having a size in the range of about 50 µm to about 300 µm. For example, in one embodiment, the filter may be configured so that the tissue composition entering the catheter after filtration has a component having a size in the range of about 150 µm to about 200 µm.

In some embodiments, the filter of the enrichment element can remove material outside the predetermined size range of about 5 µm to about 200 µm from the treated tissue to form the desired material. For example, the desired material may include tumor infiltrating lymphocytes (TIL) with an average size in the range of about 5 µm to about 200 µm. The valves 195, 196 can be placed at a predetermined distance from the collection bag. For example, the needleless valve 195 can be positioned about 20 cm from the collection bag 190. A valve such as a needleless valve can be used to add material to the collection bag 190. For example, the enzyme culture medium may be inserted into the needle-free valve 195 to add culture medium to the collection bag 190.

In some embodiments, after such a valve, there may be a predetermined amount of conduit to allow room for additional components to be welded to the cryopreservation kit. For example, after some valves, at least ten (10) cm catheter can be positioned before the next element. The conduit 199 may be sealable and/or weldable. For example, the material used for the catheter may include, but is not limited to, polyvinyl chloride (PVC) and/or other materials known in the art. In some embodiments, the catheter can be sized to fit the connector. For example, the catheter may have an inner diameter in the range of about 1.5 mm to about 4.5 mm and an outer diameter in the range of about 2.1 mm to about 6.1 mm. For example, an embodiment of a cryopreservation kit may include a catheter having an inner diameter in the range of about 2.9 mm to about 3.1 mm and an outer diameter in the range of about 4.0 mm to about 4.2 mm. The length of the catheter used in the cryopreservation kit 191 can vary, wherein the length of the individual catheter elements is in the range of about 1 cm to about 30 cm. For example, as depicted in Figure 28, the length of the individual catheter elements can vary from about 5 cm to about 20 cm.

The clamps 197, 198 as depicted in Figure 28 can be used to prevent and/or prevent the enzymatic culture medium and/or digested tissue from moving into the filter. For example, the clamp 197 can be used to prevent and/or prevent the enzymatic medium and/or digested tissue from moving into the filter before the desired filtration step. The clamp 198 can prevent and/or prevent the cryoprotectant from undesirably moving into the filter.

FIG. 29 shows a top view of an embodiment of a cryopreservation kit similar to the kit 191 shown in FIG. 28, however, the kit 201 faces downward. Figure 29 depicts the position where the collection bag 202 can be closed.

FIG. 30 shows a top view of an embodiment of an upwardly facing cryopreservation kit including a closed collection bag 206 and a cryopreservation bag 208. In some embodiments, the cryopreservation bag 208 may include ports 215, 216 that allow sampling and permit aseptic input of culture media and/or reagents into the cryopreservation bag. The cryopreservation kit 205 may include a filter 214, valves 209, 210, clamps 211, 212, and a catheter 222.

The filter 214 may be a line filter, a biological filter, a blood filter (such as a blood administration filter), and/or a line agglutinate removal filter. The filter can be configured to remove material larger than a predetermined size. For example, clumps of tissue can be separated from depolymerized tissue using a filter. The filter can be selected so that the tissue composition entering the catheter after the filter can have a component having a size in the range of about 15 µm to about 500 µm. In some embodiments, the filter can be configured such that the tissue composition entering the catheter after filtration has a component having a size in the range of about 50 µm to about 300 µm. For example, in one embodiment, the filter may be configured such that the tissue composition entering the catheter after filtration has a component having an average size in the range of about 150 µm to about 200 µm. In particular, the tissue composition that enters the catheter after filtration may have components with an average size of less than about 170 µm.

The valves 209, 210 can be placed at a predetermined distance from the collection bag. For example, the needle-free valve 209 can be positioned about 20 cm from the collection bag 206. A valve such as a needleless valve can be used to add material to the collection bag 206. For example, the enzyme culture medium can be inserted into the needle-free valve 209 to add culture medium to the collection bag 206.

In some embodiments, after such a valve, there may be a predetermined amount of conduit to allow room for additional components to be welded to the cryopreservation kit. For example, after some valves, at least ten (10) cm catheter can be positioned before the next element. The conduit 222 may be sealable and/or weldable. For example, the material used for the catheter may include, but is not limited to, PVC and/or other materials known in the art. In some embodiments, the catheter can be sized to fit the connector. For example, the catheter may have an inner diameter in the range of about 1.5 mm to about 4.5 mm and an outer diameter in the range of about 2.1 mm to about 6.1 mm. For example, an embodiment of a cryopreservation kit may include a catheter having an inner diameter in the range of about 2.9 mm to about 3.1 mm and an outer diameter in the range of about 4.0 mm to about 4.2 mm. The length of the catheters used in the cryopreservation kit 205 can vary, with the length of individual catheter elements ranging from about 1 cm to about 30 cm. For example, as depicted in Figure 30, the length of the individual catheter elements can vary from about 5 cm to about 20 cm.

The clamps 211, 212 as depicted in Figure 30 can be used to prevent and/or prevent the enzymatic medium and/or digested tissue from moving into the filter. For example, the clamp 211 can be used to prevent and/or prevent the medium enzyme solution and/or digested tissue from moving into the filter before the desired filtration step. The clamp 212 can prevent and/or prevent the cryoprotectant from undesirably moving into the filter.

FIG. 31 shows a side view of an embodiment of an upwardly facing cryopreservation kit including a closed collection bag 226 and a cryopreservation bag 228. The cryopreservation bag 228 may include a port 242. The port 242 provides access to the cryopreservation bag 228. The valves 232 and 238 and the clamps 234 and 236 can be arranged around the filter 230 and used to control the movement of the fluid in the cryopreservation kit 224.

Figure 32 shows an end view of an embodiment of the cryopreservation kit. The sealed bag 226 and filter 230 are visible. The sealed bag 226 may be coupled to the filter 230 using a tube, valve, and/or clamp.

Figure 33 shows a top view of an embodiment of the collection bag. The bag 232 is shown as open and includes indicators 234, 236 and marks 238, 240. The mark can be used to show where the part of the bag should be sealed and/or clamped. The sealed mark can be positioned close to the opening edge of the bag. Such marks can be positioned at a predetermined distance from the edge of the opening. In some embodiments, the sealed mark may be substantially parallel to the edge of the opening.

The bag 232 includes a positioner 244 and a connector 246. The connector 246 couples the bag 232 with the conduit 248. The connector 246 may allow the conduit 248 to be divided into conduits 250, 252 including clamps 254, 256 and/or ports 258, 260.

FIG. 34 shows a front view of an embodiment of a cryopreservation kit including collection bag 264, clamps 266, 268, filter 270, conduit 272, ports 274, 276, valve 278, connector 280, and cryopreservation bag 282. The collection bag and associated duct can be formed using at least some EVA material. In some embodiments, the collection bag and/or conduit may be formed of EVA. The clamps 266, 268 may be spring clamps. The connector 280 is a four-way connector and can be used to couple the catheter from the filter 270 to the valve 278, such as a needleless valve, and to couple the catheter to the cryopreservation bag 282.

35 shows a front view of an embodiment of a cryopreservation kit including collection bag 284, port 286, clamps 288, 296, valves 290, 292, filter 298, and cryopreservation bag 294. As depicted, the valves 290, 292 may be needle-free valves capable of receiving materials for use in the kit during processing. For example, the materials to be provided via the valves 290, 292 include, for example, tumor digestion medium and/or cryoprotectant or cryopreservation medium, such as dimethyl sulfoxide ("DMSO") and/or its solutions, such as 55% DMSO and 5% polydextrose cryopreservation medium (for example, BloodStor 55-5). The syringes 300 and 302 can be used to provide tumor digestion medium and 55% DMSO solution, such as 55% DMSO and 5% polydextrose cryopreservation medium via needle-free valves 290 and 292, respectively. During processing, the material can be selectively provided to the cryopreservation kit at a predetermined time. In addition, the clamp can be used to control the flow of provided materials such as tumor digestion medium and/or cryoprotective agents, such as DMSO solution can be provided to devices such as collection bags, filters and/or cryopreservation bags at a predetermined time.

Figure 36A shows a front view of an embodiment of a cryopreservation kit that can be fastened in a device such as a digester. As shown, the collection bag 304 is at least partially enclosed by the bracket 306 during use. The holder can position the collection bag 304 so that processing can occur in an efficient manner. 36A depicts a collection bag 304 having a fusion port 310 and utilizes a clamp 312 close to the fusion port 310 to reduce the pressure on the fusion port 310 during use. The tissue introduced during use can be substantially evenly distributed in the collection bag 304 so that the tissue can be processed using the paddles 314, 316 from the device. The cryopreservation bag 330 has a plurality of sections 332 each having its own port 334.

Figure 36B depicts a side view of an embodiment of a collection bag secured with a bracket. The bracket 336 can be used to fasten the collection bag. The bracket 336 includes a hinge 338, a top side 340, a bottom side 342, a clamp 344, a protrusion 346, and a latch 348. During use, the clamp 344 can be positioned close to the fusion port on the collection bag (Figure 36A). The protrusion 346 on the bracket 336 is constructed so that it will be positioned close to the surface of the collection bag and protrude upward into the collection bag during use. In some embodiments, the protrusions 346 can reduce and/or prevent movement of the tissue and/or culture medium during use to ensure that the treatment of the tissue is substantially similar along the length of the collection bag. For example, the protrusion can be constructed such that it reduces and/or prevents the sliding of tissue between the paddles (shown in Figure 36A). The bracket 336 may also include a latch 348 for securing the collection bag.

Figure 36C shows an exploded view of the clamp 344 including the ridge 350 for use with the collection bag. In detail, during use, the clamp 344 can be positioned close to the melting port on the collection bag to reduce the risk of failure of the melting port and/or the sealing port.

FIG. 37 shows a top view of an embodiment of a cryopreservation kit including collection bag 354, filter 356, valves 362, 364, clamps 358, 360, conduit 368, and cryopreservation bag 366. The length of the catheter between the various components of the cryopreservation kit 352 can vary.

FIG. 38 shows a view of an embodiment of the cryopreservation kit including the collection bag 354, the filter 356, the valves 362, 364, the clamps 358, 360, the conduit 368, and the cryopreservation bag 366 placed face down.

In a particular embodiment, two or more bags may be coupled together to ensure that the depolymerization product material can be properly stored.

In some embodiments, the present invention may include an automated device for semi-automated aseptic depolymerization, enrichment, and/or stabilization of cells and/or cell aggregates from tissues, such as solid mammalian tissues. The automation device used with the present invention may include a programmable processor and a cryopreservation kit. In some embodiments, the cryopreservation kit may be a single-use sterile kit. The present invention further relates to semi-automatic sterile tissue processing methods.

In some embodiments, bags such as collection bags may be used in the collection kit. A bag with an open end allows adding samples, such as tissue samples. The connector in the collection kit can couple the bag to the catheter. The catheter material can be sealable and/or weldable. For example, the catheter can be sealed using energy (such as heat, radio frequency, etc.). The catheter material can be made of PVA.

In some embodiments, the catheter can be coupled to a valve to allow the addition of one or more media enzyme solutions, including but not limited to collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase HI, pepsin, or Its mixture. For example, the valve may be a needleless valve.

The catheter used in the cryopreservation kit may include a catheter having an outer diameter in the range of about 3.0 mm to about 5.0 mm, wherein the inner diameter of the catheter is in a range of about 2.0 mm to about 4 mm. In particular, the catheter may have an outer diameter of 4.1 +/- 0.1 mm and an inner diameter of about 3.0 +/- 0.1 mm. The length of the catheter depends on the configuration of the collection set. For example, one embodiment of the collection kit may include a catheter with a length ranging from about 10 cm to about 20 cm.

In some embodiments of the collection kit, the prototype may include one or more clamps to prevent and/or prevent tissue and/or enzyme culture from moving. Specifically, it prevents the enzyme culture medium and/or tissue from moving into the filter before the filtration step

The present invention is further described by the following numbered paragraphs:

1. A single-use sterile kit, which includes: a disaggregation module for receiving and processing materials containing solid mammalian tissues; for filtering the disaggregated solid tissue materials and separating undisaggregated tissues and filtrate depending on the situation The enrichment module; and the stabilization module for further processing and/or storage of the depolymerized product material as appropriate, wherein each of the modules includes one or more flexible containers, the one One or more flexible containers are connected by one or more pipes adapted to enable tissue material to flow therebetween; and each of these modules includes one or more ports to permit the medium and/ Or the reagent is aseptically input into one or more flexible containers.

2. The single-use sterile kit in paragraph 1, wherein one or more flexible containers contain elastic deformable materials.

3. The single-use sterile kit according to paragraph 1 or 2, wherein one or more flexible containers of the depolymerization module include one or more sealable openings.

4. The single-use sterile kit in paragraph 3, wherein the flexible container of the depolymerization module contains a heat-sealable fusion port.

5. As in the single-use sterile kit of any of the preceding paragraphs, one or more of the flexible containers include internal rounded edges.

6. The single-use sterile kit of any of the preceding paragraphs, wherein one or more of the flexible containers of the deaggregation module includes a deaggregated surface adapted to mechanically squeeze and shear the solid tissue therein.

7. The single-use sterile kit of any of the preceding paragraphs, wherein one or more of the flexible containers of the enrichment module contains a filter that retains the retentate of the cellularized and depolymerized solid tissue.

8. The single-use sterile kit of any of the preceding paragraphs, wherein one or more of the flexible containers of the stabilization module contains a medium formulation for storing living cells in a solution form or in a cryopreservation state.

9. As the single-use sterile kit of any of the preceding paragraphs, the kit further contains digital, electronic or electromagnetic label indicators.

10. As in the single-use sterile kit in paragraph 9, where the label indicator relates to a specific program, its definition: a type of depolymerization and/or enrichment and/or stabilization process; used in their process One or more types of media; including optional freezing solutions suitable for controlled rate freezing.

11. As in the single-use sterile kit of any of the preceding paragraphs, the same flexible container can form part of one or more depolymerization modules, stabilization modules and optional enrichment modules as appropriate.

12. The single-use sterile kit of any of the preceding paragraphs, wherein the disaggregation module includes a first flexible container for receiving the tissue to be processed.

13. The single-use sterile kit of any of the preceding paragraphs, wherein the disaggregation module comprises a second flexible container, which contains a medium for disaggregation.

14. As in the single-use sterile kit of any of the preceding paragraphs, the optional enrichment module includes a first flexible container and a third flexible container for receiving enriched filtrate.

15. The single-use sterile kit of any of the preceding paragraphs, wherein the depolymerization module and the stabilization module both include a second flexible container and wherein the second container includes a digestion medium and a stabilization medium.

16. The single-use sterile kit of any of the preceding paragraphs, wherein the stabilization module includes a fourth flexible container, which includes a stabilization medium.

17. As in the single-use sterile kit of any of the preceding paragraphs, the stabilization module also includes the first flexible container and/or the third flexible container for storage and/or cryopreservation.

18. The use of a single-use sterile kit in a semi-automated process according to any of the preceding paragraphs, which is used for aseptic disaggregation, stabilization, and enrichment of mammalian cells or cell aggregates as appropriate.

19. An automated device for semi-automated aseptic depolymerization and/or enrichment and/or stabilization of cells or cell aggregates from mammalian solid tissues, comprising: a programmable processor; and as in paragraphs 1 to Any one of 17 single-use sterile kits.

20. The automated device of paragraph 19, which further includes a radio frequency identification tag reader for identifying the single-use kit.

21. The automated device of paragraph 19 or 20, wherein the programmable processor can identify the single-use sterile kit through the label and then execute the definition of the type of deaggregation, enrichment and stabilization processes and the respective processes required for them Set program for the type of medium.

22. The automated device of any preceding paragraph, wherein the programmable processor is adapted to communicate with and control one or more of the following: de-aggregation module; enrichment module; and stabilization module Group.

23. The automated device of paragraph 22, wherein the programmable processor controls the disaggregation module to enable physical and/or biological decomposition of solid tissue materials.

24. The automated device of paragraph 23, wherein the programmable processor controls the depolymerization module to enable physical and enzymatic decomposition of solid tissue materials.

25. The automated device of paragraph 24, wherein the enzymatic decomposition of the solid tissue material is performed by one or more medium enzyme solutions selected from the group consisting of collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase HI , Pepsin or mixtures thereof.

26. An automated device as in any of paragraphs 19-25, wherein the programmable processor controls the depolymerization surface in the flexible container, which mechanically squeezes and shears the solid tissue, and depolymerizes it as appropriate The surface is a mechanical piston.

27. The automation device of any one of paragraphs 19-25, wherein the programmable processor controls the stabilization module to use the enriched and deaggregated solid tissue in the programmable temperature cryogenic storage container as appropriate.

28. The automated device of any of the preceding paragraphs, wherein the device further includes one or more of the following additional components in any combination: capable of identifying the disaggregation process before transferring the disaggregated solid tissue to the optional enrichment module A sensor that has been completed in the depolymerization module; measures the amount of medium required in the container of one or more of the depolymerization module, enrichment module and/or stabilization module, and controls the material in each container A weight sensor for the transfer between the two; a sensor that controls the temperature in the container of one or more of the depolymerization module, the enrichment module and/or the stabilization module; each container that controls the culture medium in the module At least one bubble sensor for the transfer between the input and output ports; at least one pump that controls the transfer of the culture medium between the input and output ports, as the case may be, a peristaltic pump; pressure sensing to evaluate the pressure in the enrichment module One or more valves that control the tangential flow filtration process in the enrichment module; and/or one or more clamps that control the transfer of the culture medium between the input and output ports of each module.

