JP7244461B2 - Improved cell culture methods for adoptive cell therapy - Google Patents

Description

RELATED APPLICATIONS This application was filed on December 8, 2010 claiming the benefit of US Pat. This is a continuation-in-part application of WO 2005/020002 entitled "Improved Cell Culture Methods for Adoptive Cell Therapy" (hereinafter "parent application"), which is hereby incorporated by reference in its entirety into this application.

CELL CULTURE FOR CELL THERAPY TECHNICAL FIELD This invention relates generally to cell culture methods, and more particularly to cell culture for cell therapy. Additionally, the present invention relates to the production of T cells with therapeutic attributes for use in adoptive cell therapy.

Cell culture is a major contributor to the cost and complexity of cell therapy. With current methods, the cell culture process is time consuming and expensive. Typically, the in vitro culture process is performed in a stepwise manner to produce large numbers of cells. At the earliest stages, the cells of interest are a relatively small population in the cell population placed within the cell culture device. At this stage, the cell population typically includes a source of cells of interest (such as peripheral blood mononuclear cells), feeder cells that stimulate growth of the cells of interest, and/or antigen presenting. Culture devices and methods that allow the medium in which the cells reside to be substantially undisturbed are preferred, as the cells remain relatively undisturbed. Such equipment includes standard tissue culture plates, flasks and bags. Cultivation proceeds in a stepwise manner, generally depleting the cell population of the medium of growth substrates such as glucose, removing the spent medium, and replacing the spent medium with fresh medium to obtain the desired amount of cells of interest. It consists of repeating the process until the In many cases, as the population of the desired cell population increases and more growth surface is required, the cell population is transferred to another device to initiate a new stage of production. However, in conventional methods, the growth rate of the target cell population slows down as the population of the cell population on the growth surface increases. The end result is that it is very time consuming and complex to produce a sizeable population of target cells.

State-of-the-art production methods for generating T lymphocytes with antigen-specificity for Epstein-Barr virus (EBV-CTL) are an example of the complexity of production. Conventional methods for optimal expansion of EBV-CTL use standard 24-well tissue culture plates. The surface area on which the cells are placed in each well is 2 cm ² and its medium capacity is limited to 1 ml/cm ² due to gas transfer requirements. The culturing process is initiated by placing a cell population consisting of PBMCs (peripheral blood mononuclear cells) in the presence of an irradiated antigen-presenting cell line, which may be a lymphoblastoid cell line (LCL), 1×10 ⁶ PBMCs/cm ² and 2.5× ^{10 4} irradiated antigen-presenting cells/cm ² at a surface density (ie number of cells per cm 2 of growth surface) ratio of approximately 40:1. This promotes expansion of the population of EBV-CTLs in the cell population. Nine days later, EBV-CTL were again selective at a new surface density ratio of 4:1 in the presence of irradiated antigen-presenting LCL, with a minimum surface density of approximately 2.5×10 ⁵ EBV-CTL/cm ^{2 .} Multiply. Medium volume is limited to a maximum ratio of 1 ml/cm ² of growth surface area so that oxygen can reach the cells, which limits growth solutes such as glucose. As a result, the maximum achievable surface density is approximately 2×10 ⁶ EBV-CTLs/cm ² . Thus, the maximum cell proliferation per week is about 8-fold (ie, EBV-CTL 2×10 ⁶ /cm ² divided by EBV-CTL 2.5×10 ⁵ /cm ² ) or less. For continued expansion of EBV-CTLs, weekly transfer of EBV-CTLs to additional 24-well plates with antigen restimulation and twice weekly changes of medium and growth factors in each well of the 24-well plates. is necessary. 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 down, so cell injection as well as sterility assays, identity assays and potency To obtain sufficient quantities of EBV-CTL for quality control measures such as assays, these manipulations must be repeated over long production periods, often 4-8 weeks.

EBV-CTL culture is just one example of the complex cell production process inherent in cell therapy. There is a need for more practical methods of culturing cells for cell therapy that can reduce production time while simultaneously reducing production costs and complexity.

We have created a novel method to increase the population growth rate throughout production and thereby reduce the complexity and time required for cell production.

In adoptive cell therapy, T cells with natural antigen specificity (i.e., T cells against specific peptides from specific target antigens when presented in the context of specific human leukocyte antigen (HLA) alleles) are It is administered in autologous and partially HLA-matched settings to treat viral infections and target tumors. In each of these cases, T cells were injected into the recipient in which (i) the natural T cell receptor recognized the antigen of interest and (ii) the HLA alleles required to present the targeting peptide were expressed. A therapeutic benefit was obtained from the fact that it was administered.

Initial adoptive T cell transfer protocols in the allogeneic hematopoietic stem cell transplantation (HSCT) setting were based on the premise that donor peripheral blood contains T cells that can modulate antitumor and/or antiviral activity in HSCT recipients. Met. Therefore, donor lymphocyte infusion (DLI) is 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 wide range of viruses. However, while effective in treating adenovirus- and EBV-infected populations, the efficacy of this therapy is low, specific for many common acute viruses, such as rotavirus (RSV) and parainfluenza. Limited by the frequency of T cells and the relatively high frequency of alloreactive T cells. A high ratio of alloreactive T cells to virus-specific T cells suggests that higher incidence of graft-versus-host disease (GVDH) limits the acceptable DLI dose and the number of virus-specific T cells received This is a particular problem in haplocongruent transplant recipients, who limit the dose of

To preserve the benefits and improve the safety of DLI, strategies for selective inactivation or elimination of recipient-specific alloreactive T cells are being evaluated, such strategies include anergy induction. , selective alloreactive depletion to minimize the number of alloreactive T cells administered to the recipient, and the use of suicide genes to destroy off-target alloreactive T cells in vivo.

An alternative strategy for preventing and treating certain viral infections after HSCT is adoptive cell transfer of ex vivo expanded T cells with antiviral activity. Specific expansion of virus-reactive T cells has the advantage of increasing the number of infusible virus-specific T cells without increasing alloreactive T cells. Infusion of enriched antigen-specific T cells with reactivity to a particular antigen increases therapeutic efficacy while potentially reducing unwanted off-target effects such as GVHD, and this treatment modality is useful in hematologic malignancies. It has been shown to be effective and safe for the treatment of tumors and solid tumors such as melanoma and EBV-associated malignancies such as Hodgkin's lymphoma and nasopharyngeal carcinoma.

Of note, all therapies rely on the specificity of natural T cell receptors to recognize antigens in association with major histocompatibility complex (MHC) molecules via the natural T cell receptor (TCR). is required to use Therefore, therapeutic benefit per se depends on the use/administration of HLA-matched or partially-matched T cells. For example, expanding donor-derived antigen-specific melanoma-directed T cells expressing HLA haplotype (a) against GP100 (a tumor-associated antigen expressed on cancer cells) to target melanoma cells. can be done. In this context, therapeutic benefit is provided by the specific interaction of natural T-cell receptors with target antigens. However, this interaction can only occur in compatible HLA settings (ie, in the self 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 different HLA haplotypes, where the patient is matched to the most suitable T cell lineage.

In sum, in all current adoptive cell therapy applications, the therapeutic attribute of T cells that confers their therapeutic purpose is the natural antigen specificity of the donor T cells. This unique feature requires at least partial HLA matching between donor and recipient and creates the potential for off-target effects such as GVHD in allogeneic settings. Others have used complex genetic manipulations to completely eliminate donor T cell antigen receptors, and genetic reengineering of T cells to carry chimeric antigen receptors, thereby enhancing T cell intrinsic recognition. I suggest removing all abilities. However, this further complicates the T cell production process, which is already one of the major problems of adoptive cell therapy.

An entirely new approach to adoptive cell therapy that overcomes existing problems is needed to enable wider application in mainstream society. We disclose a new paradigm to modify donor T cells with therapeutic attributes that do not impair the donor T cell's antigen specificity, but render the donor T cell's natural antigen specificity irrelevant to its therapeutic purpose. Essentially, this paradigm shift opens the door to therapeutic applications of T cells in ways not previously envisioned, regardless of whether there is an HLA match between the donor and recipient. is.

U.S. Provisional Application No. 61/267761 US patent application Ser. No. 12/963,597

for cell therapy in a shorter time and in a more economical manner than currently possible by using a stepwise production process that allows the periodic re-establishment of non-traditional conditions throughout the production process. It was discovered that production of cells of Non-traditional conditions include reduced target cell surface densities (i.e., cells/cm ² ), novel target cell to antigen-presenting and/or feeder cell ratios, and/or medium volume: surface area ratios Included is the use of a growth surface composed of an enhanced gas permeable material.

Embodiments of the present invention relate to improved methods of culturing cells for cell therapy applications. These embodiments provide the ability to generate the desired number of cells of interest through the use of a variety of novel methods that allow the population of cells of interest to maintain a higher growth rate than conventional methods throughout the production process. methods that reduce the time, cost and complexity required for

One aspect of the invention relies on stepping the culture process and establishing conditions at the beginning of one or more steps that cause the growth rate of the target cell population to exceed that currently possible. Non-gas permeable growth at unconventionally low surface densities and at unconventional ratios of antigen-presenting cells (and/or feeder cells) to target cells in at least one culture stage, preferably substantially all culture stages Initial conditions are established which involve placing the cells of interest on either a surface or a gas permeable growth surface. By using novel embodiments of this aspect of the invention, the target cell population undergoes more doublings in less time than conventional methods allow, thereby reducing production time.

Another aspect of the invention relies on stepping the culture process and establishing conditions at the beginning of one or more steps such that the growth rate of the target cell population exceeds that currently possible. A condition that involves placing the cells of interest on a growth surface made of a gas permeable material at an unconventionally high medium volume:growth surface area ratio during at least one culture stage, preferably substantially all culture stages. is established. By using novel embodiments of this aspect of the invention, the target cell population undergoes more doublings in less time than conventional methods allow, thereby reducing production time.

Another aspect of the invention relies on stepping the culture process and establishing conditions for each step such that the growth rate of the desired cell population exceeds that currently possible. Unconventional delivery of antigen-presenting cells (and/or feeder cells) to cells of interest at unconventionally low surface densities (i.e., cells/cm ² ) in at least one culture stage, preferably substantially all culture stages. At a ratio, initial conditions are established that involve placing the cells of interest on a growth surface composed of a gas permeable material with an unconventionally high medium volume:growth surface area ratio. By using novel embodiments of this aspect of the invention, the target cell population undergoes more doublings in less time than conventional methods allow, thereby reducing production time.

In an embodiment of the present invention, an allogeneic T-Vehicle is created with therapeutic attributes that do not expose the recipient to graft-versus-host disease (GVHD), but have a therapeutic purpose that benefits the recipient.

In one embodiment of the present invention, T produced by a process comprising stimulating donor PBMCs or donor cord blood with antigen to activate the growth of T cells with natural antigen specificity for one or more antigens. Treatment is initiated by obtaining vehicle. By doing so, an antigen-specific T cell population is produced consisting of natural antigen receptors with antigen specificity for the antigen or antigens used to stimulate their growth. The antigen-specific T cell population does not include natural antigen receptors, but includes at least one therapeutic attribute that has a therapeutic purpose independent of the antigen specificity of the natural antigen receptors, thereby dividing the population of T-vehicles. is modified to create Then, regardless of whether the recipient cells present antigens recognized by one or more natural antigen receptors of the T-vehicle and/or the recipient cells present one or more natural antigens of the T-vehicle. The T-vehicle is delivered to a recipient who, when not presenting the antigen recognized by the receptor, can derive therapeutic benefit from the T-vehicle.

