Methods and compositions for expansion and use of allogeneic gamma / delta-t cells

Abstract

To provide methods for treating cancer.SOLUTION: Provided are methods comprising the administration of donor-derived allogeneic γδ-T cells (gamma / delta-T cells) to a subject. In such methods, at least one lymphodepletion treatment or a Hematopoietic Stem Cell (HSC) transplant is administered to the subject, followed by the subsequent administration of donor-derived allogeneic γδ-T cells. The methods also comprise expanding the donor-derived allogeneic γδ-T cells ex vivo prior to administration to the subject. Such methods comprise exposing donor-derived allogeneic γδ-T cells to one or more γδ-T cell expansion compounds. Such a therapy induces an antitumor response in the subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the fields of immunology and methods of treating cancer. [Background technology]

[0002] 2. Background of the Invention In contrast to classical (conventional) αβ T cells, which recognize specific peptide antigens presented by major histocompatibility complex (MHC) molecules, γδ T cells (gamma / delta T cells) appear to recognize general determinants expressed by cells that become dysregulated as a result of malignant transformation or viral infection (Kabelitz D. et al. Cancer Res. 2007;67(1):5-8; Silva-Santos B, et al. Eur J Immunol. 2012;42(12):3101-5; Vantourout P and Hayday A. Nature reviews Immunology 2013;13(2):88-100). γδ T cells have the innate ability to recognize and kill a broad range of tumor cell types in a manner that does not require the presence of authentic tumor-specific antigens. Thus, there is a need to develop therapeutic strategies targeting γδ T cells against a variety of cancers. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Kabelitz D. et al. Cancer Res.2007;67(1):5-8 [Non-Patent Document 2] Silva-Santos B, et al., Eur J Immunol.2012;42(12):3101-5 [Non-Patent Document 3] Vantourout P and Hayday A. Nature reviews Immunology 2013;13(2):88-100 Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention Provided herein are methods for treating cancer in a subject. Such methods include administering donor-derived allogeneic γδ T cells (gamma / delta T cells) to the subject. In such methods, at least one lymphodepletion treatment or hematopoietic stem cell (HSC) transplant is administered to the subject, followed by administration of donor-derived allogeneic γδ T cells. The methods also include ex vivo expansion of donor-derived allogeneic γδ T cells prior to administration to the subject. Such methods include exposing donor-derived allogeneic γδ T cells to one or more γδ T cell expansion compounds. Such therapy induces an anti-tumor response in the subject. Also provided is the use of donor-derived allogeneic γδ T cells in methods of treating cancer. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for treating cancer in a subject, the method comprising: (a) administering at least one lymphodepletion treatment to said subject; (b) thereafter administering at least one dose of donor-derived allogeneic γδ T-cells to said subject, whereby prior to administering said γδ T-cells to said subject, said γδ T-cells are expanded ex vivo by culturing said γδ T-cells with at least one γδ T cell expansion compound; The method, wherein said treatment results in an anti-tumor response in said subject. (Item 2) 2. The method of claim 1, wherein the lymphodepleting treatment comprises administering one or more chemotherapeutic agents. (Item 3) 3. The method of claim 2, wherein the one or more chemotherapeutic agents comprise cyclophosphamide, fludarabine or melphalan. (Item 4) 2. The method of claim 1, wherein the lymphodepletion treatment comprises low-dose total body irradiation. (Item 5) 1. A method for treating cancer in a subject, the method comprising: (a) administering a hematopoietic stem cell (HSC) transplant to the subject; (b) thereafter administering at least one dose of donor-derived allogeneic γδ T-cells to said subject, and expanding said γδ T-cells ex vivo by culturing said γδ T-cells with at least one γδ T cell expansion compound prior to administering said γδ T-cells to said subject; The method, wherein said treatment results in an anti-tumor response in said subject. (Item 6) 6. The method of any one of items 1 to 5, further comprising administering to the subject one or more of etoposide, cisplatin, doxorubicin, 5-fluorouracil, vincristine, bortezomib, oxaliplatin, or ibrutinib. (Item 7) 7. The method of any one of items 1 to 6, further comprising administering to said subject at least one dose of a therapeutically effective amount of at least one monoclonal antibody therapy. (Item 8) 8. The method of claim 7, wherein the monoclonal antibody recognizes a tumor antigen. (Item 9) 9. The method according to any one of items 1 to 8, wherein said method further comprises administering to said subject an amino-bisphosphonate prior to said administration of allogeneic γδ T-cells derived from said donor. (Item 10) 10. The method of claim 9, wherein the amino-bisphosphonate is zoledronic acid. (Item 11) 11. The method of any one of items 1 to 10, wherein the method further comprises administering one or more additional cell therapies. (Item 12) 12. The method of item 11, wherein the one or more additional cell therapies are from the same allogeneic donor as the γδ T cells. (Item 13) 13. The method according to any one of items 1 to 12, wherein the γδ T cell proliferation compound comprises a synthetic phosphoantigen. (Item 14) 14. The method of claim 13, wherein the synthetic phosphoantigen is C-HDMAPP. (Item 15) 15. The method according to any one of items 1 to 14, further comprising culturing the γδ T cells with interleukin-2. (Item 16) 16. The method of any one of items 1 to 15, further comprising culturing the γδ T cells with one or more checkpoint inhibitors prior to administration of the γδ T cells to the subject. (Item 17) 17. The method of claim 16, wherein the one or more checkpoint inhibitors comprise an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, and an anti-galectin-9 antibody, an anti-IDO antibody and / or an anti-VISTA antibody. (Item 18) Prior to administration of the γδ T cells to the subject, the γδ T cells are treated with one or more of IL-15, an anti-CD277 antibody, an anti-TGF-beta antibody, prostaglandin E 2 18. The method according to any one of items 1 to 17, further comprising culturing with an inhibitor, adenosine, osteopontin, vitamin C and / or a hypomethylating agent. (Item 19) 19. The method according to any one of items 1 to 18, wherein the subject and the donor of the γδ T cells have a complete human leukocyte antigen (HLA) mismatch. (Item 20) 20. The method of any one of items 1 to 19, further comprising repeating steps (a) and (b) one or more times, wherein the donor-derived allogeneic γδ T-cells for each subsequent administration are derived from a different donor with a complete HLA mismatch compared to the subject and the previous donor or donors. [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 shows flow cytometric analysis of ex vivo expanded donor-derived allogeneic γδ T cells cultured for 21 days with various nM concentrations of the synthetic phosphoantigen C-HDMPP. [Diagram 2] FIG. 2 shows the fold expansion of donor-derived allogeneic γδ T cells cultured with 100 nM C-HDMPP over time. [Diagram 3] Figure 3 shows the in vitro antitumor activity of ex vivo expanded human γδ T cells against human tumor cell lines. Data are presented as tumor cell lysis at an E:T ratio of 25:1. [Figure 4] Figure 4 shows that relatively resistant MDA-MB-231 tumor cells can be rendered hypersensitive to killing by ex vivo expanded γδ T cells by pre-treating the tumor cells with zoledronic acid (ZOL) for 24 hours. Data are presented as tumor cell lysis at the indicated E:T ratios. [Diagram 5] Figure 5 shows that pretreatment of a variety of different tumor cells with zoledronic acid (ZOL) for 24 hours can render relatively resistant tumor cells hypersensitive to killing by ex vivo expanded γδ T cells. Data are presented as tumor cell lysis at the indicated E:T ratios. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description of the Invention Before describing the present invention in detail, it is to be understood that this invention is not limited to particular embodiments, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0007] Many modifications and other embodiments of the inventions described herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing description. It is understood, therefore, that the invention is not limited to the specific embodiments disclosed, but that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0008] I. Overview Provided herein are methods of treating cancer using various immunological procedures in which subjects receive subsequent clinical infusions of allogeneic γδ T cells (gamma / delta T cells) obtained from the blood of an otherwise healthy donor. The purpose of introducing allogeneic γδ T cells from a healthy donor is to replace or augment tumor-killing or infection-killing γδ T cells that have otherwise become non-functional in the subject.

[0009] As used herein, a "γδ T cell" or "gamma / delta T cell" is a T cell that expresses a T cell receptor that is composed of one gamma chain and one delta chain.