29. The automated device of any of the preceding paragraphs, wherein the programmable processor is adapted to maintain the optimal storage temperature range in the stabilization module until the container is removed; or to perform a controlled freezing step.

30. The automated device of any of the preceding paragraphs, which further includes a user interface.

31. The automation device of paragraph 23, wherein the interface includes a display screen for displaying commands that guide the user to input parameters, confirm pre-programmed steps, warn of errors, or combinations thereof.

32. The automation device of any of the preceding paragraphs, wherein the automation device is adapted to be movable.

33. A semi-automatic aseptic tissue processing method, which includes: as appropriate, according to the set of any one of paragraphs 1 to 17, automatically determining the aseptic disaggregated tissue processing from the digital, electronic or electromagnetic label indicator related to the aseptic processing set Steps and related conditions; place the tissue sample in the flexible plastic container of the disaggregation module of the aseptic processing kit; and process the tissue sample by communicating with the following and controlling the following to automatically perform one or more tissue processing steps: Disaggregation module; enrichment module selected according to the situation; and stabilization module.

Procedures for collecting tumor materials, cryopreservation and TIL manufacturing

The starting material for TIL production is a depolymerized and cryopreserved cell suspension, which contains autologous TIL and tumor cells from qualified patients. An exemplary flow chart for collecting and processing tumor starting materials is provided (Figure 65).

The tumor is surgically removed and then trimmed to remove visible necrotic tissue, visible healthy (non-cancerous) tissue, adipose tissue, and excess blood. The weight of the trimmed tumor should be greater than or equal to 2 grams (≥2 grams). Tumors weighing more than 7 g can be divided into smaller parts and disaggregated individually.

Put each tumor fragment into a separate sterile bag containing culture medium, collagenase and DNase. Exemplary reagents are shown in the table below: Table 4-Disaggregation Medium Raw materials Animal/human origin supplier Available credentials Phosphate buffered saline no Life Technologies Co., Ltd. CoA 2 mM calcium chloride no Sigma-Aldrich CoA DNase 1 (deoxyribozyme alpha) U.S. approved medical products Roche Products Co., Ltd. CoA Type IV collagenase Cattle Nordmark Arzneimittel GmbH & Co. KG CoA, CoO, TSE/BSE statement BloodStor 55-5 (55% DMSO) no BioLife Solutions CoA

The bag is then heat sealed and its contents are depolymerized to produce a homogeneous cell suspension containing tumor and TIL. Depolymerization is performed by a device, such as the Tiss-U-Stor device described herein, which runs a program to use a defined compression pressure for a defined duration to deliver a defined number of repeated physical compression events to ensure that the enzyme Enter the tumor tissue, thereby speeding up the enzymatic digestion. Record the number of cycles, pressure, temperature and duration of each individual tumor.

The homogenized cell suspension was then aseptically filtered using a 200 μm filter (Baxter, RMC2159), and the filtrate was aseptically transferred to a cryopreservation bag. Aseptically add BloodStor 55-5 (Biolife Solutions, Bothell, WA) to achieve 5% DMSO. Subsequently, the Tiss-U-Stor device was used to cryopreserve the cell suspension with a defined cooling program, and the measured temperature profile of each individual cell suspension derived from each tumor part was recorded. The cryopreserved cell suspension is stored in the gas phase of liquid nitrogen.

The recommended storage conditions for cryopreserved cell suspensions are ≤-130℃.

The cell suspension is packaged in a container and transported from the clinical site to the GMP cell therapy manufacturing site by a qualified express service. The container is verified to ensure that the cryopreserved cell suspension is maintained at ≤ -130°C.

(Tiss-u-Stor)

Evaluate the weight and condition of the resected tumor. For each tumor segment, the extra material is removed and the segment is weighed. Open the CS50N bag, add up to about 7 g of tumor and then seal the bag. 15 ml of EDM digestion medium was added to the bag with 2 μl gentamicin/amphotericin/ml EDM via the needle-free port by a syringe, and then the air was removed from the bag into the syringe.

Place the tumor tissue and the disaggregation medium in the disaggregation bag in the temperature-controlled tissue disaggregator. The temperature was increased from ambient temperature to 35°C at a rate of 1.5°C/min and maintained at 35°C for a total of about 45 minutes, during which the depolymerizer was active at 240 cycles/min.

After de-aggregation, the tumor material is filtered through an in-line filter into a secondary freezing bag. 1.5 ml Blood stor (DMSO) was injected through the needle-free port and the air was removed.

Withdraw 2 ml of suspension for testing.

For the optional low-temperature storage, the low-temperature bag is loaded into a freezing cassette and the freezing cassette is placed in Via freeze. Then Via freeze is cooled to -80°C, preferably directly from 35°C to -80°C at a rate of -2°C/min.

The frozen cryogenic bag is then transferred to liquid nitrogen storage.

TIL manufacturing

Autologous tissues used for culture in the United Kingdom (UK) should comply with the HTA-GD-20 established by the Human Tissue Authority, the guidelines for the quality and safety of human tissues and cells used for patient treatment (Guide to Quality and Safety Assurance for Human Tissue and Cells for Patient Treatment), and have appropriate consent, identity chain, chain of custody and screening to confirm that the donor is hepatitis B virus, hepatitis C virus, HIV-1 and 2, HTLV-1 and 2 and syphilis were negative.

The manufacture involves overgrowth and expansion from cryopreserved cell suspensions containing TIL and tumor cells derived from excised tumors. If the tumor is larger than about 7 g, the resection process produces multiple cryopreserved cell suspensions, each of which is derived from 2 to 7 g tumor fragments. Generally, one TIL overgrowth only needs to thaw one cell suspension, while the remaining cryopreserved cell suspension is kept under GMP control and kept under the recommended storage conditions (liquid nitrogen gas phase).

In certain embodiments, the cell suspension has been filtered after depolymerization and before cryopreservation. An exemplary manufacturing procedure is shown in FIG. 66. Exemplary manufacturing raw materials are provided in the following table: Table 5-Source of raw materials Raw materials Human/animal origin supplier Available credentials T cell culture medium Humans and animals ThermoFisher Scientific CoA, CoO Fetal Bovine Serum (FBS) animal Life Technologies CoA, CoO Gentamicin/Amphotericin B, 500× no Life Technologies CoA IL-2 (Aldesleukin) unavailable Clinigen CoA Human AB serum Humanity Valley Biomedical CoA and Origin MACS GMP CD3 OKT3 antibody no Miltenyi Biotec CoA Irradiated leukocyte layer Humanity SNBTS CoA Phosphate buffered saline no Life Technologies CoA Albumin (human) 20% Humanity OctaPharma CoA and Origin CryoSure-DMSO no WAK-Chemie Medical Co., Ltd. CoA, TSE

T cell culture medium (TCM) contains albumin (human), human Holo Transferrin and animal-derived cholesterol. The source plasma used to manufacture albumin and transferrin comes from the United States and test donors for adventitious agents.

Cholesterol is derived from sheep lanolin from Australia/New Zealand, which meets the USDA regulations prohibiting ruminant raw materials from countries with reported cases of infectious spongiform encephalopathy (TSE).

Fetal Bovine Serum ( FBS) is sourced from Australia/New Zealand, which complies with USDA regulations prohibiting ruminant raw materials from countries with reported cases of infectious spongiform encephalopathy (TSE). FBS is tested in accordance with 21 CFR Part 113.47, specifically including: bluetongue virus, bovine adenovirus, bovine parvovirus, bovine respiratory fusion virus, bovine viral diarrhea virus, rabies virus, Rio virus (reovirus), cytopathic factor , Haemadsorbing agent (haemadsorbing agent). FBS was heat-inactivated at 56°C for 30 minutes and 0.1 μm filtered three times to provide two orthogonal virus removal steps.

Human AB serum comes from Valley Biomedical, an FDA registered agency (1121958). Test the hepatitis B surface antigen (HBsAg), hepatitis B virus (HBV) nucleic acid amplification test (NAT), type 1 and type 2 anti-human immunodeficiency virus (HIV), HIV-1 NAT, anti-hepatitis C virus (HCV), HCV NAT and syphilis test. Heat inactivated serum at 56°C for 30 minutes and filter with 0.1 μm.

The source, preparation, shipment and storage of the irradiated white blood cell layer: The Scottish National Blood Transfusion Service (SNBTS) screens donors, collects blood components, prepares and irradiates the white blood cell layer. SNBTS is authorized by the British Human Tissue Authority (license number 11018) in accordance with the Blood, Safety and Quality Regulations (2005) to obtain, process, test, store and distribute blood, blood components and tissues.

Healthy donor screening meets or exceeds the requirements described in Title 21 of the Code of Federal Regulations (CFR), Part 21, 1271.75, except that the donor lives in the UK. Although this presents a theoretical risk of sporadic Creutzfeldt-Jakob Disease (sCJD) or variant Creutzfeldt-Jakob Disease (vCJD), the UK has a robust national surveillance program. The most recent annual report (covering from May 1990 to December 31, 2018) (National CJD Research & Surveillance Unit, 2018) confirmed that the incidence of sCJD in the UK is comparable to that in other parts of the world (including no bovine spongiform encephalopathy (BSE)). ) Of the country) is equivalent. There were no reported cases of vCJD from 2017 to April/May 2020, and only two cases can be identified nationwide since January 1, 2012 (NCJDRSU Monthly Report, 2020). In the absence of reports since 2007, this strict surveillance network has ruled out vCJD infection by infusion transmission (National CJD Research & Surveillance Unit, 2018). Exemplary qualified donor tests (Table 7) meet 21 CFR Part 1271.85 requirements and add unnecessary hepatitis E tests. Table 6-Exemplary Donor Screening (NHSBT) Pathogen illustrate Require Hepatitis B, Hepatitis C and Hepatitis E virus Not detected/negative Every supply Human immunodeficiency virus (HIV) type 1 and type 2 Not detected/negative Every supply syphilis Not detected/negative Every supply Type 1 and Type 2 Human T Lymphotrophic Virus (HTLV) Not detected/negative The first supply and selection of subsequent supplies malaria Not detected/negative Test according to the individual circumstances of the donor Trypanosoma cruzi ( T. cruzi ) Not detected/negative or IgG positive West Nile virus Not detected/negative Cytomegalovirus (CMV) Not detected/negative or IgG positive

Authorized blood institutions to prepare clinical-grade irradiated white blood cell layers suitable for the treatment of patients with severe neutrophilic leukemia. To prepare the white blood cell layer, the blood is centrifuged to form three layers: a red blood cell layer, a white blood cell layer, and a plasma layer. The white blood cell layer from 10 donors was irradiated with 25 to 50 Gy to suppress cell growth. Prepare the clinical grade irradiated white blood cell layer and use a controlled temperature transporter including a temperature monitor to be transported by overnight express delivery to a GMP manufacturing facility. Shipment occurs the day before use during the manufacturing process.

After receiving, the white blood cell layer is kept at 15 to 30°C until used in manufacturing.

Preparation of irradiated feeder cells

The white blood cell layers from up to ten unique donors are mixed and then centrifuged by Ficoll gradient density centrifugation to collect peripheral blood mononuclear cells (PBMC). Approximately 4×10 ⁹ live leukocytes were resuspended in a closed static cell culture bag supplemented with approximately 8% human AB serum, 3000 IU/mL IL-2 and 30 ng OKT-3 in TCM. PBMC is released according to specifications. Table 7-Specifications of allogeneic PBMC stock solutions characteristic Test Methods Acceptance criteria Exterior Visual inspection ID tag Attributes Flow cytometry ≥85% live CD45+ cells Survival rate Flow cytometry Report results Total live white blood cell content Flow cytometry 2 to 4 ×10 ⁹

The sterility of PBMC and mycoplasma are also tested. Immediately before starting step 3 (day 12, panel C), remove the sample of formulated feeder cells, which includes medium, IL-2 and OKT3. Incubate and analyze this sample on the 13th, 17th, and 18th days to confirm that the feeder cells have not expanded.

Albumin (human), also known as human serum albumin (HSA), comes from a US donor. All plasma supplies were tested individually and did not respond to HBsAg, anti-HIV 1, anti-HIV 2 and anti-HCV antibodies. Each plasma pool was tested by NAT and found to be negative for HBsAg, anti-HIV 1, anti-HIV 2 and HCV-RNA. Manufacture HSA products according to GMP regulations that meet the production and testing standards of the American and European Pharmacopoeias.

TIL overgrowth

The cell suspension was inoculated at approximately 0.25×10 ⁶ to 0.75×10 ⁶ viable cells/ml to be supplemented with 10% FBS, 0.25 μg/mL amphotericin B, and 10 μg/mL gentamicin (Life Technologies, Grand Island , NY) and Interleukin-2 (IL-2; Aldesleukin) 3000 IU/mL (Clinigen, Nürnberg, Germany) in TCM and in standard cell culture conditions (37°C, 5% CO ₂ ) nourish.

On day 5, half of the medium was removed and replaced with TCM supplemented with 10% FBS, 0.50 μg/mL amphotericin B, 20 μg/mL gentamicin, and 6000 IU/mL IL-2.

On the 7th day, if the cell concentration is> 1.5×10 ⁶ viable cells/ml, the TIL overgrowth culture is diluted in a three-fold volume to maintain approximately 0.1×10 ⁶ to 2.0×10 ⁶ viable cells/ml. If the cell concentration is less than or equal to 1.5×10 ⁶ live cells/ml, replace half of the medium. In either option, the medium is TCM supplemented with 10% FBS, 0.50 μg/mL Amphotericin B, 20 μg/mL Gentamicin, and 6000 IU/mL IL-2.

On the 10th day, if the cell concentration is> 1.5×10 ⁶ viable cells/ml, the TIL overgrowth culture is diluted in a three-fold volume to maintain approximately 0.1×10 ⁶ to 2.0×10 ⁶ viable cells/ml. If the cell concentration is less than or equal to 1.5×10 ⁶ live cells/ml, replace half of the medium. In either option, the added medium is TCM supplemented with 10% FBS, 0.50 μg/mL amphotericin B, 20 μg/mL gentamicin and 6000 IU/mL IL-2.

TIL activation

Anti-CD3 antibody (OKT3) is used to provide CD3-specific stimulation to activate TIL when binding to FC receptors of irradiated feeder cells from allogeneic peripheral blood mononuclear cells (PBMC). Feeder cells provide a natural source of additional costimulation to support the added anti-CD3 (OKT-3).

On the 12th day, use about 30±10 ng/mL OKT3, about 8% human AB serum and 3000±1000 IU/mL IL-2 to add 1 to 20×10 ⁶ live T cells from TIL overgrowth step 2 Up to 2.0 to 4.0×10 ⁹ live irradiated feeder cells (section 8.1.4.4). The TIL activated cultures were incubated for 6 days under standard cell culture conditions.

TIL amplification

On day 18, activated TIL was expanded by aseptically adding the activated TIL cell suspension to a bioreactor containing T cell culture medium supplemented with approximately 8% human AB serum and 3000 IU/mL IL-2.

On day 19, TIL expansion was provided with a continuous feed of T cell culture medium supplemented with 3000 IU/mL IL-2 until collection.

TIL was collected by washing the cells with SEFIATM. The cells were concentrated by centrifugation, and then washed 2 to 4 times with phosphate buffered saline (PBS) supplemented with 1% human serum albumin (HSA). The cells are then resuspended in PBS+1% HSA to approximately 50 to 60 mL.

The washed and concentrated cells are aseptically transferred to a cryogenic bag, and a part is removed for batch release testing and sample retention. A cryoprotectant is added to obtain a formulation ^{of ≥5×10 9} living cells suspended in a PBS solution of approximately 10% DMSO and 8.5% HSA. Remove a part for batch release testing and sample retention. Cool the cryobag to -80°C.

TIL manufacturing process

The following table shows examples of process variations. Table 8-Manufacturing Process Process version v1.0 v1.1 v1.2 ITIL-168 Tumor disaggregation Artificial disaggregation Artificial disaggregation Tiss-U-Stor depolymerization Tiss-U-Stor depolymerization Starting material Fresh Cryopreservation Cryopreservation Cryopreservation TIL overgrowth 1-3 weeks 1-3 weeks 12 days 12 days Intermediate hold step Cryopreservation Cryopreservation not applicable not applicable TIL recycling 3 days 3 days not applicable not applicable Rapid amplification stage 12 days 12 days 12 days 12 days Cultivation extension 0-2 days 0-2 days not applicable not applicable final product Fresh Fresh Cryopreservation Cryopreservation

The following table shows drug information Table 8-Drug Information Product batch Process version Output (×10 ¹⁰ ) Survival rate Percentage of CD3+ cells TIL001 1.0 1.1 82 N/A TIL003 1.0 2.2 94 98 TIL005 1.0 2.0 96 N/A TIL012 1.0 3.2 95 98 TIL013 1.0 2.1 80 92 TIL014 1.0 4.4 91 95 TIL015 1.0 6.4 91 97 TIL016 1.0 5.5 93 96 TIL027 1.0 3.8 95 97 TIL032 1.0 3.7 92 99 TIL035 1.0 6.4 96 90 TIL037 1.0 2.6 92 97 TIL038 1.0 1.3 83 98 TIL039 1.1 1.2 80 93 TIL040 1.0 5.3 93 97 TIL041 1.0 3.2 93 98 TIL043 1.0 4.8 93 98 TIL054 1.1 0.82 86 91 TIL065 1.1 3.4 94 97 TIL067 1.2 3.0 91 97 TIL073 1.0 5.4 92 98 TIL077 1.2 1.0 91 97 TIL078 1.2 3.4 99 98 E2 1.2 3.5 86 97 E3 1.2 1.8 80 96 E4 1.2 1.0 88 93 E5 1.2 4.1 98 100

Comparing cryopreservation with fresh cell suspensions, the representative yield is constant as indicated by the yield of similar APIs (Figure 67A), survival rate (Figure 67B) and T cell percentage (Figure 67C).

