WO2010141093A2 - Co-signaling methods for treating cancers - Google Patents

Abstract

Provided herein is a method of treating a neoplastic disease in an individual in need of such treatment, comprising the steps of: expanding γδ T lymphocytes in vitro; priming Natural Kill cells in vitro; and administering said expanded γδ T lymphocytes and said primed Natural Kill cells to said individual, thereby resulting in a cytotoxic or cytolytic effect. Further provided is 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 yδ T lymphocytes; and administering an antibody directed against a tumor antigen associated with said neoplastic disease.

Description

CO-SIGNALING METHODS FOR TREATING CANCERS

BACKGROUND OF THE INVENTION

Cross-Reference to Related Application

This international application claims benefit of priority under 35 U. S. C. §119(e) of provisional U.S. Serial No. 61/184,000, filed June 4, 2009, now abandoned, the entirety of which is hereby incorporated by reference.

Field of the Invention

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.

Description of the Related Art

Squamous cell carcinoma of the head and neck (SCCHN) 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. Unfortunately, despite success in pre-clinical models, 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 cells¹. NK cells usually recognize and attack tumor cells that lack major histocompatibility complex (MHC) class I². Previous studies in murine tumor models clearly demonstrated that gamma delta (yδ) T lymphocytes play an important role in the regulation of antitumor NK cell function³. Specifically, yδ T lymphocytes are required for the antitumor activity of NK cells in vivo. More recently, it was demonstrated that culturing human PBMC with agents which activate yδ T lymphocytes induce NK cell mediated cytotoxicity against tumors that normally resist NK killing⁴. These findings are consistent with other studies which show that yδ T lymphocytes regulate the early phase of NK cell mediated antibacterial responses in mice⁵. Taken together, these data suggest that yδ T lymphocytes are important in the regulation of NK cell functions. yδ T cells are characterized by the expression of a T cell receptor (TCR) consisting of both gamma and delta chains⁶, and account for 1-10% of CD3⁺ cells in the peripheral blood of healthy adults⁷. Approximately 70% of yδ T lymphocytes express the Vγ2Vδ2 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. Upon stimulation, yδ 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 yδ T lymphocytes are important antitumor effector cells. The validity of this antitumor function is further supported by mouse models demonstrating that mice deficient in yδ T cells have increased sensitivity to the development of methylcholanthrene (MCA)- induced tumors¹⁴. In addition a recent clinical study showed that yδ T lymphocyte adoptive therapy for patients with advanced renal cell carcinoma was well tolerated and induced antitumor immune responses¹⁵.

The antitumor effects of yδ 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 yδ T lymphocytes cause the maturation of DC which promote development of acquired immunity¹⁶. In addition, yδ 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 yδ T cells regulate cells of the innate immune system, such as NK cells, are unclear.

There is still, however, a recognized need in the art for improved chemotherapeutics and cancer therapies. Specifically, the prior art is deficient in novel strategies to utilize both the effector and antigen presenting cell functions of NK cells and enable them to overcome immune evasion in cancer patients, such as with squamous cell carcinoma of the head and neck. The present invention fulfills this long standing need in the art.

SUMMARY OF THE INVENTION

The present invention demonstrates that yδ 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. These costimulatory effects are partially regulated by the interaction of CD137L, expressed on activated yδ T lymphocytes, with CD137, present on activated NK cells. CD137/CD137L engagement increases NKG2D expression on NK cells which augmented tumor killing. In addition, ex vivo culture of PBMC with zoledronic acid induces yδ T lymphocyte activation, resulting in enhanced NK cell mediated tumor cytotoxicity. This data define a novel mechanism through which γ6 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.

Thus, 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 γδ T lymphocytes in vitro; priming Natural Kill cells in vitro; administering said expanded γδ 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 γδ 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 yδ T lymphocytes, wherein in activation of said yδ 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 γ6 T lymphocytes; and administering an antibody directed against a tumor antigen associated with said neoplastic disease.

Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions and certain embodiments of the invention briefly summarized above are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope. Figures 1A-1C. yδ T lymphocytes increase hlgG1 induced activation of NK cells.

