WO2014130823A1 - Renforcement par les cellules dendritiques de la protection contre le cancer conférée par les cellules mononucléées stimulées par muc1 humaines - Google Patents

Renforcement par les cellules dendritiques de la protection contre le cancer conférée par les cellules mononucléées stimulées par muc1 humaines Download PDF

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WO2014130823A1
WO2014130823A1 PCT/US2014/017704 US2014017704W WO2014130823A1 WO 2014130823 A1 WO2014130823 A1 WO 2014130823A1 US 2014017704 W US2014017704 W US 2014017704W WO 2014130823 A1 WO2014130823 A1 WO 2014130823A1
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cells
dendritic cells
cancer antigen
cytotoxic
matured
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Stephen E. Wright
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Texas Tech University System
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Definitions

  • the present invention relates in general to the field of medical diagnosis and medical treatment, and more particularly, to dendritic cell enhancement of protection by human antigen stimulated mononuclear cells against cancer.
  • Immunotherapy is one such treatment in which the immune system is stimulated actively, by vaccination, or adoptively, by transfer of components of the immune system, e.g., antibodies or immune cells.
  • the immune cells which may be stimulated in culture, are generated from PBMC.
  • Cells of the immune system such as CTL, have been identified that recognize antigens, which are unique to the cancer cells, and kill them in preference to normal cells.
  • Another cancer vaccine is taught in United States Patent Application No. 20090317414 filed by Pietersz, et al., and is direct to a Cancer Vaccine Comprising A Mucin 1 (Mucl) T Cell Epitope- Derived Peptide. Briefly, the application is said to teach a cancer vaccine, and a composition for the ex vivo priming of dendritic cells, which comprises a MUC1 T cell epitope-derived peptide or peptide analogue capable of provoking a cytotoxic T cell immune response. Particular MUC 1 T cell epitope-derived peptides are disclosed.
  • DC Dendritic cells
  • BM mouse bone marrow
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • immature GM lc DC were resistant to maturation by lipopolysaccharide, TNF-a or anti-CD40 monoclonal antibodies, as the expression of co-stimulatory molecules was not increased, and stimulatory activity in oxidative mitogenesis was not enhanced.
  • IFN-gamma was found to diminish priming for IL-4 production when limiting amounts of IL-4 (100 U/ml) were used in the initial culture.
  • the article argues that the dominant effect of IL-4 in determining the lymphokine-producing phenotype of primed cells was observed with dendritic cells (DC), activated B cells, and I-Ek-transfected fibroblasts as APC.
  • DC dendritic cells
  • activated B cells activated B cells
  • I-Ek-transfected fibroblasts as APC.
  • the different APC did vary in their potency, with DC being superior to activated B cells, and that were superior to transfected fibroblasts.
  • the present invention includes a method of improving a cytotoxic T cell immune response to a cancer antigen comprising: obtaining peripheral blood mononuclear cells from a subject; incubating adherent peripheral blood mononuclear cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the adherent peripheral blood mononuclear cells into dendritic cells; and isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo.
  • the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7.
  • the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation.
  • the cancer antigen is a MUC-1 peptide.
  • the cancer antigen is selected from at least one of MelanA (MART -I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE- family members, RAGE-family members, pl5(58), CEA, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl85erbB2, pl
  • a media for incubating the dendritic cells is serum free.
  • the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4.
  • the method further comprises the step of contacting the matured dendritic cells with peripheral blood mononuclear cells that comprise immature T cells in the presence of the cancer antigen under conditions in which the immature T cells are converted into cancer antigen-specific active cytotoxic T cells.
  • Yet another embodiment of the present invention includes a method of improving a cytotoxic T cell immune response to a cancer antigen comprising: obtaining dendritic cells from a subject; incubating the dendritic cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the dendritic cells; isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo, and contacting the matured dendritic cells with peripheral blood mononuclear cells that comprise immature T cells in the presence of the cancer antigen under conditions in which the immature T cells are converted into cancer antigen-specific active cytotoxic T cells.
  • the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7.
  • the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation.