[0010] II. Methods of Treating Cancer The present invention provides a method of treating cancer in a subject, comprising first administering to the subject a lymphodepletion treatment or a hematopoietic stem cell (HSC) transplant, followed by administering to the subject at least one dose of donor-derived allogeneic γδ T cells.

[0011] By "treating" a subject with cancer is intended the administration of a therapeutically effective amount of at least one dose of donor-derived allogeneic γδ T-cells to a subject with cancer for the purpose of curing, healing, mitigating, alleviating, altering, relieving, ameliorating, improving or affecting the cancerous condition or one or more symptoms.

[0012] As used herein, "subject" refers to any animal (i.e., mammal) for which cancer treatment is desired, including, but not limited to, humans, primates, rodents, agricultural animals and livestock, including, but not limited to, dogs, cats, cows, horses, pigs, sheep, etc. In one embodiment, the subject is a mammal. In a particular embodiment, the subject is a human.

[0013] At least one dose of a therapeutically effective amount of donor-derived allogeneic γδ T cells is provided for use in the methods provided herein. As used herein, "therapeutically effective amount", "therapeutically effective dose", or "effective amount" refers to an amount that provides a therapeutic effect for a given condition and administration regimen. Thus, the phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant symptom of the host. In certain embodiments, "therapeutically effective amount" refers to an amount of donor-derived allogeneic γδ T cells that, when administered, results in a positive therapeutic response with respect to the treatment of cancer in a subject. A positive therapeutic response with respect to the treatment of cancer includes curing or ameliorating symptoms of the disease. In this context, a reduced response in a subject may be evidenced by the continuation or spread of the cancer. An improvement in a clinically significant symptom of a subject includes a decrease in tumor size, an increase in tumor necrosis, removal of the tumor from the host tissue, a decrease or amelioration of metastasis, or a decrease in any symptom associated with the cancer.

[0014] "Anti-tumor response" refers to a positive therapeutic response to the treatment of cancer, including curing or ameliorating symptoms of the disease. Anti-tumor responses in a subject include a reduction in tumor size, an increase in tumor necrosis, tumor clearance from host tissues, the presence of anti-tumor immune cells in a subject, the presence of immune cells in or adjacent to a tumor, a reduction or amelioration of metastasis, or a reduction in symptoms associated with cancer.

[0015] As demonstrated in the present invention, allogeneic γδ T cells expanded in vitro exhibit broad anti-tumor reactivity and can therefore target a broad range of tumors. Non-limiting examples of cancer types encompassed by the methods herein include carcinoma, sarcoma, leukemia, lymphoma, B-cell cancer, breast cancer, colon cancer, lung cancer, bladder cancer, pancreatic cancer, ovarian cancer, prostate cancer, brain cancer, acute lymphoblastic leukemia, and bone cancer. In one embodiment, the cancer comprises breast cancer. In another embodiment, the cancer is a hematological cancer. In a particular embodiment, the cancer is a B-cell cancer. B-cell cancers can include, for example, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, multiple myeloma, follicular lymphoma, mantle cell lymphoma, Burkitt's lymphoma, acute lymphoblastic leukemia, marginal zone lymphoma, or lymphoplasmacytic lymphoma.

[0016] A. Donor-derived allogeneic γδ T cells and their expansion The methods provided herein involve administering allogeneic γδ T cells derived from a donor. Compared to γδ T cells found in healthy donors, γδ T cells found in tumor-bearing hosts appear to be greatly reduced in number and / or functionally impaired in various important ways. Thus, in the majority of tumor-bearing hosts, the γδ T cell compartment may become irreversibly damaged or depleted, which occurs in a tumor-dependent manner. Although the mechanisms explaining these numerical or functional defects remain unclear, the potential consequences are clear. That is, strategies that rely on exploiting the innate anti-tumor properties of autologous (i.e., subject-derived) γδ T cells may prove ultimately ineffective if such defects are found to exist within the γδ T cell compartment of tumor-bearing hosts.

[0017] Provided herein is a novel method of using adoptively transferred allogeneic γδ T cells, i.e., cells obtained from an otherwise healthy donor, for the immunotherapy of malignant diseases. As used herein, "allogeneic γδ T cells" refers to γδ T cells obtained from an individual different from the subject into which the γδ T cells are to be infused. Indeed, the key to this concept is that γδ T cells obtained from a healthy donor are essentially undamaged and will be more effective against cancer cells, in contrast to γδ T cells present in a tumor-bearing host. Furthermore, donor-derived tumor-reactive γδ T cells can be easily expanded ex vivo from peripheral blood obtained from virtually any healthy donor, thus theoretically providing an unlimited supply of highly effective tumor-reactive γδ T cells that can be subsequently (and repeatedly) available for adoptive transfer into tumor-bearing hosts. As used herein, "donor-derived" refers to γδ T cells obtained from an individual different from the subject into which the γδ T cells are to be administered.

[0018] In some embodiments, the donor of the allogeneic γδ T cells is fully human leukocyte antigen (HLA) mismatched compared to the subject receiving the allogeneic γδ T cells. Full HLA mismatch means that the subject and the donor do not share any HLA antigens. In other embodiments, the donor is partially HLA mismatched. Partial HLA mismatch means that at least one HLA antigen is different between the subject and the donor. In such methods, the subject will be screened for preformed anti-HLA antibodies. If the subject is found to have preformed anti-HLA antibodies against the donor's non-shared HLA, the donor cells will not be used for that subject. In a preferred embodiment, the subject and the donor's γδ T cells have a full HLA mismatch.

[0019] Given the scarcity of γδ T cells in the blood of any person (donor or subject), the aforementioned adoptive transfer of donor-derived allogeneic γδ T cells is feasible only after a sufficient number of γδ T cells initially obtained from a healthy donor have been expanded ex vivo. Once harvested from the donor, these γδ T cells are expanded using a variety of different methods, including but not limited to those previously described by the inventors and others, e.g., Lopez RD et al., Blood. 2000; 96(12): 3827-37; Bennouna J et al., Cancer Immunol Immunother. 2008; 57(11): 1599-609; Bompas et al., J. Immunol. 2009; 10(1): 1599-609, each of which is incorporated herein by reference in its entirety. E et al., ASCO Meeting Abstracts.2006;24(18):2550;Espinosa E et al., Journal of Biological Chemistry.2001;276(21):18337-44;Gertner-Dardenne J et al., Blood.2009;Rojas RE et al., Infection&Immunity.2002;70(8):4019-27;Sicard H et al., J See Immunol. 2005;175(8):5471-80; and Squiban PJ et al., J Clin Oncol 2007;25(18suppl):3064.

[0020] The key to this concept is that, in contrast to γδ T cells present in a tumor-bearing host (i.e., patient), γδ T cells from a healthy (allogeneic) donor do indeed proliferate, whereas γδ T cells taken from a tumor-bearing patient lose the ability to proliferate and, even if they do, are much less effective at killing tumor cells. Moreover, donor-derived tumor-reactive γδ T cells can be readily expanded ex vivo from peripheral blood from virtually any healthy donor, thus theoretically providing an unlimited supply of highly effective tumor-reactive γδ T cells that can be subsequently (and repeatedly) available for adoptive transfer into a tumor-bearing host. Thus, in the methods provided herein, donor-derived allogeneic γδ T cells can be administered to a subject at least once, at least twice, at least three times, at least five times, at least ten times, at least twenty times, or as many times as necessary to induce an appropriate anti-tumor response.

[0021] In the methods provided herein, γδ T cells from an allogeneic donor are expanded ex vivo by culturing the γδ T cells with at least one γδ T cell expansion compound prior to administering the γδ T cells to a subject. By "γδ T cell expansion compound" is meant a compound that induces or enhances the proliferation of γδ T cells. In some examples, the γδ T cells may be cultured with an additional active compound, such as, for example, interleukin-2 (IL-2).

[0022] Various methods have been described for the ex vivo expansion of human γδ T cells, including but not limited to older methods first reported by R. Lopez described in WO1999046365(A1), which is incorporated herein by reference in its entirety, and newer methods such as Nieda's recently described in US20120107292(A1), which is incorporated herein by reference in its entirety. In Example 1, an exemplary method of ex vivo γδ T cell expansion is provided.

