Transfected immune cell populations and methods for their production

Abstract

The present invention provides an in vitro or ex vivo method for the transient modification of immune cells in a closed treatment system, comprising the steps of: (i) providing immune cells from a biological fluid and / or from a resected tumor of a patient; (ii) purifying the immune cells; (iii) transfecting the purified immune cells with a suppressive nucleic acid of an immunosuppressive modulator of the immune cells or with a nucleic acid of an immunopotentiator; (iv) buffering the transfected immune cells again into the physiological solution; (v) transferring the transfected immune cells into a container; wherein the transition between steps (i) to (v) is in a closed container system; and therapies using the transfected immune cells.

Description

[0001] The present invention relates to the field of cell preparation and cancer treatment methods. Background of the Invention

[0003] Cancer is the second leading cause of death worldwide and has a major impact on societies and healthcare systems around the world, causing an estimated 9.6 million deaths in 2018 (Centers for Disease Control and Prevention, 2017; International Agency for Research on Cancer, 2017), and the number of new cancer cases per year is expected to increase to nearly 30 million by 2040.

[0004] The prognosis for patients with advanced disease is poor. Improvements in conventional therapies and the development of novel targeted therapies and immunotherapies have improved outcomes for patients with advanced cancer. However, patients with extensive advanced disease may have exhausted available treatment options; in other patients, the effects of extensive disease progression and residual side effects from prior treatment regimens may preclude the use of more aggressive therapeutic agents.

[0005] The immune system can recognize and eliminate tumor cells based on the expression of tumor-specific antigens and other molecules that are abnormally expressed or induced by cellular stress. However, most tumor-reactive T cells show low affinity for tumor-associated antigens, which are mainly "self" proteins. Poor T cell function also reflects defective expression of co-stimulatory proteins and / or expression of inhibitory ligands or suppressive cytokines in the tumor microenvironment.

[0006] In addition, cancer cell immunoediting rationalizes the emergence of tumor cell variants that can resist, suppress, or avoid elimination by the immune system. In this case, some immune response against the tumor will persist, and treatments that can reinitiate a strong antitumor immune response could achieve tumor rejection.

[0007] A series of novel cancer immunotherapies have been successfully developed (Murciano-Goroff, YR, Warner, AB & Wolchok, JD The future of cancer immunotherapy: microenvironment-targeting combinations. Cell Res 30, 507–519 (2020). https: / / doi.org / 10.1038 / s41422-020-0337-2). These therapies mainly target the adaptive immune response.

[0008] One approach is treatment with monoclonal antibodies. Ipilimumab is a fully human monoclonal antibody that blocks CTLA-4, a receptor that downregulates the immune system. Ipilimumab disrupts CTLA-4 inhibition in cytotoxic T cells and enhances the immune response against tumor cells. In a phase III trial, ipilimumab was the first drug to demonstrate improvement in overall survival in patients with previously treated advanced melanoma. Adverse events associated with ipilimumab were primarily immune-related (Hoos et al., 2010). Ipilimumab was approved in Europe and the United States in 2011.

[0009] Nivolumab, Cemiplimab, Pembrolizumab, Atezolizumab, Avelumab, and Durvalumab represent a series of clinically approved monoclonal antibodies that disrupt the PD1 / PD-L1 signaling axis. This mode of action results in enhanced T cell cytotoxicity against tumor cells, showing improved cancer treatment in multiple tumor types (Murciano-Goroff et al., 2020).

[0010] Sipuleucel-T is an autologous cell-based immunotherapy for prostate cancer in which the patient's white blood cells are stimulated with a fusion protein consisting of prostatic acid phosphatase antigen and granulocyte-macrophage colony stimulating factor. This fusion protein activates antigen-presenting cells and promotes maturation. The activated blood product is then infused back, and the activated antigen-presenting cells help the patient's immune system target prostate cancer cells. Increased inflammatory cytokine production has been observed in patients treated with Sipuleucel-T (Kantoff et al., 2010).

[0011] Another approach to addressing the immune deficiencies in vivo that prevent anti-tumor responses is to activate immune cells ex vivo and then infuse them back into the patient where they can generate effective anti-tumor responses (eg WO 2009 / 073905 and WO 2012 / 089736).

[0012] Autologous cell therapy is a novel therapeutic intervention in tumor treatment that uses individual cells that are manipulated in vitro and then reintroduced into the donor. Advantages of this approach include minimizing the risk of systemic immune responses, bioincompatibility, and disease transmission associated with transplants or cells that were not cultured from the individual.

[0013] Most patients with metastatic or recurrent malignancies develop resistance to currently available treatment options and eventually succumb to their disease.

[0014] Therefore, there is a substantial unmet need for new effective and less toxic treatment strategies to improve outcomes for patients with advanced or metastatic malignancies.It is an object of the present invention to provide new cell therapies to improve outcomes for patients with advanced malignancies. Summary of the invention

[0015] T cell-based adoptive immunotherapy relies on the delivery of genetically engineered T cells to patients. This methodology has evolved dramatically over the past decade, expanding beyond T cells to other immune cells and becoming one of the most promising therapeutic strategies for treating a range of malignancies.

[0016] The present invention provides a platform method for personalized treatment of multiple solid tumors and liquid tumors. In addition, the platform technology is applicable to various tumors and targets. Specifically, the present invention is committed to activating the patient's immune system to improve or enhance the immune response to these tumors and targets.

[0017] The present invention provides modification of immune cells (leukocytes) in a cleanly interconnected closed system. The method comprises three main steps: "MAP I": (initial) purification of immune cells, "MAP II": transfection of immune cells and "MAP III": re-buffering of transfected immune cells and optional further purification.

[0018] The process enables an automated, closed leukocyte purification (MAP step I), followed by a closed, automatically performed transfection, preferably electroporation, step (MAP step II) to transfect purified autologous leukocytes, and a final rebuffering step (MAP step III).

[0019] Immune cells are provided, preferably by leukapheresis or by treating tumor fragments, and cells such as platelets and red blood cells are depleted, and immune cells are modified, preferably by electroporation. Different types of nucleic acids can be used for electroporation. Genetically modified cells, preferably electroporated immune cells, are transferred to the final formulation, and immune cells ("drug products") prepared for administration are received. This process takes about 6 hours.

[0020] The cell processing system is a closed container system and the transfection, preferably electroporation, system utilizes disposable tubing sets that are interconnected by clean welding of the immune cell purification apparatus tubing set with the transfection apparatus tubing set and the tubing set for final rebuffering.

[0021] Specifically, the present invention provides an in vitro or ex vivo method for modifying immune cells in a closed processing system, comprising the following steps: (i) providing immune cells from a biological fluid and / or from a resected tumor of a patient; (ii) purifying the immune cells (MAP step I); (iii) transfecting the purified immune cells with an inhibitory nucleic acid of an immunosuppressive regulator of the immune cells or with a nucleic acid of an immunopotentiating factor (MAP step II); (iv) purifying the transfected immune cells and / or rebuffering the transfected immune cells into a physiological solution (MAP step III); (v) transferring the transfected immune cells into a container;

[0022] wherein the transition between each two sequential steps of steps (i) to (v) is in a closed container system and / or wherein the transition from one step to another occurs in an unopened container. The immune cells may be transiently modified.

[0023] The transfected immune cells obtained in step (v) can be used as a drug, in particular for the treatment of cancer.

[0024] Checkpoint inhibitor therapy is a form of cancer immunotherapy. The therapy targets immune checkpoints, which are key regulators of the immune system that, when stimulated, can dampen the immune response to immune stimulation. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints and restore immune system function.

[0025] Cbl (Casitas B-lineage Lymphoma) protein is part of the ubiquitin ligase family, involved in cell signaling, protein ubiquitination and protein substrate degradation, and is one of the immune checkpoint inhibitors. Members of this family include RING-type E3 ligases c-Cbl, Cbl-b and Cbl-c. The E3 ubiquitin ligase Casitas B-lineage Lymphoma protein-b (Cbl-b) is a negative regulator of innate and adaptive immunity, and is also a key negative regulator of anti-tumor immunity. It limits the reactivity of most types of immune cells, especially lymphocytes and natural killer (NK) cells. Cbl-b is highly expressed in human CD4+ and CD8+ T cells, and its expression is strictly regulated by TCR, CD28 and CTLA-4, as well as other co-stimulatory and inhibitory signals. Thus, Cbl-b is involved in tumor-triggered immune evasion and has been described as a “master checkpoint” in immune function because Cbl-b inhibition simultaneously covers multiple relevant T-cell inhibitory pathways, such as through transforming growth factor β, and immune regulation through both the cytotoxic T lymphocyte-associated protein (CTLA-4) and programmed cell death ligand 1 / programmed cell death 1 (PD-L1 / PD-1) pathways. Blocking Cbl-b expression after silencing the Cbl-b gene in immune cells can enhance the activity of T cells and natural killer (NK) cells and lead to reduced tumor growth in animal models. Adoptive cell immunotherapy using Cbl-b-silenced murine T cells can significantly inhibit tumor growth in syngeneic mouse models. Cbl-b-silenced murine T cells have synergistic effects with anti-PD1, supporting the potential use of Cbl-b-silenced human T cells in combination with immune checkpoint inhibitors (CPIs).

[0026] Immune cells with at least partial Cbl-b inhibition overcome the immunosuppressive tumor microenvironment (TME).

[0027] Further immune checkpoint inhibitors are SHP-1 and SHP-2. Protein tyrosine phosphatase 1 (SHP-1) containing Src homology 2 domain is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytoplasmic protein and is therefore not suitable for antibody-mediated therapy, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies. DETAILED DESCRIPTION OF THE INVENTION

[0029]

[0026] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0030] As used herein, approximate words such as, but not limited to, "about," "substantial," or "substantially" refer to conditions that, when so modified, are understood to not necessarily be absolute or perfect, but are considered close enough to those conditions that one of ordinary skill in the art would consider to exist. The degree to which the description can be varied will depend on how much of a change can be made and still recognize that the modified feature still has the properties and capabilities required of the unmodified feature. In general, but subject to the foregoing discussion, numerical values ​​modified herein by approximate words such as "about" may vary from the stated value by, for example, ±10%.

[0031] As used herein, the terms "include," "including," "comprise," and "comprising" have the same meaning. The term "comprising" should be interpreted as an "open" transition term, which allows the inclusion of additional components in addition to those components listed. "Comprise" in relation to components connected to a range should mean that additional unlisted components are allowed, but the listed components connected to the range should be within the range and not outside the range. The term "consist," "consisting of" should be interpreted as a "closed" transition term, which does not allow the inclusion of additional components other than those listed. The term "essentially consisting of" should be interpreted as partially closed and allows only the inclusion of additional components that do not fundamentally change the properties of the listed subject matter, such as not allowing further unlisted active ingredients but allowing further non-listed auxiliary substances, such as buffer components, fillers, etc.

[0032] In the following, the term "closed system" or "closed processing system" refers to a system having a container and at least one set of pipes that is sealed from the external environment, and wherein the processing of cells occurs within the closed system without transferring the cells out of the container or set of pipes of the closed system. Similarly, when referring to the transition of cells between various method steps (particularly MAP steps I-III), the term "closed container system" refers to the following transition means, namely, comprising the container used in the step (e.g., a container for purification, a container for transfection, a container for rebuffering), and optionally a transport container between these steps. The container can be a pipe, especially in the case of a transport container. Optionally, the method steps (MAP steps) can also be performed in a pipe. Any closed system suitable for ex vivo cell processing methods can be used with the method of the present invention. Once a cell sample, such as immune cells of blood or tumor fragments, is obtained and added to a container of a closed system, the cells will not be transferred out of the closed system until the modified immune cells are ready to be administered to the patient. At least one process control can be cleanly extracted, and / or nucleic acids can be cleanly introduced into the closed system. Thus, when immune cells are transferred from one step of the modification process to another, the container of the closed system is not opened to the outside environment.

[0033] Provided are ex vivo or in vitro methods for transiently modifying immune cells, preferably unstimulated resting or activated immune cells, in a closed processing system to at least partially delete or reduce immunosuppressive modulators and / or add immune cell enhancing factors, and immune cell populations transfected in a closed system for use in methods for treating cancer.

[0034] The present invention describes novel cell therapy, in particular an ex vivo method for transiently modifying unstimulated resting or activated immune cells in a closed processing system to at least partially delete or reduce immunosuppressive regulators and / or add immune cell enhancing factors, comprising the following steps:

[0035] (i) obtaining immune cells from previously collected biological fluids and / or resected tumors from patients;

[0036] (ii) purifying immune cells;

[0037] (iii) transfecting purified immune cells;

[0038] (iv) rebuffering the transfected immune cells into physiological solution;

[0039] (v) transferring the transfected immune cells into a container, wherein the transfected immune cells are a therapeutic immune cell population;

[0040] The transition from one step to another takes place in a container that does not need to be opened.

[0041] Also provided is an in vitro or ex vivo method for modifying immune cells, comprising the following steps: (i) providing immune cells from a biological fluid and / or from a resected tumor of a patient, thereby providing a cell sample; (ii) purifying immune cells of the cell sample; (iii) transfecting the purified immune cells with an inhibitory nucleic acid of an immunosuppressive regulator of the immune cells or with a nucleic acid of an immunopotentiating factor; (iv) purifying the transfected immune cells and / or rebuffering the transfected immune cells into a physiological solution; (v) transferring the transfected immune cells to a container; wherein the transition between each two sequential steps of steps (i) to (v) is in a closed container system. The present invention provides a completely closed, automated and therefore real-time and time-saving process, without the need to open the system to an open environment, and the method provides immune cells for personalized treatment.