Optimization of cryopreservation-as a substitute for tumor materials, the separated PBMC uses the Tiss-U-Stor process and material digestion. Commercial cryopreservation reagents (CPA) were evaluated under a range of conditions to determine which reagent maximizes survival after thawing (Figure 68). The survival rates of the two CPAs, Cryostor10 and Stem Cell Banker without DMSO after thawing are similar. Subsequently, CryoStor-based DMSO was compared with Bloodstor 55-5 (a DMSO based on cryopreservation), and a higher concentration of BloodStor product was selected because it is more concentrated, thus allowing smaller cryogenic bags. Subsequently, the material is maintained at 4°C for 10 minutes, and then the temperature is reduced at a rate of -1°C/min or directly reduced from 35°C to -80°C at a rate of -2°C/min, which is relatively low temperature storage. The survival rates after thawing were similar between the two cryopreservation protocols used (Figure 69).

During the cooling period, the ice grows to release heat. Excessive cooling, a phenomenon in which the released heat appears to raise the temperature of the solution, is related to the lower recovery rate after thawing. During cryopreservation using the two schemes, self-test items record temperature data (Figure 70). Using the -1°C/min scheme, overcooling was observed in two independent operations, while the -2°C/min cooling scheme was not recorded for one overcooling event, and in the second independent operation, the overcooling event was observed relative to the alternative The program releases less heat (Figure 70).

The DP stored at low temperature is transferred to the gas phase LN2 for storage and transportation at ≤ -130°C.

The sterility of the test sample and the maintained sample were frozen using Coolcell® (Biocision, Larkspur, CA) at -80°C, and then transferred to the gas phase LN2 for storage purposes.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present invention as defined in the scope of the appended application.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present invention as defined in the scope of the appended application.

The present invention will be further illustrated in the following examples, which are given for illustrative purposes only and are not intended to limit the present invention in any way. Instance Example 1

Figure 39 shows an embodiment of the bag 400 during use. As depicted, the inner bag 400 of the device 404 such as the tray 406 is fastened by a fastening element such as a clamp 402. The tissue 408 is visible through the transparent side of the bag 400. The tube 410 is coupled to the bag 400. Example 2

Figure 40 depicts an embodiment of a bag 420 used in the present invention as described herein. As depicted, the bag 420 is fastened from the device and the tray 424 by the fastening element 422. The tissue material 424 is visible through the transparent side of the bag 420. The tube 426 is coupled to the bag 420. As shown, a fixing element 428 (specifically, adhesive tape) is used to further secure the position of the bag 400 within the tray 406. The tissue 424 is visible through the transparent side of the bag 420. As shown in FIG. 40, the bag may include a port 430 for access to the interior of the bag and/or tissue 424. Example 3- Depolymerization and cryopreservation

TIL075 is made from metastatic tumor mass (sample). The tumor samples were weighed and processed as follows. S1=1.4 g. S1 is disaggregated by automated procedures. S2=19.4 g. Separate S2, one part (approximately 7.7 g) is depolymerized by an automated procedure and the second part (approximately 12 g) is manually depolymerized.

Artificial disaggregation: Cut the tumor sample into smaller 2 to 4 mm ³ pieces and add them to a bottle of 80 ml digestion medium containing antibiotics. The bottle was placed on a shaker and depolymerized at 37°C overnight (approximately 14 hours). The digest was then filtered through netwells and a 100 μM cell filter into Falcon 50 tubes. Retain 10% of the filtered digestion for sterility testing. The remaining part was centrifuged and resuspended in 12 ml CS10 and divided into 12 frozen vials.

Tiss-U-Stor depolymerization: Open two CS50N bags with sterile scissors and cut the ends without ports. Place the S1 1.4 gm sample and the 7.7 gm portion of S2 in the CS50N bag, and seal the bag. Combine 15 ml of depolymerization medium with 30 μl of antibiotics and use a syringe to add to each of the sealed bags via the needle-free port of the bag. Transfer the bag to the tissue disaggregator loaded in ViaFreeze, and start the disaggregation protocol. The depolymerization scheme needs to increase from the ambient temperature to 35°C at a rate of 1.5°C/min, and the temperature is maintained at 35°C, while the depolymerizer is active. The deagglomerator rate was set to 240 cycles/minute. Afterwards, the temperature of ViaFreeze was kept at 35°C until the low-temperature storage step.

The bag set-up includes direct connection to the secondary cryogenic bag via a conduit through a line filter. Filter the depolymerized material in the CS50 bag into a cryogenic bag and seal the conduit connection. Slowly add 1.5 ml Blood-stor (DMSO) through the needle-free port of the cryobag, place the bag in a cassette designed for optimal heat transfer, and place the cassette back into ViaFreeze instead of the deagglomerator.

Carry out a low-temperature storage program after depolymerization. The refrigeration cycle gradually changes the ViaFreeze temperature from 35°C to -80°C at -2°C/min. Transfer the freezer bag to liquid nitrogen storage. Example 4- Depolymerization and cryopreservation

TIL077 is made from metastatic melanoma tumor mass (sample). The tumor samples were weighed and processed as follows. S1=4.6 g. S2=4.6 g.

Tiss-U-Stor depolymerization: Open two CS50N bags with sterile scissors and cut the ends without ports. Place the S1=4.6 gm sample and the S2=4.6 gm sample in the CS50N bag and seal the bag. Combine 15 ml of depolymerization medium with 30 μl of antibiotics and use a syringe to add to each of the sealed bags via the needle-free port of the bag. Transfer the bag to the tissue disaggregator loaded in ViaFreeze, and start the disaggregation protocol. The depolymerization scheme needs to increase from the ambient temperature to 35°C at a rate of 1.5°C/min, and the temperature is maintained at 35°C, while the depolymerizer is active. The deagglomerator rate was set to 240 cycles/minute. Afterwards, the temperature of ViaFreeze was kept at 35°C until the low-temperature storage step. Figure 71 shows the disaggregation record.

The bag set-up includes direct connection to the secondary cryogenic bag via a conduit through a line filter. Filter the depolymerized material in the CS50 bag into a cryogenic bag and seal the conduit connection. Slowly add 1.5 ml Blood-stor (DMSO) through the needle-free port of the cryobag, place the bag in a cassette designed for optimal heat transfer, and place the cassette back into ViaFreeze instead of the deagglomerator.

Carry out a low-temperature storage program after depolymerization. The refrigeration cycle gradually changes the ViaFreeze temperature from 35°C to -80°C at -2°C/min. Figure 71 shows the record of cryopreservation. Transfer the freezer bag to liquid nitrogen storage. Example 5- Depolymerization and cryopreservation

TIL078 is made from metastatic melanoma tumor mass (sample). The tumor samples were weighed and processed as follows. S1=11 g. S2=2 g.

Tiss-U-Stor depolymerization: Open two CS50N bags with sterile scissors and cut the ends without ports. The tumor material was separated and a 6.4 gm sample was placed in each of the two CS50N bags and the bags were sealed. Combine 15 ml of depolymerization medium with 30 μl of antibiotics and add to each of the sealed bags via the needle-free port of the bag using a syringe. Transfer the bag to the tissue disaggregator loaded in ViaFreeze, and start the disaggregation protocol. The depolymerization scheme needs to increase from the ambient temperature to 35°C at a rate of 1.5°C/min, and the temperature is maintained at 35°C, while the depolymerizer is active. The deagglomerator rate was set to 240 cycles/minute. Afterwards, the temperature of ViaFreeze was kept at 35°C until the low-temperature storage step. Figure 72 shows the record of cryopreservation.

The bag set-up includes direct connection to the secondary cryogenic bag via a conduit through a line filter. Filter the depolymerized material in the CS50 bag into a cryogenic bag and seal the conduit connection. Slowly add 1.5 ml Blood-stor (DMSO) through the needle-free port of the cryobag, place the bag in a cassette designed for optimal heat transfer, and place the cassette back into ViaFreeze instead of the deagglomerator.

Carry out a low-temperature storage program after depolymerization. The refrigeration cycle gradually changes the ViaFreeze temperature from 35°C to -80°C at -2°C/min. Figure 72 shows the record of cryopreservation. Transfer the freezer bag to liquid nitrogen storage. Example 6- Depolymerization and cryopreservation

TIL081 is made from metastatic melanoma tumor mass (sample). Update the software to include depolymerization and cryopreservation in a single solution. Figure 73 shows the records of disaggregation and cryopreservation. As in the previous example, the deaggregator was active for approximately 53 minutes. (Figure 73A, 73B). The depolymerized tissue is transferred from the depolymerized bag to the cryogenic bag via the filter, and returned to ViaFreeze for cryopreservation within about 90 minutes from the start of the depolymerization process (at this time cryogenic cooling starts). Example 7- Manufactured from vials Table 9- Cell cryopreservation and thawing Reagents/ materials Reagent manufacturer Catalog number Medium depending on cell type NA NA DPBS Sigma D8537-500ML 15 mL centrifuge tube VWR 339650 Serological pipette (Stripette) 10mL Corning CLS4101 Serological pipette 25 mL Corning CLS4251 Serological pipette 5 mL Corning CLS4051 Filtered pipette tip 1000 µL (Tips 1000 µL filtered) StarLabs S1182-1730 Trypan Blue Sigma T8154-100ML Table 10- Equipment describe manufacturer Part number Serial number/ asset number Powerpette pro 1-100 mL VWR 452-8344 NA Pipette ErgoOne 100-1000 µL Star Labs S7110-1000 NA Megafuge 40R Centrifuge Hereus 75004518 41536283 Hemocytometer Hawksley HC002 NA Water bath 12 L VWR 462-0557 BP1912001 IncuSafe CO ₂ incubator PHCBI MCO-170AIC-PE NA

The frozen vial was removed from the liquid nitrogen and placed in a 37°C water bath until the cell suspension just melted. The cell suspension was placed in a 15 mL Falcon tube and filled with PBS to 10 mL, and centrifuged at 400 g for 10 minutes. Decant the supernatant.

For cell culture, resuspend the cell aggregates in pre-warmed medium, initially in a small volume (ie, 2 to 3 mL). According to the table below, the adherent cell strain (ie, tumor strain, HEK 293) was added to the tissue flask with culture medium. Spread the non-adherent cell lines (ie T cells, TIL, Jurkat cells) at a density of 0.5 to 1×10 ^{6 cells/ml.} Place the flask in a humid 37°C incubator and change the medium every 2 to 3 days. Table 11- Cell seeding density of adherent cells in different containers Container/flask type Inoculation density Medium volume mL 24 holes 0.1×10 ⁶ 0.5 to 1 6 holes 0.5×10 ⁶ 2 to 4 T 25 0.7×10 ⁶ 4 to 6 T 75 2.1×10 ⁶ 12 to 15 T 150 4.4×10 ⁶ 25 to 30 Example 8- Manufactured by cryopreservation of depolymerized tumors .

Manufacturing process

Thaw the starting material

VIAThaw CB1000 thawing system is used to control the heating of cryopreserved samples stored in freezer bags. The cryopreserved cell suspension was thawed and then diluted in T cell culture medium (TCM) manufactured by Life Technologies (Paisley, United Kingdom). TCM contains 80% Rosewll Park Memorial Institute (RPMI) 1640 medium and 20% AIM V. The cell suspension was filtered through a 70 to 100 μm filter and centrifuged, and the supernatant was removed. The cell aggregates were resuspended in TCM (Life Technologies, Auckland, New Zealand) supplemented with 10% irradiated fetal bovine serum (FBS).

Place the depolymerized cryopreserved tumor (about 16.5 ml) in the Origin CS50 bag in the thawing tray of the VIAThaw CB1000 thawing system and raise the temperature to about 0°C. Example 9- Efficacy

The co-culture-based performance method quantifies the percentage of T cells activated by the target cell line expressing OKT3. The mechanism of action of TIL products in vivo involves the presentation of TIL peptides via pMHC-HLA, which bind to TCR in vivo. Efficacy analysis quantifies the percentage of effective T cells, defined as the total viable T cells that are positive for CD137, IFN-γ, TNFα, or CD107a when they are specifically activated by co-cultivation with the K562 cell line expressing the OCT3 antigen binding domain. T cells. The markers used to quantify the efficacy of T cells include DRAQ7, CD45, CD2, CD107a, CD137, TNF-α and IFN-γ.

To measure performance, ITIL-168 DS cells were co-cultured for about 5 hours with one of the following three cell lines: Condition 1-No stimulation-background cell activity; Condition 2-K562 cell line-background TCR-independent reactivity; Condition 3-K562 cell line expressing ScFv against OKT-3-T cell stimulation induced by TCR.

The cultured cells were analyzed by flow cytometry and gated on live white blood cells to quantitatively express T cells of at least one of the four activation markers. For stability testing, DP cells stored at low temperature were thawed, washed, and left to stand overnight.

ITIL-168 TCR efficiency is calculated as follows: Step 1) The efficiency attributable to non-specific stimulation% is obtained from Condition 2; Step 2) The efficiency attributable to CD3 specific and non-specific stimulation% is obtained from Condition 3; Step 3 ) The% potency due to CD3 specific stimulation is calculated as Condition 3-Condition 2.

For condition 2 and condition 3, the efficiency% is 100% minus the percentage of all T cells in CD137-/IFN-γ-/TNFα-/CD107a- (ie, background). This population does not produce at least one marker. Example 10-TIL overgrowth and rapid expansion

The TIL manufacturing process starts after tumor resection, disaggregation, cryopreservation, and optional packaging and shipment as appropriate. The package is transported in qualified transporters under controlled conditions and transported from the tumor treatment center to the manufacturing facility of Instil. Cryopreserved tumors and T cells are thawed under controlled conditions and diluted in 80% Losvi Park Memorial Institute supplemented with 10% FBS, amphotericin B, gentamicin, vancomycin and IL-2 (RPMI) 1640 medium and 20% AIM V composed of T cell medium (TCM) (herein referred to as ICMT).

The cells were washed by centrifugation in a closed bag, resuspended in ICMT and samples were taken for cell counting. With the goal of 0.25×10 ⁶ viable cells/ml, the cell suspension was inoculated into a culture bag with ICMT, and incubated under controlled conditions until the 8th day of the process. On day 8, a sample for cell counting was obtained and an equal volume of ICMT was added to the culture bag and incubated under controlled conditions. On day 11, the cell count was obtained and an equal volume of ICMT was added to the culture bag and incubated under controlled conditions. On day 13, the cell count was obtained, and the TIL was concentrated by centrifugation in the bag to provide 1×10 ⁶ to 20×10 ⁶ viable T cells.

Also on day 13, use anti-CD3 and irradiated feeder cells (allogeneic PBMC) and TCM containing 8% human AB serum and IL-2 (herein referred to as WTCM) to activate 1×10 ⁶ to 20×10 ⁶ cells Live overgrowth of TIL. The TIL activated cultures are grown under controlled conditions in static culture bags for up to 6 days. On the 19th day of incubation, cell counts were performed and activated TIL was inoculated into a bioreactor containing WTCM. The cells are incubated under controlled conditions for up to 6 days. On the 20th day, continuous feeding of TCM supplemented with IL-2 was provided to the TIL amplification until the collected target dose was achieved before or on the 27th day of the process.

Once the collected dose is reached, the cells are counted, washed and concentrated by centrifugation in phosphate buffered saline (PBS) supplemented with 1% human serum albumin (HSA). The TIL in the drug (DP) bag is then cooled to 2 to 8°C and mixed 1:1 with a cryoprotectant containing 16% HSA and 20% DMSO to obtain the final formulation of DP in PBS containing 8.5% HSA and 10% DMSO Things. Remove the sample volume for batch release testing, reference and backup samples.

Use a predefined program to store the prepared DP in CRF at low temperature until the product reaches the specified temperature. The cryopreserved DP is then transferred to liquid nitrogen storage, and then transported to the clinic at ≤ -130°C for administration. Table 12-Equipment Equipment/Supply Products manufacturer Model or catalog number Leukosep ficoll tube Greiner Bio-One Lrd 227288 PermaLife cell culture bag, 325 ml Origen Biomeical PL325-2G Cell culture expansion bag Charter Medical Co., Ltd. EXP-1L WAVE 10L bag Cytiva 29-1084-43 CT800.1 Sefia set Cytiva 20001 Table 13-Reagents Reagent manufacturer Catalog number batch number Expiration date T cell culture medium Life Technologies 04196658P 2021537 August 31, 2020 γFBS Irradiated Life Technologies 01190005H-RESERVE 2-2YBT2DS 2225231RP May 31, 2024 Proleukin manufacturer vial (IL-2) Clinigen Group PLC Prurigin 801313T December 31, 2020 Aliquot Il-2 stock solution N/A N/A CTU-IL2/02/09/2019 August 31, 2020 Gentamicin/Amphotericin solution (500×) Life Technologies R01510 2217613 March 30, 2021 Vancomycin manufacturer vial Bowmed Ibisqus N/A 90260 February 28, 2021 Vancomycin aliquot (50 mg/ml) N/A N/A CTU-12-06-2020 February 28, 2021 γ-irradiated human AB serum Gemini Bio-Products Co., Ltd. 100-812G H12Y00K September 30, 2020 OKT-3 manufacturer vial (1 g/ml) Miltenyi Biotec Co., Ltd. 170-076-116 6200108211 October 17, 2020 Equally divided OKT-3 N/A N/A CYU-OKT3/05/05/2020 October 17, 2020 20% human serum albumin Nova Biologics 68982-0633-02 M848B6661 November 27, 2021 CryoSure DMSO WAK-Chemie Medical Co., Ltd. WAK-DMSO-50 USP8C1S February 28, 2022 Example 11

Full-scale operation is carried out under GMP conditions. The ITIL-168 process used in these operations includes cryopreserved tumor digestion, TIL overgrowth stage (stage 1) of 0.25×10 ⁶ live cells/ml inoculation target, from TIL overgrowth to TIL rapid expansion stage ( REP) continuous processing and automatic blending of final products and cryopreservation of final medicines.