(Figure 1A) Purified NK cells and IPP+IL-2 expanded yδ T lymphocytes were co-cultured at 4:1 ratio in the presence or absence of plate-immobilized hlgG1 (2.5 μg/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 (2x10⁵ cells/well) were cultured with indicated numbers of IPP+IL-2 expanded yδ T lymphocytes in the presence of immobilized hlgG1 (2.5 μg/ml) for 48 hours. Expression of activation markers CD69 and CD54 were assessed by flow cytometry and plotted as a percentage of CD69 and CD54 positive gated NK cells. Figure 1C: (supplementary figure 2) shows that yδ T lymphocytes enhance hlgG1 induced activation of NK cells. Purified NK cells and IPP+IL-2 expanded yδ T cells were co-cultured at 4:1 ratio in the presence or absence of plate-immobilized hlgG1 (2.5 μg/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. yδ 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% yδ T lymphocytes, so the actual ratio of NK cells to IPP expanded yδ 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 + yδ T cells (right) and NK cells purified after 48 hours of culture (left) are shown. (Figure 2B) Cytolytic activity of NK cells purified after co-culture with expanded yδ T cells and immobilized hlgG1 (2.5 μg/ml) for 48 hours was analyzed in a standard 4 hour ⁵¹Cr-release assay against indicated tumor targets. Data are presented as mean±SD of triplicate samples and are representative of 7 independent experiments. * - p<0.05 compared with NK cells cultured with hlgG1 alone.

Figure 3. Cell-to-cell contact is required for the activation of NK cells by yδ T lymphocytes. Trans-well experiments were performed by culturing purified NK cells in lower wells coated with NgG 1 (2.5 μg/ml). Expanded yδ T lymphocytes were added either to the lower (cell-to-cell contact) or to the upper wells (soluble factors). The ratio of NK to yδ T cells was 4:1. After 48 hours of culture, 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 yδ T lymphocytes and NK cells. (Figure 4A) Fresh yδT lymphocytes from normal donors (top histograms) or yδT 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+γδTCR+ cells is shown. (Figure 4B) NK cells cultured with media alone or immobilized hlgG1 with or without in vitro expanded yδ 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 yδ T lymphocyte induced cytolytic activity of NK cells. (Figure 5A) Purified NK cells were co-cultured with IPP+IL-2 expanded yδT lymphocytes at 4:1 ratio in the presence of immobilized hlgG1 (2.5 μg/ml) for 48 hours. In some groups, soluble CD137lg fusion protein at 10 μg/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. (Figure 5B) Purified 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 μg/ml). In some wells containing NK cells and CD137L transfected P815 tumors, soluble CD137lg fusion protein (10 μg/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. (Figure 5C) Soluble CD137lg fusion protein (10 μg/ml) was included during the culture of purified NK cells and yδ T lymphocytes (4:1 ratio) in hlgG1 pre-coated plates for 48 hours. Cytotoxicity of NK cells re-purified after culture was analyzed in a standard 4 hour ⁵¹Cr- release assay against the TU 167 SCCHN cell line. Data are presented as mean±SD of triplicate samples and are representative of 4 independent experiments. *- p<0.05 compared with NK cells cultured in the presence of CD137lg blocking. (Figure 5D) 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 yδ 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. (Figure 6A) NK cells purified from PBMC of 11 individual donors were co-cultured in the presence of expanded γ5 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 yδ T lymphocytes was measured in a standard 4 hours ⁵¹Cr- 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 μg/ml) was added to wells containing NK cells and IPP+IL-2 expanded yδ 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. (Figure 6D) 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 yδ 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 yδ 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 μg/ml hlgG1 (isotype control) or TU 167 + 10 μg/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 μg/ml Rituximab, 15 μM Zoledronate or a combination of Rituximab with Zoledronate. The expression of CD69 on gated CD3-CD56+ NK cells was analyzed by FACS 96 hours after initiation of the cultures. A representative of 3 independent experiments is depicted. (Figure 7C) Whole PBMC were cultured with media (circles) or Zoledronate (squares). Alternatively, yδ T lymphocyte depleted PBMC was cultured with Zoledronate (triangles) for 96 hours. 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 ⁵¹Cr-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 yδ T lymphocyte depleted cultures. DETAILED DESCRIPTION OF THE INVENTION

As used herein, 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. As used 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".

As used herein, 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.

As used 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. As used herein, 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.

As used herein, the term "subject" refers to any target of the treatment. As used herein, the term "expanding γδ T lymphocytes" or term "activating γδ T lymphocytes" refers to culturing purified PBMC (1x10⁶ cells/ml) in complete media with 15 μM 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. As used herein, 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 γδ T lymphocytes, expanded in vitro in the presence isopentenylpyrophosphate (IPP), induce NK cell mediated killing of tumors which are usually resistant to NK cytolysis. The induction of cytotoxicity towards these resistant tumors requires priming of NK cells by immobilized hlgG1 and costimulation through CD137L expressed on activated γδ T lymphocytes. This costimulation increases NKG2D expression on the NK cell surface, which is directly responsible for tumor cell lysis. Moreover, culturing PBMC with zoledronic acid, a γδ T lymphocyte activating agent, enhances NK cell direct cytotoxicity and antibody-dependent cellular cytotoxicity against hematopoietic and nonhematopoietic tumors. This data reveals a novel function of human γδ 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.

