WO2001028583A2 - Melanoma vaccine and methods of making and using same - Google Patents

Abstract

An immunotherapeutic vaccine providing antigen presenting cells that have been pulsed with a disrupted cell preparation which includes enucleated cytosol and cell membranes of cancer cells infected with a recombinant vaccinia virus encoding at least one immunostimulating molecule. In a preferred embodiment, the vaccine includes autologous dendritic/monocytic cells (DC/M) that present a mixture of antigens (present in the enucleated cytosol and cell membranes) from melanoma cell lines that have been infected with a recombinant vaccinia virus encoding IL-2. In another of the preferred embodiments, the enucleated cytosol and cell membranes are from melanoma cells harvested from the patient to be treated. A method of making the vaccine and methods of using the vaccine to stimulate an anti-cancer immune response and to treat a patient with a cancer are also described.

Description

MELANOMA V ACCINE AND METHODS OF MAKING AND USING SAME

Field of the Invention

The invention generally relates to an improved therapeutic vaccine useful for treatment and prevention of a cancer Disclosed are a cancer \accine. and methods of making and using such a vaccine in a host diagnosed with a cancer The vaccine as used in accordance with the present invention comprises a new administration schedule and ingredients such as recombmant IL-2 encoding vaccinia virus and autologous dendritic monocytic cells pulsed with melanoma antigens deπved from cancerous melanoma cell lines expressing at least two HLA class I A antigens

Background of the Invention

The incidence of malignant melanoma has increased at an alarming rate

er the past few decades, and indications are that the incidence of this deadly disease will continue to nse in the future As melanoma is known to be refractory to conventional chemotherapy or radiotherapy, several alternative treatment approaches have been used for treatment of this vaπety of skm cancer Immunotherapy has been considered potentially useful for melanoma because melanoma has shown certain immunological traits, such as spontaneous regression, infiltration of lymphocytes within the tumor mass, an in vitro demonstration of anti-melanoma specific cellular responses, and evidence of responsn eness to immunomodulators such as interleukins and interferons (Mukheηi B and Chakraborty NG . Immunobiology and immunotherapy of melanoma, Curr Opin Oncol 7 175-184, 1995) For example, immunizing a patient having a melanoma with its own melanoma antigens prepared as a vaccine might induce an anti-tumor immune reaction and thus could cure such a patient from the disease

The descπption of one such vaccine can be found in U S Pat No 4.108,983 to Wallack This patent describes a first generation melanoma

V accinia Melanoma Oncolvsate (VMO), which was deπved from melanoma cells lysed by a vaccinia virus (U S Pat No 4,108,983) This vaccme and modified versions thereof were tested in multiple clinical tπals Although a clinical benefit was seen in several subsets of patients, especially young male patients with stage III (AJCC) melanoma, the vaccine did not produce a significant benefit for melanoma patients when tested as a surgical adjuvant therapy in a recently completed phase III clinical tnal. (Wallack MK, Sivanandham M, et al., Favorable clinical responses in subsets of patients from a randomized, multiinstitutional melanoma vaccine tπal, Ann. Surg. Oncol.3(2):l-8, 1996; Wallack MK, Sivanandham M, Balch CM, et al., A phase III randomized, double-blmd, multiinstitutional tπal of vaccinia melanoma oncolysate-active specific immunotherapy for patients with stage II melanoma. Cancer 75:34-42, 1995).

U.S. Pat. Nos. 5,635,188 and 5,030,621 both to Bystryn, disclose a vaccme made up of cell surface antigens of melanoma cells that are shed into the culture medium and consequently used as anti-melanoma vaccine.

Similarly, U. S. Pat No 5,484,596 to Hanna, Jr. et al. discloses a method of cancer therapy consisting of prepaπng irradiated tumor cells and injecting them as a vaccine into a human patient.

Although such immunotherapy tπals with these vaccines have shown encouraging results in some patients, e.g., partial regression of melanoma, delay in the appearance of recurrent melanoma, and an increase in overall survival as compared to standard therapy or surgery, none of the methods so far have been entirely satisfactory.

