WO2010141093A2 - Méthodes de co-signalement permettant de traiter les cancers - Google Patents

Méthodes de co-signalement permettant de traiter les cancers Download PDF

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WO2010141093A2
WO2010141093A2 PCT/US2010/001625 US2010001625W WO2010141093A2 WO 2010141093 A2 WO2010141093 A2 WO 2010141093A2 US 2010001625 W US2010001625 W US 2010001625W WO 2010141093 A2 WO2010141093 A2 WO 2010141093A2
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cells
lymphocytes
cell
tumor
expanded
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WO2010141093A3 (fr
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Scott E. Strome
Dan H. Schulze
Andrei Chapoval
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The University Of Maryland, Baltimore
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Definitions

  • the present invention relates to the fields of cancer immunology and therapeutics. Specifically, the present invention relates to a novel co-signaling method to treat certain cancers, such as squamous cell carcinoma of the head and neck.
  • Squamous cell carcinoma of the head and neck is diagnosed in over 40,000 patients per year in the United States, with an average overall 5-year survival rate of approximately 50%. Improvement of this poor prognosis will likely rely on development of novel and effective treatment strategies.
  • T cell based immunotherapeutic approaches have yet to be successfully translated into main stream clinical practice. This failure largely is due to the ability of squamous cell carcinoma of the head and neck to evade immune recognition through active immune suppression and down-regulation of HLA class I and tumor antigen processing machinery.
  • Natural killer (NK) cells contribute to innate immune responses against virally infected and neoplastic cells 1 .
  • NK cells usually recognize and attack tumor cells that lack major histocompatibility complex (MHC) class I 2 .
  • MHC major histocompatibility complex
  • y ⁇ T lymphocytes play an important role in the regulation of antitumor NK cell function 3 .
  • y ⁇ T lymphocytes are required for the antitumor activity of NK cells in vivo.
  • culturing human PBMC with agents which activate y ⁇ T lymphocytes induce NK cell mediated cytotoxicity against tumors that normally resist NK killing 4 .
  • y ⁇ T lymphocytes are important in the regulation of NK cell functions.
  • y ⁇ T cells are characterized by the expression of a T cell receptor (TCR) consisting of both gamma and delta chains 6 , and account for 1-10% of CD3 + cells in the peripheral blood of healthy adults 7 .
  • TCR T cell receptor
  • Approximately 70% of y ⁇ T lymphocytes express the V ⁇ 2V ⁇ 2 TCR and can be expanded and activated by phosphoantigens such as the cholesterol biosynthesis intermediate, isopentenylpyrophosphate (IPP), or synthetic bisphosphonates (e.g. pamidronate disodium and zoledronic acid) 8'10 .
  • IPP isopentenylpyrophosphate
  • synthetic bisphosphonates e.g. pamidronate disodium and zoledronic acid
  • y ⁇ T lymphocytes Upon stimulation, y ⁇ T lymphocytes acquire the capacity to destroy solid tumors of diverse origins such as squamous cell carcinoma of the head and neck, melanoma, colon cancer and breast carcinoma 4 11'13 , suggesting that y ⁇ T lymphocytes are important antitumor effector cells.
  • MCA methylcholanthrene
  • y ⁇ T lymphocytes The antitumor effects of y ⁇ T lymphocytes are recognized to result from both direct killing of tumor targets and trans-activation of adaptive immune responses. For example, recent data demonstrate that activated y ⁇ T lymphocytes cause the maturation of DC which promote development of acquired immunity 16 . In addition, y ⁇ T cells are known to cross-present tumor antigens (Ags) to CD8 + cytolytic T lymphocytes 17 18 . Despite their well characterized role in mediating adaptive immune responses, the mechanisms by which y ⁇ T cells regulate cells of the innate immune system, such as NK cells, are unclear.
  • y ⁇ T lymphocytes provide a costimulatory function for NK cells stimulated with suboptimal doses of immobilized human IgGL
  • Costimulated NK cells display upregulation of the activation markers CD25, CD54 and CD69 and effectively kill solid tumors which are traditionally resistant to NK mediated lysis.
  • CD137L expressed on activated y ⁇ T lymphocytes, with CD137, present on activated NK cells.
  • CD137/CD137L engagement increases NKG2D expression on NK cells which augmented tumor killing.
  • ex vivo culture of PBMC with zoledronic acid induces y ⁇ T lymphocyte activation, resulting in enhanced NK cell mediated tumor cytotoxicity.
  • This data define a novel mechanism through which ⁇ 6 T lymphocytes enhance the cytolytic function of NK cells and provide a clear opportunity to enhance existing cancer treatment strategies combining antibody-dependent cellular cytotoxicity and killing of non-opsonized tumor targets.
  • the present invention is directed to a method of treating a neoplastic disease in an individual in need of such treatment, comprising the steps of expanding ⁇ T lymphocytes in vitro; priming Natural Kill cells in vitro; administering said expanded ⁇ T lymphocytes and said primed Natural Kill cells to said individual, thereby resulting in a cytotoxic or cytolytic effect.
  • the present invention is directed further to a composition useful for treating a neoplastic disease in an individual in need of such treatment, comprising expanded ⁇ T lymphocytes; and primed Natural Kill cells.
