Improved methods of cell culture for adoptive cell therapy

Abstract

To produce therapeutic cells that can be used independent of whether or not there is an HLA match between a donor and a recipient.SOLUTION: A method comprises: stimulating donor PBMCs or donor cord blood with an antigen in order to activate the growth of T cells that have native antigen specificity to the antigen, so as to produce an antigen-specific T cell population that comprises native antigen receptors that have antigen specificity to the antigen. Then, the antigen-specific T cell population is altered to include therapeutic attributes independent of the antigen specificity of the native antigen receptors.SELECTED DRAWING: Figure 14

Description

Technical Field

[0001] Related Applications This application is a continuation-in-part of Patent Document 2 (hereinafter, the "parent application") titled "Improved Cell Culture Method for Adoptive Cell Therapy" filed on Dec. 8, 2010, which claims the benefits of Patent Document 1 titled "Improved Cell Culture Method for Adoptive Cell Therapy" filed on Dec. 8, 2009 (the entire content of which is incorporated herein by reference).

[0002] The present invention generally relates to cell culture methods, and more particularly to cell culture for cell therapy. Further, the present invention relates to the production of T cells having therapeutic attributes for use in adoptive cell therapy.

Background Art

[0003] Cell culture is a major factor in the cost and complexity of cell therapy. With current methods, the cell culture process is time-consuming and expensive. Typically, to produce a large number of cells, the in vitro culture process is carried out in a stepwise manner. At the earliest stage, the target cells are a relatively small subpopulation within the cell population placed in the cell culture device. At this stage, the cell population typically includes a source of the target cells (such as peripheral blood mononuclear cells), feeder cells that stimulate the growth of the target cells, and / or antigen presentation. Culture devices and methods that can keep the medium in which the cells are present in a largely unstirred state are preferred because the cells remain relatively unstirred. Such devices include standard tissue culture plates, flasks, and bags. The culture proceeds in stages and generally consists of depleting the medium in the cell population of growth substrates such as glucose, removing the used medium, replacing the used medium with fresh medium, and repeating the process until the desired amount of the target cells is obtained. Often, when the number of the target cell population increases and more growth area is required, the cell population is transferred to another device to start a new production stage. However, with conventional methods, the growth rate of the target cell population slows as the number of cells in the growth area increases. Ultimately, it takes a very long time and is complex to produce a fairly large population of the target cells.

[0004] A state-of-the-art production method for generating T lymphocytes with antigen specificity against Epstein-Barr virus (EBV-CTL) is an example showing the complexity of production. The conventional method for optimal proliferation of EBV-CTL uses a standard 24-well tissue culture plate. The surface area on which the cells of each well are placed is 2 cm 2 and its medium volume is limited to 1 ml / cm 2 due to the need for gas transfer. The culture process starts by placing a cell population consisting of PBMC (peripheral blood mononuclear cells) in the presence of an irradiated antigen-presenting cell line, which may also be a lymphoblastoid cell line (LCL), at a surface density ratio of about 40:1 (i.e., the number of cells per 1 cm 2 of growth area), with 1×10 6 PBMC per cm2 and 2.5 × 10 irradiated antigen-presenting cells 4 pieces / cm 2 This promotes the expansion of the EBV-CTL population in the cell population. After 9 days, EBV-CTLs were expressed in the presence of irradiated antigen-presenting LCL at a new surface density ratio of 4:1, with approximately 2.5 × 10 EBV-CTLs. 5 pieces / cm 2 Again, they are selectively grown at a minimum surface density of 1 ml / cm2. To allow oxygen to reach the cells, the medium volume must be kept at a maximum ratio of 1 ml / cm2 of growth surface area. 2 , which limits growth solutes such as glucose. As a result, the maximum achievable surface density is approximately 2 × 10 EBV-CTL. 6 pieces / cm 2 Therefore, the maximum cell proliferation per week was about 8-fold (i.e., EBV-CTL 2 × 10 6 pieces / cm 2 EBV-CTL 2.5 × 10 5 pieces / cm 2 The EBV-CTL population is expanded at a rate of 100-150% by dividing the total number of cells by 100% (divided by 0.01). Continuous expansion of EBV-CTL requires weekly transfer of EBV-CTL to additional 24-well plates with antigen restimulation and twice weekly replacement of medium and growth factors in each well of the 24-well plate. According to conventional methods, as the EBV-CTL surface density approaches the maximum possible amount per well, the growth rate of the EBV-CTL population slows, and these procedures must be repeated over a long production period, often as long as 4-8 weeks, to obtain sufficient amounts of EBV-CTL for cell injections and quality control measures such as sterility, identity and potency assays.

[0005] Cultivation of EBV-CTL is just one example of the complex cell production process inherent in cell therapy. More practical methods of culturing cells for cell therapy that can shorten production time while simultaneously reducing production costs and complexity are needed.

[0006] We have created a novel method to increase the population growth rate throughout production and, by doing so, reduce the complexity and time required for cell production.

[0007] In adoptive cell therapy, T cells with natural antigen specificity (i.e., T cells specific for a particular peptide derived from a particular target antigen when presented in the context of a particular human leukocyte antigen (HLA) allele) are administered in autologous settings and in partially HLA-matched settings to treat viral infections and target tumors. In all of these cases, a therapeutic benefit is obtained from the fact that (i) the natural T cell receptor recognized the antigen of interest and (ii) the T cells were administered to a recipient who expressed the HLA alleles necessary to present the targeted peptide.

[0008] The first adoptive T cell transfer protocol in the setting of allogeneic hematopoietic stem cell transplantation (HSCT) was based on the premise that donor peripheral blood contains T cells that can modulate anti-tumor and / or anti-viral activity in the HSCT recipient. Thus, donor lymphocyte infusion (DLI) has been widely used to confer anti-tumor immunity and, to a lesser extent, anti-viral immunity. DLI should contain tumor-specific memory T cells as well as a broad range of viruses. However, while effective in treating populations infected with adenovirus and EBV, the efficacy of this therapy is limited by low-frequency T cells specific for many common acute viruses (such as respiratory syncytial virus (RSV) and parainfluenza) and relatively high-frequency alloreactive T cells. The high ratio of alloreactive T cells to virus-specific T cells is particularly problematic in recipients of haploidentical transplants, where a higher incidence of graft-versus-host disease (GVHD) limits the tolerable DLI dose and the dose of virus-specific T cells received.

[0009] To preserve the benefits and improve the safety of DLI, strategies for the selective inactivation or removal of recipient-specific alloreactive T cells have been evaluated, and such strategies include anergy induction, selective allo-depletion to minimize the number of alloreactive T cells administered to the recipient, and the use of suicide genes to destroy alloreactive T cells that have deviated from the target in vivo.

[0010] An alternative strategy for preventing and treating specific viral infections after HSCT is adoptive cell transfer of in vitro-expanded T cells with antiviral activity. The specific expansion of virus-reactive T cells has the advantage of increasing the number of virus-specific T cells that can be infused without increasing alloreactive T cells. The infusion of enriched antigen-specific T cells with reactivity to a specific antigen potentially increases therapeutic efficacy while reducing unwanted off-target effects such as GVHD, and this treatment modality has been proven to be effective and safe for the treatment of hematological malignancies as well as solid tumors such as melanoma and EBV-related malignancies such as Hodgkin lymphoma and nasopharyngeal cancer.

[0011] Of note, in all therapies, it is necessary to use the specificity of the native T cell receptor to recognize antigens in the context of the association with major histocompatibility complex (MHC) molecules via the native T cell receptor. Thus, the therapeutic benefit itself depends on the use / administration of HLA-matched or partially matched T cells. For example, to target melanoma cells, antigen-specific melanoma-directed T cells from a donor expressing HLA haplotype (a) for GP100 (a tumor-associated antigen expressed on cancer cells) can be expanded. In this context, the therapeutic benefit is provided by the specific interaction of the native T cell receptor with the target antigen. However, this interaction can only occur in a compatible HLA setting (i.e., in an autologous setting or in the context of another individual who also expresses HLA). This approach can only be extended to treat multiple patients by generating a cell bank containing lines with various HLA haplotypes, where the patient is matched to the most suitable T cell line.

[0012] In short, in all current applications of adoptive cell therapy, the therapeutic attribute of the T cells conferring the therapeutic purpose is the natural antigen specificity of the donor T cells. This inherent feature requires at least a partial HLA compatibility between the donor and the recipient, and in an allogeneic setting, it gives rise to the possibility of off-target effects such as GVHD. Others have proposed completely eliminating the donor T cell antigen receptor by complex genetic manipulation and genetically re-engineering the T cells to carry chimeric antigen receptors, thereby completely eliminating the inherent recognition ability of the T cells. However, this further complicates the T cell production method, which is already one of the major problems of adoptive cell therapy.

[0013] To enable broader use in mainstream society, a completely new approach to adoptive cell therapy that overcomes existing problems is needed. We disclose a new paradigm for modifying donor T cells with a therapeutic attribute that does not impair the antigen specificity of the donor T cells but renders the natural antigen specificity of the donor T cells irrelevant to its therapeutic purpose. Essentially, this paradigm shift opens the door to the therapeutic use of T cells in a way that has not been envisioned before, regardless of whether there is HLA compatibility between the donor and the recipient.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Summary of the Invention

[0015] By using a stepwise production process that allows for the periodic re - establishment of conditions different from the prior art throughout the production process, it has been discovered that it is possible to produce cells for cell therapy in a more economical manner and in a shorter time than is currently possible. Conditions different from the prior art include a reduced surface density of target cells (i.e., number of cells / cm 2 ), a novel ratio of target cells to antigen - presenting cells and / or feeder cells, and / or the use of a growth surface made of a gas - permeable material with an increased medium volume:surface area ratio.

[0016] Embodiments of the present invention relate to an improved method for culturing cells for cell therapy applications. These embodiments include methods for reducing the time, cost, and complexity required to generate a desired number of target cells by using a variety of novel methods that allow a population of target cells to maintain a higher growth rate compared to conventional methods throughout the production process.

[0017] One aspect of the present invention relies on performing the culture process in steps and establishing conditions at the start of one or more steps such that the growth rate of the target cell population exceeds what is currently possible. In at least one culture step, preferably in substantially all culture steps, initial conditions are established that include plating the target cells on either a non - gas - permeable growth surface or a gas - permeable growth surface at a low surface density different from the prior art and at a ratio of antigen - presenting cells (and / or feeder cells) to target cells different from the prior art. By using the novel embodiments of this aspect of the present invention, the target cell population undergoes more doublings in a shorter time than allowed by conventional methods, thus shortening the production time.

[0018] Another aspect of the present invention relies on performing the culturing process in stages and establishing conditions at the start of one or more stages such that the growth rate of the target cell population exceeds what is currently possible. Conditions are established that include, in at least one culturing stage, preferably in substantially all culturing stages, placing the target cells on a growth surface made of a gas-permeable material at an elevated medium volume:growth surface area ratio that is different from conventional. By using the novel embodiments of this aspect of the present invention, the target cell population undergoes more doublings in a shorter time than is permitted by conventional methods, thus shortening the production time.

[0019] Another aspect of the present invention relies on performing the culturing process in stages and establishing the conditions for each stage such that the growth rate of the target cell population exceeds what is currently possible. In at least one culturing stage, preferably in substantially all culturing stages, initial conditions are established that include placing the target cells on a growth surface made of a gas-permeable material at a lower surface density (i.e., number of cells / cm 2 ) that is different from conventional, at a different ratio of antigen-presenting cells (and / or feeder cells) to the target cells that is different from conventional, and at an elevated medium volume:growth surface area ratio that is different from conventional. By using the novel embodiments of this aspect of the present invention, the target cell population undergoes more doublings in a shorter time than is permitted by conventional methods, thus shortening the production time.

