Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/46—Viral antigens
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

• the present invention relates generally to methods of culturing cells, and more specifically to culturing cells for cell therapy.
• the desired cells are a relatively small population within a composition of cells that are placed into cell culture devices.
• the composition of cells typically includes the source of the desired cells (such as peripheral blood mononuclear cells), feeder cells that stimulate growth of the desired cells, and/or antigen presenting.
• Culture devices and methods that allow the medium that cells reside in to be in a generally undisturbed state are favored since the cells remain relatively undisturbed. Such devices include standard tissue culture plates, flasks, and bags.
• the culture progresses in stages generally consisting of allowing the cell composition to deplete the medium of growth substrates such as glucose, removing the spent medium, replacing the spent medium with fresh medium, and repeating the process until the desired quantity of desired cells is obtained.
• the cell composition is moved to other devices to initiate a new stage of production as the desired cell population increases and additional growth surface is needed.
• the rate of population growth of the desired cells slows as the population of cells upon the growth surface increases. The end result is that it is very time consuming and complicated to produce a sizable population of desired cells.
• EBV-CTLs Epstein Barr virus
• the conventional method for optimal expansion of EBV-CTLs uses standard 24-well tissue culture plates, each well having 2 cm 2 of surface area for cells to reside upon and the medium volume restricted to 1 ml/cm 2 due to gas transfer requirements.
• the culture process begins by placing a cell composition comprised of PBMC (peripheral blood mononuclear cells) in the presence of an irradiated antigen presenting cell line, which may be a lymphoblastoid cell line (LCL), at a surface density (i.e.
• EBV-CTLs are selectively expanded again in the presence of irradiated antigen presenting LCL at a new surface density ratio of 4: 1 , with a minimum surface density of about 2.5x10 5 EBV-CTL/cm 2 .
• Medium volume is limited to a maximum ratio of 1 ml/cm 2 of growth surface area to allow oxygen to reach the cells, which limits growth solutes such as glucose.
• the maximum surface density that can be achieved is about 2x10 6 EBV-CTL/cm 2 .
• the maximum weekly cell expansion is about 8-fold (i.e. 2xl0 6 EBV-CTL/cm 2 divided by 2.5xl0 5 EBV-CTL/cm 2 ) or less.
• Continued expansion of EBV-CTLs requires weekly transfer of the EBV-CTLs to additional 24-well plates with antigenic re-stimulation, and twice weekly exchanges of medium and growth factors within each well of the 24-well plate.
• EBV- CTLs The culture of EBV- CTLs is but one example of the complex cell production processes inherent to cell therapy. A more practical way of culturing cells for cell therapy that can reduce production time and simultaneously reduce production cost and complexity is needed.
• the production of cells for cell therapy can occur in a shorter time period and in a more economical manner than is currently possible by using a staged production process that allows unconventional conditions to periodically be re-established throughout the production process.
• the unconventional conditions include reduced surface density (i.e. cells/cm 2 ) of desired cells, novel ratios of desired cells to antigen presenting and/or feeder cells, and/or use of growth surfaces comprised of gas permeable material with increased medium volume to surface area ratios.
• Embodiments of this invention relate to improved methods of culturing cells for cell therapy applications. They include methods that reduce the time, cost, and complexity needed to generate a desired number of desired cells by use of various novel methods that allow the desired cell population to maintain a higher growth rate throughout the production process relative to conventional methods.
• One aspect of the present invention relies on conducting the culture process in stages and establishing conditions at the onset of one or more stages that allow the growth rate of the desired cell population to exceed what is currently possible. At least one stage of culture, and preferably nearly all, establish initial conditions that include the desired cells resting either on non-gas permeable or gas permeable growth surfaces at unconventionally low surface density and at an unconventional ratio of antigen presenting cells (and/or feeder cells) per desired cell.
• the desired cell population can experience more doublings in a shorter period of time than allowed by conventional methods, thereby reducing the duration of production.
• Another aspect of the present invention relies on conducting the culture process in stages and establishing conditions at the onset of one or more stages such that the growth rate of the desired cell population exceeds what is currently possible. At least one stage of culture, and preferably nearly all, establish conditions that include the desired cells resting on a growth surface comprised of gas permeable material at unconventionally high medium volume to growth surface area ratios.
• the desired cell population can experience more doublings in a shorter period of time than is allowed by conventional methods, thereby reducing the duration of production.
• Another aspect of the present invention relies on conducting the culture process in stages and establishing conditions of each stage such that the growth rate of the desired cell population exceeds what is currently possible. At least one stage of culture, and preferably nearly all, establish initial conditions that include the desired cells resting on growth surfaces comprised of gas permeable material at unconventionally low surface density (i.e. cells/cm 2 ) with an unconventional ratio of antigen presenting cells (and/or feeder cells) per desired cell and in the presence of unconventionally high medium volume to growth surface area ratios.
• the desired cell population can experience more doublings in a shorter period of time than conventional methods allow, thereby reducing the duration of production.