Dunng the last two decades, several new lmmunomodulating cytokmes were discovered, and these cytokmes have been extensively studied for their therapeutic benefit in patients with cancer. The most studied cytokme is mterleukin-2 (IL-2), which has shown some benefit in augmenting immunity against melanoma. The benefit of IL-2 therapy is presumed to be due to the stimulation of T cells, some of which may have become toxic toward tumor cells

Generally, immunomodulating cytokmes such as IL-2 are administered either as a bolus injection or as a low dose continuous infusion (Lotze MT et al., High dose recombmant ιnterleukιn-2 m the treatment of patients with disseminated cancers, JAMA 256(22).3117-3124, 1986; West WH et al, Constant infusion of recombmant IL-2 in adoptive immunotherapy of advanced cancers, New Engl. J. Med. 316(15):898-905, 1987) However, bolus injection with a high dose of cytokines generally produces significant toxicity while low dose continuous infusion is inconvenient. A more constant level of cytokme in vivo, similar to that produced by continuous infusion of cytokine, can be

SUBSTITUTE SHEET (RULE 25) achieved using recombmant viruses or bacteria designed to produce cytokines in vivo.

The use of recombmant vectors to induce the production of cytokines in vivo falls withm the definition of gene therapy Some encouraging results have been seen using such vectors. For example, use of the IL-2 gene in recombmant vaccinia virus (rVV) seemed to reduce tumor burden in a mouse melanoma model (Sivanandham M, Scoggin SD, Sperry

RG, Wallack MK, Prospects for gene therapy and lymphokine therapy for metastatic melanoma, Ann. Plast. Surg. 28(1): 1 14-1 18, 1992). In contrast, later studies indicated that recombmant vaccinia with IL-2 had no effect For example, Qin et al. descnbe a vaccine that appeared to have some effect with rVV expressing GM-CSF, but not with rVV expressing IL-2. (Qin H, Chatterjee SK, Cancer gene therapy using tumor cells infected with recombmant vaccinia virus expressing GM-CSF, Hum. Gene Ther. 7(15): 1853-60, 1996) Similar disappointing results with rVV encoding IL-2 were observed by Shrayer et al. in their melanoma model. (Shrayer DP, Bogaars H, Heaπng VJ, Wanebo HJ, Immunization of mice with irradiated melanoma tumor cells transfected to secrete lymphokines and coupled with IL-2 or GM-CSF therapy, J. Exp. Ther. Oncol., 1(2): 126-

33, 1996).

Thus, the success of recombmant IL-2 in gene therapy strategies is unpredictable. Initial reports showed objective response rates with single- agent rIL-2 therapy m the range of 15% to 20%; however, the overall response rates m clinics were much lower than oπgmally anticipated. In addition, it appears that co-admmistration of lymphokine- activated killer (LAK) cells, generated ex vivo with rIL-2, does not enhance the response rates achieved with rIL-2 alone.

For example, U S. Pat. No 4,863,727 and 5,425,940, both to Zimmerman et al, disclose augmentation of anti-melanoma activity m mammals by admmisteπng an effective amount of IL-2 and tumor necrosis factor (TNF), or TNF and interferon (IFN)- beta, or IL-2, TNF, and IFN-beta combinations. These compositions were also suggested to be useful for treating other cancers such as leukemia, lymphoma, mastocytoma, mammary adenocarcinoma, and pharyngeal squamous cell carcinoma.

U.S. Pat. No. 5,066,489 to Paradise et al, discloses treatment of malignant melanoma by combining IL-2 with chemotherapeutic agents.

Rosenberg et al. describe a combination of IL-2 with tumor-infiltrating

SUBSTITUTE SHEET (RULE 25) lymphocytes as a means of providing an immunotherapy to patients with metastatic melanoma (Rosenberg et al , Use of tumor-infiltrating lymphocytes and ιnterleukιn-2 in the immunotherapy of patients with metastatic melanoma, New Engl J Med 319 1676- 1680,1988) Similarly, Dutcher et al descπbed combining IL-2 with IL-2-actιvated killer cells as a treatment option for metastatic melanoma (Dutcher et al , A Phase II study of high- dose continuous infusion mterleukιn-2 with lymphokine-activated killer cells m patients with metastatic melanoma, J Clin Oncol 9(4) 641-648, 1991 )