In another embodiment of the invention, made by a process comprising stimulating donor PBMC or donor cord blood with antigen to activate the growth of T cells with natural antigen specificity for one or more antigens Treatment is initiated by obtaining T vehicle. By doing so, an antigen-specific T cell population is produced consisting of natural antigen receptors with antigen specificity for the antigen or antigens used to stimulate their growth. The antigen-specific T cell population does not include natural antigen receptors, but includes at least one therapeutic attribute that has a therapeutic purpose independent of the antigen specificity of the natural antigen receptors, thereby dividing the population of T-vehicles. is modified to create A therapeutic benefit can then be derived from the T-vehicle and delivered to recipients who do not have an HLA match to the T-vehicle.

In various embodiments of the invention, the T vehicle is modified to be loaded with recombinant proteins administered as adjuvants for immunotherapy, modified with therapeutic attributes of chemotherapeutic agents for targeted cancer therapy, antimicrobial modified with therapeutic attributes of an agent to express a transgenic molecule that confers tumor specificity to cells loaded with or genetically engineered with a recombinant protein for treating autoimmune diseases; Modified with the therapeutic attribute of being engineered, loaded for in vivo imaging and/or genetically engineered.

In another embodiment of the invention, a method of producing antigen-specific T cells with antigen recognition of interest comprises placing PBMCs or cord blood in a cell culture device and adding two or more antigens into the cell culture device. to activate the growth of two or more antigen-specific T cell populations (each population capable of recognizing one of the antigens), and to allow time for antigen-specific T cells to begin expanding in population. permissive, 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 expansion, and being recognized by suitable T cell populations; This is achieved by restimulating the cultures with only the antigens that have been tested.

In another embodiment of the invention, a method of producing antigen-specific T cells with antigen recognition of interest comprises placing PBMCs or cord blood in a cell culture device and first injecting two or more antigens into the cell culture device. addition to activate the growth of two or more antigen-specific T cell populations (each population capable of recognizing one of the antigens) and a period of time for the antigen-specific T cells to begin expanding in population and separating the cultures into two or more devices, adding only one of the initial antigens into each device, which devices contain antigen-specific T cell populations suitable for continued expansion. This is accomplished by determining whether the cells are stable and terminating the culture in devices that do not contain antigen-specific T cell populations suitable for continued expansion.

In various embodiments of the invention, native antigen specificity allows recognition of only a single epitope of an antigen that is absent on normal human cells and absent on normal mammalian cells. Donor T cells are produced that have

The present invention is more fully understood upon consideration of the following detailed description of various embodiments of the invention in conjunction with the accompanying drawings.

Figure 2 shows that the antigen-specific T cell population in Example 1 undergoes at least 7 cell doublings after initial stimulation over the first 7 days. 2 shows data demonstrating the magnitude of T cell population expansion in cell populations over time as measured by the tetramer assay for Example 1. FIG. In Example 1, we show that the proliferation rate of antigen-specific T cell populations decreases over a period of 23 days. 1 is a table illustrating the difference between expected proliferation and observed fold proliferation of antigen-specific T cells in Example 1. FIG. 2 shows the presence of antigen-specific T cells after the stimulation of Example 2. FIG. In Example 2, when the surface density decreased from 1 x ¹⁰⁶ / ^cm2 to 3.1 x ¹⁰⁴ / ^cm2 while maintaining a ratio of antigen-specific T cells:antigen-presenting cells of 4:1. Shows proliferation of antigen-specific T cell populations. In Example 2, in the presence of a fixed number of antigen-presenting cells, the proliferation of antigen-specific T cell populations was measured as the surface density decreased from 1×10 ⁶ /cm ² to 3.1×10 ⁴ /cm ^{2 .} show. An example of the results obtained in continuing the study described in FIG. We further demonstrate that population expansion can be initiated at unconventionally low surface densities of target cells as long as they are in the presence of adequate supply. Histograms are shown demonstrating the ability to replicate the magnitude of population expansion of cells of interest by initiating cultures at three different cell surface densities (CTLs/cm ² ). Figure 2 shows a cross-sectional view of the gas permeability test fixture used to obtain the data. Figure 3 shows the growth curve of antigen-specific T cells produced according to the present invention compared to conventional methods as done in Example 5; In Example 5, cell viability, as measured by flow cytometric forward scatter vs. side scatter analysis, was significantly higher in antigen-specific T cells produced according to the present invention compared to conventional methods. indicates that In Example 5, we show that cell viability, as measured by annexin-PI7AAD, was significantly higher in antigen-specific T cells produced according to the invention compared to conventional methods. In Example 5, the superior proliferation of cells produced in the novel method of the present invention, as determined by daily flow cytometric analysis of CFSE-labeled cells, compared to cells cultured using conventional methods. The same cell-specific proliferation rate is shown, confirming that the increased cell proliferation rate resulted from decreased cell death. We show how EVB-CTLs were able to grow beyond what was possible in conventional methods without the need to change media. Figure 2 shows how the culture conditions of Example 6 did not alter the final cell product when EBER was assessed by Q-PCR. Figure 2 shows how the culture conditions of Example 6 did not alter the final cell product when the B cell marker CD20 was assessed by Q-PCR. Due to the very low cumulative surface density of the cells of interest and antigen-presenting cells (here, AL-CTL cells and LCL cells are combined to create a cell population with a cell surface density of 30,000 cells/cm ² ). , shows an illustrative example that experimentally demonstrated that the AL-CTL group was unable to initiate growth. Data from Example 8 are presented showing how two new methods of culturing cells produce more cells than conventional methods over a period of 23 days. 2 shows a photograph of cells cultured in the test device in Example 8. FIG. In Example 8, we show that the two new culture methods and the conventional method all produce cells with the same phenotype. In Example 8, T cells stimulated with EBV peptide epitopes derived from LMP1, LMP2, BZLF1 and EBNA1 of EBV and stained with HLA-A2-LMP2 peptide pentamer staining developed similarly to peptide-specific T cells. A representative culture with frequencies is shown. Cells maintained cytolytic activity and cell specificity and killed autologous EBV-LCL, but less killing of HLA-mismatched EBV-LCL, as assessed by the ⁵¹ Cr release assay. Methods and conventional methods are presented. FIG. 4 shows a graphical representation comparing the expansion of a population of a cell type of interest using an embodiment of the present invention with the expansion of a population of cells of interest on a growth surface under a conventional scenario. An example of the advantages that can be obtained by utilizing a growth surface comprising a gas permeable material and an unconventionally high medium volume: growth surface area ratio of greater than 1 or 2 ml/cm ² is shown. Comparing the new method of expansion of a population of target cell types under an embodiment of the present invention to a new method of expansion of a population of target cells on a growth surface based on a conventional scenario, where the cell surface density at completion is significantly greater than conventional surface densities. shows a graph display. Another novel method of cell production is shown that offers another additional advantage over conventional methods. A comparison of each production method shown in FIG. 14 is provided to demonstrate the power of the new method and why it is useful to adjust the production protocol at various stages to obtain sufficient efficiency. An example of how the production protocol can be adjusted in the novel method to gain efficiency as the production progresses is provided. Figure 2 shows test results demonstrating the inability of T-vehicle to recognize cells from mismatched allogeneic donors. Figure 10 shows the results of studies demonstrating that donor T cells can be engineered to create T-vehicles with therapeutic attributes of CD34delta-IL7 cytokine expression as measured by flow analysis. Figure 2 shows results of studies demonstrating that systemic delivery of IL7 cytokines detected more cytokines in the mouse kidney than at the tumor site. Results show that T-vehicle delivery of IL7 cytokines results in higher cytokine concentrations at tumor sites in mice compared to other organs, and how cytokine production affects tumors for at least two weeks after T-vehicle administration. , indicating whether it was persisted with Figure 2 shows the results of studies demonstrating that donor T-cells can be engineered to create T-vehicles with therapeutic attributes of CAR-PSCA as measured by flow analysis. Figure 2 shows test results demonstrating that T-vehicles with therapeutic attributes of CAR-PSCA can kill tumor cells. T-Vehicles containing receptors capable of binding IL4 are shown to be in close proximity to tumor cells expressing IL4 cytokines. We show how IL4 cytokines can be bound to T-vehicles to greatly reduce the amount of IL4 cytokines that protect tumor cells. Figure 10 shows test results demonstrating that T-Vehicles with therapeutic attributes of expressing extracellular recombinant cytokine receptor IL4R/7 can deplete IL4 cytokines. Shows how T-Vehicles loaded with chemotherapeutic agents migrate towards sites of inflammation. It shows how the recipient's immune system targets T-vehicles located at the site of tumor cells. We show how T-vehicles, under attack by the recipient's immune system, release a payload (in this case, a chemotherapeutic agent) at the site of tumor cells.

DEFINITIONS Antigen Presenting Cell (APC): A cell that acts to provoke a target cell to respond to a specific antigen.
CTL: Cytotoxic T cells.
Target cell: A specific type of cell that is targeted for increased quantity in the production process. In general, target cells are non-adherent and include regulatory T cells (Treg), 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, persistence in vivo, or safety. Cells required for clinical use can be grown using feeder cells and/or antigen-presenting cells, including PBMC, PHA blast, OKT3 T, B blast, LCL and K562 (natural or antigens and/or epitopes and co-stimulatory molecules such as 41BBL, OX40, CD80, CD86, HLA and many others), which may be peptides or other related It may or may not be pulsed with antigens that target it.
EBV: Epstein-Barr virus.
EBV-CTL: T cells that specifically recognize EBV-infected cells or cells expressing or presenting EBV-derived peptides by T cell surface receptors.
EBV-LCL: B-lymphoblastoid cell line transformed with Epstein-Barr virus.
Feeder Cells: Cells that act to increase the amount of target cells. Antigen-presenting cells can also act as feeder cells in some circumstances.
Growth Surface: The area within the culture device on which cells rest.
PBMC: Peripheral blood mononuclear cells, derived from peripheral blood, which are the source of several cells of interest and can act as feeder cells.
Responder cell (R): A cell that responds to a stimulator cell.
Static cell culture: A medium that is not agitated or mixed except when the culture apparatus is relocated for routine operation and/or when the cells are regularly supplied with fresh medium, etc. A method of culturing cells in In general, medium in static culture is usually in a quiescent state. The present invention relates to static cell culture methods.
Stimulation: The effect that antigen presentation and/or feeder cells have on target cells.
Stimulatory cell (S): A cell that affects a responding cell.
Surface Density: The amount of cells per unit area of the surface within the device on which the cells are placed.

In an attempt to find new ways to simplify the production of target cell populations 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. rice field. A number of exemplary embodiments and various aspects of the present invention are described to demonstrate how the ability to reduce production time and complexity as compared to conventional methods can be achieved.

Example 1: Demonstration of the limits of conventional methods

The data in this example is based on standard ²⁴ -well tissue culture plates (i.e. We demonstrate the limitations of conventional culture methods for producing EBV-CTL in a surface area of 2 cm ² per well.

Culture Phase 1 (Day 0): Antigen-presenting γ-irradiated (40 Gy) autologous EBV-LCL from a normal donor at a medium volume:growth surface ratio of 1 ml/cm ² and a ratio of 40:1 (PBML:LCL) of PBMC (approximately 1×10 ⁶ /ml) cell population and cells in RPMI 1640 supplemented with 45% Click medium (Irvine Scientific, Santa Ana, Calif.), 2 mM GlutaMAX-I and 10% FBS. Expansion of the EBV-CTL population was initiated by bringing the surface density of the population to about 1×10 ⁶ cells/cm ² .