• Figure 1A shows the population of antigen-specific T-cells in Example 1 undergoes at least 7 cell doublings after the initial stimulation over the first 7 days.
• Figure IB shows data demonstrating the magnitude of expansion of a T-cell population within a cell composition over time as determined by tetramer analysis for Example 1.
• Figure 1C the rate of population growth of antigen-specific T-cells diminishes over a 23 day period in Example 1.
• Figure 2 shows a table that illustrates the discrepancy between the potential expansion and observed fold expansion of antigen-specific T-cells in Example 1.
• Figure 3A shows the presence of antigen-specific T-cells following stimulations in Example 2.
• Figure 3B shows the expansion of a population of antigen-specific T-cells as surface densities diminish from lxl0 6 /cm 2 to 3.1xl0 4 /cm 2 while maintaining an antigen-specific T-cell to antigen presenting cell ratio of 4: 1 in Example 2.
• Figure 3C shows the expansion of a population of antigen-specific T-cells as surface densities diminish from lxl0 6 /cm 2 to 3.1xl0 4 /cm 2 while in the presence of a fixed number of antigen presenting cells in Example 2.
• Figure 4 shows an example of results obtained when continuing the work described in Figure 3, which further demonstrated that when desired cells need the support of other cells, unconventionally low desired cell surface density can initiate population expansion so long as desired cells are in the presence of an adequate supply of feeder and/or antigen presenting cells.
• Figure 5 shows a histogram demonstrating the ability to repeat the magnitude of the population expansion of desired cells by initiating culture at three differing cell surface densities (CTL/cm 2 ).
• Figure 6 shows a cross-sectional view of a gas permeable test fixture used to generate data.
• Figure 7A shows the growth curves of antigen-specific T cells produced in accordance with the present invention in comparison to conventional methods as undertaken in Example 5.
• Figure 7B shows that for Example 5, cell viability was significantly higher in antigen- specific T cells produced in accordance with the present invention in comparison to conventional methods as determined by flow cytometric forward vs. side scatter analysis.
• Figure 7C shows that for Example 5, cell viability was significantly higher in antigen- specific t cells produced in accordance with the present invention in comparison to conventional methods as determined by Annexin-PI 7AAD.
• Figure 7D showed that for Example 5, the superior growth of cells produced in the novel methods of the present invention exhibited the same cell specific growth rate as cell cultured using conventional methods as determined by daily flow cytometric analysis of CFSE labeled cells, confirming that the increased rate of cell expansion resulted from decreased cell death.
• Figure 8A shows how EVB-CTLs were able to expand beyond what was possible in conventional methods without need to exchange medium.
• Figure 8B shows how the culture condition of Example 6 did not modify the final cell product as evaluated by Q-PCR for EBER.
• Figure 8C shows how the culture condition of Example 6 did not modify the final cell product as evaluated by Q-PCR for B cell marker CD20.
• Figure 9 shows an illustrative example in which we experimentally demonstrated that a very low cumulative surface density of desired cells and antigen presenting cells (in this case AL-CTLs and LCLs cells combining to create a cell composition with a surface density of 30,000 cells/cm 2 ) was unable to initiate outgrowth of the AL-CTL population.
• AL-CTLs and LCLs cells combining to create a cell composition with a surface density of 30,000 cells/cm 2
• Figure 10A presents data of Example 8 that show how two novel methods of culturing cells produce more cells over a 23 day period than a conventional method.
• Figure 10B shows a photograph of cells cultured in a test fixture in Example 8.
• Figure IOC shows that in Example 8, the two novel methods of culture and the conventional method all produce cells with the same phenotype.
• FIG. 10D shows that for Example 8, a representative culture in which T-cells stimulated with EBV peptide epitopes from LMPl, LMP2, BZLFl and EBNAl of EBV and stained with HLA-A2-LMP2 peptide pentamers staining showed similar frequencies of peptide- specific T-cells.
• Figure 10E shows that for the novel methods and the conventional method of Example 8, cells maintained their cytolytic activity and specificity and killed autologous EBV-LCL, with low killing of the HLA mismatched EBV-LCL as evaluated by 5l Cr release assays.
• Figure 11 shows a graphical representation of expansion of a desired cell population on a growth surface under the conventional scenario as compared to population expansion of the desired cell type using one aspect of the present invention.
• Figure 12 shows an example of the advantages that can be obtained by utilizing a growth surface comprised of gas permeable material and an unconventionally high medium volume to growth surface area ratio beyond 1 or 2 ml/cm 2 .
• Figure 13 shows a graphical representation of a novel method of expansion of a desired cell population on a growth surface under the conventional scenario as compared to population expansion of the desired cell type under one embodiment of the present invention in which the cell surface density at the completion of is much greater than conventional surface density.
• Figure 14 shows another novel method of cell production t hat provides yet further advantages over conventional methods.
• Figure 15 shows a comparison of each production method depicted in Figure 14 to demonstrate the power of the novel method and why it is useful to adjust the production protocol at various stages to fully capture the efficiency.
