Use of cd200ar-l for enhancing adoptive t-cell therapy

Abstract

The present disclosure is generally directed to methods of injecting a subject having cancer with a vaccine comprising cancer cells isolated from the subject and CD200AR-L peptide to produce activated T cells from the subject, particularly dogs, and optionally reintroducing these activated T cells to the subject to treat the cancer.

Description

USE OF CD200AR-L FOR ENHANCING ADOPTIVE T-CELL THERAPYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 508,541, filed June 16. 2023, which is hereby incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING XML

[0002] A computer readable form of the Sequence Listing XML containing the file named "0839648.0031 Sequence Listing.xmL" which is 7,014 bytes in size (as measured in MICROSOFT WINDOWS® EXPLORER) and was created on June 13, 2024, is provided herein and is herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs: 1-7.FIELD OF DISCLOSURE

[0003] The present disclosure provides methods of producing ex vivo activated T cells from a subject having cancer, particularly dogs, and reintroducing these activated T cells to the subject to treat the cancer. The cancer can be osteosarcoma, particularly appendicular osteosarcoma.BACKGROUND OF DISCLOSURE

[0004] Osteosarcoma comprises 5-10% of canine cancers. Upwards of 75% of canine bone cancers are osteosarcomas. It is estimated that there are 8,000-10,000 new cases of canine osteosarcoma diagnosed in the U.S. annually. Current treatment options include surgery7and some form of adjuvant systemic chemotherapy. Even in combination, these provide only modest improvement in survival compared to amputation alone and are rarely curative. Further, chemotherapy can be associated with significant toxicity and may require special handling of materials. Thus, treatment of osteosarcoma in dogs represents a serious unmet medical need.

[0005] Cancer immunotherapy has gained renewed interest with the recent clinical successes achieved with immune modulating agents that are designed to overcome the cancer tissue-associated immune suppression. These agents enable the innate abilities of the human immune system to combat the cancer. Recent breakthroughs include cell-based therapies (e.g., ex vivo expanded, patient derived cancer antigen-specific T lymphocytes, T lymphocytes with genetically engineered T cell receptor (TCR), and chimeric antigen receptor (CAR)-T cell technology), oncolytic viruses (OV). and monoclonal and genetically engineered antibodies (e.g., bi-specific T cell engager (BiTE) technology and immune checkpoint inhibitors (ICI)). These therapies are intended to fight cancer more effectively at both the primary7cancer and at distantmetastases, resulting in whole-body treatments with longer-term efficacy than current therapies, e.g. chemotherapy or radiation. Despite the promise of the individual treatment strategies in certain patient populations (e.g., hematologic malignancy and melanoma), these strategies have not been proven to produce significant clinical benefit in a high proportion of treated patients with diverse cancers.SUMMARY OF DISCLOSURE

[0006] The disclosure is directed to a method of producing host cancer antigen-specific activated T cells from a subject having cancer, the method comprising: a) injecting the subject with a vaccine comprising cancer cells isolated from the subject and CD200AR-L peptide at least once; b) collecting mononuclear cells from peripheral blood from the subject; c) isolating T cells from the mononuclear cells of the subject; and d) activating the T cells ex vivo.

[0007] The disclosure is further directed to a method of treating a subject having cancer, the method comprising: a) injecting the subject with a vaccine comprising cancer cells isolated from the subject and CD200AR-L peptide at least once; b) collecting mononuclear cells from peripheral blood from the subject; c) isolating T cells from the mononuclear cells of the subject; d) activating the T cells ex vivo e) administering the activated T cells to the subject; and f) administering at least one dose of IL-2.

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety to the extent consistent herewith. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0009] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 depicts the injection sites for vaccine administration.

[0011] Figure 2 depicts the timeline of the clinical trial visits in table form.

[0012] Figure 3 depicts the timeline of the clinical trial visits graphically.DETAILED DESCRIPTION OF DISCLOSUREOVERVIEW

[0013] Exposure of a naive host to a foreign agent such as a virus results in a cascade of events that involve local inflammation at the disease site and the development of a systemic immune response that ultimately leads to the release of antigen-primed T lymphocytes and antibodies from lymphoid tissue into the blood. Unless the foreign agent has developed protective mechanisms that allow it to avoid the immune response that develops, antigen-primed T lymphocytes enter diseased tissue and differentiate into effector T lymphocytes to eliminate the agent. When the agent is one of the agents that are eliminated by T lymphocytes, i.e., obligate intracellular parasites, the T lymphocytes enter the inflamed disease tissue, encounter activated antigen-presenting cells expressing antigens from the foreign agent and are activated to differentiate into antigen-specific effector T lymphocytes. Those activated antigen-specific effector T lymphocytes then initiate a cascade of local events that results in the elimination of the foreign agent, generally by killing cells that are infected with the foreign agent.

[0014] Cancer (cancer cells are genetically foreign to the host) is a foreign agent that stimulates a chronic inflammatory7response that, rather than helping to eliminate the cancer, promotes its development and suppresses the differentiation of antigen-primed T lymphocytes into effector T lymphocytes. Generally, unless the cancer tissue is perturbed in some way, cancer fails to stimulate the development of an immune response and the cancer progresses in apparent obliviousness to the fact that cancer cells are foreign agents and have the potential to be killed and eliminated by cancer antigen-specific effector T lymphocytes. Cancer tissue contains cells, e.g., myeloid derived suppressor cells (MDSCs) and cancer-associated macrophages, which prevent the formation of effector T lymphocytes and suppress the stimulation of an immune response. Macrophages in principle have the capacity7to be stimulated to become antigen presenting cells and present cancer antigen, but, during the natural progression of a cancer, they do not develop that capacity. Macrophages are highly plastic cells that have the ability to be stimulated to express a variety of functions. Cancer-associated macrophages promote cancer tissue growth and spread rather than being involved in antigen presentation and killing of cells harboring a foreign agent ornormal cells expressing foreign antigens, e.g., cancer cells (another of their plastic functions). It is for these and other reasons that cancer progresses to kill its host.

[0015] It is possible to vaccinate a cancer patient with their own cancer cells and induce an immune response against the foreignness expressed by the cancer cells, which leads to production of large numbers of primed cancer antigen-specific T lymphocytes that leave lymphoid tissue and travel through blood to enter cancer tissue. However, that does not change the nature of the MDSCs and macrophages resident in cancer tissue, as evidenced by the failure of cancer vaccines to exhibit significant therapeutic effects. That is why cancer vaccines usually produce minimal therapeutic benefit, at best, despite the fact that exposure of the immune system to the foreignness of cancer cells results in the development of a strong systemic immune response.

[0016] The fact that cancer tissue-associated T lymphocytes fail to develop into effector T lymphocytes that produce significant therapeutic benefit can be rectified by: A) isolating the primed cancer antigen-specific T lymphocytes from cancer tissue, lymphoid tissue or preferably blood of cancer patients (preferably cancer patients vaccinated with their own malignancy), B) stimulating those primed T lymphocytes to develop into cancer antigen-specific effector T lymphocytes ex vivo and C) using adoptive cell transfer (ACT) to deliver those ex vzvo-activated cancer antigen-specific effector T lymphocytes into the patient's blood where they can travel through the blood, enter cancer tissue and initiate a cascade of effects that leads to efficient cancer cell killing and rejection of the cancer. This approach can produce durable clinical effects in a significant number of treated patients. However, not all patients that are treated by adoptive cell transfer with cancer antigen-specific effector T lymphocytes respond to the therapy and not all patients that respond are cured. This requires the additional step of adding to the vaccine comprising cancer cells isolated from the subject the CD200AR-L peptide adjuvant of the present disclosure.

[0017] This disclosure is directed to a method of producing host cancer antigen-specific activated T cells from a subject having cancer, the method comprising: a) injecting the subject with a vaccine comprising cancer cells isolated from the subject and CD200AR-L peptide at least once; b) collecting mononuclear cells from peripheral blood from the subject; c) isolating T cells from the mononuclear cells of the subject; and d) activating the T cells ex vivo.

[0018] The disclosure is further directed to a method of treating a subject having cancer, the method comprising:a) injecting the subject with a vaccine comprising cancer cells isolated from the subject and CD200AR-L peptide at least once; b) collecting mononuclear cells from peripheral blood from the subject; c) isolating T cells from the mononuclear cells of the subject; d) activating the T cells ex vzvo; e) administering the activated T cells to the subject; and f) administering at least one dose of IL-2.

[0019] The method can further comprise expanding the population of T cells ex vivo between steps c) and d).