[0020] In an embodiment of the present invention, an allogeneic T vehicle is created that has a therapeutic attribute that does not expose the recipient to graft-versus-host disease (GVHD) but has a therapeutic purpose that is beneficial to the recipient.

[0021] In one embodiment of the present invention, treatment is initiated by obtaining a T vehicle created by a process that includes stimulating donor PBMC or donor cord blood with an antigen to activate the growth of T cells having natural antigen specificity for one or more antigens. By doing so, a population of antigen-specific T cells consisting of natural antigen receptors having antigen specificity for one or more antigens used to stimulate their growth is produced. The population of antigen-specific T cells is modified to create a population of T vehicles by including at least one therapeutic attribute having a therapeutic purpose unrelated to the antigen specificity of the natural antigen receptor without including the natural antigen receptor. Then, the T vehicle is delivered to a recipient from whom a therapeutic benefit can be derived regardless of whether the recipient's cells present an antigen recognized by one or more natural antigen receptors of the T vehicle and / or when the recipient's cells do not present an antigen recognized by one or more natural antigen receptors of the T vehicle.

[0022] In another embodiment of the present invention, treatment is initiated by obtaining a T vehicle created by a process that includes stimulating donor PBMC or donor cord blood with an antigen to activate the growth of T cells having natural antigen specificity for one or more antigens. By doing so, a population of antigen-specific T cells consisting of natural antigen receptors having antigen specificity for one or more antigens used to stimulate their growth is produced. The population of antigen-specific T cells is modified to create a population of T vehicles by including at least one therapeutic attribute having a therapeutic purpose unrelated to the antigen specificity of the natural antigen receptor without including the natural antigen receptor. Then, a therapeutic benefit can be derived from the T vehicle, and the T vehicle is delivered to a recipient who does not have HLA compatibility with the T vehicle.

[0023] In various embodiments of the present invention, the T vehicle is modified to be loaded with a recombinant protein administered as an adjuvant for immunotherapy, modified with the therapeutic attributes of a chemotherapeutic agent for targeted cancer therapy, modified with the therapeutic attributes of an antibacterial agent, modified with the therapeutic attribute of expressing a transgenic molecule that confers tumor specificity to cells, modified with the therapeutic attribute of being loaded with or genetically engineered with a recombinant protein for treating autoimmune diseases, modified with the therapeutic attribute of being loaded and / or genetically engineered for in vivo imaging.

[0024] In another embodiment of the present invention, a method for producing antigen-specific T cells having the desired antigen recognition comprises placing PBMC or cord blood in a cell culture vessel, adding two or more antigens into the cell culture vessel to activate the growth of two or more antigen-specific T cell populations (each population can recognize one of the antigens), allowing a period for the antigen-specific T cells to initiate proliferation in number, evaluating the culture to determine the presence and / or amount of at least one antigen-specific T cell population, determining which T cell populations are suitable for continued proliferation, and achieved by restimulating the culture with only the antigen recognized by the suitable T cell population.

[0025] In another embodiment of the present invention, a method for producing antigen-specific T cells having the desired antigen recognition comprises placing PBMC or cord blood in a cell culture vessel, first adding two or more antigens into the cell culture vessel to activate the growth of two or more antigen-specific T cell populations (each group can recognize one of the antigens), allowing a period for the antigen-specific T cells to initiate proliferation in number, separating the culture into two or more vessels, adding only one of the initial antigens into each vessel, determining which vessel contains the antigen-specific T cell population suitable for continued proliferation, and achieved by terminating the culture in the vessel that does not contain the antigen-specific T cell population suitable for continued proliferation.

[0026] In various embodiments of the present invention, donor T cells having a natural antigen specificity that enables recognition of only a single epitope of an antigen that is not present on normal human cells and is also not present on normal mammalian cells are produced.

[0027] The present invention will be more fully understood in view of the following detailed description of various embodiments of the invention in combination with the accompanying drawings.

Brief Description of the Drawings

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[0029] Definitions Antigen - presenting cell (APC): A cell that acts to stimulate target cells to respond to a specific antigen. CTL: Cytotoxic T lymphocyte. Target cells: Specific types of cells targeted to increase in quantity in the production process. Generally, target cells are non-adherent and examples include regulatory T cells (Tregs), natural killer cells (NK), tumor-infiltrating lymphocytes (TIL), primary T lymphocytes, and various antigen-specific cells, and many others (all of which can also be genetically modified to improve function, in vivo persistence, or safety). Cells required for clinical use can be expanded using feeder cells and / or antigen-presenting cells, and such cells include PBMC, PHA blast, OKT3 T, B blast, LCL, and K562 (naturally or genetically modified to express, antigens and / or epitopes, and co-stimulatory molecules such as 41BBL, OX40, CD80, CD86, HLA, and many others), which may or may not be pulsed with peptides or other relevant antigens. EBV: Epstein-Barr virus. EBV-CTL: T cells that specifically recognize cells expressing or presenting EBV-infected cells or EBV-derived peptides via the T cell surface receptor. EBV-LCL: B lymphoblastoid cell line transformed by Epstein-Barr virus. Feeder cells: Cells that act to increase the quantity of target cells. In some situations, antigen-presenting cells can also act as feeder cells. Growth surface: The area within the culture vessel on which the cells are placed at rest. PBMC: Peripheral blood mononuclear cells derived from peripheral blood, which are a source of some target cells and can act as feeder cells. Responding cells (R): Cells that respond to stimulator cells. Static cell culture: A method of culturing cells in a medium that is not agitated or mixed, except when the location of the culture vessel is moved for routine operations and / or when fresh medium etc. is periodically supplied to the cells. Generally, the medium during static culture is usually in a stationary state. The present invention relates to a static cell culture method. Stimulation: The action exerted by antigen presentation and / or feeder cells on target cells. Stimulating cell (S): A cell that affects a responding cell. Surface density: The amount of cells per unit area of the surface within the apparatus on which the cells are allowed to settle.

[0030] When attempting to find a novel method to simplify the production of a target cell population for adoptive T cell therapy, a series of experiments were conducted that opened the door to more efficient culturing of cells for cell therapy applications. Numerous exemplary embodiments and various aspects of the present invention are described to show how the ability to reduce production time and complexity can be achieved as compared to conventional methods.

[0031] Example 1: Demonstration of the Limitations of Conventional Methods

[0032] The data of this example demonstrate the limitations of a conventional culturing method for producing EBV-CTLs in a standard 24-well tissue culture plate (i.e., the surface area per well is 2 cm 2 ) using a medium volume of 2 ml per well (i.e., the height of the medium is 1.0 cm and the medium volume:surface area ratio is 1 ml / cm 2 ).

[0033] Culture stage 1 (day 0): Starting the proliferation of the EBV-CTL population by culturing a cell population of PBMCs (about 1×10 2 cells / ml) from normal donors with antigen-presenting γ-irradiated (40 Gy) autologous EBV-LCL at a medium volume:growth surface ratio of 1 ml / cm 6 and a ratio of 40:1 (PBML:LCL), and adjusting the surface density of the cell population in RPMI 1640 supplemented with 45% Click medium (Irvine Scientific, Santa Ana, CA), 2 mM GlutaMAX-I, and 10% FBS to about 1×10 6 cells / cm 2 .

[0034] Culture stage 2 (days 9 - 16): On day 9, EBV-CTLs were recovered from the cell population created in stage 1 and seeded at 0.5×10 6 cells / cm 2Resuspended in fresh medium at the surface density, and restimulated with irradiated autologous EBV-LCL at a CTL:LCL ratio of 4:1 (CTL surface density 0.5×10 6 cells / cm 2 :LCL 1.25×10 5 cells / cm 2 ). On day 13, 1 ml out of the 2 ml of the medium volume in each well of a 24-well plate was removed and replaced with 1 ml of fresh medium containing recombinant human IL-2 (IL-2) (50 U / mL) (Proleukin; Chiron, Emeryville, CA).

[0035] Culture stage 3 (days 17 - 23): The conditions of stage 2 were repeated with IL-2 added twice a week, and the culture was terminated on day 23. Even after termination, the culture could continue with additional culture stages mimicking those of stages 2 and 3.

[0036] Cell lines and tumor cells for use as target cells in the cytotoxicity assay: BJAB (B-cell lymphoma) and K562 (chronic myelogenous leukemia) were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). All cells were maintained in RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) containing 10% heat-inactivated fetal bovine serum (FCS), 2 mM L-glutamine, 25 IU / mL penicillin, and 25 mg / mL streptomycin (all obtained from BioWhittaker, Walkersville, MD). The cells were maintained in a humidified environment containing 5% CO2 at 37°C.

[0037] Immunophenotypic examination: Cell surface: Cells were stained with phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridin chlorophyll protein (PerCP), and allophycocyanin (APC)-conjugated monoclonal antibodies (MAbs) against CD3, CD4, CD8, CD56, CD16, CD62L, CD45RO, CD45RA, CD27, CD28, CD25, and CD44 from Becton-Dickinson (Mountain View, CA, USA). The frequencies of EBV-CTL precursors were quantified using PE-conjugated tetramers (Baylor College of Medicine) and APC-conjugated pentamers (Proimmune Ltd, Oxford, UK). 10,000 and 100,000 live events for cell surface and pentamer staining, respectively, were acquired on a FACSCalibur flow cytometer and the data were analyzed using Cell Quest software (Becton Dickinson).

[0038] CFSE labeling for measuring cell division: 2×10 7 To evaluate the doubling rate, PBMC or EBV-specific CTL (EBV-CTL) were washed twice and resuspended in 850 μl of 1× phosphate-buffered saline (PBS) containing 0.1% fetal bovine serum (FBS) (Sigma-Aldrich). Prior to staining, an aliquot of carboxy-fluorescein diacetate, succinimidyl ester (CFSE) (10 mM in dimethyl sulfoxide) (Celltrace™ CFSE Cell Proliferation Kit (C34554) (Invitrogen)) was thawed, diluted 1:1000 in 1× PBS, and 150 μl of the dilution was added to the cell suspension (labeling concentration was 1 μM). Cells were incubated with CFSE for 10 minutes at room temperature. Then, 1 ml of FBS was added to the cell suspension, which was then incubated for 10 minutes at 37°C. Cells were then washed twice with 1× PBS, counted, and stimulated with the antigens described.

[0039] Annexin V-7-AAD staining: To measure the percentage of apoptotic and necrotic cells in culture, Annexin-7-AAD staining was performed according to the manufacturer's instructions (BD Pharmingen™ #559763, San Diego, CA). Briefly, EBV-CTLs from 24-well plates or G-Rex were washed with cold PBS and resuspended in 1× binding buffer at a concentration of 1×10 6 cells / ml and stained with Annexin V-PE and 7-AAD for 15 minutes at room temperature (25°C) in the dark. After incubation, the cells were immediately analyzed by flow cytometry.

[0040] Chromium release assay: As described above, the cytotoxic activity of EBV-CTLs was evaluated in a standard 4-hour 51 Cr release assay. As target cells, autologous and HLA class I and II mismatched EBV-transformed lymphoblastoid cell lines (EBV-LCLs) were used to measure killing by MHC restriction and non-restriction, and the K562 cell line was used to measure natural killer activity. Spontaneous and maximum 51 Cr release were measured using chromium-labeled target cells incubated in medium alone or with 1% Triton X-100, respectively. The mean percentage of specific lysis in triplicate wells was calculated as follows: [(experimental release - spontaneous release) / (maximum release - spontaneous release)] × 100.