• Figure 16 shows an example of how one could adjust the production protocol in the novel method to gain efficiency as production progresses.
• Antigen presenting cells Cells that act to trigger the desired cells to respond to a particular antigen.
• Desired cells The specific type of cell that that the production process aims to expand in quantity.
• the desired cells are non-adherent and examples includedie regulatory T cells (Treg), natural killer cells (NK), tumor infiltrating lymphocytes (TIL), primary T lymphocytes and a wide variety of antigen specific cells, and many others (all of which can also be genetically modified to improve their function, in-vivo persistence or safety).
• feeder cells and/or antigen presenting cells that can include PBMC, PHA blast, OKT3 T, B blast, LCLs and K562, (natural or genetically modified to express and antigen and/or epitope as well as co-stimulatory molecules such as 41BBL, OX40, CD80, CD86, HLA, and many others) which may or may not be pulsed with peptide or other relevant antigens.
• EBV Epstein Barr Virus
• EBV-CTL A T-cell that specifically recognized EBV-infected cells or cells expressing or presenting EBV-derived peptides through its T cell surface receptor.
• EBV-LCL Epstein Barr virus transformed B lymphoblastoid cell line.
• Feeder cells Cells that act to cause the desired cells to expand in quantity. Antigen presenting cells can also act as feeder cells in some circumstances.
• Growth surface The area within a culture device upon which cells rest.
• PBMCs Peripheral Blood Mononuclear Cells derived from peripheral blood, which are a source of some of the desired cells and which can act as feeder cells.
• Responder A cell that will react to a stimulator cell.
• Static cell culture A method of culturing cells in medium that is not stirred or mixed except for occasions when the culture device is moved from location to location for routine handling and/or when cells are periodically fed with fresh medium and the like. In general, medium in static culture is typically in a quiescent state. This invention is directed to static cell culture methods. Stimulated: The effect that antigen presenting and/or feeder cells have on the desired cells. Stimulator (S): A cell that will influence a responder cell.
• Surface density The quantity of cells per unit area of the surface within the device upon which the cells rest.
• EXAMPLE 1 Demonstration of limitations of conventional methods.
• the data of this example demonstrate the limits of conventional culture methods for the production of EBV-CTL in standard 24 well tissue culture plates (i.e. 2 cm 2 surface area per well) using a medium volume of 2 ml per well (i.e. medium height at 1.0 cm and a medium volume to surface area ratio of 1 ml/cm 2 ).
• Stage 1 of culture, day 0 The expansion of an EBV-CTL population was initiated by culturing a cell composition of PBMCs from normal donors (about lxlO 6 cells/ml) with antigen presenting gamma-irradiated (40 Gy) autologous EBV-LCLs at a 40: 1 ratio (PBMC:LCLs) and a medium volume to growth surface ratio of 1 ml/cm 2 thereby establishing a cell composition surface density of about lxlO 6 cells/cm 2 in RPMI 1640 supplemented with 45% Click medium (Irvine Scientific, Santa Ana, CA), with 2 mM GlutaMAX-I, and 10% FBS.
• PBMC:LCLs 40: 1 ratio
• Click medium Irvine Scientific, Santa Ana, CA
• Stage 2 of culture, day 9-16 On day 9, EBV-CTLs were harvested from the cell composition created in Stage 1, resuspended in fresh medium at a surface density of 0.5xl0 6 EBV-CTL/cm 2 and re-stimulated with irradiated autologous EBV-LCLs at a ratio 4: 1 CTL:LCL (surface density 0.5xl0 6 CTL/cm : 1.25xl0 5 LCL/cm 2 ).
• IL-2 human IL-2
• Stage 3 of culture, day 17-23 The conditions of Stage 2 were repeated with twice weekly addition of IL-2 and the culture was terminated on day 23. Although the culture was terminated, it could have been continued with additional culture stages that mimicked that of stages 2 and 3.
• BJAB a B cell lymphoma
• K562 a chronic erythroid leukemia
• Cell surface Cells were stained with Phycoerythrin (PE), fluorescein isothiocyanate (FITC), periodin chlorophyll protein (PerCP) and allophycocyanin (APC)-conjugated monoclonal antibodies (MAbs) to CD3, CD4, CD8, CD56, CD 16, CD62L, CD45RO, CD45RA, CD27, CD28, CD25, CD44 from Becton-Dickinson (Mountain View, CA, USA). PE-conjugated tetramers (Baylor College of Medicine) and APC-conjugated pentamers (Proimmune Ltd, Oxford, UK), were used to quantify EBV-CTL precursor frequencies. For cell surface and pentamer staining 10,000 and 100,000 live events, respectively, were acquired on a FACSCalibur flow cytometer and the data analyzed using Cell Quest software (Becton Dickinson).