[0042] A closed container system refers to a system that contains a cell sample (cell sample container) or a processed cell sample and all containers for reagents used in steps (ii), (iii) and (iv), and optionally an excess fluid removal container, which are all closed and not open. The container for reagents in step (ii) can be a buffer container, such as an erythrocyte lysis buffer container. The container for cell sample processing in step (ii) can be a cell purification container. The container for sample processing in step (iii) can be a transfection container. The container for reagents in step (iv) can be another buffer container. The container for cell sample processing in step (iv) can be another cell purification container. Cell sample containers, buffer containers, cell purification containers, transfection containers, another buffer container, another cell purification container, or any combination thereof can be connected with a pipeline. The connection can be permanently permeable for a cell sample or a reagent, or can be intermittently permeable for a cell sample or a reagent. Intermittent permeability can contain interruptions in permeability caused by a closing device (closing means) such as a valve. Paired closures, such as valves, can be used, and the connection is interrupted by closing two closures without opening the system to the environment. The connection can then be reconnected and restored to permeability by opening the closure. The reconnection connection is preferably sterile or clean, that is, such as by sterilization temperature treatment, such as 120°C or higher, such as in pipe welding. It is believed that the closing and opening of such closures (such as valves) without exposing the cell sample to an open environment is still an embodiment of a closed container system. This allows a transport container, for example, which can be used between different devices for performing certain steps, such as a cell purification device for step (ii), such as a leukocyte apheresis device, or an electroporator device for step (iii), or a cell purification device for step (iv). By intermittent interruptions of the connection, the device of step (ii) can be the same device as the device of step (iv). Reagents (such as buffers) can also be introduced into the closed container system and later connected in the method when required by each method step. The following connection examples can be used:

[0043] The method starts with a cell sample. The cell sample can be a sample obtained directly from a patient. Examples are serum samples, lymph samples or tumor fluid samples. It is usually not purified, but represents a body fluid from a patient. Obtaining a sample from a patient can be a step prior to the method of the invention. Typically, the method of the invention starts with a sample in a cell sample container, such as a body fluid bag.

[0044] The cell sample container (containing the cell sample) is connected to the cell purification container. The cell sample is transferred to the cell purification container by connection, such as a pipeline. The cell purification container is connected to a buffer container. The buffer container contains reagents for method step (ii), which are introduced into the cell purification container in step (ii). Once again, the connection can also be a pipeline. The buffer container is typically a storage bag or flask for holding buffer. The purification container can be a container suitable for cell purification, such as purification by leukocyte apheresis. An example is a filter container, such as a rotary membrane filter container or a countercurrent centrifugal system. The filter container contains a filter for separating immune cells from impurities in the cell sample. Impurities can be removed from the cell sample in step (ii). Impurities are, for example, platelets. For step (ii), in combination or as an alternative, it can also be erythrocyte lysis. The lysed erythrocyte fragments can be removed by filtration as separated impurities.

[0045] The purification container can be connected to the transfection container so that the purified immune cells are transferred to the transfection container for step (iii). It can be transfected with inhibitory nucleic acids or with nucleic acids of immunopotentiating factors (both referred to as transfection nucleic acids). Examples are siRNA for inhibitory nucleic acids and nucleic acids encoding T cell receptors for antigens such as tumor antigens, as immunopotentiating factors. The transfection nucleic acid can be provided in another container, such as a syringe. The other container is also connected to the transfection container as a closed system or to a transport container for transition between steps (ii) and (iii), such as a pipeline or a transport container. The closed system is connected as above, for example, by using a closure device and opening the closure device once the container is attached, thereby avoiding the opening to the open environment.

[0046] The transfection vessel can be located in a transfection device. An example of a transfection vessel can be a tube, such as for flow electroporation.

[0047] After transfection, the transfected immune cells are now in a buffer that may still contain the transfection nucleic acid and potential cell debris from the immune cells due to cell damage during transfection. The transfection nucleic acid and cell debris are removed during step (iv). Rebuffering may also be performed during step (iv) to produce a pharmaceutically acceptable physiological buffer for reinsertion, such as by infusion into a patient.

[0048] The transfection container is connected to another cell purification container. As mentioned above, the connection can be interrupted by a closure device, such as a paired closure device, to achieve transportation in a closed system. The transfected immune cells are transported to another cell purification container. Another cell purification container can be connected to another buffer container. Like the buffer container and purification container for step (ii), the connection between another cell purification container and another buffer container can also be interrupted, for example, by a closure device. These can be opened to allow reagents to flow. Further purification containers can be containers suitable for cell purification, such as purification by membrane purification. An example is a filtration container, such as a rotary membrane filtration container. The filtration container contains a filter for separating immune cells from impurities.

[0049] After purification in step (iv), the transfected and purified immune cells are transferred to a container in step (v), for example, to a drug administration container such as an infusion bag.

[0050] In a preferred embodiment of the present invention, each step follows each of the previously mentioned steps without interruption.

[0051] During steps (ii) and (iv), waste liquid may accumulate, for example by filtration. The waste liquid may be removed, for example by transferring to one or more waste containers.

[0052] The method represents a rapid, efficient and scalable in situ GMP production and handling process. Cells do not need to be cryopreserved at all. Long-term production at a central facility with complex logistical barriers is not necessary. Ex vivo or in vitro methods can be performed decentralized and local manufacturing is possible. Therefore, shipping of cryopreserved cells can also be avoided.

[0053] In addition, the protocol can be flexibly adapted to the needs of the physician and the patient. Therefore, the highest level of individualized treatment can be provided to tumor patients, and in particular, the patient's current tumor status is always treated. Tumor cells have a tendency to escape recognition by the immune system. Using the patient's current immune cells for the method of the present invention has the following advantages: the patient can be provided with a therapy with currently necessary modified autologous immune cells, which reflects the current immune response against the tumor.

[0054] Preferably, the immune cells are mononuclear or polymorphonuclear immune cells, preferably peripheral blood mononuclear cells (PBMC), preferably peripheral blood lymphocytes (PBL), natural killer (NK) cells, T cells, B cells, monocytes and / or tumor infiltrating lymphocytes (TIL) from solid tumors or tumor-associated fluids. It is also advantageous to use blood cells from recent patients for the methods and production of immune cell populations of the present invention, which are provided as a pharmaceutical product.

[0055] As used herein, the term immune cell refers to a cell population comprising, substantially consisting of, or consisting of one or more immune cells. In a preferred embodiment, the immune cell is a blood immune cell or an immune cell from a solid tumor. One or more immune cells are selected from neutrophils, antigen presenting cells, PBMCs, granulocytes, leukocytes, lymphocytes (T cells, B cells, NK cells, innate lymphocytes), monocytes, macrophages and / or dendritic cells. In a preferred embodiment, the immune cell is an autologous immune cell. In addition, many various immune cells can be used for transient silencing in a closed processing system to treat cancer.

[0056] According to the present invention, mononuclear cells are transiently transfected. Transient transfection in the context of the present invention means that nucleic acid molecules (inhibitory nucleic acids and / or nucleic acids of immune enhancing factors) are introduced into cells but are not integrated into the genome of the cells. Therefore, the introduced nucleic acid molecules cannot be inherited by their offspring.

[0057] The immune cell population is mononuclear or polymorphonuclear cells, preferably peripheral blood mononuclear cells (PBMC) and can be obtained from previously collected peripheral blood, bone marrow, cerebrospinal fluid, cerebrospinal fluid and / or tumor tissue of a subject (patient).

[0058] In another embodiment of the invention, the immune cells may be tumor infiltrating cells (TILs), which may be obtained from single cell preparations of primary tumors and / or metastases and / or spinal cord or cerebrospinal fluid.

[0059] In a further preferred embodiment, immune cells, preferably PBMCs, are obtained from a patient by apheresis, preferably by a single standard leukapheresis procedure, and the tumor infiltrating cells are obtained from processed tumor fragments or tumor-associated fluid. The leukapheresis product from each patient is received in a container, preferably in an output bag from the leukapheresis. Of the cell samples, the leukapheresis sample contains approximately 1 x 10 6 Up to 1x10 10 PBMC / kg, preferably 1.2x10 8 The target cell number is PBMC / kg. In the case of leukocyte apheresis, a large number of PBMCs can be obtained. PBMC / kg refers to the number of PBMCs per kg body weight of the patient (subject).

[0060] Thus, the immune cells comprise, consist essentially of, or consist of one or more leukapheresis products containing, for example, PBMCs or leukocytes, or processed tumor fragments, or one or more subsets thereof. The immune cell preparation may also comprise other cells, for example, non-immune cells, such as red blood cells or platelets.

[0061] In preferred embodiments, the immune cells are autologous immune cells that comprise, consist essentially of, or consist of specific immune cell types, such as T cells and / or NK cells.

[0062] For example, cells of a leukapheresis product containing PBMCs can be further processed, e.g., to enrich for certain immune cell types, e.g., by depleting other cell types, by specific stimulation, e.g., with tumor antigens or tumor cells from a subject, or by isolating certain immune cell types from an immune cell population.

[0063] In a preferred embodiment, the first process control is to cleanly extract from the single blood component output material or from the tumor resection material previously collected from the patient. White blood cell and platelet count and hematocrit and test sterility can be measured by complete blood cell count. Clean extraction control sample will not open the system to the environment. It can pass through a closed connection, such as having a closure device at a connecting device (such as a pipeline or a needle), and these closure devices are in a closed configuration when the connecting device is connected to the container, wherein the connection step is clean or aseptic, such as by sterilization, such as by increasing the temperature, such as 120 ℃ or higher, such as by a pipeline welder. Another option is to connect, such as by a membrane or a filter valve.

[0064] The immune cells of the cell sample can be purified, in particular washed, preferably with a phosphate buffered saline EDTA solution to eliminate cell debris. The washing step can be performed in several cycles.

[0065] During the first process step, MAP step I (method step (ii)), the platform method allows for red blood cell (RBC) lysis and platelet (PLT) depletion, preferably automated, if necessary, and then re-buffering of leukocytes into electroporation (EP) buffer for the following MAP step II (method step (iii)).

[0066] Before starting the leukocyte purification of MAP step I, the buffer bag and the leukocyte collection bag are cleanly connected to the corresponding ports of the disposable tubing set, which is then installed on the cell processing device.

[0067] After entering the required parameters and completing the device setup and startup procedures, the starting material containing leukocytes is cleanly connected to the input port of the tubing set. Depending on the cell processing device used, the entire contents or an aliquot of the starting material is automatically transferred to the tubing set to run MAP Step I. The successful completion of the cell processing run is reported by the internal device monitoring system for batch release.

[0068] In the detailed process of MAP Step I, several wash cycles deplete smaller sized cells and cell debris from the starting material, enabling PLT depletion, while RBCs are removed by using an incubation cycle for lysis. Lysis can be performed before or after loading the starting material. After RBC lysis, the leukocytes are washed at least once in a wash cycle and rebuffered into the respective electroporation buffer. The entire MAP Step I protocol requires ≤2 hours to collect ≤200 mL of leukocyte suspension resuspended in electroporation buffer.

[0069] In a preferred embodiment of the invention, 6 cycles of PBMC purification are performed. In cycles 1 to 3, erythrocytes are lysed and platelets are depleted, and in cycles 4 to 6, platelets are further depleted and cells are rebuffered in transfection buffer, preferably electroporation buffer.

[0070] Preferably, in the first cycle, the cell sample, preferably PBMCs, is washed with a PBS / EDTA solution.

[0071] After and / or during the purification of immune cells, preferably in a second cycle, the red blood cells (RBC) of the cell sample are lysed, preferably by using a lysis buffer having a pH value of 7 to 8, preferably a pH value of 7.4, containing chloride ions, ammonium ions, potassium ions and / or bicarbonate ions, preferably an ammonium chloride potassium lysis buffer, and platelets are depleted. When using a lysis buffer as described above, only red blood cells are lysed, while all other cells, in particular immune cells in the sample, are not damaged and remain viable.

[0072] In some embodiments of the invention, the immune cells of the cell sample can be purified and washed again, preferably in a third cycle, to deplete platelets and cell debris, preferably using a spinning membrane technique, elutriation and / or countercurrent centrifugation. Using a spinning membrane technique, preferably, the filter membrane pore size of the spin column is less than 4 μm.

[0073] Furthermore, the immune cells can be rebuffered into the transfection buffer after lysis of red blood cells and platelet depletion to produce a cell sample solution in the container that can be used for further processing, preferably in cycles 4 to 6. In these cycles, cell debris, especially platelets, can also be further depleted.

[0074] At the end of the purification and rebuffering procedure, the leukocytes resuspended in the transfection (preferably electroporation) buffer are transferred (preferably pumped) into a leukocyte collection bag which is then cleanly reconnected to the transfection (preferably electroporation) apparatus tubing set to proceed directly to MAP step II including transfection of the leukocytes by electroporation.

[0075] Preferably, at this point, the immune cells in the cell sample solution are cooled before modification, preferably before electroporation. The leukocyte bag can be stored in a refrigerator at 2°C to 8°C for at least 10 to 15 minutes to pre-cool until proceeding to MAP step II.

