WO2000050571A1 - Methode amelioree de culture de cellule t - Google Patents

Methode amelioree de culture de cellule t Download PDF

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WO2000050571A1
WO2000050571A1 PCT/US2000/004726 US0004726W WO0050571A1 WO 2000050571 A1 WO2000050571 A1 WO 2000050571A1 US 0004726 W US0004726 W US 0004726W WO 0050571 A1 WO0050571 A1 WO 0050571A1
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cells
culture
cell
cultures
patient
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E. Terry Papoutsakis
Kathleen S. Carswell
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Papoutsakis E Terry
Carswell Kathleen S
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure

Definitions

  • This invention relates to methods of culturing T cells.
  • the invention relates to an improved method of culturing T cells so as to enhance the proliferation (expansion) of the T cells.
  • the invention also relates to methods of cellular immunotherapy and gene therapy which utilize T cells.
  • Cellular immunotherapy whereby a patient is treated with large doses of autologous ex v/vo-expanded immune cells in order to eradicate aberrant cells, such as malignant or virally-infected cells, offers an alternative treatment for such diseases (see Chang etal, J. Clin. O «co/., 15(2):796-807 (1997);Plautzetal.,_ Newro_ «/r ., 89(l):42- 51 (1998); Rooney et al., Blood, 92(5): 1549-55 (1998)).
  • the effectiveness of these therapies is considered to be dose dependent, as patient survival increases proportionately with larger doses of effector cells (Cheever et al., Immunol. Rev., 157:177-94 (1997)).
  • Hematopoietic and immune cells experience a large spectrum of oxygen tension environments due to the physiology and vasculature of the bone marrow and the lympoid organs (Pennathur-Das et al., Blood, 69:899-907 (1987); Guyton et al, Textbook of Medical Physiology, W.B. Saunders Co., Philadelphia, PA, 1996).
  • the mean oxygen tension in the hematopoietic and lymphoid organ tissues is closer to 40 mm Hg (or 5% of the oxygen in the gas atmosphere), while the anatomical architecture of these organs suggests substantial intraorgan gradients that might expose cells to even lower oxygen tensions.
  • several studies have established the profound effect of lower oxygen tensions on hematopoietic cells.
  • Oxygen effects on immune cells have been examined, but with variable results.
  • Studies of myeloid hematopoietic cells have shown that greater cell proliferation is observed in a 5% O 2 environment (Roller et al, Exp. Hematol. , 20:264-70 (1992)). More recent work (Laluppa et al, Exp. Hematol., 26:835-43 (1998)) suggests that expansion and differentiation of different myeloid cell lineages are optimized under different oxygen conditions. Megakaryocytopoiesis and erythropoiesis are enhanced under high oxygen tensions (20%), while granulopoiesis is enhanced by low oxygen tensions (5%). In contrast, it has been reported that lower oxygen tensions may not have beneficial effects on lymphocyte cultures.
  • Loeffler et al. (Loeffler et al, Br. J. Cancer, 66:619-22 (1992)) reported a significant reduction of lymphocyte proliferation under 1% O 2 when compared to 20% O 2 .
  • Naldini et al. (Naldini et al, J. CellPhysiol, 173:335-42 (1997)) reported that peripheral blood mononuclear cells (PBMCs) are less susceptible to phytohemagglutinin (PHA) activation under 2% O 2 .
  • Anderson et al. (Anderson et al, J. Cell.
  • T cells experience a range of pH values in vivo as they migrate from the blood stream, into the lymph nodes, and out to the sites of infection or malignant disease.
  • Arterial blood has a normal pH of 7.4
  • other tissues have pH values in the range of 7.0 - 7.4
  • solid tumor masses which may contain tumor infiltrating lymphocytes (TIL)
  • TIL tumor infiltrating lymphocytes
  • the pHs of two commercially-available serum-free media commonly used for T cell culture were measured after equilibration in a 5% CO 2 incubator to be 7.3 (AIM V) and 7.4 (X-V-TVO 20) (see Example 2 below). Expression of certain surface receptors has been shown to be affected by pH.
  • McDowell and Papoutsakis showed that decreasing culture pH increased the CD 13 receptor surface content on HL60 cells over a range of 7.0 to 7.4 and that this increase in expression was not associated with changes in the messenger RNA levels for the receptor (McDowell et al, Biotechnol. Prog, 14(4):567-72 (1998)).
  • Katafuchi et al. (Katafuchi et al, Am. J. Physiol, 264:C1345-C1349 (1993)) demonstrated that the surface concentration of atrial natriuretic peptide (ANP) receptor, present on endothelial cells, varied more than three orders of magnitude between cells cultured at 7.0 and 7.7.
  • ADP atrial natriuretic peptide
  • Gene therapy can be defined as a therapeutic modality whereby the transfer of genetic material into some cells of a patient can bring about a therapeutic outcome of temporary or lasting duration (Anderson, Science, 256:808-813 (1992); Mulligan, Science, 260:926-932 (1993)).
  • the genetic material can be delivered to patient cells either in vivo (i.e., the cells are, and remain, part of the patient's body when the genetic material is delivered to them) or ex vivo (i.e., specific cells are removed from a patient, the genetic material is delivered to these cells, and the genetically-modified cells are then returned to the patient).
  • ex vivo gene therapy before the genetically-modified cells are returned to the patient, the cells may or may not be expanded prior to, or after, the introduction of the genetic material.