• PE Phycoerythrin
• FITC fluorescein isothio
• CFSE labeling to measure cell division To assess the doubling rate of 2 ⁇ 10 7 PBMC or EBV-specific CTLs (EBV-CTLs) were washed twice and resuspended in 850 ⁇ 1 lx phosphate-buffered saline (PBS) containing 0.1% Fetal Bovine Serum (FBS) (Sigma-Aldrich).
• PBS phosphate-buffered saline
• FBS Fetal Bovine Serum
• AnnexinV-7-AAD staining To determine the percentage of apoptotic and necrotic cells in our cultures we performed Annexin-7-AAD staining as per manufacturers' instructions (BD Pharmingen tm #559763, San Diego, CA). Briefly, EBV-CTL from the 24-well plates or the G- Rex were washed with cold PBS, resuspended in IX Binding Buffer at a concentration of l xlO 6 cells/ml, stained with Annexin V-PE and 7-AAD for 15 minutes at RT (25°C) in the dark. Following the incubation the cells were analyzed immediately by flow cytometry.
• Chromium release assay We evaluated the cytotoxic activity of EBV-CTLs in standard 4-hour 51 Cr release assay, as previously described. As desired cells we used autologous and HLA class 1 and II mismatched EBV-transformed lymphoblastoid cell line (EBV-LCL) to measure MHC restricted and unrestricted killing, as well as the K562 cell line to measure natural killer activity. Chromium-labeled desired cells incubated in medium alone or in 1% Triton X-100 were used to determine spontaneous and maximum 51 Cr release, respectively. The mean percentage of specific lysis of triplicate wells was calculated as follows: [(test counts -spontaneous counts)/(maximum counts - spontaneous counts)] x 100.
• Enzyme-Linked Immunospot (ELlspot) assay ELlspot analysis was used to quantify the frequency and function of T cells that secreted IF y in response antigen stimulation.
• CTLs were resuspended at lxl0 6 /ml in ELlspot medium [(RPMI 1640 (Hyclone, Logan, UT) supplemented with 5% Human Serum (Valley Biomedical, Inc., Winchester, Virginia) and 2-mM L-glutamine (GlutaMAX-I, Invitrogen, Carlsbad, CA)].
• the population of antigen-specific T-cells undergoes at least 7 cell doublings after the initial stimulation over the first 7 days, as shown in Figure 1A.
• a weekly T-cell expansion of 128-fold (as measured by the frequency of antigen- specific T-cells tim es the total number of cells in the cell composition).
• the frequency of tetramer positive cells after the first, second, and third stimulations is shown in Figure IB.
• RAK and QAK was 0.02% and 0.01%, respectively.
• Example 1 demonstrates that the amount of time it takes to produce the desired cells is typically delayed after roughly the first week of production since the rate of population expansion of the desired cells decreases in subsequent stages of culture.
• EXAMPLE 2 Reducing the amount of time needed to increase the desired cell population can be achieved by reducing the cell surface density of the desired cell population as the onset of any given stage or stages of culture. We hypothesized that the decreased rate of expansion of the desired cell population following the second T-cell stimulation compared to the first stimulation was due to limiting cell culture conditions that resulted in activation induced cell death (AICD).
• AICD activation induced cell death
• the EBV antigen-specific T-cell component of PBMCs represents, at most, 2% of the population and so the antigen-specific responder T-cell seeding density is less than 2xl0 4 per cm 2 , with the remaining PBMC acting as non-proliferating feeder cells (seen as the CFSE positive cells in Figure 3A) that sustain optimal cell-to-cell contact allowing proliferation of the antigen-specific CTLs.
• the majority of T-cells are antigen-specific, and although the total cell density of the composition is about the same, the proliferating cell density is 50 to 100 fold higher.
• the majority of cells proliferate and may therefore rapidly consume and exhaust their nutrients and 0 2 supply.
• EXAMPLE 3 A minimum surface density of a cell population that includes the desired cells and/or antigen presenting cells can allow outgrowth of a desired cell population that is seeded at very low surface density.
• Figure 4 shows an example of results we obtained when continuing the work described in Figure 3, which further demonstrated that when desired cells need the support of other cells, unconventionally low desired cell surface density can initiate population expansion so long as desired cells are in the presence of an adequate supply of feeder and/or antigen presenting cells.
• a total cell composition with a surface density and R:S ratio of between about l .OxlO 6 desired cells/cm 2 at an R:S ratio of 8 to 1 and merely about 3900 desired cells/cm 2 at an R:S ratio of 1 to 32 could allow desired cells to be greatly expanded to over 50 fold times the starting surface density, at which point we discontinued testing.
• EXAMPLE 4 The ability to allow a production process to repeat in stages by initiating a stage with an unconventionally low desired cell surface density, allowing population expansion, terminating the stage and repeating conditions was demonstrated to deliver repeatable outcomes.
• Example 3 We continued the assessments described in Example 3 at three of the desired cell surface densities (CTL/cm 2 ) as shown in Figure 5. Each specific seeding density was able to consistently attain the same fold expansion. The implications will be described in more detail further on as they relate to the ability to dramatically reduce the production time for a desired cell population.