[0020] The subject can be not administered glucocorticoids or other immunosuppressive drugs during the duration of the method.CANCERS AND SUBJECTS

[0021] Diseases and conditions that can be treated with the processes of the present disclosure include cancers and proliferative disorders or conditions, including the treatment of cancerous cells, neoplasms, cancers, and metastases. As used herein, "cancer" is a term for diseases caused by or characterized by any type of malignant cancer, including solid cancers, metastatic cancers, lymphatic cancers, and blood cancers. Exemplary’ cancers include, but are not limited to, leukemia, lymphoma, pancreatic cancer, lung cancer, ovarian cancer, breast cancer, cervical cancer, bladder cancer, prostate cancer, brain cancer, adenocarcinomas, liver cancer and skin cancer. Exemplary’ cancers in humans include a bladder cancer, breast cancer, prostate cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer (e.g., glioma), adenocarcinomas, lung cancer, cervical cancer, choriocarcinoma, colon and rectum cancer, connective tissue cancer, cancer of the digestive system; cancer of the small intestine and cecum; endometrial cancer, esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia; liver cancer; gall bladder cancer lung cancer (e.g., small cell and non-small cell); lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma; myeloma, neuroblastoma, oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); cancer of the salivary glands; ovarian cancer; pancreatic cancer, retinoblastoma; rhabdomyosarcoma; rectal cancer, renal cancer, cancer of the respiratory system; sarcoma, skin cancer; stomach cancer, testicular cancer, thyroid cancer; uterine cancer, cancer of the urinary system, as well as other carcinomas and sarcomas.

[0022] Exemplary cancers commonly diagnosed in dogs, cats, and other pets include, but are not limited to, lymphosarcoma, osteosarcoma, mammary cancers, mastocytoma, brain cancer,melanoma, adenosquamous carcinoma, carcinoid lung cancer, bronchial gland cancer, bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma, transmissible venereal cancer, testicular cancer, seminoma, Sertoli cell cancer, hemangiopericytoma, histiocytoma, chloroma (e.g., granulocytic sarcoma), comeal papilloma, comeal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell cancer, thymoma, stomach cancer, adrenal gland carcinoma, oral papillomatosis, hemangioendothelioma and cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma and pulmonary squamous cell carcinoma.

[0023] Preferably, the cancer is osteosarcoma. More preferably, the cancer is appendicular osteosarcoma.

[0024] The subjects include humans and non-human animals, particularly domesticated and farm animals, pets, and experimental animals, such as, chimpanzees, gorillas, horses, cats, dogs, cows, pigs, sheep, goat, mice, rabbits, chickens, rats, and guinea pigs. The subjects can be any mammal.VACCINE GENERATION AND ADMINISTRATION

[0025] The cancer cells isolated from the subject in the vaccine are irradiated to reduce their viability and / or ability to replicate.

[0026] The cancer cells can be isolated from the subject via surgery'.

[0027] The vaccine can comprise 10-100 ug / mL CD200AR-L peptide. The vaccine can preferably comprise 50 pg / mL CD200AR-L peptide. The CD200AR-L peptide can comprise a human or canine CD200AR-L peptide. The CD200AR-L peptide can comprise a portion of a full- length CD200AR-L peptide. The CD200AR-L peptide can comprise a recombinant CD200AR-L peptide.

[0028] The vaccine can comprise 1.5 x 107- 3.0 x 107cancer cells isolated from the subject.

[0029] The CD200AR-L peptide and cancer cells of the vaccine can be administered in the same dose or in separate doses. The CD200AR-L peptide and cancer cells of the vaccine can be administered at the same time or at different times. For example, the CD200AR-L peptide can be administered after the cancer cells.

[0030] The CD200AR-L can be a human CD200AR-L selected from the group consisting of IVTWQKKKAVSPENM (SEQ ID NO: 1), NITLEDEGCYMCLFN (SEQ ID NO: 2),VTFSENHGVVIQPAY (SEQ ID NO: 3), and CLFNTFGFGKISGTA (SEQ ID NO: 4). In other embodiments, the CD200AR-L can be a canine CD200AR-L: CLFNTFGSGKISGTA (SEQ ID NO: 5) or IVTWQKVKPVSLENM (SEQ ID NO: 6). The CD200AR-L can be a munne CD200AR-L: VTWQKKKAVSPENM (SEQ ID NO: 7). The CD200AR-L can be any of the peptides above or any stereoisomers thereof.

[0031] The subject can be injected with the vaccine on at least three separate days. The vaccine injections can be separated by 5 - 9 days. The vaccine injections can be separated by 7 days. The vaccine can be administered intradermally. The vaccine can be injected near a major chain of lymph nodes. The subject can be injected with the vaccine at four different injection sites (see e.g., Figure 1). The subject can be injected with the vaccine at the left and right prescapular regions and left and right popliteal regions.

[0032] The mononuclear cells can be collected 1 1-17 days after administration of the last dose of vaccine. The mononuclear cells can be collected 14 days after administration of the last dose of vaccine.EX VIVO T CELL ACTIVATION AND PROLIFERATION

[0033] Numerous immunomodulatory pathways exist that influence the proliferative abilities, the differentiation, and / or activity of T lymphocytes. Generally, those pathways are described to be either inhibiting effector functions of T lymphocytes through targeting immune checkpoint proteins, or augmenting effector functions through activation of co-stimulatory proteins that are expressed by the T lymphocytes and / or through activation of immune accessory7cells, such as macrophages, NK cells and dendritic cells.

[0034] As a consequence of the use of immunomodulatory compounds administered to patients, cancer antigen-specific T lymphocytes will be prevented from becoming exhausted / anergic, prevented from differentiation towards a regulatory phenoty pe, and / or skewed towards being able to i) expand into a higher number of effector T lymphocytes (better proliferation capacity7), and / or ii) be more potent effector cells (better anti-cancer efficacy). The immunomodulatory compound(s) can therefore lead to i) higher number and ii) extended presence of cancer antigen-specific effector T lymphocytes. Moreover, the infused T lymphocytes are prevented from becoming exhausted and / or anergic. Consequently, co-infusion of immunomodulatory7compound(s) during and / or after the adoptive T cell therapy can result in augmented anti-cancer efficacy. Post adoptive T cell immunotherapy, immunomodulatory compound(s) can be injected once or several times, such as two, three, four, five or even more times.

[0035] The mononuclear cells can be isolated from the peripheral blood by apheresis. The mononuclear cells can be isolated from the peripheral blood plasma by centrifugation. The T cells can be activated with one or more T cell activating agents selected from the group consisting of agonistic immunomodulatory compounds and antibodies.

[0036] The ex vivo activation and / or expansion process that can be used for production of adoptive T cell immunotherapy products can also benefit from immunomodulatory compound(s). In a particularly suitable embodiment, the use of agonistic compounds will activate co-stimulatory molecules on T lymphocytes (e.g., 4- IBB, 0X40, GITR, TNFRSF25, and ICOS). This results in enhanced expansion and anticancer function and can lead to augmented T lymphocyte survival and persistence in the cancer patient.

[0037] Co-inhibitory molecules such as CTLA-4 and PD-1 can be induced in activated T lymphocytes and binding of these proteins to their ligands results in inhibition of the T lymphocyte effector functions. Antibodies that block the interaction of those receptors and their ligands (checkpoint-inhibitors) are thought to prevent these inhibitory effects and thereby augment the therapeutic effects of T lymphocytes against cancer cells.

[0038] Co-stimulatory proteins include constitutively expressed surface receptors such as CD28 and CD27 and others whose expression is induced upon antigen priming of the T lymphocyte. 0X40 (CD134), ICOS (CD278), GITR (CD357), and 4-1BB (CD137) are among those inducible receptors. Agonistic (stimulating) antibodies against these co-stimulatory proteins are thought to enhance effector T lymphocyte functions and thereby improve therapeutic effects against cancer.

[0039] The peripheral blood T lymphocytes can be activated in culture with mouse monoclonal anti-CD3 (0KT3) and then stimulated to proliferate by interleukin 2, interleukin 7, interleukin 15 and / or interleukin 21. The stimulus must be capable of stimulating primed T lymphocytes to differentiate into effector T lymphocytes that maintain cancer antigen specificity and develop effector activity.

[0040] In an additional / altemative method for stimulation, agonistic immunomodulatory compounds, including, but not limited to, anti-OX40. anti-ICOS. anti-GITR. anti-CD27, or anti- 4-1BB / CD137 compounds (e.g., antibodies and / or aptamers), can be used. Another alternative method includes the use of ICI. Another alternative method includes the use of superantigens other than anti-CD3 as the T lymphocyte activator.

[0041] In yet another additional / alternative method, T lymphocyte subsets can be enriched for certain subpopulation(s) (e.g., PD-lpos T lymphocytes, CD137pos T lymphocytes, CD62Llow T lymphocytes and / or CD27pos T lymphocytes).

[0042] T lymphocytes can also be transformed with nucleic acids including (e.g., sd- rxRNA) that reduce the expression of immune checkpoint inhibitor receptors (e.g., CTLA-4; PD- 1).

[0043] Adding immunomodulatory compound(s) to the ex vivo activation / expansion of T lymphocytes can result in a more potent anti-cancer T lymphocyte immunotherapy product. The immunomodulatory compound(s) can prevent T lymphocyte exhaustion and anergy and / or result in additional stimulation, which leads to a higher number of effector T lymphocytes and / or the production of T lymphocytes that have greater potency, thereby generating a higher overall number of effector T lymphocytes.