  • a gene therapy vector is the means by which one or multiple genes are delivered to cells in vivo or ex vivo.
  • a vector is made up of genetic material suitably constructed for the introduction into the cells, and the expression of, the necessary genetic material.
  • vectors of viral and non-viral origin include vectors of viral and non-viral origin (Anderson, Science, 256:808-813 (1992); Mulligan, Science, 260:926-932 (1993); Ferrari et al, Science, 251:1363-1366 (1991); Jolly, Cancer Gene Therapy, 1:51-64 (1994); Crystal, Science, 270:404-410 (1995)) .
  • T cells are, thus, likely to be one of the most frequently used cells for ex vivo gene therapy, and T cell expansion will be necessary for most such gene therapy protocols.
  • the invention provides an improved method of culturing T cells.
  • the method comprises providing a sample of cells, at least some of which are T cells, placing the cells in a culture medium, and culturing the cells in the culture medium for a selected period of time under conditions effective to cause proliferation of the T cells.
  • the conditions effective to cause proliferation of the T cells comprise including a T cell stimulant in the culture for at least a period of time sufficient to stimulate the T cells.
  • the conditions further comprise: (i) maintaining the pH of the culture at from about 6.8 to about 7.2 during the entire length of the culture; (ii) maintaining the oxygen level of the culture at substantially less than about 20% for at least the period of time sufficient to stimulate the
  • T cells with the T cell stimulant preferably for the entire length of the culture; or (Hi) maintaining the pH of the culture at from about 6.8 to about 7.2 during the entire length of the culture and maintaining the oxygen level of the culture at substantially less than about 20% for at least the period of time sufficient to stimulate the T cells with the T cell stimulant
  • the invention also provides a method of cellular immunotherapy.
  • This method comprises administering an effective number of autologous or allogeneic T cells to a patient in need thereof, the T cells having been cultured as described in the previous paragraph.
  • the invention provides a method of gene therapy.
  • This method comprises administering an effective number of genetically-modified autologous or allogeneic T cells to a patient in need thereof, the T cells having been cultured as described above.
  • sample D as identified in Tables I-III below. Cells were stimulated six days prior to time
  • Figures 2A-B Figures 2A-B.
  • Figure 2A is a Graph showing percent of apoptotic cells (as determined by the TUNEL assay) as a function of time in cultures under 5% and 20% O 2 environments.
  • Figure 2B shows the growth curves from this culture, where the boxed time points correspond to the samples in Figure 2A.
  • ⁇ 5% O 2 and
  • Figures 3A-B Graphs showing expression kinetics of CD25 (IL-2 receptor, IL- 2R) as percent of cells brighter than an isotype control ( Figure 3 A) and amount of receptor per cell (Figure 3B) (quantified as PE molecules bound by antibody staining using flow cytometry and the Quantibrite calibration beads) on those cells designated as CD25 + in Figure 3 A. Note the log scale on the vertical axis.
  • ⁇ 5% O 2 and
  • Peripheral blood mononuclear cells from a hemochromatosis patient were stimulated simultaneously with both anti-CD3 and anti-CD28 antibodies (100 ng/ml each) and expanded under either 5% or 20% O 2 levels in the presence of IL-2, with or without 2% autologous patient plasma.
  • AIM V Peripheral blood mononuclear cells from a hemochromatosis patient (same as in the culture performed in Figure 5) were stimulated simultaneously with both anti-CD3 and anti-CD28 antibodies (100 ng/ml each) and expanded under either 5% or 20% O 2 levels in the presence of IL-2, with or without 2% autologous patient plasma.
  • O 5% O 2
  • • 20% O 2
  • Figure 7 Graph showing % apoptotic cells (as determined by the TUNEL assay) in the cultures of Figures 5 and 6.
  • O 5% O 2
  • Figures lOA-B Graphs showing slower downregulation kinetics of IL-2R expression at pH 7.0 for both percentage of IL-2R positive cells (Figure 10A) and mean fluorescence intensity of the IL-2R positive fraction ( Figure 10B) (note the log scale of the vertical axis).
  • O pH 7.0
  • ⁇ pH 7.2
  • X pH 7.4.
  • FIG 11 Graph of percent apoptotic cells (determined by A nexin V method) in a representative culture experiment showing increased levels of apoptosis at higher pH values.
  • O pH 7.0
  • ⁇ pH 7.2
  • X pH 7.4.
  • T cells are lymphocytes that mature in the thymus. They are responsible for cell-mediated immunity and act as regulators of the immune response. Mature T cells can be divided into two subsets that differ in function on the basis of surface antigenic determinants - CD4 and CD8.
  • CD4+ T cells recognize antigen associated with Class II histocompatibilty (MHC) molecules
  • CD8+ T cells recognize antigen in associated with Class I MHC molecules.
  • Most CD4+ cells are helper T cells or are responsible for delayed-type hypersensitivity reactions.
  • Most CD8+ T cells are cytotoxic and/or suppressor cells. To culture T cells according to the invention, a sample of cells containing at least some T cells is obtained.
  • T cells are found in the lymph nodes, spleen, thymus, peripheral blood, peritoneal fluid, and other tissues and fluids of the body (e.g., infiltrating T cells from tumors), and T cells from any source may be used.
  • the sample of cells is removed from the desired source, and a single-cell suspension is prepared, all by methods well known in the art. See, e.g., Freedman et al, J. Immunol. Methods, 167: 145-160 (1994); Riddell and Greenberg, J. Immunol. Methods, 128:189-201 (1990); Robinet et al, J. Hematother, 7:205-215 (1998): Chang et al, J. Clin.