[0041] Enzyme-linked immunosorbent spot (ELIspot) assay: Using the ELIspot assay, the frequency and function of T cells secreting IFNγ in response to antigen stimulation were quantified. CTL lines grown in 24-well plates or G-Rex were stimulated with irradiated LCL (40 Gy) or LMP1, LMP2, BZLF1, and EBNA1 pepmix (diluted to 1 μg / ml) (JPT Technologies GmbH, Berlin, Germany), or EBV peptides HLA-A2 GLCTLVAML=GLC, HLA-A2 CLGGLLTMV=CLG, HLA-A2-FLYALALLL=FLY, and HLA-A29 ILLARLFLY=ILL (Genemed Synthesis, Inc., San Antonio, TX), diluted to a final concentration of 2 μM, with CTL alone serving as a negative control. CTL was in ELIspot medium [RPMI 1640 (Hyclone, Logan, UT) supplemented with 5% human serum (Valley Biomedical, Inc., Winchester, VA) and 2 mM L-glutamine (GlutaMAX-I) (Invitrogen, Carlsbad, CA)] at 1 × 10 6Resuspended in / ml. A 96-well filtration plate (MultiScreen, #MAHAS4510) (Millipore, Bedford, Massachusetts) was coated overnight at 4 °C with 10 μg / mL anti-IFN-γ antibody (Catcher-mAB91-DIK) (Mabtech, Cincinnati, Ohio), then washed and blocked with ELIspot medium at 37 °C for 1 hour. Responding cells and stimulating cells were incubated on the plate for 20 hours, then the plate was washed and incubated with biotin-conjugated anti-IFN-γ monoclonal second antibody (Detector-mAB(7-B6-1-Biotin)) (Mabtech), and then incubated with avidin:biotinylated horseradish peroxidase complex (Vectastain Elite ABC Kit (Standard), #PK6100) (Vector Laboratories, Burlingame, California) and developed with AEC substrate (Sigma, St. Louis, Missouri). Each culture condition was run in triplicate. The plates were sent to Zellnet Consulting (New York, New York) for evaluation. Spot forming units (SFC) and the number of cells seeded were plotted.

[0042] Statistical analysis: Data in vitro are shown as mean ± 1 SD. Student's t-test was used to measure the statistical significance of differences between samples, and P < 0.05 was accepted as indicating a significant difference.

[0043] Under these culture conditions, as shown in Figure 1A, after the initial stimulation, the antigen-specific T cell population undergoes at least 7 cell doublings over the first 7 days. Thus, it is predicted that T cells will proliferate 128-fold in one week (obtained by multiplying the frequency of antigen-specific T cells by the total number of cells in the cell population). Figure 1B shows the frequencies of tetramer-positive cells after the first, second, and third stimulations. On day 0, the frequencies of T cells reactive to the two EBV tetramers, RAK and QAK, were 0.02% and 0.01%, respectively. After only one stimulation on day 0, by day 9, the frequencies of tetramer-positive T cells in the cell population had increased from 0.02% and 0.01% to 2.7% and 1.25%, respectively. Thus, the proportion of antigen-specific tetramer-positive T cells present in the cell population achieved increases of 135-fold and 125-fold, respectively, when measured by RAK and QAK. Also, after only one stimulation on day 0 of culture stage 1, an increase of 1.1-fold (data not shown) was observed in the surface density of cells in the cell population by day 9 (approximately 1.1×10 6 cells / cm 2 were present). Since most of the cells in the PBMC population are not specific to the stimulating antigen, little overall increase in the total cell number is observed, but as shown in Figure 1C, the growth magnification of the antigen-specific cell population in the population was approximately 280 during the first stage of culture. Unfortunately, when measured by CSFE, the number of cell doublings was the same between the second and third culture stages, but this antigen-specific T cell growth rate did not persist between the second and third culture stages, being only 5.7 in the second stage and 4.3 in the third stage. Figure 2 shows a table illustrating the difference between the expected growth and the observed growth magnification of antigen-specific T cells (n = 3).

[0044] Example 1 demonstrates that because the growth rate of the target cell population decreases in subsequent culture stages, the time required to produce the target cells typically slows down after approximately the first week of production.

[0045] Example 2: A shortening of the time required to expand the target cell population can be achieved by reducing the cell surface density of the target cell population at the start of any given one or more stages of culture.

[0046] It was hypothesized that the decreased proliferation rate of the target cell population after the second T cell stimulation compared to the first stimulation was due to limiting the cell culture conditions that result in activation-induced cell death (AICD). For example, referring to FIG. 3A, in the first stimulation, since the EBV antigen-specific T cell component of PBMC is at most 2% of the cell number, the seeding density of antigen-specific responding T cells is less than 2×10 2 per cm 4 . The remaining PBMC act as non-proliferating feeder cells (shown as CFSE-positive cells in FIG. 3A) and maintain optimal cell-to-cell contact to allow the proliferation of antigen-specific CTL. In contrast, in the second stimulation on day 9, most of the T cells are antigen-specific, the total cell density of the population is approximately the same, but the proliferating cell density is 50 - 100 times higher. As a result, in restimulation, most cells proliferate, and thus they may rapidly consume and deplete their nutrient and O2 supply.

[0047] To determine whether the limitation of culture conditions was involved in suboptimal T cell growth rates, the proliferation of activated T cells plated at lower cell densities was measured. The method was as described in Example 1 above.

[0048] As shown in FIG. 3B, in wells of a standard 24-well plate with a growth surface area of 2 cm 2 per well, activated EBV-specific T cells were seeded at doubling dilutions that produced surface densities decreasing in the range of 1×10 6 / cm 2 to 3.1×10 4 / cm 2 , while maintaining a responder cell:stimulator cell ratio (R:S) of 4:1. The maximum CTL proliferation (4.7 ± 1.1-fold) was achieved at a starting CTL surface density of 1.25×10 2 per cm 5 , but as shown in FIG. 3B, the proliferation rate decreased with further dilution. This limited dilution effect is presumably due to the lack of cell-to-cell contact, so a fixed number of feeder cells (1.25×10 5 / cm 2Plated at the surface density of EBV-LCL), together with 1×10 6 to 3.1×10 4 Doubling dilutions of EBV-CTL at the surface density up to were cultured, and cell proliferation was evaluated over 7 days. As shown in Figure 3C, CTL proliferation increased dramatically from only 2.9±0.8-fold proliferation by EBV-CTL at the surface density of 1×10 6 / cm 2 to 34.7±11-fold proliferation by EBV-CTL at the surface density of 3.1×10 4 / cm 2 . Importantly, this change in culture conditions did not alter the cell function or antigen specificity (data not shown). Therefore, the activated antigen-specific T cell population can grow larger than that allowed by the conventional culture method. It should be noted that the maximum surface density (1.7 - 2.5×10 6 / cm 2 ) achieved after stimulation was the same regardless of the starting surface density.

[0049] Therefore, the conventional culture conditions are limiting, and it is shown that in order for the target cell population to exceed the surface density limit of the conventional method, the medium volume:growth surface area ratio must be higher than the conventional 1 ml / cm 2 . Furthermore, by reducing the surface density of the target cell population at the start of any culture stage below the conventional method, the proliferation of antigen-specific CTL can be improved up to about 34-fold. This has a significant derivative effect in cell therapy where the cell amount at the start of production is often quite limited. For example, by distributing a limited amount of target cells at a reduced surface density over an increased surface area, the growth rate of the population becomes dramatically higher compared to the conventional surface density, and a larger target cell population can be achieved in a shorter time.

[0050] Example 3: The minimum surface density of a cell population containing target cells and / or antigen-presenting cells enables the growth of a target cell population seeded at a very low surface density.

[0051] Figure 4 shows an example of the results obtained by continuing the study described in Figure 3, and further demonstrates that when target cells require the support of other cells, as long as the target cells are in the presence of a sufficient supply of feeder cells and / or antigen-presenting cells, they can initiate proliferation of the population at a lower target cell surface density, different from the conventional situation. In these experiments, the R:S ratio was 8:1 and the target cells were approximately 1.0×10 6 cells / cm 2 and the R:S ratio was 1:32 and simply the target cells were approximately 3900 cells / cm 2 and the entire cell population with the surface density and R:S ratio between them was continuously demonstrated how it could significantly proliferate the target cells until exceeding 50 times the starting surface density, and the test was interrupted at that point.

[0052] Example 4: The ability to start a stage at a lower target cell surface density, different from the conventional situation, proliferate the population, end the stage, and repeat the conditions to enable a stepwise repetition of the production process was demonstrated to produce reproducible results.

[0053] As shown in Figure 5, the evaluation described in Example 3 was continued at three target cell surface densities (number of CTLs / cm 2 ). Each specific seeding density was able to consistently achieve the same growth magnification. This suggestion will be described in more detail in relation to the ability to dramatically shorten the production time of the target cell population.

[0054] Example 5: Culturing target cells on a growth surface made of a gas-permeable material while simultaneously increasing the medium volume:growth surface area ratio increases the number of times the target cell population can double in a given culture stage compared to the conventional method and increases the achievable surface density.

[0055] Cell lines and tumor cells, immunophenotyping, CFSE labeling, annexin V-7-AAD staining, chromium release assay, enzyme-linked immunosorbent spot (ELIspot) assay, retrovirus production and transduction of T lymphocytes, and statistical analysis were as described in Example 1.

[0056] A test instrument (hereinafter generally referred to as "G-Rex") was constructed as shown in FIG. 6. The bottom 20 of each G-Rex 10 is made of a gas-permeable silicone membrane, and the thickness of this membrane was approximately 0.005 - 0.007 inches. U.S. Patent Publication No. 2005 / 0106717 (hereinafter referred to as Wilson '717), which is co-pending, is one of many other sources of information regarding the use of alternative gas-permeable materials, and those skilled in the art can obtain knowledge about the shape, characteristics, and other useful features of gas-permeable culture instruments that are beneficial for many embodiments of the present invention therefrom. In Example 3, the growth area of the G-Rex (referred to as "G-Rex 40") was 10 cm 2 and a cell population (shown as element 30) was placed statically thereon. The characteristics of the cell population varied through experiments as described herein. The medium volume (shown as element 40) was 30 mL unless otherwise specified, creating a medium volume:growth area ratio of 3 ml / cm 2 .

[0057] Activated EBV-specific CTL and irradiated autologous EBV-LCL were cultured in the G-Rex 40 instrument with a CTL:LCL ratio of the conventional 4:1. EBV-CTL was seeded in the G-Rex 40 at a surface density of 5×10 5 cells / cm 2 , and the growth rate of the EBV-CTL group was compared with that of EBV-CTL seeded at the same surface density in a standard 24-well plate with a medium volume:growth area of 1 ml / cm 2 . After 3 days, as shown in FIG. 7A (p = 0.005), the EBV-CTL in the G-Rex 40 increased from 5×10 5 / cm 2 to a median of 7.9×10 6 / cm 2 (range 5.7 - 8.1×10 6 / cm 2 ) without changing the medium. In contrast, the EBV-CTL cultured in the conventional 24-well plate for 3 days decreased from a surface density of 5×10 5 / cm 2 to a median of 1.8×10 6 / cm 2 (range 1.7 - 2.5×106 / cm 2 ) only increased up to. By replenishing the medium in G-Rex40, the surface density could be further increased, but replenishing the medium or IL2 in a 24-well plate could not increase the cell surface density. For example, the EBV-CTL surface density was 9.5×10 6 cells / cm 2 (range 8.5×10 6 ~11.0×10 6 / cm 2 ) and further increased (data not shown) after replenishing the medium and IL-2 on day 7 in G-Rex40.