[0044] Inhibitory' pathways can be attacked by immunomodulatory compounds that are known as immune checkpoint inhibitors. These compounds block the interaction of immune checkpoint proteins and their ligands; whereas compounds that activate co-stimulatory molecules on T lymphocytes act as agonists. Therefore, immunomodulatory compounds bind to proteins on T lymphocytes or other cells (such as cancer cells, macrophages, antigen-presenting cells (dendritic cells), MSDCs, or NK cells) or ligands thereof, which results in the modulation of effector cell T lymphocyte and effector T lymphocyte-activated bystander cell activity. Immunological targets that bind such compounds include, but are not limited to CTLA4, CD28, CD80 (B7-1), CD86 (B7-2), PD-1, PD-L1 (B7-H1), PD-L2 (B7-DC), 4-1BB (CD137 / TNFRSF9), 4-1BB ligand, 0X40 (CD134 / TNFRSF4), 0X40 ligand (CD252 / TNFSF4) ICOS, ICOS ligand, GITR (CD357 / TNFRSF18), GITR ligand (TNFSF18), CD27, CD70, TNFRSF25 (DR3), TL1A (TNFSF15), CD40 (TNFRSF5), CD40L (TNFSF5), HVEM (TNFRSF14). CD160, LIGHT (HVEML / TNFSF14), BTLA, Siglecs, LAG3, TIM3 (HAVCR2), phosphatidylserine, galectins, B7-H3 (CD276), B7-H4 (VTCN1), VISTA, HHLA2, TMIGD2, Butyrophilm-like proteins, BTNL2, TIGIT, CD155 (PVR), CD226 (DNAM1), CD96, CD112 (PVRL2 / nectin2), CD113 (PVRL3 / nectin3), nectins CD25, CD30, VEGF. VEGFR, Neuropilin, IDO, TGF0, CD39, CD73, Adenosine, ADORA2A (A2A), IL-10, IL-27, CXCR4, CXCL12 KIRs (e g., KIR2DL1, KIR2DL2, KIR2DL3), C-type lectins (e.g., NKG2A, NKG2D), MICA, MICB, ILT / LIR protein family members, CD244, CD48 CSF1R, SIRPA (CD172a), CD47 and TLR (TLR1-11).

[0045] Immunomodulatory compounds include, but are not limited to, Ipilimumab, Tremelimumab, Galiximab, Gilvetmab, TGN1412, Pembrolizumab (MK-3475. Lambrolizumab),Nivolumab (ONO-4538, MDX1106, BMS936558), Atezolizumab (MPDL3280A), MEDI4736, Avelumab (MSB0010718C), PDR001, Pidilizumab (CT-011), MEDI0680 (AMP-514). AUNP- 12, BMS-936559 (MDX1105), Urelumab, PF-05082566, BMS-663513, MEDI6383, MEDI6469, MOXR0916, GSK3174998, GSK3359609, TRX518, Varlilumab (CDX1127), CP-870893, BMS- 986016, IMP321, Bavituximab, MGA271, Bevacizumab, MNRP1685A, INCB024360, Galunisertib, Ulocuplumab, BKT140, Larilumab, IPH2101, IPH2201, Emactuzumab (RG7155), CC-90002, and the TLR-agonists; triacyl lipoproteins, heat shock proteins, peptidoglycans, lipoproteins, HMGB1 (high mobility group box 1-amphoterin), lipoteichoic acid, self dsRNA, viral dsRNA, fibrinogen, lipopolysaccharides, heparin sulfate, RSV fusion protein, hyaluronic acid, paclitaxel, flagellin, triacyl lipoproteins, zymosan, self DNA, viral or bacterial DNA and profilin.ADMINISTRATION OF ACTIVATED T CELLS

[0046] Diphenhydramine and / or maropitant citrate can be administered to the subject prior to administering the activated T cells. They can be administered 30 to 60 min prior to administering the activated T cells.

[0047] Additional CD200AR-L peptide can be added with the administration of the activated T cells.

[0048] The method can further comprise an additional administration of the vaccine one day before or after administering the activated T cells to the subject.

[0049] The activated T cells can be administered 5-9 days after collecting the peripheral blood from the subject. The activated T cells can be administered 7 days after collecting the peripheral blood from the subject. The activated T cells can be administered intravenously. The activated T cells can be administered via a non-peristaltic pump through a 170-120 pm filter into the subject's bloodstream. The activated T cells can be administered over at least 30 minutes. The activated T cells can be administered over 30 to 60 minutes.

[0005] The IL-2 can be administered at a dose of around 20,000 lU / kg body weight. The at least one dose of IL-2 can be administered subcutaneously. The at least one dose of IL-2 can be at least two doses, at least three doses, or at least five doses. The at least one dose of IL-2 can be administered 24-48 hours following infusion of the activated T cells. The at least one dose of IL-2 can comprise more than one dose, and subsequent doses following the first dose are delivered at 24-72 hour intervals.OPTIONAL ADDITIONAL THERAPEUTIC STEPS AND / OR ANTI-CANCER AGENTS

[0050] The method can further comprise a cancer tumor being removed from the subject via surgery.

[0051] Any therapeutic or anti-cancer agent can be used as a second therapeutic or anticancer agent in a combined cancer treatment method. The method can include administering one or more therapeutic compounds to the subject in addition to administering an adoptive T cell immunotherapy or a lurality thereof to a subject. Therapeutic compounds can act independently, or in conjunction with the adoptive T cell immunotherapy, for anti-cancer therapeutic effects.

[0052] Therapeutic compounds also include, but are not limited to, chemotherapeutic agents, nanoparticles, radiation therapy, siRNA molecules, enzyme / pro-drug pairs, photosensitizing agents, toxins, microwaves, a radionuclide, an angiogenesis inhibitor, a mitosis inhibitor protein (e.g., cdc6), an anti-cancer oligopeptide (e.g., antimitotic oligopeptides, high affinity7cancer-selective binding peptides), a signaling modulator, anti-cancer antibiotics, immunomodulatory7compounds or a combination thereof.

[0053] Exemplary photosensitizing agents include, but are not limited to, for example, indocyanine green, toluidine blue, aminolevulinic acid, texaphyrins, benzoporphyrins, phenothiazines, phthalocyanines, porphyrins such as sodium porfimer, chlorins such as tetra(m- hydroxyphenyljchlorin or tin(IV) chlorin e6, purpurins such as tin ethyl etiopurpurin, purpurinimides, bacteriochlorins, pheophorbides. pyropheophorbides or cationic dyes. In one example, an adoptive T cell therapy is administered to a subject having a cancer or cancer metastasis in combination with a photosensitizing agent.

[0054] Radionuclides, which depending upon the radionuclide, amount and application can be used for diagnosis and / or for treatment. They include, but are not limited to, for example, a compound or molecule containing Phosphorus, Cobalt. Yttrium. Technitium. Palladium, Ruthenium, Indium, Lutetium, Iodine, Iodine, Cesium, Samarium, Rhenium, Rhenium, Iridium, Gold, Astatine, Bismuth or Bismuth. In one example, an adoptive T cell immunotherapy is administered to a subject having a cancer or cancer metastasis in combination with a radionuclide.

[0055] Toxins include, but are not limited to, chemotherapeutic compounds such as, but not limited to, 5-fluorouridine, calicheamicin and maytansine. Signaling modulators include, but are not limited to, for example, inhibitors of macrophage inhibitory7factor, toll-like receptor agonists and stat 3 inhibitors. In one example, an adoptive T cell immunotherapy is administered to a subject having a cancer or cancer metastasis in combination with a toxin or a signaling modulator.

[0056] Combination therapy between chemotherapeutic agents and therapeutic adoptive T cell immunotherapy can be effective / curative in situations when single agent treatment is not effective. Chemotherapeutic compounds include, but are not limited to, alkylating agents such as thiotepa, temozolomide and cyclophosphamide; alkyd sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodepa, carboquone, meturedepa and uredepa; ethylenimine and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novobiocin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carubicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin. olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine. floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defosfamide; demecolcine; diaziquone; efl ornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide-K; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2' ,2"- trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacyto sine; cyto sine arabino side; cyclopho sphamide; thiotepa; taxoids, e.g., paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; Navelbine; Novantrone; teniposide; daunomycin; aminopterin; Xeloda; ibandronate: CPT11; topoisomeraseinhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; esperamycins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as antiestrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide. leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Such chemotherapeutic compounds that can be used herein include compounds whose toxicities preclude use of the compound in general systemic chemotherapeutic methods. Chemotherapeutic agents also include new classes of targeted chemotherapeutic agents such as, for example, imatinib (sold by Novartis under the trade name Gleevec in the United States), gefitinib (developed by Astra Zeneca under the trade name Iressa) and erlotinib. Particular chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-S vincristine, prednisone, doxorubicin and L-asparaginase; mechlorethamine, vincristine, procarbazine and prednisone (MOPP). cyclophosphamide, vincristine, procarbazine and prednisone (C-MOPP), bleomycin, vinblastine, gemcitabine, Toceranib phosphate (Palladia®), Verdinexor (LAVERDIA™-CA1; a selective inhibitor of CRMl-mediated nuclear export (SINE)), Rabacfosadine (Tanovea®; a DNA polymerase inhibitor), Tigilanol tiglate (Stelfonta®), Gilvetmab, Oncept®, and 5-flurouracil. Exemplary chemotherapeutic agents are, for example, cisplatin, carboplatin, oxaliplatin, DWA2114R. NK121, IS 3 295. and 254-S. In a non-limiting example, a vaccinia virus is administered to a subject having a cancer or cancer metastasis in combination with a platinum coordination complex, such as cisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-S.