  • the cell suspension containing T cells and other cells, can be used as such in the culture, or the T cells can be purified, partially or completely, as is also well known in the art.
  • the source of the cells will be peripheral blood in many cases, and the most common, and the currently preferred, method of T cell purification for peripheral blood is apheresis (a density gradient-based separation of peripheral blood yielding T cells in the mononuclear cell fraction which may be achieved using a continuous process). See, e.g., Peshwa et al, Biotechnol. Bioeng., 50:529-540 (1996); Robinet et al., J. Hematother.,
  • T cell purification can be performed using antibody-based techniques to selectively capture the desired T cell fraction or to remove undesired populations, such as B cells. See, e.g., Levine et al., J. Hematother., 7:437-448 (1998).
  • Subpopulations of T cells can also be used in the cultures of the invention. Methods of preparing subpopulations of T cells are well known. For instance, antibody-based methods employing antibodies to a selected T cell surface antigen (e.g., CD4 or CD8) can be used to purify a subpopulation of T cells (e.g., anti-CD4 antibodies can be used to purify either the CD4+ or CD8+ subpopulation).
  • a selected T cell surface antigen e.g., CD4 or CD8
  • anti-CD4 antibodies can be used to purify either the CD4+ or CD8+ subpopulation.
  • the present invention provides a method of culturing the sample of cells under conditions effective to enhance the proliferation of the T cells in it as compared to standard T cell cultures.
  • the conditions that have been found to enhance the proliferation of T cells are: (i) culturing the T-cell-containing sample at an optimum pH; (ii) culturing the T-cell-containing sample at an optimum oxygen level; or, preferably, (iii) culturing the T-cell-containing sample at both an optimum pH and an optimum oxygen level.
  • the optimum pH for cultures containing T cells has been found to be from about
  • the pH of the culture should be measured and maintained on a regular basis. Generally, measurement of the pH once per day will be sufficient. Methods for continuous pH measurement and control using probes inserted into the culture vessel are well known. See, e.g., McDowell and Papoutsakis, Biotechnol. Prog, 14: 567-572 (1998).
  • the pH can also be measured by taking a sample of the culture medium and measuring its pH with a pH meter. The pH of the culture should be adjusted as needed to keep the pH in the optimum pH range during the entire culture period.
  • the pH can be adjusted by adding base or acid, such as 1 MNaOH or 1M HCl
  • the pH can also be adjusted by the addition or removal of CO 2 in media containing a bicarbonate buffer.
  • the pH of the fresh culture medium should be measured and adjusted to be within the optimum pH range, if necessary.
  • the optimum oxygen level for T cell cultures has been found to be substantially less than about 20% O 2 , the level of oxygen employed in standard T cell cultures.
  • the oxygen level employed for the T cell cultures of the invention is from about
  • the oxygen level of the culture should be kept at the optimum level for at least the period of T cell stimulation (see below). After this time, the oxygen level can be increased (up to about 20%), but it is preferably kept at the optimum level for the entire period of the culture.
  • substantially less than about 20% O 2 is meant that level of oxygen below 20% O 2 giving statistically significant better T cell proliferation than obtained using 20% O 2 .
  • about 5% O 2 etc. is meant 5% + the measurement error of the machine being used to measure oxygen level.
  • Percent (%) oxygen refers to the oxygen concentration in the gas phase with which the culture medium is equilibrated (i.e., the culture headspace or incubator environment).
  • T cell cultures are cultured in an incubator having an atmosphere consisting of 5% CO 2 and 95% air, which has an oxygen concentration of about 20%.
  • an inert gas such as N 2
  • N 2 an inert gas
  • Incubators containing oxygen sensors and mechanisms for controlling oxygen concentration are commercially available from, e.g., Forma Scientific.
  • the oxygen level of the T cell culture should be in equilibrium with that of the atmosphere in the incubator.
  • the oxygen level of the culture may be measured on regular basis during the culture period.
  • culturing T-cell-containing cultures using an optimum pH and an optimum oxygen level enhances the proliferation of the T cells in the cultures as compared to standard T cell cultures.
  • culturing of peripheral blood mononuclear cells (PBMC) at a pH of about 7.0 or about 7.2 for the entire length of the culture resulted in over a three-fold greater expansion of the T cells as compared to culturing PBMC at a pH of about 1.4.
  • Standard T cell cultures are performed at a pH of about 7.3-7.4.
  • culturing PBMC at an oxygen level of about 5% over the entire length of the culture resulted in a 5.8-fold greater expansion of the cultures as compared to culturing PBMC at an oxygen level of about 20%.
  • Standard T cell cultures are performed at an oxygen level of about 20%. It is expected that the use of both an optimum pH and an optimum oxygen level will give multiplicative results (e.g., about 17-18 fold greater expansion for PBMC as compared to standard culture conditions).
  • the conditions, equipment, media, and other reagents used in the T cell cultures of the invention will be those well known in the art. See Freedman et al., J. Immunol. Methods, 167:145-160 (1994) for a review of these conditions, equipment, media, and reagents. Some of these conditions and reagents will be discussed briefly.
  • the cultures are initiated using a particular inoculum density.