ITIL-168 is a tumor infiltrating lymphocyte (TIL) therapy for adult patients with advanced melanoma who have relapsed from at least one previous therapy line or are difficult to treat with at least one previous therapy line. ITIL-168 consists of a single infusion of autologous T cells isolated from the patient's cancer tissues and ex vivo expansion and intravenous administration. Process improvements have been identified and implemented over time, and the improved process is called ITIL-168. The table summarizes the variations and implementation of the manufacturing process. Table 14- Overview of Manufacturing Process Development Process steps Unit operation / change MS v1.0 MS v1.1 UTIL-01 ITIL‑168 process Tumor digest preparation Tumor disaggregation Artificial disaggregation in the bottle Artificial disaggregation in the bottle Automatic depolymerization in the bag ( using Tiss-U-Stor device) Automatic depolymerization in the bag (using Tiss-U-Stor device) Tumor digestion preparation Non-cryogenic preservation Non-cryogenic preservation Cryopreservation Cryopreservation TIL overgrowth Culture container for tumor digest Open process in disk Open process in disk Open process in disk The sealing process in the bag Inoculation density 1×10 ⁶ live cells/ml target 1×10 ⁶ live cells/ml target 0.5×10 ⁶ live cells/ml target 0.25×10 ⁶ live cells/ml target Cell count test method Hemocytometer Flow cytometry Flow cytometry Flow cytometry Material Gentamicin and Amphotericin B Gentamicin and Amphotericin B Gentamicin and Amphotericin B Gantamycin, Amphotericin B and Vancomycin Material Heat inactivation, and 0.1 µm filter FBS Heat inactivation, and 0.1 µm filter FBS Heat inactivation, and 0.1 µm filter FBS Heat inactivation, and 0.1 µm filter irradiated FBS TIL REP Material Heat inactivation, and 0.1 µm filter human AB donor Heat inactivation, and 0.1 µm filter human AB donor Heat inactivation, and 0.1 µm filter human AB donor Thermally inactivated , and 0.1 µm filtered irradiated human AB donor TIL overgrowth to REP After TIL overgrowth, cryopreservation, thawing/washing and recycling Low-temperature storage and preservation steps and thawing 1-3 days after recycling Low-temperature storage and preservation steps and thawing 1-3 days after recycling Continuous processing without cryopreservation Continuous processing without cryopreservation Collect to drug formulation drug Haemonetics Cell Saver 5 (manually adjusted to 270 mL) Haemonetics Cell Saver 5 (manually adjusted to 270 mL) Haemonetics Cell Saver 5 (manually adjusted to 270 mL) Cytiva Sefia S-2000 (automatically adjust to 110 mL) Drug blending drug Non-cryogenic preservation Non-cryogenic preservation Cryopreservation Cryopreservation

A summary of the ITIL-168 manufacturing process used in the development and operation of the two processes is shown in Table 15. Two process development operations, labeled Operation 1 (TIL065) and Operation 2 (Biopartners 9251), were carried out on a full scale under GMP conditions and used excess tumors collected from patients and tumors from the supplier Biopartners.

During the development and operation of these two processes, in-process tests for bioburden and final product sterility, endotoxin, mycoplasma and appearance tests are not performed, because these operations are mainly intended to evaluate the efficiency of the manufacturing process and product quality after the process has been improved. , And to act as a manufacturing operator training operation under GMP conditions before the process verification operation. Table 15 -Process flow chart of ITIL-168 manufacturing process Process steps sky Unit operation Process control describe Receive and release Acceptance, inspection and release of cryopreserved tumor digest ● Acceptance, inspection and release of cryopreserved tumor digest ↓ TIL overgrowth 1 Low-temperature preservation of tumor digestion thawing and washing ● Thaw, wash and dilute tumor digests in a medium supplemented with FBS, IL-2 and antimicrobial reagents ↓ 1 TIL overgrowth inoculation → cell counts ● Inoculate washed cells in one or more culture bags in a medium supplemented with FBS, IL-2 and antimicrobial reagents ↓ 1 TIL overgrowth cultivation ● Cultivate the washed cells in one or more culture bags in a medium supplemented with FBS, IL-2 and antimicrobial reagents for up to 12 days ↓ 8 TIL overgrowth medium supplement → cell counts ● TIL continues to expand in the medium supplemented with FBS, IL-2 and antimicrobial reagents ↓ 11 TIL overgrowth medium supplement → cell counts ● TIL continues to expand in the medium supplemented with FBS, IL-2 and antimicrobial reagents ↓ 13 TIL overgrowth enrichment → cell counts ● Concentration of TIL by centrifugation in one or more bags ↓ TIL rapid expansion stage 13 TIL activation → cell counts ● TIL activation with anti-CD3 and irradiated feeder cells in a medium containing human AB serum and IL-2 in one or more culture bags for up to 6 days ● If available, store excess TIL at low temperature ↓ 19 TIL inoculation in bioreactors → cell counts ● TIL inoculated in the bioreactor in the medium supplemented with human AB serum and IL-2 for up to 8 days ↓ 20-27 TIL amplification (perfusion) in a bioreactor → cell counts ● TIL amplification in a bioreactor bag with continuous feed of medium supplemented with IL-2 ↓ collect X ¹ Collection, washing and concentration cell counts ● Wash to reduce impurities and concentrate TIL ↓ Drug blending X ¹ Dispensing drugs → Cell count, dose, survival rate, attributes/purity, potency ● TIL preparation using cryoprotectant ↓ Cryopreservation X ¹ Cryopreservation of drugs → temperature ● Controlled rate freezing of medicines ↓ Release and delivery Drug storage, packaging and transportation → temperature ● Store the product at ≤-130℃ and release it ● Ship to clinical/infusion center

TIL overgrowth and REP were performed as in Example 10 using the materials shown in Table 12 and Table 13.

For the two operations (operation 1 and operation 2), according to the batch manufacturing record (BMR), for the TIL overgrowth stage or stage 1, on day 1, day 8, day 11, and day 13, and for TIL fast In the expansion phase (REP) or phase 2, the total CD3+ cell count was measured on the 13th, 19th, 22nd and 25th days. Figures 76A and 76B show the total CD3+ cell counts of the two operations in the TIL overgrowth phase (phase 1) and TIL REP phase (phase 2), respectively. The data shown in Figure 76B shows that for the two operations, >1×10 ¹⁰ CD3+ cells were achieved at the end of the REP phase, resulting in two that meet the dose acceptance criteria of ^{5×10 9} to 5×10 ^{10 CD3+ cells} batch.

For the two operations on Day 1, Day 8, Day 11, Day 13, and Day 25, the survival rate (percentage of viable CD3+ cells) was also measured. Figure 76C shows the improved survival rate during the manufacturing process and towards the end of the REP phase and both operations meet the final product criterion of >70%.

For the two operations, calculate the rapid expansion stage (REP) expansion multiple from the cell count data. In addition, the final product quality attributes of the two process development operations were also evaluated, such as dosage, survival rate, potency, T cell phenotype, and T cell subset.

The data presented in Table 16 shows that after the process improvement, the ITIL-168 manufacturing process is similar to the historical process and produces the final product quality attributes that meet the specifications. Table 16 - ITIL-168 Manufacturing Process Efficiency and Product Quality Attributes Operation Amplification factor during REP (absolute) Dose ( total live CD3+ cells) Survival rate (%) Performance ¹ (%) Acceptance standard/specification requirements NA 5×10 ⁹ to 5×10 ¹⁰ ≥ 70 ≥ 40 Observed historical range 395-7526 (n=22) 7.90×10 ⁹ to 6.25×10 ¹⁰ (n=23) 80-99 (n=23) History retention in the process tested Operation 1 1350 3×10 ¹⁰ 90 63.2 Operation 2 1700 2×10 ¹⁰ 88 65.2 ¹ Potency is calculated as the frequency of all live CD2+ cells that are positive for one or more of CD137, CD107a, TNF-α and IFN-γ Example 12- Investment

therapy

The subject received a chemotherapy regimen of cyclophosphamide and fludarabine for lymphocytic depletion. Therapies are designed to reduce the influence of suppressor cells such as regulatory T cells and increase the performance of lymphocyte growth-promoting cytokines (eg, IL-7 and IL-15). Start the hydration regimen before and during the lymphocyte depletion chemotherapy. Antimicrobial and antifungal prophylaxis are started before lymphocyte depletion chemotherapy is started. Assess and manage fever and neutropenia. Start non-steroidal antiemetic therapy before lymphocyte depletion chemotherapy and continue as needed.

Lymphocyte depletion chemotherapy is administered as follows. The dosage of cyclophosphamide and fludarabine was calculated based on the weight assessment obtained at the baseline visit. In obese individuals (body mass index> 35), actual body weight is used. The dosage of cyclophosphamide is based on body weight, and the dosage of fludarabine is based on body surface area. The dose can be rounded according to the practice of dose grouping. The following table shows the recommended dosage, route of administration, infusion volume and duration: Table 17- Lymphocyte depletion chemotherapy regimens sky drug dose way Invest in -7 Fludarabine 25 mg/m ² IV In 10-100ml 0.9% NaCl, after about 30 minutes Cyclophosphamide 60 mg/kg IV In 500ml 0.9% NaCl, after about 1 hour -6 Fludarabine 25 mg/m ² IV In 10-100ml 0.9% NaCl, after about 30 minutes Cyclophosphamide 60 mg/kg IV In 500ml 0.9% NaCl, after about 1 hour -5 Fludarabine 25 mg/m ² IV In 10-100ml 0.9% NaCl, after about 30 minutes -4 Fludarabine 25 mg/m ² IV In 10-100ml 0.9% NaCl, after about 30 minutes -3 Fludarabine 25 mg/m ² IV In 10-100ml 0.9% NaCl, after about 30 minutes -2 Withdrawal day -1 Withdrawal day Table 18- Fludarabine Dose Adjustment Creatinine clearance rate (measured by Cockcroft-Gault formula) Fludarabine dosage ＞/= 70 mL/min 25 mg/m ² 51-69 mL/min 20 mg/m ²

The individual was pre-administered with antihistamine and acetaminophen before TIL infusion. The contents of the infusion bag are infused using a non-leukocyte depleting filter (for example, a pipeline/catheter filter of >/=170 microns). Individuals receive up to 8 doses of intravenous IL-2 for post-infusion support. IL-2 was administered after completion of the TIL infusion, starting on day 0 and continuing to day 4. Example 13- Treatment Results

A total of 44 patients with metastatic cutaneous melanoma underwent tumor resection and started TIL overgrowth production (stage 1). Of these 44 patients, 42 individual patients completed stage 1, and 2 failed attempts. Thirty-one patients continued REP manufacturing (Phase 2). A TIL overgrowth stage 1 manufacturing failed and implemented the revised stage 1 manufacturing process, which achieved a successful stage 2 manufacturing. Then treat the patient. The remaining 12 patients were not selected to start REP for the following reasons: 8 patients were not suitable for TIL therapy due to clinical deterioration between patients' status; 2 patients no longer needed TIL due to clinical improvement of other therapies; 1 One patient could not secure the funding for the treatment; and one patient failed due to lack of tumor tissue on the excised sample. Four patients were successfully manufactured, however, these patients were deemed not clinically suitable for TIL therapy and were therefore untreated.

Among the 44 tumors removed, 2 failed to manufacture, achieving a 95% manufacturing success rate. Twenty-seven patients were treated with TIL products prepared using standard manufacturing processes. At the completion of TIL manufacturing, 6 of these patients were deemed clinically unsuitable for the complete treatment regimen and received significantly lower doses of modulated chemotherapy and post-infusion IL-2, and were therefore excluded from the analysis. One patient underwent tumor resection, which did not meet the criteria of the initial standard TIL overgrowth manufacturing step (stage 1). Therefore, the initial modified stage 1 can indeed achieve a rapid expansion scheme (stage 2) and final product deployment, despite the extremely low final cell dose (1.7×10 ⁹ ). Because this product is produced using a modified manufacturing process and produces a low cell dose, it is not considered to represent a licensed MS process, and therefore clinical data is excluded from the analysis.

Collect and analyze the demographic data, baseline patient characteristics, treatment details and treatment, and clinical efficacy and safety results of the remaining 21 patients. As of the analysis cut-off date, these patients have a median possible follow-up time of 52.2 months (range: 4.6, 98.8 months) from the date of TIL infusion.

Among these 21 patients, the majority (71%) were men, and the median age at the time of TIL treatment was 45 years (range: 16, 68). At baseline, all patients had stage IV metastatic cutaneous melanoma, with a median value of 39 months since the original diagnosis of melanoma (range: 8, 177). Most (67%) patients had lesions reported in more than 3 disease sites, including 7 patients (33%) who had brain metastases recorded during TIL treatment. The median number of previous systemic therapies was 2 (range: 1, 9). Fifty-two percent (52%) of patients had BRAF mutations, all of whom had received BRAF inhibitors in the presence or absence of MEK inhibitors and experienced progression under BRAF inhibitors. All but two patients (90%) had received at least one previous checkpoint inhibitor, and 12 (57%) had received a PD-1 inhibitor (nivolumab or peclizumab ( pembrolizumab)). In addition, 8 (38%) received ipilimumab and nivolumab or peclizumab sequentially, and 4 (19%) received both ipilimumab and nivolumab anti. Before tumor removal for TIL production, 20 (95%) had relapsed or refractory progressive melanoma, and 1 (5%) stopped treatment before TIL therapy due to intolerance.

Immediately before receiving TIL, 10 (48%) patients had elevated serum lactose dehydrogenase (LDH) levels, of which 7 (33%) were between 1 and 2 times the upper limit of normal range (ULN) and 3 patients (14%) 2 times higher than ULN. For 20 patients, the baseline tumor burden measured by the total lesion size (SLD) of the target lesion is available; the median baseline SLD is 100 mm (range: 13,281). TIL treatment

All 21 patients received 2 doses of cyclophosphamide and 5 doses of fludarabine as modulating chemotherapy before TIL infusion. The median total number of infused TIL cells was 31.9×10 ⁹ (range: 7.9×10 ⁹ , 62.5×10 ⁹ ). The total number of IL-2 dose medians was 8 (range: 4, 11). The patients stayed in the hospital for a median of 10 days (range: 7, 15). Three (14%) patients were admitted to the ICU during the treatment phase.

Reports clinically significant AEs during the TIL treatment phase. Common AEs (≥10%) reported during the modulating chemotherapy phase included neutropenia (43%) and nausea (19%), and were generally consistent with the side effects profile of these chemotherapy agents.

Common AEs that occur after TIL infusion include thrombocytopenia (62%), fever (57%), rigors (43%), tachycardia (29%), and neutrophil deficiency (29 %), pulmonary edema (24%), vascular leakage (24%), rash (19%), atrial fibrillation (14%), cardiovascular instability (14%), chest infection (14%), and edema (14%) ( Table 19 ). These AEs are consistent with those reported in other TIL trials (Dafni et al., 2019; Rohaan et al., 2018).

Patients who failed in stage 1 of the manufacturing process but were treated with products produced by the modified manufacturing process died on the 6th day after TIL therapy due to the massive tumor burden exacerbated by renal failure, fluid overload, and possible sepsis. Table 19. TIL AE (all treated individuals) after the onset of infusion AE item -n (%) All treated individuals (N=21) Thrombocytopenia 13 (61.9) fever 12 (57.1) Shudder 9 (42.9) Neutrophil deficiency 6 (28.6) Tachycardia 6 (28.6) Pulmonary Edema 5 (23.8) Vascular leakage 5 (23.8) rash 4 (19.0) Atrial fibrillation 3 (14.3) Cardiovascular instability 3 (14.3) Chest infection 3 (14.3) Edema 3 (14.3) confusion 2 (9.5) Hypokalemia 2 (9.5) Low blood pressure 2 (9.5) Neurological deficit 2 (9.5) Kidney damage 2 (9.5) Respiratory tract sepsis 2 (9.5) epilepsy 2 (9.5) septicemia 2 (9.5) Vitiligo 2 (9.5) Weight gain 2 (9.5) Wheezing 2 (9.5) cough 1 (4.8) diarrhea 1 (4.8) language disability 1 (4.8) Implantation syndrome 1 (4.8) Hallucinations 1 (4.8) Lethargy 1 (4.8) PICC tube infection 1 (4.8) Pleural effusion 1 (4.8) Infectious pneumonia (pneumonia) 1 (4.8) Non-infectious pneumonia (pneumonitis) 1 (4.8) Breathing problems 1 (4.8) Rapid breathing 1 (4.8)

Measure the peripheral blood count during the treatment phase. At the beginning of modulating chemotherapy, a tendency to decrease in neutrophils, platelets, lymphocytes, white blood cell counts, and hemoglobin was observed. The blood count and hemoglobin content generally reach their lowest point 1 to 4 days after TIL infusion. The return of blood counts to baseline levels is usually observed approximately 7 days after the TIL infusion date.