[0023] A variety of γδ T cell expansion compounds have been developed, including synthetic phosphoantigens (BrHPP; Phosphostim, U.S. Patent Nos. 7,109,183; 7,625,879; and 6,660,723; each of which is incorporated herein by reference in its entirety), that were specifically developed for either in vivo activation of endogenous γδ T cells when the compounds are administered to a patient as drugs, or for ex vivo expansion of γδ T cells initially harvested from a patient as described by Salot (U.S. Patent Application Publication No. 2005196385(A1), which is incorporated herein by reference in its entirety). Non-limiting examples of other γδ T cell expansion compounds intended for the same clinical use include: the synthetic phosphoantigen C-HDMAPP ("Picostim") described in U.S. Pat. No. 7,399,756 and European Patent No. 1408984; polymorphs of C-HDMAPP (WO 2010029062); and "Mayoly" (U.S. Pat. No. 8017596(B2)), each of which is incorporated herein by reference in its entirety. Additionally, amino-bisphosphonates such as pamidronate (Aredia) or zoledronate (Zometa), also known as zoledronic acid, can also be used to expand γδ T cells in the methods provided herein.

[0024] Amino-bisphosphonates act indirectly on γδ T cells by releasing isopentenyl pyrophosphate (IPP) from bystander cells present in PBMC cultures, which induces proliferation of γδ T cells. This can result in highly variable, inefficient, and unpredictable γδ T cell proliferation. In contrast, synthetic phosphoantigens, such as C-HDMAPP, act directly on γδ T cells, allowing for precise concentration optimization and predictable proliferation of γδ T cells. In one embodiment, the γδ T cell proliferation compound comprises a synthetic phosphoantigen. In a particular embodiment, the synthetic phosphoantigen is C-HDMAPP.

[0025] In some embodiments, γδ T cell expansion compounds can be used in combination with other compounds. Such other compounds can enhance the expansion of γδ T cells when used in combination with one or more γδ T cell expansion compounds. Other compounds can enhance the survival of γδ T cells during ex vivo expansion. Still other compounds can enhance the survival of γδ T cells in vivo after being introduced into humans. Still other compounds can promote preferential expansion of distinct functional phenotypes of γδ T cells that are more capable of killing tumors or homing to tumor sites in tissues due to preferential increased expression of certain homing receptors.

[0026] In one embodiment, donor-derived allogeneic γδ T cells are expanded in vitro by culturing γδ T cells with at least one phosphoantigen and an anti-CD277 antibody, which may be any anti-CD277 antibody, including but not limited to, the anti-CD277 antibodies described in US Patent Publication No. 2015 / 0353643, the entire contents of which are incorporated herein by reference, such as the 20.1 antibody.

[0027] In other embodiments, donor-derived allogeneic γδ T cells are expanded ex vivo by culturing γδ T cells with at least one phosphoantigen and one or more of the following substances: cytokines, including but not limited to interleukin-2 (IL-2), IL-12, IL-15, IL-17, IL-33 and synthetic agonists thereof; e.g., anti-CD277, as described in U.S. Patent Application Publication No. 2015 / 0353643; 20.1 antibodies, immune checkpoint inhibitors including but not limited to checkpoint inhibitor antibodies including but not limited to anti-PD-1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, antibodies against LAG3, antibodies against TIM-3, anti-galaectin-9 antibodies, anti-IDO antibodies and anti-VISTA antibodies; "epigenetic modifiers" including but not limited to decitabine (a hypomethylating agent) and azacytadine (a hypomethylating agent) and vitamin C; mTOR inhibitors including but not limited to rapamycin (also known as sirolimus) and everolimus; and various other additives including but not limited to antibodies against TGF-beta; prostaglandin E2 inhibitors, GM-CSF, G-CSF, and osteopontin.

[0028] In one embodiment, the donor-derived allogeneic γδ T cells are cultured with interleukin-2 (IL-2). In another embodiment, the donor-derived allogeneic γδ T cells are cultured with one or more checkpoint inhibitors prior to administering the γδ T cells to a subject. In a particular embodiment, the one or more checkpoint inhibitors comprise an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, and an anti-galaectin-9 antibody, an anti-IDO antibody, and / or an anti-VISTA antibody.

[0029] In another embodiment, donor-derived allogeneic γδ T-cells are cultured with one or more of IL-15, anti-CD277 antibody, anti-TGF-beta antibody, prostaglandin E2 inhibitor, adenosine, osteopontin, vitamin C, and / or hypomethylating agents prior to administration of the γδ T-cells to the subject.

[0030] None of the above compounds or methods have been developed for use in the ex vivo expansion of allogeneic γδ T-cells obtained from healthy donors, with the subsequent transfer of these cells into a subject as provided in the therapeutic methods provided herein.

[0031] The tumor microenvironment (TME) has the ability to suppress the function of tumor-infiltrating lymphocytes, including γδ T cells, which may lead to the loss or reduction of antitumor immune responses. In particular, overcoming this TME-mediated immunosuppression remains a major challenge for cell-based immunotherapy. After expansion, γδ T cells can be modified to directly or indirectly disrupt or inhibit specific immunosuppressive pathways within the TME. This can be achieved by a variety of methods, including, but not limited to, gene editing using CRISPR technology to render γδ T cells resistant to TME-mediated immunosuppression; disrupting receptors on γδ T cells that respond to soluble immunosuppressive factors within the TME; or by disrupting receptors / ligands on γδ T cells that bind to contact-dependent factors present on various cells within the TME. Non-limiting examples include disruption of receptors for TGF-beta or PD-1.

[0032] B. Adoptive transfer of expanded allogeneic donor-derived γδ T cells The methods of treating cancer provided herein require the subject to accept allogeneic donor-derived γδ T cells by performing an initial immunological procedure or treatment. Means for making the subject accept donor-derived allogeneic γδ T cells include, but are not limited to, performing an allogeneic hematopoietic stem cell (HSC) transplant using HSCs initially obtained from a suitable bone marrow donor or lymphodepletion by lymphodepletion treatment. Without these initial immunological procedures (treatments), donor-derived γδ T cells would be immediately rejected by the subject.

[0033] Provided herein are two distinct but related immunological strategies / platforms that specifically enable the adoptive transfer of tumor-reactive γδ T cells from healthy donors to tumor-bearing hosts (subjects).

[0034] 1. Hematopoietic Stem Cell (HSC) Transplantation Platform A first platform for enabling subsequent treatment of cancer by adoptive transfer of donor-derived allogeneic γδ T-cells for use in the methods provided herein involves administering a hematopoietic stem cell (HSC) transplant to the subject prior to administering the donor-derived allogeneic γδ T-cells. As used herein, "hematopoietic stem cell (HSC) transplant" refers to the transplantation of multipotent hematopoietic stem cells. Hematopoietic stem cells can be obtained, for example, from bone marrow, peripheral blood, or umbilical cord blood.

[0035] In one embodiment, a method of treating cancer comprises first administering a hematopoietic stem cell (HSC) transplant to a subject. In such a method, the subject is then administered at least one dose of donor-derived allogeneic γδ T cells. The γδ T cells used in such a method are expanded ex vivo by culturing with at least one γδ T cell expansion compound before administering the γδ T cells to the subject. Thus, this treatment induces an anti-tumor response in the subject.

[0036] Also described herein is the use of donor-derived allogeneic γδ T cells in a method for treating cancer. The use comprises first administering a hematopoietic stem cell (HSC) transplant to a subject; then administering at least one dose of donor-derived allogeneic γδ T cells to the subject, where the γδ T cells are expanded ex vivo by culturing the γδ T cells with at least one γδ T cell expansion compound before administering the γδ T cells to the subject. In such a use, the treatment induces an anti-tumor response in the subject.

[0037] In another embodiment, a method of treating cancer comprises first administering a hematopoietic stem cell (HSC) transplant to a subject. In such methods, the subject is subsequently administered a combination therapy of at least one dose of donor-derived allogeneic γδ T cells and at least one dose of a therapeutically effective amount of at least one monoclonal antibody treatment. The γδ T cells used in such methods are expanded ex vivo by culturing with at least one γδ T cell expansion compound prior to administering the γδ T cells to the subject.