  • the inoculum density refers to the quantity of mononuclear cells (MNCs) per unit volume in the cell sample used to initiate the cultures. Methods of determining the number of MNCs present in a sample are well known in the art. Preferably, the MNCs are counted using a hemocytometer, Coulter Counter or similar apparatus. The inoculum density will vary depending on the source of the cells, the culture device, and the other culture conditions. Acceptable inoculum densities are known or can be determined empirically.
  • Culture media suitable for T cell cultures are well known. Either serum-containing or serum-free medium can be used for the T cell cultures. Serum-containing medium generally gives higher total cell expansion.
  • serum-free medium Preferably, however, serum or plasma obtained from the patient that is to receive the T cells (autologous serum or plasma) will be used for clinical applications.
  • allogeneic serum or plasma can be used in cultures for clinical applications.
  • the culture medium should be adjusted to the optimum pH and/or optimum oxygen level prior to adding cells.
  • T cell stimulants are materials which stimulate the proliferation of T cells (T cell activation) .
  • T cell stimulants, effective amounts of them, and methods of using them are well known.
  • polyclonal mitogens such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Staphylococcal enterotoxin B (SEB)
  • PHA phytohemagglutinin
  • Con A concanavalin A
  • PWM pokeweed mitogen
  • SEB Staphylococcal enterotoxin B
  • antibodies specific for antigens on the surfaces of T cells whose binding stimulates T cells can be used. Suitable antibodies include anti-CD3 antibodies and anti-CD28 antibodies.
  • T cell stimulants further include antigen-presenting cells.
  • Suitable antigen-presenting cells include dendritic cells, macrophages, B cells, and tumor cells, which may or may not be genetically modified (Peshwa et al. , Biotechnol. Bioeng. ,
  • T cell stimulant e.g., a combination of PHA and an anti-CD3 antibody or a combination of an anti-CD3 antibody and an anti-CD28 antibody.
  • the T cell stimulant must be kept in the culture for at least a time sufficient to stimulate the T cells. Such times are known or can be determined empirically.
  • a cytokine is also preferably used in the T cell cultures. Cytokines activate T cells and/or promote the growth and/or proliferation of T cells. Many suitable cytokines are known in the art. Preferred and crucial is interleukin 2 (IL-2) which promotes the growth and proliferation of all T cells. Recombinant IL-2 is commercially available from Chiron Corp. IL-2 is also a component of T cell conditioned media, also known as autologous lymphokine mixtures, which can be used in the culture of T cells (Gold et al, Eur. J. Cancer, 31 A:698-708 (1995)).
  • IL-2 interleukin 2
  • Recombinant IL-2 is commercially available from Chiron Corp.
  • IL-2 is also a component of T cell conditioned media, also known as autologous lymphokine mixtures, which can be used in the culture of T cells (Gold et al, Eur. J. Cancer, 31 A:698-708 (1995)).
  • cytokines that have been shown to have a positive effect on T cell proliferation and can be used in T cell cultures, include IL-l ⁇ (Baxevanis et al, Br. J. Cancer, 70:625-630 (1994), IL-4 (Kawakami et al, J. Immunother. , 14:336- 347 (1993), and IL-12 (Kobayasbi et al, J. Exp. Med, 127:827-845 (1989)). Effective amounts of cytokines are known or can be determined empirically.
  • Suitable protocols for feeding and/or subdividing T cell cultures are known in the art or can be determined empirically. In general, the higher the inoculum density and the higher the cell density present in a culture, the more often feeding and/or subdivision of the culture (whether static or stirred) will be required.
  • the T-cell-containing cultures of the invention are cultured for a selected period of time.
  • the selected period of time may be a set period of time (e.g. , 14-21 days) or may be the period of time necessary to obtain a desired number of T cells.
  • the number of T cells in the culture will be monitored periodically during the culture (e.g., daily) by methods well known in the art (see, e.g., the Examples below). Stimulation of the T cells with one or more T cell stimulants as described above induces proliferation which will typically last for 2-3 weeks, and can be repeated if desired.
  • T cells from both autologous and allogeneic sources, can be used in cellular immunotherapy and gene therapy protocols.
  • T cell therapies see Greenberg and Riddell, Science,
  • Atypical protocol would include harvesting the starting cell material (usually peripheral blood) from a patient, purifying the mononuclear cells (this fraction contains the T cells) by apheresis and then culturing the cells as described above. Serum-free medium can be used to culture the T cells, but, preferably, the patient's plasma or serum is added to the culture medium at an effective concentration (2% - 5% has been found to give good results). The cells would be cultured until the desired number of T cells is obtained.
  • the cells would be washed and resuspended in saline, or another similar solution, for administration to the patient.
  • the cells could be genetically modified prior to commencing the culture, after stimulation with the T cell stimulant(s), or after the culture is completed.
  • Example 1 Effect of Oxygen Level on T Cell Cultures
  • PBMCs were stimulated with either PHA, an anti-CD3 monoclonal antibody (mAb), or a combination of an anti-CD3 mAb and an anti-CD28 mAb 5% (low) or 20% (high) oxygen atmospheres.
  • mAb monoclonal antibody
  • IL-2 low or high oxygen conditions.
  • levels of proliferation, apoptosis and expression of certain cell-surface receptors and metabolic rates were determined. It was found that T cells stimulated and grown under 5% O 2 exhibited higher proliferation rates and a mean of 5.8 fold greater total expansion over T cells grown under 20% O 2 .
  • RESULTS AND MATERIALS Cells and culture conditions.