Culture Stage 2 (Days 9-16): On day 9, EBV-CTLs are harvested from the cell population generated in Stage 1 and resuspended in fresh medium at a surface density of 0.5×10 ⁶ cells/cm ^{2 .} They were clouded and restimulated with irradiated autologous EBV-LCL at a 4:1 CTL:LCL ratio (0.5×10 ⁶ surface density of CTL/cm ² : 1.25×10 ⁵ LCL/cm ² ). On day 13, remove 1 ml of the 2 ml medium volume in each well of the 24-well plate and add recombinant human IL-2 (IL-2) (50 U/ml) (Proleukin; Chiron, Emeryville, Calif.). was replaced with 1 ml of fresh medium containing

Culture Phase 3 (Days 17-23): Phase 2 conditions were repeated with IL-2 additions twice a week and cultures terminated on Day 23. Once terminated, the culture could be continued with further culture stages mimicking the cultures of stages 2 and 3.

Cell lines and tumor cells for use as target cells in cytotoxicity assays: BJAB (B-cell lymphoma) and K562 (chronic erythroleukemia) from the American Type Culture Collection (ATCC), Rockville, MD, USA obtained. All cells contained 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 25 IU/mL penicillin and 25 mg/mL streptomycin (all from BioWhittaker, Walkersville, Md.). Cultures were maintained in RPMI 1640 medium (GIBCO-BRL, Gaithersburg, Md.). Cells were maintained in a humidified environment containing 5% _CO2 at 37°C.

Immunophenotyping:
Cell surface: phycoerythrin (PE), fluorescein isothiocyanate (FITC), periodin chlorophyll protein (PerCP) and CD3, CD4, CD8, CD56, CD16, CD62L, CD45RO from Becton-Dickinson (Mountain View, CA, USA) , CD45RA, CD27, CD28, CD25, CD44 with allophycocyanin (APC)-conjugated monoclonal antibodies (MAbs). PE-linked tetramer (Baylor College of Medicine) and APC-bound pentamers (Proimmune Ltd, Oxford, UK) were used to quantify the frequency of EBV-CTL precursors. 10,000 and 100,000 live events for cell surface and pentamer staining were acquired on a FACSCalibur flow cytometer, respectively, and data were analyzed using Cell Quest software (Becton Dickinson).

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

Annexin V-7-AAD Staining: To determine 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, Calif.). gone. Briefly, EBV-CTL from 24-well plates or G-Rex were washed with cold PBS, resuspended in 1× binding buffer at a concentration of 1×10 ⁶ cells/ml, and incubated at room temperature in the dark. Stained with Annexin V-PE and 7-AAD for 15 minutes at (25° C.). After incubation, cells were immediately analyzed by flow cytometry.

Chromium Release Assay: Cytotoxic activity of EBV-CTL was assessed in a standard 4 hour ⁵¹ Cr release assay as previously described. As cells of interest, autologous and HLA class I and II mismatched EBV-transformed lymphoblastoid cell lines (EBV-LCL) were used to measure MHC-restricted and unrestricted killing, and the K562 cell line was used to measure natural Killer activity was measured. Spontaneous and maximal ⁵¹ Cr release were measured using chromium-labeled cells of interest incubated with medium alone or with 1% Triton X-100, respectively. The average percentage of specific lysis of triplicate wells was calculated as follows: [(number tested-spontaneous number)/(maximum number-spontaneous number)] x 100.

Enzyme-linked immunospot (ELIspot) assay: ELIspot analysis was used to quantify the frequency and function of T cells that secreted IFNγ in response to antigenic stimulation. CTL lines grown in 24-well plates or G-Rex were treated 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-FLYALLALL=FLY and HLA-A29 ILLARLFLY=ILL (Genemed Synthesis, Inc., San Antonio, Tex.) and diluted to a final concentration of 2 μM. and only CTL served as a negative control. CTLs were supplemented with ELIspot medium [5% Human Serum (Valley Biomedical, Inc., Winchester, VA) and 2 mM L-glutamine (GlutaMAX-I) (Invitrogen, Carlsbad, CA). RPMI 1640 (Hyclone, Logan, Utah)] at 1 x ¹⁰⁶ /ml. A 96-well filtration plate (MultiScreen, #MAHAS4510) (Millipore, Bedford, Mass.) was washed overnight at 4° C. with 10 μg/mL anti-IFN-γ antibody (Catcher-mAB91-DIK) (Mabtech, Cincinnati, OH). Coated, then washed and blocked with ELIspot medium for 1 hour at 37°C. Responder and stimulator cells were incubated on plates for 20 hours, then plates were washed and plated with biotin-conjugated anti-IFN-γ monoclonal secondary antibody (Detector-mAB (7-B6-1-Biotin)) (Mabtech). followed by incubation with avidin:biotinylated horseradish peroxidase complex (Vectastain Elite ABC Kit (standard), #PK6100) (Vector Laboratories, Burlingame, Calif.) and AEC substrate (St. Louis, Mo.). (Sigma, Inc.). Each culture condition was performed in triplicate. Plates were sent to Zellnet Consulting (New York, NY) for evaluation. Spot forming units (SFC) and input cell numbers were plotted.

Statistical Analysis: In vitro data are presented as mean ± 1 SD. Student's t-test was used to determine the statistical significance of differences between samples, and P<0.05 was accepted as indicating a significant difference.

Under these culture conditions, after initial stimulation, the antigen-specific T cell population undergoes at least 7 cell doublings over the first 7 days, as shown in FIG. 1A. Therefore, T cells are expected to expand 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). The frequencies of tetramer-positive cells after the first, second and third stimulations are shown in FIG. 1B. 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 a single stimulation on day 0, the frequency of tetramer-positive T cells in the cell population increased from 0.02% and 0.01% to 2.7% and 1.25% by day 9. each increased. Thus, the proportion of antigen-specific tetramer-positive T cells present in the cell population achieved a 135- and 125-fold increase as measured by RAK and QAK, respectively. There was also a 1.1-fold increase (data not shown) in the surface density of cells in the cell population by day 9 after a single stimulation on day 0 of culture phase 1 (approximately 1 .1×10 ⁶ /cm ² were present). Since the majority of cells in the PBMC population are not specific for the stimulating antigen, little overall increase in total cell number is observed, but the fold proliferation of the antigen-specific cell population in the population is shown in Figure 1C. was about 280 during the first stage of culture. Unfortunately, the number of cell doublings was the same during the second and third culture stages, as measured by CSFE, but this antigen-specific T cell proliferation rate persisted during the second and third culture stages. No, it was only 5.7 in the second stage and 4.3 in the third stage. FIG. 2 shows a table illustrating the difference between expected proliferation and observed fold proliferation of antigen-specific T cells (n=3).

Example 1 demonstrates that the time taken to produce target cells typically slows after about the first week of production because the target cell population slows down in subsequent culture stages. .

Example 2: Reducing the time required to expand a cell population of interest can be achieved by reducing the cell surface density of the cell population of interest at the beginning of any given stage or stages of culture. be.

We hypothesized that the reduced proliferation rate of the target cell population after the second T cell stimulation compared to the first stimulation was due to limiting cell culture conditions that lead to activation-induced cell death (AICD). For example, referring to FIG. 3A, at the first stimulation, the EBV antigen-specific T cell component of PBMC is at most 2% of the population, so the seeding density of antigen-specific responding T cells is 2 per cm ² . It is less than ×10 ⁴ . The remaining PBMC act as non-proliferating feeder cells (shown as CFSE-positive cells in FIG. 3A) and maintain optimal cell-to-cell contacts that allow expansion of antigen-specific CTLs. In contrast, at the second stimulation on day 9, the majority of T cells are antigen-specific, with similar total cell densities in the population, but 50-100 fold higher proliferating cell densities. As a result, most cells proliferate on restimulation and thus can rapidly consume and deplete their nutrient and _O2 supply.

To determine whether limiting culture conditions were responsible for suboptimal T cell growth rates, we measured proliferation of activated T cells plated at lower cell densities. The method is as described in Example 1 above.

As shown in Figure 3B, 1 x 10 cells were placed in the wells of a standard 24-well plate, each well having a growth surface area of 2 ^cm2 , while maintaining a 4:1 responder:stimulator ratio (R:S). Activated EBV-specific T cells were seeded at doubling dilutions yielding decreasing surface densities ranging from ⁶ /cm ² to 3.1×10 ⁴ /cm ² . Maximum CTL expansion (4.7±1.1-fold) was achieved at a starting CTL surface density of 1.25×10 ⁵ per cm ² , but as shown in FIG. 3B, further dilution reduced the expansion rate to Decreased. We speculate that this limited dilution effect is probably due to the lack of cell-to-cell contact, so a fixed number of feeder cells (EBV-LCL plated at a surface density of 1.25×10 ⁵ /cm ² ) Doubling dilutions of EBV-CTL at surface densities from 1 x 10 ⁶ to 3.1 x 10 ⁴ were cultured in parallel with cells and cell proliferation was assessed over 7 days. As shown in FIG. 3C, CTL proliferation increased from a mere 2.9±0.8-fold proliferation by EBV-CTL at a surface density of 1×10 ⁶ /cm ² to a surface density of 3.1×10 ⁴ /cm ^{2 .} A dramatic increase was observed up to 34.7±11-fold expansion by EBV-CTL at . Importantly, this change in culture conditions did not alter cell function or antigen specificity (data not shown). Therefore, the activated antigen-specific T cell population can be expanded to a greater extent than conventional culture methods allow. Of note, the maximum surface densities achieved after stimulation (1.7-2.5×10 ⁶ /cm ² ) were the same regardless of the starting surface density.

Therefore, the conventional culture conditions are limited, and in order for the target cell group 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 . This indicates that it should not. Furthermore, by reducing the surface density of the target cell population at the start of any culture step, compared to conventional methods, expansion of antigen-specific CTLs can be enhanced by up to about 34-fold. This has considerable ramifications in cell therapy, where the amount of cells at the start of production is often very limited. For example, by distributing a limited amount of cells of interest over an increased surface area at a reduced surface density, population growth rates are dramatically increased compared to conventional surface densities, resulting in more A larger target cell population can be achieved in a short time.

Example 3: Minimal surface density of cell populations containing cells of interest and/or antigen-presenting cells allows growth of cell populations of interest seeded at very low surface densities.

FIG. 4 shows an example of the results obtained by continuing the study described in FIG. Alternatively, as long as there is a sufficient supply of antigen-presenting cells, it has been demonstrated that population growth can be initiated at an unconventionally low target cell surface density. In these experiments, there was a difference between about 1.0×10 ⁶ cells of interest/cm ² at an R:S ratio of 8:1 and only about 3900 cells of interest/cm ² at an R:S ratio of 1:32. We continued to demonstrate how the entire cell population with surface density and R:S ratio could significantly expand the cells of interest to more than 50 times the starting surface density, at which point the study was discontinued. .

Example 4: The ability to start the stage at unconventionally low target cell surface densities, expand the population, terminate the stage, and repeat the conditions to enable stepwise repetition of the production process. , has been demonstrated to produce repeatable results.

The evaluation described in Example 3 was continued at three target cell surface densities (CTLs/cm ² ), as shown in FIG. Each specific seeding density was able to consistently achieve the same fold growth. This implication will be described in more detail in connection with the ability to dramatically shorten the production time of the cell population of interest.

Example 5: Culturing target cells on a growth surface made of a gas permeable material while at the same time increasing the medium volume:growth surface area ratio significantly increases the target cell population at a given culture stage compared to conventional methods. increases the number of times that can be doubled and increases the achievable surface density.

For cell lines and tumor cells, immunophenotyping, CFSE labeling, annexin V-7-AAD staining, chromium release assays, enzyme-linked immunospot (ELIspot) assays, retroviral production and transduction of T lymphocytes, and statistical analysis. was as described in Example 1.