Because of the broad activation nature of T cells, the anti-tumor response generated by IL-2 is not very specific For that reason, IL-2-based therapy has not proven to be very effectι\ e In addition, the doses of IL-2 required bv these IL-2 treatment methods appear toxic to treated patients (Siegel et al , lnterleukιn-2 toxicity, J Clin Oncology, 9 694- 704, 1991 ) Based on these observations, it was believed that a more specific immune response can be generated by combining IL-2 with tumor specific antigens U S Pat No 5,290,551 to Berd, discloses the treatment of melanoma with a vaccine compnsing irradiated autologous melanoma tumor cells conjugated to a hapten and combining the vaccme with IL-2

U S Pat No 5,478,556 to Elliott et al , discloses vaccination of cancer patients using tumor-associated antigens mixed with IL-2 and granulocyte-macrophage colony stimulating factor (GM-CSF)

The importance of antigen presenting (APC) or accessory cells in inducing specific cellular immune response was postulated long ago Among many types of accessory cells are dendritic cells (DC), which are denved from vaπous cell lineages such as bone marrow stem cells, macrophages and lymphocytes The DC stimulate cytotoxic and helper T-cells by expressing high levels of HLA class I and class II antigens and the T-cell co-stimulatory factors CD80, CD86, ICAM-1 and LFA-3 DC also secrete cytokines such as IL-12, IL-15 and IFN-gamma, which have been shown to be useful for the expansion of stimulated T- cells DC-based immunotherapies have also been studied m patients with cancers and viral diseases To elicit anti-tumor immune response, vaπous cell types have been employed as cellular adjuvants with tumor antigens, and recently several groups have shown that

4 SUBSTITUTE SHEET (RULE 25) dendritic cells (DC), cultured with tumor cell lysates, synthetic tumor antigens, or peptides purified from tumor cells, induced rather significant anti-tumor immunity in vivo. In all of these approaches, the DC were pulsed with an exogenous source of antigen. Alternative methods were also proposed consisting of genetically engineering DC to express tumor antigens. The expression of tumor antigens by DC is a potent method of inducing tumor antigen-specific responses in vivo. (See Pardoll DM, Cancer vaccines. Nat. Med. 4(5 Suppl): 525-31 , 1998). Several melanoma-specific antigens were identified recently, e.g., MART-1/Melan A, gplOO, tyrosinase, MAGE-1, MAGE-3, and others. They were accordingly used to elicit anti-tumor immune reaction through presentation via DC. Some studies used not only peptides but unfractionated tumor cell lysates as well (Abdel-Wahab

Z, DeMatos P. Hester D, Dong XD, and Seigler HF, Human dendritic cells, pulsed with either melanoma tumor cell lysates or the gplOO peptide (280-288), induce pairs of T-cell cultures with similar phenotype and lytic activity, Cell. Immunol. 186(l):63-74, 1998).

Thus, despite the fact that vaccination with autologous DCs was determined to be safe, the efficacy of such vaccines was not any better than with conventional melanoma vaccines. For example, the objective responses were evident only in 5 out of 16 patients with metastatic melanoma. (Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D, Vaccination of melanoma patients with peptide- or tumor lysate pulsed dendritic cells, Nat. Med. 4(3):328-332, 1998). This and other similar approaches have failed to improve DC-based vaccines and to enhance the chances of survival of melanoma patients. Despite many studies relating to melanoma vaccine development, so far little real progress has been achieved. Clearly, there is a need for an effective melanoma vaccine useful to slow the progression of, if not cure, melanoma in a higher proportion of patients than in any earlier described vaccines. U.S. Pat. No. 5,788,963 to Muφhy et al., discloses methods and compositions for using human DC to activate T cells for immunotherapeutic responses against primary and metastatic prostate cancer. Other DC based approaches have also been described, e.g. Hsu et al. (Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells, Nat. Med. 2(l):52-58, 1996). Thus, while multiple approaches to melanoma (and cancer generally) have been undertaken during the last twenty years, and some progress made, the problem has remained Melanoma continues to be on the πse and refractory to available therapies (Vil kka K, Pyrhonen S Cytokme therapy of malignant melanoma Ann Med 28(3) 227-233, 1996)

Thus, the present invention provides an improved melanoma vaccine that provides a better success rate than the melanoma therapies existing in the pπor art