  • Cells for this study were obtained from two different populations. Samples of leukapheresis (continuous-flow process for blood cell separation) products from non-hematological cancer patients who had undergone stem cell mobilization procedures consisting of treatment with G-CSF with or without chemotherapy (Response Oncology, Memphis, TN) were used, as they represent a potentially clinically relevant population. Samples were also obtained from otherwise healthy donors undergoing therapeutic phlebotomies for treatment of hemochromatosis
  • the mobilized apheresis product samples were from patients with non-hematological malignant disease, and although their potential exposure to chemotherapeutic agents could alter lymphocyte proliferation and susceptibility to oxidative damage, this nonetheless represents a population of significant interest as potential recipients of adoptive immunotherapy.
  • the PBMCs from the hemochromatosis patient samples were collected using a Histopaque density gradient, and were washed in culture medium prior to stimulation, which removes most of the iron- enriched serum and erythrocytes.
  • Cells were seeded at lxl 0 6 cells/ml and cultured for 5- 10 days in T-flasks using RPMI with 100 U/ml IL-2 (Chiron: Emeryville, CA), 10% FBS (Hyclone: Logan, UT), 2 mM glutamine, 1 mM sodium pyruvate, 0J mM non-essential amino acids, 25 mMHEPES, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and either 5 ⁇ g/ml PHA or 20 ng/ml of soluble anti-human CD3 monoclonal antibody (Pharmingin:
  • Counting of nucleated cells was carried out using a Coulter Multisizer (Coulter Electronics, Hialeah, FL) after treatment with Cetrimide (Sigma, St. Louis, MO) to lyse the cells and release the nuclei.
  • the impact of oxygen level on T cell proliferation was assessed based on differences in proliferation rate and total fold expansion resulting from the initial stimulation with PHA or anti-CD3 mAb. These differences can be readily observed on plots of T cell expansion versus time. Cells were stimulated only once, and experiments concluded when cell proliferation stopped (usually 2 to 3 weeks). Total fold expansion was calculated based on percentage of CD3 + cells in the initial blood sample.
  • Glucose and lactate metabolic analysis were determined using a YSI model 2700 Biochemistry Analyzer (Yellow Springs Instrument
  • the yield coefficient describing the ratio of lactate production to glucose consumption, Y, ⁇ was calculated as q la6A I .
  • the yield coefficients ⁇ *____ & and Y mmo i kc/ce u were calculated as the respective ratios of changes in cell, glucose, or lactate concentrations over specific time intervals.
  • TUNEL assay Flow cytometric determination of the apoptotic fraction of cells was performed using the terminal deoxy transferase mediated dUTP nick end labeling (TUNEL) assay (Li et al, Cytometry, 20:172-180 (1995)) (In Situ Cell Death Detection Kit, Fluorescein, Boehringer Mannheim, Indianapolis, IN). The basis of this assay is enzymatic incorporation of fluorescein-conjugated nucleotides into the DNA strand breaks characteristic of apoptotic cells.
  • TUNEL terminal deoxy transferase mediated dUTP nick end labeling
  • cells were washed in PBS containing 1% BSA, fixed in 1% methanol-free formaldehyde, washed in PBS, and then resuspended in 70% ethanol and stored at -20 °C until further processing. After all of the samples had been collected, cells were rewashed in PBS with 1% BSA, then resuspended in the label and enzyme solutions from the kit, and incubated at 37 °C for 1 hr. Cells were then washed and resuspended in PBS with 1% BSA for analysis by flow cytometry. As a positive control, cells were incubated in DNAse (lmg/ml) following the ethanol permeabilization to induce strand breaks.
  • a volume of 8 ⁇ l of the dye solution was mixed with 60 ⁇ l of cell suspension, and viewed using a hemacytometer and fluorescence microscope (Diastar, Reichert-Jung). At least 200 cells were counted per sample at each timepoint.
  • PBA containing an amount of monoclonal antibody (Becton Dickinson: San Jose, CA) that had been previously titrated to be saturating (CD3 - 25 ⁇ l, CD25 - 15 ⁇ l, CD28 - 20 ⁇ l, CD62L - 20 ⁇ l).
  • Samples were incubated at 4 °C for 30 minutes, washed twice more with cold PBA, and then resuspended in 500 ⁇ l PBA for immediate analysis.
  • Flow cytometric measurements were performed using a Becton Dickinson FACScan cytometer equipped with a 15 mW, 488 nm air cooled argon-ion laser. Approximately 7500 cells were analyzed per sample.
  • QuantiBRITE Becton Dickinson fluorescence quantitation beads were used at each acquisition session in order to provide a consistent calibration measure to relate fluorescence intensity to the number of phycoerythrin (PE) molecules conjugated to the cell by the antibody staining process. While this does not necessarily give the exact number of receptors per cell (as the ratio of antibody binding to receptor is not known), the number of PE molecules per cell is proportional to the number of receptors per cell. This allowed for direct quantitative comparison of values of fluorescence intensity from samples stained and analyzed on different days in order to obtain kinetic information about surface receptor expression levels.
  • PE phycoerythrin
  • PBMCs were activated with either PHA or anti-CD3 mAb and then cultured under 5% or 20% O 2 gas- phase environments. After approximately a week, half of the cells were maintained in the original culture environment and the other half was switched to the alternate O 2 condition.
  • Figures 1 A-B are representative examples of these "crossover" cultures which show the increased rate and greater extent of proliferation achieved by activating and culturing T cells in the 5% O 2 environment for both PHA and anti-CD3 mAb activated cells.