  • the present invention is directed further still to a method of enhancing NK cell cytotoxicity in an individual in need of such treatment, comprising the step of: administering a compound to said individual that activates y ⁇ T lymphocytes, wherein in activation of said y ⁇ T lymphocytes results in enhancement of NK cell cytotoxicity.
  • the present invention is directed further still to a method of treating a neoplastic disease in an individual in need of such treatment, comprising the steps of: administering a compound to said individual that activates ⁇ 6 T lymphocytes; and administering an antibody directed against a tumor antigen associated with said neoplastic disease.
  • FIG. 1A Purified NK cells and IPP+IL-2 expanded y ⁇ T lymphocytes were co-cultured at 4:1 ratio in the presence or absence of plate-immobilized hlgG1 (2.5 ⁇ g/ml) for 48 hours. The expression of CD69 was analyzed by flow cytometry. Histograms represent gated CD3-CD56+ NK cells.
  • Figure 1 B Purified NK cells (2x10 5 cells/well) were cultured with indicated numbers of IPP+IL-2 expanded y ⁇ T lymphocytes in the presence of immobilized hlgG1 (2.5 ⁇ g/ml) for 48 hours.
  • FIG. 1C shows that y ⁇ T lymphocytes enhance hlgG1 induced activation of NK cells.
  • Purified NK cells and IPP+IL-2 expanded y ⁇ T cells were co-cultured at 4:1 ratio in the presence or absence of plate-immobilized hlgG1 (2.5 ⁇ g/ml) for 48 hours and the expression of CD54 was analyzed by flow cytometry. Histogram represents gated CD3-CD56+ NK cells. A representative data from twenty independent experiments is shown.
  • Figures 2A-2B y ⁇ T lymphocytes induce NK cell mediated cytotoxicity against various tumor cell lines.
  • Figure 2A Purified NK cells were cultured with bulk IPP expanded PBMC at a 2:1 ratio for 48 hours (i.e. 2 NK cells/1 IPP expanded PBMC).
  • IPP expanded PBMC in these experiments contained 70-80% y ⁇ T lymphocytes, so the actual ratio of NK cells to IPP expanded y ⁇ T lymphocytes was approximately 2/0.8.
  • NK cells were re-purified from the cultures by immunomagnetic depletion of non-NK cells. Representative dot plots of NK + y ⁇ T cells (right) and NK cells purified after 48 hours of culture (left) are shown.
  • FIG. 3 Cell-to-cell contact is required for the activation of NK cells by y ⁇ T lymphocytes.
  • Trans-well experiments were performed by culturing purified NK cells in lower wells coated with NgG 1 (2.5 ⁇ g/ml). Expanded y ⁇ T lymphocytes were added either to the lower (cell-to-cell contact) or to the upper wells (soluble factors). The ratio of NK to y ⁇ T cells was 4:1.
  • the expression of CD69 and CD54 was analyzed by flow cytometry. The bar diagrams depict the percentage of CD69 and CD54 expressing cells in gated NK populations. Representative data from 1 of 3 independent experiments is shown.
  • Figures 4A-4B Expression of costimulatory ligands and receptors on y ⁇ T lymphocytes and NK cells.
  • Figure 4A Fresh y ⁇ T lymphocytes from normal donors (top histograms) or y ⁇ T lymphocytes expanded in the presence of IPP+IL-2 (lower histograms) were stained with mAb specific for CD80, CD86, CD252 (OX40L) and CD137L (41 BBL). The expression of indicated costimulatory ligands on gated CD3+ ⁇ TCR+ cells is shown.
  • NK cells cultured with media alone or immobilized hlgG1 with or without in vitro expanded y ⁇ T lymphocytes for 48 hours were stained with mAbs specific for CD28, CD152 (CTLA-4), CD134 (OX40) and CD137 (4-1 BB). Overlays of histograms representing gated CD3-CD56+ NK cells are shown. Depicted data represent 1 of 5 independent experiments.
  • Figures 5A-5D Blocking of CD137L partially inhibits y ⁇ T lymphocyte induced cytolytic activity of NK cells.
  • Figure 5A Purified NK cells were co-cultured with IPP+IL-2 expanded y ⁇ T lymphocytes at 4:1 ratio in the presence of immobilized hlgG1 (2.5 ⁇ g/ml) for 48 hours. In some groups, soluble CD137lg fusion protein at 10 ⁇ g/ml was added to block CD137 receptor and ligand interactions.
  • the bar diagram represents the percentage of cells expressing CD54 in gated CD3-CD56+ NK cell population. Representative data from 1 of 4 independent experiments is shown.
  • NK cells were co- cultured with either mock (left histograms) or CD137L transfected P815 (central histograms) at a 4:1 ratio in the presence of immobilized hlgG1 (2.5 ⁇ g/ml).
  • soluble CD137lg fusion protein (10 ⁇ g/ml) was added (right histograms). After 48 hours of culture, cells were stained for CD54 and CD25. Histograms represent cells gated on CD56+CD3- NK population.