• EXAMPLE 5 Culturing desired cells on a growth surface that is comprised of gas permeable material while simultaneously increasing the medium volume to growth surface area ratio increases the number of times a desired cell population can double in a given stage of culture relative to conventional methods and increases the surface density that is attainable.
• G-Rex Test fixtures (hereinafter generically referred to as "G-Rex") were constructed as shown in Figure 6. Bottom 20 of each G-Rex 10 was comprised of gas permeable silicone membrane, approximately 0.005 to 0.007 inches thick. Pending U.S. Patent Application 10/961 ,814 to Wilson is among many other sources of information relating to the use of alternative gas permeable materials and can be used to educate skilled artisans about gas permeable culture device shapes, features, and other useful characteristics that are beneficial to many of the embodiments of this invention.
• G-Rex (referred to as "G-Rex40”) had a growth surface area of 10 cm 2 , upon which a cell composition (shown as item 30) rested, the characteristics of the cell composition varied throughout the experiment as described within. Medium volume (shown as item 40) unless otherwise indicated was 30 mL, creating a medium volume to growth surface area ratio of 3 ml/cm 2 .
• EBV-specific CTL and irradiated autologous EBV-LCLs at the conventional 4: 1 ratio of CTL:LCL were cultured in G-Rex40 devices.
• EBV-CTLs were seeded at a surface density of 5x10 5 cells/cm 2 in the G-Rex40 and the rate of EBV-CTL population expansion was compared with EBV-CTL seeded at the same surface density in a standard 24-well plate with a medium volume to growth surface area of 1 ml/cm 2 .
• EBV-CTLs in the G-Rex40 had increased from 5xl0 5 /cm 2 to a median of 7.9x10 6 /cm 2 (range 5.7 to 8.1xl0 6 /cm 2 ) without any medium exchange.
• EBV-CTLs cultured for 3 days in conventional 24-well plates only increased from a surface density of 5xl0 5 /cm 2 to a median of 1.8xl0 6 /cm 2 (range 1.7 to 2.5x10 6 /cm 2 ) by day 3.
• EBV- CTL surface density could be further increased by replenishing medium whereas cell surface density could not be increased by replenishing medium or IL2 in the 24-well plate.
• EBV- CTL surface density further increased in the G-Rex40 to 9.5x10 6 cells/cm 2 (range 8.5 xl0 6 to 1 1.0 xl0 6 /cm 2 ) after replenishing the medium and IL-2 on day 7 (data not shown).
• T-cells were labeled with CFSE on day 0 and divided between a G-Rex40 device with a 40 ml medium volume and a 24 well plate with each well at a 2 ml medium volume.
• Daily flow cytometric analysis demonstrated no differences in the number of cell divisions from day 1 to day 3. From day 3 onwards, however, the population of desired cells cultured in the G-Rex40 continued to increase at a rate that exceeded the diminishing rate of the 2 ml wells, indicating that the culture conditions had become limiting as shown in Figure 7D.
• the large population of desired cells in the G-Rex40 test fixtures resulted from a combination of decreased cell death and sustained proliferation relative to conventional methods.
• EXAMPLE 6 By use of unconventionally high ratios of medium volume to growth surface area and use of growth surfaces comprised of gas permeable material, the need to feed culture during production can be reduced while simultaneously obtaining unconventionally high desired cell surface density. This was demonstrated through use of G-Rex test fixtures for the initiation and expansion of EBV:LCLs.
• G-Rex2000 refers to device as described in Figure 8, the exception being the bottom is comprised of a 100 cm 2 growth surface area and a 2000 ml medium volume capacity is available. EBV-LCLs were cultured in and expand in the G-Rex2000 without changing the cell phenotype.
• EBV-LCL were plated into a G-Rex2000 at a surface density of lxl 0 5 cells/cm 2 along with 1000 ml of complete RPMI medium to create a medium volume to surface area ratio of 10 ml/cm 2 .
• EBV-LCL were plated into a T175 flask at a surface density of 5xl0 5 cells/cm 2 along with 30 ml of complete RPMI medium to create a medium volume to surface area ratio of about 0.18 ml/cm 2 .
• the EBV-LCL cultured in G-Rex2000 expanded more than those in the T175 flask without requiring any manipulation or media change. This culture condition did not modify the final cell product as evaluated by Q-PCR for EBER and B cell marker CD20 as presented in Figure 8B and Figure 8C.
• EXAMPLE 7 When sufficient feeder and/or antigen cells are not present at the onset of culture, desired cells may not expand. However, the cell composition can be altered to include an additional cell type acting as feeder cells and/or antigen presenting cell to allow expansion.
• Figure 9 shows an illustrative example in which we experimentally demonstrated that a very low cumulative surface density of desired cells and antigen presenting cells (in this case AL-CTLs and LCLs cells combining to create a cell composition with a surface density of 30,000 cells/cm 2 ) was unable to initiate outgrowth of the AL-CTL population.