Summary of the Invention

The present invention provides a therapeutic composition made up of antigen presenting cells that have been pulsed with a disrupted cell preparation The disrupted cell preparation includes enucleated cytosol and cell membranes of cancer cells that have been infected with a recombmant vaccinia virus that encodes for at least one lmmunostimulating molecule In one embodiment, the invention provides a therapeutic vaccme in which autologous dendπtic/monocytic cells (DC/M) present a mixture of antigens (present in a preparation of enucleated cytosol and cell membranes) that are from cells from cancer cell lines that have been infected with a recombmant vaccinia virus encoding IL-2 In another embodiment, the enucleated cytosol and cell membranes are from cancer cells harvested from a patient's own cancer In yet another embodiment, the antigen presenting cells are HLA-matched dendntic/monocytic cells for the host receiving the vaccine According to the present invention the enucleated cytosol is substantially free of cell nuclei In addition, the cancer cell membranes of the invention contain at least two and preferably more than two HLA class I A antigens

The present invention also provides an immunotherapeutic vaccine having two parts The first part of the vaccme involves admimstenng a recombmant vaccinia virus encoding at least one lmmunostimulating molecule The second part of the vaccme provides antigen presenting cells that have been pulsed with a preparation of antigens from cancer cells infected with a recombmant vaccinia virus encoding at least one lmmunostimulating molecule In a preferred embodiment, the recombmant vaccinia virus of the first part of the vaccme encodes IL-2 and the second part of the vaccine compπses autologous DC/M that have been pulsed with enucleated cytosol and cell membranes from cancer cells of cancer cell lines which have been infected with recombmant vaccinia virus encoding IL-2 The invention further provides a method of prepaπng an immunotherapeutic vaccine useful for creating an anti-cancer immune response or treating a host for cancer This method includes the following steps (1) contacting cancer cells with a recombmant vaccinia virus encoding an lmmunostimulating molecule, (n) disrupting the vaccinia virus- contacted cancer cells to obtain a preparation of enucleated cytosol and cell membranes from the vaccinia-mfected cancer cells, and (in) pulsing antigen presenting cells with the preparation for a time sufficient for the antigens in the preparation to be presented on the surface of the APC

Also included in the invention is a method for eliciting an anti-cancer immune response in a subject by admimstenng a therapeuticallv effective amount of a composition including antigen presenting cells that have been pulsed with a preparation including enucleated cvtosol and cell membranes of cancer cells infected with a recombmant vaccinia virus encoding for at least one lmmunostimulating molecule

In addition, the invention provides for a method of eliciting an anti-cancer immune response in a subject by (I) admimstenng a recombmant vaccinia virus encoding at least one lmmunostimulating molecule, and (n) admimstenng a composition providing antigen presenting cells pulsed with a preparation including enucleated cytosol and cell membranes of cancer cells infected with a recombmant vaccinia virus encoding for at least one lmmunostimulating molecule In yet another preferred embodiment, the first part of the vaccme is administered approximately thirty (30) minutes pπor to the second part and in substantially the same location on the patient

The present invention also provides a method of treating a subject for cancer by (l) admimstenng a recombmant vaccinia virus encoding at least one lmmunostimulating molecule, and (n) admimstenng a composition compπsing antigen presenting cells pulsed with a preparation including enucleated cytosol and cell membranes of cancer cells infected with a recombmant vaccinia virus encoding at least one lmmunostimulating molecule In yet another preferred embodiment, the first part of the vaccme is administered approximately thirty (30) minutes pπor to the second part and in substantially the same location on the patient

Brief Description of Drawings

7 SUBSTITUTE SHEET (RULE 25) Fig. 1 is a flow chart illustrating the steps in the preparation of CVACII vaccine. Fig. 2 shows relative efficacy of various vaccine preparations in protecting against development of tumor.

Fig. 3 shows percent of survival of mice treated with seven different vaccine preparations and with a no-treatment protocol.

Fig. 4 demonstrates that vaccine protection correlates with induction of CD8- positive cytolytic cells.

Fig. 5 shows cytotoxicity tests resulting from treatment with CVACII vaccine and control vaccine preparation consisting of DC/M pulsed with tumor sonicate only. Fig. 6 shows HLA restricted proliferation of PBL and CD8 cells from a representative patient when exposed to various melanoma antigens (MART-1 , Mel-9, Mel- 2, and own tumor cells) as tested before and after vaccination.