[0057] Exemplary anti-cancer antibiotics include, but are not limited to, anthracy clines such as doxorubicin hydrochloride (adriamycin), idarubicin hydrochloride, daunorubicin hydrochloride, aclarubicin hydrochloride, epirubicin hydrochloride and pirarubicin hydrochloride, phleomycins such as phleomycin and peplomycin sulfate, mitomycins such as mitomycin C, actinomycins such as actinomycin D, zinostatinstimalamer and polypeptides such as neocarzinostatin. In one example, a vaccinia virus is administered to a subject having a cancer or cancer metastasis in combination with an anti-cancer antibiotic.

[0058] In one example, nanoparticles can be designed such that they cany7one or more therapeutic agents provided herein. Additionally, nanoparticles can be designed to carry a molecule that targets the nanoparticle to the cancer cells and / or the infused T lymphocytes. In onenon-limiting example, nanoparticles can be coated with a radionuclide and, optionally, an antibody immunoreactive with a cancer-associated antigen.

[0059] Therapeutic compounds that can act in conjunction with the adoptive T cell immunotherapy to increase the proliferation, cancer cell killing or immune response eliciting properties of an adoptive T cell immunotherapy are compounds that can alter gene expression, where the altered gene expression can result in an increased killing of cancer cells or an increased anti-cancer immune response in the subject. A gene expression-altering compound can, for example, cause an increase or decrease in expression of one or more genes, including endogenous viral genes and / or exogenous viral genes, genes of the adoptively transferred T lymphocytes, and / or genes of cells that are present in cancer tissue (e.g., cancer cells, stromal cells, including but not limited to, cancer associated fibroblasts, endothelial cells, dendritic cells, macrophages, myeloid-derived suppressor cells). For example, a gene expression-altering compound can induce or increase transcription of a gene in effector T lymphocytes such as an exogenous gene that can cause cell lysis or cell death that can provoke an immune response that can catalyze conversion of a prodrug-like compound, or that can inhibit expression of a cancer cell gene. Any of a wide variety of compounds that can alter gene expression are known in the art, including IPTG, RU486, and epigenetic modulators. Exemplary genes whose expression can be up-regulated include proteins and RNA molecules, including toxins, enzy mes that can convert a prodrug to an anti-cancer drug, cytokines, transcription regulating proteins, siRNA and ribozymes. Any of a variety of compounds that can reduce or inhibit gene expression can be used in the methods provided herein, including siRNA compounds, transcriptional inhibitors or inhibitors of transcriptional activators. Exemplary genes whose expression can be down-regulated include proteins and RNA molecules, including adoptive T cell therapy proteins or RNA that suppress lysis, nucleotide synthesis or proliferation, and cellular proteins or RNA molecules that suppress cell death, immunoreactivity, lysis, or adoptive cell therapy replication.

[0060] In another example, therapeutic compounds that can act in conjunction with the adoptive T cell therapy to increase the proliferation, cancer cell killing, or immune response eliciting properties of an adoptive T cell immunotherapy are compounds that can interact with an expressed gene product, and such interaction can result in an increased killing of cancer cells or an increased anti-cancer immune response in the subject. A therapeutic compound that can interact with an expressed gene product can include, for example, a prodrug or other compound that has little or no toxicity or other biological activity in its subject-administered form, but after interact on with a virally expressed gene product, the compound can develop a property that results in cancercell death, including but not limited to, cytotoxicity, ability to induce apoptosis, or ability to trigger an immune response. Once the enzyme is introduced into the cancer cells, an inactive form of a chemotherapy drug (i.e., a prodrug) is administered. When the inactive prodrug reaches the cancer cells, the enzyme converts the prodrug into the active chemotherapy drug, so that it can kill the cancer cell. Thus, the treatment is targeted only to cancer cells. The prodrug can be administered concurrently with, or sequentially to, the adoptive T cell immunotherapy. A variety of prodruglike substances are known in the art and an exemplary set of such compounds are disclosed elsewhere herein, where such compounds can include gancyclovir, 5 -fluorouracil, 6-methylpurine deoxyriboside, cephalosporin-doxorubicin, 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl- L-glutamic acid, acetaminophen, indole-3 -acetic acid, CB 1954, 7-ethyl-10-[4-(l-piperidino)-l- piperidino]carbonyloxycamptothecin, bis-(2-chloroethyl)amino-4-hydroxyphenyl- aminomethanone 28, l -chloromethyl-5-hydroxy-l ,2-dihydro-3H-benz[e]indole, epirubicin- glucuronide, 5'-deoxy-5-fluorouridine, cytosine arabinoside, linamarin, and a nucleoside analogue (e.g., fluorouridine, fluorodeoxyuridine, fluorouridine arabinoside, cytosine arabinoside, adenine arabinoside, guanine arabinoside, hypoxanthine arabino side, 6-mercaptopurineriboside, theoguanosine riboside, nebularine, 5-iodouridine, 5-iododeoxyuridine, 5-bromodeoxyuridine, 5- vinyldeoxyuridine, 9-[(2 -hydroxy )ethoxy]methylguanine (acyclovir), 9-[(2-hydroxy-l- hydroxymethyl)-ethoxy]methylguanine (DHPG), azauridien, azacytidine, azidothymidine, dideoxyadenosine, dideoxycytidine, dideoxyinosine, dideoxyguanosine, dideoxythymidine, 3'- deoxyadenosine, 3 '-deoxy cytidine. 3'-deoxyinosine. 3 '-deoxy guanosine, 3 '-deoxy thymidine).

[0061] In addition to combination therapy between chemotherapeutic agents and adoptive T cell therapy provided herein, other more complex combination therapy strategies could be applied as well. For example, a combination therapy can include chemotherapeutic agents, therapeutic antibodies, and an adoptive T cell immunotherapy provided herein. Alternatively, another combination therapy can be the combination of radiation, therapeutic antibodies, and an adoptive T cell immunotherapy provided herein. Therefore, the concept of combination therapy also can be based on the application of an adoptive T cell immunotherapy provided herein along with one or more of the following therapeutic modalities, namely, chemotherapeutic agents, radiation therapy, therapeutic antibodies, hyper- or hypothermia therapy, siRNA, diagnostic / therapeutic bacteria, diagnostic / therapeutic mammalian cells, immunotherapy (including immunomodulatory compounds), and / or targeted toxins (delivered by antibodies, liposomes and nanoparticles).

[0062] Many cancers are treated with radiation therapy either as a primary therapeutic intervention or as a method for eliminating cancer cells remaining at sites of surgical cancer removal. The wide use of radiation treatment stems from the ability of gamma-irradiation to induce irreversible damage in targeted cells. Ionizing radiation triggers apoptosis, the intrinsic cellular death machinery in cancer cells, and the activation of apoptosis seems to be the principal mode by which cancer cells die following exposure to ionizing radiation. Low dose radiation has immunostimulatory effects on cancer tissue, thereby enhancing the cancer cell killing abilities of adoptively transferred cancer antigen-specific effector T lymphocytes.

[0063] For combination therapies with chemotherapeutic compounds, dosages for the administration of such compounds are known in the art or can be determined by one skilled in the art according to known clinical factors (e.g., subject's species, size, body surface area, age. sex, immune competence, and general health, duration and route of administration, the kind and stage of the disease, for example, cancer size, and other treatments or compounds, such as other chemotherapeutic drugs, being administered concurrently). In addition to the above factors, adoptive T cell immunotherapy, as can be determined by one skilled in the art.

[0064] As will be understood by one of skill in the art, the optimal treatment regimen will vary and it is within the scope of the treatment methods to evaluate the status of the disease under treatment and the general health of the patient prior to, and following one or more cycles of combination therapy in order to determine the optimal therapeutic combination.