[0058] To understand the mechanism behind the superior cell growth in G-Rex devices, on day 5 of culture, the viability of OKT3-stimulated peripheral blood T cells was evaluated using forward versus side scatter analysis by flow cytometry. EBV-CTL could not be evaluated in this assay because residual irradiated EBV-LCL that interfered with the analysis was present in the culture. As shown in Figure 7B, the cell viability was significantly higher in the culture of G-Rex40 (the viability in G-Rex40 was 89.2% and in the 24-well plate was 49.9%). Then, the cultures were analyzed daily for 7 days using annexin-PI 7AAD to discriminate between live cells and apoptotic / necrotic cells, and as shown in Figure 7C, it was consistently observed that the viability of T cells growing in the 24-well plate was lower compared to the viability of T cells growing in G-Rex. These data indicate that the cumulative improvement in the survival of proliferating cells contributed to the increase in cell number in G-Rex devices compared to 24-well plates.

[0059] To determine whether there is also a contribution from the increased number of cell divisions in the G-Rex versus a 24-well plate, T cells were labeled with CFSE on day 0 and divided into G-Rex40 with a medium volume of 40 ml and a 24-well plate with a medium volume of 2 ml in each well. As a result of performing flow cytometry analysis every day, it was demonstrated that there was no difference in the number of cell divisions from day 1 to day 3. However, after day 3, as shown in Fig. 7D, the population of target cells cultured in G-Rex40 continued to increase at a rate exceeding the decline rate in the 2 ml wells. This indicates that the culture conditions were limited. Therefore, the large population of target cells in the G-Rex40 test device was obtained from the combination of reduced cell death and sustained proliferation compared to the conventional method.

[0060] Example 6: By using a growth surface composed of a high medium volume: growth area ratio and a gas-permeable material that is different from the conventional one, it is possible to reduce the need to supply nutrients to the culture during production while obtaining a high target cell surface density that is different from the conventional one.

[0061] This was demonstrated by using the G-Rex test device for the initiation and proliferation of EBV:LCL. In this example, G-Rex2000 refers to the device as described in Fig. 8, excluding that the bottom has a growth area of 100 cm 2 and the available medium volume is 2000 ml. EBV-LCL was cultured in G-Rex2000 and proliferated without changing the cell phenotype. Along with 1000 ml of complete RPMI medium, EBV-LCL was plated in G-Rex2000 at a surface density of 1×10 5 cells / cm 2 to make the medium volume: surface area ratio 10 ml / cm 2 For comparison, along with 30 ml of complete RPMI medium, EBV-LCL was plated in a T175 flask at a surface density of 5×10 5 cells / cm 2 to make the medium volume: surface area ratio 0.18 ml / cm 2It was thus. As shown in Fig. 8A, EBV-LCL cultured in G-Rex2000 grew larger than that cultured in a T175 flask without requiring any operation or medium change. As shown in Figs. 8B and 8C, when EBER and the B cell marker CD20 were evaluated by Q-PCR, the final cell product was not modified under this culture condition.

[0062] Example 7: If there are not enough feeder and / or antigen cells at the start of culturing, the target cells may not grow. However, the cell population can be modified to include additional cell types that act as feeder cells and / or antigen-presenting cells for growth.

[0063] Fig. 9 shows an exemplary embodiment experimentally demonstrating that the growth of the AL-CTL group could not be initiated because the cumulative surface density of the target cells and antigen-presenting cells was very low (in this case, combining AL-CTL and LCL cells to create a cell population with a surface density of 30,000 cells / cm 2 However, by modifying the population to include other cell types that function as feeder cells, this same cell population could be grown. In this case, a feeder layer of three different forms of irradiated K562 cells with a surface density of approximately 0.5×10 6 cells / cm 2 was evaluated. In each case, the AL-CTL group grew from the initial cell population shown in the first column of the histogram, from a surface density of just 15,000 cells / cm 2 to 4.0×10 6 cells / cm 2It changed over 14 days to the surface density. Also, in contrast to the addition of the third cell type, it was demonstrated that similar favorable results were obtained by the increase in the LCL group. To demonstrate that a very low target cell population can be used to initiate growth when the cell population contains a sufficient number of feeder and / or antigen-specific cells, the high surface density used for LCL or K562 was arbitrarily selected. If the feeder cells are in short supply, expensive, or cumbersome to prepare, it is recommended to reduce their surface density to less than 0.5×10 6 cells / cm 2 . Generally, and as has been demonstrated, when antigen-presenting cells and / or feeder cells are present in the cell population, the additional surface density of the antigen-presenting cells and / or feeder cells and the target cells should preferably be at least about 0.125×10 6 cells / cm 2 to create the surface density in a cell population sufficient to initiate the growth of the target cell population. Also, to achieve continuous growth beyond the limits of the standard surface density, in this example, a growth surface made of a gas-permeable material was used together with a medium volume:surface area ratio of 4 ml / cm 2 .

[0064] Example 8: By reducing the surface density of the target cells, modifying the responder:stimulator ratio, increasing the medium:growth surface area ratio, and periodically distributing the cells in a low-surface-density culture onto a growth surface made of a gas-permeable material, more target cells can be produced in a shorter time compared to other methods, and the production process is simplified.

[0065] To further evaluate the ability to simplify and shorten the production of target cells, the initiation and growth of EBV-CTL were performed using a G-Rex test device. In this example, G-Rex500 refers to a device as described in FIG. 6, except that the bottom consists of a growth surface area of 100 cm 2 and the usable medium volume is 500 ml.

[0066] As an initial stage of EBV-CTL production, PBMCs were seeded in a G-Rex40 at a surface density of 1×10 6 / cm 2 (total = 10 2 PBMCs distributed over the growth area of 10 cm 7 of G-Rex40), and they were stimulated with EBV-LCL at a PBMC:EBV-LCL ratio of 40:1. For CTL production, this 40:1 ratio is preferred for the first stimulation to maintain the antigen specificity of the responding T cells. After the initial culture stage, the second stage was started on day 9. Here, 1×10 7 responding T cells were transferred from the G-Rex40 to a G-Rex500 test device. To initiate the second culture stage, when 200 mL of CTL medium was placed in the G-Rex500, the medium volume:surface area ratio at the start of the second stage was 2 ml / cm 2 , and the height of the medium was 2.0 cm higher than the area of the growth surface. The surface density of the target cells at the start of the second stage was CTL 1×10 5 cells / cm 2 , and the surface density of the antigen-presenting cells was LCL 5×10 5 cells / cm 2 , so the target cell:antigen-presenting cell ratio was 1:5, different from the conventional one. This cell surface density and R:S ratio in the second stage resulted in consistent EBV-CTL proliferation in all donors screened. Four days later (day 13), when IL-2 (50 U / ml - final concentration) was directly added to the culture and 200 ml of fresh medium was added as well, the medium volume:surface area ratio became 4 ml / cm 2 . On day 16, the cells were harvested and measured. The median surface density of the obtained CTLs was 6.5×10 2 per 1 cm 6 (range of 2.4×10 6 ~3.5×10 7 ).

[0067] By using a growth surface made of a gas-permeable material, compared to the conventional protocol, there was an increase in the medium volume:surface area ratio (i.e., greater than 1 ml / cm 2 ), and a decrease in the cell surface density (i.e., less than 0.5×10 6 / cm 2Less than) and responder cells: Modification of the stimulator cell:responder cell ratio (less than 4:1) is possible, and the production time is shortened. Figure 10A shows a comparison of the G-Rex method of Example 8 with the use of the conventional method of Example 1 and the G-Rex method described in Example 5. As shown, the conventional method required 23 days to confer the same number of target cells that could be conferred in about 10 days in either G-Rex method. After 23 days, the G-Rex method of Example 8 was able to produce 23.7-fold more target cells than the G-Rex method of Example 5, and 68.4-fold more target cells than the conventional method of Example 1. Furthermore, when the culture was split when the cell surface density exceeded 7×10 6 / cm 2 , the target cells continued to divide until days 27 - 30 without the need for further antigen-presenting cell stimulation.

[0068] CTLs in G-Rex could not be clearly visualized using an optical microscope, but CTL clusters could be seen by eye or with an inverted microscope. The appearance of the cells on days 9, 16, and 23 of the culture is shown in Figure 10B. As shown in Figure 10C, culturing in G-Rex did not change the phenotype of the expanded cells, with more than 90% of the cell population being CD3+ cells (G-Rex vs 24-well being 96.7 ± 1.7 vs 92.8 ± 5.6), and these were mainly CD8+ (62.2% ± 38.3 vs 75% ± 21.7). Evaluation of the activation markers CD25 and CD27 and the memory markers CD45RO, CD45RA, and CD62L demonstrated that there were no substantial differences between the EBV-CTLs expanded under each culture condition. Antigen specificity was also not affected by the culture conditions as measured by ELISpot and pentamer analysis. Figure 10D shows a representative culture of T cells stimulated with EBV peptide epitopes from LMP1, LMP2, BZLF1, and EBNA1 and stained with HLA-A2-LMP2 peptide pentamer staining, showing a similar frequency to peptide-specific T cells. Furthermore, as shown in Figure 10E, 51When evaluated by a Cr release assay, the expanded cells maintained their cytolytic activity and specificity, killing autologous EBV-LCLs (G-Rex vs 24-well plate, 62% ± 12 vs 57% ± 8 at an E:T ratio of 20:1), and there was less killing of HLA-mismatched EBV-LCLs (15% ± 5 vs 12% ± 7 at a ratio of 20:1).

[0069] Examination of various novel methods for improved cell production for cell therapy: Examples 1-8 have been presented to demonstrate to those skilled in the art how to promote and simplify the production of cells for cell therapy research and clinical applications using various conditions such as a decrease in the surface density of the target cell population at the start of the production cycle, a decrease in the surface density ratio between responder cells and stimulator cells, a growth surface made of a gas-permeable material and / or an increase in the medium volume: growth surface area ratio. Examples 1-8 relate to the production of antigen-specific T cells, but these novel culture conditions are applicable to many important floating cell types relevant clinically (or required for preclinical evidence in a concept mouse model), such as regulatory T cells (Tregs), natural killer cells (NK), tumor-infiltrating lymphocytes (TILs), primary T lymphocytes, a wide range of antigen-specific cells, and many others (all of which can also be genetically modified to improve their function, persistence in vivo, or safety). Cells can be expanded using feeder cells and / or antigen-presenting cells, which can include PBMC, PHA blasts, OKT3 Ts, B blasts, LCLs, and K562s (naturally or genetically modified to express antigens and / or epitopes, as well as costimulatory molecules such as 41BBL, OX40L, CD80, CD86, HLA, and many others), which may or may not be pulsed with peptides and / or related antigens.

[0070] Lower initial surface density compared to the prior art: One aspect of the present invention is the discovery that a lower target cell surface density can reduce the production time compared to conventional methods. Thus, the target cells can have a greater difference between the maximum cell surface density and the minimum cell surface density than is allowed by conventional methods. Although the growth rate of the target cell population has begun to decline, if the amount of target cells is not yet sufficient to complete production, it is preferable to redistribute the target cells again at a low starting surface density on an additional growth surface made of a gas-permeable material.

[0071] To illustrate how a novel cell production method that depends on a lower surface density is applicable at the start of any given culture stage, an example is described here. Figure 11 shows a graphical representation of the growth of a target cell population on a growth surface based on a conventional scenario, compared to the growth of an individual number of a target cell type using one aspect of the present invention. In this novel method, the surface density of the target cells at the start of the production stage is smaller than the conventional surface density. To focus on the advantages of this novel method, this description does not touch on the process of initially obtaining the target cell population. To make it easier for those skilled in the art to confirm the relative time advantages of this novel method, the culture "Day" starts from "0". In this example, each production cycle of the conventional method starts with a conventional target cell surface density of 0.5×10 6 cells / cm 2 , but each production cycle of this example starts with a much lower target cell surface density of 0.125×10 6 cells / cm 2 that is different from the prior art. Thus, in this example, a surface area that is 4 times larger than the surface area required by the conventional method (i.e., 500000 / 125000) is required to start the culture. In this example, the target cells of the conventional method reach a maximum surface density of 2×10 6 cells / cm 2 in 14 days. Thus, a growth area of 1 cm 2 is seeded with 2×10 6 cells / cm 2 , and those cells are then redistributed onto a growth area of 4 cm 2 to a conventional starting density of 0.5×10 6 cells / cm 2(i.e., 4 cm 2 × 0.5 × 10 6 cells = 2 × 10 6 cells) can be used to continue production. This cycle is repeated for an additional 14 days, at which point the maximum cell surface density is reached again, and each of the growth areas of 4 cm 2 has 2.0 × 10 6 cells, i.e., a total of 8.0 × 10 6 cells are seeded, and then these cells are distributed over a growth area of 16 cm 2 and the growth cycle is repeated such that a total of 32 × 10 6 cells are seeded in 42 days.