[0038] In another embodiment, a method of treating cancer comprises first administering a hematopoietic stem cell (HSC) transplant to a subject. In such methods, the subject is then administered at least one dose of donor-derived allogeneic γδ T cells. The γδ T cells used in such methods are expanded ex vivo by culturing with at least one phosphoantigen and an anti-CD277 antibody prior to administering the γδ T cells to the subject.

[0039] In conventional HSC transplantation, antitumor effects are provided by high-dose chemotherapy and / or radiation delivered as part of transplant conditioning. However, it is also clear that secondary nonspecific immune-mediated graft-versus-tumor effects also contribute to disease control. Although the mechanisms by which this occurs are poorly understood, it is clear that (allogeneic) immune effector cells derived from a qualified donor play an important role in the graft-versus-tumor effects seen in the setting of allogeneic HSC transplantation in select diseases.

[0040] Indeed, given the potent antitumor effects of donor-derived immunity, it is not uncommon to perform donor lymphocyte infusion (DLI) after allogeneic HSC transplantation to induce (promote) or maintain post-transplant remission in certain diseases. However, this is generally performed by introducing crude, unfractionated preparations of donor-derived peripheral blood lymphocytes that contain primarily αβ T cells. Thus, not unexpectedly, such procedures commonly result in the development of sometimes life-threatening graft-versus-host disease (GVHD) in the recipient (15).

[0041] Given the potent innate antitumor properties of γδ T cells and their inability to cause GVHD, donor-derived allogeneic γδ T cells administered as highly purified γδ T cell donor lymphocyte infusion (γδ T cell DLI) after allogeneic HSC transplantation are ideal cells to be introduced in such situations.

[0042] In essence, the allogeneic HSC transplantation procedure itself will be relegated to a supportive role serving as a therapeutic platform for the subsequent adoptive transfer of tumor-reactive donor-derived allogeneic γδ T cells (γδ T cell DLI).

[0043] 2. Lymphocyte Depletion Platform A second platform to enable subsequent treatment of cancer by adoptive transfer of donor-derived allogeneic γδ T-cells for use in the methods provided herein involves administering a lymphodepletion treatment to the subject prior to administering the donor-derived allogeneic γδ T-cells.

[0044] It is envisioned that through deliberate immunosuppression of the subject using a variety of treatments, including but not limited to administration of chemotherapy or other compounds as well as radiation, the subject may be rendered temporarily receptive to donor-derived allogeneic γδ T cells that retain potent anti-tumor properties.

[0045] As used herein, "lymphodepletion" refers to the depletion of lymphocytes in a subject. Lymphodepletion can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more depletion of lymphocytes in a subject. "Lymphocyte" refers to a type of white blood cell that is part of the immune system. Lymphocytes include, for example, various types of B cells, T cells, or natural killer (NK) cells. Lymphodepletion can be achieved by administering at least one lymphodepletion treatment. As used herein, "lymphodepletion treatment" refers to any treatment that results in lymphodepletion in a subject. Lymphocyte depletion treatments include, but are not limited to, methylprednisolone, radiation, low-dose total body irradiation, etoposide, cisplatin, doxorubicin, 5-fluorouracil, vincristine, bortezomib, oxaliplatin, or lymphocyte depletion chemotherapy agents, including cyclophosphamide, fludarabine, or melphalan. In one embodiment, lymphocyte depletion treatments include administering one or more chemotherapeutic agents. In a particular embodiment, the chemotherapeutic agent includes cyclophosphamide, fludarabine, or melphalan. In another embodiment, lymphocyte depletion treatments include low-dose total body irradiation.

[0046] Although the above HSC transplantation platforms are clearly logical, from a clinical perspective, such an approach, which requires the performance of allogeneic HSC transplantation, may not be appropriate for many subjects. Therefore, a second platform, lymphodepletion, was developed, which may have much greater clinical applicability.

[0047] The lymphodepletion platform establishes temporary, rather than permanent, donor-host immune tolerance prior to delivery of allogeneic γδ T cells, obviating the need for allogeneic HSC transplantation.

[0048] The lymphodepletion platform is designed to accommodate eventual rejection of adoptively transferred, donor-derived allogeneic γδ T cells. In (temporarily) lymphodepleted hosts, adoptively transferred allogeneic γδ T cells are nevertheless capable of mediating antitumor effects, especially when delivered repeatedly on a regular basis.

[0049] In pilot studies in animal models, the inventors found that a functional window of opportunity could be established during which adoptively transferred allogeneic γδ T cells could mediate a measurable antitumor effect in lymphodepleted tumor-bearing hosts. This functional window of opportunity could be created in a clinically relevant manner, for example, using cyclophosphamide, a drug that is both immunosuppressive (i.e., lymphodepleting) and, under certain circumstances, effective against a variety of cancers.

[0050] In one embodiment, the method of treating cancer comprises first administering a lymphodepletion treatment to a subject. In such methods, the subject is then administered at least one dose of donor-derived allogeneic γδ T cells. The γδ T cells used in such methods are expanded ex vivo by culturing with at least one γδ T cell expansion compound prior to administering the γδ T cells to the subject.

[0051] Also described herein is the use of donor-derived allogeneic γδ T cells in a method for treating cancer.The use comprises administering at least one lymphodepletion treatment to a subject; then administering at least one dose of donor-derived allogeneic γδ T cells to a subject, wherein the γδ T cells are expanded ex vivo by culturing the γδ T cells with at least one γδ T cell expansion compound before administering the γδ T cells to a subject.In such a use, the treatment induces an anti-tumor response in the subject.

[0052] In another embodiment, the method of treating cancer comprises first administering a lymphodepletion treatment to the subject. In such a method, the subject is subsequently administered a combination therapy of at least one dose of donor-derived allogeneic γδ T cells and at least one dose of a therapeutically effective amount of at least one monoclonal antibody treatment. The γδ T cells used in such a method are expanded ex vivo by culturing with at least one γδ T cell expansion compound before administering the γδ T cells to the subject. Thus, the combination therapy induces an anti-tumor response in the subject.

[0053] In another embodiment, the method of treating cancer comprises first administering a lymphodepletion treatment to the subject. In such methods, the subject is then administered at least one dose of donor-derived allogeneic γδ T-cells. The γδ T-cells used in such methods are expanded in vitro by culturing with at least one phosphoantigen and an anti-CD277 antibody prior to administering the γδ T-cells to the subject.

[0054] In all the above embodiments, a particular strategy for selecting a suitable donor cell product for use with a given patient involves screening the donor and patient for human leukocyte antigen (HLA) type (both class I and II), where the cell product generated from each donor (which can be produced in large quantities and then cryopreserved in single dose units) is fully characterized with respect to HLA; and / or screening the patient for anti-HLA antibodies, for example using standard Luminex-based assays. One strategy for selecting a compatible donor-derived γδ T-cell product for use with a given patient involves identifying a donor-derived cell product as "suitable" or "compatible" for use with a given patient if the donor-derived cell product does not share HLA with the patient and / or if the patient does not have anti-HLA antibodies against the non-shared HLA of the donor (cell product), to avoid immediate antibody-mediated (humoral) rejection of the donor cells.

[0055] The lymphodepletion procedure allows the HLA-mismatched donor's γδ T cells to be accepted temporarily. This occurs by suppressing the host's cellular immune response, which would recognize and immediately reject the HLA-mismatched γδ T cells. Because the subject is screened for the absence of anti-HLA antibodies against the selected donor, there is no humoral (antibody-mediated) rejection of the donor γδ T cells. Thus, the lymphodepleted state is only temporary, and the subject eventually regains immune function and rejects the γδ T cells of a given HLA-mismatched donor. However, the method only requires that the HLA-mismatched γδ T cells be present long enough to be able to kill tumor cells before rejection occurs.

[0056] After rejection of donor-derived allogeneic γδ-T cells, the lymphodepletion procedure followed by administration of allogeneic donor-derived γδ T cells can be repeated. In such a method, lymphodepletion will again be administered to the subject, but the administered γδ T cells will be from a different HLA-mismatched donor. In some embodiments, the subject and the second γδ T cell donor will have a complete or partial HLA mismatch. The cycle of lymphodepletion and administration of donor-derived allogeneic γδ T cells may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times. In such a method, each subsequent donor similarly does not share HLA with any of the previous donors.