[0076] According to the present invention, after purification and resuspension of immune cells, a second in-process control can be cleanly extracted from the cell bag containing the purified and resuspended mononuclear cells in the cell sample solution to determine, for example, the activity and efficacy of immune checkpoint inhibition, in particular to measure the default state of immune cell activity with respect to transfected gene expression and efficacy, for example, with respect to IL-2 production when the cells are transfected with cbl-b siRNA.

[0077] Modification, in particular transfection, of immune cells in a cell sample can be performed by microinjection, liposomes, electroporation, particle guns, magnetic-assisted transfection, sonoporation, nanoparticles and / or cell squeezing techniques.

[0078] Using an automated, closed electroporation system, large numbers of leukocytes can be treated by electroporation, preferably continuous flow electroporation.

[0079] In an alternative embodiment of the present invention, immune cells can also be electroporated in a single cuvette in a closed system.

[0080] In a preferred embodiment of the present invention, the modification of immune cells is carried out by using flow electroporation of siRNA for at least one immune checkpoint inhibitor. Flow electroporation technology uses electric charge to produce the reversible permeability of cell membrane. This allows molecules (such as nucleic acids) to be transferred into cells, thereby creating cell therapy. In an alternative embodiment of the present invention, the electroporation of immune cells can be carried out in a large capacity cuvette.

[0081] Transfection of immune cells with at least one nucleic acid (preferably DNA, mRNA or siRNA) and / or peptide nucleic acid (PNA), preferably electroporation, results in modification of the gene expression pattern of one or more immune checkpoint inhibitor genes, chemokine receptors, cytokines and / or chimeric antigen receptors.

[0082] As used herein, the term "nucleic acid" refers to a nucleic acid that is substantially free of other macromolecules such as lipids, polymers, and proteins. Nucleic acids, such as self-replicating RNA, are not formulated with other macromolecules to improve cellular uptake. Thus, nucleic acids are not encapsulated in, adsorbed on, or bound to liposomes, microparticles or nanoparticles, cationic emulsions, and the like.

[0083] Alternative forms of RNA, called small interfering or "siRNA," can affect cells differently than mRNA, preventing the production of normal proteins rather than triggering their production. There are two forms of siRNA therapy: antisense oligonucleotides (ASOs) and RNA interference (RNAi). RNA silencing is a conserved biological response to double-stranded RNA molecules that regulate gene expression. ASOs and RNAi are short RNA sequences that can specifically bind to a target sequence and block normal protein production.

[0084] In RNA interference technology, siRNA binds to the RNA-induced silencing complex ("RISC"), where one strand (the "passenger strand" or "sense strand") is displaced and the remaining strand (the "guide strand" or "antisense strand") cooperates with RISC to bind to complementary RNA (target RNA). Once bound, the target RNA is cleaved by the RNA endonuclease Argonaute (AGO) in RISC and then further degraded by RNA exonucleases.

[0085] Key challenges in developing oligonucleotide therapies (e.g., siRNA therapies) include (i) poor compound stability, (ii) low efficiency of in vivo delivery to target cells, and (iii) side effects such as "off-target" gene silencing and undesirable immune stimulation. Among them, the most significant obstacle is the targeted delivery of RNA and subsequent cellular uptake. In order to overcome some of these obstacles, various chemical modifications of oligonucleotides have been attempted, including (i) sugar modifications, (ii) internucleotide linkage modifications, and (iii) nucleobase modifications. Although these chemical modifications lead to enhanced stability and reduced immunogenicity of RNA, these modifications are still insufficient to deliver these large, negatively charged macromolecules across the negatively charged phospholipid bilayer of the cell membrane and into the cytoplasm.

[0086] For siRNAs used as therapeutics, structural modifications are essential to stabilize the molecule and prevent its destruction. A hallmark of RNA silencing is a class of short RNAs that are processed from double-stranded precursors by the enzyme Dicer.

[0087] There are a variety of approaches being used to treat cancer, including mRNA vaccines targeting cancer-associated antigens and siRNA that silences cancer genes. A 2010 clinical trial demonstrated that the siRNA approach can prevent cancer growth (Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA, Yen Y, Heidel JD, Ribas A. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature. 2010 Apr 15;464(7291):1067-70.).

[0088] Gene modification by siRNA is transient and in contrast to other cell therapies where long-term persistence of gene-modified cells is observed.

[0089] After the discovery of RNA interference, research focused on opportunities to actively interfere with target protein production by using small (about 22 nucleotides) siRNA molecules. Making these siRNAs stable and resistant to degradation by enzymes presents another challenge. The use of siRNAs in therapeutic approaches has the following advantages: by making cells permeable, they can be easily introduced into cells. Transient permeability can be achieved by methods such as extrusion, electroporation, or chemistry. siRNAs are designed to bind specifically to their target sequences, thereby preventing the silencing of other genes ("off-target" effects), a phenomenon commonly seen in other cancer therapies. Although the target gene is transiently inhibited, minimizing safety concerns, RNAi is expected to be used to treat diseases in many different areas, including immuno-oncology. In this case, siRNA silencing of novel immune checkpoints (such as Cbl-b) reactivates the anti-tumor immune response and triggers the destruction of cancer cells.

[0090] The siRNA of the present invention targets Cbl-b, wherein the siRNA is selected from the group consisting of a plurality of siRNA sequences targeting Cbl-b.

[0091] In a preferred embodiment, at least one gene or portion of a gene for one or more immune checkpoint inhibitors is silenced or reduced, and / or at least one gene or portion of a gene for one or more chemokine receptors, cytokines and / or chimeric antigen receptors or a combination thereof is inserted into at least a portion of the transfected immune cell population.

[0092] This transient transfection results in transient target gene silencing, because neither the siRNA nor the mRNA molecules are stably integrated into the target cell genome and are degraded after a certain period of time, allowing the target gene expression to resume.

[0093] After removing the sample for process control, the syringe containing the nucleic acid payload, siRNa or mRNA or DNA, is connected to the leukocyte bag and the payload is transferred into the leukocyte bag for subsequent electroporation.

[0094] After payload transfer, the electroporation device tubing set connected to the leukocyte collection bag was installed on the electroporation device to run closed, automated electroporation.

[0095] Serial electroporations were performed automatically to process the leukocyte suspension within a time frame of approximately one minute per electroporation.

[0096] Drug substance (DS), defined as autologous leukocytes transfected with the payload, is collected in the leukocyte bag for further processing.

[0097] The DS is further processed after an optional holding time of 30 to 60 minutes at room temperature depending on the device to generate the drug product (DP) by cleanly reconnecting the leukocyte bag containing the DS to the cell processing device tubing set for final formulation. No defined DS holding or storage time applies.

[0098] The processing pathways for cell processing devices and electroporation devices are programmed by the respective equipment manufacturers or during method development. All programs are access controlled and an audit trail can be used to track changes.

[0099] In some embodiments of the present invention, the one or more immune checkpoint genes are selected from the group comprising Cbl-b, PD1, PD-L1, PD-L2, CASP, CTLA4, FoxP3, LAG-3, TIM-3, TIGIT, A2AR, KIR, BTLA, VISTA, B7-H3, B7-H4, SHIP, SHP-1, SHP-2, IL-1R8, NKG2A, CD96, CD112R, IDO, IRG-1, STAT-3, JAKs, Arg-1, Nos-2, Cish, TGFb, PKA, TNFRSF, preferably, encoding human Cbl-b and / or SHIP genes or combinations thereof. The target nucleic acid sequence can be a sequence or a combination thereof of a further immunosuppressive regulator, chemokine receptor and / or cytokine. Any of these immune checkpoint genes, immunosuppressive regulators, and chemokine receptors can be targeted by transfected nucleic acids, especially inhibitory nucleic acids.

[0100] In another embodiment of the present invention, the transfected nucleic acid encodes at least one gene selected from TMEM222, SOCS1, CDKN1, CD5, UBASH3A, RASA2, CD5, TNFAIP3, TCEB2, NR2F6, TET2, BiTE or a combination thereof. These are examples of nucleic acids of immune enhancing factors.

[0101] The present invention may further comprise CAR-T cells engineered by mRNA transfection, for example, expressing chimeric antigen receptors (CARs) against EGFR, NY-ESO, CEA, GD2 to transiently express proteins.

[0102] Blocking Cbl-b function thus activates adaptive and innate anti-tumor mechanisms, leading to more effective tumor destruction. Cbl-b-silenced PBMCs represent a novel approach with the concept of enhancing anti-tumor immune responses, thereby improving cancer therapy.

[0103] Cbl-b is known as a major immune checkpoint that regulates various immune pathways in a cell type-dependent manner.

[0104] Regulation of Cbl-b is central to combating tumor cells in a wide range of tumor indications. Cbl-b acts as a negative regulator and major signaling hub for tumor-specific immune cell activation. Cbl-b can be silenced by binding to Cbl-b mRNA via siRNA. Cbl-b deficiency activates a variety of immune cells, such as helper T cells, cytotoxic T cells, B cells, and NK cells, to destroy tumors.

[0105] The following sections describe the role of Cbl-b in anti-tumor and autoimmunity. The Cbl-b gene encodes a member of the E3 ubiquitin ligase that mediates the attachment of ubiquitin to various secondary signaling molecules within immune cells. Along this line, Cbl-b regulates various immune stimulatory pathways in a cell type-dependent manner and has therefore been described as a "master checkpoint" of immune function.

[0106] Cbl-b plays a role in both the adaptive immune system (T and B cells) and the innate immune system (natural killer (NK) cells and monocytes). Therefore, blocking its activity in T cells and other immune cell subtypes can effectively enhance tumor antigen-specific activity and recognition of general tumor cell features.

[0107] In T cells, E3 ubiquitin ligase activity limits cell activation and can promote anergy (T cell failure to respond to self-antigens, Schmitz, 2009). Ubiquitin ligation to secondary signaling molecules disrupts T cell activation via the major histocompatibility complex (MHC) / T cell receptor (TCR). Deficiency in Cbl-b eliminates the need for CD28 co-stimulation for downstream activation in CD3+ T cells, thus establishing a positive role for Cbl-b in the induction and maintenance of peripheral T cell tolerance (Jeon et al., 2004). Therefore, silencing of Cbl-b promotes T cell activation.

[0108] Tumors lack co-stimulatory proteins that bind CD28, which helps minimize T cell activation; in addition, CD28 expression is often lost on T cells that differentiate into effector cells after exposure to repeated stimulation (Topp et al., 2003). Disruption of Cbl-b expression in such tumor-reactive T cells may provide a unique strategy to increase autocrine interleukin 2 (IL-2) production, thereby promoting proliferation and survival after tumor recognition in the absence of co-stimulatory molecules. This approach may also lower the threshold for T cell activation, allowing low-affinity T cells to respond to tumors that express low amounts of antigen.

[0109] In a preferred embodiment of the present invention, cells are transiently transfected with Cbl-b siRNA molecules by electroporation. Cbl-b checkpoint inhibitors have unique advantages over traditional checkpoint blockade. Since other checkpoint inhibitors mainly act on T cells, Cbl-b inhibitors show universal effects based on the reactivation of anti-tumor immunity of various cell types: CD4+ T cells, CD8+ T cells, NK, NKT, B cells, antigen presenting cells (APC).

[0110] Drug products containing silenced Cbl-b cells could be used as monotherapy or in combination with other immune checkpoint inhibitors or other anti-tumor therapies.

[0111] Any siRNA and mRNA of the immune checkpoint inhibitor can be used for electroporation at a concentration of 1.0 to 10 mg / mL, preferably 5.0 mg / mL in phosphate-buffered saline.

[0112] The siRNA is injected into the container containing the cell sample solution, preferably using a syringe.

[0113] According to the present invention, siRNA is injected through a 0.2 μm filter connected to a wipeable Luer secondary process control withdrawal port, thereby ensuring a closed manufacturing process and avoiding the insertion of particles.

[0114] In some embodiments of the invention, the combination of immune cells and siRNA in a transfection buffer, preferably an electroporation buffer, is processed in an automated electroporation device in multiple electroporation cycles.

[0115] Preferably, the time frame of each electroporation cycle is 10 seconds to 5 minutes, preferably 30 seconds to 2 minutes, most preferably 1 minute.

[0116] The mRNA silencing efficiency of transfected mononuclear cells exceeded 15% compared with that of untreated mononuclear cells.

[0117] In the final rebuffering process step (step iv), further depletion of residual undesirable cells and cell debris is performed, and then the leukocytes are rebuffered into the final solution for administration, preferably a 0.9% NaCl solution containing a stabilizer such as albumin or a carbohydrate source. In addition, the electroporation buffer as a drug matrix and excess free payload are greatly reduced.

[0118] The collected leukocyte suspension is then adjusted to a target volume containing a specified number of cells for infusion.

[0119] Purified transfected immune cells (drugs) can be stored in bags directly removed after the last manufacturing step and / or can be infused from bags directly removed after the last manufacturing step.

[0120] An aliquot of the clean draw drug product is used for subsequent release testing. After the aliquot is removed, the remaining volume defines the maximum manufacturable dose (MMD) of leukocytes for possible transfusion.

[0121] In the final step (v), the immune cells can be transferred to a container, preferably an infusion bag to receive the drug. Preferably, the autologous transfected immune cells are rebuffered into a final formulation, which is a physiological formulation with stabilizers, preferably a 0.9% NaCl solution containing 1% to 4% autologous human serum, preferably a 0.9% NaCl solution containing 0.7 g / l to 4.5 g / l human serum albumin, for improving the viability and stability of the drug.