[0065] Therapeutic compounds also include, but are not limited to. compounds that exert an immunotherapeutic effect, stimulate or suppress the immune system, carry a therapeutic compound, or a combination thereof. Optionally, the therapeutic agent can exhibit or manifest additional properties, such as, properties that permit its use as an imaging agent, as described elsewhere herein. Such therapeutic compounds include, but are not limited to. anti-cancer antibodies, radiation therapy, siRNA molecules and compounds that suppress the immune system (i.e. immunosuppressors, immunosuppressive agents) or compounds that stimulate the immune system (i.e., immuno-modulators, including but not limited to, checkpoint inhibitors and costimulatory molecule agonists). In some cases, it is desirable to administer an immunosuppressive agent to a subject to suppress the immune system prior to the administration of the adoptive T cell immunotherapy in order to minimize any adverse reactions to the adoptive T cell immunotherapy. Exemplary immunosuppressive agents include, but are not limited to, glucocorticoids, alkydating agents, antimetabolites, and immunosuppressive antibodies.

[0066] Immunotherapy also includes for example, immune-stimulating molecules (protein-based or non-protein-based), cells and antibodies. Immunotherapy treatments can include stimulating immune cells to act more effectively or to make the cancer cells or cancer-associated antigens recognizable to the immune system (i.e., break tolerance).

[0067] Cytokines and grow th factors include, but are not limited to, interleukins, such as, for example, interleukin-1, interleukin-2, interleukin-6 and interleukin-7, interleukin- 12, interleukin-21, tumor necrosis factors, such as tumor necrosis factor alpha (TNF-a), interferons such as interferon gamma (IFN-y), granulocyte macrophage colony stimulating factors (GM-CSF), angiogenins, and tissue factors. Immune checkpoint modulators and immunostimulators include compounds that bind to proteins on T lymphocytes or other cells (such as cancer cells, antigen- presenting cells, macrophages, dendritic cells, MSDCs, or NK cells) or ligands thereof, which results in the modulation of effector T lymphocyte activity. Immunological targets that bind such compounds include, but are not limited to CTLA4, CD28, CD80 (B7-1), CD86 (B7-2), PD-1, PD- L1 (B7-H1), PD-L2 (B7-DC), 4-1BB (CD137 / TNFRSF9), 4-1BB ligand, 0X40 (CD134 / TNFRSF4), 0X40 ligand (CD252 / TNFSF4) ICOS, ICOS ligand, GITR (CD357 / TNFRSF18), GITR ligand (TNFSF18), CD27, CD70, TNFRSF25 (DR3), TL1A (TNFSF15), CD40 (TNFRSF5), CD40L (TNFSF5) HVEM (TNFRSF14), CD160, LIGHT (HVEML / TNFSF14), BTLA, Siglecs, LAG3, TIM3 (HAVCR2), Phosphatidylserine, galectins, B7-H3 (CD276), B7-H4 (VTCN1), VISTA, HHLA2, TMIGD2, Butyrophilin-like proteins, BTNL2, T1GIT, CD155 (PVR), CD226 (DNAM1). CD96. CD112 (PVRL2 / nectin2). CD113 (PVRL3 / nectin3), nectins CD25, CD30, VEGF, VEGFR, Neuropilin, IDO, TGFn, CD39, CD73, Adenosine, ADORA2A (A2A), IL-10, IL-27, CXCR4, CXCL12 KIRs (e.g., KIR2DL1, KIR2DL2, KIR2DL3). C-type lectins (e.g., NKG2A, NKG2D), MICA, MICB. ILT / LIR protein family members, CD244, CD48 CSF1R, SIRPA (CD172a), CD47 and TLRs (e.g. TLR1-11). Immune checkpoint modulators and immunostimulatory compounds include, but are not limited to, Ipilimumab, Tremelimumab, Galiximab, TGN1412, Pembrolizumab (MK-3475, Lambrolizumab), Nivolumab (ONO-4538, MDX1106, BMS936558), Atezolizumab (MPDL3280A), MEDI4736, Avelumab (MSB0010718C), PDR001, Pidilizumab (CT-011), MEDI0680 (AMP-514), AUNP-12, BMS-936559 (MDX1105), Urelumab, PF-05082566, BMS- 663513, MEDI6383, MEDI6469, MOXR0916, GSK3174998, GSK3359609, TRX518, Varlilumab (CDX1127), CP-870893, BMS-986016, IMP321, Bavituximab, MGA271, Bevacizumab, MNRP1685A, INCB024360, Galunisertib, Ulocuplumab, BKT140. Larilumab, IPH2101. IPH2201, Emactuzumab (RG7155). CC-90002 and TLR agonists.

[0068] Anti-cancer antibodies include, but are not limited to, 3F8, 8H9, Abagovomab, Abituzumab, Adecatumumab, Afutuzumab, Alacizumab pegol, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anetumab ravtansine, Apolizumab, Arcitumomab, Ascrinvacumab, Atezolizumab, Bavituximab, Bectumomab, Belimumab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Cantuzumab ravtansine, Capromab pendetide, Carlumab. Catumaxomab, cBR96-doxorubicin immunoconjugate. Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab, Dacetuzumab, Dalotuzumab, Daratumumab, Demcizumab, Denintuzumab mafodotin, Derlotuximab biotin, Detumomab, Drozitumab, Durvalumab, Dusigitumab, Ecromeximab, Edrecolomab, Elgemtumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enavatuzumab. Enfortumab vedotin, Enoblituzumab, Ensituximab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, FBTA05, Ficlatuzumab, Figitumumab, Flanvotumab, Galiximab, Ganitumab, Gemtuzumab ozogamicin, Gilvetmab, Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Icrucumab, Igovomab, IMAB362, Imalumab, Imgatuzumab, Indatuximab ravtansine, Indusatumab vedotin, Inotuzumab ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Labetuzumab, Lambrolizumab, Lexatumumab, Lifastuzumab vedotin, Lilotomab satetraxetan, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab, Matuzumab, Milatuzumab, Minretumomab. Mirvetuximab soravtansine, Mogamulizumab, Moxetumomab pasudotox, Nacolomab tafenatox, Naptumomab estafenatox, Namatumab, Necitumumab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Obinutuzumab, Ocaratuzumab, Ofatumumab, Olaratumab, Onartuzumab, Ontuxizumab, Oportuzumab monatox, Oregovomab. Otlertuzumab, Panitumumab, Pankomab, Parsatuzumab, Pasotuxizumab, Patritumab, Pembrolizumab, Pemtumomab, Pertuzumab, Pidilizumab, Pinatuzumab vedotin, Polatuzumab vedotin, Pritumumab, Racotumomab, Radretumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Sacituzumab govitecan, Samalizumab, Satumomab pendetide, Seribantumab. SGN-CD19A, SGN-CD33A, Sibrotuzumab, Siltuximab, Sofituzumab vedotin, Tabalumab, Tacatuzumab tetraxetan. Taplitumomab paptox, Tarextumab, Tenatumomab, Teprotumumab, Tetulomab, TGN1412, Ticilimumab (=tremelimumab), Tigatuzumab, TNX-650, Tovetumab, Trastuzumab, Trastuzumab emtansine, TRBS07, Tremelimumab, Tucotuzumab celmoleukin, Ublituximab, Urelumab, Vandortuzumab vedotin, Vantictumab. Vanucizumab, Veltuzumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, and Zatuximab.

[0069] Monoclonal antibodies are of particular interest for treating cancer because of the specificity of binding to a unique antigen and the ability to produce large quantities of the agent in the laboratory for mass distribution. Monoclonal antibodies can be engineered to act in the same way as immune system proteins: that is, to seek out and kill foreign matter in the body, such as viruses. Monoclonal antibodies can be designed to recognize epitopes on the surface of cancer cells. The antibodies specifically bind to the epitopes and either kill the cancer cells or deliver a therapeutic agent to the cancer cell. Methods of conjugating therapeutic agents to antibodies are well-known in the art. Different antibodies have to be made for different types of cancer; for example, Rituximab recognizes CD20 protein on the outside of non-Hodgkin's lymphoma cells; ADEPT is a treatment using antibodies that recognize bowel (colon) cancer; and Trastuzumab (Herceptin) recognizes breast cancer cells that produce too much of the protein HER 2 ("HER 2 positive”). Thus, the adoptive cell immunotherapy provided herein can be administered concurrently with, or sequentially to, one or more monoclonal antibodies in the treatment of cancer. In one example, additional therapy is administered in the form of one or more of any of the other treatment modalities provided herein.

[0070] Thus, provided herein are methods of administering to a subject one or more immuno-modulating / therapeutic compounds that can act in conjunction with the adoptive T cell immunotherapy to stimulate or enhance the immune system, thereby enhancing the effect of the adoptive T cell immunotherapy. Such immunotherapy can be either delivered as a separate therapeutic modality or could be encoded (if the immunotherapy is protein-based) by the administered adoptive T cell immunotherapy.

[0071] Biological therapies are treatments that use natural body substances or drugs made from natural body substances. They can help to treat a cancer and control side effects caused by other cancer treatments such as chemotherapy. Biological therapies are also sometimes called Biological Response Modifiers (BRM's), biologic agents or simply ’‘biologies” because they stimulate the body to respond biologically (or naturally) to cancer. Immunotherapy is treatment using natural substances that the body uses to fight infection and disease. Because it uses natural substances, immunotherapy is also a biological therapy. There are several types of drugs that come under the term biological therapy: these include, for example, monoclonal antibodies, cancer vaccines, growth factors, cancer growth inhibitors, anti-angiogenic factors, interferon alpha, interleukin-2, gene therapy and BCG vaccine. One exemplary' vaccine is Oncept®, a canine melanoma DNA vaccine.