Fig. 7 shows HLA restricted cytotoxicity profile of a patient as tested at pre- and post- vaccination (at biweekly intervals) time periods.

Detailed Description of the Invention The present invention relates to an improved immunotherapeutic vaccine useful for treating a host diagnosed with cancer, e.g., melanoma, as well as methods of making and using the vaccine and various components of the vaccine. The term "HLA" means human leukocyte antigen and is equivalent to the term

"major histocompatibility complex" (MHC) molecule. In general, class 1 molecules are MHC-encoded peptides that are associated with β2-microglobulin, while class 2 molecules have two non-covalently associated MHC encoded peptides. Class 1 (HLA-A, B, C) and 2 (HLA-D or DR, DQ, DP) molecules, when on the cell surface, are capable of presenting "antigens" or "immunogens"-molecules which elicit an immune response. The term

"immune response" is commonly associated with the activation of immune cells. However, the opposite is true as well when an immunogen causes immune tolerance or anergy. The term "HLA-matched" means those HLA antigens from one individual are essentially similar to HLA antigens from another individual. The term "autologous cells" means that the cells are an individual's own cells.

Dendritic cells (DC) are one subtype of "antigen presenting cells" (APC) capable of eliciting an immune response or reaction mediated in part by "cytotoxic T lymphocytes" (CTL) CTL are cells capable of killing or suppressing the growth of cells having response-e citmg antigens The term "CTL" usually refers to CD8+ T-cells, although CD4+ T-cells and "natural killer" (NK) cells can also display cytolytic activity When CTL are activated or pπmed with lymphokines such as IL-2 they are also called lymphokine activated killer cells or "LAX" cells The term CD, e g , CD8 or CD4, stands for cluster of differentiation and usually represents an immune marker used to distinguish different types of cells The term "antigen presenting cells" usually means specialized lymphoid cells such as dendπtic cells, B cells, and monocytic cells, which are capable to induce T cell activation The term "monocytes" refers to cells related to "macrophages" and thev represent vet another type of APC, which mainly re-activate previously sensitized CTL DC, however, appear to activate functionally-native or unpπmed T cells Both DC and monocytes are needed to activate and to sustain immune cell responses In addition to differences in immune phenotype, these two types of APC differ moφhologically, DC having more pronounced "hairy" or "dendntic" phenotype However, in contrast to monocytes, the exact ongm of DC is still unclear as they can be obtained from a vanety of tissues, e g , peπpheral blood, spleen, thyrnus, bone marrow, lymph nodes, or skin Monocytes can be obtained essentially from the same sources Some DC are also known as "veiled" cells found in the blood or as "Langerhans cells" usually found in the epidermis

The term "lmmunostimulating molecule" refers to cytokines, hematopoietic growth factors, and melanoma immunogens The term "cytokme" refers to bioactive molecules derived from cells and capable of affecting cells' behavior, e g , growth, migration, killing capacity, differentiation, secretion, etc The term "lymphokine" means essentially same as the cytokme but usually refers to bioactive molecules deπved from lymphocytes and affecting predominantly the behavior of lymphocytes

The term "immunotherapeutic vaccme," as opposed to the notion of a "prophylactic vaccme," means a vaccme administered to treat and/or prevent further progression of the disease in a host already diagnosed with the disease The term "admi stenng" means any method of providing a host m need thereof with a vaccine, including oral, intranasal, topical, transdermal, parenteral, e g , intravenous, subcutaneous, mtradermal, intramuscular, and other means of delivery known in the art.

The term "melanoma" means a malignant skin cancer or tumor of varying degree of severity and having the tendency to spread or "metastasize" in advanced stages of the disease. The term "cancer" or "neoplasm" generally means a malignant disease and is characterized by an uncontrolled growth of "tumor" or cancer cells. Tumors may spread locally as a primary tumor mass or spread to the distant parts of the body, i.e., metastasize.