[0122] In parallel with the rebuffering step, the immune cells are purified and further depleted of residual platelets and cell debris, preferably using a spin membrane technique and / or elutriation. In addition, the electroporation buffer and excess siRNA are reduced to receive a therapeutic immune cell population for the final drug product.

[0123] In a preferred embodiment of the present invention, the cell concentration in the final solution is about 5×10 5 Up to 5x10 10 , preferably 5x106 Up to 5x10 7 Immune cells / mL.

[0124] Most preferably, the concentration of transfected cells in the final solution is 5x10 5 to 5x10 6 Up to 5x10 7 mononuclear cells / mL and construct the drug.

[0125] Preferably, the drug contains more than 40% mononuclear cells among the leukocytes.

[0126] It is also possible to collect the transiently modified immune cells of the final formulation directly in the infusion bag, while the cell aggregates larger than 200 μm are filtered through the membrane, so that the cell aggregates larger than 200 μm do not reach the final infusion bag. The membrane improves product analysis, because the collected samples do not contain any inhomogeneities, and the visual inspection of the drug product will show at most only very small aggregates, even at the end of the shelf life.

[0127] According to the present invention, at least one container, preferably a bag with immune cells and / or at least one buffer container, at least one pipe set connecting the containers, and / or at least one removal port, preferably for process control, are cleanly interconnected using a sterile pipe welder.

[0128] According to the present invention, immune cells can be expanded in a closed system before and / or after immune cell transfection, preferably electroporated to obtain more number of cells. Generally, unstimulated cells are used for the present invention. The source material of the inventive method is preferably provided by leukocyte apheresis, which allows to gently separate high PBMC quantity (also referred to as "leukocyte bag" or "leukocyte apheresis product") from the patient. The present invention is effective in the case of using PBMC without the need for a cell amplification step. Therefore, the process of the present invention requires about 6 hours, and the autologous modified cells are re-infused into the patient within the same day. But for example, when tumor infiltrating cells (TIL) are used to implement the method of the present invention, these cells will have to be expanded and therefore can be stimulated or activated. The amplification of immune cells can be carried out before immune cell transfection, or can be carried out after immune cell transfection, or can be carried out before and after immune cell transfection.

[0129] The present invention also relates to an immune cell population for use in a method for treating cancer, wherein the immune cell population can be obtained by the method of the present invention. For example, the method performed in a closed system comprises:

[0130] (i) obtaining immune cells from previously collected biological fluids and / or resected tumors from patients;

[0131] (ii) purifying immune cells;

[0132] (iii) transfecting purified immune cells;

[0133] (iv) rebuffering the transfected immune cells into physiological solution;

[0134] (v) transferring the transfected immune cells into a container, wherein the transfected immune cells are a therapeutic immune cell population;

[0135] Wherein the transition from one step to another step occurs in an unopened container or in a closed container system. Immune cell populations (drugs) can be generated and tested within about 5 hours to be reinfused into the patient, and the acquisition of cells (preferably leukocyte apheresis) is not included in these 5 hours. It is administered as a freshly manufactured cell suspension, neither cryopreserved, nor ex vivo or in vitro amplified, and must be reinfused into the patient before cell viability and efficacy are reduced. In addition, rapid release testing allows the drug to be reinfused within 2 hours after completing drug manufacturing. In the case of reinfusion of TIL, the expansion of cells is advantageous.

[0136] In a preferred embodiment of the invention, after transfection, preferably after electroporation, more than 30% of the immune cells (preferably NK cells, T cells and B cells) of the biological sample contain siRNA, mRNA or DNA.

[0137] The immune cell population of this embodiment is used for autologous cell therapy, which uses at least one immune checkpoint inhibitor selected from the group comprising Cbl-b, PD-1, PD-L1, PD-L2, CASP, CTLA4, FoxP3, LAG-3, TIM-3, TIGIT, A2AR, KIR, BTLA, VISTA, B7-H3, B7-H4, SHIP, SHP-1, SHP-2, IL-1R8, NKG2A, CD96, CD112R, CD160, CD244, IDO, IRG-1, STAT-3, JAKs, Arg-1, Nos-2, Cish, TGFb, PKA, TNFRSF or a combination thereof to treat various cancers.

[0138] Immune cell populations are used to treat cancer.

[0139] Preferably, the immune cell population is provided for the treatment of leukemia, head and neck cancer, glioblastoma multiforme (GBM), lung cancer, colon cancer, pancreatic cancer. At least all solid tumors and blood tumors

[0140] Treatment is available in both early and late stages.

[0141] The invention further describes methods of administering a population of immune cells to treat or prevent a neoplastic disease in a subject.

[0142] The immune cell population is administered in at least one dose, and one dose to be administered to a patient contains 5x10 6 Up to 5x10 10 , preferably 5x10 7 Up to 5x10 8 transfected PBMC / kg or TIL / kg, so that the newly cultured immune cells due to systemic and / or intratumoral and / or intranodal reinfusion travel through the blood to seek tumor cells. / kg refers to each kg of the patient's body weight.

[0143] According to the present invention, the patient receives multiple administrations of the immune cell population intravenously or intratumorally or intranodally, preferably 1 to 10, preferably 3 to 5, and the modified immune cell infusions are administered at intervals of 2 to 10 weeks, preferably 3 to 7 weeks, most preferably 4 weeks, for at least 2, preferably 3 cycles.

[0144] The transfected immune cell populations produced according to the method of the invention are preferably used for the treatment of tumor diseases, but can also be used for prevention in special patient populations.

[0145] The drug comprises autologous human PBMCs transfected in vitro or in vitro with small interfering RNA, which can silence Cbl-b messenger RNA. Cbl-b-silenced PBMCs are returned to the patient, where they can respond to tumor antigen stimulation by enhanced proliferation and cytokine production. Cbl-b-silenced cytotoxic T cells and NK cells can directly mediate tumor cell killing. Cbl-b-silenced B cells and mononuclear cells can act as antigen presenting cells and trigger a stronger immune response to tumor antigens.

[0146] The cell therapy of the present invention has multiple advantageous properties compared to other cell therapies. The genetic modification is transient, can treat solid and liquid tumors, and activate innate and adaptive immune cells.

[0147] The manufacturing process is affordable, real-time and more scalable without being expensive and time-consuming.

[0148] Treatment with the modified immune cells was safe and well tolerated and did not show any toxicity.

[0149] Leukocyte apheresis can be performed at the production site or at the clinical treatment site. Cell samples do not need to be transported.

[0150] The flexibility of RNAi technology may expand the applicability of cell therapy to additional immune checkpoints and holds potential for the development of new therapeutic approaches.

[0151] The present invention is further described below by means of the accompanying drawings and examples, but the present invention is not limited to these embodiments.

[0152] Attached pictures:

[0153] Figure 1 . sfFACS data show the composition of the drug;

[0154] Figure 2 . sfFACS data illustrate the characterization of drugs;

[0155] Figure 3 . sfFACS data illustrate the impurities of drug products;

[0156] Figure 4 . A flow chart of the fully closed manufacturing process for the production of the drug;

[0157] Figure 5 . Immune subtype distribution in PBMCs electroporated with Cbl-b siRNA;

[0158] Figure 6 . Screening of optimized siRNAs for silencing Cbl-b in PBMCs;

[0159] Figure 7 Cytokine induction as a parameter for Cbl-b silencing efficiency in PBMCs;

[0160] Figure 8 . Compare the IL-2 fold increase of the drug to the no-pulse control as measured by ELISA;

[0161] Fig. 9 . Percentage of Cbl-b RNA expression of drug compared to no pulse control;

[0162] Fig.10 . Use qPCR to measure the percentage of Cbl-b silencing comparing drug and no-pulse control;

[0163] Fig.11 Comparison of Cbl-b silencing percentage at protein level between DP and NPC using WB analysis;

[0164] Fig.12 Quantification of Cbl-b in DP and NPC cell lysates using LC-MS / MS;

[0165] Fig.13 IFN-γ production in response to peptide stimulation;

[0166] Fig.14 . eGFP fluorescence in eGFP-mRNA-transduced leukocytes quantified by flow cytometry;

[0167] Fig.15 Protein expression of TCEB2 / ELOB and Cbl-b in leukocytes transduced with 4x siRNA pools;

[0168] Fig.16 Protein expression of TCEB2 / ELOB and Cbl-b in leukocytes transduced with four different siRNAs;

[0169] Fig.17 IL-2 and IFN-γ production by leukocytes silenced for TCEB2 (pool of four siRNAs) or Cbl-b as measured by ELISA;

[0170] Fig.18 IL-2 and IFN-γ production by leukocytes silenced for TCEB2 (single siRNA: TCEB2-08–TCEB2-11) or Cbl-b as measured by ELISA;

[0171] Fig.19 . mRNA expression of TCEB2 / ELOB and Cbl-b in leukocytes transduced with 4x siRNA pools;

[0172] Fig. 20 . mRNA expression of TCEB2 / ELOB and Cbl-b in leukocytes transduced with four different siRNAs.

[0173] Experimental and Examples

[0174] Human peripheral blood mononuclear cell experiment

[0175] In humans, Cbl-b deficiency enhances the antitumor activity of T cells and NK cells, resulting in enhanced expression of inflammatory cytokines and activation markers as well as proliferation and antitumor cytotoxicity upon T cell receptor (TCR) stimulation. In addition, T cells are resistant to TGF-β-mediated immunosuppression. NK cell activation is increased upon cytokine stimulation or tumor cell contact.

[0176] Primary human T cells were isolated from Cbl-b-silenced PBMCs by magnetic enrichment and stimulated with anti-TCR and anti-differentiation cluster CD28 agonistic antibodies (anti-CD3 / CD28 stimulation). Treatment resulted in enhanced IFN-γ and IL-2 production by T cells. In addition, Cbl-b silencing in human T cells also induced increased secretion of IL-12 and IL-17.

[0177] Primary human T cells were isolated from PBMCs, labeled with carboxyfluorescein succinimidyl ester (CFSE; a proliferation indicator), and transfected with control or Cbl-b siRNA. The resulting cells were stimulated with anti-CD3 / CD28 antibodies, and CFSE levels showed higher levels of cell counts at lower CFSE concentrations, indicating that cell proliferation reduced dye levels in daughter cells. Neither cytokine production nor cell proliferation was induced in unstimulated T cells, indicating that Cbl-b silencing only enhances T cell activity in the presence of antigen stimulation and does not lead to universal systemic activation of all T cells.

[0178] Transient transfection of PBMC with Cbl-b siRNA results in efficient Cbl-b elimination from both T cells and NK cells. In the presence or absence of antibodies recognizing Her2 and IL-2 and IL-12 (NK cell stimulating cytokines), PBMC transfected with Cbl-b siRNA were incubated with SKBR3 breast cancer cells for 4 hours. Although PBMC is usually a very poor effector cell in cytotoxicity assays, the PBMC silenced by Cbl-b showed enhanced SKBR3 cell killing in the presence of anti-Her2 antibodies, IL-2 and IL-12.

[0179] Animal tumor experiments

[0180] In mouse models, it can be shown that Cbl-b deficiency protects mice in various tumor models, in which tumor antigen-specific T cells mediate long-term protection, highly reactive NK cells kill primary and metastatic tumor cells, adoptive cell therapy with T cells or NK cells eradicates established tumors, and T cells and NK cells are resistant to PD-1 / PD-L1, CTLA-4 / B7, and TGF-β-mediated immunosuppression.

[0181] A mouse melanoma tumor model was used to ensure that the results seen in human PBMCs silenced in vitro with Cbl-b might be translatable to cancer immunotherapy in patients. T cells were prepared from normal mice and silenced using a protocol similar to that described in WO2009 / 073905. Transfected T cells were adoptively transferred into tumor-bearing (B16 melanoma expressing ovalbumin) mice along with dendritic cells loaded with ovalbumin. Tumor-inoculated mice received T / dendritic cell transfers 7 and 14 days after tumor cell inoculation. Tumor size was measured up to 1 month after tumor cell inoculation. The results showed that adoptive transfer of Cbl-b-silenced mouse CD8+ T cells significantly reduced tumor burden (Hinterleitner R, Gruber T, Pfeifhofer-ObermairC, et al. Adoptive transfer of siRNA Cblb-silenced CD8+ T lymphocytes augments tumor vaccine efficacy in a B16 melanoma model. PLoS One. 2012;7(9):e44295.doi:10.1371 / journal.pone.0044295).

[0182] Five days after the adoptive cell transfer event, T cells were isolated from tumor-draining lymph nodes of mice that had received transferred Cbl-b-silenced T cells and re-challenged with tumor cell-loaded dendritic cells. Sustained T cell activity was demonstrated as evidenced by the ability to continue to produce both IFN-γ and IL-2.

[0183] There were no overt signs of autoimmunity in a range of tissues in mice administered CD8+ T cells transfected with Cbl-b siRNA.

[0184] In mice bearing B16-SIY melanoma lesions, administration of 5x10 T cells three times was compared with administration of control siRNA-transfected T cells. 6 T cells transfected with Cbl-b siRNA resulted in a significant reduction in tumor volume. In a similar study for B16-SIY melanoma, mice were administered Cbl-b siRNA T cells twice (on days 9 and 14), and lymph nodes were harvested on day 21 and the characteristics of immune cells were analyzed. For CD4+ T cells, CD8+ T cells, and NK cells isolated from lymph nodes, the expression of programmed cell death 1 (PD-1) was significantly lower than that of untreated animals and animals receiving control siRNA-transfected T cells. Since PD-1 is involved in downregulating the immune system, this would indicate that Cbl-b silencing can also disrupt mechanisms that are usually involved in suppressing the inflammatory activity of immune cells at the tumor site.