Implement recent changes in the manufacturing process to improve robustness and achieve multi-center clinical trials using centralized manufacturing. In this update, the digested tumor material is stored at low temperature to extend its stability. Importantly, in the four patients treated with the products prepared under pre-cryogenic storage, the observed AE profiles were similar to those of other patients treated in the series (Table 20) and were compared with those reported in clinical trials of other TIL products. The AE profiles are generally the same. Table 20. AEs after TIL infusion (individuals treated with Cryo-in products) AE item-n (%) All treated individuals (N=4) Thrombocytopenia 4 (100) fever 2 (50.0) rash 2 (50.0) Shudder 2 (50.0) Low blood pressure 1 (25.0) Kidney damage 1 (25.0) Vascular leakage 1 (25.0) Vitiligo 1 (25.0)

Fifteen of the 21 patients were evaluated for disease by continuous CT and/or MRI scans including radiological measurements of the target lesion. Among these patients, the quantitative response rate (reaction confirmation not required) was 53%, including 2 (13%) patients who achieved CR and 6 (40%) patients who achieved PR (Table 21). Table 21. Summary of the best overall response (Efficacy Evaluable Analysis Set) Evaluable analysis set of efficacy (N=15) Best overall response Complete response (CR) 2 (13.3) 95% CI (Clopper-Pearson method) 1.7, 40.5 Partial Response (PR) 6 (40.0) 95% CI (gram-picfar) 16.3, 67.7 Stable disease (SD) 3 (20.0) 95% CI (gram-picfar) 4.3, 48.1 Progressive disease (PD) 4 (26.7) 95% CI (gram-picfar) 7.8, 55.1 Response rate (CR+PR) 8 (53.3) 95% CI (gram-picfar) 26.6, 78.7 Disease control rate (CR+PR+SD) 11 (73.3) 95% CI (gram-picfar) 44.9, 92.2

Based on both quantitative and qualitative responses, the response rate including all patients was 57%, including 3 (14%) patients who achieved CR and 9 (43%) patients who achieved PR. Two additional patients have developed resistance to the BRAF inhibitor dabrafenib and are undergoing disease progression under therapy before switching to TIL therapy. Stop dabrafenib just before TIL therapy and restart it about 1 to 2 weeks after TIL to prevent the rapid tumor growth that usually accompanies the interruption of dabrafenib. Each of these 2 patients achieved a qualitative response after TIL (1 persistent CR and 1 PR). Two patients subsequently discontinued dabrafenib once they responded after TIL. Because these patients all suffer from diseases that have become refractory to dabrafenib, it is reasonable to conclude that the clinical benefits they experience after TIL are due to TIL rather than the temporary recovery of dabrafenib. Therefore, a sensitivity analysis is performed on the response, including these patients as responders. In this sensitivity analysis, the response rate was 14/21 (67%), including 4 (19%) complete responders and 10 (48%) partial responders (Table 22). Table 22-Summary of the best overall response and sensitivity analysis (all treated individuals) All treated individuals (N=21) Best overall response Complete response (CR) 4 (19.0) 95% CI (gram-picfar) 5.4, 41.9 Partial Response (PR) 10 (47.6) 95% CI (gram-picfar) 25.7, 70.2 Stable disease (SD) 4 (19.0) 95% CI (gram-picfar) 5.4, 41.9 Progressive disease (PD) 3 (14.3) 95% CI (gram-picfar) 3.0, 36.3 Response rate (CR+PR) 14 (66.7) 95% CI (gram-picfar) 43.0, 85.4 Disease control rate (CR+PR+SD) 18 (85.7) 95% CI (gram-picfar) 63.7, 97.0

Responses are generally based on important baseline and disease characteristics, including age, number of disease sites, number of previous treatment lines, previous BRAF inhibitors, previous PD-1 inhibitors, baseline brain metastases, and baseline tumor burden consistent across all subgroups. Notably, among the 4 patients treated with the manufacturing process most similar to ITIL-168, the overall response rate (75%) and CR rate (25%) were consistent with the broader population. Among the 15 patients with quantitative responses based on CT and/or MRI scans, 14 had detailed tumor measurements and the largest percentage of tumor reduction relative to baseline is presented in a waterfall chart (Figure 74). A patient has the best overall response to PD, but no post-treatment target disease variable measurements (measured by observing the progress of new lesions) have been reported and therefore are not shown in the figure.

According to the quantitative response data (N=15), the median progression-free survival (PFS) time was 6.7 months. Among them, 4 patients had sustained responses (2 CR and 2 PR), and there was no follow-up therapy at the end of the analysis. Based on quantitative and qualitative response data (N=21), the median PFS time was 6.7 months, and 5 individuals had sustained responses (3 CR and 2 PR) without any follow-up therapy. The median overall survival (OS) time for all 21 treated patients was 21.3 months (Figure 75A). The median OS time of 15 patients with quantitative response data was 16 months (Figure 75B). However, the median OS time for non-responders (based on quantitative response only, N=8), while the median OS time for non-responders (N=7) was 6.5 months (Figure 75C). Example 14- Genetically modified TIL Table 23 - Reagents and Equipment Reagent manufacturer Catalog number 15 mL polypropylene centrifuge tube Appleton Woods AB031 50 mL polypropylene centrifuge tube Appleton Woods AB028 Dulbecco's Phosphate Buffered Saline Sigma-Aldrich D8537-24X500ML Fetal Bovine Serum (heat inactivated) Sigma-Aldrich F9665-500ML TCM- CT4834/GIBCO CUSTOM P158718 Gibco Penicillin-streptomycin Sigma-Aldrich P0781-100ML TC 6-hole plate StarLab CC7682-7506 Sterile 1.5 mL Eppendorf tube (Eppendorf) StarLab S1615-5510 Non-TC flat bottom 96-well plate Falcon 353072 96-well U chassis Falcon 351177 FACS tube SLS 352063 TC 24 hole plate StarLab CC7682-7524 Microplate for suspension culture, 96-well, bottom F Grenier, Bio-One 655185 T cell TransACT (TM), human Miltenyi 130-111-160 Gentamicin and Amphotericin Invitrogen (ThermoFisher Scientific) 10184583 Aldesleukin IL-2 Novartis PL-00101/0936 Heraeus Megafuge 40R, refrigerated centrifuge Thermo Scientific 75004518 IncuSafe CO2 incubator PHCBI MCO-170AIC-PE NovoCyte 3005 Flow Cytometer System (CE-IVD) Agilent Technologies 2010064D NovoExpress software Agilent Technologies

The frozen vial of tumor digest was removed from the liquid nitrogen storage and thawed in a 37°C water bath until the cell suspension just melted (D1). The cell suspension was transferred to a 15 mL Falcon tube, filled to 10 mL with PBS, centrifuged at 400 g for 5 minutes and the supernatant was decanted.

The cell aggregates were resuspended in pre-warmed appropriate T cell culture medium, and trypan blue was used to count the cells to determine the survival rate. Resuspend the cells at a density of 1×10 ^{6 cells/ml.}

Resuspend the cells to be cultured without activation at 0.5×10 ⁶ cells/ml and place 2 ml (1×10 ⁶ cells) on a 24-well tissue culture dish with IL-2 (3000 IU/mL) In the hole. The cells were cultured in a humidified 37°C incubator until transduction, and IL-2 (3000 IU/mL) was added every 2 to 3 days.

For cells to be transduced on D3 and D4, cell activation occurs on D1. For cells to be transduced on D7 and D8, cell activation occurs on D5.

For TIL activation, 0.5×10 ⁶ cells/ml are placed in a 24-well tissue culture dish with 3000 IU/mL IL-2. Add 10 μL of T cell TransACT (TM) (1:1 ratio) per 1×10 ^{6 cell TIL suspension and incubate the cells in a 37°C incubator for 48 hours}

First day of transduction (D3 or D7)

Collect the cells from the 24-well plate into a 15 mL Falcon tube, fill with 10 mL TCM and spin at 400 g for 5 minutes. The cells were counted using trypan blue ^{and resuspended at 1×10 6} cells/ml.

Use 1×10 ⁵ cells (100 µL)/96-well flat plate wells for each transduction method. For transduction in a 24-well plate, place 1×10 ⁶ cells/well (500 µL). If transducing in a 6-well plate, place 5×10 ⁶ cells/well (2 mL).

Prepare bean virus (MOI5) and IL-2 (3000 IU/mL) by resuspending in TCM to ^{the final value of 100 µl/10 5} cells/condition (or appropriate density and volume for 24-well and 6-well plates) ) The basic mixture. Prepare the basic mixture volume for the number of wells + 1 to account for pipetting losses.

For NT cells (hypothetical (MOCK)), a basic mixture of TCM and IL-2 (3000 IU/mL)/100 µL was prepared in a 96-well flat plate. For 24-well and 6-well plates, resuspend hypothetical T cells in 500 µL and 2 mL with IL-2 (3000 IU/mL), respectively.

Remove the supernatant from the cells in the Eppendorf or 15 mL Falcon tube and resuspend the cells in an appropriate 100 µL base mixture (or 24-well and 6-well plates) ^{for every 1×10 5 cells depending on the conditions} The appropriate density and volume).

Correspondingly, the conditions were appropriately resuspended and cells were transferred to non-TC flat bottom 96-well, 24-well or 6-well plates.

Add 200 μL of PBS to the surrounding wells in the 96-well plate transduction to prevent evaporation.

The cells were incubated overnight in a humidified 37°C incubator.

The second day of transduction (D4 or D8)

Collect the cells by resuspending up and down from a 96-well flat pan and transferring to a 96-well U pan. (Collection from 24-well or 6-well discs is performed in 15 mL Falcon tubes.) Rotate the disc at 400 g for 5 minutes and wash the cells with TCM.

For each transduction method, use 1×10 ⁵ cells (100 µL)/96-well flat plate wells. For transduction in a 24-well plate, place 1×10 ⁶ cells/well (500 µL). If transducing in a 6-well plate, place 5×10 ⁶ cells/well (2 mL).

Prepare bean virus (MOI5) and IL-2 (3000 IU/mL) by resuspending in TCM to ^{the final value of 100 µl/10 5} cells/condition (or appropriate density and volume for 24-well and 6-well plates) ) The basic mixture. Prepare the basic mixture volume for the number of wells + 1 to account for pipetting losses.

For NT cells (hypothetical), prepare a basic mixture of TCM and IL-2 (3000 IU/mL)/100 µL for a 96-well flat plate. For 24-well and 6-well plates, resuspend hypothetical T cells in 500 µL and 2 mL with IL-2 (3000 IU/mL), respectively.

Remove the supernatant from the cells in the Eppendorf or Falcon tube ^{and resuspend the cells in the appropriate 100 µL basic mixture for every 1×10 5} cells depending on the conditions (or the appropriate 24-well and 6-well plates Density and volume).

Correspondingly, the conditions were appropriately resuspended and cells were transferred to non-TC flat bottom 96-well, 24-well or 6-well plates. Add 200 μL of PBS to the surrounding wells in the 96-well plate transduction to prevent evaporation. The cells were incubated overnight in a humidified 37°C incubator.

The next day, the cells were transferred to fresh medium with IL-2 (3000 IU/mL) in a new 96-well round dish, 24-well or 6-well dish, and incubated in a humidified 37°C incubator for 72 hours.

The final volume of the 96-well plate is 200 μL/well; the final volume of the 24-well plate is 2 mL/well; the final volume of the 6-well plate is 5 mL/well. Add IL-2 (3000 IU/mL) every 2 to 3 days.

For D3+D4 transduction at D8 and for D7+D8 transduction at D12, cells are stained for transduction efficiency.

TIL overgrowth

The hypothetical and transduced cells are maintained in a 96-well U-chassis until they are placed in the REP.

For cell maintenance, half of the medium is removed and replaced with fresh TCM and IL-2 (3000 IU/mL) every 2 to 3 days. For 96-well plates, remove and replace 100 μl of medium to a final volume of 200 μL. For 24-well plates, remove and replace 1 mL of medium to a final volume of 2 mL. For 6-well plates, remove and replace 1 mL of medium to a final volume of 2 mL.

REP starts on D13 (12 days of overgrowth).

The present invention is further described by the following numbered paragraphs:

1. A method for isolating a therapeutic population of unmodified tumor infiltrating lymphocytes (UTIL) stored at low temperature, which comprises: (a) aseptically disaggregating tumors resected from individuals to produce disaggregated tumors, in which the tumors are fully disaggregated, So that the cell suspension can be stored at low temperature; (b) on the same day as step (a), cryopreservation depolymerized tumor by cooling or maintaining at low temperature; (c) storing cryopreserved depolymerized tumor as appropriate; (d) by Culture the disaggregated tumor in a cell culture medium containing IL-2 to perform the first expansion to produce a first population of UTIL; (e) by adding additional IL-2, OKT-3 and antigen-presenting cells (APC) The first population of UTIL is cultivated together for second expansion to produce the second population of TIL; and (f) the second population of UTIL is collected and/or cryopreserved.

2. The method of paragraph 1, wherein the depolymerization inclusions understand polymerization, enzymatic depolymerization, or physical and enzymatic depolymerization.

3. The method of paragraph 1 or 2, wherein the cooling is performed at a controlled rate.

4. The method of paragraph 3, wherein the controlled rate freezing is from about -2°C/min to about -60°C.

5. The method of any of paragraphs 1-5, wherein the disaggregated tumor is cellularized.

6. The method of any of paragraphs 1-5, wherein the disaggregated tumor is purified.

7. The method of any of paragraphs 1-6, wherein a single cell suspension is provided after step (a).

8. The method of any one of paragraphs 1-7, wherein the first population of UTIL is about 1 to 20 million UTILs.

9. The method of any one of paragraphs 1-8, wherein step (d) further comprises growing UTIL from the tumor starting material and then rapidly amplifying it in step (e).

10. The method of paragraph 9, wherein step (d) is performed for about two weeks and step (e) is performed for about two weeks.

11. The method according to any one of paragraphs 1-10, wherein step (d) and/or step (e) further comprises adding IL-7, IL-12, IL-15, IL-18, IL-21 or combination.

12. The method of any one of paragraphs 1-11, which further comprises step (g) suspending the second population of UTIL.

13. The method of paragraph 12, wherein the suspension is in buffered saline, human serum albumin, and dimethylsulfoxide (DMSO).

14. The method of any one of paragraphs 1-13, wherein step (f) is cryopreservation and further includes the final step of thawing UTIL.

15. The method of paragraph 14, wherein the thawed UTIL is prepared to be infused in a single dose without further modification.

16. A therapeutic population of cryopreserved unmodified tumor infiltrating lymphocytes (UTIL) obtained by the method as in any one of paragraphs 1 to 15.

17. The therapeutic population of paragraph 16, wherein the population comprises about 5×10 ⁹ to 5×10 ¹⁰ T cells.

18. A cryopreservation bag for the therapeutic population of paragraph 16 or 17.

19. The cryopreservation bag of paragraph 18, which is used for intravenous infusion.

20. A method of treating cancer, which comprises administering the therapeutic population of paragraph 14 or 15 or the cryopreservation bag of paragraph 18 or 19.

21. The method of paragraph 20, wherein the cancer is bladder cancer, breast cancer, cancer caused by human papilloma virus, cervical cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), lung cancer, melanoma, Ovarian cancer, non-small cell lung cancer (NSCLC), kidney cancer or renal cell carcinoma.

The present invention is further described by the following numbered paragraphs:

1. A method for isolating a therapeutic population of unmodified tumor-infiltrating lymphocytes (UTIL) stored at low temperature, which comprises: (a) excising the tumor from an individual; (b) storing the excised tumor in a single-use sterile kit, The sterile kit includes: a depolymerization module for receiving and processing materials containing solid mammalian tissues; an enrichment module for filtering depolymerized solid tissue materials and separating undisaggregated tissues and filtrate; and A stabilization module for further processing and/or storage of depolymerized product materials as appropriate, wherein each of the modules includes one or more flexible containers, the one or more flexible containers by One or more tubing connections adapted to enable tissue material to flow therebetween; and each of the modules therein includes one or more ports to permit aseptic input of culture media and/or reagents to one or more flexible (C) Aseptically disaggregate and resect the tumor in the disaggregation module to produce a disaggregated tumor. If the resected tumor can be stored at low temperature without cell damage, it will be fully disaggregated; (d) In the stabilization module Cryopreservation of the disaggregated tumor; (e) by culturing the disaggregated tumor in a cell culture medium containing IL-2 for the first expansion to produce a first population of UTIL; (f) by adding extra IL-2 , OKT-3 and antigen-presenting cells (APC) cultivate the first population of UTIL together for second expansion to produce the second population of TIL; (g) Collect and/or cryopreserve the second population of UTIL. In some embodiments, step a) is optional.

2. The method of paragraph 1, wherein the depolymerization inclusions understand polymerization, enzymatic depolymerization, or physical and enzymatic depolymerization.

3. The method of paragraph 1 or 2, wherein the disaggregated tumor is cellularized.

4. The method of any of paragraphs 1-3, wherein a single cell suspension is provided after step (c).

5. The method of any one of paragraphs 1-4, wherein the first group of UTILs is about 1 to 20 million UTILs.

6. The method of any one of paragraphs 1-5, wherein step (e) further comprises growing UTIL from the excised tumor starting material followed by rapid expansion in step (f).

7. The method of paragraph 6, wherein step (e) is performed for about two weeks and step (f) is performed for about two weeks.

8. The method of any one of paragraphs 1-7, wherein step (e) and/or step (f) further comprises adding IL-7, IL-12, IL-15, IL-18, IL-21 or combination.