• AL-CTLs and LCLs cells combining to create a cell composition with a surface density of 30,000 cells/cm 2
• this same cell composition could be made to grow by altering the composition to include another cell type acting as a feeder cell.
• the additive surface density of the antigen presenting cells and/or feeder cells and the desired cells should preferably be at least about 0.125xl0 6 cells/cm 2 to create enough surface density in the cell composition to initiate the expansion of the desired cell population. Also, to attain the continued expansion beyond standard surface density limits, the use of growth surfaces comprised of gas permeable material was used in this example along with a medium volume to surface area ratio of 4 ml/cm 2 .
• EXAMPLE 8 Reduced desired cell surface densities, altered responder cell to stimulatory cell ratios, increased medium to growth surface area ratios, and periodic distribution of cells at a low surface density culture onto growth surfaces comprised of gas permeable material allow more desired cells to be produced in a shorter period of time and simplifies the production process when compared to other methods.
• G-Rex500 refers to device as described in Figure 6, the exception being the bottom is comprised of a 100 cm 2 growth surface area and a 500 ml medium volume capacity is available.
• a second stage was initiated on day 9, wherein lxl 0 7 responder T-cells were transferred from the G-Rex40 to a G-Rex500 test fixture.
• stage two of culture 200 ml of CTL medium was placed in the G-Rex500, creating a medium volume to surface area ratio at the onset of stage two of 2 ml/cm 2 medium height at 2.0 cm above the growth surface area.
• the surface density of desired cells at the onset of stage two was lxl 0 5 CTL/cm 2 with antigen presenting cells at a surface density of 5x10 5 LCL/cm 2 , thereby creating a non-conventional 1 :5 ratio of desired cells to antigen presenting cells.
• This stage two cell surface density and R:S ratio produced consistent EBV-CTL expansion in all donors screened.
• IL-2 50U/ml - final concentration
• 200 ml of fresh medium bringing medium volume to surface area ratio to 4 ml/cm 2
• the cells were harvested and counted.
• the median surface density of CTLs obtained was 6.5xl0 6 per cm 2 (range 2.4xl0 6 to 3.5xl0 7 ).
• FIG. 10A shows the comparison of this G-Rex approach of Example 8 to the use of conventional methods of Example 1 and the G-Rex approach described in Example 5. As shown, the conventional method needed 23 days to deliver as many desired cells as could be delivered in either G-Rex method in about 10 days.
• Example 8 After 23 days, the G-Rex approach of Example 8 was able to produce 23.7 more desired cells than the G-Rex method of Example 5 and 68.4 times more desired cells than the conventional method of Example 1. Furthermore, the desired cells continued to divide until day 27-30 without requiring additional antigen presenting cell stimulation provided the cultures were
• FIG. 10D shows a representative culture in which T-cells stimulated with EBV peptide epitopes from LMP1, LMP2, BZLF1 and EBNA1 and stained with HLA-A2-LMP2 peptide pentamers staining showed similar frequencies of peptide-specific T-cells. Further, the expanded cells maintained their cytolytic activity and specificity and killed autologous EBV- LCL (62% ⁇ 12 vs.
• Examples 1 - 8 have been presented to demonstrate to skilled artisans how the use of various conditions including reduced surface density of the desired cell population at the onset of a production cycle, reduced surface density ratios between responder cells and stimulating cells, growth surfaces comprised of gas permeable materials, and/or increased medium volume to growth surface area ratios can be used to expedite and simplify the production of cells for research and clinical application of cell therapy.
• Examples 1 - 8 were related to the production of antigen specific T cells, these novel culture conditions can be applied to many important suspension cell types with clinical relevance (or required for pre-clinical proof of concept murine models) including regulatory T cells (Treg), natural killer cells (NK), tumor infiltrating lymphocytes (TIL), primary T lymphocytes, a wide variety of antigen specific cells, and many others (all of which can also be genetically modified to improve their function, in-vivo persistence or safety).
• regulatory T cells Teg
• NK natural killer cells
• TIL tumor infiltrating lymphocytes
• primary T lymphocytes a wide variety of antigen specific cells, and many others (all of which can also be genetically modified to improve their function, in-vivo persistence or safety).
• Cells can be expanded with feeder cells and/or antigen presenting cells that can include PBMC, PHA blast, O T3 T, B blast, LCLs and 562, (natural or genetically modified to express and antigen and/or epitope as well as co-stimulatory molecules such as 41BBL, OX40L, CD80, CD86, HLA, and many others) which may or may not be pulsed with peptide and/or a relevant antigen.
• feeder cells and/or antigen presenting cells can include PBMC, PHA blast, O T3 T, B blast, LCLs and 562, (natural or genetically modified to express and antigen and/or epitope as well as co-stimulatory molecules such as 41BBL, OX40L, CD80, CD86, HLA, and many others) which may or may not be pulsed with peptide and/or a relevant antigen.
• One aspect of the present invention is the discovery that production time can be reduced relative to conventional methods by the use of lower desired cell surface density.