  • the cancer antigen is a MUC-1 peptide.
  • the cancer antigen is selected from at least one of MelanA (MART -I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE- family members, RAGE-family members, pl5(58), CEA, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum
  • a media for incubating the dendritic cells is serum free.
  • the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4.
  • Yet another embodiment of the present invention includes a composition for activating a cytotoxic T cell response comprising: isolated adherent peripheral blood mononuclear cells from a subject incubated with a cancer antigen and GM-CSF matured in the absence of IL-4, wherein the matured adherent peripheral blood mononuclear cells stimulate cytotoxic T cells specific for the cancer antigen in vivo.
  • the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7.
  • the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation.
  • the cancer antigen is a MUC-1 peptide.
  • the cancer antigen is selected from at least one of MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE- family members, BAGE-family members, GAGE-family members, RAGE-family members, pl5(58), CEA, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1
  • a media for incubating the dendritic cells is serum free.
  • the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4.
  • Yet another embodiment of the present invention is an isolated dendritic cell made by the method of obtaining dendritic cells from a subject; incubating the dendritic cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the dendritic cells; and isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo.
  • Figures 1A-C Influence of IL-4 on the generation of DC for stimulation of PBMC to produce CTL.
  • Dendritic cells DC were generated and maturated with or without IL-4. Percentage of specific lysis was performed on day 8 by XTT assay (MCF-7 (1A)) and alamarBlue® assays (K562 (IB), RAJI (1Q). EffectonTarget (E:T) ratios are 1.25, 2.5, 5 and 10: 1. Bars are standard error.
  • FIG. 2 Tumor specific target, MCF-7, and nonspecific, NK target, K562, and LAK target, RAJI, specific lysis by CTL used for protection study.
  • MUCl loaded-DC (-) IL-4 were added to PBMC at 1 : 100 ratio on days 0 and 7. Percentage of specific lysis was performed on day 8 by XTT assay (MCF-7) and alamarBlue® assays (K562, RAJI). Lysis of the cell lines was determined at 10: 1 effectontarget (E:T) ratio. Bars are standard error. Standard error bars are hidden in some of the symbols. The results are from two independent studies.
  • FIG. 3 Cytokine production by CTL used for protection study.
  • Figure 4 Enhancement of MUCl stimulated CTL protection against MCF-7 tumor development by antigen presenting cells.
  • Groups and treatment Group 1, None; Group 2, MC + Unloaded DC on days 0,2,4,9, 14, 19; Group 3, MC + MlLDC(-)IL-4 on days 0,2,4,9, 14, 19; Group 4, CTL; Group 5, CTL + M1LPBMC on days 0, 14; Group 6, CTL + M1LPBMC on days 0,2,4,9, 14, 19; Group 7, CTL + MlLDC(-)IL-4 on days 0,14; Group 8, CTL + MlLDC(-)IL-4 on days 0,2,4,9, 14,19.
  • MC mononuclear cells, peripheral blood
  • CTL cytotoxic T- lymphocytes
  • DC Densiclear Cells
  • MlLDC(-)IL-4 mimucin 1 -loaded DC generated without IL- 4
  • M1LPBMC mimucin 1 -loaded peripheral blood mononuclear cells
  • 5x 106 MCF-7 cells/mouse subcutaneously other cell types, 5x 107 MC or CTL, with or without 5x 105 unloaded DC, MUCl loaded-DC (-) IL-4 or MUCl loaded- PBMC, were injected intraperitoneally on the days indicated. Mice were observed up to thirty- one days after the MCF-7 injection for tumor development.
  • DC Dendritic cells
  • APC antigen-presenting cells
  • PBMC peripheral blood mononuclear cells
  • CTL cytotoxic T-lymphocytes
  • Mucin 1 (MUCl), a glycoprotein, found on the cell surface of adenocarcinomas, was used to load DC.
  • MUCl loaded-DC generated without IL-4 (MUCl loaded-DC (-) IL-4) were superior to DC produced with IL-4 (MUCl loaded-DC (+) IL-4) in stimulating PBMC to kill MCF-7 in vitro.
