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• • C—CHEMISTRY; METALLURGY
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  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • C12N5/0693—Tumour cells; Cancer cells
  • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
• • 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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]

Definitions

• the invention relates generally to the modification of cytotoxic T cells by treatment with a selected cytokine, and to the use of such modified cells in cancer therapy.
• the human T cell line TALL-104 (CD3/TCRc + CD8 + CD16 ⁇ ) [A. Cesano et al , In Vitro Cell. Dev. Biol., 28A:648 (1992); A. Cesano et al , J. Immunol. , 151:2943-2957 (1993); and A. Cesano et al , Cancer Immunol. Immunoth.. .40:139 (1995)] is endowed with MHC non-restricted killer activity and has been reported as useful, when lethally irradiated, against a broad range of tumors across several species, while sparing cells from normal tissues.
• unmodified TALL-104 cells are available from the American Type Culture Collection, 10801 University Boulevard Manassas, VA 20110- 2209 under Accession Number CRL 11386 and are described in U.S. Patent No. 5,272,082. These cells may be preferably modified by lethal ⁇ -irradiation and/or by stimulation in the cytokine Interleukin 2 (IL-2) or Interleukin 12 (IL-12) to provide them with an increased cytotoxicity against tumor and virus-infected targets.
• IL-2 cytokine Interleukin 2
• IL-12 Interleukin 12
• one modification step includes in vi tro treatment of the TALL-104 cells with one or both of the two interleukins, recombinant human (rh) IL-2 and rhIL-12.
• rh recombinant human
• IL-2 and IL-12 can induce the cell line's cytotoxic activity.
• these cytokines are used together to modify the cell line, the modified cell line displays additive or increased cytotoxic effects. This results in a significant increase in cytotoxic activity and recycling capability, ultimately leading to 100% elimination of tumor targets at an E:T ratio ⁇ 0.1:1 [Cesano et al , J.
• Another known modification step involves the exposure of the TALL-104 cell l ne to lethal irradiation to confer irreversible loss of growth capability with full retention of cytotoxic activity, both in vi tro and in vi vo .
• This is achieved by subjecting the cell line to ⁇ - ⁇ rrad ⁇ at ⁇ on just prior to its use.
• the cells are irradiated at 4000 rads using a j Cs source.
• irradiation of TALL-104 cells provides a modified cytotoxic cell line that has lost its proliferative ability and, therefore, the possibility of growing in an unrestrained fashion in the recipient organism.
• TALL-104 cells have been used in methods for the treatment of various cancers in humans and animals. See, also, US Patent Nos . 5,683,690; 5,702,702 and 5,820,856, and International patent publication No. WO98/48630, all incorporated herein by reference.
• TALL-103/2 cells Other cytotoxic cells have also been described, such as the TALL-103/2 cells. See, US Patent No. 5,272,082 and A. Cesano et al , J. Immunol, 151:2943-2957 (1993); S. Visonneau et al , Cell Immunol., 165:252-265 (1995); and A. Cesano et al , J. Immunol. , 160: 1106-1115 (1998).
• TALL-103/2 cells, stimulated with IL-2 or IL-12 have been noted to have a limited spectrum of tumor target reactivities and display low levels of killing. These cells do not grow m severe combined lmmuno-deflcient (SCID) mice. Thus, at present, TALL-103/2 cells have not appeared promising for clinical use.
• Interleukm-15 is a relatively novel T cell growth factor that shares some activities and receptor components with IL-2 [US Patent No. 5,747,024; J. G. Gin et al , J. Leuko . Biol., 57 (5) :763-6 (May 1995); L. S. Quinn et al , Endocrinol. , 136 ( 8 ): 3669-72 (Aug. 1995)].
• IL-15 utilizes the ⁇ and ⁇ chains of the IL-2 receptor for signal transduction, but uses a different subunit ( ⁇ ) to bind to the cells.
• the expression pattern of IL-15 ⁇ receptor is distinct from that of IL-2 ⁇ receptor.
• IL-15 has been shown to induce LAK cell functions in vi tro at high doses of about 100 ng/ml by a CD18-dependent, perfo ⁇ n-related mechanism [A. M. Gamero et al , Cancer Res ⁇ , 55.(21) : 4988-94 (Nov. 1995)].