The term "enucleated cytosol" refers to the cytoplasmic contents of a cell from which the nucleus has been removed with minimal rupture of the nucleus. Hence, enucleated cytosol is substantially free of nuclei. As used herein, "pulsing" means to provide an antigen presenting cell with an antigen, or an immunogen, or a preparation containing antigens or immunogens, e.g., such as a preparation of tumor antigens derived from a tumor. In a preferred embodiment, antigens, immunogens, or a preparation is bound to or taken up by the APC, for processing into peptides to be delivered to the plasma membrane as a peptide-MHC or peptide-HLA complex. When this complex is contacted by an immune cell, e.g., CTL, it will prime these cells to recognize and kill tumor cells carrying a similar antigen or immunogen. By pulsing APC with tumor antigens, the immunogenicity of these antigens is improved.

The term "CVACII vaccine" encompasses two components: the first is a live recombinant vaccinia virus encoding a cytokine, e.g., human IL-2 (rIL-2VV), which is preferably injected first, and the second component comprises dendritic and/or monocyte cells (DC/M) pulsed with cancer and/or melanoma antigens derived from cancer and/or melanoma cells or cell lines exposed to a vaccinia virus encoding a cytokine, e.g., rlL- 2VV. The second component is preferably administered about 30 minutes after rIL-2VV. The term "VV" stands for vaccinia virus and term "rVV" stands for recombinant vaccinia virus, which encodes an extraneous gene foreign to said virus.

The term "vaccine" as used herein includes a therapeutic or immunotherapeutic vaccine. In an embodiment of the invention, the vaccine is used in a host already diagnosed with cancer and can be administered to stimulate an immune response against a poorly immunogenic tumor. The immune response can lead to reduced tumor growth and spread, elimination of tumor cells by cellular and humoral immune responses, and/or prevention or delay of tumor recurrence upon partial or complete remission of the cancer. One aspect of the invention is directed to a therapeutic composition of antigen presenting cells pulsed with a preparation of tumor antigens found in an enucleated cytosol and cell membranes of cancer cells, e.g., melanoma cell lines, which were non- cytolytically infected with recombinant vaccinia virus encoding an immunostimulating molecule, e.g. such as a cytokine, IL-2, a hematopoietic factor, or a tumor immunogen. In a preferred embodiment the APC are the host's own or HLA-matched antigen presenting cells, e.g., dendritic and/or monocytic cells. The composition may contain cancer cell membranes containing at least two and preferably more than two HLA class I A antigens. In a preferred embodiment of the invention melanoma cells such as Mel-2, Mel-3, Mel-4, Mel-6, and Mel-9 melanoma cell lines are used. HLA-matched dendritic and/or monocytic cells provided by a donor are also contemplated as useful constituents of this vaccine.

The instant invention is directed to administering rVV encoding at least one immunostimulating molecule such as a cytokine, a hematopoietic growth factor or a melanoma immunogen. The present invention contemplates that the vaccinia virus includes genes encoding cytokines and hematopoietic growth factors such as FLT-3 or

FLT-3/FLK-2 ligand, GM-CSF, G-CSF, IL-2, IL-3, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, stem cell factor, various interferons, or a combination thereof. The practitioner will appreciate that these cytokines can stimulate the immune system of a host in a manner similar to IL-2 action. The present invention also contemplates the use of recombinant vaccinia virus encoding melanoma immunogens such as MAGE-1, MAGE-3, BAGE, GAGE, PRAME and NY-ESO-1 antigens; melanocyte differentiation antigens such as tyrosinase, Melan- A/MART-1, gplOO, TRP-1 and TRP-2; mutated or aberrantly expressed antigens such MUM-1, CDK4, beta-catenin, gplOO-in 4, p.15 and N-acetylglucosaminyltransferase; and other suitable antigens like B7-1, TA-90, lysosome-associated membrane protein (LAMP), melanocyte-stimulating hormone receptor (MC1R), p90 calnexin, and other antigens known in the art. These immunogens or antigens may provide further benefit in the instant composition by adding an additional challenge(s) to a host's immune response.

A preferred embodiment of the present invention is termed CVACII. In this embodiment the vaccinia virus (VV) used is a recombinant virus containing a gene encoding human IL-2. In addition, the APC in the CVACII embodiment are preferably pulsed with preparations from any one of five human melanoma cell lines or cell lines expressing more than one HLA class I A antigen. Finally, the patient's own dendritic cells as well as monocytes can be used as APC in CVACII.