[0185] In the mouse hepatocellular carcinoma model, mice were inoculated with Hepa1-6 cells and then treated with 5x10 6 T cells that had been previously transfected with Cbl-b siRNA were treated for 7 days. Administration of Cbl-b-transfected T cells reduced tumor volume by 87% by day 14 compared with animals given control-transfected T cells; the inhibitory effect on tumor growth was evident over the 23-day study period.

[0186] In the mouse colon cancer model, mice were inoculated with MC38 cells and then treated with 5x10 6 T cells that had been previously transfected with Cbl-b siRNA were treated for 7 days. By day 14, administration of Cbl-b-transfected T cells reduced tumor volume by 41% compared with animals given control-transfected T cells; the inhibitory effect on tumor growth was evident over the 20-day study period.

[0187] Thus, in a murine model, reduced levels of PD-1-expressing immune cells in mice receiving Cbl-b-silenced T cells could be observed, as well as antitumor activity of Cbl-b-silenced T cells against MC38 colon cancer.

[0188] Tumor growth in Cbl-b knockout mice was inhibited, and tumor volume was reduced after adoptive cell therapy targeting Cbl-b (Loeser, S. et al. (2007). Spontaneous tumor rejection by Cbl-b–deficient CD8 + T cells. JEM (204), S. 879-891).

[0189] Specifications of the drug product produced by the method of the present invention

[0190] Identity and vitality

[0191] To quantify the closed manufacturing process, the identity and viability (% of 7-AAD negative cells among CD3+, CD14+, CD16+, CD19+ cells) and purity (% of CD3+, CD14+, CD16+, CD19+ cells) of all live cells were tested by flow cytometry for 12 runs of drug product. In addition, impurities were measured as a percentage and CD235+ RBCs within CD45 positive leukocytes, CD41+ PLTs and residual CD3 / CD14 / CD16 / CD19 negative cells were considered as putative granulocytes. Mean ± SD is shown. WBC: white blood cell, RBC: red blood cell, PLT: platelet.

[0192] In all experiments, the starting material consisted of an average of approximately 73% viable leukocytes. Figure 1 and Table 1 depicts sfFACS data showing the composition of the drug.

[0193] Table 1. sfFACS data showing drug composition

[0194]

[0195] Characterization

[0196] By using sfFACS, the drug was analyzed based on its cellular composition involving effector cells (percentage of CD45 positive cells). About 50% of the cells were T cells, while monocytes, B cells and NK cells were about less than 20%. Figure 2 and Table 2 describe sfFACS data illustrating drug characterization.

[0197] Table 2. sfFACS data illustrating drug characterization

[0198]

[0199] Impurity Characterization

[0200] In addition, all cells were characterized to determine the amount of impurities, such as red blood cells and platelets. A large number of platelets (44.5%) and approximately 11.7% of red blood cells were detected. Figure 3 and Table 3 describe sfFACS data illustrating the characterization of drug product impurities.

[0201] Table 3. sfFACS data illustrating characterization of drug product impurities.

[0202]

[0203] Example 1: Production of Cbl-b-silenced PBMCs

[0204] Autologous cell therapy using Cbl-b checkpoint inhibitors for the treatment of various cancers is described. Example 1 describes autologous cell therapy targeting Cbl-b with siRNA based on highly specific activation of tumor-reactive blood cells.

[0205] This example evaluates the effect of autologous cell immunotherapy of patients with advanced solid tumors using PBMCs derived from the patient's own cells, which were transiently transfected with Cbl-b siRNA molecules ex vivo, and Cbl-b was silenced to reduce Cbl-b protein levels. The transfected PBMCs were then administered back to the patient. The transfected and purified PBMCs are also referred to as the produced drug.

[0206] Immune cells are sampled, for example, by leukapheresis, Cbl-b is silenced in the immune cells, and the activated cells can be reinfused into the patient. The drug is an autologous cellular immunotherapy derived from the patient's own peripheral blood mononuclear cells (PBMCs) that are transiently transfected with Cbl-b-specific small interfering RNA (siRNA) molecules that are able to reduce Cbl-b protein levels.

[0207] The described cell processing system efficiently transfers Cbl-b specific siRNA into purified PBMCs in a closed system. The Cbl-b siRNA transfected PBMCs are administered back to the patient.

[0208] Cbl-b is a key negative regulator of antitumor immunity. It limits the responsiveness of most types of immune cells, especially lymphocytes and NK cells. A series of nonclinical data demonstrate that Cbl-b inhibition simultaneously overrides multiple relevant immune “checkpoints” and modulates sensitivity to regulatory T cells, inhibition through transforming growth factor β, and immune regulation through two pathways: cytotoxic T lymphocyte-associated protein (CTLA)-4 and programmed cell death ligand 1 (PD-L1) / PD-1. Adoptive cell immunotherapy using Cbl-b-silenced murine T cells can significantly inhibit tumor growth in syngeneic mouse models. Cbl-b-silenced murine T cells have synergistic effects with anti-PD1, supporting the potential use of Cbl-b-silenced human T cells in combination with immune checkpoint inhibitors (CPIs) (Tang et al., 2019).

[0209] The drug product comprises autologous human PBMCs transfected ex vivo with siRNA that silences Cbl-b messenger RNA. The Cbl-b-silenced PBMCs are returned to the patient, where they can respond to tumor antigen stimulation by enhanced proliferation and cytokine production. Cbl-b-silenced cytotoxic T cells and NK cells can directly mediate tumor cell killing. Cbl-b-silenced B cells and mononuclear cells can act as antigen presenting cells and trigger a stronger immune response against tumor antigens.

[0210] PBMCs were transfected with candidate siRNAs by electroporation and the mRNA was taken up into immune cells. Inhibition of Cbl-b would elicit an immune response upon cell activation. The strong correlation of Cbl-b silencing in humans with enhanced immune response at both the mRNA and protein levels could be read out by cytokine secretion of IL-2, IFN-γ and TGF-β as well as TCR stimulation.

[0211] A high transfection rate of >95% was achieved for various immune cells associated with anti-tumor activity, among which the Cbl-b silencing efficiency in immune cells was >90%.

[0212] Cbl-b-silenced primary human T cells isolated from human PBMCs of healthy donors showed higher expression of activation markers and increased secretion of IL-2 and IFN-γ after in vitro T cell receptor stimulation.

[0213] Cbl-b-silenced human NK cells were more responsive to tumor cell contact, with enhanced NK cell activation by tumor cell contact, secretion of higher levels of cytotoxic granzyme B, and more efficient killing of tumor cells.

[0214] In preclinical studies, Cbl-b-deficient immune cells led to the shrinkage and complete disappearance of tumor grafts in mice, durable anti-tumor memory, and growth inhibition of non-injected tumors. It is believed that inactivating Cbl-b in PBMCs and returning the cells to patients induces a de novo anti-tumor immune response, resulting in improved priming and activation of tumor-specific cytotoxic CD8+ T cells and increased infiltration of CD8+ T cells into tumors. Therefore, silencing cells with Cbl-b siRNA and using transfected cells in therapy may be effective in treating tumors that are primarily unresponsive (refractory) to anti-PD-1 / L1 checkpoint inhibitors as well as tumors with acquired resistance to checkpoint inhibitors.

[0215] Description of the manufacturing process and process controls

[0216] For cell therapy, each batch is defined by the amount of freshly manufactured, non-cryopreserved, autologous PBMCs containing Cbl-b silencing siRNA as required for a single treatment cycle per patient. PBMCs are obtained from a single standard leukapheresis procedure for each patient, resulting in approximately 200 mL containing no less than 0.9x10 target cells per kg body weight. 8 PBMCs were processed in a completely closed system using no less than 0.9 x 10 per kg body weight. 8 The leukocyte apheresis product of an appropriate amount of PBMCs is performed.

[0217] Preferably, leukapheresis and manufacturing of the transfected cells as a pharmaceutical product are performed at the same location. Thus, no transportation is required between the collection site and the manufacturing site.

[0218] Transfected cells were manufactured using a fully contained, aseptically interconnected, three-step manufacturing process (MAP) using the LOVO PBMC Purification Device (Fresenius Kabi AG, Bad Homburg, Germany) and the MaxCyteExPERT TM GTx Transfection System (MaxCyte, Inc. Gaithersburg, MD, USA).

[0219] Figure 4 A flow chart of a fully closed manufacturing process for producing a drug product is shown, including Step I, Step II, and Step III.

[0220] The diagram includes manufacturing steps I 1, II 2 and III 3.

[0221] Before the manufacturing process begins, samples, preferably unstimulated immune cells from biological fluids and / or from tumors resected from patients, are collected and prepared in step 4, either by leukapheresis or processing of tumor samples. When tumor resection is performed, the tumor sample is processed into single cells and will be further processed.

[0222] In step 5, the sample, in particular the immune cells, is purified and resuspended, and then in step 6, transfection of the sample, in particular transfection of the purified immune cells, is performed, preferably by electroporation.

[0223] The transfected cells are obtained in step 7 and can be resuspended in electroporation buffer after final collection into the container.

[0224] In the next step 8 , the transfected immune cells are transferred to a container, preferably an infusion bag, and the immune cells are rebuffered into the final formulation, and finally, in the last step 9 , the drug product for infusion is obtained in the container.

[0225] Controls 10, 11, 12 and 13 were performed and they were taken from step 4, control IPC01 10 after step 5, control IPC02 11 and S01 12 were performed from step 9, control S02 13.

[0226] The manufacturing process (MAP) for generating transfected cells includes MAP step I 1, PBMC purification and resuspension, followed by MAP step II 2, PBMC electroporation.

[0227] PBMCs from patients undergoing leukapheresis are the starting material shown in step 4 of the flow chart ( Figure 4 ). Before the manufacturing process begins, a sample is cleanly removed from the starting material (i.e., leukapheresis product) (control IPC01 10) to measure PBMC number and content, preferably by complete blood count (CBC) to measure WBC number and hematocrit, and tested for sterility as shown in flowchart step 10. The CBC parameters are required for programming the LOVO device of MAP step I 1.

[0228] In MAP step I, PBMCs were purified and resuspended by adding PBS / EDTA solution, potassium ammonium chloride lysis buffer, and electroporation buffer, as shown in step 5 of the flowchart.

[0229] MAP Step II 2 includes transfection with Cbl-b siRNA by electroporation, as shown in step 6 of the flowchart. The duration is 20 + / - 5 minutes, approximately 1x10 10 Purified PBMCs were efficiently silenced for Cbl-b expression, showing comparable silencing efficiency at the mRNA level compared to AmaxaNucleofector 4D. TM By GTx electroporation, an average silencing of 65% of mRNA levels resulted in a reduction of Cbl-b protein below the detection limit of a mass spectrometry (MS)-based Cbl-b assay. Furthermore, effective silencing was associated with a robust and consistent induction of IL-2 release that was an average of 8-fold compared to non-silenced, no-pulse controls.

[0230] MAP Step I 1 and Step II 2 are cleanly connected to each other without reprocessing of intermediate PBMCs. Used buffers and siRNA are cleanly introduced into the manufacturing process.

[0231] During the cell transfection process, the control sample IPC02 as shown in step 11 of the flowchart is cleanly removed from the cell bag containing the purified PBMC, which contains the specification control sample S01 before electroporation in MAP step II 2 as shown in step 12 of the flowchart. IPC02 also serves as a no pulse control (NPC) for measuring the immune default state of PBMC activity (Cbl-b expression) and efficacy (IL-2 secretion) before siRNA transfection by PBMC electroporation as S01 12 in MAP step II 2.

[0232] Both Cbl-b and IL-2 levels of the no pulse control 12 provide information on activity and potency in a given final drug product.

[0233] Therefore, the manufacturing process implements an automated, closed PBMC purification step, as described in MAP Step I 1, followed by an automated, closed PBMC electroporation step, as described in MAP Step II 2, to transfect purified autologous PBMCs from the apheresis product with Cbl-b specific siRNA.

[0234] Control IPC01 10 of the starting material was used as an input control to determine leukocyte and platelet counts and hematocrit. Control IPC02 11 was drawn as a no pulse control (NPC) and served only as a specification control S01 12 for the final drug release. Control S01 1 defined the patient's immune status prior to Cbl-b silencing and was therefore used to normalize activity and potency measurements of the drug.

[0235] Electroporation of purified leukopak PBMCs to introduce Cbl-b siRNA may result in cell damage, thereby reducing cell viability and cell aggregation. Therefore, the majority of cells after transfection 6 consist of non-viable and aggregated cells and electroporated granulocytes, erythrocytes and platelets, which are largely depleted during the final MAP step III, including and as shown in step 8 of the flowchart, wherein the transfected immune cells are rebuffered to the final formulation and receive the drug product in the final container, as shown in step 9 of the flowchart.

[0236] Cell aggregation by macroscopic visual inspection (Appearance) and PBMC viability by flow cytometry (Identity) could not be tested in the transfected product, so these two parameters were only specified in the transfected and purified drug product, as shown in steps 9 and 13 of the flowchart, respectively. Impurities such as granulocytes or non-nucleated cells such as PLTs and RBCs were specified on the drug product, as shown in step 9 of the flowchart, and were assessed by flow cytometry as PBMC-negative cells among all blood cells, which functioned as an indirect purity measure. Excipient buffers and siRNA were strongly depleted in the final drug product formulation.