  • a corollary in vivo protection study was performed by injecting non-obese, diabetic - severe, combined immuno-deficient (NOD-SCID) mice subcutaneously with the human breast cancer cell line MCF-7.
  • mice were injected intraperitoneally (IP) with CTL and/or PBMC, which had been loaded with MUCl, as well as MUCl loaded-DC (-) IL-4 on different schedules.
  • Current methods of treating breast cancer such as chemotherapy and radiotherapy cause many unwanted side effects, including secondary cancers and senescence of normal cells because they are not specific to cancer cells.
  • Immunotherapy is one such treatment in which the immune system is stimulated actively, by vaccination, or adoptively, by transfer of components of the immune system, e.g., antibodies or immune cells.
  • the immune cells which may be stimulated in culture, are generated from PBMC.
  • Cells of the immune system such as CTL, have been identified that recognize antigens, which are unique to the cancer cells, and kill them in preference to normal cells.
  • TAA tumor-associated antigens
  • cancer antigens and “tumor-associated antigens” refer to proteins and/or peptides processed from proteins and presented by antigen presenting cells to T cells that include, but are not limited to Muc-1, MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE-family members, RAGE- family members, pi 5(58), CEA, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER- 2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl85erbB2, pl80erbB-3, c- met, n
  • Mucins are polymorphic, O-linked glycosylated proteins expressed on the surface of ductal epithelial cells.
  • MUC1 is characterized by a 20 amino acid sequence that is tandem repeated 25- 100 times in each molecule.
  • Adenocarcinomas are cancers of secretory organs and are typically deficient in glycosyltransferases that glycosylate mucin. As a result, adenocarcinoma cells should produce altered protein trafficking that would increase antigen processing. 7 This would cause MUCl of adenocarcinomas to be recognized as a novel antigen and serve as a target for CTL.
  • T-cells have been primed with DC in vivo, 1 with virus, 14 antigens in liposomes 15 or DNA 16 in SCID mice. Addition of peptides during the maturation of DC results in an effective loading of these peptides into MHC I complexes. 17 DC loaded with TAA, e.g., MUCl, can elicit specific tumor-reactive T-cells, e.g., breast cancer specific CTL. In addition, prolonged in vivo antigen stimulation enhances killing of tumor by CTL. 18 This may be explained by the observation that DC restimulation maintains protective memory CTL in a viral mouse model. 19
  • the present inventors have previously used MUCl to stimulate PBMC that showed specific killing of human breast cancer cells in vitro 3 and in vivo? 0
  • the current studies were performed to determine the effects of human DC on CTL specific lysis of human breast cancer cells.
  • the present inventors compared DC generated with or without IL-4.
  • IL-4 is incorporated to reduce the production of macrophages. 21 ' 22
  • the rationale for not using IL-4 is that this cytokine favors the development of type 2 immune responses, at the expense of the development of type 1 immune responses, 23"25 which is the desired cellular immune response.
  • CCL3/MIP-1 and CCL4/MIP-1 are not induced in human primary monocytes when granulocyte-macrophage colony-stimulating factor (GM-CSF) is combined with IL-4. 26 These chemokines are required for DC to polarize Type 1 T cells. 27 In addition, DC generated with GM-CSF alone have been shown to function to stimulate CTL. 28 The optimum method for generating DC was then used to generate the CTL for the in vivo study. The ability of MUCl loaded-DC to enhance CTL killing of human tumor cells was evaluated in a NOD-SCID mouse model.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • MUC1 peptide MUCl-mtri peptide G APPAHGV APDNRPAP (SEQ ID NO.: l) (S2.12- N; T3, l 1, 16-N) 5 was custom synthesized by American Peptide Co., Inc..
  • DC Dendritic Cells
  • the non-adherent fractions were centrifuged and placed into a second set of 225 cm 2 tissue culture flasks for adherence. After 1-hour incubation, the non-adherent fraction in the second set of flasks was removed and 75 mL of fresh AIM-V ® serum-free lymphocyte medium was added. Both sets of flasks were incubated at 37°C and 5% CO 2 . MUCl peptide ( ⁇ g/mL final concentration) was added on day 0 of culture. Human recombinant GM-CSF (500 IU/mL final concentration) (Biosource International Inc., catalog number PHC2015) was added on days 0, 3, and 7.