[0057] In one embodiment, the method comprises administering at least one lymphodepletion treatment to the subject; and thereafter administering to the subject at least one dose of donor-derived allogeneic γδ T-cells, where the γδ T-cells are expanded ex vivo by culturing the γδ T-cells with at least one γδ T-cell expansion compound prior to administering the γδ T-cells to the subject, and the method is repeated one or more times, where the donor-derived γδ T-cells for each subsequent administration are derived from a different donor having a complete HLA mismatch compared to the subject and the previous donor or donors.

[0058] A bank of γδ T cells that maximizes a patient's chances of obtaining a compatible cell product can be generated by creating a repository of γδ T cells derived from individuals with rare HLA types, increasing the likelihood that a given patient does not share HLA with a given donor. The bank of γδ T cells can also include γδ T cells that have been pre-screened for their in vitro anti-tumor activity against a panel of human tumor cell lines. While γδ T cells exhibit killing power against most tumor cell lines, cells from some donors may be more effective against certain classes of cancers, or even against certain types of cancer. In one embodiment, a method is provided for selecting γδ T cells from a bank of γδ T cells for use in treating cancer in a subject. The selection method includes selecting γδ T cells from a donor with a specific HLA type that is completely mismatched with the subject's HLA. In some embodiments, the HLA type of the selected γδ T cells is a complete HLA mismatch compared to the HLA of a previous donor used for the same subject. The selection method may also include selecting γδ T cells that are highly effective against the type of cancer being treated.

[0059] C. Monoclonal antibody therapy In some embodiments, donor-derived allogeneic γδ T-cells are administered as a combination therapy comprising a therapeutically effective amount of at least one monoclonal antibody therapy. A variety of antibodies can be used in the methods of treating cancer provided herein. The various antibodies disclosed herein and for use in the methods provided herein can be produced using antibody production methods known to those skilled in the art.

[0060] The term "antibody" is used in the broadest sense and includes fully assembled antibodies, antibody fragments capable of binding antigen (e.g., Fab, F(ab')2, Fv, single chain antibodies, diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, and the like), and recombinant peptides including the above.

[0061] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 10:1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0062] In one embodiment, the antibody is a monoclonal antibody. By "monoclonal antibody" is intended an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, such as those produced by a clonal population of B cells, and should not be construed as requiring production of the antibody by a particular method.

[0063] For example, the monoclonal antibodies used according to the methods provided herein may be produced by the hybridoma method first described in Kohler et al. (1975) Nature 256:495, or a modification thereof. Generally, mice are immunized with a solution containing an antigen. Immunization can be performed by mixing or emulsifying the antigen-containing solution in saline, preferably an adjuvant such as Freund's complete adjuvant, and parenterally injecting the mixture or emulsion. Methods of immunization known in the art can be used to obtain monoclonal antibodies. After immunizing the animal, the spleen (and optionally several large lymph nodes) are removed and dissociated into single cells. The spleen cells can be screened by applying a cell suspension to a plate or well coated with the antigen of interest. B cells expressing membrane-bound immunoglobulin specific for the antigen will bind to the plate and not be washed away. The resulting B cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas and cultured in a selective medium. The resulting cells are plated by serial dilution and assayed for the production of antibodies that specifically bind the antigen of interest (and that do not bind to unrelated antigens). Selected monoclonal antibody (mAb)-secreting hybridomas are then cultured in vitro (in tissue culture bottles or hollow fiber reactors) or in vivo (as ascites in mice).

[0064] Alternatively, the monoclonal antibodies may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol. Biol. 222:581-597; and U.S. Pat. No. 5,514,548.

[0065] "Epitope" means a portion of an antigen molecule to which an antibody is raised and to which the antibody binds. Epitopes can include linear amino acid residues (i.e., the residues in the epitope are arranged one after the other in a linear fashion), non-linear amino acid residues (referred to herein as "non-linear epitopes" - these epitopes are not arranged in a continuous fashion), or both linear and non-linear amino acid residues.

[0066] Furthermore, the term "antibody" as used herein includes chimeric antibodies and humanized monoclonal antibodies. By "chimeric" antibodies is intended an antibody that is most preferably derived using recombinant deoxyribonucleic acid technology and contains both human (including immunologically "related" species, e.g., chimpanzees) and non-human components. Thus, the constant region of the chimeric antibody is substantially identical to the constant region of a natural human antibody. The variable region of the chimeric antibody is most preferably derived from a non-human source and has the desired antigen specificity. The non-human source may be a vertebrate source that can be used to generate antibodies against materials containing tumor antigens or tumor antigen polypeptides. Such non-human sources include, but are not limited to, rodents (e.g., rabbits, rats, mice, etc.; see, e.g., U.S. Pat. No. 4,816,567) and non-human primates (e.g., Old World monkeys, apes, etc.; see, e.g., U.S. Pat. Nos. 5,750,105 and 5,756,096).

[0067] In some embodiments, monoclonal antibody therapy includes humanized antibodies. "Humanized" refers to forms of antibodies that contain minimal sequence derived from non-human immunoglobulin sequences. Thus, such "humanized" antibodies may include antibodies in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species.

[0068] Depending on the amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, IgG1 and IgG3 isotypes have ADCC (antibody-dependent cell-mediated cytotoxicity) activity.

[0069] The monoclonal antibody for use in the methods provided herein can be any monoclonal antibody of various antibody classes. In one embodiment, the monoclonal antibody is an IgG class antibody. In other embodiments, the monoclonal antibody can be an IgM, IgE, IgD, or IgA class monoclonal antibody. In certain embodiments, the antibody is an IgG isotype, such as IgG1, IgG2, IgG3, or IgG4.

[0070] The monoclonal antibodies used in the methods provided herein can recognize any antigen, hi some embodiments, the monoclonal antibodies recognize tumor antigens. Any known therapeutic monoclonal antibody can be used in the combination therapy provided herein.

[0071] For example, therapeutic antibodies against CD20 are known in the art for treating various types of B-cell malignancies, including, for example, rituximab (Rituxan) (see, e.g., Weiner, GJ. (2010) Semin Hematol. 47(2):115-123). In one embodiment, the monoclonal antibody comprises a CD20 antibody. In a particular embodiment, the monoclonal antibody therapy comprises rituximab.

[0072] In another non-limiting example, therapeutic antibodies against HER-2, such as Herceptin (Trastuzumab), are known in the art and are used to treat breast cancer (see, e.g., Capietto AH et al. (2011) J Immunol. 187(2):1031-8). In one embodiment, the monoclonal antibody comprises a HER-2 antibody. In a particular embodiment, the monoclonal antibody therapy is Herceptin (Trastuzumab).

[0073] Another non-limiting example includes checkpoint inhibitor antibodies, including but not limited to nivolumab (Opdivo), pembrolizumab (Keytruda), or ipilimumab (Yervoy).

[0074] In some embodiments, two or more monoclonal antibody therapies may be administered. In certain embodiments, two or more monoclonal antibody therapies may be administered to a subject.

[0075] In another embodiment, the therapeutic antibody may be a tribody. Tribody refers to a multifunctional antibody derivative. Tribodies may contain an antigen binding domain and / or an effector domain, including but not limited to an immunotoxin, an immunocytokine, an enzymatic function, or any functional domain. In one embodiment, the tribody contains one or more effector domains that enhance the anti-tumor activity of γδ T cells. In another embodiment, the tribody contains one or more antigen binding domains specific for one or more tumor antigens. In a particular embodiment, the tribody contains one or more antigen binding domains that bind HER2 and a CD16 binding domain, including but not limited to the [(HER2)2xCD16] tribody described in Oberg et al. (2018) Front. Immunol. 9:814, the entire contents of which are incorporated herein by reference.