[0072] Growth factors are natural substances that can for example stimulate the bone marrow to make blood cells. Recombinant technology can be used to generate growth factors which can be administered to a subject to increase the number of white blood cells, red blood cells and stem cells in the blood. Growth factors used in cancer treatment to boost white blood cells include granulocyte colony stimulating factor (G-CSF) also called filgrastim (Neupogen) or lenograstim (Granocyte) and GM-CSF. also called molgramostim or sargramostim (Leukine). Thus, the adoptive T cell therapy provided herein can be administered concurrently with, or sequentially to, a growth factor such as GM-CSF, interleukin 2, interleukin 7, interleukin 15 and / or interleukin 21, in the treatment of cancer.

[0073] Cancer growth inhibitors use cell-signaling molecules which control the growth and multiplication of cells, such as cancer cells. Drugs that block these signaling molecules can stop cancers from growing and dividing. Cancer growth factors include, but are not limited to, tyrosine kinases. Thus, drugs that block tyrosine kinases are tyrosine kinase inhibitors (TKIs). Examples of TKIs include, but are not limited to, Toceranib phosphate (Palladia®), Erlotinib (Tarceva. OSI — 774), Iressa (Gefitinib, ZD 1839) and Imatinib (Glivec, STI 571). Another type of growth inhibitor is Bortezomib (Velcade) for multiple myeloma and for some other cancers. Velcade is a proteasome inhibitor. Proteasomes are found in all cells and help break down proteins in cells. Interfering with the action of proteasomes causes a buildup of proteins in the cell to toxic levels; thereby killing the cancer cells. Cancer cells are more sensitive to Velcade than normal cells. Thus, the adoptive T cell immunotherapy provided herein can be administered concurrently with, or sequentially to, a cancer growth inhibitor, such as Velcade, in the treatment of cancer.

[0074] Cancers need a blood supply to expand and grow their own blood vessels as they get bigger. Without its own blood supply, a cancer cannot grow due to lack of nutrients and oxygen. Anti-angiogenic drugs stop cancers from developing their own blood vessels. Examples of these types of drugs include, but are not limited to. Thalidomide, mainly for treating myeloma but also in trials for other types of cancer, and Bevacizumab (Avastin). Thus, the adoptive T cell immunotherapy provided herein can be administered concurrently with, or sequentially to, an anti- angiogenic drug in the treatment of cancer.

[0075] Interferon-alpha (IFN-a) is a natural substance produced in the body, in very small amounts, as part of the immune response. IFN-a is administered as a treatment to boost the immune system and help fight cancers such as renal cell (kidney) cancer, malignant melanoma, multiple myeloma and some types of leukemias. IFN-a works in several ways: it can help to stop cancer cells growing, it can also boost the immune system to help it attack the cancer, and it can affectthe blood supply to the cancer cells. Thus, the adoptive T cell immunotherapy provided herein can be administered concurrently with, or sequentially to, IFN-a in the treatment of cancer.

[0076] Gene therapy involves treating cancer by blocking abnormal genes in cancer cells, repairing or replacing abnormal genes in cancer cells, encouraging even more genes to become abnormal in cancer cells so that they die or become sensitive to treatment, using oncolytic viruses to carry treatment-activating enzymes into the cancer cells, or combination thereof. As a result, cancer cells die due to damage in the cell. Cancer cells develop as a result of several types of mutations in several of their genes. Targeted genes include, but are not limited to, those that encourage the cell to multiply (i.e., oncogenes), genes that stop the cell multiplying (i.e., cancer suppressor genes) and genes that repair other damaged genes. Gene therapy can involve repair of damaged oncogenes or blocking the proteins that the oncogenes produce. The cancer suppressor gene, p53, is damaged in many human cancers. Oncolytic viruses have been used in to deliver an undamaged p53 gene into cancer cells, and early clinical trials are now in progress looking at treating cancers with modified p53-producing oncolytic viruses. Gene therapy could be used to replace the damaged DNA repairing genes. In an alternative example, methods of increasing DNA damage within a cancer cell can promote death of the cancer cell or cause increased susceptibility of the cancer cell to other cancer treatments, such as radiotherapy or chemotherapy. Thus, the adoptive T cell immunotherapy provided herein can be administered concurrently with, or sequentially to, any of the gene therapy methods provided herein or known in the art in the treatment of cancer.

[0077] The gene product or therapeutic agent could be given as a separate modality, i.e., not necessarily to be encoded or carried by the adoptive T cell immunotherapy. This can be give prior to, concurrently, or after adoptive T cell immunotherapy treatment.

[0078] Effective delivery of each component of the combination therapy is an important aspect of the methods provided herein. In accordance with one aspect, the modes of administration discussed below exploit one or more of the key features: (i) delivery' of an adoptive T cell immunotherapy provided herein to the cancers by a mode of administration designed to achieve highest titer of adoptively transferred T cells and greatest therapeutic effect; (ii) delivery of any other mentioned therapeutic modalities to the cancer by a mode of administration to achieve the optimal therapeutic effect. The dose scheme of the combination therapy administered is such that the combination of the two or more therapeutic modalities is therapeutically effective. Dosages will vary in accordance w ith such factors as the age, health, sex, size and weight of the patient, theroute of administration, the toxicity of the drugs, frequency of treatment and the relative susceptibilities of the cancer to each of the therapeutic modalities.

[0079] As used in this application, including the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise, and are used interchangeably with "at least one" and "one or more."

[0080] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.EXAMPLES

[0081] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the preceding description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0082] The following examples detail a designed clinical trial for generation of activated T cells and their use in the treatment of appendicular osteosarcoma.EXAMPLE 1: CLINICAL TRIAL PATIENT SELECTIONRationale for the general study design

[0083] This study is designed to evaluate the safety and effectiveness of a vaccination with an CD200AR-L adjuvant. This adjuvant has previously been shown to downregulate the host CD200 and PDL-1 immune checkpoint proteins. It is hypothesized that the downregulation of these checkpoint proteins will improve the host immune response to the autologous vaccine. To assess efficacy in treating appendicular osteosarcoma, the immunotherapy plus CD200AR-L will be compared to prior results from clinical trials evaluating immunotherapy without adjuvant and to historical published results using chemotherapies as treatment.Animal selection and identification

[0084] The study uses client-owned dogs that have been diagnosed with osteosarcoma, are under veterinary care at the study site, and for which owner informed consent has been obtained. Dogs remain housed with their owners who report back to the clinic for scheduled treatments and observations by the treating veterinarian. Dogs of any breed, sex, or age may be eligible for inclusion.Patient inclusion criteria

[0085] An informed consent form is signed by the client prior to entry of the patient into the study and before any study procedures are performed.

[0086] Surgically resectable cancer has been clinically diagnosed and confirmed through central pathology as appendicular osteosarcoma.

[0087] Cancer tissue specimen has been aseptically collected.

[0088] The dog is a male (either sexually intact or neutered) or female (either sexually intact or neutered) that is not pregnant and not lactating.

[0089] The dog has not previously received any treatment other than surgery for their osteosarcoma.

[0090] The dog is sufficiently manageable and cooperative to be able to conform to study procedures.

[0091] Clinical results with the following ranges: ALT is < 3X upper limit of normal laboratory range. Serum creatinine is < 1.5X the upper limit of the normal laboratory range. Hematocrit is > 28%. Hemoglobin is > l Og / dL. White blood cell count is > 3000 cells / microliter (mcL) of blood. Platelet count is > 100,000 platelets / mcL of blood. Lymphocyte count is > 1000 cells / mcL of blood. Animal must weigh at least 15 kg at screening. The client is willing to present the dog on schedule for timed clinic visits, as required by the protocol.Patient exclusion criteria

[0092] The patient has received glucocorticoids or other immunosuppressive agent within the 14 days prior to enrollment.

[0093] The patient has other co-morbidities that might reasonably be expected to interfere with the dog’s ability to tolerate or complete the medical procedures required in the study.

[0094] The patient has metastatic disease, including any detected via 3-view thoracic radiographs (lateral, L and R, and ventral).

[0095] The patient has another uncontrolled medical condition not in remission as determined by investigator.

[0096] The patient’s owners have familial, sociological or geographical conditions that do not permit adequate medical follow-up and compliance with the study protocol.

[0097] A dog is considered enrolled in the study only when it has met all inclusion and no exclusion criteria.Prior Concomitant Medications

[0098] Medications given within the last 30 days prior to enrollment will be recorded.Prohibited medications

[0099] Glucocorticoids or any other immunosuppressive drugs are not permitted from Day -14 until 30 days after end of treatment. If necessary, and at the clinician’s discretion,dexamethasone (0.25 mg / kg. IV, qd) is permitted to mitigate serious adverse events following T cell infusion.