[0072] The novel method shown in Figure 11 creates a starting surface density that is different from the conventional method of depositing 500,000 target cells per cm 2 at the start of production. Instead, 500,000 cells are equally distributed over a growth area of 4 cm 2 to create a lower starting surface density of 125,000 target cells / cm 2 on day 0. In the example, the novel method has a growth rate that begins to decline exactly on day 7, as in the conventional method. The cells in the novel method have a surface density of 1 × 10 6 cells / cm 2 . Thus, at the point when the growth rate begins to decline, this culture stage yields 4 × 10 6 cells, which are then redistributed over a growth area of 32 cm 2 to create a starting surface density of 0.125 × 10 6 cells / cm 2 (i.e., 32 cm 2 × 0.125 × 10 6 cells = 4 × 10 6 cells) with which production in stage 2 can be continued. The production cycle or stage is repeated for an additional 7 days until day 14, at which point the cell surface density reaches its maximum again, with each growth area of 32 cm 6 containing 1.0 × 10 2 target cells, and a total of 32 × 10 6Individual cells are produced. It should be noted that, as with the conventional method, at the end of each production cycle, it is a matter of how the new method imparts a multiple obtained by dividing the final surface density by the starting surface density. However, by reducing the starting cell surface density before the cells enter growth production and by completing each production stage, the time is dramatically shortened. In this example, by reducing the target cell surface density (in this case, to 0.125×10 6 cells / cm 2 or less), it describes how the same amount of target cells are imparted in only 33% of the time taken by the conventional method (14 days vs. 42 days) compared to the conventional cell surface density.

[0073] 0.125×10 6 cells / cm 2 The starting surface density was used to quantify the advantages, but those skilled in the art should realize that this example of the present invention demonstrates that the production time is shortened by any reduction below the conventional cell surface density. Furthermore, those skilled in the art will recognize that the cell growth rate and the point at which cell growth decline occurs described in the present method and other novel methods presented herein are described for illustrative purposes only, and the actual ratios will vary for each application based on a wide range of conditions such as the composition of the medium, cell type, etc. Furthermore, those skilled in the art will recognize that the advantage of this aspect of the present invention is the shortening of the production time resulting from having a cell surface density lower than the conventional cell surface density in any particular application, and that the specific conventional surface density used in this exemplary embodiment may vary from application to application for a given application.

[0074] Therefore, one aspect of the method of the present invention will be described here when there is a desire to minimize the production time of a given amount of target cells present in a cell population by using a reduced cell surface density. The target cells should be deposited on the growth surface at a low cell surface density different from the conventional one so as to satisfy the following: a. The target cells are at most 1 ml / cm when the growth surface is not made of a gas-permeable material in the presence of antigen-presenting cells and / or feeder cells. 2The medium volume:surface area ratio, and when the growth surface consists of a gas-permeable material, it is at most 2 ml / cm 2 The medium volume:surface area ratio, b. The preferred surface density condition at the start of the production cycle is such that the target cell surface density is preferably less than 0.5×10 6 cells / cm 2 and more preferably is decreased as described in FIG. 4, c. The surface density of the target cells + the surface density of the antigen-presenting cells and / or feeder cells is preferably at least about 1.25×10 5 cells / cm 2 .

[0075] When attempting to make the surface density of the antigen-presenting cells and / or feeder cells even lower than 1.25×10 5 cells / cm 2 it is advisable to demonstrate based on the above examples that the growth of the target cell population is not limited. Based on the objective of demonstrating that when increasing by sufficiently supplying antigen-presenting cells and / or feeder cells, growth of the target cell population can be achieved at a lower density different from the conventional one, 1.25×10 5 cells / cm 2 was selected.

[0076] The use of a growth surface consisting of a gas-permeable material and a higher medium volume:growth surface area ratio can simplify and shorten production. Another aspect of the present invention is the discovery that production time is shortened by repeating a production cycle that uses a growth surface consisting of a gas-permeable material and a medium volume:growth surface area ratio that exceeds the conventional ratio, and increases the amount of the growth surface area used over time.

[0077] Here, exemplary examples are presented showing how these conditions can shorten the production time. FIG. 12 shows a growth surface consisting of a gas-permeable material and 1 or 2 ml / cm 2To reinforce the discussion by showing an example of the advantages obtained by using a high medium volume: growth area ratio that is different from the prior art and exceeds it. The following discussion is intended to demonstrate to those skilled in the art how several options, including shortening the production time, reducing the amount of growth area used, and / or reducing labor and contamination risks, can be utilized by using such a method. Those skilled in the art will recognize that Figure 12 and the related discussion are merely examples and do not limit the scope of the present invention.

[0078] The cell population containing the target cell group in this exemplary embodiment is considered to consume approximately 1 ml per "X" hours. Figure 12 shows two production processes named "conventional method" and "new method". At the start of growth, each process begins with target cells at a surface density of 0.5×10 6 / cm 2 However, the growth surface of the new method is made of a gas-permeable material, and its medium volume: surface area ratio is 2 ml / cm 2 whereas the conventional method is 1 ml / cm 2 At time "X", the target cell group of the conventional method reaches a surface density plateau of 2×10 6 / cm 2 and depletes the nutrients, but the additional medium volume of the new method allows growth to continue, and the target cell surface density becomes 3×10 6 / cm 2 If the new method continues, it will be 4×10 6 / cm 2Reach the surface density. Thus, many advantageous options arise. The new method may produce more cells than the conventional method and finish before time "X", may produce about 1.5 times as many cells as the conventional method and finish at time "X", or may take twice as long but without the need to operate the supply equipment, produce twice as many target cells as the conventional method, and continue until the nutrients are depleted. To collect the same number of cells by the conventional method, the cells must be recovered, and when the process is restarted, labor and the potential risk of contamination are added. Since cell therapy applications typically cannot start without a certain number of cells, in the conventional method, the option of simply increasing the surface area at the start of production is not allowed.

[0079] Figure 13 continues the example of Figure 12 to show how multiple production cycles can be even more advantageous. Figure 13 shows a graphical representation of the growth of the target cell population on the growth surface by the conventional method compared to the growth of the target cell population of the target cell type by one new method of the present invention, and the surface density of the new method is higher than that of the conventional method. To focus on this embodiment, this description does not touch on the process of obtaining the target cell population. To make it easier for those skilled in the art to confirm the relative time advantages of this aspect of the present invention, the culture "day" starts from "0". In this example, on "Day 0", both cultures are started using the conventional target cell surface density of 0.5×10 5 cells / cm 2 . This exemplary embodiment is such that the growth surface of the conventional method also consists of a gas-permeable material. However, the medium volume:growth surface ratio of the conventional method is 1 ml / cm 2 compared to 4 ml / cm 2 of the new method. As shown in Figure 13, the growth rate of the target cell population by the conventional method begins to decline when the surface density reaches about 1.5×10 6 cells / cm 2 in about 4 days, and reaches a maximum surface density of 2×10 6 cells / cm 2 in 14 days. At that point, the target cell population is at a surface density of 0.5×10 2 / cm 6 in 1.0 ml / cm 2 of fresh medium and 4 cm2 is distributed to the growth area, the production cycle starts again, and in another 14 days, the surface density reaches 2×10 6 cells / cm 2 and reaches 8×10 6 target cells in 28 days. In comparison, the target cell population of the new method has a surface density of approximately 3.6×10 6 cells / cm 2 when the growth rate begins to decline, and can reach a maximum surface density of 4×10 6 cells / cm 2 in 28 days. However, to accelerate production, the cycle is terminated while the growth rate of the target cell population is still high. Thus, in approximately 10 - 11 days, 3×10 6 cells are redistributed onto a growth area of 6 cm 2 in 4.0 ml / cm 6 of fresh medium at a surface density of 0.5×10 2 / cm 2 and the production cycle starts again. The target cell population reaches a surface density of approximately 3×10 6 cells / cm 2 in another approximately 10 - 11 days and provides 18×10 6 target cells in approximately 21 days. Thus, the new method produced more than twice the number of target cells in approximately 75% of the time compared to the conventional method.

[0080] A cell surface density of more than 10×10 6 cells / cm 2 was achieved on the growth surface made of a gas - permeable material. This demonstrates that the use of the high - surface - density aspect of the present invention is not limited to the density described in this example.

[0081] Therefore, another example of the method of the present invention is described below when there is a desire to minimize the time to produce a given amount of target cells present in a cell population by using a reduced cell surface density: a. Seeding target cells on a growth surface area made of a gas - permeable material at a medium volume: surface area ratio of at least 2 ml / cm 2 in the presence of antigen - presenting cells and / or feeder cells, b. Establish favorable surface density conditions at the start of the production cycle such that the target cell surface density is within the conventional density of about 0.5×10 6 cells / cm 2 ; c. Enable the target cell population to grow beyond the conventional surface density of about 2×10 6 cells / cm 2 ; and d. If more target cells are required, redistribute the target cells to an additional growth surface made of a gas-permeable material and repeat steps a - d until sufficient target cells are obtained.

[0082] Using these novel methods, further advantages can be achieved by combining the attributes for starting the following cultures. That is, using a low surface area different from the conventional one, using a novel surface density ratio of target cells and / or feeder cells, utilizing a growth surface area made of a gas-permeable material, using a high medium volume:growth surface area ratio different from the conventional one, and performing production in cycles. The said conditions can be changed in any production cycle to achieve the desired results, such as balancing the shortening of production time, utilization of surface area, supply frequency, etc.

[0083] Figure 14 shows another novel method that provides further advantages compared to the conventional method. Those skilled in the art will recognize that, similar to the other exemplary embodiments described herein, the description herein does not limit the scope of the invention, but instead serves to explain a method for obtaining the advantage of improving production efficiency.

[0084] In this example, the target cells double weekly under conventional conditions. For ease of confirmation by those skilled in the art of the relative time advantage of this embodiment, the culture "Day" starts from "0". Also, for the sake of simplifying this example, the matters previously described regarding the feeder and / or antigen-presenting cell surface density ratio will not be repeated. By way of illustration, assume that on "Day 0" of production, there is a starting target cell population of 500,000 with a doubling time of 7 days under conventional conditions. The conventional method is 0.5×10 6cells / cm 2 surface density and 1 ml / cm 2 medium volume:surface area ratio. As shown, when the target cell population reaches a surface density of 2×10 6 cells / cm 2 , the cells are distributed onto additional surface area at a surface density of 0.5×10 6 cells / cm 2 and a new production cycle is initiated. The novel method of this example starts with a surface density of 0.06×10 6 cells / cm 2 , a growth surface area made of a gas-permeable material and a medium volume:surface area ratio of 6 ml / cm 2 . As shown, when the population approaches the start of the growth plateau, the cells are redistributed onto more growth surface area. In this case, in the conventional method, a plateau is initiated when the cell surface density approaches 1.5 times the medium volume:surface area ratio (i.e., 1.5×10 6 cells / ml), so the population is determined to have reached the plateau. Thus, on approximately day 9, the cells are distributed onto a growth surface area of 36 cm 6 at a surface density of about 4.5×10 2 cells / cm 2 and a new production cycle is initiated.