Although the embodiment exemplified herein encompasses melanoma therapy, one skilled in the art would recognize that the principles disclosed are equally applicable to a variety of other malignant tumors including but not limited to squamous cell carcinoma, lung cancers, breast cancers, head and neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers, prostatic cancers, ovarian cancers, bladder cancers, other types of skin cancers, brain cancers, angiosarcomas, mast cell tumors, primary hepatic cancers, pancreatic cancers, gastrointestinal cancers, renal cell carcinomas, lymphomas, and hematopoietic neoplasias.

Although preferred cells for use in human melanoma vaccines are from the Mel- series (e.g., Mel-2, Mel-3, Mel-4, Mel-6, Mel-9), other melanoma cell lines can be used. Such cell lines can be established de novo from tumor biopsies of melanoma patients or can be selected from already existing sources. For example, cell lines designated as FM3,

FM6, FM9, FM28, FM37, FM45, FM55p, FM55M1 and FM55M2 were established by Kirkin et al. from eight metastatic tumors and one primary tumor of seven different patients (Kirkin, A.F., Petersen, T.R., Olsen, A.C., Li, L., thor Straten, P., Zeuthen, J. Generation of human-melanoma-specific T lymphocyte clones defining novel cytolytic targets with panels of newly established melanoma cell lines, Cancer Immunol.

Immunother. 41(2):71-81, 1995). Procedures for establishing melanoma lines are routine and well known by those of ordinary skill in the art. It will be appreciated that similar method will be applicable to the selection or establishment of transformed cell lines corresponding to tumors and cancer cells of other cell types. It will also be appreciated that where common tumor antigens are involved, vaccines can be developed from cell lines which are of different origin that the cancer to be treated.

In preferred embodiments of the present invention the selected melanoma cell lines provide at least two HLA class I antigens, preferably HLA-A2 and/or Al. In general, HLA-A2 expression is predominant in melanoma patients and plays the critical role in HLA class I restricted CTL killing of melanomas. However, some patients may express other HLA alleles. Accordingly, melanoma cell lines should preferably express more than above two HLA antigens. More preferably they should express a third HLA-A antigen and preferably this antigen is A3 antigen.

In a preferred embodiment of the instant invention, the DC are used in combination with other types of antigen presenting cells such as monocytes (M). It is preferable that DC/M cells are used freshly although one can freeze them according to established methods (e.g., U.S. Pat. No. 5,788,963) and use them whenever it is necessary. According to a preferred embodiment, DC/M cells are obtained from a patient's own blood. According to another embodiment DC/M cells are obtained from an HLA-matched donor. In addition to melanoma therapy, the instant invention provides a method of treating metastatic melanoma especially those affecting lung, liver, brain, and being either cutaneous or subcutaneous. The instant invention is also applicable to other types of cancer. In a preferred embodiment, these types of cancer may comprise fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, Kaposi's sarcoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma, or leukemia. Accordingly DC/M cells can be prepared from patients suffering from above types of cancer and are pulsed with corresponding tumor antigens which can be obtained from a patient's own tumor or from established cell lines of the same type as the tumor in need of therapy.

In embodiments of the invention, extracts for use in pulsing of DC/M cells are prepared from transformed cell lines which have been infected with recombinant vaccinia virus. Preferred extracts are those from which nuclear material has been removed so that the preparations comprise enucleated cytosol and cell membranes from recombinant or vaccinia virus infected cells. For example, a cell suspension of melanoma cells is exposed to a rIL-2VV preparation at a ratio of about 10 cells to about 1 PFU of the rIL-2VV. In accordance with the invention, the ratio of cancer cells to virus can vary and can be anywhere between about 1000-0.001 cells to about 1 PFU of virus. After a short incubation penod, to avoid virus-induced cell lysis, preferably 4-36 hrs, more preferably 18-30 hrs and even more preferably 24 hrs in a CO_: incubator, the melanoma cells are separated from the culture supernatant. This can be accomplished, for example, by spinning at approximately 1,200 rpra for 10 mm in a refrigerated centπfuge. Other means of separating cells and culture medium are well known in the art and can be employed