• IL-15 is produced by monocytes and dendritic cells and has been shown to induce cytokine production in human T helper cells, and adhesion receptor redistribution in T lymphocytes. It has been described to stimulate proliferation of y ⁇ T cells and act synergistically with other stimuli in inducing lymphokine production thereby [See, also, W. E. Carson et al , J. Clin. Invest.
• IL-15 has also been described as a vaccine adjuvant [US Patent No. 5,747,024], a therapeutic [US Patent No. 5,660,824], and an inducer of angiogenesis [A. L. Angiolillo et al , Biochem. Biophys . Res. Comm. , 233(1): 231-7 (Apr. 7, 1997)].
• IL-15 has been said to have IL-2-l ⁇ ke stimulating activities on T lymphocytes and NK cells [P. Allavena et al , J. Leuko . Biol . , 61(6):729-35 (June 1997); J. P. DiSanto, Current Biol. , 7(7):R424-6 (July 1, 1997); R. Evans et al . , Cell. Immunol. , 179 (1) : 66-73 (Jul. 10, 1997) ] .
• the invention provides a method of modifying, or reversibly modifying, the phenotype and function of cytotoxic T cells while retaining the cytotoxicity of the cells comprising the steps of:
• step (c) optionally repeating steps (a) and (b) a selected number of times.
• the invention provides a method of modifying a cytotoxic T cells while retaining the cytotoxicity of the cells comprising the steps of:
• step (c) optionally repeating steps (a) and (b) a selected number of times .
• the first and second modified cells from either method above demonstrate a change in at least one characteristic, such as increased proliferation, differentiation, growth, phenotype, adhesion molecule expression, biodistribution, cytokine production profile, level of cytotoxic activity, and tumor target spectrum.
• the cells are TALL-104 cells or TALL-103/2 cells.
• TALL-104 cells are cultured in an effective amount of IL-15, wherein said cells grow at a rate faster than when stimulated by IL- 2, and have an altered phenotypic profile; and then the IL-15 stimulated TALL-104 cells are cultured in an effective amount of IL-2.
• the modification of cell characteristics is accomplished by first culturing TALL-104 cells in an effective amount of IL-2 and then culturing the IL-2 stimulated TALL-104 cells in an effective amount of IL-15.
• the invention provides a method of modifying TALL-104 cells comprising culturing TALL- 104 cells in an effective amount of IL-15, wherein said cells grow at a rate faster than when stimulated by IL-2, and have an altered phenotypic, cytotoxic and cytokine profile.
• the modified cells have an increased level of cytotoxicity or another change in a characteristic such as increased proliferation, differentiation, growth, phenotype, adhesion molecule expression, biodistribution, cytokine production profile, and tumor target spectrum.
• the cytokine profile includes increased expression of IL-10, GM-CSF, TNF-cc and TNF- ⁇ and decreased expression of gamma mterferon (IFN- ⁇ ) by the modified TALL- 104 cells.
• the modified phenotype includes increased expression of the cytotoxic adhesion/activation marker CD56 and/or decreased expression of the adhesion molecule CD38.
• the invention provides a method for increasing the levels of cytotoxic activity and spectrum of tumor target recognition of TALL-103/2 cells comprising culturing TALL-103/2 cells in an effective amount of IL-15, wherein said cells grow at a faster rate and have an expanded tumor target spectrum of cytotoxicity than when stimulated by IL-2.
• the invention provides modified TALL-104 cells, which are produced by stimulating said cells in an effective amount of IL-15.
• the invention provides modified TALL-103/2 cells having an increased cytotoxicity, which are produced by stimulating said cells in an effective amount of
• Fig. 1 is a graph which illustrates that IL-15 supports greater TALL-104 cell proliferation in vi tro .
• the symbol D indicates the 5 ng/ml dosage of IL-15; the symbol 0 indicates the 100 U/ml dosage of IL-2. Growth of cells is measured by a metabolic surrogate marker, lactate (mg/dl), over days in culture.
• Fig. 2 is a bar graph illustrating that upon expansion of TALL-104 cells in vivo in SCID mice and re- adaptation to tissue culture conditions, IL-15 induces quicker differentiation of TALL-104 cells into cytotoxic cells in comparison to the effects of IL-2.
• Undifferentiated TALL-104 cells extracted from SCID mouse spleens, were cultured for one week with either IL-2 or IL-15, and then tested for cytotoxicity against either K562 or Raji tumor cells . Cytotoxicity is demonstrated by percent lysis of the tumor cells.