  • NK cells were purified from PBMC of healthy donors and co-cultured with irradiated mock or CD137L transfected P815 cells at a 4:1 ratio. Expanded y ⁇ T lymphocytes were used as a positive control for NK cell activation. After 48 hours of culture, NK cells were repurified and used as effectors against TU 167 SSCHN target cells. Data are presented as mean ⁇ SD of triplicate samples and representative of 3 independent experiments. * - p ⁇ 0.05 compared to NK cells cultured with mock transfected P815. Figures 6A-6D. CD137 ligation on NK cells results in enhanced NKG2D expression which is involved in tumor cell killing.
  • NK cells purified from PBMC of 11 individual donors were co-cultured in the presence of expanded ⁇ 5 T lymphocytes (4:1 ratio) on plates pre-coated with hlgG1. After 48 hours of culture, the expression of NKG2D was analyzed on NK cells. Dots represent individual values of NKG2D expression on gated NK cells. Horizontal lines represent average values of NKG2D expression in indicated groups.
  • Figure 6B Cytotoxic activity of NK cells purified after 48 hours of culture with in vitro expanded y ⁇ T lymphocytes was measured in a standard 4 hours 51 Cr- release assay against TU167 SCCHN.
  • Blocking anti-NKG2D antibodies or isotype control IgG were added into the wells containing purified NK cells and TU167 targets for the duration of the cytotoxicity test. Data are presented as mean ⁇ SD of triplicate samples and are representative of 2 independent experiments. *-p ⁇ 0.05 compared with isotype control.
  • Figure 6C CD137lg (10 ⁇ g/ml) was added to wells containing NK cells and IPP+IL-2 expanded y ⁇ T lymphocytes. After 48 hours of culture, cells were stained with anti-NKG2D mAb. The histograms depict NKG2D expression on gated CD56+CD3- NK cells. Numbers in brackets indicate MFI of NKG2D expression.
  • NK cells Purified NK cells were cultured with irradiated mock or CD137L transfected P815 cells (4:1) on plates pre- coated with hlgGL After 48 hours the expression of NKG2D was analyzed by FACS. Expanded y ⁇ T lymphocytes were used as a positive control for NK cells activation. Numbers in brackets indicate MFI of NKG2D expression.
  • Figures 7A-7C Zoledronate, a y ⁇ T lymphocyte activating agent, enhances NK cell activation and cytotoxicity.
  • Figure 7A Purified NK cells were cultured for 96 hours in the presence media, TU 167 cells alone, TU 167 + 10 ⁇ g/ml hlgG1 (isotype control) or TU 167 + 10 ⁇ g/ml Cetuximab. CD137 expression on CD56+ NK cells was analyzed by FACS. Two representative experiments are shown.
  • Figure 7B Whole PBMC were incubated in the presence of media, 10 ⁇ g/ml Rituximab, 15 ⁇ M Zoledronate or a combination of Rituximab with Zoledronate.
  • NK cells were purified from the groups described above. NK cell direct cytotoxicity (left plots) or antibody-dependent cellular cytotoxicity (right plots) was measured in a standard 4 hour 51 Cr-release assay against TU167 SCCHN or Ramos B cell lymphoma targets. Data are presented as mean ⁇ SD of triplicate samples and are representative of 2 independent experiments. * - p ⁇ 0.05 compared with NK cells purified from y ⁇ T lymphocyte depleted cultures. DETAILED DESCRIPTION OF THE INVENTION
  • the term “a” or “an”, when used in conjunction with the term “comprising” in the claims and/or the specification, may refer to “one", but it is also consistent with the meaning of "one or more”, “at least one", and “one or more than one”. Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any device, compound, composition, or method described herein can be implemented with respect to any other device, compound, composition, or method described herein.
  • the term “or” in the claims refers to “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or”.
  • the term "contacting" refers to any suitable method of bringing one or more of the compounds described herein with or without one or more other therapeutic agents into contact with one or more cancer cells or a tumor comprising the same. In vitro or ex vivo this is achieved by exposing the cancer cells or tumor to the compound(s)/therapeutic agent(s) in a suitable medium. For in vivo applications, any known method of administration is suitable as described herein.
  • the terms “effective amount”, “pharmacologically effective amount” or “therapeutically effective amount” are interchangeable and refer to an amount that results in an antiproliferative effect against cancer cells in vitro or an improvement or remediation in the cancer in vivo. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure of the cancer.
  • the term “treating” or the phrase “treating a cancer” includes, but is not limited to, halting the growth of the cancer, killing the cancer, or reducing the size of the cancer. Halting the growth refers to halting any increase in the size or the number of or size of the cancer cells or to halting the division of the cancer cells. Reducing the size refers to reducing the size of the tumor associated with the cancer or the number of or size of the cancer cells.
  • the term "subject” refers to any target of the treatment.
  • the term “expanding ⁇ T lymphocytes” or term “activating ⁇ T lymphocytes” refers to culturing purified PBMC (1x10 6 cells/ml) in complete media with 15 ⁇ M isopentyl pyrophosphate (IPP) and 100 U/ml human recombinant IL-2 for an appropriate period of time, such as 14 days. Fresh complete medium and IL-2 supplement at 100 U/ml was added every 3 days.