Thus, in one embodiment of the present invention, there is provided a method of treating a neoplastic disease in an individual in need of such treatment, comprising the steps of: expanding γδ T lymphocytes in vitro; priming Natural Kill cells in vitro; and administering said expanded γδ T lymphocytes and said primed Natural Kill cells to said individual, thereby resulting in a cytotoxic or cytolytic effect. Methods of expanding γδ T lymphocytes are well known in the art. Representative compounds useful to expand γδ 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). Methods of priming γδ 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. Generally, administration of said Natural Kill cells results in co-stimulation through CD137L expressed on activated γδ T lymphocytes and increases NKG2D expression on the NK cell surface leading to tumor cell lysis. In a preferred embodiment, the expanded γδ T lymphocytes and said primed Natural Kill cells are co-cultured prior to administering to said individual. Preferably, the primed Natural Kill cells and said expanded γδ T lymphocytes are co-cultured in a ratio of from about 2:1 to about 10:1. A person having ordinary skill in this art can readily determined neoplastic diseases to which the methods of the present invention would be useful, 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. In another embodiment of the present invention, there is provided a composition useful for treating a neoplastic disease in an individual in need of such treatment, comprising: expanded γδ T lymphocytes; and primed Natural Kill cells. Expansion of γδ 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. In one preferred embodiment, the expanded γδ 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.

In yet another embodiment of the present invention, there is provided 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 yδ T lymphocytes, wherein in activation of said yδ T lymphocytes results in enhancement of NK cell cytotoxicity. Preferably, the γδ 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).

In yet another embodiment of the present invention, there is provided 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 yδ T lymphocytes; and administering an antibody directed against a tumor antigen associated with said neoplastic disease. Generally, activation of said yδ T lymphocytes results in enhancement of NK cell cytotoxicity. Representative compounds that activate γδ T lymphocytes including but not limited to isopentenylpyrophosphate, zoledronic acid, human recombinant IL-2, and phosphoantigens. In one embodiment of this method of the present invention, the antigen is CD20 on B cells and the antibody may be but is not limited to rituximab, tositumomab, ibritumomab, ocrelizumab, ofatumumab. In another embodiment of this method of the present invention, the tumor antigen is epidermal growth factor receptor and antibody may be but is not limited to cetuximab, panitumumab, and zalutumumab. In another embodiment of this method of the present invention, 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. In another embodiment of this method of the present invention, 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. In another embodiment of this method of the present invention, 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. In another embodiment of this method of the present invention, the tumor antigen is a T cell lymphoma antigen and the antibody may be but is not limited to Denileukin difitox. The following example(s) are given for the purpose of illustrating various embodiments of the invention and do not limit the present invention in any fashion.

EXAMPLE 1

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 described³². 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 μg/ml) and 10 mM HEPES (all purchased from Gibco). To ensure the purity of original and cultured tumor cell lines we performed haplotyping using a PCR kit (One Lambda Inc., Canoga Park, CA) and/or flow cytometry staining with antibodies against HLA class I (BD Biosciences, San Jose, CA).

EXAMPLE 2

Antibodies and fusion proteins Fluorochrome-conjugated mAbs against the following Ags were purchased from vendors and used according to the manufacturers instructions: yδ 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.

EXAMPLE 3

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 4⁰C for 30 min and re washed in PBS. For intracellular cytokine analysis, cells were cultured with various stimuli and 3μM monensin (Golgi stop) was added during the last 4 hours of culture. The cells were stained with mAb against cell surface molecules (e.g. γδT^"CR, CD3, CD56), fixed and permeabilized using the BD Cytofix/Cytoperm Kit as described by the manufacturer (BD Biosciences). After permeabilization, the cells were stained with PE-conjugated mAb specific to IFN-γ and TNF-α. To determine granzyme A and B expression, permeabilized cells were stained with PE-conjugated anti-human Granzyme A and B or the appropriate isotype control (BD Biosciense).

In most flow cytometry samples, at least 3x10⁴ gated NK cells or γ5 T lymphocytes (defined as CD3- CD56+ and CD3+ and yδ TCR+, respectively) were acquired using a BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ). All samples were analyzed using FACS Diva software (Becton Dickinson).