9. The method of any one of paragraphs 1-7, which further comprises step (h) suspending the second population of UTIL.

10. The method of paragraph 9, wherein the suspension is in buffered saline, human serum albumin, and dimethylsulfoxide (DMSO).

11. The method of any one of paragraphs 1-9, wherein step (g) is cryopreservation and further includes the final step of thawing UTIL.

12. The method of paragraph 10, wherein the thawed UTIL is prepared to be infused in a single dose without further modification.

13. A therapeutic population of cryopreserved UTIL obtained by the method of any one of paragraphs 1 to 11.

14. The therapeutic population of paragraph 13, wherein the population contains about 5×10 ⁹ to 5×10 ¹⁰ T cells.

15. A cryopreservation bag for the therapeutic population as described in paragraph 13 or 14.

16. The cryopreservation bag of paragraph 15, which is used for intravenous infusion.

17. A method of treating cancer, which comprises administering the therapeutic population of paragraph 13 or 14 or the cryopreservation bag of paragraph 15 or 16.

18. The method of paragraph 17, wherein the cancer is bladder cancer, breast cancer, cancer caused by human papilloma virus, cervical cancer, head and neck cancer (including head and neck squamous cell carcinoma [HNSCC]), lung cancer, melanoma , Ovarian cancer, non-small cell lung cancer (NSCLC), kidney cancer or renal cell carcinoma.

19. The method of paragraph 1, wherein one or more of the flexible containers of the sterile kit comprises an elastically deformable material.

20. The method of paragraph 1, wherein one or more of the flexible containers of the disaggregation module of the sterile kit includes one or more sealable openings.

21. The method of paragraph 20, wherein the flexible container of the depolymerization module of the sterile kit includes a heat-sealable melting port.

22. The method of paragraph 1, wherein one or more of the flexible containers of the sterile kit includes internal rounded edges.

23. The method of paragraph 1, wherein one or more of the flexible containers of the depolymerization module of the sterile kit includes a depolymerization surface adapted to mechanically squeeze and shear the solid tissue therein.

24. The method of paragraph 1, wherein one or more of the flexible containers of the enrichment module of the sterile kit includes a filter that retains the retentate of the cellularized and deaggregated solid tissue.

25. The method of paragraph 1, wherein one or more of the flexible containers of the stabilization module of the sterile kit contains a medium formulation for storing living cells in a solution form or in a cryopreservation state.

26. The method of paragraph 1, wherein the sterile kit further includes a digital, electronic or electromagnetic tag identifier.

27. The method of paragraph 26, wherein the label identifier of the sterile kit is related to a specific program, and its definition: a type of depolymerization and/or enrichment and/or stabilization process; one of these processes is used Or multiple types of media; including optional freezing solutions suitable for controlled rate freezing.

28. The method of paragraph 1, wherein the same flexible container can form one or more of the de-aggregation module, the stabilization module, and the enrichment module selected according to the situation.

29. The method of paragraph 1, wherein the disaggregation module of the sterile kit includes a first flexible container for receiving the tissue to be processed.

30. The method of paragraph 1, wherein the disaggregation module of the sterile kit comprises a second flexible container which contains a culture medium for disaggregation.

31. The method of paragraph 1, wherein the optional enrichment module of the sterile kit includes a first flexible container and a third flexible container for receiving the enriched filtrate.

32. The method of paragraph 1, wherein the depolymerization module and the stabilization module of the aseptic kit both include a second flexible container and wherein the second container includes a digestion medium and a stabilization medium.

33. The method of paragraph 1, wherein the stabilization module of the sterile kit includes a fourth flexible container, which includes a stabilization medium.

34. The method of paragraph 1, wherein the stabilization module of the sterile kit also includes a first flexible container and/or a third flexible container for storage and/or cryopreservation.

35. A method for isolating a therapeutic population of unmodified tumor-infiltrating lymphocytes (UTIL) stored at low temperature, which comprises: (a) removing tumors from an individual; (b) applying to cells or cell aggregation from solid tissues from mammals The semi-automated aseptic disaggregation and/or enrichment and/or stabilization of the body is stored in an automated device for resection of the tumor. The automated device includes a programmable processor and a single-use sterile kit. The sterile kit includes: A depolymerization module for receiving and processing materials containing solid mammalian tissue; an enrichment module for filtering depolymerized solid tissue materials and separating undepolymerized tissue and filtrate; and for further processing and/ Or stabilization modules for storing depolymerized product materials, where each of the modules includes one or more flexible containers that are adapted to enable tissue materials to be One or more pipe connections flowing therebetween; and each of its modules includes one or more ports to permit aseptic input of culture media and/or reagents into one or more flexible containers; (c) aseptic Disaggregate resection of the tumor to produce depolymerized tumors. If the resected tumor can be stored at a low temperature without cell damage, it will be fully depolymerized; (d) Store the depolymerized tumor at low temperature in the stabilization module; (e) The disaggregated tumor is cultured in a cell culture medium containing IL-2 to perform the first expansion to produce a first population of UTIL; (f) by combining with additional IL-2, OKT-3 and antigen-presenting cells (APC) Cultivating the first population of UTIL for second expansion to produce the second population of TIL; (g) collecting and/or cryopreserving the second population of UTIL. In some embodiments, step a) is optional.

36. The method of paragraph 35, wherein the automated device further includes a radio frequency identification tag reader for identifying the sterile kit.

37. The method of paragraph 36, wherein the programmable processor of the automated device can identify the sterile kit through the label and then execute the definition of the types of deaggregation, enrichment and stabilization processes and the types of the respective media required for these processes Package program.

38. The method of paragraph 35, wherein the programmable processor of the automated device is adapted to communicate with and control one or more of the following: de-aggregation module; enrichment module; and stabilization Module.

39. The method of paragraph 38, wherein the programmable processor of the automated device controls the disaggregation module to enable the physical and/or biological decomposition of the solid tissue material.

40. The method of paragraph 39, wherein the programmable processor controls the depolymerization module to enable physical and enzymatic decomposition of solid tissue materials.

41. The method of paragraph 40, wherein the enzymatic decomposition of the solid tissue material is performed by one or more medium enzyme solutions selected from the group consisting of collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase HI, pepsin and mixtures thereof.

42. The method of paragraph 35, wherein the programmable processor controls the disaggregation surface in the deagglomerated flexible container, which mechanically squeezes and shears the solid tissue, where the disaggregation surface is a mechanical piston as the case may be.

43. The method of paragraph 35, wherein the programmable processor controls the stabilization module to use the enriched and deaggregated solid tissue in the programmable temperature cryopreservation container as appropriate.

44. The method of paragraph 35, wherein the automated device further includes one or more of the following in any combination: capable of identifying whether the depolymerization process has been performed before transferring the depolymerized solid tissue to the optional enrichment module The sensor completed in the disaggregation module; measures the amount of culture medium required in the container of one or more of the disaggregation module, the enrichment module and/or the stabilization module, and controls the amount of material between the respective containers Transfer weight sensor; sensor that controls the temperature in the container of one or more of the deaggregation module, enrichment module and/or stabilization module; controls the input and input of the culture medium in each container in the module At least one bubble sensor for transfer between output ports; at least one pump that controls the transfer of culture medium between input and output ports, peristaltic pumps as appropriate; pressure sensors for evaluating the pressure in the enrichment module; control One or more valves in the tangential flow filtration process in the enrichment module; and/or one or more clamps that control the transfer of the culture medium between the input and output ports of each module.

45. The method of paragraph 35, wherein the programmable processor of the automated device is adapted to maintain the optimal storage temperature range in the stabilization module until the container is removed; or perform a controlled freezing step.

46. The method of paragraph 35, wherein the automated device further includes a user interface.

47. The method of paragraph 46, wherein the interface includes a display screen for displaying commands that guide the user to input parameters, confirm pre-programmed steps, warn of errors, or combinations thereof.

48. The method of paragraph 35, wherein the automated device is adapted to be movable.

49. A semi-automatic sterile tissue processing method for separating the therapeutic population of UTIL, which includes the following steps: (a) Automatically determine the sterile disaggregated tissue processing steps from the digital, electronic or electromagnetic tag identifiers associated with the sterile processing kit And related conditions. The sterile kit includes: a depolymerization module for receiving and processing materials containing solid mammalian tissue; Set modules; and stabilization modules for further processing and/or storage of depolymerized product materials as appropriate, wherein each of the modules includes one or more flexible containers, the one or more The flexible container is connected by one or more pipes adapted to enable tissue material to flow therebetween; and each of the modules in it includes one or more ports to permit the aseptic input of culture medium and/or reagents into a Or multiple flexible containers; (b) excise the tumor from the individual; (c) place the tumor in the flexible plastic container of the disaggregation module of the sterile kit; (d) communicate with the following and control the following Automatically execute one or more tissue processing steps to treat tumors: disaggregation module; in which aseptically disaggregate and resect the tumor to produce disaggregated tumors, among which, if the resected tumor can be stored at low temperature without cell damage, it will be fully disaggregated; optional The enrichment module, in which the depolymerized tumor is filtered to remove the depolymerized solid tissue material and the undepolymerized tissue and filtrate are separated; the stabilization module, in which the depolymerized tumor is stored at low temperature; (e) by including IL- 2. Cultivate the disaggregated tumor in the cell culture medium for the first expansion to produce the first population of UTIL; (f) by culturing UTIL with additional IL-2, OKT-3 and antigen presenting cells (APC) The first population is subjected to the second amplification to produce the second population of TIL; and (g) the second population of UTIL is collected and/or cryopreserved.

The present invention is further described by the following numbered paragraphs:

1. A flexible container for treating tissues, comprising: one or more layers made of sealable polymer, wherein at least three edges of the flexible container are sealed during manufacture; on the flexible container The opening edge, through which tissue material is inserted during use; and one or more connectors, which are configured to couple the flexible container to at least one element via a catheter; wherein the section near the opening edge is in the tissue material After being placed in a flexible container, it is sealed to form a sealed mouth.

2. The flexible container according to paragraph 1, wherein the sealing opening includes an area at least 3 mm wide, which is parallel to the opening edge and spaced apart from the opening edge of the flexible container.

3. The flexible container according to paragraph 1, further comprising a clamp which has a protrusion and is arranged close to the sealing port and is further spaced apart from the opening edge of the flexible container than the sealing port.

4. The flexible container as in paragraph 3, wherein during use, the combination of the sealing port and the clamp is configured to withstand the 100 N force applied to the flexible container.

5. The flexible container as in paragraph 3, wherein during use, the combination of the sealing port and the clamp is configured to withstand the 75 N force applied to the flexible container.

6. The flexible container of paragraph 1, wherein the sealing opening includes an area at least 5 mm wide, which is parallel to the opening edge and spaced apart from the opening edge of the flexible container.

7. The flexible container of paragraph 1, wherein the flexible container is used to depolymerize tissue materials.

8. The flexible container of paragraph 1, wherein the flexible container is used to depolymerize tissue materials, filter depolymerized tissue materials, and separate undisaggregated tissue and filtrate.

9. The flexible container of paragraph 1, which further comprises an elastic deformable material.

10. The flexible container of paragraph 1, which further includes one or more indicators.

11. The flexible container as in paragraph 1, which further contains one or more marks.

12. The flexible container of paragraph 1, wherein the sealing port is formed by a heat sealer operating under a predetermined pressure, a predetermined temperature and a predetermined time range.

13. The flexible container of paragraph 1, wherein the flexible container is configured to be used with a device that mechanically squeezes the tissue material placed in the flexible container.

14. The flexible container of paragraph 1, wherein the flexible container is configured to cut tissue materials.

15. The use of the flexible container according to paragraph 1 in a semi-automated or automated process, which is used for aseptic depolymerization, stabilization, and enrichment of mammalian cells or cell aggregates as appropriate.

16. A system for self-organized extraction of desired materials, comprising: a set, which includes: a depolymerized flexible container; a stabilized flexible container; and a depolymerized flexible container or a stabilized flexible container At least one indicator label on at least one of the containers, which can provide at least one of tissue source, tissue state, or identifier; capable of processing at least some tissue in a depolymerized flexible container to form a solution of the treated fluid Aggregating element; an enriching element capable of accumulating at least some of the treated fluid to form a desired material; a stabilizing element capable of storing part of the desired material in a stabilized flexible container; and a depolymerizing element or stabilizing element At least one indicator tag reader on at least one of the elements can provide at least one of a source of tissue or a state of tissue at the stabilization element.

17. The system of paragraph 15, wherein the desired material comprises tumor infiltrating lymphocytes (TIL).

18. The system of paragraph 15, wherein one or more types of media are used in the process by means of depolymerization elements and stabilization elements.

19. The system of paragraph 15, further comprising a cryopreservation medium for use in a stabilization element capable of freezing at a controlled rate.

20. The system of paragraph 15, wherein the deagglomerated flexible container comprises a deagglomerated bag with an open edge that is sealed during use, and the stabilized flexible container is a stabilized bag.

21. An automated device for semi-automated aseptic disaggregation and/or enrichment and/or stabilization of cells or cell aggregates from mammalian solid tissues, comprising: a programmable processor; and including as in paragraph 1 At least one of the flexible containers from any one of to 15 serves as a set of disaggregated flexible containers.

22. The automated device of paragraph 21, which further includes an indicator tag reader.

23. The automated device of paragraph 21, which further includes a radio frequency identification tag reader for identifying the kit components.

24. The automated device of paragraph 21, wherein the programmable processor is able to identify the kit components via tags and then execute programs that define the types of de-aggregation, enrichment, and stabilization processes and the types of individual media required for these processes.

25. The automation device of paragraph 21, wherein the programmable processor controls the disaggregation element of the automation device, so that the solid tissue can be physically and/or biologically decomposed in the depolymerization flexible container.

26. The automated device of paragraph 25, wherein the programmable processor controls the depolymerization surface close to the depolymerization flexible container, which mechanically squeezes and shears the solid tissue placed in the depolymerization flexible container, depending on The situation where the depolymerization surface is a mechanical piston.

27. The automation device of paragraph 21, wherein the programmable processor controls the depolymerization element of the automation device to enable physical and enzymatic decomposition of solid tissue in the depolymerization flexible container.

28. The automated device of paragraph 27, wherein the enzymatic decomposition of the solid tissue is performed by one or more medium enzyme solutions selected from the group consisting of collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase HI, Pepsin or mixtures thereof.

29. The automated device of paragraph 21, wherein the device includes at least two of the following: a depolymerization element; an enrichment element; and a stabilization element; and wherein the programmable processor is adapted to be compatible with one or more of the following Communicate and control one or more of the following: depolymerization element; enrichment element; and stabilization element.

30. The automated device according to any one of paragraph 29, wherein the programmable processor controls the stabilizing element to use the programmable temperature to cryopreserve the enriched and depolymerized solid tissue in the cryopreservation container as appropriate.

31. The automated device of any of paragraph 29, wherein the device further comprises one or more of the following additional components in any combination: capable of identifying the solution before transferring the depolymerized solid tissue to the enrichment element that is optionally present A sensor to determine whether the polymerization process has been completed in the depolymerization element; measure the amount of culture medium required in the container of one or more of the depolymerization element, enrichment element, and/or stabilization element, and control the material in each A weight sensor for transfer between different containers; a sensor that controls the temperature in one or more of the depolymerization element, enrichment element, and/or stabilization element; each container that controls the culture medium in the element At least one air bubble sensor for the transfer between the input and output ports; at least one pump that controls the transfer of the culture medium between the input and output ports, as the case may be, a peristaltic pump; a pressure sensor for evaluating the pressure in the enrichment element One or more valves that control the tangential flow filtration process in the enrichment element; and/or one or more clamps that control the transfer of the culture medium between the input and output ports of each element.

32. The automated device of paragraph 29, wherein the programmable processor is adapted to maintain the optimal storage temperature range in the stabilizing element until the container is removed; or to perform a controlled freezing step.

33. The automated device of any of the preceding paragraphs, which further includes a user interface.

34. The automation device of paragraph 26, wherein the interface includes a display screen for displaying commands that guide the user to input parameters, confirm pre-programmed steps, warn of errors, or combinations thereof.

35. The automation device of paragraph 21, wherein the automation device is adapted to be movable.

36. An automatic tissue processing method, comprising: automatically determining conditions and related conditions for the processing steps from digital, electronic or electromagnetic label indicators related to the set; the flexibility of placing the tissue sample in the set In the container; and

Seal at least one edge of the flexible container; process the tissue sample by communicating with the indicator and controlling the flexible container to automatically perform one or more tissue processing steps; and filter at least a portion of the processed tissue sample to produce a filtered fluid ; And provide at least some of the filtered fluid to cryogenic storage flexible container.

37. The method of paragraph 31, wherein the processing includes stirring, extracting, and enzymatically digesting at least a portion of the tissue sample in the flexible container.

38. The method of paragraph 31, wherein the processing includes stirring, extracting, and enzymatically digesting at least a portion of the tissue sample in the flexible container and causing extraction of the desired material.

39. The method of paragraph 31, wherein the processing includes stirring, extracting, and enzymatically digesting at least a portion of the tissue sample in the flexible container and causing the extraction of tumor infiltrating lymphocytes (TIL).

40. The method of paragraph 31, wherein the flexible container comprises a heat-sealable material.

41. The method of paragraph 31, wherein the flexible container comprises at least one of EVA, vinyl acetate and polyolefin polymer blend or polyamide. * * *

Therefore, the preferred embodiments of the present invention are described in detail. It should be understood that the present invention defined by the above paragraphs should not be limited to the specific details set forth in the above description, because many modifications may be made without departing from the spirit or scope of the present invention. Obvious change.