  • the term “priming NK cells” refers to culturing NK cells on plates coated with human IgGI (hlgG1) or tumor cells opsonized with Ab recognizing tumor associated antigens.
  • Natural killer (NK) cells are innate effector lymphocytes which control the growth of MHC class I negative tumors.
  • the present invention demonstrates that ⁇ T lymphocytes, expanded in vitro in the presence isopentenylpyrophosphate (IPP), induce NK cell mediated killing of tumors which are usually resistant to NK cytolysis.
  • IPP isopentenylpyrophosphate
  • the induction of cytotoxicity towards these resistant tumors requires priming of NK cells by immobilized hlgG1 and costimulation through CD137L expressed on activated ⁇ T lymphocytes. This costimulation increases NKG2D expression on the NK cell surface, which is directly responsible for tumor cell lysis.
  • culturing PBMC with zoledronic acid enhances NK cell direct cytotoxicity and antibody-dependent cellular cytotoxicity against hematopoietic and nonhematopoietic tumors.
  • This data reveals a novel function of human ⁇ T lymphocytes in the regulation of NK cell mediated cytotoxicity and provide a rationale for the utilization of strategies to manipulate the CD137 pathway to augment innate antitumor immunity.
  • a method of treating a neoplastic disease in an individual in need of such treatment comprising the steps of: expanding ⁇ T lymphocytes in vitro; priming Natural Kill cells in vitro; and administering said expanded ⁇ T lymphocytes and said primed Natural Kill cells to said individual, thereby resulting in a cytotoxic or cytolytic effect.
  • Methods of expanding ⁇ T lymphocytes are well known in the art. Representative compounds useful to expand ⁇ T lymphocytes selected from the group consisting of human recombinant IL-2 in combination with phosphoantigens (i.e. isopentenylpyrophosphate) or bisphosphonates (i.e. zoledronic acid).
  • ⁇ T lymphocytes are well known in the art.
  • Representative compounds useful to prime Natural Kill cells include but are not limited to immobilized human IgGI or tumor specific monoclonal antibodies.
  • administration of said Natural Kill cells results in co-stimulation through CD137L expressed on activated ⁇ T lymphocytes and increases NKG2D expression on the NK cell surface leading to tumor cell lysis.
  • the expanded ⁇ T lymphocytes and said primed Natural Kill cells are co-cultured prior to administering to said individual.
  • the primed Natural Kill cells and said expanded ⁇ T lymphocytes are co-cultured in a ratio of from about 2:1 to about 10:1.
  • neoplastic diseases including but not limited to squamous cell carcinoma of the head and neck, melanoma, breast cancer, B cell lymphoma, T cell lymphoma and colon cancer lines.
  • a composition useful for treating a neoplastic disease in an individual in need of such treatment comprising: expanded ⁇ T lymphocytes; and primed Natural Kill cells. Expansion of ⁇ T lymphocytes and priming of Natural Kill cells may be accomplished as described supra. Preferably, this increases NKG2D expression on the NK cell surface leading to tumor cell lysis.
  • the expanded ⁇ T lymphocytes and said primed Natural Kill cells are co-cultured in a ratio of from about 2:1 to about 10:1.
  • This composition would be useful in treating neoplastic disease including but not limited to squamous cell carcinoma of the head and neck, melanoma, breast cancer, B cell lymphoma, T cell lymphoma, and colon cancer lines.
  • a method of enhancing NK cell cytotoxicity in an individual in need of such treatment comprising the step of: administering a compound to said individual that activates y ⁇ T lymphocytes, wherein in activation of said y ⁇ T lymphocytes results in enhancement of NK cell cytotoxicity.
  • the ⁇ T lymphocytes are activated in the presence of a compound selected from the group consisting of human recombinant IL-2 in combination with phosphoantigens (i.e. isopentenylpyrophosphate) or bisphosphonates (i.e. zoledronic acid).
  • a method of treating a neoplastic disease in an individual in need of such treatment comprising the steps of: administering a compound to said individual that activates y ⁇ T lymphocytes; and administering an antibody directed against a tumor antigen associated with said neoplastic disease.
  • activation of said y ⁇ T lymphocytes results in enhancement of NK cell cytotoxicity.
  • Representative compounds that activate ⁇ T lymphocytes including but not limited to isopentenylpyrophosphate, zoledronic acid, human recombinant IL-2, and phosphoantigens.
  • the antigen is CD20 on B cells and the antibody may be but is not limited to rituximab, tositumomab, ibritumomab, ocrelizumab, ofatumumab.
  • the tumor antigen is epidermal growth factor receptor and antibody may be but is not limited to cetuximab, panitumumab, and zalutumumab.
  • the tumor antigen is carcinoembryonic antigen, 17-1 A, colon cancer-specific antigen-3, colon cancer-specific antigen-4 or colon cancer-specific antigen-3 and the antibody may be but is not limited to edrecolomab.
  • the tumor antigen is selected from the group consisting of MUC-1 , epithelial tumor antigen, or HER-2 and the antibody may be but is not limited to trastuzumab.
  • the tumor antigen is tyrosinase, melanoma associated antigen, or CTLA- 4, CD137, and the antibody may be but is not limited to ipilimumab, and tremelimumab.