[0100] Immunomodulating drugs, herbal supplements, and other naturopathic therapies are not permitted from Day 0 until 30 days post T cell infusion.EXAMPLE 2: CLINICAL TRIAL VACCINE AND ACTIVATED T CELL PRODUCTION AND ADMINISTRATIONSurgery

[0101] Surgical removal of the primary’ bone cancer by amputation of the affected limb is the recommended first-line treatment for canine bone cancer. As a palliative treatment, surgery reduces or eliminates bone pain and eliminates the risk of fracture. Surgery is required to obtain cancer tissue to be used to manufacture the attenuated autologous cancer cell product that is an essential component of the vaccine. In this study, a limb-sparing approach will be conducted where the limb is not amputated. Instead, surgery is performed to remove all gross cancer tissue from the limb and harvest cancer tissue for vaccines. Using this approach, it is hoped to remove all gross tissue and preserve the affected limb. While potentially permitting the preservation of the affected limb, this limb-sparing technique does carry’ some potentially significant risks including limb fracture after surgery’, post-surgical infection at site, and / or significant pain and discomfort to the patient. Careful monitoring and management of these limb-spare dogs will be needed.

[0102] Limb-sparing surgery’ of the affected limb will be performed by treating veterinarians. A small portion of the malignant tissue is placed in formalin for histopathological processing. The remainder (majority) is aseptically transferred to a sterile specimen container with sterile cell culture medium. Also, 20 mL of blood are collected into two 10 mL red top Vacutainer® blood tubes. A specimen label containing all relevant patient information is placed on each container including the patient’s study accession number. The cancer tissue specimen and blood tubes are shipped to the processing center in a refrigerated transport container. The specimens are shipped by overnight courier for arrival within 48 hours of surgery. Autologous Vaccines Vaccine production

[0103] Upon receipt at the processing center, the specimen is processed to produce the vaccine serial. Vaccines are stored at ultra-low temperature until shipped to the treatment site for vaccine administration. After processing, the frozen vaccines are shipped to the treatment site where they are stored frozen until the vaccines are administered.

[0104] Vaccine carrier and cryovials are labelled with the patient name and identification number. The cryovials include attenuated autologous cancer cell vaccine. One cryovial equals one vaccine.Vaccine administration

[0105] Immediately prior to vaccination, a vial containing the vaccine serial is removed from the freezer or dry ice, thawed and evenly distributed into four 1.0 mL tuberculin syringes. Vaccines are administered intradermally in all 4 injection sites during each visit (see Figure 1). Visits are separated by 7 ± 2 days.Timing and number of autologous vaccines

[0106] As autologous vaccines are only generated from available tissue harvested from each patient, there may be occasions in which the material harvested is inadequate to generate 3 vaccines immediately post-harvest.

[0107] Initiation of the treatment protocol (first vaccination) may be delayed if there is a need to culture the harvested cancer cells to generate sufficient cells for vaccines. Harvested cells will be cultured to obtain sufficient cells for vaccine manufacture. The first vaccination visit marks the start of the treatment schedule timeline (see Figures 2 and 3). Injection sites

[0108] Specific vaccination sites allow the contents of the vaccine to drain into major chains of lymph nodes and to travel to where cancer antigens can be exposed to maximal numbers of lymph node T cells. The vaccinations are delivered into the following areas as shown in Figure 1.

[0109] First injection is in the right prescapular region. Second injection is in the left prescapular region. Third injection is in the right popliteal region. Fourth injection is in the left popliteal region.Mononuclear cell collection by apheresis

[0110] Standard apheresis procedures are employed by clinical staff trained on the performance of a mononuclear cell (MNC) collection using a standard apheresis instrument. The purpose of the instrument is to collect MNCs from peripheral blood. The apheresis collection bag is shipped to the processing center in a refrigerated transport container. T cell InfusateT cell infusate production

[0111] Upon receipt at the processing center, the apheresis specimen is used to produce an autologous activated T cell infusate. The infusion bag containing the activated T cellinfusate and accompanying transfer documentation are shipped to the treatment site in a refrigerated transport container for IV infusion.Premedicants prior to T cell infusion

[0112] Diphenhydramine: 2 mg / kg is administered intramuscularly 30 to 60 minutes prior to T cell infusion to minimize inflammatory t pe reactions to the activated T cell infusion.

[0113] Maropitant citrate: 1 mg / kg is administered subcutaneously 30 to 60 minutes prior to T cell infusion to prevent vomiting.Activated T cell infusion

[0114] The activated T cell infusate is intravenously administered using standard blood transfusion procedures. The cells are infused via anon-peristaltic pump through a 170 - 210 pm filter into the subject’s bloodstream over a minimum of 30 minutes. Most infusions are completed within 30 to 60 minutes.IL-2 Treatment

[0115] IL-2 is used as an immunomodulator to increase the immune response. IL- 2 (5X 20,000 lU / Kg body weight per dose q48 hours) is injected subcutaneously beginning 24 - 48 hours following infusion of activated T cells in order to continue to stimulate T cell multiplication in vivo. Subsequent doses of IL-2 are delivered at 48-hour intervals. A non- therapeutic dose of IL-2 is used which has been associated with minimal adverse effects in dogs. The dosing regimen is designed to continue exposure of infused effector T cells to IL-2 for a prolonged period of time after the administered T cells would have entered cancer tissue, thereby potentially augmenting the anti-cancer effect generated by those T cells.

[0116] The first of five IL-2 injections (20,000 lU / Kg per dose) is administered subcutaneously beginning 24 to 48 hours following completion of infusion of the activated T cell infusate. The subsequent IL- 2 injections (20,000 lU / Kg per dose) are administered 24 to 72 hours (2 ± 1 day) following the preceding injection. There will be 5 total doses administered.EXAMPLE 3: CLINICAL TRIAL TIMELINE

[0117] The clinical trial timeline is depicted in Figure 2 and Figure 3. Eligibility Screen

[0118] Informed consent is reviewed and signed by the client prior to surgery.

[0119] 3-view thoracic radiographs (lateral, L and R. and ventral) are evaluated for metastatic disease.

[0120] The following items are shipped for processing: cancer tissue collected from surgery and red-topped blood tubes containing ~20 mL of blood collected for vaccine preparation (two 10 mL tubes).

[0121] Cancer diagnosis will be confirmed through central pathology as osteosarcoma. The cancer specimen must meet tissue acceptance criteria.

[0122] The patient must meet inclusion / exclusion criteria.

[0123] Demographic information is obtained.

[0124] A physical examination (with vitals: pulse, respiration, temperature, and weight) is performed. Medical history and pre-existing medical conditions are recorded. Medications, including supplements and herbal formulations, for the previous thirty days are recorded. A quality of life questionnaire (QoL) is filled out. A chemistry profile is determined: albumin, alkaline phosphatase, ALT, GGT, BUN, calcium, chloride, CO2, creatinine, glucose, potassium, sodium, phosphorous, total bilirubin, and total protein. Complete blood count (CBC) with differential and platelet count is obtained.(Visit 1) First vaccination (Study Day 7)

[0125] A physical examination is conducted with vitals: pulse, respiration, temperature, and weight. A standard chemistry profile is obtained. Complete blood count (CBC) with differential and platelet count is obtained. The first vaccine is administered (performed 7-14 days following surgery). A quality of life questionnaire (QoL) is filled out.(Visit 2) Second vaccination (Study Dav 14)

[0126] The second vaccine is administered (performed 7 days (± 2 days) following the first vaccination). New or changed medical events and medications are recorded. Adverse events are reviewed and recorded. A quality of life questionnaire (QoL) is filled out.(Visit 3) Third vaccination (Study Dav 21)

[0127] The third vaccine is administered (performed 7 days (± 2 days) following the second vaccination). New or changed medical events and medications are recorded. Adverse events are reviewed and recorded. A quality of life questionnaire (QoL) is filled out.(Visit 4) Apheresis (Study Dav 35)

[0128] Apheresis is performed 14 ± 3 days following the third vaccination. Mononuclear cells are collected by apheresis. The apheresis collection bag is shipped for processing.(Visit 5) T cell infusion (Study Day 42) 7 ± 2 days following the apheresis

[0129] A physical examination is conducted with vitals: pulse, respiration, temperature, and weight. Complete blood count (CBC) with differential and platelet count is obtained prior to T cell infusion. New or changed medical events and medications are recorded. Adverse events are reviewed and recorded. A quality of life questionnaire (QoL) is fdled out.

[0130] T cell infusion is initiated within 8 hours following receipt of the T cell infusate at the site. The T cell infusate is intravenously infused over a period not less than 30 minutes. Most infusions are completed within 60 minutes. The patient will be monitored for adverse events in clinic for 4-6 hours following infusion.(Visits 6-10) IL-2 injections (Study Days 43-51)

[0131] The first of five IL-2 injections (20,000 lU / Kg per dose) are administered subcutaneously by the clinician beginning 24 to 48 hours following infusion of the activated T cell infusate. The subsequent IL-2 injections (20,000 lU / Kg per dose) are administered 24 to 72 hours (2 ± 1 day) following the preceding injection. At the clinician's discretion, owners may be instructed to administer these subcutaneous injections themselves at the appropriate times.(Visit 11-16) Follow-up Visits (Study Davs 56, 146, 236, 326, 390, 450 [± 7 days!)