1a: container 1b: end 1c: hole 1d: location 1e: edge 1f: Catheter 1g: catheter 1h: catheter 2: set 2a: filter 2b: filter 2c: filter 3a: Medium 3b: Enzyme, medium 3c: solution, medium 4: bag, element 4a: Filter unit 4b: filter 4c: Filter unit 4d: filter 4e: filter 5: bag, syringe 5a: Fixture 5b: Fixture 5c: Fixture 5d: fixture 5e: fixture 5f: Fixture 5g: fixture 5h: fixture 5i: Fixture 5j: fixture 5k: fixture 5l: fixture 5m: fixture 5n: fixture 5o: fixture 5p: fixture 5q: Fixture 5r: fixture 5s: fixture 5t: fixture 6: bag, element 6a: container 6b: hole 7: bag 7a: container 7b: hole 7c: Edge 7d: Catheter 7e: Catheter 7f: Catheter 8: Strips, sealing ports, components 8a: Filter unit 8b: filter 8c: Valve 9: Sealing port, filter 9a: Filter unit 9b: filter 9c: Valve 10: bag, catheter 10a: container 10b: hole 10c: Edge 10e: Catheter 10f: Catheter 10g: cover 10h: Connector 11: opening, duct 11a: container 11b: hole 11d: Catheter 11e: Catheter 11f: Catheter 12: Valve, cavity 12a: container 12b: Fixed parts 12c: space 12d: location 13: valve, conduit 13a: peg 13b: peg 13c: Fusion splicer 13d: Module 13e: Module 13f: surface 13g: fixture 13h: pump 13i: locator 13j: fixture 13k: Valve 13l: peg 13m: fusion splicer 13n: cutter 13o: module 14: Peripheral, fixture 15: Connector 16: port 17: shunt connection, shunt 18: corner hole 19: Valve 19a: Valve 19b: Valve 19c: Valve 20: frame, port 20': frame 21: section 22: hole, bag 23: edge, locator 24: pegs, connectors 25: Catheter 26: window, section, end 27: Indicator 28: indicator 29: mark 30: shell, cover, bag 31: Edge 32: port 33: locator 34: Connector 35: Catheter 36: section, indicator 37: indicator 38: indicator 39: mark 40: freezer, bag 41: Edge 43: locator 44: Connector 46: End, catheter 50: heat sealing machine, bag 52: Connector 54: Catheter 55: Heat sealing machine 60: fixture, bag 61: Wedge formation 62: Rod, connector 63: protrusion 64: Rod, part 65: Vertex 66: screw, part, edge 67: Channel 68: Groove 69: Ridge Formation 70: bag 72: Connector 74: Part 76: part 80: bag 82: Connector 84: end 86: Edge 90: bag 92: Connector 94: end 96: Edge 100: device, bag 100': device 101: Edge 102: end 103: locator 104: Connector 105: Catheter 106: Connector 107: Indicator 108: indicator 109: Mark 110: shell, port 111: Back Wall 112: Chassis, fixture 113: Motor 114: Motor unit, motor and gearbox, port 115: Transmission 116: crank 118: connecting rod 120: body, bag 121: Edge 122: pivot, end 123: locator 124: pivot, connector 125: Catheter 126: Rod 127: indicator 128: Rod, indicator 129: Mark 130: pole, bag 131: Part 132: Rod 133: Locator 134: feet, assembly, connector 135: Catheter 136: feet, assembly 137: Indicator 138: bottom plate, indicator 140: bag, sealed mouth, bottom plate 141: Mark 142: frame, mark, line 143: compartment, locator 144: frame, compartment 145: section 146: spring, compartment 147: District 148: area, port 149: port 150: board, base, port 151: Surface 152: surface, bag 156: Seal mouth 158: section 160: locator 162: bag 164: Valve 166: Catheter 170: bag 172: Catheter 174: Catheter 176: Catheter 178: Valve 180: bag 182: Catheter 184: Catheter 186: Catheter 188: Valve 190: bag 191: set 192: bag 193: Indicator 194: Indicator 195: Valve 196: Valve 197: Fixture 198: Fixture 199: Catheter 200: device, filter 201: Set 202: bag 205: set 206: bag 208: bag 209: Valve 210: shell, valve 211: Fixture 212: Fixture 213: Motor 214: motor unit, filter 215: Port 216: port 220: Institution 221: controller, axis 222: toothed belt, catheter 223: Axis 224: Camshaft, set 225: driven wheel 226: bracket, bag 227: driven wheel 228: bracket, bag 229: guide 230: cam, filter 231: Spring 232: cam, valve, bag 234: feet, clamps, indicators 236: feet, clamps, indicators 238: valve, marking 240: mark 241: Membrane 242: port 244: Locator 246: Connector 248: area, duct 250: plate, duct 251: Surface 252: surface, duct 254: Fixture 255: Hinge 256: sensor, fixture 258: port 260: Port 264: bag 266: Fixture 268: Fixture 270: filter 272: Catheter 274: port 276: port 278: Valve 280: Connector 282: bag 284: bag 286: port 288: Fixture 290: Valve 292: Valve 294: bag 296: Fixture 298: filter 300: Syringe 302: Syringe 304: bag 306: Bracket 310: Fusion interface 312: Fixture 314: Paddle 316: Paddle 330: bag 332: section 334: Fixture 336: Bracket 338: Hinge 340: side 342: side 344: Fixture 346: Prominence 348: Lash 350: spine 352: set 354: bag 356: filter 358: Fixture 360: Fixture 362: Valve 364: Valve 366: bag 368: Catheter 400: bag 402: Fixture 408: Organization 410: Catheter 420: bag 422: component 424: Pallets, materials, organization 426: Catheter 428: component 430: port C: Arrow D: Arrow E: Enzyme F: Arrow T: sample, tissue U: Arrow

The patent or application contains at least one color drawing. After applying and paying the necessary fees, the Patent Office will provide a copy of this patent or patent application publication with one or more color drawings.

Given by way of example, but not intended to limit the present invention only to the specific embodiments described, the following embodiments can be best understood in combination with the accompanying drawings.

Figure 1 is a schematic diagram of a flexible container used for depolymerization and digestion of solid tissue materials.

Figure 2A is a schematic diagram of a series of filtration modules that guide digested solid tissue materials to subsequent modules or waste containers.

Figure 2B is a schematic diagram of a flexible container for enriching cells after digestion and removal of waste materials.

2C is a schematic diagram of another embodiment of a flexible container for enriching cells after digestion and removal of waste materials.

Figure 3A is a schematic diagram of a flexible container for stabilizing cells after deaggregating solid tissue materials and/or enriching cells.

3B is a schematic diagram of another embodiment of a flexible container containing a connection with an additional flexible container for stabilizing cells through cryopreservation after deaggregating solid tissue materials and/or enriching cells.

Figure 4 is a schematic diagram of the sterile kit.

Fig. 5 is a bar graph indicating various disaggregation times in the range of several seconds to several hours, and the observed fold change of the cell population obtained from the disaggregation process.

6 is a diagram describing a semi-automatic sterile tissue processing method using multiple flexible containers for different starting solutions as part of a module for the process of depolymerization and stabilization.

Figure 7 is a diagram describing how a flexible container containing a culture medium for the process can be shared among the modules of the aseptic processing kit and method.

Figure 8 depicts a general overview of the method used to generate TIL.

Figure 9 depicts an overview of the collection and processing of tumor starting materials.

Figure 10 depicts an overview of the TIL manufacturing process.

Figure 11A shows a view of an embodiment of a kit for processing and storing tissue materials.

Figure 11B shows a view of an embodiment of a kit for processing and storing tissue materials.

Figure 11C shows a view of an embodiment of a kit for processing and storing tissue materials.

Figure 11D shows a view of an embodiment of a kit for processing and storing tissue materials.

Figure 12A shows a perspective view of an embodiment of the collection bag.

Figure 12B shows a perspective view of an embodiment of the collection bag.

Figure 12C shows a perspective view of an embodiment of the collection bag.

Figure 12D shows a perspective view of an embodiment of the collection bag.

Figure 12E shows a perspective view of an embodiment of the collection bag.

Figure 13A shows a front view of an embodiment of the collection bag.

Figure 13B shows a front view of an embodiment of the collection bag.

Figure 13C shows a front view of an embodiment of the collection bag.

Figure 13D shows a front view of an embodiment of the collection bag.

Figure 13E shows a front view of an embodiment of the collection bag.

Figure 14 shows a rear view of the embodiment of the collection bag.

Figure 15 shows a side view of an embodiment of the collection bag.

Figure 16A shows a top view of an embodiment of the collection bag.

Figure 16B shows a bottom view of an embodiment of the collection bag.

Figure 17A shows a top view of an embodiment of a partially open tissue collection bag for sealing tissue therein for processing of the present invention, wherein the bag has a sealing edge.

Figure 17B shows a bottom view of an embodiment of an open tissue collection bag for sealing tissue therein for processing of the present invention, wherein the bag has a sealing edge.

Figure 18A shows a top view of an embodiment of a partially open tissue collection bag for sealing tissue therein for processing of the present invention.

Figure 18B shows a top view of an embodiment of a completely open tissue collection bag used to seal tissue therein for the treatment of the present invention.

Figure 19A shows a top view of an embodiment of a partially open tissue collection bag for sealing tissue therein for processing of the present invention, wherein the bag has a sealing edge of a predetermined width.

Figure 19B shows a top view of an embodiment of a completely open tissue collection bag for sealing tissues therein for processing of the present invention, wherein the bag has a sealed edge of a predetermined width.

Figure 20A shows a front view of an embodiment of the collection bag.

Figure 20B shows a front view of an embodiment of the collection bag.

Figure 20C shows a front view of an embodiment of the collection bag.

Figure 20D shows a front view of an embodiment of the collection bag.

Figure 20E shows a front view of an embodiment of the collection bag.

Figure 21A shows a front view of an embodiment of the collection bag.

Figure 21B shows a front view of an embodiment of the collection bag.

Figure 21C shows a front view of an embodiment of the collection bag.

Figure 21D shows a front view of an embodiment of the collection bag.

Figure 21E shows a front view of an embodiment of the collection bag.

Figure 22A shows a front view of an embodiment of the collection bag.

Figure 22B shows a front view of an embodiment of the collection bag.

Figure 22C shows a front view of an embodiment of the collection bag.

Figure 22D shows a front view of an embodiment of the collection bag.

Figure 23 shows a front view of an embodiment of the collection bag.

Figure 24 shows a front view of an embodiment of the collection bag.

Figure 25 shows a front view of an embodiment of the collection bag.

Figure 26 shows a front view of an embodiment of a collection bag coupled to a catheter and port.

Figure 27A shows a front view of an embodiment of the collection bag before use.

Figure 27B shows a front view of an embodiment of a collection bag that has been sealed, for example, after the material is deposited in the bag.

Fig. 28 shows a top view of an embodiment of a face-up cryopreservation kit including an open collection bag and a cryopreservation bag.

FIG. 29 shows a top view of an embodiment of a cryopreservation kit that includes a collection bag indicating where it should be closed and a cryopreservation bag facing down.

Fig. 30 shows a top view of an embodiment of a face-up cryopreservation kit including a closed collection bag and a cryopreservation bag.

Figure 31 shows a side view of an embodiment of an upwardly facing cryopreservation kit including a closed collection bag and a cryopreservation bag.

Figure 32 shows an end view of an embodiment of the cryopreservation kit.

Figure 33 shows a top view of an embodiment of a collection bag including a logo coupled to a catheter.

Figure 34 shows a front view of an embodiment of a cryopreservation kit including a collection bag, a filter, and a cryopreservation bag.

Figure 35 shows a front view of an embodiment of a cryopreservation kit including a collection bag, a filter, and a cryopreservation bag.

Figure 36A shows a front view of an embodiment of a cryopreservation kit including a collection bag, a filter, and a cryopreservation bag.

Figure 36B shows a side view of an embodiment of a collection bag using clamps, hinges and latches and rod fastenings positioned close to the surface of the collection bag during use.

Figure 36C shows an exploded view of the clamp on the collection bag.

Figure 37 shows a front view of an embodiment of a cryopreservation kit including a collection bag, a filter, and a cryopreservation bag.

Figure 38 shows a front view of an embodiment of a cryopreservation kit including a collection bag, a filter, and a cryopreservation bag.

Figure 39 shows a front view of an embodiment of a collection bag secured by a clamp.

Figure 40 shows a front view of an embodiment of the collection bag.

Figure 41 shows a front view of a treading device used to disaggregate tissue into individual cells or cell aggregates in a closed sample container.

Figures 42 and 43 show the device of Figure 41 in two different operating positions; Figure 44 shows a plan view of the device shown in the previous figures.

Figure 45 shows another plan view of an alternative configuration of the device.

Figures 46, 47 and 48 show three different configurations of sample containers suitable for use with the devices of Figures 41 to 45.

Figure 49 shows the sample bag prepared for use.

Figure 50 depicts the bag sealing jig.

Figures 51A, 51B, and 51C show alternative ways of sealing the sample bag.

Figures 52, 53, and 54 show the equipment and techniques used to prepare bags for use.

Figure 55 shows that the sample bag or container is loaded into the harness.

Figures 56, 57, and 58 show the equipment used to divide the depolymerized sample.

Figures 59, 60, and 61 show equipment for controlling the temperature of samples or divided samples.

Figures 62 to 64 show another embodiment of the pedaling device.

Figure 65 is an exemplary flow chart for collecting, processing, and cryopreserving tumor tissue.

Figure 66 is an exemplary flow chart for the production of TIL from processed and cryopreserved tumor tissue.

Figure 67 compares the yield (Figure 67A), survival percentage (Figure 67B) and CD3+ T cell percentage (Figure 67C) of cryopreserved and fresh disaggregated cell suspensions.

Figure 68A and 68B compare the survival rate of PBMC cryopreserved with commercially available cryopreservation agents.

Figure 69 compares the digestion, followed by maintaining the material at 4°C for 10 minutes, and then lowering the temperature at a rate of -1°C/min or directly reducing it from 35°C to -80°C at a rate of -2°C/min. Survival rate of preserved PBMC.

Figure 70 compares the sample bag records following the plan of maintaining the material at 4°C for 10 minutes, then lowering the temperature at a rate of -1°C/min or directly at a rate of -2°C/min from 35°C to -80°C的温度。 The temperature.

Figure 71 depicts the deaggregation and cryopreservation of TIL077: (A) deaggregator speed set point; (B) deaggregator speed record; (C) temperature set point (deaggregation); (D) low temperature disc temperature record (solution Poly); (E) temperature set point (low temperature storage); (F) temperature record (low temperature storage); (G) set point cooling rate; (H) low temperature disk cooling rate record.

Figure 72 depicts the Tiss-U-Stor depolymerization and cryopreservation of TIL078 (1 of 2 bags): (A) deagglomerator speed set point; (B) deagglomerator speed record; (C) temperature set point (Depolymerization); (D) Low temperature disk temperature record (depolymerization); (E) Temperature set point (low temperature storage); (F) Temperature record (low temperature storage); (G) Set point cooling rate; (H) low temperature Record the disk cooling rate.

Figure 73 depicts the Tiss-U-Stor depolymerization and cryopreservation of TIL078 in a continuous process: (A) deagglomerator speed set point; (B) deagglomerator speed record; (C) temperature set point (depolymerization and cryopreservation ); (D) Low temperature disk temperature record (deaggregation and cryopreservation); (E) Cooling rate set point (depolymerization and (low temperature storage); (F) Low temperature disk cooling rate record (deaggregation and (low temperature storage).

Figure 74 depicts a waterfall chart showing the best overall response and the percentage change in tumor burden. CR, complete response; PD, progressive disease; PR, partial response; SD, stable disease. Tumor burden is defined as the total diameter of the target lesion; the change in tumor burden is defined as the change from baseline to the lowest point after baseline. In both CR patients, a minimum post-baseline SLD of 0 was used. These patients did not report the target disease variable measurement value (no disease or metastasis was observed by CT/MRI scan) at the time of the CR assessment. One of the individuals with the best overall response to PD did not report any post-treatment target disease variable measurement (by observing the progress of the new lesion determination) and therefore is not shown in the figure.

Figure 75 depicts the total survival time. (A) The median overall survival (OS) time for all 21 treated patients was 21.3 months. (B) The median OS time of 15 patients with quantitative response data was 16 months. (C) The median OS time for non-responders (N=7) was 6.5 months. Those who did not get a response (based on quantitative response only, N=8) median OS time.

Figure 76 depicts the characteristics of the manufactured TIL. (A) Cell count during the TIL overgrowth phase (phase 1) of full-scale ITIL-168 GMP operation. (B) Cell count during the TIL REP phase (phase 2) of the full-scale ITIL-168 GMP operation. (C) Survival percentage (% of live CD3+ cells) during full-scale ITIL-168 GMP operation.