• the open bar indicates IL-2 treatment; the strippled bar indicates IL-15 treatment.
• TALL-104 cells stimulated in IL-15 also show higher levels of cytotoxic molecules such as perforin, serine esterases (SE) and TIA-1, an apoptosis inducing molecule.
• Fig. 3 is a graph indicating that IL-15 induces higher expression of cytotoxic adhesion/activation marker CD56, both as percent of positive cells in the total TALL-104 population and at the single cell level, as the number of molecules present on each cell (higher density) .
• the symbol ⁇ indicates IL-2; the symbol ⁇ C indicates IL-15. Results are plotted as ⁇ CD56+ cells over time (days) in culture.
• Fig. 4 is a graph demonstrating that undifferentiated TALL-104 cells freshly obtained from the SCID mouse have a higher expression of CD2 and that with time in culture CD2 surface levels decline. However, this decline is slower in TALL-104 cells grown in IL-15 than in IL-2.
• the symbol ⁇ indicates IL-2; the symbol 0 indicates IL-15. Results are plotted as % CD2+ cells over time (days) in culture .
• Fig. 5 is a graph demonstrating that TALL-104 cells grown in IL-15 have lower expression of the adhesion molecule CD38.
• the symbol ⁇ indicates IL-2; the symbol 0 indicates IL-15.
• Results are plotted as % CD38+ cells over time (days) in culture.
• FIG. 6 is a bar graph illustrating the comparative effects of IL-15 and IL-2 on the induction of GM-CSF from TALL-104 cells.
• On the X axis are the stimuli used to trigger GM-CSF production.
• the graph shows that TALL-104 cells grown in IL-15 have a baseline level of GM-CSF production, and respond to OKT3 monoclonal antibody with higher production of GM-CSF than cells grown in IL-2.
• Fig. 7 is a bar graph illustrating the comparative effects of IL-15 and IL-2 on the induction of IL-10 from TALL-104 cells.
• On the X axis are the stimuli used to trigger IL-10 production.
• the graph shows that TALL-104 cells grown in IL-15 have a baseline level of IL-10 production, and respond to OKT3 monoclonal antibody with higher production of IL-10 than cells grown in IL-2.
• Fig. 8 is a bar graph illustrating the comparative effects of IL-15 and IL-2 on the induction of TNF- from TALL-104 cells.
• On the X axis are the stimuli used to trigger TNF- ⁇ production.
• the graph shows that TALL-104 cells grown in IL-15 have a baseline level of TNF- ⁇ production, and respond to OKT3 monoclonal antibody with higher production of TNF- ⁇ than cells grown in IL-2.
• Fig. 9 is a bar graph illustrating the comparative effects of IL-15 and IL-2 on the induction of TNF- ⁇ from TALL-104 cells.
• On the X axis are the stimuli used to trigger TNF- ⁇ production.
• the graph shows that TALL-104 cells grown in IL-15 have a baseline level of TNF- ⁇ production, and respond to OKT3 monoclonal antibody with higher production of TNF- ⁇ than cells grown m IL-2.
• Fig. 10 is a bar graph illustrating the comparative effects of IL-15 and IL-2 on the induction of IFN- ⁇ from TALL-104 cells.
• On the X axis are the stimuli used to trigger IFN- ⁇ production.
• the graph shows that TALL- 104 cells grown in IL-15 have a baseline level of IFN- ⁇ production, and respond to OKT3 monoclonal antibody with lower production of IFN- ⁇ than cells grown in IL-2.
• Fig. 11 is a graph which illustrates that IL-15 supports the growth of TALL-103/2 cells in culture.
• the symbol ⁇ indicates the dosage of IL-2 in U/ml; the symbol ⁇ indicates the dosage of IL-15 in ng/ml. Growth of cells is indicated by cpm in 3 H-TdR proliferation assays.
• the X axis shows the concentration of the cytokmes.
• Fig. 12 is a bar graph demonstrating that IL-15 (strippled bars) supports the cytotoxic phenotype of TALL- 103/2 cells and broadens the spectrum of target recognition by these cells.
• the cells were cultured for a week in either IL-15 or IL-2 (clear bars), and then exposed to K562 tumor cells (sensitive target) and H160 or Daudi tumor cells (resistant targets) . Cytotoxicity is measured by % lysis of the target cells in l Cr release assays.