[0132] 3- view thoracic radiographs (lateral, L and R, and ventral) are generated.A physical examination is conducted with vitals: pulse, respiration, temperature, and weight. New or changed medical events and medications are recorded. Adverse events are reviewed and recorded. A quality of life questionnaire (QoL) is filled out.(Visit 17) End of Study Visit (Study Day 540 [± 7 days])

[0133] 3- view thoracic radiographs (lateral, L and R, and ventral) are generated.A physical examination is conducted with vitals: pulse, respiration, temperature, and weight. New or changed medical events and medications are recorded. Adverse events are reviewed and recorded. A quality of life questionnaire (QoL) is filled out.EXAMPLE 4: CLINICAL TRIAL ANALYSISPrimary study endpoint

[0134] The primary' outcome for each dog will be overall survival time defined as the number of days between clinical diagnosis (date of first radiograph that suggested osteosarcoma) and death. Median survival times will be determined using Kaplan-Meier analysis of study survival data. Surviving dogs and dogs lost to follow-up at the study reporting date will be censored in the Kaplan-Meier analyses. Efficacy results of the vaccine / adjuvant therapy will be compared to results from prior studies which used vaccine alone. Additionally, benchmarkcomparisons to historical chemo therapeutics used to treat OSA may be conducted to evaluate efficacy.Secondary Study Endpoints

[0135] Disease-Free Interval (DFI): Disease-free interval is defined as time in days from diagnosis to detection of metastatic disease. This assessment will be conducted via 3-view thoracic radiographs and clinical evaluation of patient (e.g. integumentary tumors). These evaluations occur periodically or can be unscheduled if the clinician suspects metastatic disease. Median DFI will be assessed via Kaplan-Meier analysis, with dogs in remission at the study reporting date in the Kaplan-Meier analyses.

[0136] Additional statistical evaluations may also be conducted.Safetv

[0137] Adverse events will be assessed throughout the study by recording clinical signs, performing physical examinations and performing clinical laboratory tests. Patients are monitored for adverse events during the study and for 30 days following removal from study for any reason. All adverse events will be collected and reported in the final report.Study Design

[0138] Single-arm, open-label, single-site clinical field study.Study standard

[0139] This study will be conducted in accordance with the protocol and good clinical practice (GCP) as defined by the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) topic GL9 and USDA- CVB Memorandum 800.301.Masking

[0140] No masking / blinding will be used in this open label study. Rationale: the primary variable and secondary variables for this study are survival and disease progression, which are unlikely to be impacted by knowledge of the treatment received.Statistical Criterion

[0141] Overall survival is the most objective method to assess the impact of any cancer treatment and is the factor of most important clinical relevance to most owners, when compared to other treatment modalities. One-year survival time, denoted as yes or no, is a commonly used surrogate for overall survival.

[0142] All enrolled animals which receive any portion of therapy will be evaluated as the "intention to treat" group, whereas those animals which complete all aspects of assigned therapy will be evaluated as the ‘'per protocol" group.

Claims

WHAT IS CLAIMED IS:

1. A method of producing host cancer antigen-specific activated T cells from a subject having cancer, the method comprising: a) injecting the subject with a vaccine comprising cancer cells isolated from the subject and CD200AR-L peptide at least once; b) collecting mononuclear cells from peripheral blood from the subject; c) isolating T cells from the mononuclear cells of the subject; and d) activating the T cells ex vivo.

2. A method of treating a subject having cancer, the method comprising: a) injecting the subject with a vaccine comprising cancer cells isolated from the subject and CD200AR-L peptide at least once; b) collecting mononuclear cells from peripheral blood from the subj ect; c) isolating T cells from the mononuclear cells of the subject; d) activating the T cells ex vivo, e) administering the activated T cells to the subj ect; and f) administering at least one dose of IL-2.

3. The method of claim 2, wherein diphenhydramine and maropitant citrate are administered to the subject 30 to 60 min prior to administering the activated T cells.

4. The method of claim 2 or 3, wherein additional CD200AR-L peptide is added with the administration of the activated T cells.

5. The method of any one of claims 2-4, wherein the method further comprises an additional administration of the vaccine one day before or after administering the activated T cells to the subject.

6. The method of any one of claims 2-5, further comprising a cancer tumor being removed from the subject via surgery.

7. The method of any one of claims 2-6, wherein the activated T cells are administered 5-9 days after collecting the peripheral blood from the subj ect.

8. The method of any one of claims 2-7, wherein the activated T cells are administered 7 days after collecting the peripheral blood from the subject.

9. The method of any one of claims 2-8, wherein the activated T cells are administered intravenously.

10. The method of any one of claims 2-9, wherein the activated T cells are administered via a non- peristaltic pump through a 170-120 pm filter into the subject's bloodstream.1 1. The method of any one of claims 2-10, wherein the activated T cells are administered over at least 30 minutes.

12. The method of any one of claims 2-11. wherein the activated T cells are administered over 30 to 60 minutes.

13. The method of any one of claims 2-12, wherein the IL-2 is administered at a dose of 20,000 IU / kg body weight.

14. The method of any one of claims 2-13, wherein the at least one dose of IL-2 is administered subcutaneously.

15. The method of any one of claims 2-14, wherein the at least one dose of IL-2 is at least five doses.

16. The method of any one of claims 2-15, wherein the at least one dose of IL-2 is administered 24-48 hours following infusion of the activated T cells.

17. The method of any one of claims 2-16, wherein the at least one dose of IL-2 comprises more than one dose, and subsequent doses following the first dose are delivered at 24-72 hour intervals.

18. The method of any one of claims 1-17, further comprising expanding the population of T cells ex vivo between steps c) and d).

19. The method of any one of claims 1-18. wherein the subject is a mammal.

20. The method of any one of claims 1-19, wherein the subject is a human or canine.

21. The method of any one of claims 1 -20, wherein the cancer is selected from the group consisting of lymphosarcoma, osteosarcoma, mammary cancers, mastocytoma, brain cancer, melanoma, adenosquamous carcinoma, carcinoid lung cancer, bronchial gland cancer, bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma, transmissible venereal cancer, testicular cancer, seminoma, Sertoli cell cancer, hemangiopericytoma, histiocytoma, chloroma (e.g., granulocytic sarcoma), comeal papilloma, comeal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell cancer, thymoma, stomach cancer, adrenal gland carcinoma, oralpapillomatosis, hemangioendothelioma and cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma, and pulmonary squamous cell carcinoma.

22. The method of any one of claims 1-21, wherein the cancer is appendicular osteosarcoma.

23. The method of any one of claims 1-22, wherein the cancer cells isolated from the subject in the vaccine are irradiated.

24. The method of any one of claims 1-23. wherein the subject is not administered glucocorticoids or other immunosuppressive drugs during the duration of the method.

25. The method of any one of claims 1-24, wherein the cancer cells isolated from the subject are isolated via surgery.

26. The method of any one of claims 1-25, wherein the vaccine comprises 50 pg / mL CD200AR- L peptide.

27. The method of any one of claims 1-26, wherein the CD200AR-L peptide comprises a human or canine CD200AR-L peptide.

28. The method of any one of claims 1-27, wherein the CD200AR-L peptide comprises a portion of a full-length CD200AR-L peptide.

29. The method of any one of claims 1-28, wherein the CD200AR-L peptide comprises a recombinant CD200AR-L peptide.

30. The method of any one of claims 1-29, wherein the mononuclear cells are isolated from the peripheral blood by apheresis.

31. The method of claim 1-29, wherein the mononuclear cells are isolated from the peripheral blood plasma by centrifugation.

32. The method of any one of claims 1-31, wherein the T cells are activated with one or more T cell activating agents selected from the group consisting of agonistic immunomodulatory compounds and antibodies.

33. The method of any one of claims 1-32, wherein the subject is injected with the vaccine on at least three separate days.

34. The method of claim 33, wherein the vaccine injections are separated by 5 - 9 days.

35. The method of claim 33 or 34, wherein the vaccine injections are separated by 7 days.

36. The method of any one of claims 1-35. wherein the vaccine is administered intradermally.

37. The method of any one of claims 1-36, wherein the vaccine is injected near a major chain of lymph nodes.

38. The method of any one of claims 1-37, wherein the subject is injected with the vaccine at four different injection sites.

39. The method of any one of claims 1-38, wherein the subject is injected with the vaccine at the left and right prescapular regions and left and right popliteal regions.

40. The method of any one of claims 1-39, wherein the mononuclear cells are collected 11-17 days after administration of the last dose of vaccine.

41. The method of any one of claims 1-40, wherein the mononuclear cells are collected 14 days after administration of the last dose of vaccine.

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