  • the CTL were generated from PBMC and grown in a gas-permeable hydrophobic bag for eight days.
  • PBMC were cultured at 2* 10 6 cells/mL in AIM- V ® serum-free lymphocyte medium and maintained in a 37°C humidified and 5% CO 2 atmosphere.
  • IL-2 (Cetus, GenWayBio, Inc., catalog number GWB-76974E) was added twice per week at 100 IU/mL.
  • PBMC were stimulated with MUCl (1 ⁇ g/mL final concentration) 42 or MUCl loaded-DC(-)IL-4 (1 : 100 ratio) on days 0 and 7 of culture.
  • PBMC stimulated under these optimized conditions 41 are primarily CD+4 T-lymphocytes with the remainder being CD+8 T-lymphocytes.
  • 3 PBMC from both subjects were stimulated separately and used individually in the mouse studies.
  • MUC1 loading of PBMC (MUC1 loaded-PBMC).
  • PBMC were loaded with MUC1 peptide (1 ⁇ g/mL final concentration) and maintained in a flask at 37°C humidified and 5% CO 2 atmosphere for two hours.
  • Cytotoxicity assays In order to evaluate the ability of CTL to lyse specific, as well as nonspecific target cell lines in vitro, two cytotoxicity assays were used during this study: XTT assay and alamarBlue ® assay. From our previously unpublished work, it was shown that the XTT assay worked better with solid tumor adherent cell lines and the alamarBlue ® assay worked well with the hematopoietic suspension cell lines.
  • MCF-7 (ATCC HTB-22) breast cancer, K562 (ATCC CCL-243) erythroleukemia and RAJI (ATCC CCL-86) B-cell lymphoma cell lines were obtained from, and cultured as recommended, by the American Type Culture Collection (Manassas, VA, USA). MCF-7 expresses hypoglycosylated mucin, 8 and was used as the target cell line in a XTT assay (Roche Diagnostics Corp., catalog number 1 1465015001). MCF-7 was cultured in Dulbecco's Modified Eagle's Medium (Gibco-BRL, Life Technologies, Inc.
  • K562 a natural killer/lymphokine-activated killer sensitive target 43 cell line, and RAJI, a natural killer-relatively resistant/lymphokine-activated killer- sensitive target 44 cell line were used as target cell lines in an alamarBlue ® assay (Biosource International Inc., catalog number NC9423932).
  • K562 and RAJI were cultured in RPMI-1640 (Gibco-BRL, Life Technologies, Inc. catalog number 11875093) supplemented with 10% fetal bovine serum and 1% L-glutamine. All the cells were maintained in a 37°C humidified and 5% CO 2 atmosphere.
  • the cell lines were seeded into separate 96-well tissue culture plates. 5> ⁇ 10 3 target cells (MCF-7, K562, or RAJI) were added to each well except the minimum wells, to which no cells were added. Washed effector CTL were added to each well in three effector cell to target cell (E:T) ratios: 10: 1, 5: 1, and 2.5: 1. The effector cells alone were seeded at the corresponding numbers per well as background. Six maximum wells were set up with the target cells only. The CTL from each subject was analyzed separately, in triplicate wells for each ratio. XTT assay. The XTT assay was used to evaluate the ability of CTL to kill the specific target cells, MCF-7, per the manufacturer's instructions.
  • the XTT assay is a non-radioactive, colorimetric assay using XTT labeling reagent, sodium 3'-[l-(phenylamino-carbonyl)-3,4- tetrazolium] -bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate, 45 which is cleaved into formazan dye only by metabolically active cells. It is used for the determination of cellular proliferation, viability and activation in response to growth factors, cytokines, nutrients, and cytotoxicity.