A test fixture (hereafter generically referred to as "G-Rex") was constructed as shown in FIG. The bottom 20 of each G-Rex 10 consisted of a gas permeable silicone membrane, which had a thickness of approximately 0.005-0.007 inches. Co-pending U.S. Patent Publication No. 2005/0106717 (hereafter referred to as Wilson '717) is one of many other sources of information regarding the use of alternative gas permeable materials, and one of ordinary skill in the art can find it here. Knowledge of the shape, properties and other useful features of gas permeable culture devices can be obtained that are beneficial to many of the embodiments of the present invention. In this Example 3, the G-Rex (referred to as “G-Rex 40”) growth surface area was 10 cm ² , and the cell population (shown as element 30) was placed thereon. Cell population characteristics varied throughout the experiment as described herein. The medium volume (designated as element 40) was 30 mL unless otherwise stated, yielding a medium volume:growing surface area ratio of 3 ml/cm ² .

Activated EBV-specific CTL and irradiated autologous EBV-LCL were cultured in G-Rex40 instruments at a conventional 4:1 CTL:LCL ratio. EBV-CTL were seeded at a surface density of 5×10 ⁵ cells/cm ² in G-Rex 40 and the growth rate of the EBV-CTL population was measured in standard 24-well plates with a media volume of 1 ml/cm ² for growth surface area. EBV-CTL seeded at the same surface density were compared. After 3 days, EBV-CTL in G-Rex 40 increased from 5×10 ⁵ /cm ² to 7.9×10 ⁶ /cm 2 without medium change, as shown in FIG. 7A (p=0.005). up to a median of ² (range 5.7-8.1×10 ⁶ /cm ² ). In contrast, EBV-CTL cultured in conventional 24-well plates for 3 days had a median surface density of 5×10 ⁵ /cm ² to 1.8×10 ⁶ /cm ² by day 3 (range 1 .7-2.5×10 ⁶ /cm ² ). Supplementing medium in G-Rex40 could further increase surface density, but supplementing medium or IL2 in 24-well plates failed to increase cell surface density. For example, EBV-CTL surface density was 9.5×10 ⁶ cells/cm ² (range 8.5×10 ⁶ -11.0×10) in G-Rex 40 after supplementation with medium and IL-2 on day 7. 10 ⁶ /cm ² ) (data not shown).

Survival of OKT3-stimulated peripheral blood T cells using flow cytometric forward versus side scatter analysis on day 5 of culture to understand the mechanism behind the predominant cell proliferation in the G-Rex device. rate was evaluated. EBV-CTL could not be evaluated in this assay because residual irradiated EBV-LCL was present in the cultures that interfered with analysis. As shown in FIG. 7B, cell viability was significantly higher in G-Rex40 cultures (89.2% viability in G-Rex40 and 49.9% in 24-well plates). there were). Cultures were then analyzed daily for 7 days using annexin-PI 7AAD to discriminate between viable and apoptotic/necrotic cells, and the viability of T cells proliferating in G-Rex, as shown in Figure 7C. A consistently lower viability of T cells growing in 24-well plates was observed compared to . These data indicate that the cumulatively improved survival of proliferating cells contributed to the increased number of cells in the G-Rex device compared to 24-well plates.

To determine if there is also a contribution from increased cell division numbers in G-Rex vs. 24-well plates, T cells were labeled with CFSE on day 0 and treated with G-Rex40 in a medium volume of 40 ml. , divided into 24-well plates with a medium volume of 2 ml in each well. Daily flow cytometric analysis demonstrated no difference in the number of cell divisions from day 1 to day 3. However, from day 3 onwards, the population of target cells cultured in G-Rex40 continued to increase at a rate that exceeded the rate of decline in 2 ml wells, as shown in FIG. 7D. This indicates that the culture conditions were limited. Thus, the large population of target cells in the G-Rex40 test device resulted from a combination of reduced cell death and sustained proliferation compared to conventional methods.

Example 6: The use of an unconventionally high medium volume:growth surface area ratio and a growth surface made of a gas permeable material reduces the need to feed the culture during production while simultaneously can obtain different high target cell surface densities.

This was demonstrated by using the G-Rex test fixture for initiation and proliferation of EBV:LCL. In this example, G-Rex 2000 refers to a device as described in FIG. 8, except that the base consists of a growth surface area of 100 cm ² and a usable medium volume of 2000 ml. EBV-LCL were cultured in G-Rex 2000 and proliferated without changing cell phenotype. EBV-LCLs were plated in G-Rex 2000 at a surface density of 1×10 ⁵ cells/cm ² with 1000 ml of complete RPMI medium to give a medium volume:surface area ratio of 10 ml/cm ² . For comparison, EBV-LCL were plated in T175 flasks at a surface density of 5×10 ⁵ cells/cm ² with 30 ml of complete RPMI medium, resulting in a medium volume:surface area ratio of 0.18 ml/cm ² . As shown in FIG. 8A, EBV-LCL cultured in G-Rex 2000 outgrew those cultured in T175 flasks without requiring any manipulation or media change. As shown in Figures 8B and 8C, EBER and the B-cell marker CD20 were assessed by Q-PCR and the final cell product was not altered in this culture condition.

Example 7: Target cells may not proliferate if sufficient feeder and/or antigen cells are not present at the start of the culture. However, the cell population can be modified to include additional cell types that act as feeder cells and/or antigen presenting cells for expansion.

Figure 9 shows that the cumulative surface density of target cells and antigen-presenting cells is very low (in this case, AL-CTL and LCL cells are combined to create a cell population with a surface density of 30,000 cells/cm ² ). , shows an illustrative example that experimentally demonstrated the inability of the AL-CTL population to initiate growth. However, this same cell population could be grown by modifying the population to include other cell types that function as feeder cells. In this case, feeder layers of three different morphologies of irradiated K562 cells with a surface density of approximately 0.5×10 ⁶ cells/cm ² were evaluated. In both cases, the AL-CTL population expanded from the initial cell population shown in the first column of the histogram, from a surface density of only 15000 cells/cm ² to a surface density of 4.0×10 ⁶ cells/cm ^{2 .} changed over 14 days. We also demonstrated that expansion of the LCL population, as opposed to the addition of a third cell type, yielded similar favorable results. Used for LCL or K562 to demonstrate that very low target cell populations can be used to initiate growth when the cell population contains sufficient numbers of feeder and/or antigen-specific cells We arbitrarily chose a high surface density that If feeder cells are in short supply, are expensive, or are cumbersome to prepare, it is recommended to reduce their surface density to less than 0.5×10 ⁶ cells/cm ² . In general, and as has been demonstrated, when antigen-presenting cells and/or feeder cells are present in a cell population, the additional surface density of antigen-presenting cells and/or feeder cells and cells of interest increases the proliferation of the cell population of interest. should preferably be at least about 0.125×10 ⁶ cells/cm ² to produce a surface density in the cell population sufficient to initiate . Also, to achieve continued growth beyond the limits of standard surface densities, this example used a growth surface composed of a gas permeable material with a medium volume:surface area ratio of 4 ml/cm ² .

Example 8: Reducing the Surface Density of Target Cells, Altering the Responder:Stimulator Ratio, Increasing the Medium:Growth Surface Area Ratio, and Cultivating Low Surface Density onto a Growth Surface Consisting of a Gas Permeable Material By periodically distributing the cells at , more cells of interest can be produced in a shorter time than other methods, simplifying the production process.

To further assess the ability to simplify and shorten the production of target cells, the G-Rex test instrument was used to initiate and expand EBV-CTL. In this example, G-Rex 500 refers to a device as described in FIG. 6, except that the base consists of a growth surface area of 100 cm ² and a usable medium volume of 500 ml.

As an initial step in EBV-CTL production, PBMCs were seeded into G-Rex 40 at a surface density of 1×10 ⁶ /cm ² (total = 10 ⁷ PBMCs distributed over 10 cm ² of growth surface area of G-Rex 40). , stimulated them with EBV-LCL at a PBMC:EBV-LCL ratio of 40:1. For CTL production, this 40:1 ratio is preferred in the first stimulation to maintain the antigen specificity of the responding T cells. After the initial culture phase, the second phase was started on day 9. Here, 1×10 ⁷ responding T cells were transferred from the G-Rex40 to the G-Rex500 test fixture. To initiate the second culture stage, 200 mL of CTL medium was placed in the G-Rex 500, resulting in a medium volume: surface area ratio of 2 ml/cm ² at the start of the second stage and a medium height equal to the area of the growth surface. 2.0 cm higher than The surface density of target cells at the start of the second stage was CTL 1×10 ⁵ cells/cm ² , and the surface density of antigen-presenting cells was LCL 5×10 ⁵ cells/cm ^{2 .} was 1:5, which is different from the conventional one. This second stage cell surface density and R:S ratio resulted in consistent EBV-CTL expansion in all donors screened. After 4 days (day 13), IL-2 (50 U/ml—final concentration) was added directly to the cultures, as well as 200 ml of fresh medium, resulting in a medium volume:surface area ratio of 4 ml/cm ² . . On day 16, cells were harvested and counted. The median surface density of the CTLs obtained was 6.5×10 ⁶ per cm ² (ranging from 2.4×10 ⁶ to 3.5×10 ⁷ ).

The use of a growth surface made of gas permeable material has resulted in increased medium volume:surface area ratios (i.e. greater than 1 ml/ ^cm2 ), decreased cell surface densities (i.e. 0 .5×10 ⁶ /cm ² ) and modified responder:stimulator cell ratios (less than 4:1), reducing production time. FIG. 10A shows the comparison of the G-Rex approach of Example 8 with the use of the conventional method of Example 1 and the G-Rex approach described in Example 5. FIG. As shown, the conventional method required 23 days to deliver the same number of target cells that could be delivered in about 10 days for either G-Rex method. After 23 days, the G-Rex method of Example 8 was able to produce 23.7 times more target cells than the G-Rex method of Example 5, and 68.4 times more than the conventional method of Example 1. We were able to produce twice as many target cells. Furthermore, when the cultures were split when the cell surface density exceeded 7×10 ⁶ /cm ² , target cells continued to divide up to 27-30 days without the need for further antigen-presenting cell stimulation. rice field.

Although it was not possible to clearly see the CTLs in the G-Rex using a light microscope, CTL clusters could be seen by eye or by an inverted microscope. The appearance of the cells on days 9, 16 and 23 of culture is shown in Figure 10B. As shown in FIG. 10C, culturing in G-Rex did not alter the phenotype of the expanded cells, with over 90% of the cell population being CD3+ cells (G-Rex versus 96.7±1 in 24 wells). .7 vs 92.8±5.6), which were predominantly CD8+ (62.2%±38.3 vs 75%±21.7). Evaluation of activation markers CD25 and CD27 and memory markers CD45RO, CD45RA and CD62L demonstrated no substantial differences between EBV-CTLs grown under each culture condition. Antigen specificity was also unaffected by culture conditions, as determined by ELIspot and pentamer analysis. FIG. 10D. Representative T cells stimulated with EBV peptide epitopes from LMP1, LMP2, BZLF1 and EBNA1 and stained with HLA-A2-LMP2 peptide pentamer stain show similar frequency to peptide-specific T cells. It shows a good culture. Furthermore, as shown in FIG. 10E, the expanded cells maintained their cytolytic activity and specificity and killed autologous EBV-LCL (G-Rex vs. 24-well plates) as assessed by the ⁵¹ Cr release assay. , 62% ± 12 vs. 57% ± 8 at an E:T ratio of 20:1), killing less HLA-mismatched EBV-LCL (15% ± 5 vs. 12% ± 7 at a ratio of 20:1).