[0085] Figure 15 tabulates the comparison of each production method shown in Figure 14 and grows in stages to demonstrate the strength of the novel method and the reason why it is advisable to adjust the production protocol at various stages to fully obtain its effects. The novel method overwhelms the conventional method only after just the second stage of the production cycle is completed, and it should be noted that it provides approximately 1.37 times more cells with only 61% of the required surface area in just half the time. However, in the third stage of the production cycle, attention should also be paid to how the cells increase quantitatively and correspondingly the surface area increases. Thus, to achieve the optimal effect level for any given process, the production cycle should be modeled to predict the method of adjusting the initial cell surface density and / or the final cell surface density throughout each cycle of the process.

[0086] As an example, FIG. 16 shows an example of how variable quantities can be changed in a novel method in order to gain efficiency as production progresses. For example, the starting surface density in cycle 3 increases from 0.06 to 0.70 cells / cm 2 and the final surface density can change from 4.5 to 7.5 cells / cm 2 . The increase in the final surface density is such that the medium volume:surface area ratio becomes a larger number than the initial 6 ml / cm 2 . As the medium volume:surface area ratio increases, the time that the cycle remains in the rapid growth phase (i.e., the population growth before the plateau phase) becomes longer. In this case, the rapid growth phase was further completed over 5 days to increase the medium volume:surface area ratio to approximately 8 ml / cm 2 . By doing so, in this example, more than 3 trillion cells can be produced at a reasonable surface area in 34 days. For example, an apparatus was fabricated and tested on a growth surface of approximately 625 cm 2 made of a gas-permeable material. This is clearly a cell production technique superior to the conventional method.

[0087] Thus, another preferred embodiment of the method of the present invention is described herein when there is a desire to minimize the time to produce a given amount of target cells present in a cell population by using a reduced cell surface density: a. Seeding target cells on a growth surface area made of a gas-permeable material at a medium volume:surface area ratio of at least 2 ml / cm 2 in the presence of antigen-presenting cells and / or feeder cells, b. Establishing, at the start of the production cycle, a preferred surface density condition such that the target cell surface density is less than the conventional density, preferably in the range of about 0.5×10 6 cells / cm 2 to about 3900 cells / cm 2 and the total of the target cells and antigen-presenting cells and / or feeder cells is at least about 1.25×10 5 cells / cm 2 , c. About 2×10 6 cells / cm 2Enabling the growth of the target cell population beyond the conventional surface density. d. When more target cells are required, redistribute the target cells onto an additional growth surface made of a gas-permeable material and repeat steps a-d until sufficient target cells are obtained.

[0088] The disclosure of the present invention advances the field of adoptive cell therapy by creating a new class of therapeutic cells called T vehicles. T vehicles consist of a population of T cells that do not have the inherent risk of GVHD and are further modified to include one or more therapeutic attributes that can act for treatment, providing therapeutic benefits to the recipient. Since T vehicles do not have the natural ability to cause GVHD disease, they are an ideal biological transport vehicle with any number of countermeasures to combat a wide range of medical conditions and diseases. The present invention discloses a method for producing and using T vehicles with therapeutic attributes to confer the health benefits of adoptive cell therapy to the recipient without the inherent risk of GVHD present in state-of-the-art methods. Importantly, since the therapeutic purpose of T vehicles is completely unrelated to the antigen specificity of the natural T cell receptor, T vehicles function contrary to state-of-the-art adoptive cell therapy. Those skilled in the art should recognize throughout the present disclosure and the presented exemplary embodiments that the therapeutic attributes of T vehicles do not include the natural antigen receptors of T vehicles.

[0089] T vehicles are produced by stimulating donor PBMC or donor cord blood with an antigen to activate the growth of donor T cells with natural antigen specificity to the antigen, thereby producing a population of antigen-specific T cells containing antigen receptors with antigen specificity to the antigen. By selecting antigens that do not exist on normal cells, a population of T cells with antigen receptors that cannot recognize normal cells can be created. By modifying natural T cells with one or more therapeutic attributes that ignore the therapeutic benefits that would be obtained from the antigen specificity of natural T cells and do not include the natural antigen receptor recognition ability, a group of T vehicles can be created that have purposes unrelated to their antigen-specific recognition and do not inherently tend to cause or are unable to cause GVHD.

[0090] The T vehicle may contain two or more natural antigen-specific T cell populations. However, since the T vehicle does not rely on their natural antigen specificity for its therapeutic purpose, the T vehicle can be infused into the recipient regardless of whether the recipient's serum type shows positive compatibility with one or more natural antigen receptors of the T vehicle. Also, since the one or more natural T cell populations from which the T vehicle is obtained do not carry the inherent risk of GVHD, an important attribute of the T vehicle is their ability to be used in the setting of HLA incompatibility. This can enable the establishment of an allogeneic bank of T vehicles that can benefit a wide society without the HLA compatibility restrictions required by state-of-the-art methods. If the T vehicle has a natural antigen specificity that is HLA-incompatible with the recipient, the natural T cell receptor of the T vehicle can recognize the recipient's cells and cannot cause GVHD. Nevertheless, since the T vehicle has been modified to have a therapeutic attribute that does not rely on the natural antigen receptor to achieve its therapeutic purpose, it initiates its therapeutic activity completely in the setting of HLA incompatibility. However, the T vehicle is not limited to use in the setting of HLA incompatibility. By creating a T vehicle consisting of T cells with natural antigen receptors with a highly restricted antigen specificity for antigens not expressed in normal cells, it is possible to avoid the onset of GVHD disease despite a partial HLA compatibility between the recipient and the natural antigen specificity of the T vehicle. To enable the T vehicle to be used in the setting of HLA compatibility or HLA incompatibility, it is preferred that the natural antigen specificity of the T vehicle causes the T vehicle to recognize an antigen that is not present in normal cells, preferably normal human cells, and it is even more preferred that it can recognize only a single epitope of an antigen that is not present in normal mammalian cells.

[0091] If the T vehicle is HLA-incompatible with the recipient, the recipient is expected to initiate an intense immune response that will ultimately eliminate the T vehicle. Therefore, by administering one or more additional doses of the T vehicle, the therapeutic objective of the T vehicle can be continued. This process can be continued as necessary to obtain the desired therapeutic objective. In a preferred method, since each dose of the T vehicle differs in terms of HLA, the patient's immune system must re-prepare itself each time it attempts to attack a new dose of the T vehicle, thereby keeping the intervals between each dose of the T vehicle approximately equal.

[0092] Methods for producing T cells having natural antigen receptors with highly restricted antigen specificity: Historically, it has not been practically possible to produce T cell populations at the scale required for widespread use in adoptive cell therapy. The state-of-the-art production methods for expanding T cell populations into therapeutically useful doses are not very practical and difficult to handle, thus limiting cell therapy to very small populations that must be handled in a few very specialized institutions. The basic attribute of T vehicles is that their natural T cell properties essentially do not expose the recipient to GVHD. The natural antigen specificity of T vehicles preferably only causes them to recognize antigens that do not exist on normal cells, more preferably normal human cells, and even more preferably only a single epitope of an antigen that does not exist on normal mammalian cells. Therefore, the effective production of these cells is the basis for the widespread use of methods involving T vehicles. Such T cells exist only at very low, sometimes undetectable frequencies in donor PBMC or cord blood. Thus, the problems inherent in state-of-the-art T cell production methods are exacerbated when attempting to generate T cell populations most suitable for use in T vehicles.

[0093] We have discovered methods and apparatus as described in U.S. Patent Application No. 13 / 475,700, filed May 18, 2012, entitled "Improved Cell Culture Methods for Adoptive Cell Therapy" (hereinafter referred to as Vera'700), which is incorporated herein by reference. This is contrary to state-of-the-art methods and is capable of efficiently producing T cells that have natural properties that essentially do not expose the recipient to GVHD. In so doing, the long periods required for the practical production of T cells, which are found at low frequencies in donor PBMC or cord blood, are satisfied. Further, when combined with the novel concept of T cells having therapeutic attributes not inherent to the natural antigen specificity of T cells, the production of T vehicles that function as biological carriers becomes possible and practical.

[0094] In one exemplary method, at the start of culturing, two or more selected antigens are presented to PBMC or cord blood (i.e., the original reservoir of antigen-specific T cells) to stimulate the growth of two or more distinct antigen-specific T cell populations, each expressing an antigen receptor for one of the presented antigens. The intention is to later select the most abundant and / or desirable natural T cell population for production and terminate the others. As the culture progresses after initiation, various T cell populations that react to various antigens may exhibit various levels of population expansion, depending on the size of their original populations. Further, some or all of them may continue to be undetectable. After a while, the culture is evaluated for the acceptable growth of a T cell population that reacts to any of the selected antigens. Such an evaluation may be for only one population specific to one antigen or for further populations specific to further antigens. When one antigen-specific T cell population demonstrates acceptable expansion, adding the antigen it recognizes into the instrument restimulates that particular T cell population and ultimately kills the remaining T cells, while the T cell population of particular interest continues to expand. However, when two or more T cell populations demonstrate acceptable expansion, there are two options: 1) the culture can be restimulated with only the antigen to which a particular T cell population is reacting (thereby terminating the expansion of the less abundant T cell populations), or 2) the culture can be divided into two or more culture vessels. Each vessel receives a single antigen different from the antigens of all other vessels and grows only one T cell population within each vessel, with all but the most abundant culture ultimately being terminated. Preferably, all culture vessels are gas permeable and are of the type described in co-pending U.S. Patent Publication Nos. 2005 / 0106717A1 (Wilson et al.) (hereinafter referred to as Wilson ‘717) and 2008 / 0227176A1 (Wilson et al.) (hereinafter referred to as Wilson ‘176), both of which are incorporated herein by reference, and rely on the method of Vera ‘700.

[0095] In a further example, the PBMC population present in the culture vessel can present antigen A, antigen B, and antigen C. After a while, the culture can be evaluated for the presence and / or proliferation of populations of individuals reactive to antigen A, B, or C. If the antigen-specific population reactive to antigen A is the only population not showing an acceptable frequency and / or number of individuals proliferating, that population can be terminated by restimulation with only antigens B and C. Alternatively, if the antigen-specific populations reactive to antigens B and C are growing approximately equally but it is unclear which will continue to grow at the best ratio, the culture can be divided into two vessels, with the intention of ultimately continuing production in one vessel and terminating production in the other. The first vessel receives antigen B and the second vessel receives antigen C. T cells presenting antigen specificity for antigen B proliferate in the first vessel, while T cells presenting antigen specificity for antigen C ultimately die out. The opposite occurs in the second vessel. Eventually, at some point after the start of the culture in the first and second vessels, consideration of the frequency and / or population size can be initiated with the aim of terminating the culture that is least efficient in the proliferation of the desired T cell population. Those skilled in the art should recognize that the main advantage of using multiple antigens rather than just one at the start of the culture is to increase the likelihood of finding a T cell population with suitable antigen specificity and growth rate. Further, by using multiple antigens in one vessel instead of multiple vessels with one antigen each, PBMC or cord blood, medium, cytokines, laboratory space, labor, and biohazard waste space are used more efficiently.