[0237] Both cell processing systems use a single-use tubing set for each patient batch, which is clean welded from the LOVO tubing set to the ExPERT TM The GTx tubing sets were interconnected without additional PBMC reprocessing. The PBMC and buffer bags as well as the sterile sample removal ports for control starting material / leukapheresis products and controls IPC01 10 and IPC02 11 were cleanly connected using a Terumo sterile tubing welder (TerumoBCT, Belgium).

[0238] All procedures were performed using single-use equipment sterilized by electron beam.

[0239] The manufacturing process requires bagging of RBC lysis buffer and electroporation buffer, which is performed under clean conditions. When the current manufacturing process is set up, Cbl-b siRNA is injected into the LOVO output PBMC bag of MAP step I 1 through a sterile filter connected to the control IPC01 10 withdrawal port through a wipeable Luer port in a laminar airflow cabinet, followed by the electroporation process. According to GMP standards, the laminar airflow cabinet is regularly monitored by a sedimentation plate.

[0240] MAP step II 2 and MAP step III 3 of drug product manufacturing are also cleanly interconnected without intermediate immune cell (preferably PBMC) reprocessing. Drug product controls S02 13 are cleanly extracted from drug product for measuring quality control parameters, namely appearance, identity, quantity, purity, safety, activity and potency.

[0241] Figure 5 The immune subtype distribution of PBMCs electroporated with Cbl-b siRNA using LOVO and MaxCyte electroporation devices is shown, and the immune cell subtype distribution measured by lineage marker expression of CD14 (monocytes), CD3 (T cells), CD16 (NK cells) and CD19 (B cells) obtained by flow cytometry in the apheresis product undergoing the manufacturing process is shown. The processed PBMCs underwent a final purification and rebuffering step as it has been established for final drug product formulation (n = 11 healthy donors).

[0242] Starting materials for drug substance manufacturing (Leukopak)

[0243] Collect the patient's PBMCs by leukapheresis according to standard protocols (e.g., Terumo Optia apheresis device) to enrich for lymphocytes and monocytes and deplete for granulocytes, platelets, and erythrocytes. Leukapheresis protocols should yield no less than 0.9 x 10 per kg body weight. 8 PBMCs (target: 1.2 per kg body weight + 0.3 x 10 8 PBMC), which was measured by complete blood count (CBC) as input control 10, to further specify leukopaks (PBMC content <70% (percentage of PBMC to total WBC), hematocrit <3.5%, and platelet count <2.0x10 6 PCT / µL). Prior to collection, the following serological viral safety tests were performed for patient safety screening: HIVI / II, HBV, HCV, and syphilis). PBMCs were collected using ACD-A buffer containing citric acid, dextrose monohydrate, sodium citrate dihydrate, and water, which was diluted 1:12 during the leukapheresis procedure to give a final concentration of the ACD-A component in the leukopaks.

[0244] Store the patient's finished leukocyte apheresis product at 5±3°C without shaking for ≤24 hours.

[0245] MAP Step I – LOVO – PBMC purification and re-buffering:

[0246] The present invention allows for automated erythrocyte lysis and platelet depletion, followed by rebuffering of PBMCs into electroporation buffer for subsequent MAP step II. In this closed-channel set, PBMCs are purified and resuspended in electroporation buffer (MaxCyte, USA), while the incoming components of apheresis buffer, PBS / EDTA wash buffer (CliniMACS buffer, Miltenyi, Germany) and ammonium chloride potassium lysis buffer (ACK) (Thermo Scientific, Massachusetts) are efficiently depleted.

[0247] The LOVO system allowed standardized isolation of PBMCs to similar numbers as Ficoll separation (p>0.05), which was highly operator dependent.

[0248] Before starting the PBMC purification of MAP step I 1, the buffer bag and the PBMC collection bag were cleanly connected to the corresponding ports of the disposable LOVO tubing set, which was subsequently mounted on the LOVO apparatus.

[0249] Prior to starting the LOVO procedure, the cell strainer for filtering cell aggregates upstream of the final container closure system bag needs to be inserted into the disposable tubing set in the correct orientation. The weld must be opened correctly to ensure free flow of drug product to the final seal. After ingestion of the leukopak PBMC into the Lovo tubing set, rinse the leukopak container bag with PBS / EDTA. During this step, the bag can be agitated to minimize cell loss.

[0250] To program the LOVO device, leukopak starting material is specified by testing white blood cell count (WBC) and hematocrit (HCT) counts. Leukopak starting material is verified to contain 0.9–1.5x10 8 PBMC. When the PBMC content exceeds 1.5x10 per kg body weight 8 PBMCs, assuming a theoretical maximum body weight of 100 kg, this would exceed the maximum number of PBMCs that can be processed in MAP step I 1 (1.5x10 10 PBMCs). Therefore, in this case, the calculated excess volume was removed from the leukopak bag before connection to the LOVO device.

[0251] After entering the desired LOVO program parameters and completing the LOVO setup and startup procedures, the leukopak bag is cleanly connected to the input port at the LOVO tubing system. The entire leukopak content is then automatically transferred to the tubing system to run MAP step I 1. The successful completion of the LOVO run recorded by the internal device monitoring system is reported for batch release.

[0252] In the detailed procedure of MAP Step I, six wash cycles achieve PLT depletion by spin membrane filtration to deplete smaller sized cells and cell debris from 200 mL of leukopak starting material, while RBCs are removed by an incubation cycle using an RBC lysing agent (potassium ammonium chloride lysis buffer). After adding the ACK lysis buffer, the operator is prompted to mix the incubation bag without delay. Only after confirmation, the 10-minute countdown for incubation begins. Any delay may increase the incubation time and may result in cell loss. After the RBC lysis wash cycle, the PBMCs are washed with PBS / EDTA buffer and rebuffered into MaxCyte electroporation buffer. The entire MAP Step I procedure requires ≤1 hour to collect ≤55 mL of PBMCs resuspended in electroporation buffer.

[0253] At the end of the sample purification and resuspension protocol, PBMCs resuspended in electroporation buffer were pumped into the PBMC collection bag and cleanly reconnected to the ExPERT TM GTx pipeline set to proceed directly to MAP step II (MaxCyte ExPERT via electroporation TM GTx - PBMC transfection). At this point, place the processing assembly with the attached PBMC bag in a refrigerator at 2-8°C for 10 to 15 minutes to pre-chill until proceeding to MAP Step II.

[0254] MAP Step II – MaxCyte ExPERT by electroporation TM GTx - PBMC transfection

[0255] Using ExPERT TM The GTx electroporation system can automatically process large numbers of PBMCs by continuous electroporation in a closed system. Before starting the electroporation protocol, cleanly remove the process control IPC02 (Specification Control Sample S01) from the PBMC bag through a wipeable Luer port in a laminar flow hood to collect a no pulse control (NPC) before electroporation. Store the control sample S01 at room temperature for drug product (DP) specification testing until drug product manufacturing is completed and drug product sample S02 is removed.

[0256] After sampling the in-process control IPC01, the entire contents of one vial of ready-to-use Cbl-b siRNA were injected into the PBMC bag using a syringe through a 0.2 µm syringe filter connected to the wipeable Luer in-process control (IPC01) sampling port, thus ensuring a closed MAP.

[0257] After siRNA injection, connect the ExPERTTM GTx Tubing Set Installed into MaxCyteExPERT TM Closed-circuit automated electroporation was performed on the GTx transfection system.

[0258] Initiate siRNA transfection by electroporation by starting the designated electroporation protocol ("Expanded T Cell 3pro") using the MaxCyte ExPERTTM GTx Transfection System electroporation software. Electroporation parameters were according to the manufacturer's instructions.

[0259] Automatically perform up to 19 consecutive electroporations to process up to 55 mL of PBMC suspension in a 20 + / - 5 minute time frame, enabling the production of a batch of ≤56 mL of suspended transfected cells.

[0260] Autologous PBMCs transfected with Cbl-b siRNA were resuspended in electroporation buffer after final collection in PBMC bags from ExPERT TM An integral part of the GTx pipeline group.

[0261] siRNA

[0262] The human Cbl-b gene is defined by the UniGene entry in GenBank Hs.430589. The mRNA sequence of Cbl-b is defined by GenBank entry NM_170662.3, single nucleotide polymorphism information was retrieved from GenBank, and all relevant polymorphisms were confirmed by sequencing.

[0263] siRNA allowed sustained Cbl-b silencing (for no less than 4 days) and cytokine induction (especially IFN-γ expression).

[0264] Figure 6 Optimized siRNAs when isolating PBMCs and silencing Cbl-b are illustrated. 65 siRNAs were designed, synthesized, and then screened for Cbl-b silencing and induced cytokine production in PBMCs. siRNAs that both induced IFN-γ expression and caused strong Cbl-b silencing were selected for resynthesis. After stimulation with anti-CD3 / 28, IFN-γ production (ELISA) and Cbl-b levels (icFACS) in T cells were determined and expressed relative to a reference commercial siRNA (Dharmacon). The frame highlights siRNAs that show strong silencing efficiencies associated with a strong increase in IFN-γ production.

[0265] PBMCs were isolated and Cbl-b was silenced, followed by stimulation with anti-CD3 / 28 antibodies, as described in Figure 7IL-2 production was determined by ELISA 3 days after stimulation. Grey and black bars indicate independent replicates for transfection and stimulation. Ctrl: control electroporation with non-target siRNA.

[0266] Sixty siRNAs were synthesized and screened for Cbl-b silencing and cytokine production enhancement by TCR-stimulated PBMCs, as shown in Table 4.

[0267] Table 4. List of modified nucleotide sequences

[0268]

[0269] 1 = dT (deoxythymidine)

[0270] 2 = 2'-FU

[0271] 3 = 2'-FC

[0272] 4 = 2'-FA

[0273] 5 = 2'-OMe-U

[0274] 6 = 2'-OMe-A

[0275] 7 = 2'-OMe-C

[0276] 8 = 2'-OMe-G

[0277] The silencing efficacy of siRNA can be different, and the persistence in the cell is also different. In general, modifications such as methylation or fluorinated bases enhance stability, but excessive modification reduces silencing efficiency. Ideally, siRNA causes efficient and lasting silencing, thereby achieving enhanced cell stimulation for a sustained period of time and also for cells stimulated by APC in vivo only within a few days after administration. Therefore, an experiment was established to modify and monitor the early (1st day) and late (7th day) levels of Cbl-b listed in Table 4 and the consequences of Cbl-b silencing on T cell activation.

[0278] PBMCs were isolated from healthy donors (buffy coat) by Ficoll centrifugation. For transfection, cells were electroporated using an Amaxa Nucleofector device (pulse program Y01) with a 0.2 cm gap width cuvette. These cells were mock transfected or transfected with the corresponding Cbl-b siRNA (6 μM). The cells were left to stand overnight in Xvivo15 serum-free medium. On day 1 (d1), cells were harvested, counted and divided into 4 equal parts, intracellular staining and flow cytometry analysis of Cbl-b levels (co-stained CD4 and CD8), and RT-PCR analysis of Cbl-b mRNA expression, anti-CD3 / 28 stimulation (overnight), followed by ELISA detection of cytokine (IL-2, IFN-g and TNF-a) production, and cell surface expression of activation markers (CD25, CD69) was analyzed by flow cytometry. The remaining cells were cultured for another 6 days in the presence of recombinant human IL-2 (rhu IL-2, 5 ng / mL). On day 7 (d7), these remaining cells were harvested and subjected to icFACS, RT-PCR, and ELISA / FACS analysis as on day 1 (d1). When sufficient cells were available, Western blot analysis was performed for Cbl-b protein. Western blot data from day 7 confirmed the icFACS data.

[0279] Most modified siRNAs could enhance IL-2 production due to enhanced T cell activation by Cbl-b silencing.

[0280] Some modified siRNAs could enhance T cell activation through sustained Cbl-b silencing over 7 days, thereby enhancing IL-2 production.

[0281] The best Cbl-b siRNA was unique in the human transcriptome as could be confirmed by blast searches of homologous sequences.

[0282] To make siRNA, sense and antisense single strands were chemically synthesized using a commercially available OP100 synthesizer (Biospring Gesellschaft für Biotechnologie mbH, Frankfurt, Germany). After synthesis, the crude oligonucleotides were cut and deprotected and then purified by AEX-HPLC. After purification, the single strands were desalted, concentrated, annealed and lyophilized.

[0283] Symbiosis Pharmaceutical Services (Sterling, Scotland) processed siRNA into a ready-to-use formulation (“formulated siRNA”) at a concentration of 5.0 mg per 1 mL of phosphate-buffered saline, which can be used to transfect approximately 1.2 × 108 / kg PBMC from leukopak input material. siRNA undergoes a final sterile filtration step and is then aseptically transferred into glass vials. Stoppered ("ready-to-use") glass vials are stored at -20 ± 5 °C. siRNA frozen at the beginning of the process is thawed at room temperature.

[0284] After 30 to 60 minutes of room temperature holding time in the PBMC bag, the transfected PBMCs were further processed for purification and resuspension by cleanly reconnecting the PBMC bag to the LOVO tubing set for final formulation.