• Fig. 13 is a bar graph which illustrates the comparative effect of IL-2 and IL-15 on the induction of IFN ⁇ in TALL-103/2 cells. The X axis shows the cytokine dosage.
• Fig. 14 is a bar graph which illustrates the comparative effect of IL-2 and IL-15 on the induction of TNF- ⁇ in TALL-103/2 cells.
• the X axis shows cytokine dosage.
• IL-15 induces higher levels of TNF- ⁇ at the concentration of 10 ⁇ g/ml.
• Fig. 15 is a bar graph which illustrates the comparative effect of IL-2 and IL-15 on the induction of TNF- ⁇ in TALL-103/2 cells. Similar levels of TNF- ⁇ are induced by the two cytokines.
• Fig. 16 is a bar graph which illustrates the comparative effect of IL-2 and IL-15 on the induction of IL- 10 in TALL-103/2 cells.
• IL-15 is a better inducer of IL-10 as compared to IL-2.
• Cytotoxic T cell lines such as TALL-104
• TALL-104 have found use in clinical settings, such as the treatment of cancers, when administered m vi vo, or when employed in ex vi vo therapeutic regimens.
• Still other cytotoxic T cell lines, such as TALL-103/2 could be clinically useful if their target specificity was broadened and their growth in culture improved.
• the inventors have now discovered novel methods for increasing the cytotoxicity of these cells, altering their phenotypes and spectrum of target recognition, and, increasing their yield in culture. Such modifications can be introduced to TALL-104 or TALL-103/2 cells, and are anticipated to be introduced to other cytotoxic T cells by stimulating the cells in IL-15, rather than, or in addition to, IL-2.
• Such IL-15 stimulation may optionally be followed by exposing the stimulated TALL- 104 cell line to lethal irradiation to confer irreversible loss of growth capability with retention of cytotoxic activity, both m vi tro and m vi vo .
• lethal irradiation to confer irreversible loss of growth capability with retention of cytotoxic activity, both m vi tro and m vi vo .
• This may be achieved by subjecting the cell line to ⁇ -irradiation just prior to its use.
• the cells are irradiated at 4000 rads using a n, Cs source, similar to the process described in International Patent Publication No. W094/26284.
• Such irradiation of the IL-15 stimulated TALL-104 cells provides a modified cytotoxic cell line that has lost its proliferative ability and, therefore, the possibility of growing in an unrestrained fashion in the recipient organism.
• TALL-104 cells are prepared as follows.
• TALL-104 cells (ATCC CRL 11386) are exponentially grown in tissue culture in the presence of recombinant human (rh) IL-15.
• the resulting proliferation of the cytokine stimulated TALL-104 cells (as measured by 3 H-TdR uptake) is higher at plateau doses of IL-15 than at plateau doses of IL-2.
• a "plateau dose” is the dose at which maximal activity is reached, e.g., the optimal dose (see Fig. 1).
• These cells also demonstrate increased ability to adhere to plastic m vi tro (and potentially to endothelium m vi vo) by increase in expression of adhesion molecules.
• modified TALL-104 cells also demonstrate increased cytotoxic function, as shown by higher levels of killing, increased spectrum of tumor target recognition, and a quicker and more effective kinetic of induction of lytic proteins, such as PFP, SE1 and SE2, and TIA1.
• Cells grown in an optimal dose of IL-15 generally show higher levels of cytotoxic activity, as compared to the same cells m an optimal dose of IL-2 (see Fig. 2, which demonstrates a significant increase in cytotoxicity against NK-sensitive K562 cells and NK-resistant Raji cells, as compared to the same TALL-104 cells stimulated in IL-2) .
• the cells also demonstrate an increased expression of the cytotoxic adhesion marker CD56 (Fig. 3).
• TALL-104 cells grown in IL-15 have higher baseline levels of cytokmes and respond to stimuli, such as antibodies and target cells, producing higher levels of cytokmes than TALL-104 grown in IL-2, with the exception of gamma interferon (IFN- ⁇ ), which is produced in higher levels by stimulation of TALL-104 cells in IL-2.
• IFN- ⁇ gamma interferon
• TALL-104 cells may be grown in IL-15 simply to increase the yield thereof, and then grown in IL-2 to reproduce the IL-2 cytotoxic phenotype previously used in clinical therapies for cancer.