  • the cells were then transferred into spinner flasks in incubators with 2.5%, 5%, and 20% O 2 .
  • AO/EtBr assays during the cultures. Apoptosis timepoints measured prior to 4 days following stimulation were excluded due to the large amount of cell death from non-T cell populations. Average results show an increase in apoptosis levels in the 20% O 2 cultures of 2.9 fold based on the TUNEL assay, and 1.4 fold based on the AO EtBr assay. The extent of the increase in apoptosis levels in the 20% O 2 cultures (as measured by the
  • the mean for the non-hematological cancer samples is a 4.4 fold increase, while the mean for the hemochromatosis patient samples is 1.8. It is not clear at this point why this difference is observed.
  • O 2 level alters the levels of expression of the IL-2 receptor (CD25Y Whether the expression levels of several surface receptors known to be important for in vivo T cell function were affected by O 2 concentration was also examined.
  • CD3, CD28, and CD62L expression was carried out using flow cytometry over the course of the cell expansion.
  • CD25 is the interleukin-2 receptor alpha chain (IL-2R- ⁇ ). Stimulation of T cells upregulates the expression of CD25, with peak levels occurring between days 2 and 8 depending upon the stimulation method (Hviid et al, J. Clin. Lab. Immunol., 40: 163-71 (1993); Biselli et al, Scand. J. Immunol., 35:439-447 (1992); Caruso et al, Cytometry, 27:71-76 (1997)). CD25 expression levels then decrease back to amounts equivalent to background (isotype control) staining over the next several weeks of culture.
  • IL-2R- ⁇ interleukin-2 receptor alpha chain
  • Figure 3A shows that as CD25 is downregulated over the course of the culture, T cells growing under 5% O 2 had a greater fraction of cells which were positive for CD25 (defined as staining brighter than an isotype control) than the 20% O 2 cultures.
  • Figure 3B shows that the CD25 receptor content
  • CD28 is an important costimulatory molecule expressed on the majority of T cells and its expression is enhanced by activation (June et al., Immunol. Today, 11:211-216 (1990); Turka et al, J. Immunol, 144:1646-1653 (1990)).
  • CD62L also known as L-selectin, is a key protein involved in regulation of trafficking of T cells to the lymph nodes. Comparison of expression levels between T cells grown under 5% and 20% O 2 revealed no consistent trends for either CD28 or CD62L (data not shown).
  • O-, level affects the metabolism of T cells.
  • Basic knowledge regarding the metabolism of T cells in culture is important for further optimization of culture media and feeding strategies for T cell ex vivo expansion, therefore examined the effect of O 2 level on on the metabolic characteristics of T cells in culture was examined.
  • Important metabolic parameters including the specific rates of glucose uptake (q ⁇ and lactate production (q ⁇ ), as well as several key yield coefficients (Y hc/ u , Y ce iw mmo i g i u .
  • Y mmo ii ac ⁇ ii are vital for understanding the basic metabolism and the impact of O 2 level (Miller and Reddy, in Doyle and Griffiths, eds., Cell and Tissue Culture: Laboratory Procedures in Biotechnology, pages 133-159 (John Wiley & Sons, New York, 1998)). These parameters were examined from cultures of T cells stimulated and grown under either 5% or 20%> O 2 . Over the duration of a culture, as the proliferation of the cells slowed, q ⁇ and q ⁇ decreased by as much as 10 fold. Both parameters were consistently higher in the culture stimulated and cultured under 5% O 2 .
  • T cells Expansion of each subtype, or both subtypes, of T cells is likely to be useful in various cellular immunotherapies and gene therapies (Greenberg and Riddell, Science, 285:546- 551 (1999)).
  • cellular immunotherapies and gene therapies will require the use of clinically approved serum-free media, such as AIM V.
  • serum-free media such as AIM V.
  • autologous patient plasma or serum has been used to enhance the expansion and improve the cellular properties of T cells (Trimble et al, Biotechnol. Bioeng, 50:521-528 (1996)).
  • CD 8+ T cells with or without plasma, as compared to the high, standard oxygen level (20%).
  • T cell proliferation may also be dependent on the expressed surface concentration of IL-2 receptor, with those expressing higher surface concentrations entering the cell cycle more rapidly (Cantrell et al, Science, 224:1312-1316 (1984); Burke et al., Cell Immunol, 178:42-52 (1997)). Therefore, T cells cultured under 5% O 2 , which possess a higher CD25 surface receptor content per cell, may be proliferating faster and also adding to this effect.
  • Bioeng., 42:339- 50 (1993) reported increasing specific glucose uptake and lactate production rates with decreasing pO 2 for a recombinant CHO cell line grown in continuous culture. The data also show an increased specific glucose uptake and lactate production rates in cultures grown at low (5%) O 2 level as compared to the cultures grown at high (20%) O 2 tension. The variation in the rates of metabolism of T lymphocytes over the course of expansion is quite dramatic and it is important to understand the kinetics of this process as one attempts to develop optimal feeding protocols. The observation that proliferating lymphocytes have increased metabolic activity is well known (Brand et al, Immunobiol, 173:23-34 (1986); O'Rourke and Rider, Biochem. Biophys.
  • the reported value is the percentage of apoptotic cells as detected by either the flow cytometry-based TUNEL assay orthe fluoresence microscopy-based (AO/EtBr) assay.
  • the values are the means of all measurements during that culture. No measurements were made during the first four days of culture due to the extensive apoptotic cell death of non-T cell populations.