[0285] MAP Step III - Drug Product Formulation

[0286] To ensure fully closed manufacturing, a new LOVO disposable catheter set was used for each patient batch. Prior to starting the process, the ExPERT from MAP Step II was TM The output PBMC bag of the GTx tubing set (specified drug substance) was directly connected to the LOVO tubing set and mounted on the LOVO device. A Terumo sterile tubing welder (TerumoBCT, Belgium) was used to cleanly connect the PBMC and buffer bags and the withdrawal port to withdraw the sterile drug control (S02) or cleanly remove excess PBMC for cell administration. All steps were performed using disposable equipment sterilized with electron beam sterilization.

[0287] The transfected cells were formulated for intravenous infusion in 0.9% w / v sodium chloride solution containing 0.7 g / l human serum albumin (HSA), which further depletes residual PLTs and cell debris. In addition, the electroporation buffer and excess free siRNA, which serve as a matrix for the transfected cells, were strongly reduced. The concise excipients selected for the formulation protect the transfected cells from degradation and aggregation and are used for parenteral dosage forms of the cell product. The final cell concentration was adjusted to 4.5 x 10 per kg body weight in 200 mL of physiological sodium chloride solution containing 0.7% human serum albumin. 7 PBMCs. The number of PBMCs administered per dose determines the total infusion volume. The entire MAP step III procedure takes ≤12 minutes to collect ≤200 mL of PBMCs resuspended in 0.9% physiological NaCl + HSA (0.7 g / l) solution (i.e., the drug), which is then adjusted to a target volume containing a defined number of PBMCs for infusion. The drug is finally stored and infused from the bag directly detached after the last manufacturing step. 16 mL of drug control S02 is aseptically removed for immediate release testing. After removing SO2, the highest possible total volume of ≤184 mL can be obtained, which therefore contains the maximum manufacturable dose (MMD) of PBMCs for possible infusion.

[0288] The drug product was administered immediately after manufacturing and release testing (no cryopreservation was required). The amount of PBMCs administered per dose was determined according to the treatment regimen, preferably one dose contained 5 x 10 6 Up to 5x10 10 pcs, preferably 5x10 7 Up to 5x10 8 The total volume used for infusion is defined by the number of transfected PBMCs / kg. The majority of PBMCs consist of T cells and NK cells.

[0289] The target volume for a single drug infusion depends on the selected PBMC target dose defined by the administration group of a given PBMC quantity / kg patient weight increment in the current clinical research scheme. Therefore, the target number of PBMC is calculated by multiplying the per kg dose by the patient weight. In addition, the actual PBMC concentration (PBMC / mL) in the drug determined by the CBC count of control sample S02 allows the calculation of the target volume for infusion containing the PBMC quantity defined. The target volume is multiplied by a density correction factor (according to the EDQM blood component preparation, use and quality assurance guidelines, the 20th edition (Pharmacopoeia, 2015)) to calculate the target drug bag weight (plus empty bag weight tare). This value allows the drug for infusion to be finally adjusted.

[0290] Technically, the drug product is reduced to the target amount by transferring the overage into a sterile connected overload bag. The remaining PBMC suspension in the drug product bag is weighed using a qualified scale to accurately adjust the target drug product weight for infusion. The reported target drug product weight should not deviate by more than ±10% from the calculated drug product weight containing the exact amount of PBMCs for administration.

[0291] The manufacturing process from PBMC purification from patient blood (leukopak) to the final formulation of Cbl-b silenced PBMC (drug product) by cell transfection is semi-automated and can be performed without the need for isolated intermediates. No cell holding time or additional storage of transfected cells is required to shorten the manufacturing process time and maximize the number of viable PBMCs that can be re-administered to the patient, thereby obtaining robust PBMC viability and the maximum manufacturable dose, which is considered to be the critical cell dose for effective treatment.

[0292] Drug efficacy depends primarily on the presence of T cells and NK cells after the final formulation of the drug for infusion. Within the processing of apheresis products starting with apheresis, cell destruction showed an average of 43% T cells and 20% NK cells in the final drug product.

[0293] Drug product characteristics are measured by release testing prior to administration and further testing that can only be completed after infusion. Drug product release testing prior to patient infusion includes appearance (visual inspection), identity (PBMCs among nucleated cells), quantity (drug product containing the target number of PBMCs to be infused), viability (live PBMCs), purity (PBMCs as a percentage of all blood cells), and safety (endotoxin content). Release testing prior to administration is performed by the manufacturer. Further drug product testing completes post-infusion characterization by further determining sterility, activity (activation of Cbl-b expression in drug product), and potency (activation of IL-2 secretion in drug product).

[0294] The adjusted drug product was administered within 6 hours of manufacturing without further cellular manipulation of the same cell bag.

[0295] The mRNA silencing results were confirmed at the protein level by immunoblotting using a Cbl-b-specific antibody showing an average silencing rate of 100%. This correlated with an average reduction from 4.06 ng Cbl-b / 100 µg total protein in S01 to 0.89 ng Cbl-b / 100 µg total protein in S02 as measured by mass spectrometry analysis of trypsin-digested PBMC total protein.

[0296] LOVO PBMC Purification Device and MaxCyte ExPERT TM GTx Transfection System Both devices are programmed by the respective equipment manufacturers or during method development.

[0297] Specifications of the drug produced by siRNA-mediated cbl-b silencing using the method of the present invention

[0298] The drug product (DP) is a suspension of live leukocytes from an individual patient that has been transfected with small interfering RNA (siRNA) to reduce Cbl-b expression. The transfected autologous leukocytes are infused back into the patient. Each batch is defined as a single treatment cycle per patient using freshly manufactured (not cryopreserved) DP.

[0299] Cbl-b is considered to be a negative regulator of innate and adaptive immunity and is therefore involved in tumor-induced immune evasion. Therefore, blocking Cbl-b function by silencing Cbl-b mRNA in leukocytes may lead to the reactivation of immune processes and thus help eradicate tumor cells. Along these lines, administration of autologous Cbl-b-silenced leukocytes represents a novel approach conceived to enhance anti-tumor immune responses, leading to a new cancer therapy.

[0300] Effectiveness

[0301] Interleukin-2 enzyme-linked immunosorbent assay (ELISA)

[0302] Drug samples and unpulsed control samples (NPC) from 12 MAP runs (n=12 healthy individuals) were analyzed for IL-2 secretion by ELISA using supernatants from anti-CD3 / 28 stimulated cultures. Fold induction of IL-2 in DP relative to NPC, as reported in DP specifications, shows maximum and minimum induction. Figure 8 and Table 5 show the fold increase in IL-2 as mean ± SEM comparing the drug product (DP) to the no pulse control (NPC) measured by ELISA.

[0303] Table 5. Comparison of IL-2 fold increase measured by ELISA for drug product (DP) and no pulse control (NPC).

[0304]

[0305] active

[0306] Silencing efficiency was assessed by measuring drug Cbl-b mRNA expression by qPCR and correspondingly measuring Cbl-b protein availability by western blot analysis. Of note, stimulation drives Cbl-b expression, thereby facilitating detection of Cbl-b, so cells were stimulated with anti-CD3 / CD28.

[0307] RNA isolation and qPCR

[0308] Gene silencing using siRNA results in the specific degradation of sequence-complementary messenger RNA, leading to the accumulation of the appropriate protein. Therefore, measuring specific mRNA by using quantitative real-time PCR (qPCR) is a suitable method to quantify the efficacy of siRNA silencing. In these experiments, the average silencing efficiency at the mRNA level was detectable at 63.9%, with a mean of 1.3% at 1.5 ng / mL. Fig. 9 and Fig.10 and shown in Tables 6 and 7. Fig. 9 The percentage of Cbl-b RNA expression of the drug (DP) relative to the no pulse control (NPC) is depicted, and Fig.10 Depicted is a comparison of the percentage of Cbl-b silencing of DP and NPC measured by qPCR.

[0309] Table 6. Relationship between Cbl-bRNA expression of drug (DP) and non-pulse control (NPC)

[0310]

[0311] Table 7. Comparison of the percentage of Cbl-b silencing in DP and NPC measured by qPCR

[0312]

[0313] Western blotting

[0314] As mentioned above, detection of Cbl-b protein by Western blot analysis is a key analytical method to evaluate the activity of the manufacturing process of transfected cells. Of note, stimulation drives Cbl-b expression, thereby facilitating detection of Cbl-b, so cells were stimulated with anti-CD3 / CD28.

[0315] like Fig.11 As shown in Table 8, after anti-CD3 / CD28 stimulation, almost 100% Cbl-b silencing efficiency was detected at the protein level.

[0316] Table 8. Comparison of protein level Cbl-b silencing percentages between DP and NPC using WB analysis.

[0317]

[0318] LC-MS / MS-based quantification of Cbl-b

[0319] Detection of low molecular weight levels such as endogenous levels of Cbl-b in complex matrices such as cell lysates requires specialized analytical procedures to overcome those limitations. Nanomolecular range quantification of Cbl-b protein by mass spectrometry was performed using a sensitive and selective protein quantification assay for Cbl-b and sequential LC-MS / MS-based quantification of a selected tryptic peptide for a targeted quantitative approach. Significant silencing efficacy was observed in those cell lysates comparing NPC and DP, demonstrating Fig.12 And the effectiveness of the method of the present invention shown in Table 9.

[0320] Table 9. Quantification of Cbl-b in DP and NPC cell lysates using LC-MS / MS

[0321]

[0322] The results showed that the drug induced long-lasting tumor-specific T cell immunity, such as Fig.13 Shown are IFN-γ production in response to stimulation with peptides corresponding to NY-ESO, survivin and telomerase of PBMCs collected from selected patients before treatment and at 9, 17, 15 and 29 weeks (W).

[0323] The present invention shows the effect of administering autologous peripheral blood mononuclear cells, in which the E3 ubiquitin protein ligase Casitas-B-lineage lymphoma protein-b has been silenced ex vivo in patients with advanced solid tumors in which well-established standard of care drugs are no longer effective.

[0324] To investigate the effects of the drug (cbl-b silenced PBMCs) on humans, it was evaluated in two Phase 1 clinical studies in cancer patients. The open-label Phase 1 study was primarily designed to evaluate the safety and tolerability of transfected PBMCs in patients with inoperable, recurrent or metastatic malignant solid tumors who were considered incurable, unresponsive to ongoing treatment or had no standard treatment available. In addition, the study was designed to determine if there was sufficient evidence of immune activity for clinical development.

[0325] The first Phase I study (CCCWFU 99114) was a single-dose study designed to characterize the toxicity profile and determine the maximum tolerated dose (MTD). Three patients received a single dose of 5x10 5 transfected PBMC / kg, 3 patients received a dose of 1x10 6 transfected PBMC / kg, and 10 patients received a dose of 5x10 6 transfected PBMC / kg. Patients had colorectal cancer, renal cell carcinoma, and pancreatic cancer. Transfected PBMCs were generally well tolerated, with most patients experiencing mild to moderate chills after completing the infusion. No dose-limiting toxicity was observed. No immediate hypersensitivity reactions occurred, and there was no evidence of autoimmune side effects. Therefore, this study established the feasibility and safety of administering the drug.

[0326] The second Phase I study (CCCWFU 03716) was a multiple-dose study designed to characterize the toxicity profile. The highest dose used in the first study (5x10 6 The dose is 1000 transfected PBMC / kg. Patients will receive 3 intravenous PBMC infusions, 4 weeks apart. Nine patients were treated with transfected PBMCs; 7 patients received all 3 infusions, 1 patient received 2 infusions, and 1 patient received 1 infusion. Patients had pancreatic, colon, or rectal cancer. The drug was generally well tolerated, with mild to moderate chills in most patients. No dose-limiting toxicity was observed. No autoimmune toxicity or hypersensitivity reactions were detected.

[0327] Results from the IIT study showed that infusion of transfected PBMCs was safe and well tolerated with single and multiple doses, and no serious effects were reported. Clinical studies have shown that silencing of Cbl-b in patient PBMCs resulted in enhanced secretion of inflammatory cytokines IFN-γ and IL-2 following in vitro TCR stimulation. Durable responses of PBMCs to common tumor antigens were observed to last at least 6 months. To date, efficacy in reducing tumor burden has not been shown in clinical studies due to the small number of patients enrolled, the advanced stage of cancer in the patients, and the possible suboptimal administration of transfected PBMCs as the MTD has not yet been determined.

[0328] Four of 16 single-dose patients and three of 11 multiple-dose patients showed stable tumor growth (approximately 30% of patients).

[0329] Further studies are designed to evaluate the safety, tolerability, efficacy, and immunological effects of the treatment in patients with advanced solid tumors who are no longer responsive to established standard treatments, building on experience gained from two previous Phase I studies.

[0330] This is an open-label, multicenter, dose escalation and expansion study in patients with advanced solid tumors. The study will be conducted in two parts. Part A is a Phase 1 dose exploration part in patients with solid tumors. Three dose levels of the drug will be evaluated using a 3+3 design. Part B is a preliminary dose expansion study of the RP2D, with 15 patients in each of the three tumor types (lung cancer, colorectal cancer, and head and neck cancer) being treated.

[0331] The first part of this study is intended to evaluate the safety, tolerability and effectiveness of transfected PBMCs and identify RP2D. Anti-tumor efficacy will also be evaluated. Patients with advanced solid tumors will use a 3+3 design to sequentially distribute increasing doses of medicines, in which the well-established standard therapeutic drugs are no longer effective (e.g., progress or refractory in at least two previous systemic treatment lines). Dose escalation requires at least three patients to be treated and observe at least 3 weeks after the first dose.

[0332] Patients with lung, colorectal, or head and neck cancer were included in this part of the study.