  • the plate was set up as previously described above the day before the XTT assay was run. Fifty ⁇ L of XTT labeling mixture (5mL XTT labeling reagent with 100 ⁇ ⁇ electron coupling reagent) were added to all wells of the plate. The plate was incubated in a 37°C humidified and 5% CO 2 atmosphere for four hours. The optical density (OD) was then measured by using a spectrophotometer (Dynatech MR 5000, Dynatech Laboratories Inc.). The plate was read at a wavelength of 450 nm with a reference wavelength of 630 nm, and background absorbance was subtracted.
  • XTT labeling mixture 5mL XTT labeling reagent with 100 ⁇ ⁇ electron coupling reagent
  • the maximum XTT was determined as the mean of the six wells containing only target cells and the minimum was determined as the mean of the six wells containing only medium.
  • the nonspecific formation of formazan attributable to the presence of effector cell was determined from the wells containing effector cells alone.
  • the percent specific lysis (%SL) was calculated as follows: 46
  • Alamar Blue ® assay 47 was used to evaluate the ability of CTL to kill the non-specific targets cells K562 and RAJI per the manufacturer's instructions.
  • the internal environment of a proliferating cell is more reduced than that of non-proliferating cell 48
  • AlamarBlue ® which can be reduced by the metabolic intermediates, is useful in monitoring cell proliferation because their reduction is accompanied by a measurable shift in color.
  • the alamarBlue ® reacts with the cells, it accepts electrons and changes in color from the oxidized indigo blue, non-fluorescing state to the reduced fluorescing pink state.
  • the alamarBlue ® reduction was evaluated by measuring absorbance spectrophotometrically and the calculation of the percentage of alamarBlue ® reduction is as follows according to the manufacturer's protocol:
  • ⁇ ⁇ ⁇ and ⁇ ⁇ ⁇ 2 represent the molar extinction coefficient of alamarBlue at 570 and 630 nm, respectively, in the oxidized forms.
  • ⁇ and ⁇ 2 represent absorbance of test wells at 570 and 630 nm, respectively.
  • a and ⁇ ° ⁇ 2 represent absorbance of untreated positive growth control wells at 570 and 630 nm, respectively.
  • the values of percentage difference in alamarBlue ® reduction were corrected for background values of untreated positive growth controls.
  • the plates were set up as previously described above the same day as the alamarBlue ® assay was run. Twenty ⁇ ⁇ of alamarBlue ® was added to all wells. After overnight incubation in a 37°C humidified and 5% C0 2 atmosphere, the OD was read with a spectrophotometer at wavelengths of 570 and 630 nm.
  • ELISA Cytokine Assay The amount of cytokine, tumor necrosis factor-alpha (BD Pharmingen Inc., catalog number 550610), interferon-gamma (BD Pharmingen Inc., catalog number 550612), IL-10 (BD Pharmingen Inc., catalog number 550613), and GM-CSF (Biosource International Inc., catalog number 555126), present in the supernatant samples was determined by the use of enzyme linked-immunosorbent assay kits per manufacturer's instructions. The supernatant from each subject's CTL was analyzed separately, in triplicate.
  • mice Female NOD-SCID mice (Jackson Laboratory, Bar Harbor, ME, USA), 6-12 weeks of age were injected subcutaneous ly, in the back of the neck, with O. lmL of phosphate-buffered saline: matrigel (Gibco BRL, Life Technologies, Inc. catalog number A1413202) at 1 : 1 ratio containing 5> ⁇ 10 6 MCF-7. Washed 5x l0 7 mononuclear cells (peripheral blood) (MC) or CTL were injected IP with or without washed 5x l0 5 unloaded DC, MUC1 loaded-DC (-) IL-4 or MUC1 loaded-PBMC according to schedules in the figure legend for Figure 4.
  • MC peripheral blood
  • Control animals received phosphate-buffered saline or the above-mentioned cells individually. Each mouse was checked for tumor development three times per week for one month. Animal care was in accordance with institutional guidelines. At all times, the inventors followed the Guidelines for Ethical Conduct in the Care and Use of Animals (www.apa.org/science/anguide.html) by the APA Board of Scientific Affairs Committee on Animal Research and Ethics.