  • ⁇ sample from hemochromatosis patient therapeutic phlebotomy product
  • the given ratio is the mean ratio of CD25 expression for 5% 0 2 : 20% O z averaged over all of the timepoints taken during a culture ⁇ standard deviation.
  • the overall means are the averages of the means from each culture ⁇ SEM.
  • PBMCs were stimulated with PHA and cultured at pH values of 7.0, 7.2, or 7.4.
  • the effects of pH on the cells were studied over the two to three weeks of proliferation resulting from the PHA stimulation, rather than using single timepoint assays, in order to examine the culture kinetics over realistic time scales for ex vivo expansion.
  • a greater than three fold increase in the proliferation capacity of the T cells was observed for the pH 7.0 and 7.2 cultures as compared to the pH 7.4 cultures.
  • the culture pH also affected the kinetics of the Interleukin-2 receptor (IL-2R)(CD25) downregulation process.
  • the faster receptor downregulation in both the pH 7.2 and 1.4 cultures resulted in a greater than two fold higher fraction of IL-2R + cells in the pH 7.0 cultures.
  • Culture pH also significantly increased the fraction of apoptotic cells in the higher pH cultures, with 27% more apoptosis in the pH 7.4 cultures than the 7.2 cultures, and 49% more apoptosis in the pH 7.4 cultures than the 7.0 cultures.
  • PBMCs Peripheral blood mononuclear cells
  • N NaOH or 1 N HCl in predetermined amounts based on titrations performed in the same 5% CO 2 environments used in the culture experiments.
  • the media were allowed to equilibrate in the 5% CO 2 incubators overnight before use. Once the cells had begun proliferating they were subcultured to a density of 5x10 4 cells/ml using the above pH- adjusted, pre-equilibrated media without PHA.
  • the cultures were maintained between 5xl0 4 and 2.5xl0 5 cells/ml throughout the experiment to avoid accumulation of lactic acid, which would alter the pH.
  • the cultures were maintained in a 37 °C incubator with an atmosphere of 5% CO 2 in air and 95% relative humidity. Unless otherwise noted, all reagents were from Sigma Chemical Co. (St. Louis, MO).
  • Counting of nucleated primary cells was performed using a Coulter Multisizer (Coulter Electronics, Hialeah, FL) after treatment with Cetrimide (Sigma) to lyse the cells and release the nuclei. Viabilities were measured using the trypan blue dye exclusion method. pH Measurements. Samples of the cultures were taken once a day and measured immediately using a pH meter (Corning 340, Corning, NY). The measured values were within 0.05 pH units of the targeted values. Prior to each subculturing, the pH of the feed media was also verified.
  • Flow cytometric measurements were performed using a Becton Dickinson FACScan cytometer equipped with a 15 mW, 488 nm air cooled argon-ion laser. Approximately 10,000 cells were analyzed per sample. Data acquisition was performed using the FACScan Research software and then analyzed using CellQuest version 1.2 (Becton
  • the QuantiBRITE (Becton Dickinson) fluorescence quantitation beads were used at each acquisition session in order to provide a consistent calibration measure to relate fluorescence intensity to the number of PE molecules conjugated to the cell by the antibody staining process. While this does not necessarily give the exact number of receptors per cell (as the ratio of antibody binding to receptor is not known), the number of PE molecules per cell is proportional to the number of receptors per cell. This allowed for direct quantitative comparison of values of fluorescence intensity from samples stained and analyzed on different days in order to obtain kinetic information about surface receptor expression levels. Propidium iodide (PI) (2 ⁇ g/ml) was added to one unstained sample on each day in order to exclude dead cells from the analyses.
  • PI polyidium iodide
  • Annexin V-FITC (Pharmingen, San Diego, CA) was used in a flow cytometric-based assay to determine the fraction of apoptotic cells in the T cell cultures.
  • Annexin V is a phospholipid binding protein that has high affinity for phosphatidylserine (PS).
  • PS phosphatidylserine
  • Annexin V-FITC was used together with PI to distinguish between nonviable cells and viable apoptotic cells.
  • the ratios were calculated based on the total cell expansion values for each culture.
  • the ratios of both percentage of positive cells and PE molecules per cell were calculated between the pH conditions for each of the multiple timepoints at which they were measured during the cultures.
  • the apoptosis statistical analysis was also performed on ratios calculated from the multiple measurements made during the experimental cultures.
  • RESULTS Proliferation of Primary Human T Cells is Affected by Culture pH. T cells from eight different donor samples were stimulated with PHA and expanded in media at pH values of either 7.0, 7.2, 1.4.
  • FIGS 9A-B show the proliferation profiles from two representative samples.
  • Figure 9A shows the results from a culture with relatively low expansion levels
  • Figure 9B shows the results from a sample with relatively high expansion levels.
  • the trend that the PBMC samples cultured at the high pH, 1.4, show lower total expansion is consistent for samples with widely varying degrees of expansion.
  • Both cultures ( Figures 9A-B) also show that during the initial days of culture, the rates of proliferation are similar between the different pH cultures.
  • IL-2 Receptor Expression is Altered by Variations in Culture pH.
  • Theinterleukin- 2 receptor (IL-2R, CD25) is a surface protein expressed on T cells which is critical for proliferation of the cells.