Explore various novel methods for improved cell production for cell therapy: lowering the surface density of the target cell population at the beginning of the production cycle, lowering the surface density ratio between responder and stimulator cells, How can various conditions, such as increasing the growth surface and/or medium volume:growth surface area ratio of gas permeable materials, be used to facilitate and simplify the production of cells for cell therapy research and clinical applications? Examples 1-8 have been presented to demonstrate to those skilled in the art that this can be achieved. Although Examples 1-8 are directed to the production of antigen-specific T cells, these novel culture conditions have a number of important suspension cell types such as regulatory T cells (Treg), natural killer cells (NK), tumor infiltrating lymphocytes (TIL), primary T lymphocytes, a wide range of antigen-specific cells and many others All can also be genetically modified to improve function, persistence in vivo or safety). Cells can be grown using feeder cells and/or antigen-presenting cells, which include PBMC, PHA blast, OKT3 T, B blast, LCL and K562 (natural or genetically modified to express antigens and/or epitopes and co-stimulatory molecules such as 41BBL, OX40L, CD80, CD86, HLA and many others) when pulsed with peptides and/or related antigens. Sometimes they do, sometimes they don't.

Unconventional Low Initial Surface Density: One aspect of the present invention is the discovery that lower target cell surface densities can reduce production time compared to conventional methods. Thus, cells of interest can have a greater difference between maximum and minimum cell surface densities than conventional methods allow. If the growth rate of the target cell population begins to slow down, but the amount of target cells is still not sufficient to finish production, the target cells can be grown again at a lower starting surface density on an additional growth surface consisting of a gas permeable material. Redistribution is preferred.

An example is described here to illustrate how novel cell production methods that rely on lower surface densities at the start of any given culture stage can be applied. FIG. 11 shows a graphical representation of the growth of a cell population of interest on a growth surface under a conventional scenario compared to population growth of a cell type of interest using an embodiment of the present invention. In this new method, the surface density of the cells of interest at the beginning of the production phase is less than the conventional surface density. In order to focus on the advantages of this novel method, this description does not address the process of obtaining the target cell population initially. The culture "Day" starts at "0" so that those skilled in the art are more likely to see the relative time advantage of this new method. 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 uses a different and much lower target cell density. Start with a cell surface density of 0.125×10 ⁶ cells/cm ² . Thus, in this example, four times more surface area (ie, 500,000/125,000) is required to initiate the culture than the surface area required by conventional methods. 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 1 cm ² growth area gives 2×10 ⁶ cells/cm ² , and those cells are then redistributed over a 4 cm ² growth area to achieve a conventional starting density of 0.5×10 ⁶ cells/cm 2 . ² (ie, 4 cm ² ×0.5×10 ⁶ cells=2×10 ⁶ cells) can be used to continue production. This cycle was repeated for a further 14 days, at which point the maximum cell surface density was reached again, with each 4 cm ² growth surface area giving 2.0×10 ⁶ cells, for a total of 8.0×10 ⁶ cells. The cells are then distributed over a growth area of 16 cm ² and the growth cycle is repeated to give a total of 32×10 ⁶ cells in 42 days.

The novel method shown in FIG. 11 distributes 500,000 cells equally over a 4 cm ² growth area, instead of using the conventional method of depositing 500,000 cells of interest on 1 cm ² at the start of production. It yields an unconventionally low starting surface density of 125,000 cells of interest/cm ² in the eye. In the example, the new method has a growth rate that begins to decline at just 7 days, similar to the conventional method. Cells in the new method have a surface density of 1×10 ⁶ cells/cm ² . Thus, when the growth rate started to slow down, this culture step yielded 4×10 ⁶ cells, which were then redistributed over a 32 cm ² growth area, resulting in a starting surface density of 0.125× Production can be continued in Stage 2 with 10 ⁶ cells/cm ² (ie, 32 cm ² ×0.125×10 ⁶ cells=4×10 ⁶ cells). The production cycle or stage was repeated for an additional 7 days, through day 14, at which point each growth surface area of 32 cm ² containing 1.0×10 ⁶ cells of interest again reached maximum cell surface density, with only a few 14 days gives rise to a total of 32×10 ⁶ cells. Note how, like the conventional method, the new method provides a multiple of the final surface density divided by the starting surface density at the end of each production cycle. However, by reducing the starting cell surface density and finishing each production step before the cells enter growth production, the time is reduced dramatically. In this example, by reducing the target cell surface density (in this case, to 0.125×10 ⁶ cells/cm ² ), compared to conventional cell surface densities, only 33% of the time required by the conventional method is achieved. It describes how the same amount of target cells was given in % (14 days vs. 42 days).

Although the advantage was quantified using a starting surface density of 0.125×10 ⁶ cells/cm ² , one skilled in the art will appreciate that this example of the invention reduces production time with any reduction below conventional cell surface densities. It should be noted that demonstrating that Furthermore, one skilled in the art will appreciate that the cell growth rate and the point in time at which a decrease in cell growth occurs as described in the present method and other novel methods presented herein are described for illustrative purposes only, and actual ratios will vary in each application based on a wide range of conditions such as medium composition, cell type, and the like. Furthermore, those skilled in the art will appreciate that an advantage of this aspect of the invention in any particular application is the reduced production time resulting from lower cell surface densities than conventional cell surface densities, and in this illustrative example It will be appreciated that the particular conventional surface density used may vary from application to application for a given application.

Accordingly, one aspect of the method of the present invention is described herein where there is a desire to minimize the production time of a given amount of cells of interest present in a cell population by using a reduced cell surface density. The cells of interest should be deposited on the growth surface at unconventionally low cell surface densities such that:
a. Cells of interest are grown in the presence of antigen-presenting cells and/or feeder cells at a medium volume:surface area ratio of up to 1 ml/cm ² if the growth surface is not made of a gas permeable material, and the growth surface is gaseous. a medium volume: surface area ratio of at most 2 ml/cm ² if made of permeable material;
b. Preferred surface density conditions at the start of the production cycle are target cell surface densities of preferably less than 0.5×10 ⁶ cells/cm ² , more preferably reduced as described in FIG.
c. The surface density of cells of interest plus surface density of antigen-presenting cells and/or feeder cells is preferably at least about 1.25×10 ⁵ cells/cm ² .

Based on the above examples, it was demonstrated that when the surface density of antigen-presenting cells and/or feeder cells is further lowered below 1.25×10 ⁵ cells/cm ² , the proliferation of the target cell population is not limited. It's good to Based on the objective of demonstrating that the growth of target cell populations can be achieved at unconventional low densities when expanded by adequate supply of antigen-presenting cells and/or feeder cells, 1.25×10 ⁵ pieces/cm ² was selected.

Production can be simplified and shortened by the use of growth surfaces made of gas permeable materials and higher medium volume: growth surface area ratios. Another aspect of the invention uses a growth surface made of gas permeable material and a production cycle that uses a medium volume: growth surface area ratio that exceeds conventional ratios and increases the usage of the growth surface area over time. It is a discovery that repetition shortens the production time.

An illustrative example is now presented showing how these conditions can reduce production time. FIG. 12 is discussed to illustrate an example of the advantages obtained by utilizing a growth surface composed of a gas permeable material and an unconventionally high medium volume: growth surface area ratio of greater than 1 or 2 ml/cm ² . It is for reinforcement. The following discussion demonstrates how several options are available using such methods, including reduced production time, reduced growing surface area usage, and/or reduced labor and contamination risk. It is intended to demonstrate to those skilled in the art how Those skilled in the art will recognize that FIG. 12 and related discussion are merely examples and do not limit the scope of the invention.

It is assumed that the cell population containing the target cell population in this illustrative example consumes approximately 1 ml per "X" hours. FIG. 12 shows two production processes labeled "Conventional Method" and "New Method". At the start of growth, each process starts with target cells at a surface density of 0.5×10 ⁶ /cm ² . However, the growth surface of the new method consists of a gas permeable material and its medium volume to surface area ratio is 2 ml/cm ² as compared to 1 ml/cm ² for the conventional method. At time "X", the target cell population of the conventional method reaches a surface density plateau of 2 x ¹⁰⁶ / ^cm2 and is depleted of nutrients, while the additional medium volume of the new method allows continued growth and a target cell surface density of is 3×10 ⁶ /cm ² . If the new method continues, a surface density of 4×10 ⁶ /cm ² is reached. This gives rise to many advantageous options. The new method may produce more cells than the conventional method and terminate before time "X", and may produce about 1.5 times more cells than the conventional method and terminate at time "X." Alternatively, it may take twice as long, but produce twice as many target cells as conventional methods without the need to manipulate delivery equipment, and continue until nutrients are exhausted. In order for conventional methods to harvest the same number of cells, the cells must be harvested and restarting the process adds labor and possible contamination risk. Since cell therapy applications typically cannot begin with a fixed number of cells, conventional methods do not allow the option of simply increasing the surface area at the start of production.

Figure 13 continues the example of Figure 12 to show how more than one production cycle can be even more advantageous. Figure 13 shows a graphical representation of the expansion of a target cell population on a growth surface by conventional methods compared to expanding a population of the target cell type by one of the novel methods of the present invention; The surface density is higher than that of conventional methods. In order to focus on this embodiment, this description does not mention the process of obtaining the target cell population. Culture "days" start at "0" so that those skilled in the art can more easily ascertain the relative time advantages of this aspect of the invention. In this example, both cultures are initiated on "day 0" using a conventional target cell surface density of 0.5 x ¹⁰⁵ cells/ ^cm2 . In this exemplary embodiment, the conventional growth surface is also made 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 FIG. 13, the target cell group of the conventional method began to decrease in growth rate when the surface density was about 1.5×10 ⁶ cells/cm ² in about 4 days, and reached a maximum surface density of 2 in 14 days. It reaches x10 ⁶ /cm ² . At that point, the target cell population was distributed over a 4 cm ² growth area at a surface density of 0.5×10 ⁶ /cm ² in 1.0 ml/cm ² fresh medium, and the production cycle began again for another 14 days. to reach a surface density of 2×10 ⁶ cells/cm ² in 28 days, giving 8×10 ⁶ target cells. By comparison, the population of cells of interest for the new method began to decrease in growth rate at approximately 10-11 days at a surface density of approximately 3.6×10 ⁶ cells/cm ² , reaching a maximum surface density of 4× at 28 days. 10 ⁶ pieces/cm ² could be reached. However, in order to accelerate production, the cycle is terminated while the growth rate of the target cell population is still high. Thus, in about 10-11 days, 3×10 ⁶ cells were redistributed over a growth area of 6 cm ² at a surface density of 0.5×10 ⁶ /cm ² in 4.0 ml/cm ² of fresh medium. , the production cycle begins again and the target cell population reaches a surface density of about 3×10 ⁶ cells/cm ² in about another 10-11 days, giving 18×10 ⁶ target cells in about 21 days. Thus, the new method produced more than double the number of cells of interest approximately 75% of the time compared to the conventional method.

Cell surface densities in excess of 10×10 ⁶ cells/cm ² could be obtained on growth surfaces composed of gas permeable materials. This demonstrates that the use of high surface density embodiments of the present invention is not limited to the densities described in this example.

Thus, another example of the method of the invention, where there is a desire to minimize the time to produce a given amount of cells of interest present in a cell population by using a reduced cell surface density. are listed below:
a. seeding the cells of interest onto a growth surface area composed of a gas permeable material in the presence of antigen-presenting cells and/or feeder cells at a medium volume:surface area ratio of at least 2 ml/cm ² ;
b. Establishing 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. allowing the population of cells of interest to grow beyond conventional surface densities of about 2×10 ⁶ cells/cm ² , and d. If more cells of interest are desired, redistributing the cells of interest onto an additional growth surface of gas permeable material and repeating steps ad until sufficient cells of interest are obtained.