EXAMPLE 4 vδ T cell expansion and activation

Buffy coats from healthy donors were purchased through Biologic Specialty Corp. (Colmar, PA) as approved under the University of Maryland IRB exempt. For expansion of yδ T cells, whole PBMC were separated on a Ficoll gradient (Amersham Biosciences, Piscataway, NJ) and 1x10⁶ cells/ml were cultured in complete media with 15 μM 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. After 14 days of culture, cells were harvested and the percentage of yδ T cells was measured by flow cytometry. The percent of yδ T lymphocytes in IPP expanded cultures varied from 60% to 90%, and the range for individual experiments is reported in the figure legends, yδ T lymphocytes were not purified prior to co-culture with NK cells.

Alternatively, PBMC isolated from buffy coats (3x10⁶ cells/ml) were cultured with 15 μM Zometa (Novartis) alone or in the presence of 10 μg/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.

EXAMPLE 5

Immunomaqnetic bead purification of NK cells

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%.

EXAMPLE 6 NK cell and vδ T lymphocyte coculture

Human IgGI (hlgG1) was immobilized on plastic culture plates by incubating hlgG1 (2.5 μg/ml) in PBS at 4°C overnight, a condition that provides stable attachment of lgs on neutral plastic substrates. Purified NK cells (2x10⁶ cells/well) and IPP-expanded yδ T lymphocytes (1x10⁶ cells/well) were co cultured in 1 ml of RPMI in 24 well cell plates (Falcon) precoated with 10 μg/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. In some experiments purified NK cells (2x10⁶ cells/well) were cocultured with mock or CD137L transfected P815 cells (1x10⁶ cells/well). In blocking experiments, human soluble recombinant Ig fusion proteins or mAbs (i.e. CD137lg, CD152lg, CD134lg) at 10 μg/ml were included at the onset of the NK and yδ T cell cocultures. After 48 hours, NK cells were purified and tested for cytotoxicity against SCCHN targets. In some experiments purified NK cells (1x10⁶ cells/ml) were cultured with live TU167 (0.5x10⁶ cells/ml) alone, in the presence of 10 μg/ml hlgG1 (isotype control) or Cetuximab (Bristol-Myers). Activation of NK cells was confirmed by FACS.

EXAMPLE 7 Transwell coculture

Purified NK cells (2x10⁶ cells/well) were resuspended in 1 ml of RPMI and placed in 24-well plates pre coated with 10 μg/ml hlgGL IPP-expanded yδ T lymphocytes were resuspended at 0.5x10⁶ cells/ml and 0.5 ml of cells were added into the Transwell (Costar) with a polycarbonated membrane (pore diameter 0.4 μM) 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.

EXAMPLE 8

Cytotoxicity assay

NK cell cytotoxicity was measured using a standard ⁵¹Cr-release assays, as described³³. Briefly, target cells (2x10⁶ in 0.3 ml of complete media) were incubated for 90 min at 37⁰C in 5% CO₂ with 150 μCi of ⁵¹Cr (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 5x10⁴ cells/ml in complete media. Labeled targets cells were cultured for 30 min with 4 μg/ml Retuximab or Cetuximab. Effector NK cells were purified from γό T lymphocyte co culture or from cultured PBMC by immunomagnetic MACS NK negative selection kit (MiltenyiBiotec). Serial dilutions of effector cells (100 μl/well) were added into each well of 96-well V-bottomed plates (Corning, NY). Aliquots of ⁵¹Cr-labeled target cells (100 μl/well) were dispensed into wells containing effector cells. The plates were centrifuged at 200 rpm for 2 min and incubated at 37⁰C in 5% CO₂. After 4 hours of incubation, the plates were centrifuged again at 13,000 rpm for 5 min and 100 μl aliquots of the supematants from each well were transferred to a new plate containing 100 μl/well of Optiphase Supermix scintillation fluid (Perkin Elmer, Boston, MA). Radioactivity was measured using 1450 Microbeta counter (Wallac, Turku Finland). In some experiments anti-NKG2D or isotype control mouse IgG were added at 10 μg/ml, 15 min before the addition of labeled target cells. The percentage of specific cytotoxicity was calculated as (experimental release - spontaneous release)/(maximum release - spontaneous release) x 100. Spontaneous release was determined by incubating the targets with 100 μl of complete media. Maximum release was determined by incubating the target cells with 100 μl of 0.5% Triton-X.

EXAMPLE 9 Statistical analysis

All cytotoxicity data were analyzed by using the Student's test, whereby p<0.05 indicated that the value of the test sample was significantly different from the relevant control.