[0076] D. Combination Therapy The methods provided herein may include combination therapy of donor-derived allogeneic γδ T cells with one or more therapeutic agents. The term "combination" or "combination therapy" is used herein in its broadest sense to mean that a subject is treated with at least two therapeutic regimens. The timing of administration of different therapeutic regimens can be varied, provided that a beneficial effect of the combination of these therapeutic regimens is achieved. Treatment with donor-derived allogeneic γδ T cells in combination with one or more therapeutic agents may be simultaneous (concurrent), sequential (sequential), or a combination thereof. Thus, a subject receiving combination therapy may receive both donor-derived allogeneic γδ T cells and one or more therapeutic agents at the same time (i.e., simultaneously) or at different times (i.e., on the same day or sequentially in any order on different days), as long as the therapeutic effect of the combination of both is elicited in the subject receiving the treatment. When donor-derived allogeneic γδ T-cells or one or more therapeutic agents are administered simultaneously, they may be administered as separate pharmaceutical compositions, each containing either donor-derived allogeneic γδ T-cells or a therapeutic agent, or as a single pharmaceutical composition containing both of these agents. In some embodiments, the one or more therapeutic agents are administered as a pretreatment prior to administration of donor-derived allogeneic γδ T-cells. The one or more therapeutic agents may be administered to the subject at least 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, or more prior to administration of donor-derived allogeneic γδ T-cells.

[0077] The combination therapy provided herein may be accomplished intermittently. By "intermittent combination therapy" is intended a period of combination therapy with donor-derived allogeneic γδ T-cells and one or more therapeutic agents, followed by a rest period, followed by another period of combination therapy with donor-derived allogeneic γδ T-cells and one or more therapeutic agents, etc.

[0078] In the methods provided herein, donor-derived allogeneic γδ T cells may be combined with one or more therapeutic agents to enhance the in vivo killing activity and / or homing ability of γδ T cells. Thus, tumors, including tumors that are relatively resistant to treatment, may be sensitized to killing by γδ T cells. γδ T cell sensitizing agents include, but are not limited to, chemotherapeutic agents including etoposide, cisplatin, doxorubicin, 5-fluorouracil, vincristine, bortezomib, and oxaliplatin; small molecules including ibrutinib; therapeutic antibodies including rituximab, trastuzumab, nivolumab, pembrolizumab, and ipilimumab; or amino-bisphosphonates including zoledronic acid. In one embodiment, the methods provided herein further comprise administering to the subject one or more of etoposide, cisplatin, doxorubicin, 5-fluorouracil, vincristine, bortezomib, oxaliplatin, or ibrutinib.

[0079] The methods provided herein include pre-treating a subject with an amino-bisphosphonate prior to administration of donor-derived allogeneic γδ T cells. As described elsewhere herein, amino-bisphosphonates (such as zoledronic acid) can act indirectly on γδ T cells by causing bystander cells to release isopentenyl pyrophosphate (IPP). This occurs because zoledronic acid inhibits the enzyme farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate biosynthetic pathway, in bystander cells. Disruption of the pathway leads to the accumulation of IPP, which is eventually released from the cells and then stimulates γδ T cells. Tumor cells exposed to aminobisphosphonates (such as zoledronic acid) can also release IPP and associated phosphoantigens. Because tumor cells already preferentially produce IPP as a result of a dysregulated state, the effect of aminobisphosphonates is even greater. This enhanced release of IPP by tumor cells first exposed to aminobisphosphonates renders these cells highly susceptible to killing by γδ T cells. Moreover, this IPP functions as a chemoattractant for γδ T cells, making them more potent in vivo. In one embodiment, the method of treating cancer further comprises administering an amino-bisphosphonate to the subject prior to administering the donor-derived allogeneic γδ T cells. In a particular embodiment, the amino-bisphosphonate is zoledronic acid.

[0080] Donor-derived allogeneic γδ T cells may be administered as a combination therapy with other cell therapies to enhance anti-tumor activity. Donor-derived allogeneic γδ T cells may be administered simultaneously or sequentially with any number of other cell types, including but not limited to γδ T cells of the Vδ1 variant, or any γδ T cell subset that is non-Vδ2; NK cells; or CAR-T cells (derived from αβ-T cells or γδ T cells). In an embodiment, the method provided herein further comprises administering one or more additional cell therapies. In some methods, the one or more additional cell therapies are derived from the same allogeneic donor as the γδ T cells.

[0081] As described elsewhere herein, the methods provided herein may include a combination therapy of donor-derived allogeneic γδ-T cells and monoclonal antibody therapy that allows targeted killing of tumor cells in a subject with cancer. In such a method, a monoclonal antibody against a surface tumor antigen can redirect donor-derived allogeneic γδ T cells to the tumor. Although γδ T cells have innate anti-tumor activity independent of ADCC, activation of γδ T cells with at least one γδ T cell expansion compound (i.e., any of the various γδ T cell expansion compounds provided herein) increases the expression of Fc receptors, thereby increasing the likelihood that γδ T cells will bind to the Fc portion of an antibody. In this way, the combination therapy may enhance ADCC mediated by a therapeutic antibody by specifically directing γδ T cells to tumor targets. See, e.g., Gertner-Dardenne J et al. (2009) Blood. 113: 4875-4884; and Capietto AH et al. (2011) J. Immunol. 187(2): 1031-8; each of which is incorporated herein by reference in its entirety. In one embodiment, the method provided herein further comprises administering to the subject at least one dose of a therapeutically effective amount of at least one monoclonal antibody treatment. In certain embodiments, the monoclonal antibody recognizes a tumor antigen.

[0082] In one embodiment, a method for treating cancer in a subject is provided, comprising administering to the subject at least one lymphodepletion treatment; thereafter administering to the subject a combination therapy of at least one dose of donor-derived allogeneic γδ T cells and at least one dose of a therapeutically effective amount of at least one monoclonal antibody treatment, where the γδ T cells have been expanded ex vivo by culturing the γδ T cells with at least one γδ T cell expansion compound prior to administering the γδ T cells to the subject; said combination therapy results in an anti-tumor response in the subject. In another embodiment, a method for treating cancer in a subject is provided, comprising administering to the subject a hematopoietic stem cell (HSC) transplant; thereafter administering to the subject a combination therapy of at least one dose of donor-derived allogeneic γδ T cells and at least one dose of a therapeutically effective amount of at least one monoclonal antibody treatment, where the γδ T cells have been expanded ex vivo by culturing the γδ T cells with at least one γδ T cell expansion compound prior to administering the γδ T cells to the subject; said combination therapy results in an anti-tumor response in the subject.

[0083] In some embodiments, the combination therapy of donor-derived allogeneic γδ T cells and monoclonal antibody therapy results in an anti-tumor response that is greater than the anti-tumor response observed with donor-derived allogeneic γδ T cell therapy or monoclonal antibody therapy alone. By "greater than" it is meant that the combination therapy results in a statistically significant increase in the anti-tumor response compared to either γδ T cell therapy or monoclonal antibody therapy alone. The anti-tumor response of the combination therapy can be at least a statistically significant increase of 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more compared to the anti-tumor response of either γδ T cell therapy or monoclonal antibody therapy alone.

[0084] In other embodiments, combination therapy of at least one dose of donor-derived allogeneic γδ T cells and at least one dose of a therapeutically effective amount of at least one monoclonal antibody treatment induces antibody-dependent cellular cytotoxicity (ADCC).

[0085] In a particular embodiment, the donor-derived allogeneic γδ T-cells and a therapeutically effective amount of at least one monoclonal antibody treatment are administered simultaneously to the subject, while in another embodiment, the donor-derived allogeneic γδ T-cells and a therapeutically effective amount of at least one monoclonal antibody treatment are administered at different times to the subject.

[0086] In a further embodiment, the subject is a mammal. In yet a further embodiment, the subject is a human.

[0087] The ex vivo expanded donor-derived allogeneic γδ T cells provided herein may be pre-coated with a monoclonal antibody prior to administering the γδ T cells to a subject. In such methods, the γδ T cells are cultured with an antibody to allow binding of Fc receptors on the γδ T cells to the Fc portion of the antibody for subsequent administration to a subject. The γδ T cells and the antibody may be cultured for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours or more, as long as the γδ T cells are bound to the antibody. Assays for measuring binding of antibodies to Fc receptors on γδ T cells are known in the art and include, for example, flow cytometry, radioligand binding assays, fluorescent binding assays or colorimetric binding assays.

[0088] In some embodiments, multiple administrations of antibody-coated γδ T-cells may be administered to a subject during the course of cancer treatment. In other embodiments, a subject receiving antibody-coated γδ T-cells may also receive donor-derived allogeneic γδ T-cells, monoclonal antibody therapy and / or a combination of donor-derived allogeneic γδ-T-cells and monoclonal antibody therapy as provided herein.