  • the tumor antigen is a T cell lymphoma antigen and the antibody may be but is not limited to Denileukin difitox.
  • Tumor cell lines Squamous cell carcinoma head and neck tumor cell lines TU167, TU159 and
  • MDA1986 were provided by Dr. Gary dayman (M. D. Anderson Cancer Center). 012SCC was provided by Dr. Bert O'Malley (University of Pennsylvania).
  • the K562 cell line was purchased from American Type Culture Collection (ATCC CCL-213). CD137L transfected and mock transfected P815 cell lines were established as described 32 . All tumor cell lines and peripheral blood mononuclear cells (PBMC) were cultured in complete RPMI 1640 media (Gibco, Grand Island, NY) supplemented with 10% FBS (Atlanta Biologicals, Atlanta, GA), 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 ⁇ g/ml) and 10 mM HEPES (all purchased from Gibco).
  • Antibodies and fusion proteins Fluorochrome-conjugated mAbs against the following Ags were purchased from vendors and used according to the manufacturers instructions: y ⁇ TCR, CD56, CD3, CD69, CD54, CD40L, CD80, CD86, CD28, CD94, CD161 , CD16, CD152, CD278, CD279, CD134, CD137, CD252, CD137L, IFN-g, TNF-a (BD Biosciences); CD44, CD46, NKG2D (Biolegend). Blocking experiments with NKG2D and CD54 were purchased from R&D System USA. Human IgGI was obtained from Sigma Aldrich. Human soluble recombinant CD137lg, CD134lg, CD152lg fusion proteins were purchased from R&D System USA.
  • Flow cytometry All Ab staining for cell surface markers was performed according to the following protocol. The cells were washed once in PBS containing 1% FBS and 0.05% NaN3, incubated with appropriate amounts of mAb at 4 0 C for 30 min and re washed in PBS. For intracellular cytokine analysis, cells were cultured with various stimuli and 3 ⁇ M monensin (Golgi stop) was added during the last 4 hours of culture. The cells were stained with mAb against cell surface molecules (e.g. ⁇ T " CR, CD3, CD56), fixed and permeabilized using the BD Cytofix/Cytoperm Kit as described by the manufacturer (BD Biosciences).
  • mAb against cell surface molecules e.g. ⁇ T " CR, CD3, CD56
  • the cells were stained with PE-conjugated mAb specific to IFN- ⁇ and TNF- ⁇ .
  • PE-conjugated anti-human Granzyme A and B or the appropriate isotype control (BD Biosciense).
  • Buffy coats from healthy donors were purchased through Biologic Specialty Corp. (Colmar, PA) as approved under the University of Maryland IRB exempt.
  • whole PBMC were separated on a Ficoll gradient (Amersham Biosciences, Piscataway, NJ) and 1x10 6 cells/ml were cultured in complete media with 15 ⁇ M isopentyl pyrophosphate (IPP) (Sigma) and 100 U/ml human recombinant IL-2 (Tecin, Biological Resources Branch, National Institutes of Health, Bethesda, MD). Fresh complete medium and IL-2 supplement at 100 U/ml was added every 3 days.
  • IPP isopentyl pyrophosphate
  • IL-2 Tecin, Biological Resources Branch, National Institutes of Health, Bethesda, MD
  • PBMC isolated from buffy coats (3x10 6 cells/ml) were cultured with 15 ⁇ M Zometa (Novartis) alone or in the presence of 10 ⁇ g/ml Rituximab (Genentech). Activation of NK cells was verified by FACS or NK cells were purified from the cultures using magnetic beads and used in cytotoxicity assay.
  • NK cells were isolated from fresh PBMC by negative selection using MACS NK cell isolation kit (MiltenyiBiotec) according to the manufacturer's instructions. The purity of the resulting cell populations was checked routinely by flow cytometry. NK cell purity generally exceeded 97%.
  • Human IgGI (hlgG1) was immobilized on plastic culture plates by incubating hlgG1 (2.5 ⁇ g/ml) in PBS at 4°C overnight, a condition that provides stable attachment of lgs on neutral plastic substrates.
  • Purified NK cells (2x10 6 cells/well) and IPP-expanded y ⁇ T lymphocytes (1x10 6 cells/well) were co cultured in 1 ml of RPMI in 24 well cell plates (Falcon) precoated with 10 ⁇ g/ml of hlgGL After 48 hours of culture, NK cells were assessed by flow cytometry and/or purified using MACS negative isolation kits for analysis of cytolytic activity.
  • NK cells 2x10 6 cells/well
  • mock or CD137L transfected P815 cells 1x10 6 cells/well
  • human soluble recombinant Ig fusion proteins or mAbs i.e. CD137lg, CD152lg, CD134lg
  • NK cells were purified and tested for cytotoxicity against SCCHN targets.
  • NK cells (1x10 6 cells/ml) were cultured with live TU167 (0.5x10 6 cells/ml) alone, in the presence of 10 ⁇ g/ml hlgG1 (isotype control) or Cetuximab (Bristol-Myers). Activation of NK cells was confirmed by FACS.