EXAMPLE 10 yδ T lymphocytes activate hlgG1 primed NK cells

Long term (14 day) culture of PBMC with IPP + IL-2 results in the induction of NK cell mediated cytotoxicity against tumors that are normally resistant to NK cell killing⁴. It was also observed that IPP did not stimulate NK cells directly and yδ T lymphocytes were critical for IPP-induced NK cell cytotoxicity. Usually NK cells do not survive for a long period of time without cytokine support¹⁹. Therefore, in order to understand the mechanisms of γ6 T lymphocyte mediated NK cell activation in a more physiological model, in this study a short term culture of fresh NK cells purified from PBMC of healthy donors and yδ T lymphocytes expanded in vitro was used. As shown in Figure 1A, fresh NK cells cultured with media or yδ T lymphocytes alone do not express CD69, a molecule associated with NK cell activation. In contrast, consistent with published data²⁰, after incubating in wells pre-coated with hlgG1 , 25% of NK cells express CD69 on their surface. Adding yδ T lymphocytes to hlgG1 coated wells further increases the expression of CD69 on NK cells to 45.9% (Fig. 1A). A similar pattern of expression was seen for CD54, another marker of NK cell activation (Fig. 1C). Neither immobilized hlgG1 alone nor in combination with NK cells induced activation of yδ T lymphocytes, as measured by the expression of activation markers. Since short term (48 hours) culture of yδ T lymphocytes without hlgG1 did not result in the activation of NK cells, plastic immobilized hlgG1 was included in all subsequent experiments as a putative initial signal for NK cell priming. An optimal ratio for NK cell activation by yδ T lymphocytes was also determined. As shown in Figure 1b culturing 2x10⁵ NK cells with 10⁵ or 5x10⁴ T lymphocytes (2:1 and 4:1 ratio, respectively) induced significant increases in CD69 and CD54 expression on NK cells primed with NgG 1 , while ratios of 20:1 (10⁴ IPP expanded yδ T lymphocytes) failed to increase the expression of activation markers on NK cells. Therefore, in subsequent experiments a ratio of 4:1 was used. These results indicate that in vitro expanded yδ T lymphocytes stimulate the activation of NgG 1 primed human NK cells.

EXAMPLE 11 vδ T lymphocytes enhance NK cell mediated antitumor cytotoxicity In order to determine if yδ T lymphocyte stimulation of hlgG1 primed NK cells enhances their cytolytic activity, purified NK cells were stimulated with media, NgG 1 or IPP expanded yδ T lymphocytes in the presence of immobilized NgGL After 48 hours, NK cells were re-purified by negative selection, enabling a highly pure population of "untouched" NK cells (>99%) for functional analysis (Fig. 2a). 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. However yδ 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 cytotoxicity, was also significantly increased by yδ T lymphocytes. The activation of NK cells was independent of donor HLA-type, since both autologous and allogeneic yδ T lymphocytes enhanced cytolytic activity. This lack of HLA restriction in NK cell activation by yδ T lymphocytes was very reproducible and observed in more than 20 independent experiments. Based on these findings, in subsequent studies NK cells and yδ 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 γ5 T lymphocytes enhance direct NK cell mediated cytolytic activity against hematopoietic and nonhematopoietic tumors.

EXAMPLE 12

Cell-to-cell contact is essential for NK cell activation by yδ T lymphocytes

Soluble factors produced by yδ T lymphocytes are responsible for activation of NK cells in long term culture⁴. To ascertain whether the enhanced NK cell activation by yδ T lymphocytes in short term culture is mediated by cell-to-cell contact or soluble factors, a transwell system was used. Purified NK cells were placed in lower wells coated with hlgG1 and IPP-expanded yδ T lymphocytes were added to either the lower or the upper wells. As expected, NK cells co-cultured with immobilized hlgG1 and yδ T lymphocytes, placed in lower wells, showed increased expression of the CD69 and CD54 activation markers (Fig. 3). When NK cells were separated from yδ T lymphocytes by a membrane with 0.4 μm pore size there was no increase in the expression of activation markers. These data indicate that, in contrast to the long-term exposure data, cell-to-cell contact is required for NK cell activation by yδ T lymphocytes during short term interaction.

EXAMPLE 13 Expression of costimulatory molecules on activated yδ T lymphocytes and NK cells

Ligands belonging to the B7 and TNF super families are essential for costimulation of immune cells²¹'²². Having demonstrated the activation of hlgG1 primed NK cells by yδ 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 yδ T lymphocytes was analyzed.