• desired cells are able to have a greater numerical difference between their minimum and maximum cell surface densities than conventional methods allow.
• the desired cells are re-distributed upon additional growth surfaces comprised of gas permeable material at low starting surface density once again.
• Figure 11 shows a graphical representation of expansion of a desired cell population on a growth surface under the conventional scenario as compared to population expansion of the desired cell type using one aspect of the present invention.
• the surface density of desired cells at the onset of a production stage is less than conventional surface density.
• this explanation does not describe the process of initially obtaining the desired cell population.
• the 'Day" of culture starts at "0" to allow skilled artisans to more easily determine the relative time advantages of this novel method.
• each production cycle of the conventional method begins at a conventional surface density of 0.5x10 6 desired cells/cm 2 while each production cycle of this example begins at a much lower and unconventional surface density of 0.125x10 6 desired cells/cm 2 .
• 4 times more surface area i.e. 500,000/125,000
• the desired cells of the conventional method reaches a maximum surface density of 2xl0 6 cells/cm 2 in 14 days.
• 1 cm 2 of growth area delivers 2xl0 6 cells/cm 2 which are then re-distributed onto 4 cm 2 of growth area so that production can be continued using the conventional starting density of 0.5x10 6 cells/cm 2 (i.e.
• the novel method depicted in Figure 11 instead of using the conventional method of depositing 500,000 desired cells onto 1 cm 2 at the onset of production, distributes the 500,000 cells equally onto 4 cm 2 of growth area to create at unconventionally low starting surface density of 125,000 desired cells/cm 2 on Day 0.
• the novel method as with the conventional method, has its growth rate about to diminish on Day 7.
• Cells in the novel method are at a surface density of lxlO 6 cells/cm 2 .
• this stage of culture has produced 4xl0 6 cells that are then re-distributed onto 32 cm 2 of growth area so that production in Stage 2 can be continued using the starting surface density of 0.125xl 0 6 cells/cm 2 (i.e.
• the advantage of this aspect of the present invention is the production time reduction resulting from the reduction of cell surface density below that of conventional cell surface density in any particular application, wherein the particular conventional surface density used in this illustrative example may vary from application to application.
• Desired cells should be deposited upon a growth surface at an unconventionally low cell surface density such that: a. the desired cells are in the presence of antigen presenting cells and/or feeder cells and with medium volume to surface area ratio of up to 1 ml/cm 2 if the growth surface is not comprised of gas permeable and up to 2 ml/cm 2 if the growth surface is comprised of gas permeable, and b.
• the preferred surface density conditions at the onset of a production cycle being such that the target cell surface density is preferably less than 0.5x10 6 cells/cm 2 and more preferably diminishing as described in Figure 4, and c.
• the surface density of the desired cells plus the surface density of the antigen presenting cells and/or feeder cells is preferably at least about 1.25 xlO 5 cells/cm 2 .
• growth surfaces comprised of gas permeable material and higher medium volume to growth surface area ratios can simplify and shorten production.
• Another aspect of the present invention is the discovery that the use of growth surfaces comprised of gas permeable material and medium volume to growth surface area ratios that exceed conventional ratios, and repeated cycles of production that increase the amount of growth surface area used over time will reduce production duration.
• Figure 12 augments the discussion to show an example of the advantages that can be obtained by utilizing a growth surface comprised of gas permeable material and an unconventionally high medium volume to growth surface area ratio beyond 1 or 2 ml/cm 2 .
• the discussion that follows is intended to demonstrate to skilled artisans how, by use of such a method, several options become available including reducing production time, reducing the amount of growth surface area used, and/or reducing labor and contamination risk. Skilled artisans will recognize that Figure 12 and associated discussion is merely an example, and does not limit the scope of this invention.
• the cell composition containing the desired cell population in this illustrative example is assumed to consume about 1 ml per "X" period of time.
• Figure 12 shows two production processes, labeled “conventional method” and "novel method.”
• each process begins with desired cells at a surface density of 0.5xl0 6 /cm 2 .
• the growth surface of in the novel method is comprised of gas permeable material and medium volume to surface area ratio is 2 ml/cm 2 as opposed to the conventional method of 1 ml/cm 2 .
• time period "X" the desired cell population of the conventional method has a reached a surface density plateau of 2xl0 6 /cm 2 and is depleted of nutrients while the additional medium volume of the
• novel method has allowed growth to continue and desired cell surface density is 3x10 /cm . If the novel method continues, it reaches a surface density of 4xl0 6 /cm 2 . Thus, many beneficial options accrue.
• the novel method can be terminated prior to time "X" with more cells produced than the conventional method, can be terminated at time "X" with about 1.5 times more cells produced than the conventional method, or can continue until the medium is depleted of nutrients with 2 times many desired cells produced as the conventional method in twice the time but without any need to handle the device for feeding. In order for the conventional method to gather as many cells, the cells must be harvested and the process reinitiated, adding labor and possible contamination risk. Since cell therapy applications typically only are able to start with a fixed number of cells, the conventional method does not allow the option of simply increasing surface area at the onset of production.