  • NK cells (2x10 6 cells/well) were resuspended in 1 ml of RPMI and placed in 24-well plates pre coated with 10 ⁇ g/ml hlgGL IPP-expanded y ⁇ T lymphocytes were resuspended at 0.5x10 6 cells/ml and 0.5 ml of cells were added into the Transwell (Costar) with a polycarbonated membrane (pore diameter 0.4 ⁇ M) permeable for soluble factors. Cells, separated by a transwell, were cultured for 48 hours as previously described and expression of activation markers was analyzed by flow cytometry.
  • NK cell cytotoxicity was measured using a standard 51 Cr-release assays, as described 33 . Briefly, target cells (2x10 6 in 0.3 ml of complete media) were incubated for 90 min at 37 0 C in 5% CO 2 with 150 ⁇ Ci of 51 Cr (GE Healthcare, Piscataway, NJ). The labeled cells were then washed twice with media and incubated for an additional 30 min to reduce background radioactivity. Cells were then washed two more times and adjusted to a concentration of 5x10 4 cells/ml in complete media. Labeled targets cells were cultured for 30 min with 4 ⁇ g/ml Retuximab or Cetuximab.
  • Effector NK cells were purified from ⁇ T lymphocyte co culture or from cultured PBMC by immunomagnetic MACS NK negative selection kit (MiltenyiBiotec). Serial dilutions of effector cells (100 ⁇ l/well) were added into each well of 96-well V-bottomed plates (Corning, NY). Aliquots of 51 Cr-labeled target cells (100 ⁇ l/well) were dispensed into wells containing effector cells. The plates were centrifuged at 200 rpm for 2 min and incubated at 37 0 C in 5% CO 2 .
  • EXAMPLE 10 y ⁇ T lymphocytes activate hlgG1 primed NK cells
  • EXAMPLE 11 v ⁇ T lymphocytes enhance NK cell mediated antitumor cytotoxicity
  • purified NK cells were stimulated with media, NgG 1 or IPP expanded y ⁇ T lymphocytes in the presence of immobilized NgGL
  • NK cells were re-purified by negative selection, enabling a highly pure population of "untouched" NK cells (>99%) for functional analysis (Fig. 2a).
  • NK cells Use of these NK cells as effectors against various tumor cell lines revealed that cells cultured with media or immobilized hlgG1 alone did not kill SCCHN (TU167, 012SCC, MDA1986), melanoma (Mel526), breast cancer (MDA MB231 and MCF-7), B cell lymphoma (Daudi), or T cell lymphoma (Jurkat) tumor cell lines.
  • SCCHN TU167, 012SCC, MDA1986)
  • melanoma Melanoma
  • Mel526 breast cancer
  • MDA MB231 and MCF-7 B cell lymphoma
  • Daudi B cell lymphoma
  • T cell lymphoma Jurkat tumor cell lines.
  • y ⁇ T lymphocytes significantly increased the lytic activity of hlgG1 primed NK cells against the above cell lines (Fig. 2b).
  • the killing of colon cancer lines (HCT 116) that appears to be sensitive to NK cell mediated
  • NK cells The activation of NK cells was independent of donor HLA-type, since both autologous and allogeneic y ⁇ T lymphocytes enhanced cytolytic activity. This lack of HLA restriction in NK cell activation by y ⁇ T lymphocytes was very reproducible and observed in more than 20 independent experiments. Based on these findings, in subsequent studies NK cells and y ⁇ T lymphocytes derived from the PBMC of different donors were used, enabling access to a sufficient numbers of NK cells for functional and phenotypic analysis. Overall these data suggest that IPP activated ⁇ 5 T lymphocytes enhance direct NK cell mediated cytolytic activity against hematopoietic and nonhematopoietic tumors.
  • Soluble factors produced by y ⁇ T lymphocytes are responsible for activation of NK cells in long term culture 4 .
  • a transwell system was used. Purified NK cells were placed in lower wells coated with hlgG1 and IPP-expanded y ⁇ T lymphocytes were added to either the lower or the upper wells.
  • EXAMPLE 13 Expression of costimulatory molecules on activated y ⁇ T lymphocytes and NK cells
  • Ligands belonging to the B7 and TNF super families are essential for costimulation of immune cells 21 ' 22 . Having demonstrated the activation of hlgG1 primed NK cells by y ⁇ T lymphocytes requires cell-to-cell contact, the expression of costimulatory molecules on these cells was characterized. First, the expression of known costimulatory ligands on y ⁇ T lymphocytes was analyzed.
  • y ⁇ T lymphocytes in unstimulated PBMC did not express CD80, CD86, CD252 (OX40L) or CD137L (4-1 BBL) on their surface.
  • stimulation of PBMC with IPP and IL-2 for 14 days induced the expression of CD86 (86%), CD252 (12%), and CD137L (58%).
  • CD134 (OX40) and CD137 (4-1 BB) expression were enhanced on the surface of NK cells cultured with immobilized hlgG1 and this expression was further augmented by the addition of y ⁇ T lymphocytes (Fig. 4b).