As shown in Figure 4a, yδ T lymphocytes in unstimulated PBMC did not express CD80, CD86, CD252 (OX40L) or CD137L (4-1 BBL) on their surface. However, stimulation of PBMC with IPP and IL-2 for 14 days induced the expression of CD86 (86%), CD252 (12%), and CD137L (58%). Moreover, 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 yδ T lymphocytes (Fig. 4b). No expression of CD28, CD152 (CTLA-4), CD278 (ICOS) and CD279 (PD-1) were observed on NK cells even after co-culture with yδ T lymphocytes in the presence of immobilized hlgG1 (Fig. 4b). EXAMPLE 14

Activation of NK cells by vδ T lymphocytes is partially mediated by CD137/CD137L interactions

The fact that NK cell costimulation with γ6 T lymphocytes and IgGI induces CD137 and CD134 suggested that some of the observed antitumor effects might be mediated by TNF superfamily members. To determine if CD134 and/or CD137 are involved in the activation of NK cells by yδ 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 yδ T lymphocytes. However CD137lg partially inhibited CD54 expression on NK cells (Fig. 5a). These results suggest that stimulation of hlgG1 primed NK cells by yδ T lymphocytes involves CD137.

To confirm the role of CD137 in the activation of NK cells, purified 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 yδ T lymphocytes.

EXAMPLE 15 CD137 mediates the induction of NK cell cytotoxicity by vδ T lymphocytes

Whether CD137 engagement enhances the cytolytic potential of NK cells cultured with yδ T lymphocytes was investigated. Reproducibly, hlgG1 alone did not induce cytolytic function of NK cells while addition of yδ 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 yδ 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.

To confirm the role of CD137 signaling in the activation of NK cell mediated cytotoxicity, purified NK cells were cultured with CD137L transfected P815 cells. Data presented in Figure 5d indicate that NK cells cultured with NgG 1 and yδ 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. These data suggest that CD137 is at least partially involved in the regulation of NK cell cytolytic activity costimulated by yδ T lymphocytes.

EXAMPLE 16 hlgG1 primed. CD137 costimulated NK cells utilize NKG2D for tumor cvtolysis

The mechanism of tumor killing by NK cells cultured in the presence of yδ T lymphocytes was examined. It is well known that NKG2D regulates NK cell cytotoxicity against many tumors²³. 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 yδ T lymphocytes (Fig. 6a). Furthermore, the cytolytic activity of NK cells cultured with yδ 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.

Many squamous cell carcinoma of the head and neck tumors express ULBP-2 and ULBP-3, NKG2D ligands, suggesting a role of NKG2D in SCCHN killing by NK cells. Results presented on Figure 6b indicate that anti-NKG2D mAb blockade reduced the cytolysis of TU167 tumors by NK cells cultured with yδ T lymphocytes from 27% to 17% at 20:1 E:T ratio. These observations indicate that NK cells co-cultured with yδ T lymphocytes may kill SCCHN tumors by recognizing NKG2D ligands.

To verify the role of CD137 engagement in the induction of NKG2D expression on NK cells cultured with yδ T lymphocytes CD137lg fusion protein was used to block CD137/CD137L interaction. The addition of CD137lg decreased yδ 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 yδ T lymphocytes. EXAMPLE 17

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 yδ T lymphocyte induced activation. In order to provide a platform for translating these observations, a clinically applicable system for immobilizing human IgG in vivo and providing simulatanoeus yδ T lymphocyte activation was developed. First, 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. These data suggest that mAbs which target tumor associated antigens, when composed of the appropriate IgG isotype, can also provide a first signal for NK activation. In order to build on this finding, whether clinically relevant agents which activate yδ

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 yδ T lymphocytes, results in notable upregulation of CD69 on NK cells (Fig. 7b). Interestingly, culturing PBMC with zoledronic acid alone also induces NK cell activation (Fig. 7b).

To verify the effects of yδ T lymphocytes activation on NK cell cytotoxicity in physiological conditions, whole PBMC were cultured with Zometa. 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. As shown in Figure 7, incubation of PBMC with Zometa significantly increases direct cytolytic activity of NK cells against SCCHN (TU 167) and B cell lymphoma (Ramos) targets. Interestingly depletion of yδ T lymphocytes prior to culturing PBMC with Zometa significantly reduced but did not completely abrogate the effects of Zometa on direct NK cytotoxicity.

In order to determine whether the interplay of Zometa and opsonized tumor can enhance killing of SCCHN, a combinatorial approach was employed. As expected, higher levels of NK mediated cytotoxicity against TU167 and Ramos targets in the presence of appropriate Abs (Cetuximab and Rituximab, respectively) was observed. Nevertheless, culturing PBMC with Zometa significantly enhanced NK mediated ADCC. Moreover depletion of yδ T lymphocytes from PBMC before the addition of Zometa reduced NK killing to the level of cytotoxicity observed in NK cells purified from PBMC cultured without Zometa (Fig. 7). This observation indicates that yδ T lymphocytes are critical for the Zometa induced increase of NK killing of SCCHN and B cell lymphoma. Furthermore, these results define the physiological and clinical relevance of the interaction between yδ T lymphocytes and NK cells for the regulation of direct and antibody dependent NK cytotoxicity.