  • Interleukin-2 is the principle growth factor for primary T cells. Upon activation by either an antigen presenting cell or polyclonal mitogens such as PHA or an anti-CD3 monoclonal antibody, T cells experience a dramatic upregulation of IL-2R with peak levels occurring between days 2 and 8 depending upon the stimulation method (Biselli et al, Scand.
  • IL-2R downregulation process are affected by the extracellular pH of the culture.
  • IL-2R The expression of IL-2R was examined by flow cytometry and was quantified in two ways. First the percentage of the cells which were positive (defined as staining brighter than the isotype control) in their expression of IL-2R was determined. Then for that positive fraction, the average fluorescence intensity was measured and used along with the Quantibrite PE calibration beads to determine a mean number of PE molecules per cell (bound by the IL-2R antibody staining), which is proportional to the number of IL-2 receptors per cell.
  • Figures 10A-B show the IL-2R expression kinetics data from a representative pH experiment. The changes in both the percentage of positive cells (Figure 10A) and fluorescence intensity quantified as PE molecules per cell ( Figure 10B) are depicted.
  • the IL-2R expression data were also analyzed based on the relative differences between the 7.0, 7.2, and 7.4 pH cultures using the following ratios: 7.0:7.2, 7.0:7.4, and 7.2:7.4. For each of seven experimental cultures, multiple timepoints were analyzed for IL-2R expression. The ratios of expression levels were calculated for both the percentage of positive cells and the fluorescence intensity of the IL-2R positive cells.
  • CD3 Expression is not Altered by Variations in Culture pH.
  • the CD3 receptor complex is the primary signal transduction molecule which is triggered upon binding of the T cell receptor with the MHC/peptide complex on a target cell, initiating activation and proliferation of the T cell.
  • the effect of pH on the expression of CD3 was examined over the course of six experimental cultures. The expression of CD3 was evaluated in terms of fluorescence intensity, which was translated to PE molecules per cell using the Quantibrite calibration beads. No significant effect of pH on the expression of CD3 was found over the range of pH values in this study (7.0 - 1.4) (data not shown).
  • T Cell Apoptosis Levels are Affected by pH. The levels of apoptosis over the course of the cultures were also examined. The flow cytometry-based Annexin V assay was used to quantify the fraction of cells which had undergone extemalization of phosphatidylserine to the outer leaflet of the plasma membrane, an early marker for apoptosis. Apoptosis levels were measured daily from days 5-12, where day 0 is the day of initial stimulation. The data from one of these cultures is shown in Figure 11. Earlier days were not included because of the large extent of cell death from the non-T cell fraction of the PBMC starting samples.
  • the apoptosis data were collected for two sample cultures and analyzed based on the relative differences between percentages of apoptotic cells at the three different pH culture conditions.
  • the effect of extracellular culture pH on the downregulation kinetics of the IL-2R may offer some insight as to the mechanism behind the observed pH effects on T cell proliferation. For the pH 7.0 cultures, the IL-2R is maintained in greater numbers on a greater fraction of the cells for a longer period during culture. This correlates with the greater expansion observed in the pH 7.0 cultures.
  • the pH 7.4 cultures show faster IL-2R downregulation kinetics, such that more of the cells become negative for IL-2R faster during culture, which also seems to be reflected in the slower growth seen at the end of the high pH cultures.
  • the behavior of the pH 7.2 cultures however is more difficult to explain, as they have the greater expansion profiles similar to the low pH cultures but the kinetic behavior of IL-2R expression is similar to that of the more rapidly downregulating pH 7.4 cultures.
  • the fact that similar effects of pH were not observed for the expression of CD3 shows that the alterations in IL-2R expression are not the result of global changes in T cell protein expression due to pH.

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Abstract

L'invention concerne une méthode d'amélioration de la stimulation et de la prolifération de cellules T. Cette méthode consiste à cultiver une culture contenant des cellules T à un PH variant entre environ 6,8 et 7,2 pendant toute la durée de la culture, et/ou à cultiver la culture à un taux d'oxygène pratiquement inférieur à environ 20%, de préférence aux alentours de 5 %, pendant au moins le temps nécessaire à la stimulation de la culture. Cette invention a également trait à une méthode d'immunothérapie cellulaire et de thérapie génique. Ces méthodes consistent à administrer un nombre efficace des cellules T autologues ou allogéniques à un patient qui en a besoin, selon la méthode de cette invention.
PCT/US2000/004726 1999-02-23 2000-02-22 Methode amelioree de culture de cellule t WO2000050571A1 (fr)

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US9687510B2 (en) 2014-09-04 2017-06-27 The Johns Hopkins University Activation of marrow infiltrating lymphocytes in hypoxic alternating with normoxic conditions

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US9687510B2 (en) 2014-09-04 2017-06-27 The Johns Hopkins University Activation of marrow infiltrating lymphocytes in hypoxic alternating with normoxic conditions
US10172887B2 (en) 2014-09-04 2019-01-08 The Johns Hopkins University Activation of marrow infiltrating lymphocytes in hypoxic alternating with normoxic conditions
US10406178B2 (en) 2014-09-04 2019-09-10 The Johns Hopkins University Activation of marrow infiltrating lymphocytes in hypoxic alternating with normoxic conditions
US11160830B2 (en) 2014-09-04 2021-11-02 The Johns Hopkins University Activation of marrow infiltrating lymphocytes in hypoxic alternating with normoxic conditions
US11406666B2 (en) 2014-09-04 2022-08-09 The Johns Hopkins University Activation of marrow infiltrating lymphocytes in hypoxic alternating with normoxic conditions

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