[0333] The incidence of dose-limiting toxicities (DLTs) will be assessed within 3 weeks after the first dose of treatment, but all available safety data will be considered when making cohort expansion and / or dose escalation decisions.

[0334] In dose level 1, three patients received three doses of 5x10 6 transfected PBMC / kg at 3-week intervals.

[0335] In dose level 2, three patients received three doses of 1.5x10 7 transfected PBMC / kg at 3-week intervals.

[0336] In dose level 3, three patients received three doses of 4.5x10 7 transfected PBMC / kg at 3-week intervals.

[0337] In the second part of this study, evidence of the desired pharmacodynamic effect in peripheral blood or tumor should also be evident at the recommended phase 2 dose (RP2D). For example, a drug product can be considered immunologically active if PBMC IFN-γ production is increased in response to anti-cluster of differentiation (CD) 3 / CD28 stimulation or one or more tumor antigens in at least two treated patients compared with baseline.

[0338] For each tumor type selected in Part B (Expansion Phase), 15 patients will be enrolled and receive drug treatment at RP2D, i.e., there will be 3 parallel cohorts of 15 patients each (45 patients total in Part B).

[0339] Three patients were treated in Cohort 1 (dose level 1) of Part A, and three patients were treated in Cohort 2 (dose level 2), all of whom received at least one dose of Cbl-b-silenced PBMCs. Two patients received six infusions (one patient received dose level 1 and one patient received dose level 2), one patient received three infusions (dose level 1), two patients received two infusions (dose level 1 and dose level 2), and one patient received one infusion at dose level 2. Overall, Cbl-b-silenced PBMCs were well tolerated in these six patients, and no DLTs were observed. The study was terminated because feasibility was demonstrated and the available preliminary data suggested a positive benefit-risk ratio for treatment with Cbl-b-silenced PBMCs. No additional patients were treated.

[0340] The study showed that after transient Cbl-b silencing, immune responses were enhanced and durable, and two patients who received six infusions showed disease stabilization for limited periods of time before they eventually showed disease progression.

[0341] DP infusions were safe and well tolerated, and no immediate hypersensitivity reactions or evidence of autoimmunity were observed. No dose-limiting toxicities were observed.

[0342] Furthermore, IL-2 fold induction (DP vs. non-DP) positively correlated with the presence of T cells (but not granulocytes or myeloid-derived suppressor cells) in DP. IL-2 fold induction also significantly correlated with the percentage of cbl-b silencing determined by quantitative PCR.

[0343] Cbl-b-silenced PBMCs exhibited a significant increase in proliferation and production of certain cytokines, such as interferon gamma (IFN-g) and interleukin (IL)-2, upon stimulation. Importantly, neither proliferation nor cytokine production was induced in unstimulated T cells, suggesting that silencing Cbl-b enhances T cell activity only in the setting of antigenic stimulation. This approach is superior to general systemic activation of all lymphocytes or systemic administration of cytokines, which are often associated with severe toxicity and may also have negative immunomodulatory effects. Specifically, Cbl-b deficiency can enhance the in vitro antitumor activity of T cells and NK cells, resulting in: enhanced expression of inflammatory cytokines and activation markers when stimulated by the T cell receptor (TCR); enhanced proliferation and antitumor cell cytotoxicity; resistance to transforming growth factor β-mediated immunosuppression (T cells); and increased activation of NK cells after cytokine stimulation or tumor cell contact.

[0344] Patients received multiple cycles of GMP-produced drugs at local centers. After silencing of human Cbl-b, the adaptive and innate immune systems were reactivated. This was measured by enrichment of tumor-specific immune cells, increased IL-2 levels and IFN-γ levels, persistent immune responses of PBMCs to common tumor antigens after TCR stimulation, memory cell effects of anti-tumor-specific immune cells, clinical anti-tumor activity, and very good safety.

[0345] Example 2: Production of SHP-1 and SHP-2 silenced PBMCs

[0346] Similar to Example 1, in addition to using siRNA encoding Cbl-b, siRNA encoding SHP-1 / PTPN6 and SHP-2 / PTPN11 can also be used to produce a drug using the method of the present invention.

[0347] The selected siRNA oligonucleotides show the following sequences.

[0348] SHP-1 / PTPN6

[0349]

[0350] SHP-2 / PTPN11

[0351]

[0352] Example 3: Transduction of leukocytes with large RNA and DNA

[0353] To validate the methods of the present invention for transducing nucleic acids larger than siRNA, leukocytes were purified as described in MAP Step I and electroporated with mRNA encoding enhanced green fluorescent protein (eGFP, TriLink Biotechnologies) or a DNA plasmid vector encoding GFP using a closed automated process as described in MAP Step II. After electroporation, leukocytes were cultured for up to 48 hours and analyzed for GFP expression by flow cytometry on a Fortessa LSR X-20 (BD Bioscience) after 24 and 48 hours, as described in Fig.14 shown.

[0354] Fig.14 eGFP fluorescence in leukocytes transduced with eGFP-mRNA quantified by flow cytometry after (A) 24 h or (B) 48 h culture time. Dashed lines represent non-electroporated controls, black outlines represent GFP fluorescence in live monocytes, CD8+ T cells, CD4+ T cells, NK cells, B cells, or CD14+ monocytes.

[0355] Example 4: Silencing the intracellular molecule TCEB2 / Elongin-B

[0356] In addition to silencing the primary immune checkpoint Cbl-b, the same siRNA approach was used to silence TCEB2 / Elongin-B / ELOB, which was identified in a CRISPR screen (Shifrut et al. 2018, Cell 175, 1958–1971, December 13, 2018: Genome-wide CRISPR Screens in Primary Human TCells Reveal Key Regulators of Immune Function. DOI: https: / / doi.org / 10.1016 / j.cell.2018.10.024) as inhibiting T cell proliferation after TCR stimulation. A siRNA pool containing 4 different siRNAs was purchased from Dharmacon (on-target plus human ELOB gene ID#6923) and used as a pool ( Fig.15 ) or used alone ( Fig.16 In addition, a pool of four siRNAs can also be used in combination with Cbl-b-silencing. After electroporation, cells were cultured for 48 h and ELOB mRNA and protein expression were analyzed using qRT-PCR and Western blotting, respectively ( Fig.15 ). Fig.15Depicted is the protein expression of TCEB2 / ELOB and Cbl-b in leukocytes transduced with a 4x siRNA pool targeting TCEB2 / ELOB or Cbl-b or a combination thereof. Protein expression was visualized using Western blotting after 48 hours of culture in the presence or absence of CD3 / CD28 stimulation. Fig.16 Depicted are protein expressions of TCEB2 / ELOB and Cbl-b in leukocytes transduced with four different siRNAs targeting TCEB2 / ELOB (TCEB2-08-TCEB2-11) or with Cbl-b targeting siRNA. After 48 hours of culture, protein expression was visualized using Western blotting.

[0357] In addition, the supernatants of CD3 / CD28 stimulated cells were analyzed for IL-2 and IFN-γ production ( Fig.17 and Fig.18 ).

[0358] Fig.17 Depicted are IL-2 and IFN-γ production by TCEB2 (four siRNA pools) or Cbl-b silenced leukocytes measured in supernatants by ELISA 48 hours after CD3 / CD28 stimulation.

[0359] Fig.18 Depicted are IL-2 and IFN-γ production by leukocytes silenced for TCEB2 (single siRNA: TCEB2-08–TCEB2-11) or Cbl-b in supernatants measured by ELISA 48 hours after CD3 / CD28 stimulation.

[0360] Fig.19 Depicted are mRNA expression of TCEB2 / ELOB and Cbl-b in leukocytes transduced with a 4x siRNA pool targeting TCEB2 / ELOB or a single siRNA targeting Cbl-b or a combination thereof. After a 48 hour culture period in the presence or absence of CD3 / CD28 stimulation, mRNA levels were analyzed using qRT-PCR.

[0361] Fig. 20 Depicted are the mRNA expression of TCEB2 / ELOB and Cbl-b in leukocytes transduced with four different siRNAs targeting TCEB2 / ELOB (TCEB2-08-TCEB2-11). mRNA levels were analyzed using qRT-PCR after 48 hours of culture time.

[0362] Reference Mark List

[0363] 1 Manufacturing process step I

[0364] 2 Manufacturing process step II

[0365] 3 Manufacturing process step III

[0366] 4 Sample preparation

[0367] 5. Sample purification and resuspension

[0368] 6. Sample Transfection

[0369] 7. Immune cells transfected with nucleic acids

[0370] 8 Final sample preparation

[0371] 9. Get the medicine ready for infusion

[0372] 10Comparison with IPC01

[0373] 11Compare with IPC02

[0374] 12 Comparison S01

[0375] 13 Comparison S02

Claims

1. An in vitro or ex vivo method for modifying immune cells, comprising the following steps: (i) providing immune cells from biological fluids and / or from resected tumors of patients, thereby providing cell samples; (ii) purifying the immune cells from the cell sample; (iii) transfecting the purified immune cells with an inhibitory nucleic acid of an immunosuppressive regulator of the immune cells or with a nucleic acid of an immune enhancing factor; (iv) purifying the transfected immune cells and / or rebuffering the transfected immune cells into a physiological solution; (v) transferring the transfected immune cells into a container; The transition between each two sequential steps of steps (i) to (v) is in a closed container system.

2. The method according to claim 1, wherein the immune cells are mononuclear or polymorphonuclear immune cells, preferably peripheral blood mononuclear cells (PBMCs), particularly preferably peripheral blood lymphocytes (PBLs), natural killer (NK) cells, T cells, B cells, monocytes and / or tumor infiltrating lymphocytes (TILs) from solid tumors or from tumor-associated fluids.

3. The method of claim 2, wherein the peripheral blood mononuclear cells (PBMCs) are obtained from the patient by apheresis, preferably by a single standard leukapheresis procedure, and wherein the tumor infiltrating cells are obtained from processed tumor fragments or from tumor-associated fluid.

4. The method according to any one of claims 1 to 3, wherein after and / or during purification of the immune cells, the red blood cells of the cell sample are lysed with a buffer having a pH value of 7 to 8, preferably a pH value of 7.4, the buffer containing chloride ions, ammonium ions, potassium ions and / or bicarbonate ions, preferably by using potassium ammonium chloride lysis buffer, and / or the platelets in the cell sample are depleted.

5. The method according to any one of claims 1 to 4, wherein the transfection of the immune cells is performed by microinjection, liposomes, electroporation, particle gun, magnetic-assisted transfection, sonoporation and / or cell squeezing technology.

6. A method according to claim 5, wherein the transfection of the purified immune cells is performed with at least one nucleic acid, preferably DNA, mRNA or siRNA and / or PNA, which results in modification of the gene expression pattern of one or more immune checkpoint inhibitors, chemokine receptors, cytokines and / or chimeric antigen receptors.

7. The method according to any one of claims 1 to 6, wherein the transfection of the purified immune cells with siRNA for silencing at least one immune checkpoint inhibitor or mRNA encoding an immune enhancing factor is performed by electroporation, preferably by flow electroporation or by large volume electroporation.

8. The method of claim 6 or 7, wherein the one or more immune checkpoint inhibitors are selected from the group consisting of Cbl-b, PD-1, PD-L1, PD-L2, CASP, CTLA4, FoxP3, LAG-3, TIM-3, TIGIT, A2AR, KIR, BTLA, VISTA, B7-H3, B7-H4, SHIP, SHP-1, SHP-2, IL-1R8, NKG2A, CD96, CD112R, CD160, CD244, IDO, IRG-1, STAT-3, JAKs, Arg-1, Nos-2, Cish, TGFb, PKA, TNFRSF, or a combination thereof.

9. The method according to any one of claims 1 to 8, wherein re-buffering the transfected immune cells comprises re-buffering into a physiological fluid with a stabilizer, preferably re-buffering into a 0.9% NaCl solution containing 1% to 4% autologous human serum, preferably a 0.9% NaCl solution containing 0.7 g / l to 4.5 g / l human serum albumin.

10. The method according to any one of claims 1 to 9, wherein in parallel with the rebuffering step the immune cells are purified and further depleted of platelets and cell debris, preferably using a spinning membrane technique, elutriation and / or counter-flow centrifugation.

11. The method according to any one of claims 1 to 10, wherein the closed container system comprises at least one container, preferably a bag with immune cells and / or at least one buffer container, and / or at least one tubing set connected to the containers, and / or at least one removal port, preferably for in-process control; preferably, wherein the containers, tubing sets and / or removal ports are cleanly interconnected, preferably by using a sterile tubing welder.

12. An immune cell population for use in a method for treating cancer, wherein the immune cell population is obtained by the method according to at least any one of claims 1 to 11.

13. The immune cell population for use according to claim 12, wherein after transfection, preferably electroporation, more than 15% of the immune cells, preferably NK cells, T cells and B cells, in the biological sample contain siRNA, mRNA or DNA.

14. The immune cell population for use according to claim 12 or 13, wherein the immune cell population is administered in at least one dose, and one dose contains 5 x 10 6 Up to 5x10 10 pcs, preferably 5x10 7 Up to 5x10 8 transfected PBMCs or TILs.

15. The immune cell population for use according to any one of claims 12 to 14, wherein the patient receives a single or multiple administration of the immune cell population intravenously, intratumorally and / or intranodally, preferably 1 to 10 doses, preferably 3 to 5 doses, and the doses are administered at intervals of 2 to 10 weeks, preferably 3 to 7 weeks, most preferably 4 weeks, for at least 2.

Images