• one may grow TALL-104 cells in IL-15 and use the IL-15 phenotype where enhanced adhesion to endothelium is desired in some clinical applications .
• the inventors have determined that one may reversibly switch the IL-15 and IL-2 phenotypes of TALL-104 by sequential growth of the cells in one and then the other of these two cytokmes, as desired.
• the biodistribution of the TALL-104 cells may also be affected differently by the two cytokmes, based on the different levels of expression of adhesion molecules.
• TALL- 103/2 cells may also be modified by stimulation in IL-15.
• the TALL-103/2 cells will grow more rapidly in culture (Fig. 11) when stimulated with IL-15.
• Fig. 12 shows the results of stimulation of TALL 103/2 cells with IL- 15 vs. IL-2.
• the IL-15 TALL 103/2 cells are able to recognize and kill HL60 and Daudi cells, against which the IL-2 stimulated TALL 103/2 cells were not cytotoxic.
• IL-15 alters the cytokine production by the cells. See, for example, Figs. 12-16, which showing cytokmes that are likely to be involved in the anti-tumor activity of the killer cells. Thus, changing the cytokine profile can result in clinical changes, both in terms of efficacy and/or toxicity.
• IL-15 may be employed in a method for reversibly altering the phenotype of cytotoxic T cells by culturing said cells in IL-15, thereby obtaining a high yield of a cell having a first phenotype; followed by culturing these cells in IL-2, thereby altering the first phenotype to a second phenotype.
• the second phenotype may be returned to the first phenotype by further culturing in IL-15 again, if desired.
• the IL-15 phenotypes are characterized by enhanced growth kinetics, increased cytotoxicity, enhanced cytokine production, and, likely, increased adhesion to vasculature.
• IL-15 stimulated cytotoxic T cells may then be employed in methods for in vi vo and ex vi vo therapy of cancer, and for other uses for which TALL-104 cells are known, as described in the US patents incorporated by reference above.
• modified cells may also be employed as research reagents, as reagents for screening the effect of proposed developmental drugs on their cytotoxic activity, as reagents for the study of their expression of adhesion molecules or cell surface markers, as well as for the production of cytokmes or other biological molecules expressed by the modified cells.
• Iscove's modified Dulbecco ' s medium Gibco-BRL, Grand Island, NY
• 10% heat-mactivated fetal bovine serum Sigma
• 100 U/ml rhIL-2 Choiron Therapeutics, Emeryville, CA
• rh IL-15 1-5 ⁇ g/ml
• the cells were then examined for differences in characteristics such as growth, phenotype, cytokine profile, and cytotoxicity, biodist ⁇ bution and tumor target spectrum of the cultures, and the results were reported in Figs. 1-10.
• TALL-104 cells grown in the plateau dose (5 ng/ml) of IL-15 proliferate m vi tro faster than do the same cells grown in the plateau dose (100 U/ml) of IL-2.
• the IL-2- or IL-15- treated TALL-104 cells were expanded in vi vo in SCID mice and re-adapted to tissue culture conditions. IL-15 induced quicker differentiation of TALL-104 cells into cytotoxic cells in comparison to the effects of IL-2.
• Undifferentiated TALL-104 cells extracted from SCID mouse spleens, were cultured for one week with either IL-2 or IL-15, and then tested for cytotoxicity against either K562 or Raji tumor cells.
• the IL-2-treated TALL-104 cells were only marginally ( ⁇ 10%) cytotoxic, as demonstrated by percent lysis of the tumor cells, for K562 cells.
• the TALL-104 cells stimulated with IL-15 lysed about 40% more K562 tumor cells.
• the IL-2-treated cells lysed no Raji cells, whereas the IL-15 treated TALL-104 cells lysed almost 60% of these cells. See Fig. 2.
• the IL-15 treated cells also showed higher levels of cytotoxic molecules, such as perforin, serine esterases (SE) and TIA-1, an apoptosis inducing molecule.
• Phenotype TALL-104 cells treated with IL-15 express higher levels of the cytotoxic/adhesion/activation marker CD56, both as percent of positive cells in the total TALL-104 population and at the single cell level, as the number of molecules present on each cell (higher density) , than do TALL-104 cells treated with IL-2. See Fig. 3.