Claims (87)

A method for preparing a therapeutic population of tumor infiltrating lymphocyte (TIL), which comprises: (a) Aseptically disaggregate a tumor resected from an individual, thereby producing a depolymerized tumor product, wherein the tumor line is sufficiently depolymerized so that the depolymerized tumor product can be stored at a low temperature; (b) Cool the depolymerized tumor product to a suitable cryopreservation temperature, (c) Perform the first expansion by culturing the cryopreserved depolymerized tumor product in a cell culture medium containing IL-2 to produce a first population of TIL; (d) performing a second expansion by culturing the first population of TIL together with additional IL-2, OKT-3 and antigen presenting cells (APC) to produce a second population of TIL; and (e) Collecting and/or cryopreserving the second population of TIL; Wherein the depolymerization includes enzymatic depolymerization and/or biopolymerization, wherein the biopolymerization includes repeated application of physical pressure to the resectioned tumor; The steps (a) to (e) are executed in a closed system. The method of claim 1, wherein the depolymerization comprises repeated application of physical pressure 120 to 360 times ^{per minute at a maximum of 6 N/cm 2} , and more preferably 3 N/cm ^2. The method according to any one of claims 1 or 2, wherein the depolymerized tumor product comprises a single cell suspension. The method according to any one of claims 1 to 3, wherein the resected tumor is not fragmented before disaggregation. The method according to any one of claims 1 to 4, wherein step (a) is carried out at a temperature suitable for enzymatic digestion. The method according to any one of claims 1 to 5, wherein step (b) comprises directly cooling the depolymerized tumor product to the cryopreservation temperature. The method according to any one of claims 1 to 6, wherein the depolymerization time period is 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less. Such as the method of any one of claims 1 to 7, wherein the disaggregation is continuous or is performed for a period of at least one minute. The method of any one of claims 1 to 8, wherein the tumor is not impregnated. The method of any one of claims 1 to 9, which comprises cooling the depolymerized tumor product in a controlled temperature device programmed to lower the temperature at a constant rate. Such as the method of claim 10, wherein the cryogenic storage temperature is -80℃±10℃ and the device is programmed to 1℃/min or 1.5℃/min or 2℃/min or 1℃/min±0.5℃/min Or 1℃/min±0.5℃/min or 2℃/min±0.5℃/min to lower the temperature. Such as the method of any one of claims 1 to 11, wherein the TILs comprise UTIL, or wherein the TILs comprise MTIL. The method according to any one of claims 1 to 12, wherein the first group of TILs is about 1 to 20 million TILs. The method according to any one of claims 1 to 13, wherein step (c) includes growing TIL to generate the first population, and the second amplification in step (d) includes rapid amplification. The method according to any one of claims 1 to 14, wherein step (c) is performed for about two weeks and step (d) is performed for about two weeks. Such as the method of any one of claim 1 to 15, wherein the cultivation in step (c) and/or step (d) includes adding IL-7, IL-12, IL-15, IL-18, IL-21 or Its combination. The method according to any one of claims 1 to 16, which comprises placing the resected tumor tissue in a flexible container with a depolymerization fluid, sealing the container, and subjecting the tumor tissue to physical and/or enzymatic depolymerization, And cryopreservation of the disaggregated tumor tissue. A therapeutic population of cryopreserved tumor infiltrating lymphocytes (TIL), which is obtained by the method according to any one of claims 1 to 17. The therapeutic population of claim 18, wherein the population contains about 5×10 ⁹ to 5×10 ¹⁰ T cells. A cryopreservation bag for therapeutic groups such as claim 18 or 19. Such as the cryopreservation bag of claim 20, which is used for intravenous infusion. A method for preparing a therapeutic population of tumor infiltrating lymphocytes (TIL), which comprises: (a) Aseptically disaggregate the tumor resected from the individual to produce a depolymerized tumor product, wherein the tumor is sufficiently depolymerized so that the depolymerized tumor product can be stored at low temperature; (b) Perform the first expansion by culturing the cryopreserved depolymerized tumor product in a cell culture medium containing IL-2 to produce a first population of TIL; (c) Performing a second expansion by culturing the first population of TIL together with additional IL-2, OKT-3 and antigen-presenting cells (APC) to produce a second population of TIL; and (d) Collecting and/or cryopreserving the second population of TIL; Wherein the depolymerization includes enzymatic depolymerization and/or biopolymerization, wherein the biopolymerization includes repeated application of physical pressure to the resectioned tumor; The steps (a) to (d) are executed in a closed system. The method of claim 22, wherein the depolymerization comprises repeated application of physical pressure 120 to 360 times ^{at 6 N/cm 2} at most, and more preferably 3 N/cm ^{2 per minute.} The method of any one of claims 22 or 23, wherein the depolymerized tumor product comprises a single cell suspension. The method of any one of claims 22 to 24, wherein the resected tumor is not fragmented before disaggregation. The method according to any one of claims 22 to 25, wherein step (a) is carried out at a temperature suitable for enzymatic digestion. The method according to any one of claims 22 to 26, wherein the depolymerization time period is 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less. Such as the method of any one of claims 22 to 27, wherein the disaggregation is continuous or for a period of at least one minute. The method of any one of claims 22 to 28, wherein the tumor is not impregnated. The method of any one of claims 22 to 29, which comprises cooling the depolymerized tumor product in a controlled temperature device programmed to lower the temperature at a constant rate. Such as the method of claim 30, wherein the cryogenic storage temperature is -80℃±10℃ and the device is programmed to 1℃/min or 1.5℃/min or 2℃/min or 1℃/min±0.5℃/min or 1℃/min±0.5℃/min or 2℃/min±0.5℃/min to lower the temperature. Such as the method of any one of Claims 22 to 31, wherein the TILs comprise UTIL, or wherein the TILs comprise MTIL. The method of any one of claims 22 to 32, wherein the first group of TILs is about 1 to 20 million TILs. The method according to any one of claims 22 to 33, wherein step (b) includes growing TIL to generate the first population, and the second amplification in step c) includes rapid amplification. The method of any one of claims 22 to 34, wherein step (b) is performed for about two weeks and step (c) is performed for about two weeks. Such as the method of any one of claim 22 to 35, wherein the cultivation in step (b) and/or step (c) includes adding IL-7, IL-12, IL-15, IL-18, IL-21 or Its combination. The method according to any one of claims 22 to 36, which comprises placing the resected tumor tissue in a flexible container with a depolymerization fluid, sealing the container, and subjecting the tumor tissue to physical and/or enzymatic depolymerization, And cryopreservation of the disaggregated tumor tissue. A therapeutic population of cryopreserved tumor infiltrating lymphocytes (TIL), which is obtained by the method according to any one of Claims 22 to 37. The therapeutic population of claim 38, wherein the population contains about 5×10 ⁹ to 5×10 ¹⁰ T cells. A cryopreservation bag for therapeutic groups such as claim 38 or 39. Such as the cryopreservation bag of claim 40, which is used for intravenous infusion. A method for preparing a therapeutic population of tumor infiltrating lymphocytes (TIL) in a closed system, which comprises: (a) (i) cryopreservation to remove the tumor and disaggregate the cryopreserved tumor, or (ii) Disaggregate the tumor removed and store the disaggregated tumor at low temperature, or (iii) cryopreservation to remove the tumor and process the tumor into multiple tumor fragments, or (iv) The excised tumor is processed into multiple tumor fragments and the tumor fragments are stored at low temperature, (b) Perform the first expansion by culturing the cryopreserved depolymerized tumor product in a cell culture medium containing IL-2 to produce a first population of TIL; (c) Performing a second expansion by culturing the first population of TIL together with additional IL-2, OKT-3 and antigen-presenting cells (APC) to produce a second population of TIL; and (d) Collect and/or cryopreserve this second population of TIL. The method of claim 42, wherein the depolymerization inclusions understand polymerization, enzymatic depolymerization, or physical and enzymatic depolymerization. The method of any one of claims 42 or 43, wherein the disaggregation comprises repeated application of physical pressure to the resected tumor. The method according to any one of claims 42 to 44, wherein the depolymerization comprises repeated application of physical pressure 120 to 360 times ^{per minute at a maximum of 6 N/cm 2} , more preferably 3 N/cm ^2. The method according to any one of claims 42 to 45, wherein the substance understands that the polymer includes extrusion and shearing. The method of any one of claims 42 to 46, wherein the cryopreserved depolymerized tumor comprises a single cell suspension. The method according to any one of claims 42 to 47, wherein the depolymerization is carried out at a temperature suitable for enzymatic digestion. The method according to any one of claims 42 to 48, wherein cryopreservation comprises directly cooling the resected or depolymerized tumor to a set cryopreservation temperature. The method of any one of claims 42 to 49, which comprises cooling the resected or disaggregated tumor in a controlled temperature device programmed to lower the temperature at a constant rate. Such as the method of claim 50, wherein the cryogenic storage temperature is -80℃±10℃ and the device is programmed to 1℃/min or 1.5℃/min or 2℃/min or 1℃/min±0.5℃/min Or 1℃/min±0.5℃/min or 2℃/min±0.5℃/min to lower the temperature. A method for a therapeutic population of TIL isolated from tumor tissues excised from an individual, which comprises: (a) placing the excised tumor tissue in an automated device including a programmable processor and a single-use sterile kit to perform semi-automated aseptic disaggregation of the tumor tissue, wherein the sterile kit includes a closed system; wherein This sterile kit contains: Disaggregation module for receiving and processing materials containing tumor tissue; Optional enrichment module for filtering depolymerized solid tissue materials and separating undepolymerized tissues and filtrate; and A stabilization module for further processing and/or storage of depolymerized product materials as appropriate, Wherein each of the modules includes one or more flexible containers, and the one or more flexible containers are connected by one or more pipes adapted to enable the tissue material to flow therebetween ;and Wherein each of the modules includes one or more ports to allow the aseptic input of culture medium and/or reagents into the one or more flexible containers; (b) Aseptically disaggregate the resected tumor, thereby producing a disaggregated tumor, (c) Perform the first expansion by culturing the disaggregated tumor in a cell culture medium containing IL-2 to produce a first population of UTIL; (d) Performing a second expansion by culturing the first population of UTIL together with additional IL-2, OKT-3 and antigen-presenting cells (APC) to produce a second population of TIL; and (f) Collect and/or cryopreserve the second population of UTIL. The method of claim 32, wherein the disaggregation comprises repeatedly applying physical pressure to the resected tumor. The method according to any one of claim 52 or 53, wherein the depolymerization comprises repeated application of physical pressure 120 to 360 times ^{at most 6 N/cm 2} , and more preferably 3 N/cm ^{2 per minute.} The method of any one of claims 52 to 54, wherein the resected tumor is not fragmented before disaggregation. The method of any one of claims 52 to 55, wherein the tumor tissue is not impregnated. The method according to any one of claims 52 to 56, wherein the depolymerization is performed for 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less. The method according to any one of claims 52 to 57, wherein after step (b), the method comprises (b′) cryopreserving the depolymerized tumor in the stabilization module. The method of claim 58, wherein step (b') comprises directly cooling the depolymerized tumor product to the cryopreservation temperature. Such as the method of claim 59, wherein the cryogenic storage temperature is -80℃±10℃ and the device is programmed to 1℃/min or 1.5℃/min or 2℃/min or 1℃/min±0.5℃/min Or 1℃/min±0.5℃/min or 2℃/min±0.5℃/min to lower the temperature. Such as the method of any one of claims 52 to 60, wherein the automated device further includes one or more of the following in any combination: A sensor capable of identifying whether the depolymerization process has been completed in the depolymerization module before transferring the depolymerized solid tissue to the enrichment module that is optionally selected; A weight sensor that measures the amount of culture medium required in the container of one or more of the depolymerization module, the enrichment module, and/or the stabilization module, and controls the transfer of materials between the respective containers ； A sensor that controls the temperature in the container of one or more of the depolymerization module, the enrichment module, and/or the stabilization module; At least one air bubble sensor that controls the transfer of the culture medium between the input and output ports of each container in the module; At least one pump that controls the transfer of culture medium between the input and output ports, peristaltic pumps as appropriate; A pressure sensor that evaluates the pressure in the enrichment module; Control one or more valves in the tangential flow filtration process in the enrichment module; and/or One or more clamps for controlling the transfer of the culture medium between the input and output ports of each module. The method of any one of claim 52 to 61, wherein the programmable processor controls the depolymerization module to enable physical and enzymatic decomposition of the solid tissue material, and/or wherein the programmable processor controls the The stabilization module preserves the depolymerized solid tissue enriched in the container at a low temperature. The method according to any one of claims 52 to 62, wherein the disaggregation module is repeatedly squeezed and sheared from the tumor tissue 120 to 360 times ^{per minute at 6 N/cm 2} , more preferably 3 N/cm ^{2, and} The stabilization module cools the depolymerized tumor tissue to -80℃±10℃ and the device is programmed to 1℃/min or 1.5℃/min or 2℃/min or 1℃/min±0.5℃ /min or 1℃/min±0.5℃/min or 2℃/min±0.5℃/min to lower the temperature. A flexible container suitable for a closed system for separating TIL from therapeutic groups, It is used to treat tissues, and the flexible container contains: One or more layers made of a sealable polymer, wherein at least three sides of the flexible container are sealed during manufacture; An open side of the flexible container, through which tissue material is inserted during use; and One or more connectors configured to couple the flexible container to at least one element via a conduit; The section close to the edge of the opening is sealed after the tissue material is placed in the flexible container to form a sealed port. Such as the 64 flexible container of claim, wherein the sealing port is formed by a heat sealer operating under a predetermined pressure, a predetermined temperature and a predetermined time range. The flexible container according to any one of claim 64 or 65, wherein the flexible container is configured to be used with a device that mechanically squeezes the tissue material placed in the flexible container. The flexible container according to any one of claims 64 to 66, wherein the flexible container is adapted to repeatedly apply a physical pressure of 120 to 360 ^{at a maximum of 6 N/cm 2} per minute, more preferably 3 N/cm ² Second-rate. A system for extracting TIL from tumor tissue, which comprises: Set, which contains: Disaggregated flexible container; Stabilize flexible containers; and At least one indicator label on at least one of the depolymerized flexible container or the stabilized flexible container, which can provide at least one of a tissue source, a tissue state, or an identifier; Capable of processing at least some tissues in a depolymerized flexible container to form a depolymerized element of treated fluid; Capable of enriching at least some of the treated fluid to form an enrichment element of the desired material; A stabilizing element capable of storing part of the desired material in the stabilizing flexible container and controlling freezing as appropriate; and At least one indicator tag reader located on at least one of the depolymerization element or the stabilization element can provide at least one of a tissue source or the tissue state in the stabilization element. A method of treating cancer in an individual, which comprises: (a) Aseptically disaggregate the tumor resected from the individual to produce a depolymerized tumor product, wherein the tumor line is sufficiently depolymerized so that the depolymerized tumor product can be stored at low temperature; (b) Cool the depolymerized tumor product to a suitable cryopreservation temperature, (c) Perform the first expansion by culturing the cryopreserved depolymerized tumor product in a cell culture medium containing IL-2 to produce a first population of TIL; (d) Performing a second expansion by culturing the first population of TIL together with additional IL-2, OKT-3 and antigen-presenting cells (APC) to produce a second population of TIL; (e) Collecting and/or cryopreserving the second population of TIL; and (f) The second group of TIL administered to the individual; Wherein the depolymerization includes enzymatic depolymerization and/or biopolymerization, wherein the biopolymerization includes repeated application of physical pressure to the resectioned tumor; The steps (a) to (e) are executed in a closed system. Such as the method of claim 69, wherein the depolymerization comprises repeated application of physical pressure 120 to 360 times ^{per minute at 6 N/cm 2} at most, more preferably 3 N/cm ^2. The method of any one of claims 69 or 70, wherein the depolymerized tumor product comprises a single cell suspension. The method of any one of claims 69 to 71, wherein the resected tumor is not fragmented before disaggregation. The method according to any one of claims 69 to 72, wherein step (a) is carried out at a temperature suitable for enzymatic digestion. The method according to any one of claims 69 to 73, wherein step (b) comprises directly cooling the depolymerized tumor product to the cryopreservation temperature. The method according to any one of claims 69 to 74, wherein the depolymerization period is 90 minutes or less, or 75 minutes or less, or 60 minutes or less, or 50 minutes or less. The method according to any one of claims 69 to 75, wherein the disaggregation is continuous or for a period of at least one minute. The method of any one of claims 69 to 76, wherein the tumor is not impregnated. The method of any one of claims 69 to 77, which comprises cooling the depolymerized tumor product in a controlled temperature device programmed to lower the temperature at a constant rate. Such as the method of claim 78, wherein the cryogenic storage temperature is -80℃±10℃ and the device is programmed to 1℃/min or 1.5℃/min or 2℃/min or 1℃/min±0.5℃/min Or 1℃/min±0.5℃/min or 2℃/min±0.5℃/min to lower the temperature. Such as the method of any one of claims 69 to 79, wherein the TILs comprise UTIL, or wherein the TILs comprise MTIL. The method of any one of claims 69 to 80, wherein the first group of TILs is about 1 to 20 million TILs. The method according to any one of claims 69 to 81, wherein step (c) includes growing TIL to generate the first population, and the second amplification in step (d) includes rapid amplification. The method according to any one of claims 69 to 82, wherein step (c) is performed for about two weeks and step (d) is performed for about two weeks. The method according to any one of claims 69 to 83, wherein the cultivation in step (c) and/or step (d) comprises adding IL7, IL-12, IL-15, IL-18, IL-21 or a combination thereof . The method according to any one of claims 69 to 84, which comprises placing the resected tumor tissue in a flexible container with a depolymerization fluid, sealing the container, and subjecting the tumor tissue to physical and/or enzymatic depolymerization, And cryopreservation of the disaggregated tumor tissue. The method of any one of claims 69 to 85, wherein the second population of TILs comprises about 5×10 ⁹ to 5×10 ¹⁰ T cells. The method according to any one of claims 69 to 86, wherein the cancer is bladder cancer, breast cancer, cancer caused by human papilloma virus, cervical cancer, head and neck cancer (including head and neck squamous cell carcinoma) squamous cell carcinoma; HNSCC), lung cancer, melanoma, ovarian cancer, non-small-cell lung cancer (NSCLC), kidney cancer or renal cell carcinoma.

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