Discussion

Although the role of CD137 signaling in the activation of cytolytic αβ T lymphocytes is well established²⁴, the present invention provides the first evidence that CD137 engagement co-stimulates antitumor function of hlgG1 primed NK cells. These findings suggest that two signals are required for the optimal activation of NK cells in a manner similar to the two signal model established for αβ T lymphocyte activation. This idea is relevant to the improvement of mAb-based cancer immunotherapy where a combination of mAb specific to tumor Ag(s) with CD137 cross-linking, induces direct NK cell mediated tumor lysis. Specifically, these findings suggest that combination of Ab based cancer immunotherapies with either adoptive transfer of in vitro expanded yδ T lymphocytes or systemic injection of yδ T lymphocyte stimulating agents (zoledronic acid)^8"10 may improve clinical outcome through the enhancement of direct and antibody-dependent cellular cytotoxicity (ADCC) tumor killing by NK cells.

While CD137/CD137L interactions partially account for the activation potential of yδ T lymphocytes on hlgG1 stimulated NK cells, other costimulatory molecules are likely to also play a role. Although it is reported that NK cell activation can be triggered by CD80 and CD86²⁵'²⁶, this study did not reveal known receptors for these ligands on the surface of stimulated NK cells. Similarly, CD152lg did not block the activation of NK cells by yδ T lymphocytes, suggesting that CD80 and CD86 are not involved in the activation of NK cells. Furthermore, in contrast to previous reports describing the presence of functional ICOS on murine NK cells, there was no expression of ICOS on human NK cells²⁷. Taken in concert, this data suggests that other costimulatory and/or adhesion molecules expressed on the surface of activated yδ T lymphocytes are involved in the activation of NK cells antitumor properties. The identification of these costimulatory molecules may improve Ab and NK cell based cancer immunotherapy.

Activated NK and CD8+ αβ T cells express the NKG2D receptor which recognizes specific ligands (ULBPs and MIC A/B) presented on tumors²⁸. It has been shown that CD137 regulates the expression of NKG2D in human cord blood CD8 T lymphocytes²⁴. This data indicates that CD137 engagement is important for the induction of NKG2D receptor expression on NK cells by yδ T lymphocytes. Moreover anti-NKG2D mAb significantly inhibits the cytolytic potential of yδ 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. However it is conceivable that other molecules expressed on yδ T lymphocyte activated NK cells are also involved in the killing of tumors, since anti-NKG2D mAb blocking did not completely abrogate cytolytic activity.

Previous data indicate that long term culture (14 days) of PBMC with agents specifically activating yδ T lymphocytes (i.e. IPP + IL-2) increases the cytolytic activity of NK cells⁴. This NK cell activation was mediated by soluble factors released by yδ T lymphocytes during the culture. In contrast, these current results indicate that activation of NK cells in short term culture (48 hours) with expanded yδ T lymphocytes requires cell-to- cell contact and priming with immobilized hlgGl These findings suggest pleiotropic mechanisms of NK cell functions regulation by activated yδ T lymphocytes. The direct physiological role of NK and yδ T cells interaction in diseases and the maintenance of immune responses remain to be determined.

The clinical significance of yδ 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 yδ T lymphocytes are more important for the regulation of antibody-dependent cellular cytotoxicity since yδ 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 yδ 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 summary, in vitro expanded yδ T lymphocytes improves adaptive immune responses against tumor Ags, by effectively presenting tumor Ags to conventional αβ T lymphocytes^{17 18}. This data indicates that in vitro culture with yδ T lymphocyte activating agents (IPP or Zometa) can also improve antitumor innate function, as determined by increased NK cell cytotoxicity. Thus activation of yδ T lymphocytes in vivo or adoptive transfer of in vitro expanded yδ T lymphocytes has the potential to improve existing strategies for cancer immunotherapy. In particular a combination of tumor specific mAbs that engage Fc receptors on NK cells (Cetuximab or Rituximab)²⁹ and yδ 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. Of perhaps greater import, 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. The following references were cited herein:

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Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are incorporated by reference herein to the same extent as if each individual publication was incorporated by reference specifically and individually. One skilled in the art will appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a neoplastic disease in an individual in need of such treatment, comprising the steps of: expanding γδ T lymphocytes in vitro; priming Natural Kill cells in vitro; and administering said expanded γδ T lymphocytes and said primed Natural Kill cells to said individual, thereby resulting in a cytotoxic or cytolytic effect.