• Undifferentiated TALL-104 cells freshly obtained from the SCID mice have a high expression of CD2. With time in culture, the CD2 surface levels decline. However, this decline when compared for TALL-104 cells grown in IL-2 or IL-15 as described above, was demonstrated to be slower in the IL-15 stimulated cells. See Fig. 4.
• TALL-104 cells grown in IL-15 were also shown to have lower expression of the adhesion molecule CD38 than TALL-104 cells grown in IL-2. See Fig. 5.
• D Cytokine Profi l e
• TALL-104 cells grown in either IL-2 or IL-15 as above, were stimulated to trigger cytokine production with OKT3 (anti-CD3), Moon-1 (anti-CD31), or IB4 (anti-CD38) monoclonal antibodies or by exposure to K562 cells .
• TALL-104 cells grown in IL-15 have a baseline level of GM-CSF production, and respond to OKT3 monoclonal antibody with a significantly higher production of GM-CSF than cells grown in IL-2.
• TALL-104 cells grown in IL-15 have a baseline level of IL-10 production, and respond to OKT3 monoclonal antibody with significantly higher production of IL-10 than cells grown in IL-2.
• TALL-104 cells grown in IL-15 have a baseline level of IL-10 production, and respond to OKT3 monoclonal antibody with significantly higher production of IL-10 than cells grown in IL-2.
• TALL-104 cells grown in IL-15 have a baseline level of IL-10 production, and respond to OKT3 monoclonal antibody with significantly higher production of IL-10 than cells grown in IL-2.
• IL-15 have a baseline level of TNF- ⁇ production, and respond to OKT3 monoclonal antibody with higher production of TNF- ⁇ than cells grown in IL-2.
• TALL-104 cells grown in IL-15 have a baseline level of TNF- ⁇ production, and respond to OKT3 monoclonal antibody with higher production of TNF- ⁇ than cells grown in IL-2.
• TALL-104 cells grown in IL-15 have a baseline level of IFN- ⁇ production, and respond to OKT3 monoclonal antibody with lower production of IFN- ⁇ than cells grown in IL-2.
• TALL-103/2 cells were grown in endotoxin-free Iscove's modified Dulbecco 's medium (Gibco-BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Sigma) and 100 U/ml rhIL-2 (Chiron Therapeutics, Emeryville, CA) or rh IL-15 (1-5 ⁇ g/ml) [R&D Systems] in a humidified incubator at 37°C with 10% C0 2 in tissue culture for two weeks. The cells were then examined for differences in characteristics such as growth, phenotype, cytokine profile, and cytotoxicity, biodistribution and tumor target spectrum of the cultures, and the results were reported in Figs. 1-10. A . Phenotype
• the surface phenotype of the cultured cells were compared to determine the effect of the two cytokmes.
• the results are reported below in Table I as % positive cells; the mean fluorescence intensity (on a scale with the upper limit of 200) is in parentheses and provides an indication of the antigen density, i.e., the number of molecules/cell .
• TALL-103/2 cells grown in IL-15 showed greater proliferation than the cells grown in IL-2 at stimulating cytokine doses greater than 1 ng/ml IL-15. See Fig. 11. C. Target Spectrum
• TALL-103/2 cells were cultured for a week in either IL-15 or IL-2, and then exposed to K562 tumor cells, HL60 tumor cells or Daudi tumor cells. Cytotoxicity was measured by Z lysis of the target cells in 51 Cr release assays. As demonstrated in Fig. 12, the IL-15 treated cells caused lysis of all three tumor cell types. The IL-2 treated cells were cytotoxic only for the K562 cells. Thus, the method of this invention supported the cytotoxic phenotype of TALL-103/2 cells and broadened the spectrum of target recognition by these cells.
• the IL-2-treated and IL-15-treated TALL-103/2 cells were also evaluated for dose-dependent cytokine production. As revealed by Figs. 13-16, the IL-15 induces production of cytokmes from the cells, which is different from that produced by stimulating the cells with IL-2.
• IL-2 stimulation induces better expression of IFN ⁇ in TALL-103/2 cells, than does IL-15 stimulation (Fig. 13) .
• IL-15 induces higher levels of TNF- ⁇ at the concentration of 10 ⁇ g/ml (Fig. 14), than does IL-2 stimulation.
• IL-15-st ⁇ mulated TALL-103/2 cells produce greater amounts of IL-10 at concentrations over 10 ng/ml IL- 15 (Fig. 16) .

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