With these novel methods, additional advantages can be achieved by combining the following culture-initiating attributes. using non-traditional low surface areas; using novel target and/or feeder cell surface density ratios; utilizing growth surface areas made of gas permeable materials; Utilizing the capacity:growing surface area ratio and producing in cycles. Such conditions can be varied in any production cycle to achieve desired results, such as balancing production time reduction, surface area utilization, feed frequency, and the like.

FIG. 14 illustrates another novel method that provides additional advantages over conventional methods. Those skilled in the art will appreciate that this description, as well as other exemplary embodiments described herein, do not limit the scope of the invention, but instead describe how to obtain the advantages of increased production efficiency. You will recognize that you have a role to play.

In this example, the cells of interest are doubling weekly under conventional conditions. The culture "Day" starts at "0" so that one skilled in the art can easily ascertain the relative time advantages of this embodiment. Also, in order to simplify this example, what was said above regarding feeder and/or antigen-presenting cell surface density ratios will not be repeated. For purposes of illustration, assume that on "day 0" of production there is a starting population of 500,000 target cells with a doubling time of 7 days in conventional conditions. Conventional methods start with a surface density of 0.5×10 ⁶ cells/cm ² and a medium volume:surface ratio of 1 ml/cm ² . As shown, when the desired cell population reaches a surface density of 2×10 ⁶ cells/cm ² , the cells are distributed over an additional surface area at a surface density of 0.5×10 ⁶ cells/cm ² and the production cycle is repeated. start fresh. The novel method of this example starts with a surface density of 0.06×10 ⁶ cells/cm ² , a growth area composed of gas permeable material and a medium volume:surface area ratio of 6 ml/cm ² . As shown, as the population nears the onset of a growth plateau, it redistributes cells to more growth area. In this case, in the conventional method, the population starts to plateau when the cell surface density approaches 1.5 times the medium volume: surface area ratio (i.e., 1.5 × ¹⁰ cells/ml). determined to have reached Thus, on about day 9, the cells are distributed over a 36 cm ² growth area at a surface density of about 4.5×10 ⁶ cells/cm ² and the production cycle begins anew.

FIG. 15 tabulates a comparison of each production method shown in FIG. 14, demonstrating the power of the new method and why it is advisable to adjust the production protocol at various stages to fully take advantage of it. It proliferates in stages. Note that the new method overwhelms the conventional method after only the second stage of the production cycle is completed, yielding almost 1.37 times more cells with only 61% surface area required in only half the time. should. However, it should also be noted how in the third stage of the production cycle the cells are increased in quantity, with a corresponding increase in surface area. Thus, in order to predict how the initial and/or final cell surface density will be adjusted throughout each cycle of the process to achieve optimal efficacy levels for any given process, the production cycle should be modeled.

As an example, FIG. 16 shows an example of how variables can be changed in the novel method to gain efficiency as production progresses. For example, the starting surface density in cycle 3 may increase from 0.06 to 0.70 counts/cm ² and the final surface density may vary from 4.5 to 7.5 counts/cm ² . An increase in final surface density means that the medium volume: surface area ratio will be a number greater than the initial 6 ml/cm ² . The higher the medium volume: surface area ratio, the longer the cycle remains in the rapid growth phase (ie, population growth before the plateau interval). In this case, the rapid growth phase was completed for an additional 5 days to increase the medium volume:surface area ratio to approximately 8 ml/cm ² . By doing so, the present example is able to produce over 3 trillion cells in a reasonable surface area in 34 days. For example, devices were constructed and tested on approximately 625 cm ² of growth surface of gas permeable material. This is clearly a better cell production approach than conventional methods.

Thus, another preferred practice of the method of the invention when there is a desire to minimize the time to produce a given amount of cells of interest present in a cell population by using a reduced cell surface density. The morphology is described here:
a. seeding the cells of interest onto a growth surface area composed of a gas permeable material in the presence of antigen-presenting cells and/or feeder cells at a medium volume:surface area ratio of at least 2 ml/cm ² ;
b. Target cell surface density is less than conventional density, preferably within the range of about 0.5×10 ⁶ target cells/cm ² to about 3900 target cells/cm ² , and target cells and antigen-presenting cells and/or feeders Establishing favorable surface density conditions at the start of the production cycle such that the total cells are at least about 1.25×10 ⁵ cells/cm ² ;
c. allowing the population of cells of interest to grow beyond conventional surface densities of about 2×10 ⁶ /cm ² ;
d. If more cells of interest are desired, redistributing the cells of interest onto an additional growth surface of gas permeable material and repeating steps ad until sufficient cells of interest are obtained.

The present disclosure advances the field of adoptive cell therapy by creating a new class of therapeutic cells called T vehicles. The T-vehicle is further modified to comprise a population of T cells that have no inherent risk of GVHD and to include one or more therapeutic attributes that can act to treat and provide therapeutic benefit to the recipient. . T-vehicles have no natural ability to cause GVHD disease, making them ideal biological delivery vehicles with any number of countermeasures capable of combating a wide range of medical conditions and diseases. The present invention provides methods of producing and using T-vehicles with therapeutic attributes to confer to recipients the health benefits of adoptive cell therapy without the inherent risks of GVHD present in state-of-the-art methods. disclose. Importantly, T-vehicles work in opposition to state-of-the-art adoptive cell therapy, as their therapeutic purpose is completely independent of the antigen specificity of the native T-cell receptor. Those skilled in the art should recognize that throughout this disclosure and the exemplary embodiments presented, the therapeutic attributes of T-vehicles do not include the natural antigen receptors of T-vehicles.

The T-vehicle stimulates donor PBMCs or donor cord blood with antigen to activate the development of donor T cells with natural antigen specificity for the antigen, thereby generating antigen containing antigen receptors with antigen specificity for the antigen. Produced by producing specific T-cell populations. By selecting an antigen that is not present on normal cells, it is possible to create a population of T cells that have antigen receptors that cannot recognize normal cells. By modifying natural T cells with one or more therapeutic attributes that ignore the therapeutic benefit that would derive from the antigen specificity of natural T cells and that do not include the ability to recognize the natural antigen receptor, these antigens A group of T-Vehicles can be created that have a purpose independent of specific recognition and are inherently not prone or unable to cause GVHD.

Although T-vehicles can include more than one natural antigen-specific T-cell population, T-vehicles do not rely on their natural antigen specificity for their therapeutic purposes, thus T-vehicles are highly sensitive to the recipient's serotype. Recipients can be infused regardless of whether they display a positive match for one or more of the natural antigen receptors of the T-vehicle. Important attributes of T-vehicles also include their ability to be used in HLA-mismatched settings, since the one or more natural T-cell populations from which they are derived do not carry an inherent risk of GVHD. This allows the establishment of a homogenous bank of T-vehicles that can serve the wider community without the HLA-matching limitations required by state-of-the-art methods. If the T-vehicle has a natural antigen specificity that is HLA-mismatched to the recipient, the natural T-cell receptors of the T-vehicle cannot recognize the recipient's cells and cause GVHD. Nevertheless, T-vehicles have been engineered to have therapeutic attributes that are independent of the natural antigen receptor to achieve their therapeutic goals, thus initiating their therapeutic activity in completely HLA-mismatched settings. . However, T-vehicles are not limited to use in HLA-mismatched settings. Part between the recipient and the natural antigen specificity of the T-vehicle by creating a T-vehicle consisting of T-cells that have natural antigen receptors with highly restricted antigen specificity for antigens not normally expressed by cells. Development of GVHD disease can be avoided despite significant HLA matching. In order for the T-vehicle to be used in HLA-matched or HLA-mismatched settings, it is preferred that the natural antigen specificity of the T-vehicle allows the T-vehicle to recognize antigens not present in normal cells, preferably normal human cells. Even more preferably, it can recognize only a single epitope of the antigen that is not present in mammalian cells.

If the T-vehicle is HLA-mismatched to the recipient, the recipient is expected to mount a vigorous immune response that will eventually eliminate the T-vehicle. Thus, one or more additional T-vehicle doses can be given to continue the therapeutic purpose of the T-vehicle. This process can be continued as necessary to achieve the desired therapeutic goal. In a preferred method, since each dose of T-vehicle is different in terms of HLA, the patient's immune system must reprime itself each time it is about to challenge a new dose of T-vehicle, thereby The intervals between each dose of T-vehicle are kept approximately equal.

Methods for producing T cells with natural antigen receptors with highly restricted antigen specificity: Historically, it has been difficult to produce T cell populations on the scale necessary for their widespread use in adoptive cell therapy. It was actually impossible. State-of-the-art production methods for expanding T-cell populations into therapeutic doses of the desired size are too impractical and cumbersome to apply cell therapy to extremely small populations that must be handled by a few highly specialized institutions. limit. A fundamental attribute of T-vehicles is that their natural T-cell properties inherently do not expose the recipient to GVHD. Preferably, the natural antigenic specificity of the T-vehicles only allows them to recognize antigens that are absent from normal cells, more preferably normal human cells, and only recognizes a single epitope of antigens that are absent from normal mammalian cells. Even more desirable to be recognized, the efficient production of these cells underlies the widespread use of methods involving T-vehicles. Such T cells are only present at very low and 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 trying to generate T-cell populations most suitable for use in T-vehicles.

We have disclosed U.S. patent application Ser. We have discovered a method and apparatus as described in (incorporated herein by reference). This is contrary to state-of-the-art methods and effectively produces T cells with natural properties that essentially do not expose the recipient to GVHD. In doing so, the long term required for practical production of the T cells found at low frequency in donor PBMC or cord blood is met. Furthermore, when combined with novel concepts of T cells possessing therapeutic attributes not inherent in the natural antigen specificity of T cells, the production of T vehicles that function as biological carriers becomes possible and practical.

In one exemplary method, at culture initiation, two or more selected antigens are present in two or more unique antigen-specific T cell populations (each population expressing an antigen receptor for one of the antigens presented). are presented to PBMC or cord blood (ie, the original reservoir of antigen-specific T cells) to stimulate the growth of T cells. The intention is to later select the most abundant and/or desirable population of natural T cells for production and terminate the others. As the culture progresses after initiation, different T cell populations responding to different antigens can exhibit different levels of population expansion, depending on the size of their original population. Moreover, some or all of them may remain undetectable. After a period of time, the cultures are evaluated for acceptable growth of T cell populations that respond to any of the selected antigens. Such evaluation may relate to just one population specific to one antigen or to additional populations specific to additional antigens. When one antigen-specific T-cell population has demonstrated acceptable proliferation, simply adding the antigen it recognizes into the device will restimulate that particular T-cell population to ultimately convert the remaining T-cells. T cell populations of particular interest continue to proliferate. However, when more than one T cell population has demonstrated acceptable proliferation, there are two options: 1) the culture can be restimulated only with the antigen to which the particular T cell population is responding. (thereby terminating the proliferation of less abundant T cell populations), or 2) splitting the culture into two or more cultureware. Each device receives a single antigen, distinct from that of all other devices, allowing only one T cell population to grow within each device, and all but the most abundant cultures are eventually terminated. Preferably, all cultureware is gas permeable and is described in co-pending US Patent Publications 2005/0106717A1 (Wilson et al.) (hereinafter Wilson '717) and 2008/0227176A1 (Wilson et al.) (hereinafter Wilson '176). ) (both of which are incorporated herein by reference) and rely on the method of Vera '700.