  • IL-4-stimulated PBMC There was increased type 1 cytokines production from MUCl loaded-DC (-) IL-4-stimulated PBMC used for the in vivo protection studies.
  • Tumor necrosis factor-alpha went from 0 pg/mL on day 0 to 19.7 on day 3, and 146.9 on day 8;
  • Interferon-gamma went from 8.4 pg/mL on day 0 to 117 on day 3, and 406 on day 8;
  • GM-CSF went from 0 pg/mL on day 0 to 107.8 on day 3, and 159.4 on day 8.
  • IL-10 which may be a type 1 or type 2 cytokine and is produced by inducible regulatory T lymphocytes (Figure 3).
  • Groups 5 - 8 received CTL on day 0 plus APC on two different schedules.
  • MUCl loaded-PBMC were injected IP on days 0 and 14 (group 5) or days 0, 2, 4, 9, 14, and 19 (group 6) as a control for DC.
  • MUCl loaded-DC (-) IL-4 were injected IP on days 0 and 14 (group 7) or days 0, 2, 4, 9, 14, and 19 (group 8) to determine the optimum schedule of DC administration.
  • PBMC may be stimulated in vivo by MUCl loaded- DC (-) IL-4 to kill tumor cells.
  • Group 4 which received CTL only on day 0, exhibited 50% protection (5 mice without tumors out of 10).
  • Group 5 which received CTL on day 0 and MUCl loaded-PBMC on days 0 and 14, exhibited 50% protection (5 mice without tumors out of 10).
  • Group 6 which received CTL on day 0 and MUCl loaded- PBMC on days 0, 2, 4, 9, 14 and 19, exhibited 56% protection (5 mice without tumors out of 9).
  • MUC1 loaded-DC (-) IL-4 on days 0, 14 (group 7) or days 0, 2, 4, 9, 14 and 19 (group 8) enhanced protection compared to the other groups. There was no significant difference between groups receiving CTL plus MUC1 loaded-DC(-)IL-4 on days 0, 14 (group 7) or days 0, 2, 4, 9, 14 and 19 (group 8) statistically, but the group that received six days of stimulation showed 100% tumor protection. This shows that multiple and/or extended stimulation of CTL with MUC1 loaded-DC (-) IL-4 is optimum.
  • MCF-7 cell-injected mice were protected from tumor growth when MUC1 loaded- DC(-)IL-4 were injected along with CTL, implies that MUC1 loaded-DC(-)IL-4 enhanced the in vivo killing of tumor cells and are required, in addition to CTL, to obtain 100% protection from tumor growth.
  • a similar in vivo study with mouse cells showed DC enhanced CTL killing of tumor. 32
  • the present study is novel in that human cells were used.
  • compositions of the invention can be used to achieve methods of the invention.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • the present invention may also include methods and compositions in which the transition phrase "consisting essentially of or "consisting of may also be used.
  • words of approximation such as, without limitation, "about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
  • the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as "about” may vary from the stated value by at least ⁇ 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Gendler S Taylor-Papadimitriou J, Duhig T, Rothbard J, Burchell J. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J Biol Chem 1988; 263: 12820-3.

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Abstract

L'invention porte sur des compositions et des procédés qui permettent d'améliorer la réponse immunitaire cytotoxique à un antigène du cancer, lesquels procédés consistent à : obtenir des cellules mononucléées de sang périphérique chez un sujet; incuber les cellules mononucléées adhérentes de sang périphérique avec l'antigène du cancer et GM-CSF en l'absence d'IL-4 pour faire mûrir lesdites cellules mononucléées adhérentes de sang périphérique en cellules dendritiques; et isoler les cellules dendritiques mûres, les cellules dendritiques mûres stimulant les cellules T cytotoxiques spécifiques de l'antigène du cancer in vivo.
PCT/US2014/017704 2013-02-22 2014-02-21 Renforcement par les cellules dendritiques de la protection contre le cancer conférée par les cellules mononucléées stimulées par muc1 humaines WO2014130823A1 (fr)

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