[0096] The selection of preferred native antigen specificities of T vehicles is described herein: It is preferred that only the native antigen specificities of T vehicles recognize antigens that are not present in normal cells, more preferably normal human cells, and even more preferably recognize only a single epitope of an antigen that is not present in normal mammalian cells, although this is not limiting and there are many suitable attributes of native antigen receptors that those skilled in the art should consider. Many options and characteristics are suitable. By way of example, the native antigen specificities of T vehicles can consist of two or more T cell populations having native antigen specificities. The native antigen specificities of T vehicles can be for self or non-self antigens; reptiles, amphibians, fish or birds; invertebrates such as sponges, coelenterates, worms, arthropods, mollusks or echinoderms; bacteria, fungi, parasites and sponges; viruses including but not limited to adenovirus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus (Adv), respiratory syncytial virus (RSV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), BK virus, JC virus, influenza, H1N1, parainfluenza, herpes simplex virus (HSV), varicella-zoster virus (VZV), parvovirus B19, coronavirus, metapneumovirus, bocavirus or K1 virus / WU virus; or for whole antigens or single epitopes of survivin, gp100, tyrosinase, SSX2, SSX4, CEA, NY-ESO-1, PRAME, MAGE-A1, MAGE-A3, MAGE-A4, claudin-6, cyclin-B1, Her2 / neu-ErbB2, histone H1.2, histone H4, mammaglobin-A, Melan-A / MART-1, Myc, p53, ras, PSA, PSMA, PSCA, Sox2, stromelysin-3, Trp2, WT1, proteinase 3, Muc1, alpha-fetoprotein, CA-125, bcr-abl, hTERT or prostate acid phosphatase-3.

[0097] To promote the growth of a suitable natural T cell population of donor cells, one of ordinary skill in the art would review U.S. Patent Publication No. 2011 / 0182870A1 (hereinafter referred to as Leen‘870, which is incorporated herein by reference), and consider the use of dendritic cells, monocytes, macrophages, B cells, T cells, PBMCs or artificial antigen presenting cells such as modified k562 (any of which can present the antigen of interest and give rise to the antigen specificity of the desired natural donor T cell population and thus the natural antigen specificity of the T vehicle) for stimulation; the use of antigens to induce the desired immune response in donor cells by cell lysates containing the antigen of interest, purified proteins containing the antigen of interest, recombinant proteins containing the antigen of interest, plasmid DNA encoding the antigen of interest, plasmid RNA encoding the antigen of interest, and / or peptide libraries containing the antigen of interest and / or one or more single synthetic peptides containing the antigen of interest.

[0098] The production of the T cell population is preferably initiated using the method of Vera‘700 and / or those presented herein, and more preferably initiated using gas-permeable culture devices of the types described in Wilson‘717 and Wilson‘176. One of ordinary skill in the art should recognize that the various methods in the described series of examples are more or less suitable depending on the specific purpose of each application. For example, various surface densities, medium heights, medium volume:growth surface area ratios, etc. are available, and similarly, stimulation at various frequencies and concentrations by cytokines such as IL2, IL15, IL21, IL12, IL7, IL27, IL6, IL18 and / or IL4, and the use of repeated in vitro stimulation with any antigen source in combination with any of the antigen presentation methods are also possible, and can be initiated with or without cell sorting by methods including but not limited to γ-ray capture, magnetic separation, single cell cloning and / or flow cytometry.

[0099] Example 9: A T vehicle having a natural T cell receptor that recognizes the CMV epitope NLV is unable to recognize non-self cell targets.

[0100] Antigen-specific T cells having natural antigen specificity against NLV-CMV were expanded from a frequency of 0.03% to 87% in PBMC in 12 days by the method described above. These cells were then placed in cultures containing cells obtained from three HLA-mismatched donors presenting the NLV peptide of the target CMV antigen.

[0101] Figure 17 demonstrates how T vehicles, despite having full functionality as demonstrated by their ability to recognize and kill autologous cells presenting the NLV peptide ("Auto4 pep") and avoid killing autologous cells not presenting the NLV peptide (Auto4), are unable to recognize cells from mismatched allogeneic donors, and shows whether they express the NLV peptides ("allo1" and "allo1 pep", "allo2" and "allo2 pep", "allo3" and "allo3 pep").

[0102] Selection and generation of one or more desired therapeutic attributes: There are a wide variety of options for modifying a population of antigen-specific T cells to include at least one therapeutic attribute. The following examples, while non-limiting, are intended to enable one of ordinary skill in the art to appreciate how the selection of therapeutic attributes is determined by the therapeutic objective and why the therapeutic attributes and their therapeutic objectives are independent of the antigen specificity of the T vehicle's natural antigen receptor.

[0103] Example 10: T vehicles loaded with recombinant proteins, administered as adjuvants for immunotherapy.

[0104] Immunotherapy is a class of treatments designed to elicit or amplify a patient's immune response. Examples include administration of vaccines designed to activate an immune response against tumor antigens expressed on cancer cells or delivery of T cells or NK cells expanded ex vivo. Recombinant proteins such as cytokines like IL2, IL7, GM-CSF are administered systemically to promote the growth, proliferation, persistence, and / or function of these cells in vivo, but systemic administration of some cytokines (e.g., IL2) is associated with in vivo toxicities including severe mucositis, nausea, diarrhea, edema, dyspnea, liver and kidney dysfunction, and proliferation of regulatory T cells that impair the function of induced / infused T cells. Administration of T vehicles loaded with recombinant proteins including cytokines can migrate to the site of inflammation and overcome such toxicities by directly delivering these recombinant proteins to the site of inflammation induced by immunotherapy.

[0105] One skilled in the art should recognize that T vehicles can be used for the purpose of delivering such cytokines instead of traditional non-specific systemic administration. For example, experiments were initiated to create a T vehicle that can produce the cytokine IL7 and express truncated CD34Δ. This truncated CD34Δ can be used to detect the proportion of transduced cells and select transgenic populations. In this case, as shown in Figure 18, donor T cells with 98% natural antigen specificity against the NLV epitope of the CMV virus were successfully modified to create a T vehicle with the therapeutic attribute of CD34Δ-IL7 cytokine expression when measured by flow analysis. Furthermore, testing demonstrated that only the T vehicle modified with the retroviral vector (CD34Δ-IL7 cytokine) could produce IL7 as detected by ELISA.

[0106] To evaluate a therapeutic T vehicle modified with a retroviral vector (CD34Δ - IL7 cytokine) regarding the in vivo effects and distribution of the IL7 cytokine, mice were divided into two groups (5 mice per group). In Group 1, tumor-bearing mice were treated with 2000 ng of IL7 cytokine administered systemically by intravenous injection. In Group 2, mice were treated with a single intravenous injection of 10E+06 T vehicle. Subsequently, randomly selected subjects from each group were sacrificed at week 1 and week 2, and the IL7 cytokine concentrations at various locations such as the heart, liver, kidney, spleen, peritoneum, tumor, and blood were evaluated by ELISA.

[0107] Figure 19A shows the IL7 cytokine accumulation at various locations for Group 1. ELISA analysis of the IL7 cytokine demonstrated that higher cytokine levels were detected in the kidney and that they were below the detection limit at the tumor site.

[0108] Figure 19B shows the IL7 cytokine accumulation at various locations for Group 2. ELISA analysis of the IL7 cytokine demonstrated that the cytokine concentration was higher at the tumor site compared to other organs and that cytokine production was sustained in the tumor for at least 2 weeks after T vehicle administration. Thus, the T vehicle was able to migrate to the tumor site and preferentially deliver the cytokine IL7 for an extended period. This clearly demonstrates that, due to the ability of the T vehicle to have a therapeutic attribute of delivering cytokines, superior therapeutic advantages are obtained compared to the state-of-the-art delivery methods of systemically administered cytokines. As expected, the in vivo presence of the T vehicle is limited, as indicated by the decrease in cytokine concentration between week 1 and week 2. Since the T vehicle does not remain within the recipient, this can be considered an additional advantage of the T vehicle. Preferably, additional doses of the T vehicle are administered as needed until the treatment outcome is met, and if additional doses are not administered, the T vehicle is removed from the recipient.

[0109] Example 11: Donor T cells can be modified to create a T vehicle whose therapeutic attribute is a chimeric antigen receptor (CAR) that targets a specific antigen.

[0110] Donor T cells (evaluated by pentamer analysis) having 98% of T cells with natural antigen specificity for the epitope NLV of virus CMV can be transduced to create a T vehicle having the therapeutic attribute of expressing a CAR capable of recognizing prostate stem cell antigen (PSCA). The therapeutic purpose of the T vehicle is the destruction of prostate tumor cells. As shown in Figure 20, in quadrant E2, 57.23% of the donor T cells were successfully modified to create a T vehicle having the therapeutic attribute of CAR-PSCA when measured by flow analysis. To test the killing efficacy of the T vehicle containing the therapeutic attribute of CAR-PSCA, unmodified donor T cells and T vehicles created by modifying donor T cells with CAR-PSCA were cultured at a 1:1 ratio with target cells that are antigen PSCA positive (GFP+) or PSCA negative (mOrange+). After 72 hours of culture, the number of remaining PSCA-positive tumor cells was measured by flow analysis. Figure 21 shows the experimental results at 72 hours. Quadrant A1 represents the number of PSCA-negative cells, quadrant A3 represents the number of T vehicles, and quadrant A4 represents the number of PSCA-positive tumor cells. As expected, after 72 hours, the unmodified donor T cells did not modify the original culture population. However, in contrast, the T vehicle expressing CAR-PSCA was able to almost completely eliminate the entire population of PSCA-positive tumor cells, while simultaneously demonstrating excellent selectivity for the PSCA antigen by leaving the PSCA-negative cells intact. This clearly demonstrates the ability of the T vehicle to produce a therapeutic benefit independent of its natural antigen specificity.

[0111] Example 12: Donor T cells can be modified to create a T vehicle whose therapeutic attribute is a receptor capable of depleting undesirable cytokines in the recipient.

[0112] Tumor cells are protected from the immune system by producing immunosuppressive cytokines that suppress the anti-tumor effects of endogenous T cells. Donor T cells are modified to create a T vehicle that has the therapeutic attribute of expressing whatever specific cytokine receptor is needed to endow the therapeutic purpose of attracting unwanted specific cytokines from the tumor, thereby having the therapeutic advantage of making the tumor environment more permissive to immunotherapy strategies. FIGS. 22A and 22B are diagrams depicting such a process. In the depiction of FIG. 22A, a T vehicle containing the therapeutic attribute of a receptor capable of binding IL4 is in proximity to tumor cells expressing the IL4 cytokine. In the depiction of FIG. 22B, the IL4 cytokine is bound to the T vehicle, and the amount of the IL4 cytokine that protects the tumor cells is significantly reduced. It should be noted how the therapeutic attribute, therapeutic purpose, and therapeutic advantage of the T vehicle do not include and are independent of the natural antigen receptor of the T vehicle. The ability of a T vehicle having the therapeutic attribute of expressing the extracellular recombinant cytokine receptor IL4R / 7 was evaluated, and experiments were conducted to deplete the IL4 cytokine. The T vehicle was conditioned by modifying donor T cells having natural specificity for the NLV epitope of the CMV virus. 5E+05 T vehicles were cultured in a 2 ml dose of medium in the presence of 2000 pg / ml of IL4 in a 24-well plate and compared to donor T cells. Then, the concentration of the cytokine IL4 was evaluated by ELISA at 24, 48, and 72 hours. The results are shown in FIG. 23. Since the reduction in the immunosuppressive tumor growth factor IL4 cytokine at 72 hours was significant, the T vehicles were clearly able to achieve their therapeutic purpose. Conversely, donor T cells (i.e., the histogram labeled "unmodified T vehicle") showed no ability to reduce the presence of IL4.

[0113] One of ordinary skill in the art should recognize that by virtue of the T vehicle having multiple therapeutic attributes, it becomes capable of achieving the therapeutic purpose intended to confer therapeutic advantages to the recipient. Here, the disclosed possibilities are augmented by several further examples.