[0089] III. Method of Administration The methods of treating cancer provided herein can include administration of the treatment via any parenteral route, including, but not limited to, intramuscular, intraperitoneal, intravenous, and the like.

[0090] Furthermore, as used herein, "pharmaceutically acceptable carriers" are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M or 0.05M phosphate buffer or 0.8% saline. Furthermore, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, collating agents, inert gases, and the like.

[0091] Controlled or sustained release compositions include formulations in lipophilic depots (e.g., fatty acids, waxes, oils). Also encompassed herein are particulate compositions coated with polymers (e.g., poloxamers or poloxamines) and compounds bound to antibodies against tissue-specific receptors, ligands or antigens, or bound to ligands of tissue-specific receptors. Other embodiments of the compositions presented herein incorporate protective coatings, protease inhibitors or penetration enhancers in particulate form for various routes of administration, including parenteral, pulmonary, nasal and oral.

[0092] When administered, compounds are often rapidly cleared from mucosal surfaces or circulation and may therefore elicit relatively short-lived pharmacological activity. As a result, relatively large doses of bioactive compounds may need to be administered frequently to maintain therapeutic efficacy. Compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in the blood after intravenous injection than the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications may increase the solubility of the compounds in aqueous solutions, eliminate aggregation, increase the physical and chemical stability of the compounds, and may also significantly reduce the immunogenicity and reactivity of the compounds. As a result, the desired in vivo bioactivity may be achieved by administering such polymeric compound abducts less frequently or in lower doses than the unmodified compounds.

[0093] Dosage of antibody therapy. Sufficient amounts include, but are not limited to, about 1 μg / kg to about 100 μg / kg, about 100 μg / kg to about 1 mg / kg, about 1 mg / kg to about 10 mg / kg, 10 mg / kg to about 100 mg / kg, about 100 mg / kg to about 500 mg / kg, or about 500 mg / kg to about 1000 mg / kg. This amount may be 10 mg / kg. Pharmaceutically acceptable forms of the composition include pharma- ceutically acceptable carriers.

[0094] The preparation of therapeutic compositions containing active ingredients is well known in the art. Generally, such compositions are prepared as an aerosol of the polypeptide delivered to the nasopharynx, or as an injectable, either in liquid solution or suspension, although solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. Preparations can also be emulsified. The active therapeutic ingredient is often mixed with an excipient that is pharma-ceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if necessary, the composition can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient.

[0095] The active ingredient can be formulated in the therapeutic composition as a neutralized pharma-ceutically acceptable salt form.Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptides), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. The salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0096] One or more components of the therapeutic composition provided herein can be introduced parenterally, transmucosally, for example, orally, nasally, pulmonary, or rectally, or transdermally.Preferably, administration is parenteral (e.g., via intravenous injection), including, but not limited to, intraarteriolar, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.The term "unit dose" when used in relation to the therapeutic composition provided herein refers to a physically separate unit suitable as a unit dose for humans, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect in association with a diluent; i.e., carrier, or vehicle.

[0097] In another embodiment, the active compound may be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al., "Liposomes in the Therapy of Infectious Disease and Cancer", Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0098] In yet another embodiment, the therapeutic compound may be delivered in a controlled release system. For example, the therapeutic composition may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see "Medical Applications of Controlled Release", Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); "Controlled Drug Bioavailability, Drug Product Design and Performance", Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the tumor, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (1990) Science 249:1527-1533.

[0099] The subject for which administration of the active ingredient as above is an effective treatment regimen for cancer is preferably human, but can be any animal.Therefore, as those skilled in the art can easily understand, the method and pharmaceutical composition provided herein are particularly suitable for administration to any animal, particularly mammal, including but not limited to domestic animals such as cat or dog subjects, livestock such as but not limited to cattle, horse, goat, sheep and pig subjects, wild animals (whether wild or zoo), research animals such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, i.e. animals for veterinary use.

[0100] In the therapeutic methods and compositions provided herein, a therapeutically effective dose of the active ingredient is provided. The therapeutically effective dose can be determined by the ordinarily skilled medical practitioner based on the characteristics of the patient, such as age, weight, sex, symptoms, comorbidities, other diseases, etc., as is well known in the art. Furthermore, as further routine research is conducted, more specific information will become available regarding the appropriate dosage levels for the treatment of various symptoms in various patients, and the ordinarily skilled practitioner can ascertain suitable administration, taking into account the recipient's treatment context, age, and overall health. In general, for intravenous injection or infusion, the dosage may be lower than for intraperitoneal, intramuscular, or other routes of administration. The administration schedule may vary depending on the circulating half-life and the formulation used. The composition is administered in a manner compatible with the formulation to be administered in a therapeutically effective amount. The precise amount of active ingredient required to be administered depends on the judgment of the practitioner and is specific to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, more preferably from 1 to several milligrams of active ingredient per kilogram of individual body weight per day, and will also vary depending on the route of administration. Suitable regimens for initial administration and booster injections are also variable, but typically an initial administration is followed by repeated administrations at intervals of one or more hours, followed by subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain a blood concentration of 10 nanomolar to 10 micromolar is contemplated.

[0101] Administration with other compounds For the treatment of cancer, the active ingredient can be administered in combination with one or more pharmaceutical compositions used in the treatment of cancer, including, but not limited to, chemotherapeutic agents. Administration can be simultaneous (e.g., administration of a mixture of the active ingredient and the chemotherapeutic agent) or sequential.

[0102] Those skilled in the art will recognize that the therapeutic methods disclosed herein can be used before, after, or concurrently with other forms of tumor treatment, which may include chemotherapy regimens or radiation treatments.

[0103] Also contemplated are dry powder formulations containing at least one protein provided herein and another therapeutically active drug, such as a chemotherapeutic agent.

[0104] Contemplated for use herein are oral solid dosage forms, which are generally described in Chapter 89 of Remington's Pharmaceutical Sciences, 18th Edition, 1990 (Mack Publishing Co. Easton PA 18042), incorporated herein by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Liposomal or proteinoid encapsulation may also be used to formulate the compositions (e.g., as proteinoid microspheres, as reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may also be used, with the liposomes being derivatized with various polymers. The solid dosage forms may be formulated into solid forms (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for therapeutic use is provided in Marshall, K. In: "Modern Pharmaceutics" Edited by GS Banker and CT Rhodes Chapter 10, 1979, which is incorporated herein by reference. In general, the formulation includes one or more components (or chemically modified forms thereof) that provide protection against the stomach environment and release of the biologically active substance in the intestine, and inactive ingredients.

[0105] Oral dosage forms of the derivatized component(s) described above are also specifically contemplated. The component(s) may be chemically modified such that oral delivery of the derivative is effective. In general, the chemical modifications contemplated are to attach at least one moiety to the component molecule itself, which (a) inhibits proteolysis; (b) allows for uptake from the stomach or intestine into the bloodstream. Also desired is an increase in the overall stability of the component(s) and an increase in circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and polyproline. Abuchowski and Davis (1981) "Soluble "Polymer-Enzyme Abducts" In: "Enzymes as Drugs", Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383; Newmark et al. (1982) J. Appl. Biochem. 4:185-189. Other polymers which can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. As noted above, preferred for pharmaceutical applications are polyethylene glycol moieties.

[0106] For the component (or derivative), the location of release may be the stomach, the small intestine (the duodenum, jejunum, or ileum), or the large intestine. One skilled in the art has available formulations that will not dissolve in the stomach, but will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the protein (or derivative) or by release of the bioactive material beyond the stomach environment, such as in the intestine.

[0107] To ensure adequate gastric resistance, a coating impermeable to at least pH 5.0 is essential. Examples of the more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings can be used as mixed films.

[0108] A coating or mixture of coatings may also be used on tablets that are not intended for protection from the stomach. This may include sugar coatings, or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutics, i.e. powders. For liquid forms, a soft gelatin shell may be used. Shell material for cachets may be thick starch or other edible paper. For pills, lozenges, molded tablets, or tablet triturates, moist massing techniques may be used.

[0109] The peptide therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets with a particle size of about 1 mm. Formulation of the material for capsule administration can be as a powder, lightly compressed plugs, or even tablets. The therapeutic can be prepared by compression.