By way of further example, a population of PBMCs present in a culture device can present antigen A, antigen B and antigen C. After some time, the culture can be evaluated for the presence and/or growth of populations reactive with antigen A, B or C. If the antigen-specific population reactive to antigen A is the only population that does not exhibit acceptable frequency and/or population growth, terminate that population by restimulation with antigen B and antigen C only. can be made Alternatively, if the antigen-specific populations reactive to Antigen B and Antigen C are growing approximately equally, but it is unclear which will continue to grow at the best ratio, eventually continue production in one device. , the culture can be split into two instruments with the intention of terminating the production of the other instrument. A first device receives antigen B and a second device receives antigen C. T cells exhibiting antigen specificity for antigen B proliferate in the first device, while T cells exhibiting antigen specificity for antigen C eventually die out. The opposite occurs within the second instrument. Eventually, at some point after initiation of culture in the first and second devices, frequency and/or population size studies are initiated with the goal of terminating cultures that are least efficient in expanding the population of T cells of interest. obtain. Those skilled in the art will recognize that a major advantage of using multiple antigens rather than just one antigen at culture initiation is to increase the likelihood of finding T cell populations with suitable antigen specificity and growth rate. should. Furthermore, by using multiple antigens in one device instead of multiple devices with one antigen, PBMC or cord blood, media, cytokines, laboratory space, labor and biohazardous waste space can be more efficiently used. used.

The selection of the preferred natural antigen specificity of the T-vehicle is described here: it is preferred that the natural antigen specificity of the T-vehicle alone allows them to recognize antigens that are not present in normal cells, more preferably normal human cells. Even more preferably, it is capable of recognizing only a single epitope of the antigen that is not present in mammalian cells, although this is non-limiting and there are many favorable attributes of natural antigen receptors that should be considered by those skilled in the art. Many options and properties are suitable. By way of example, the natural antigen specificity of a T-vehicle can consist of two or more T cell populations with natural antigen specificity. The natural antigen specificities of T-vehicles are self or non-self antigens; reptiles, amphibians, fish or birds; invertebrates such as sponges, coelenterates, worms, arthropods, mollusks or echinoderms; Fungi, Parasites and Sponges; Adenovirus, Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Adenovirus (Adv), Respiratory Syncytial Virus (RSV), Human Herpes Virus 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 viruses including but not limited to K1 virus/WU virus; Din-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 prostatic acid phosphatase-3 all antigens or a single epitope.

To promote the growth of the appropriate natural T-cell population of donor cells, those skilled in the art may refer to US Patent Publication No. 2011/0182870A1 (hereafter referred to as Leen '870), which is incorporated herein by reference. Dendritic cells, monocytes, macrophages, B cells, T cells, PBMCs or artificial antigen-presenting cells such as modified k562 (all of which present antigens of interest and are antigen-specific of interest in natural donor T cell populations) cell lysate containing the antigen of interest, purified protein containing the antigen of interest, recombinant protein containing the antigen of interest, in donor cells with plasmid DNA encoding the antigen of interest, plasmid RNA encoding the antigen of interest, and/or a peptide library containing the antigen of interest and/or one or more single synthetic peptides containing the antigen of interest The use of antigens to induce the desired immune response should also be considered.

The production of T cell populations is preferably initiated using the methods of Vera '700 and/or those presented herein, more preferably gas permeable cells of the type described in Wilson '717 and Wilson '176. It starts with the use of culture equipment. Those skilled in the art should recognize that a variety of methods in the series of examples described are more or less suitable for the particular objectives of each application. For example, various surface densities, medium heights, medium volume:growth surface area ratios, etc. are available, as well as IL2, IL15, IL21, IL12, IL7, IL27, IL6, IL18 and/or IL4. Stimulation with cytokines at varying frequencies and concentrations, and repeated in vitro stimulation with any antigen source in combination with any antigen presentation method can also be used, such as gamma-ray capture, magnetic separation. Cells can be initiated with or without sorting by methods including, but not limited to, single cell cloning and/or flow cytometry.

Example 9: T-vehicles with natural T-cell receptors that recognize the CMV epitope NLV are unable to recognize non-self cell targets.

Antigen-specific T cells with natural antigen specificity for NLV-CMV were expanded to frequencies of 0.03% to 87% in PBMC in 12 days by methods previously described. These cells were then placed in culture with cells obtained from three HLA-mismatched donors presenting the NLV peptide of the target CMV antigen.

Figure 17 shows how T vehicles fail to recognize cells from mismatched allogeneic donors, the ability to recognize and kill autologous cells presenting the NLV peptide ("Auto4 pep") and not presenting the NLV peptide (Auto4). Despite their complete functionality as demonstrated by their ability to avoid autologous cell death, the NLV peptides (“allo1” and “allo1 pep”, “allo2” and “allo2 pep” , “allo3” and “allo3 pep”).

Selection and creation of one or more therapeutic attributes of interest: There are a wide range of options for modifying the antigen-specific T cell population to include at least one therapeutic attribute. The following non-limiting examples show how the choice of therapeutic attribute depends on the therapeutic objective, and why the therapeutic attribute and its therapeutic objective are independent of the antigen specificity of the T vehicle natural antigen receptor. It is intended to make one skilled in the art aware of whether

Example 10: Recombinant protein-loaded T-Vehicle administered as an immunotherapy adjuvant.

Immunotherapy is a class of therapy 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 ex vivo expanded T cells or NK cells. Recombinant proteins such as cytokines such as IL2, IL7, GM-CSF are administered systemically to promote the growth, proliferation, persistence and/or function of these cells in vivo, although some cytokines ( For example, systemic administration of IL2) causes severe mucositis, nausea, diarrhea, edema, dyspnea, hepatic and renal dysfunction, and proliferation of regulatory T cells that impair the function of the induced/infused T cells in vivo. associated with toxicity in Administration of T-vehicles loaded with recombinant proteins containing cytokines overcomes such toxicity by translocating to the site of inflammation and delivering these recombinant proteins directly to the site of immunotherapy-induced inflammation. obtain.

Those skilled in the art should recognize that T-vehicles can be used to target the delivery of such cytokines instead of traditional non-specific systemic administration. For example, experiments were undertaken to create a T vehicle capable of producing the cytokine IL7 and expressing truncated CD34Δ. This truncated CD34Δ can be used to detect the percentage of transduced cells and select transgenic populations. In this case, donor T cells with 98% native antigen specificity for the NLV epitope of CMV virus, as measured by flow analysis, have the therapeutic attribute of CD34Δ-IL7 cytokine expression, as shown in FIG. It was successfully modified to create a T-vehicle. Furthermore, studies demonstrated that only T-vehicles modified with a retroviral vector (CD34Δ-IL7 cytokine) were able to produce IL7, as detected by ELISA.

To evaluate therapeutic T-vehicles modified with a retroviral vector (CD34Δ-IL7 cytokine) with respect to in vivo effects and distribution of IL7 cytokines, mice were divided into two groups (1 group per group). 5). 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+06T vehicle. Subjects randomly selected from each group were then sacrificed at weeks 1 and 2 and IL7 cytokine concentrations at various locations such as heart, liver, kidney, spleen, peritoneum, tumor and blood were measured by ELISA. evaluated.

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

FIG. 19B shows IL7 cytokine accumulation at various locations for Group 2. FIG. ELISA analysis of IL7 cytokines demonstrated higher cytokine concentrations in tumor sites compared to other organs, demonstrating that cytokine production was sustained in tumors for at least 2 weeks after T-vehicle administration. Therefore, T-vehicle could migrate to the tumor site and preferentially deliver the cytokine IL7 for a long time. This clearly demonstrates that the ability of T-vehicles with therapeutic attributes to deliver cytokines provides superior therapeutic advantages over state-of-the-art delivery methods for systemically administered cytokines. . As expected, the in vivo presence of T-vehicle is limited, as indicated by the decrease in cytokine levels between weeks 1 and 2. This can be seen as a further advantage of T-vehicles as they do not remain in the recipient. Preferably, further doses of the T-vehicle are administered as needed until a therapeutic outcome is met, and when no further doses are administered, the T-vehicle is removed from the recipient.

Example 11: Donor T cells can be modified to generate T vehicles whose therapeutic attributes are chimeric antigen receptors (CARs) targeting specific antigens.

Donor T cells (assessed by pentamer analysis), with 98% of T cells having natural antigen specificity for the epitope NLV of the virus CMV, express CARs capable of recognizing prostate stem cell antigen (PSCA). can be transduced to create a T-vehicle with therapeutic attributes of The therapeutic goal of T-vehicles is destruction of prostate tumor cells. As shown in FIG. 20, in quadrant E2, 57.23% of donor T cells were successfully modified to make T-vehicles with therapeutic attributes of CAR-PSCA as measured by flow analysis. rice field. To test the killing efficacy of T-vehicles containing therapeutic attributes of CAR-PSCA, unmodified donor T cells and T-vehicles made by modifying donor T cells with CAR-PSCA were tested against the antigen PSCA-positive (GFP+ ) or PSCA-negative (mOrange+) target cells at a 1:1 ratio. After 72 hours of culture, the number of residual PSCA-positive tumor cells was determined by flow analysis. Figure 21 shows the results of the experiment at 72 hours, where quadrant A1 represents PSCA-negative cell counts, quadrant A3 represents T-vehicle counts, and quadrant A4 represents PSCA-positive tumor cells. represents a number. As expected, unmodified donor T cells did not modify the original culture population after 72 hours. Conversely, however, CAR-PSCA-expressing T-vehicles were able to nearly eradicate entire PSCA-positive tumor cell populations, while at the same time being superior to PSCA antigens by leaving PSCA-negative cells intact. Demonstrated choice. This clearly demonstrates the ability of T-vehicles to produce therapeutic benefits independent of their natural antigenic specificity.

Example 12: Donor T-cells can be engineered to create T-vehicles that are receptors capable of depleting cytokines whose therapeutic attributes are undesirable in the recipient.

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 engineered to create T-vehicles with the therapeutic attribute of expressing whatever specific cytokine receptor is required to confer the therapeutic goal of drawing unwanted specific cytokines from the tumor. and thereby have the therapeutic advantage of making the tumor environment more tolerant to immunotherapeutic strategies. Figures 22A and 22B are diagrams depicting such a process. In the depiction of Figure 22A, a T vehicle containing therapeutic attributes of a receptor capable of binding IL4 is in close proximity to tumor cells expressing the IL4 cytokine. In the depiction of FIG. 22B, the T-vehicle is bound by IL4 cytokines and the amount of IL4 cytokines protecting tumor cells is greatly reduced. It should be noted how the therapeutic attributes, therapeutic objectives and therapeutic benefits of T-vehicles do not involve and are independent of the natural antigen receptors of T-vehicles. Experiments were performed to evaluate the ability of T-vehicles to have therapeutic attributes to express the extracellular recombinant cytokine receptor IL4R/7 and to deplete IL4 cytokines. T-vehicles were prepared by modifying donor T-cells with natural specificity for NLV epitopes of CMV virus. 5E+05T vehicle was cultured in a 2 ml volume of medium in the presence of 2000 pg/ml IL4 in 24 well plates and compared to donor T cells. The concentration of cytokine IL4 was then assessed by ELISA at 24, 48 and 72 hours. The results are shown in FIG. The T-vehicles were clearly able to achieve their therapeutic goals, as the reduction in the immunosuppressive tumor growth factor IL4 cytokine at 72 hours was significant. Conversely, donor T cells (ie, the histogram labeled "Unmodified T-Vehicle") showed no ability to reduce the presence of IL4.

Those skilled in the art should recognize that T-Vehicles possess a number of therapeutic attributes that enable them to achieve therapeutic goals intended to impart therapeutic benefit to the recipient. We will now augment the disclosed possibilities with some further examples.