• Figure 13 continues the example of Figure 12 to show how more than one production cycle can be of further benefit.
• Figure 13 shows a graphical representation of expansion of a desired cell population on a growth surface under the conventional method as compared to population expansion of the desired cell type under one novel method of the present invention in which the surface density of the novel method exceeds surface density of the conventional method.
• the 'Day" of culture starts at "0" to allow skilled artisans to more easily determine the relative time advantages of this aspect of the invention. In this example, both cultures are initiated using conventional desired cell surface density of 0.5x10 5 cells/cm 2 at "Day 0".
• the growth surface of the conventional method is also comprised of gas permeable material.
• the medium volume to growth surface ratio in the conventional method is 1 ml/cm 2 as opposed to 4 ml/cm 2 in the novel method.
• the desired cell population in the conventional method begins to diminish in growth rate when it is at a surface density of about 1.5xl0 6 cells/cm 2 in about 4 days and reaches a maximum surface density of 2x10 6 cells/cm 2 in 14 days.
• the desired cell population is distributed to 4 cm 2 of growth area at a surface density of 0.5x10 6 /cm 2 in fresh medium at 1.0 ml/cm 2 and the production cycle begins again, reaching a surface density of 2x10 6 cells/cm 2 in another 14 days and delivering 8xl0 6 desired cells in 28 days.
• the desired cell population in the novel method begins to diminish in growth rate when it is at a surface density of about 3xl0 6 cells/cm 2 in roughly about 10 to 11 days and could reach a maximum surface density of 4xl0 6 cells/cm 2 in 28 days.
• the cycle ends when the desired cell population is still in a high rate of growth.
• the 3x10 6 cells are re-distributed to 6 cm 2 of growth surface area at a surface density of 0.5x10 6 /cm 2 in fresh medium at 4.0 ml/cm 2 and the production cycle begins again, with the desired cell population reaching a surface density of 3xl0 6 cells/cm 2 in roughly another 10 to 1 1 days and delivering 18xl0 6 desired cells around 21 days.
• the novel method has produced over 2 times the number of desired cells as compared to the conventional method.
• Figure 14 shows another novel method in which still further advantages relative to conventional methods are obtained.
• skilled artisans will recognize that the description herein does not limit the scope of this invention, but instead acts to describe how to attain advantages of improved production efficiency.
• desired cells are doubling weekly in conventional conditions.
• the 'Day" of culture starts at "0" to allow skilled artisans to more easily determine the relative time advantages of this embodiment.
• issues previously described related to feeder and/or antigen presenting cell surface density ratios are not repeated to simplify this example.
• the conventional method begins with a surface density of 0.5xl0 6 cells/cm 2 and a medium volume to surface area ratio of
• the novel method of this example begins with a surface density of 0.06x10 6 cells/cm 2 , a growth surface area comprised of gas permeable material, and a medium volume to surface area ratio of 6 ml/cm 2 . As shown, when the population is nearing the start of a growth plateau, cells are redistributed to more growth surface area.
• the population is determined to be reaching plateau from noting that plateau is initiated in the conventional method when cell surface density approaches 1.5 times the medium volume to surface area ratio (i.e. about 1.5xl0 6 cells/ml).
• the medium volume to surface area ratio i.e. about 1.5xl0 6 cells/ml.
• Figure 15 tabulates a comparison of each production method depicted in Figure 14, and extends to stages to demonstrate the power of the novel method, and why it is wise to adjust the production protocol at various stages to fully capture the efficiency.
• the novel method overpowers the conventional method after completing just the second stage of the production cycle, delivering nearly 1.37 times more cells in only about half the time with just 61% of the surface area requirement.
• the third stage of the production cycle creates a massive increase in cells and a corresponding increase in surface area.
• Figure 16 shows an example of how one could alter variables in the novel method to gain efficiency as production progresses.
• an increase in the starting surface density of cycle 3 from 0.06 to 0.70 cell/cm 2 and a change to the final surface density from 4.5 to 7.5 cells/cm 2 can be undertaken.
• Increasing the final surface density is a matter of increasing the medium volume to surface area ratio beyond the initial 6 ml/cm 2 to a greater number. The greater the medium volume to surface area, the longer the cycle remains in rapid growth phase (i.e. the population expansion prior to plateau). In this case we have allowed 5 extra days to complete the rapid growth phase and raised the medium volume to surface area ratio to about 8 ml/cm 2 .
• the target cell surface density is less than the conventional density, preferably at between about 0.5x10 6 desired cells/cm 2 an d about 3900 desired cells/cm 2 an d total number of desired cells and antigen presenting cells and/or feeder cells being at least about 1.25xl0 5 cells/cm 2 , and c. allowing the desired cell population to expand beyond the conventional surface density of about 2xl0 6 cells/cm 2 , and d. if more of the desired cells are wanted, redistributing the desired cells to additional growth surface comprised of gas permeable material and repeating steps a-d until enough desired cells are obtained.

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