  • Activation of NK cells by v ⁇ T lymphocytes is partially mediated by CD137/CD137L interactions
  • NK cell costimulation with ⁇ 6 T lymphocytes and IgGI induces CD137 and CD134 suggested that some of the observed antitumor effects might be mediated by TNF superfamily members.
  • CD134 and/or CD137 are involved in the activation of NK cells by y ⁇ T lymphocytes fusion proteins were used to block engagement of CD134 and CD137 with their cognate ligands.
  • Addition of CD152lg (used as negative control) or CD134lg fusion proteins into the culture did not inhibit the activation of NK cells by y ⁇ T lymphocytes.
  • CD137lg partially inhibited CD54 expression on NK cells (Fig. 5a).
  • NK cells were cultured with irradiated P815 cells expressing CD137L. Culturing purified NK cells with mock transfected P815 cells in the presence of immobilized hlgG1 did not induce the expression of CD54 or CD25 (Fig. 5b). In contrast CD137L expressing P815 tumors significantly increased the expression of activation markers on NK cells. The inclusion of the CD137lg fusion protein to cultures containing NK cells and CD137L transfected P815 tumors, completely abrogated the expression of CD54 and CD25 (Fig. 5b), indicating that CD137lg fusion protein blocks CD137/CD137L engagement. Overall, these results demonstrate that CD137/CD137L interactions are at least partially involved in the activation of NK cells by y ⁇ T lymphocytes.
  • CD137 mediates the induction of NK cell cytotoxicity by v ⁇ T lymphocytes
  • CD137 engagement enhances the cytolytic potential of NK cells cultured with y ⁇ T lymphocytes was investigated. Reproducibly, hlgG1 alone did not induce cytolytic function of NK cells while addition of y ⁇ T lymphocytes significantly increased killing of SCCHN targets (Fig. 5c). The addition of soluble CD137lg fusion protein decreased the cytolytic activity of NK cells cultured in the presence of immobilized hlgG1 and y ⁇ T lymphocytes by 40%, suggesting that CD137 engagement is important for the regulation of NK cell cytolytic function. CD152lg fusion protein containing the same Fc portion did not inhibit the induction of NK cell cytotoxicity.
  • NK cells were cultured with CD137L transfected P815 cells.
  • Data presented in Figure 5d indicate that NK cells cultured with NgG 1 and y ⁇ T lymphocytes induced 29% cytotoxicity against SCCHN cells at 20:1 effectortarget ratio. Thirteen percent cytotoxicity was observed in NK cells cultured with CD137L transfected P815 while only 6% cytotoxicity was mediated by NK cells cultured with mock P815.
  • CD137 costimulated NK cells utilize NKG2D for tumor cvtolysis
  • NK cells cultured in the presence of y ⁇ T lymphocytes The mechanism of tumor killing by NK cells cultured in the presence of y ⁇ T lymphocytes was examined. It is well known that NKG2D regulates NK cell cytotoxicity against many tumors 23 . Resting NK cells express a considerable amount of NKG2D on their surface. There were reproducible increases in the expression of NKG2D on the surface of NK cells from 11 different donors cultured with immobilized hlgG1 and y ⁇ T lymphocytes (Fig. 6a). Furthermore, the cytolytic activity of NK cells cultured with y ⁇ T lymphocyte correlated with the levels of NKG2D expression. In contrast, there was no expression of other well-characterized NK cell receptors (i.e. CD16, NKp30, NKp44, NKp46, CD94 CD161) on stimulated NK cells.
  • NK cell receptors i.e. CD16, NKp30, NKp44, NKp46, CD94 CD161
  • CD137lg fusion protein was used to block CD137/CD137L interaction.
  • the addition of CD137lg decreased y ⁇ T lymphocyte induced expression of NKG2D on NK cells from 82.8% (MFI 19,522) to 30.5% (MFI 3,872) (Fig. 6c), indicating that CD137 engagement is important for the induction of NKG2D expression.
  • Experiments utilizing CD137L transfected P815 cells further confirmed the involvement of CD137 signaling in the NKG2D expression (Fig. 6d). Overall these data indicate that CD137 engagement plays a significant role in the control of NKG2D expression which is important for tumor killing by NK cells cultured with expanded y ⁇ T lymphocytes.
  • Zoledronic acid enhances both direct NK cytotoxicity and ADCC against SCCHN and lymphoma
  • the present invention suggests that priming of NK cells by immobilized hlgG1 induces CD137 expression which is important for y ⁇ T lymphocyte induced activation.
  • a clinically applicable system for immobilizing human IgG in vivo and providing simulatanoeus y ⁇ T lymphocyte activation was developed.
  • whether opsonized tumor could serve as a platform for IgG immobilization was evaluated. The results shown on Figure 7a indicate that the EGFR positive SCCHN cell line,
  • TU167 when opsonized with the anti-EGFR mAb (Cetuximab), used clinically for the treatment of patients with SCCHN, induces expression of CD137 on NK cells.
  • T lymphocytes can be used in combination with mAb opsonized tumors to enhance tumor killing through direct cytolysis and antibody-dependent cellular cytotoxicity was evaluated.