T-Vehicles modified with therapeutic attributes of chemotherapeutic agents for targeted treatment of cancer: breast, prostate, pancreatic, liver, lung cancer using various chemotherapeutic or anti-tumor agents , brain tumors, leukemia, lymphoma, melanoma and myeloma. Most chemotherapy is delivered intravenously, but many drugs are administered orally and then circulate throughout the body. Chemotherapeutic agents work by killing rapidly dividing cells, one of the main characteristics of most cancer cells. This means that chemotherapy also damages rapidly dividing cells under normal circumstances, such as cells in the bone marrow, gastrointestinal tract and hair follicles. Consequently, the most common side effects of chemotherapy are myelosuppression (decreased blood cell production and hence immunosuppression), mucositis (inflammation of the gastrointestinal mucosa) and alopecia (hair loss). Chemotherapy-induced nausea and vomiting are also common side effects of treatment. Administration of T-vehicles loaded with these agents has the potential to counteract these toxicities. This can occur by including chemotherapeutic agents in T-vehicles and injecting them into the recipient. This causes the T vehicle to migrate to the site of inflammation (cancer) and lower the chemotactic gradient. In this way, the chemotherapeutic agent is placed in close proximity to the tumor cells, as opposed to being administered systemically to the recipient. In the case of HLA mismatch, the recipient's immune system mounts an attack against the T-vehicles so that they are destroyed, but no chemotherapeutic agent is released at the tumor cell site. Thus, the load (ie, chemotherapeutic agent) is deposited directly at the target site, as opposed to systemic administration, thereby reducing off-target toxicity associated with chemotherapy.

This process is as shown in Figures 24A, 24B and 24C. As shown in FIG. 24A, chemotherapeutic drug-loaded T-vehicles migrate toward the site of inflammation (i.e., tumor cells) and due to HLA mismatch between T-vessel and recipient cells, Natural antigen receptors do not recognize recipient cells and reach tumor cells without causing GVHD. As shown in FIG. 24B, the recipient's immune system targets T-vehicles that are placed at the site of tumor cells. As shown in Figure 24C, under attack by the recipient's immune system, the T vehicle releases the chemotherapeutic agent at the site of the tumor cell, thereby avoiding the off-target toxicity inherent in state-of-the-art chemotherapy delivery methods. do.

T-Vehicles Modified with Antimicrobial Therapeutic Attributes: Antimicrobials are substances that kill or inhibit the growth of microorganisms such as bacteria, fungi or protozoa. These agents are typically administered systemically and can be delivered more targeted when loaded onto a T vehicle that has the ability to return to the site of inflammation to deliver their load.

T-vehicles modified with therapeutic attributes to produce recombinant proteins administered as adjuvants for immunotherapy: As well as being loaded with exogenous recombinant proteins, T-vehicles may be viral (e.g. adenoviral, retroviral , lentivirus, etc.) or non-viral transfection techniques can be used to genetically modify and transgenically express recombinant proteins, including cytokines, chemokines, enzymes, tumor antigens and cytokine receptors, which are also It can also be designed to act as an adjuvant to other immunotherapeutic interventions, eg, to improve T cell persistence, promote proliferation, or induce homing.

T-vehicles modified with therapeutic attributes to express transgenic molecules that confer cells with tumor specificity: Similarly, T-vehicles can be modified with recombinant proteins such as cytokines, and T-vehicles can also be modified with viruses ( For example, adenoviral, retroviral, lentiviral, etc.) or non-viral transfection techniques can be used to genetically modify and transgenically express the chimeric T-cell receptor (CAR).

T-Vehicles modified with therapeutic attributes loaded with or genetically modified with recombinant proteins for autoimmune disease treatment It arises from an appropriate immune response. In other words, the immune system mistakes parts of the body for pathogens and attacks its own cells. This may be limited to some organs. Administration of T-Vehicles loaded with recombinant proteins such as IL10, TGFB, IL13 cytokines, which suppress inflammation, results in the delivery of recombinant proteins when needed rather than distributing these recombinant proteins indiscriminately. Direct delivery to the site of inflammation may overcome such autoimmune effects.

T-vehicles can be genetically modified to express suicide genes: T-vehicles can be incorporated with safety-switch or suicide genes for rapid and complete elimination of injected cells. The gene can be turned on should toxicity occur. The most effective of the suicide genes is thymidine kinase from herpes simplex type I (HSV-tk). This enzyme phosphorylates the non-toxic prodrug ganciclovir, which is then phosphorylated by an endogenous kinase to GCV-triphosphate, which upon incorporation into DNA results in chain termination and single-strand breaks. , 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 acts as a suicide switch when fused to an FK binding protein (FKBP) mutant that binds the chemical inducer dimerization (CID) AP1903, a synthetic agent with proven safety in healthy volunteers. can. Administration of this small molecule results in 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, elimination of 90% of transduced cells may not be sufficient to ensure the safety of genetically modified cells in vivo. Transgenic expression of the CD20 molecule, normally expressed on B cells, has also been postulated as a suicide gene for T cell therapy. This strategy relies on the clinical availability of a widely used humanized anti-CD20 antibody (rituximab) to eliminate both neoplastic and normal B cells expressing the CD20 antigen. Thus, infusion of T cells transgenically expressing human CD20 followed by in vivo administration of rituximab effectively eliminates the infused T cell population, but also eliminates normal B cells. It will be done. Thus, T-vehicles can be modified to express one or a combination of these different suicide genes to control clearance and delivery of the payload.

T-Vehicles genetically modified and/or modified with loaded therapeutic attributes for in vivo imaging: Positron Emission Tomography (PET) produces three-dimensional images or photographs of functional processes within the body nuclear medicine imaging. This system detects gamma-ray pairs emitted indirectly by positron-emitting radionuclides (tracers) that are introduced onto biologically active molecules in the body. A three-dimensional image of the tracer concentration in the body is then constructed by computer analysis. Due to the ability of the T-vehicle to migrate to the tumor site, the T-vehicle can be loaded with radioisotopes that allow the location of the tumor site to be detected and determined in vivo.

Similarly, iodine-123 (123I or I-123) is a radioisotope of iodine used in nuclear medicine imaging, such as single-photon emission computed tomography (SPECT). This is the isotope of choice for diagnostic testing for thyroid disease. A half-life of approximately 13.3 h (hours) makes it ideal for 24 h (hours) iodine uptake studies, and 123 I has other advantages for imaging thyroid tissue and thyroid cancer metastases. Because iodine is selectively captured in "iodine scavenging" by hydrogen peroxide produced by the enzyme thyroid peroxidase (TPO), it can be safely used for imaging or treating thyroid tumors. Thus, the T-vehicle can be modified with thyroid peroxidase (TPO) which can be used to image/kill the T-vehicle after scavenging iodine.

Those skilled in the art should recognize that therapeutic attributes of T-Vehicles for any given therapeutic purpose can be produced by a number of 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) electroporation using transposons and transposases (e.g. Sleeping Beauty) and/or Genetic modification by non-viral vectors, including the use of DNA and/or RNA vectors incorporated by physical and/or chemical techniques such as lipofection and/or Piggybac technology; and/or c) T alter vehicle migration, incorporate suicide genes, improve recipient immune reconstitution (e.g., cytokine production), and/or induce direct antiviral or antitumor effects (e.g., chimeric antigen receptors), or genetic modification to include one or more transgenes that suppress the immune response for autoimmune disease therapy, and/or d) CCR1, CCR2, CCR3, CCR4, CCR5, CCR6 to improve T vehicle translocation; Genetic modifications to improve T-vehicle translocation by expression of one or more chemokine receptors such as CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3-A, CXCR5, CXCR6, _CX3CR1 and/or XCR1 and/or e) by T vehicle expression of one or more cytokines such as GM-CSF, TNFα, INFγ, IL2, IL8, IL15, IL7, IL12, IL21 or IL26, or the co-stimulatory molecules CD80, CD86, 41BBL, genetic modification to improve immune reconstitution in the recipient by expression or overexpression of OX40L; and/or f) by expression of one or more suicide genes such as the thymidine kinase TK gene, CD20, CD19, i-caspase 9. genetic modification to induce T-vehicle death, and/or g) i) an extracellular spacer using CH2CH3 sequences from the IgG-FC region, or ii) containing sequences of CD28, CD4, CD3 or CD8. with a transmembrane component including, but not limited to, iii) CD3 (endodomain-encoded), or iv) CD3 (endodomain-encoding cytokine receptor or a specific ligand linked by expression of a natural ligand such as a cytokine). such as chimeric antigen receptors (CARs) that recognize tumor targets via single-chain variable fragments (scFv) isolated from the antibodies of expression of one or more transgenes, genetic modification to induce a direct antiviral or antitumor effect, including but not limited to, and/or h) e.g., IL4, IL6, IL10, IL13, TFGβ, etc. Genetic modifications to suppress immune responses for autoimmune disease treatment by expression of transgenes that produce one or more immunosuppressive cytokines, or by expression of competing ligands such as CTLA-4, PD1.

Those skilled in the art should recognize that the therapeutic purposes of T-Vehicles can range widely, 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) organisms that tolerate carrying 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) have no therapeutic purpose but have secondary benefits including but not limited to in vivo identification and imaging that allow identification of sites of metastatic disease. as a biological vehicle for carrying chemical compounds of

Each application, patent, and article cited herein, and each document or reference cited in each of these applications, patents, and articles, including during the prosecution of each issued patent, ; "Application Citation"), pending U.S. Patent Publication Nos. 2005/0106717 and 2008/0227176, which correspond to and/or claim priority from any of these applications and patents; Each of the PCT and foreign applications or patents that file, as well as each document cited or referred to in each application citation, is hereby expressly incorporated by reference.

Any documents incorporated by reference above are limited so as not to incorporate subject matter that is contrary to the explicit disclosure herein. Any document incorporated by reference above is further limited such that no claim contained in that document is incorporated by reference herein. Any document incorporated by reference above is further restricted not to be incorporated herein by reference unless any of the definitions given therein are expressly incorporated herein.

In interpreting the claims of the present invention, the provisions of 35 U.S.C. 112, sixth paragraph should not be invoked unless the specific terms "means for" or "step for" appear in the claim. is clearly intended.

Those skilled in the art will recognize that many modifications can be made to this disclosure without departing from the spirit of the inventions described herein. Accordingly, there is no intention to limit the scope of the invention to the described embodiments and examples. Rather, the scope of the invention should be construed by the appended claims and their equivalents.

Claims (7)

A genetically modified human T cell for adoptive cell therapy, comprising:
said T cells have an HLA that does not match the HLA of the patient into which said T cells are transferred;
said T cells have a natural T cell receptor that recognizes the NLV epitope of the CMV virus;
said T cells are expressing a recombinant protein;
A genetically modified human T cell, wherein said recombinant protein is IL7, a chimeric antigen receptor specific for prostate stem cell antigen, or IL4R/IL7R. 2. The genetically modified human T cell of claim 1, wherein
A genetically modified human T cell , wherein said recombinant protein is IL7 . 2. The genetically modified human T cell of claim 1, wherein
A genetically modified human T cell , wherein said recombinant protein is a chimeric antigen receptor specific for prostate stem cell antigen . 2. The genetically modified human T cell of claim 1, wherein
A genetically modified human T cell , wherein said recombinant protein is IL4R/IL7R . 5. A genetically modified human T cell according to any one of claims 1 to 4 ,
Genetically modified human T cells with a suicide gene. 6. The genetically modified human T cell of claim 5 ,
A genetically modified human T cell, wherein the suicide gene is a gene that expresses thymidine kinase, Fas-related death domain-containing protein, or caspase-9 from herpes simplex virus type 1. 7. The genetically modified human T cell of claim 6 ,
A genetically modified human T cell, wherein said T cell has been virally transfected.

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