[0110] Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposomal or microsphere encapsulation) and then further included within an edible product, such as a refrigerated beverage, that contains colorants and flavoring agents.

[0111] The amount of therapeutic agent can be diluted or increased with inert materials. These diluents can include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, and starch. Certain inorganic salts, including calcium triphosphate, magnesium carbonate, and sodium chloride, can also be used as bulking agents. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

[0112] Disintegrants can be included when formulating a therapeutic agent into a solid dosage form. Materials used as disintegrants include starches, including but not limited to Explotab, a commercially available disintegrant based on starch. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge, and bentonite can all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gums can be used as disintegrants and binders, and these can include powdered gums such as agar, Karaya, or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials derived from natural products such as gum arabic, tragacanth, starch, and gelatin. Others include methylcellulose (MC), ethylcellulose (EC), and carboxymethylcellulose (CMC). Both polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) can be used in alcoholic solutions to granulate the therapeutic agent.

[0113] Antifriction agents may be included in the formulation of the therapeutic agent to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic agent and the die wall, and these may include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes.Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000 may also be used.

[0114] Glidants may also be added that may improve the flow properties of the drug in the formulation and aid in rearrangement during compression. Glidants may include starch, talc, pyrogenic silica, and hydrated silica aluminate.

[0115] Surfactants may be added as wetting agents to help dissolve the therapeutic agent in the aqueous environment. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents may be used and may include benzalkonium chloride or benzethomium chloride. A list of potential non-ionic detergents that can be included in the formulation as surfactants is lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. These surfactants may be present in the formulation of the protein or derivative alone or as a mixture in different ratios.

[0116] Additives that potentially enhance uptake of the protein (or derivative) are, for example, the fatty acids oleic acid, linoleic acid and linolenic acid.

[0117] Administration of donor-derived γδ T cells. In general, the γδ-T cell composition is prepared in a form suitable for its administration route, for example, in the form of an injection, transfusion, or similar liquid or solution. The liquid or solution form, including the injection, can be prepared in the same manner as the various conventional pharmaceutical products as described above are prepared. The carrier used can be any of the various pharma- ceutically acceptable carriers (diluents) well known in the art. Non-limiting examples thereof are PBS and RPMI1640. In preparing the above liquid or solution forms, various techniques currently commonly used in preparing various infusions can be used. The γδ T cell composition may be prepared immediately before use. The γδ T cell composition is administered at a respective predetermined dose via one or more predetermined administration routes according to the methods described herein. For example, the number of cells to be infused into a subject can be about 1×10 per infusion. 3 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 10 , about 1×10 5 ~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 10 , about 1×10 7 ~Approx. 1×10 10 , about 1×10 8 ~Approx. 1×10 10 , about 1×10 3 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 6 ~Approx. 1×10 9 , about 1×10 4 ~Approx. 1×10 8 , about 1×10 6 ~Approx. 1×10 8 , or approximately 1 × 10 5 ~Approx. 1×10 7 The dose can be in the range of

[0118] It is recognized that the method of treatment may include a single administration of a therapeutically effective amount of the treatment or multiple administrations of a therapeutically effective amount of the treatment. Furthermore, the treatment may be achieved by varying the dosage as well as the administration regimen.

[0119] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Additionally, it should be understood that all base or amino acid sizes and all molecular weights or molecular weight values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes.

[0120] The subject matter of the present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0121] Example 1 - Generation of in vitro expanded human γδ T cells using C-HDMAPP Starting material: PBMCs from healthy donors Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood obtained by venipuncture from healthy individuals. Fresh blood was collected using standard aseptic techniques. PBMCs were isolated using standard density gradient centrifugation. PBMCs were washed with PBS and then resuspended in complete RPMI-1640 supplemented with 10% characterized fetal bovine serum and PCN+streptomycin.

[0122] Ex vivo expansion of human γδ T cells Cell culture was initiated by seeding PBMCs in tissue culture flasks supplemented with recombinant human IL-2. Proliferation of γδ T cells was initiated by addition of C-HDMPP. Cultures were maintained at 37°C and 5% CO2 in an incubator located within a BSL2 containment environment. Fresh medium was added as needed to maintain a cell concentration of approximately 1x10 6Cells / ml were maintained at 100 mM C-HDMPP. Human IL-2 was added every 5 days. Cultures were maintained for 21 days at which point cultures were analyzed by flow cytometry. Figure 1 shows that C-HDMPP used at nanomolar concentrations can be titrated to promote optimal γδ T cell proliferation while avoiding overstimulation. At 100 nM C-HDMPP, cultures were 96% or greater pure γδ T cells with viability of 96% or greater. Figure 2 shows the fold expansion of γδ T cells cultured for 21 days with 100 nM C-HDMPP.

[0123] Example 2 - Cryopreservation After 21 days, cell clusters in culture are gently disrupted, transferred to 50 ml conical tubes, and pelleted by centrifugation. The supernatant is discarded and the cell pellet is resuspended in complete RPMI, then centrifuged again at 300 x g for 15 min at 20 °C. The cell pellet is resuspended in cell culture medium containing 10% DMSO. The cells are transferred in 1 ml or 2 ml aliquots to sterile cryovials. The cryovials are placed in a controlled rate freezing device (-1 °C / min freezing container) and transferred to a -80 °C freezer and subsequently to a liquid nitrogen storage system for storage in the liquid nitrogen vapor phase at -135 °C.

[0124] Thaw and incubate overnight with IL-2 prior to use One day before use, remove the cryovials containing γδ T cells from liquid nitrogen storage and transport them on dry ice to the BSL2 laboratory. Partially thaw the cells in a 37 °C water bath. Fill the partially thawed vials with chilled medium dropwise. Add the contents of each vial dropwise into a 50 mL tube containing medium. After centrifugation, cell pellets are collected at 1 × 10 6 Resuspend in complete RPMI (supplemented with 10% FBS) containing human IL-2 at a concentration of 100 cells / ml. Maintain cells for 24 h at 37 °C and 5% CO2 in a BSL2 lab incubator. The next day, assess cells by flow cytometry to confirm viability and purity. Wash cells (to remove IL-2) and resuspend at a final concentration appropriate for intended use (in vitro studies, in vivo xenografts, or human clinical use).

[0125] Example 3 - Antitumor activity of ex vivo expanded human γδ T cells Human γδ T cells were expanded using the methods described in Example 1. After 21 days in culture, the cells were used as effector cells (killer cells) for in vitro cytotoxicity assays against selected tumor target cells. Each of the indicated human tumor cell lines was first labeled with carboxyfluorescein succinimidyl ester (CFSE) using standard methods. Effector cells and CFSE-labeled tumor cells were co-cultured for 4 hours at various effector-to-target (E:T) ratios. Once the co-culture was completed, propidium iodide was added to the cell mixture, which was then analyzed by multicolor FACS. Gating only on CFSE-positive events (tumor cells) determined the percentage of PI-positive tumor cells (dead cells), allowing the calculation of specific tumor cell death. Results in Figure 3 are shown as tumor cell lysis at an E:T ratio of 25:1. Figure 3 shows that expanded γδ T cells have antitumor activity against a variety of tumor cells, including cells from hematological malignancies and solid tumors.

[0126] Example 4 - Sensitization of tumor cells to killing by γδ T cells Human γδ T cells were expanded using the methods described in Example 1. Expanded γδ T cells were used as effector (killer) cells in an in vitro cytotoxicity assay against selected tumor target cells. 24 hours prior to the killing assay, human tumor cell lines were cultured in the presence of various concentrations (0 μM; 10 μM or 50 μM) of zoledronic acid (ZOL), respectively. Cells were carefully washed before use in the killing assay. Effector and tumor cells were then co-cultured for 4 hours at various effector-to-target (E:T) ratios. Specific tumor cell death was calculated using either a FACS-based method (described in Example 3) or a commercially available LDH release assay (Promega LDH-Glo). Results in Figures 4 and 5 are shown as tumor cell lysis at the indicated E:T ratios. These results indicate that exposure to low concentrations of zoledronic acid renders various tumor cells highly sensitive to killing by γδ T cells. This includes tumor cells that are relatively resistant to γδ T cells.

[0127] All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0128] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

[Claim 1] The invention described in this specification.

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