  • Co-culture of PBMC with Rituximab (a clinical grade mAb recognizing CD20 on B cells) and Zometa (zoledronic acid), a bisphosphonate approved for clinical use which induces activation of y ⁇ T lymphocytes results in notable upregulation of CD69 on NK cells (Fig. 7b).
  • culturing PBMC with zoledronic acid alone also induces NK cell activation (Fig. 7b).
  • NK cells were purified from stimulated cultures and their direct cytotoxicity and antibody-dependent cellular cytotoxicity was evaluated in standard 4 hour Cr-release assays.
  • incubation of PBMC with Zometa significantly increases direct cytolytic activity of NK cells against SCCHN (TU 167) and B cell lymphoma (Ramos) targets.
  • TU 167 SCCHN
  • Ramos B cell lymphoma
  • CD137 engagement co-stimulates antitumor function of hlgG1 primed NK cells.
  • CD137/CD137L interactions partially account for the activation potential of y ⁇ T lymphocytes on hlgG1 stimulated NK cells, other costimulatory molecules are likely to also play a role.
  • NK cell activation can be triggered by CD80 and CD86 25 ' 26 , this study did not reveal known receptors for these ligands on the surface of stimulated NK cells.
  • CD152lg did not block the activation of NK cells by y ⁇ T lymphocytes, suggesting that CD80 and CD86 are not involved in the activation of NK cells.
  • ICOS in contrast to previous reports describing the presence of functional ICOS on murine NK cells, there was no expression of ICOS on human NK cells 27 .
  • Activated NK and CD8+ ⁇ T cells express the NKG2D receptor which recognizes specific ligands (ULBPs and MIC A/B) presented on tumors 28 .
  • CD137 regulates the expression of NKG2D in human cord blood CD8 T lymphocytes 24 .
  • This data indicates that CD137 engagement is important for the induction of NKG2D receptor expression on NK cells by y ⁇ T lymphocytes.
  • anti-NKG2D mAb significantly inhibits the cytolytic potential of y ⁇ T lymphocyte stimulated NK cells against tumor cell lines, suggesting that the increased NKG2D expression mediated by CD137, augments the cytolytic potential of NK cells.
  • other molecules expressed on y ⁇ T lymphocyte activated NK cells are also involved in the killing of tumors, since anti-NKG2D mAb blocking did not completely abrogate cytolytic activity.
  • y ⁇ T lymphocytes and NK cell interaction were confirmed by experiments utilizing PBMC cultured with clinically applicable reagents for the treatment of patients with SCCHN and lymphoma.
  • the present invention indicates that culturing PBMC with Zometa, increases direct and Ab dependent NK cytotoxicity against SCCHN and lymphoma targets. It is conceivable that y ⁇ T lymphocytes are more important for the regulation of antibody-dependent cellular cytotoxicity since y ⁇ T cell depletion had only partial impact on zoledronic acid induced direct NK cell cytotoxicity. However other molecular and cellular targets of zoledronic acid which are involved in the regulation of direct NK cell cytotoxicity remain to be determined. Overall this data suggests that administration of y ⁇ T cell activating agents may improve antitumor effects of Cetuximab and Rituximab used for the treatment of patients with SCCHN and B cell lymphoma, respectively.
  • in vitro expanded y ⁇ T lymphocytes improves adaptive immune responses against tumor Ags, by effectively presenting tumor Ags to conventional ⁇ T lymphocytes 17 18 .
  • This data indicates that in vitro culture with y ⁇ T lymphocyte activating agents (IPP or Zometa) can also improve antitumor innate function, as determined by increased NK cell cytotoxicity.
  • y ⁇ T lymphocyte activating agents IPP or Zometa
  • activation of y ⁇ T lymphocytes in vivo or adoptive transfer of in vitro expanded y ⁇ T lymphocytes has the potential to improve existing strategies for cancer immunotherapy.
  • a combination of tumor specific mAbs that engage Fc receptors on NK cells (Cetuximab or Rituximab) 29 and y ⁇ T lymphocytes activating agents approved for clinical use e.g.
  • Zometa 30 ' 31 may improve existing cancer immunotherapy by stimulating both the adaptive and innate antitumor immunity.
  • this activation strategy may overcome pre-existing defects in NK cell function recognized to exist in patients with large tumor burdens, further augmenting the clinical utility of this strategy.

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Abstract

La présente invention concerne une méthode de traitement d'une maladie néoplasique chez un individu ayant besoin d'un tel traitement, ladite méthode comprenant les étapes consistant à multiplier des lymphocytes T γδ in vitro ; à sensibiliser des cellules NK in vitro ; et à administrer lesdits lymphocytes T γδ multipliés et lesdites cellules NK sensibilisées audit individu, avec pour résultat un effet cytotoxique ou cytolytique. L'invention concerne, en outre, une méthode de traitement d'une maladie néoplasique chez un individu ayant besoin d'un tel traitement, ladite méthode comprenant les étapes consistant à administrer audit individu un composé capable d'activer les lymphocytes T yδ, puis un anticorps dirigé contre un antigène tumoral associé à ladite maladie néoplasique.
PCT/US2010/001625 2009-06-04 2010-06-04 Méthodes de co-signalement permettant de traiter les cancers WO2010141093A2 (fr)

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