2. The method of claim 1 , wherein said γδ T lymphocytes are expanded in the presence of a compound selected from the group consisting of isopentenylpyrophosphate, zoledronic acid, and human recombinant IL-2.

3. The method of claim 1 , wherein said Natural Kill cells are primed with immobilized human IgGI or tumor cells opsonized with antibody recognizing tumor assotiated antigens.

4. The method of claim 1 , wherein administration of said Natural Kill cells results in co-stimulation through CD137L expressed on activated γδ T lymphocytes.

5. The method of claim 4, wherein said co-stimulation increases NKG2D expression on the NK cell surface leading to tumor cell lysis.

6. The method of claim 1 , wherein said expanded γδ T lymphocytes and said primed Natural Kill cells are co-cultured prior to administering to said individual.

7. The method of claim 6, wherein said primed Natural Kill cells and said expanded γδ T lymphocytes are co-cultured in a ratio of from about 2:1 to about 10:1.

8. The method of claim 1 , wherein said neoplastic disease is selected from the group consisting of squamous cell carcinoma of the head and neck, melanoma, breast cancer, B cell lymphoma, T cell lymphoma, and colon cancer lines.

9. A composition useful for treating a neoplastic disease in an individual in need of such treatment, comprising: expanded γδ T lymphocytes in vitro; and primed Natural Kill cells in vitro.

10. The composition of claim 9, wherein said γδ T lymphocytes are expanded in the presence of a compound selected from the group consisting of isopentenylpyrophosphate, zoledronic acid, and human recombinant IL-2.

11. The composition of claim 9, wherein said Natural Kill cells are primed with immobilized human IgGI or tumor cells opsonized with antibody recognizing tumor assotiated antigens.

12. The composition of claim 9, wherein said composition increases NKG2D expression on the NK cell surface leading to tumor cell lysis.

13. The composition of claim 9, wherein said expanded γδ T lymphocytes and said primed Natural Kill cells are co-cultured.

14. The composition of claim 13, wherein said primed Natural Kill cells and said expanded γδ T lymphocytes are co-cultured in a ratio of from about 2:1 to about 10:1.

15. The composition of claim 9, wherein said neoplastic disease is selected from the group consisting of squamous cell carcinoma of the head and neck, melanoma, breast cancer, B cell lymphoma, T cell lymphoma, and colon cancer lines.

16. 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 yδ T lymphocytes, wherein in activation of said yδ T lymphocytes results in enhancement of NK cell cytotoxicity.

17. The method of claim 16, wherein said γδ T lymphocytes are activated in the presence of a compound selected from the group consisting of isopentenylpyrophosphate, zoledronic acid, and human recombinant IL-2.

18. 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 yδ T lymphocytes; and administering an antibody directed against a tumor antigen associated with said neoplastic disease.

19. The method of claim 18, wherein in activation of said yδ T lymphocytes results in enhancement of NK cell cytotoxicity.

20. The method of claim 18, wherein said compound that activates γδ T lymphocytes is selected from the group consisting of isopentenylpyrophosphate, zoledronic acid, and human recombinant IL-2.

21. The method of claim 18, wherein said tumor antigen is CD20 on B cells.

22. The method of claim 21 , wherein said antibody is selected from the group consisting of rituximab, tositumomab, ibritumomab, ocrelizumab, and ofatumumab.

23. The method of claim 18, wherein said tumor antigen is epidermal growth factor receptor.

24. The method of claim 23, wherein said antibody is selected from the group consisting of cetuximab, panitumumab, and zalutumumab.

25. The method of claim 18, wherein said tumor antigen is carcinoembryonic antigen, 17-1A, colon cancer-specific antigen-3, colon cancer-specific antigen-4 and colon cancer-specific antigen-3.

26. The method of claim 25, wherein said antibody is edrecolomab.

27. The method of claim 18, wherein said tumor antigen is selected from the group consisting of MUC-1 , epithelial tumor antigen, and HER-2.

28. The method of claim 27, wherein said antibody is trastuzumab.

29. The method of claim 18, wherein said tumor antigen is tyrosinase, melanoma associated antigen, CTLA-4 and CD137.

30. The method of claim 29, wherein said antibody is selected from the group consisting of ipilimumab, and tremelimumab.

31. The method of claim 18, wherein said tumor antigen is a T cell lymphoma antigen.

32. The method of claim 31 , wherein said antibody is Denileukin difitox.