[0114] T vehicle modified with the therapeutic attributes of chemotherapeutic agents for targeted cancer therapy: Various chemotherapeutic agents or antitumor drugs are used to treat various types of cancers such as breast cancer, prostate cancer, pancreatic cancer, liver cancer, lung cancer, brain tumors, leukemia, lymphoma, melanoma, and myeloma. Most chemotherapy is delivered intravenously, but many drugs are administered orally and then circulate in the body. Chemotherapeutic agents act by killing cells that divide rapidly (one of the main characteristics of most cancer cells). This means that chemotherapy also damages cells that divide rapidly under normal circumstances (such as cells in the bone marrow, digestive tract, and hair follicles). As a result, the most common side effects of chemotherapy are myelosuppression (decrease in blood cell production, thus immunosuppression), mucositis (inflammation of the digestive tract mucosa), and alopecia (hair loss). Nausea and vomiting induced by chemotherapy are also common treatment side effects. Administration of T vehicles loaded with these agents has the potential to counteract these toxicities. This can occur by incorporating chemotherapeutic agents into the T vehicle and injecting them into the recipient. Thereby, the T vehicle migrates to the inflamed (cancer) site and reduces the chemotactic gradient. In this way, the chemotherapeutic agent is placed in close proximity to the tumor cells, in contrast to when it is administered systemically to the recipient. In the case of HLA incompatibility, the recipient's immune system initiates an attack against the T vehicles, and they are destroyed, but the chemotherapeutic agent is not released at the tumor cell site. Thus, the payload (i.e., the chemotherapeutic agent) is directly deposited at the target site, unlike systemic administration, thereby reducing the off-target toxicity associated with chemotherapy.

[0115] This process is as shown in FIGS. 24A, 24B, and 24C. As shown in FIG. 24A, the T vehicle loaded with the chemotherapeutic agent moves towards the inflammatory site (i.e., tumor cells), and due to the HLA incompatibility between the T vehicle and the recipient cells, the natural antigen receptor of the T vehicle does not recognize the recipient cells and reaches the tumor cells without causing GVHD. As shown in FIG. 24B, the recipient's immune system targets the T vehicle placed at the site of the tumor cells. As shown in FIG. 24C, under the attack by the recipient's immune system, the T vehicle releases the chemotherapeutic agent at the site of the tumor cells, thereby avoiding the off-target toxicity inherent in the state-of-the-art chemotherapy delivery methods.

[0116] T vehicle modified with the therapeutic attribute of an antibacterial agent: An antibacterial agent is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoa. These agents are typically administered systemically and can be delivered in a more targeted manner when loaded onto a T vehicle that has the ability to return to the inflammatory site to deliver their payload.

[0117] T vehicle modified with the therapeutic attribute of producing a recombinant protein administered as an adjuvant for immunotherapy: Similar to being loaded with an exogenous recombinant protein, the T vehicle can be genetically modified using viral (e.g., adenovirus, retrovirus, lentivirus, etc.) or non-viral transfection techniques to transgenically express recombinant proteins including cytokines, chemokines, enzymes, tumor antigens, and cytokine receptors, and these can also be designed to act as adjuvants for other immunotherapy interventions, for example, to improve T cell persistence, promote proliferation, and induce homing.

[0118] T vehicle modified with a therapeutic attribute of expressing a transgenic molecule that confers tumor specificity: Similarly, the T vehicle can be modified with a recombinant protein such as a cytokine, and the T vehicle can also be genetically modified using viral (e.g., adenovirus, retrovirus, lentivirus, etc.) or non-viral transfection techniques to transgenically express a chimeric T cell receptor (CAR).

[0119] T vehicle modified with a therapeutic attribute of being loaded with a recombinant protein for the treatment of autoimmune diseases or being genetically modified with the protein: Autoimmune diseases result from an inappropriate immune response of the body against substances and tissues that normally exist within the body. In other words, the immune system mistakes a part of the body for a pathogen and attacks its own cells. This can be limited to some organs. Administration of a T vehicle loaded with recombinant proteins such as the cytokines IL10, TGFB, IL13 that suppress inflammation can overcome such autoimmune effects by directly delivering the recombinant proteins to the site of inflammation when needed, rather than distributing these recombinant proteins indiscriminately.

[0120] T vehicles can be genetically modified to express suicide genes: To rapidly and completely eliminate the injected cells, T vehicles can be incorporated with safety switches or suicide genes, which can be activated in case of toxicity. The most effective suicide genes are thymidine kinases from herpes simplex virus type I (HSV-tk). This enzyme phosphorylates the non-toxic prodrug ganciclovir, which is then phosphorylated by endogenous kinases to form GCV-triphosphate, which causes chain termination and single-strand breaks when incorporated into DNA, thereby killing dividing cells. Several phase I-II studies have shown that administration of ganciclovir can safely eliminate transplanted HSV-tk-modified cells in vivo. More recently, inducible Fas, Fas-associated death domain-containing protein (FADD), and caspase 9 have been considered as alternative non-immunogenic suicide genes. Each of these molecules can act as a suicide switch when fused to an FK-binding protein (FKBP) mutant that binds the dimerizing chemical inducer (CID) AP1903, a synthetic agent that has been proven safe in healthy volunteers. Administration of this small molecule causes cross-linking and activation of pro-apoptotic target molecules. Up to 90% of T cells transduced with inducible Fas or FADD undergo apoptosis after exposure to CID. Although promising, eliminating 90% of the transduced cells may be insufficient to ensure the safety of genetically modified cells in vivo. Transgenic expression of the CD20 molecule, which is normally expressed on B cells, is also hypothesized to be a suicide gene for T cell therapy. This strategy relies on the clinical availability of the humanized anti-CD20 antibody (rituximab), which is widely used to eliminate both tumorous B cells and normal B cells that express the CD20 antigen. Thus, by injecting T cells that transgenically express human CD20 and then administering rituximab in vivo, the injected T cell population is effectively eliminated, but normal B cells are also eliminated. Therefore, T vehicles can be modified to express one or a combination of these different suicide genes to control the elimination and delivery of the payload.

[0121] T vehicles genetically modified for in vivo imaging and / or modified with therapeutic attributes loaded thereon: Positron emission tomography (PET) is a nuclear medicine imaging method that produces three-dimensional images or photographs of functional processes within the body. This system detects pairs of gamma rays indirectly emitted by positron-emitting radionuclides (tracers) introduced onto biologically active molecules within the body. A three-dimensional image of the tracer concentration within the body is then constructed by computer analysis. Due to the ability of T vehicles to migrate to tumor sites, T vehicles can be loaded with radioisotopes that enable the detection and determination of the location of tumor sites in vivo.

[0122] Similarly, iodine 123 (123I or I-123) is a radioactive isotope of iodine used in nuclear medicine imaging methods such as single photon emission computed tomography (SPECT). This is the most suitable isotope for diagnostic tests for thyroid diseases. With a half-life of approximately 13.3 h (hours), which is ideal for a 24 h (hours) iodine uptake test, 123I has other advantages for imaging thyroid tissue and thyroid cancer metastases. Since iodine is selectively trapped in "iodine trapping" by hydrogen peroxide produced by the enzyme thyroid peroxidase (TPO), it can be safely used for imaging or treating thyroid tumors. Thus, T vehicles can be modified with thyroid peroxidase (TPO) that can be used to image / kill T vehicles after iodine trapping.

[0123] Those skilled in the art should recognize that the therapeutic attributes for any given therapeutic purpose of T vehicles can be created by many techniques including, but not limited to, the following techniques: a) Genetic modification with viral vectors such as retroviruses, adenoviruses, adeno-associated viruses or lentiviruses, and / or b) Genetic modification using non-viral vectors, including the use of DNA and / or RNA vectors incorporated by physical and / or chemical techniques such as electroporation and / or lipofection methods using transposons and transposases (e.g., Sleeping Beauty), and / or Piggybac technology, and / or c) Genetic modification to incorporate one or more transgenes to modify T vehicle trafficking, incorporate suicide genes, improve recipient immune reconstitution (e.g., cytokine production), and / or induce a direct anti-viral or anti-tumor effect (e.g., chimeric antigen receptors) or suppress the immune response for the treatment of autoimmune diseases, and / or d) Genetic modification to improve T vehicle trafficking by the expression of one or more chemokine receptors such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3-A, CXCR5, CXCR6, CX3CR1, and / or XCR1, and / or e) Genetic modification to improve recipient immune reconstitution by the expression of one or more cytokines such as GM-CSF, TNFα, INFγ, IL2, IL8, IL15, IL7, IL12, IL21, or IL26 in T vehicles, or by the expression or overexpression of co-stimulatory molecules CD80, CD86, 41BBL, OX40L, and / or f) Genetic modification to induce the death of T vehicles by the expression of one or more suicide genes such as the thymidine kinase TK gene, CD20, CD19, i-caspase 9, and / or g) i) An extracellular spacer using a CH2CH3 sequence derived from the IgG-Fc region, or ii) a transmembrane component including but not limited to the sequences of CD28, CD4, CD3 or CD8, and iii) linked to the CD3ζ endodomain, or iv) isolated from a specific antibody linked by the expression of a cytokine receptor or a natural ligand such as a cytokine encoding the CD3ζ endodomain, the expression of one or more transgenes such as a chimeric antigen receptor (CAR) that recognizes a tumor target via a single-chain variable fragment (scFv), including but not limited to genetic modifications for inducing direct antiviral or antitumor effects, and / or h) Genetic modifications for suppressing the immune response for the treatment of autoimmune diseases by the expression of one or more immunosuppressive cytokines such as IL4, IL6, IL10, IL13, TGFβ or by the expression of competing ligands such as CTLA-4, PD1.

[0124] Those skilled in the art should recognize that the therapeutic purposes of the T vehicle can be within a wide range including but not limited to any of the following: a) As a biological vehicle for carrying DNA, RNA, recombinant proteins, peptides or aptamers, b) As a biological vehicle for carrying chemical compounds, c) As a biological vehicle that allows the carrying of chemical compounds with therapeutic purposes including but not limited to chemotherapeutic agents, small molecules, nanoparticles, hormone agonists or antagonists, antiviral agents, antifungal agents, antiparasitic agents, and / or d) As a biological vehicle for carrying one or more chemical compounds that have no therapeutic purpose but have secondary advantages including but not limited to in vivo identification and imaging that enable the identification of metastatic disease sites.

[0125] Each application, patent, and paper cited in this specification, as well as each document or reference cited in each of these applications, patents, and papers (including during the examination of each issued patent; "application cited documents"), U.S. Patent Publication Nos. 2005 / 0106717 and 2008 / 0227176, each PCT and foreign application or patent corresponding to and / or claiming priority from any of these applications and patents, and further each document cited or referred to in each application cited document are hereby expressly incorporated by reference into this specification.

[0126] None of the documents incorporated by reference as above are to be construed as incorporating by reference any subject matter that would conflict with the express disclosure herein. None of the documents incorporated by reference as above are further construed as incorporating by reference any claim contained in such document that is not incorporated by reference herein. None of the documents incorporated by reference as above are further construed as incorporating by reference any definition given in such document that is not expressly included herein.

[0127] In interpreting the claims of the present invention, it is clearly intended that the provisions of 35 U.S.C. § 112, paragraph 6, should not be invoked unless the specific terms "means for" or "step for" are recited in the claims.

[0128] One of ordinary skill in the art will recognize that many changes can be made to the present disclosure without departing from the spirit of the invention described herein. Accordingly, it is not intended to limit the scope of the present invention to the embodiments and examples described. Rather, the scope of the present invention should be construed by the appended claims and their equivalents.

Claims

[Claim 1] 1. A genetically modified T cell for adoptive cell therapy comprising: the T cells are derived from human T cells that have a native T cell receptor that recognizes an antigen of fungal or viral origin; The T cells are infused into a patient with an HLA that does not match that of the T cells.

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