The separated melanoma cells are collected and disrupted by mechanical, chemical or physical means. A vanety of methods are known and can be employed These can include repeated freezing and thawing, high pressure (French press), Dounce homogenizer, microwave or ultrasound irradiation, vaπous detergents, or any other methods known in the art. The preferred method is a high frequency vibration or sonication method using a probe somcator In preferred embodiments of the invention, cells are disrupted but nuclei remain substantially mtact. The condition of the disrupted cells is monitored, for example, with a microscope. The disrupted cells are then treated to remove nuclei, for example, by centnfugation at 800 φm for 10 mm. The remaining cellular mateπal compπses vaccinia virus particles, enucleated cytosol and cell membranes, and is used for pulsing of DC/M

In an embodiment where sonication is employed to disrupt cells, the cellular material is the melanoma sonicate (MS) In certain embodiments of the invention, the virus collected from the culture supernatant is added back to the cellular matenal before pulsing of DC/M In a preferred embodiment, the combination of MS and recombinant virus from the supernatant is referred to as rIL-2VV-MS. Pπor to pulsing of DC/M, the MS or rIL-2VV-MS can be further treated to inactivate virus particles, for example, by exposure to ultraviolet light.

Pulsing of DC/M involves contacting DC/M with the cellular mateπal recovered from the disrupted cells In a preferred embodiment, contacting is for a penod of time sufficient for processing and presentation of tumor and vaccinia virus antigens by the

DC/M. Methods for pulsing immune system cells for presentation of antigen are well known to those of skill in the art.

Preferably, antigen presenting cells are obtained from the patient. The use of a patient's own or autologous APC and preferably DC, provides an opportunity for devising an individualized therapeutic approach. (Celluzzi, CM., Falo, L.D. Jr. Physical interaction between dendritic cells and tumor cells results in an immunogen that induces protective and therapeutic tumor rejection. J. Immunol. 160(7):3081-3085, 1998).

A method of making the instant composition is also disclosed. In a preferred method, the composition is prepared by growing tumor cells or tumor cell lines as the source of enucleated cytosol and cell membranes; contacting the cells with a recombinant vaccinia virus encoding an immunostimulating molecule, e.g., IL-2, in a serum free medium; sonicating or disrupting substantially intact vaccinia-infected cells to cause cells' break-down (cell sonicate); spinning cell debris to separate from cell nuclei; collecting the sonicate containing enucleated cytosol, vaccinia virus, and cell membranes; inactivating, e.g., irradiating the sonicate with ultraviolet light; pooling more or less equal volumes of sonicates from different tumor cells; adjusting the volume of sonicate to about ten million original cells per ml; dispensing each 1 ml volume of pooled sonicate into sterile glass vials; freezing and storing said vials at -70°C; retrieving dendritic and/or monocyte cells from an HLA-matched donor or from a host diagnosed with a cancer and growing said cells in culture (ex vivo); mixing or pulsing dendritic and/or monocytic cells with thawed supernatant of cancer cells in a serum free medium; and collecting pulsed dendritic and/or monocytic cells.

It is preferable that the DC/M are administered from freshly prepared cells. However, one can freeze the cells according to techniques well known in the art (U.S. Pat. No. 5,788,963) and subsequently use the DC/M when needed. In another embodiment, the invention provides a method for eliciting an anti- cancer immune response, comprising administering, to a host diagnosed with a cancer, an effective amount of a live recombinant vaccinia virus encoding an immunostimulatory molecule, such as a cytokine, e.g., IL-2, and an effective amount of antigen-presenting cells. Prior to administration antigen-presenting cells are pulsed with an enucleated cytosol and cell membranes from cancer cells infected with a recombinant vaccinia virus encoding the same or another immunostimulatory molecule. Also contemplated by the invention, is a method for treating a human host diagnosed with a cancer, e g , melanoma by administering, preferably subcutaneously (s c), a live recombinant vaccinia virus encoding an immunostimulatory molecule such as a cytokine, e.g., IL-2, and injecting, preferably into substantially the same site, a therapeutic composition prepared in accordance with the instant invention

In a preferred embodiment the effective amount of a live recombinant vaccinia virus encoding an immunostimulating molecule comprises an amount ranging from 10⁴ to 10⁹ plaque forming units (PFU) per injection Preferably, effective amounts are between about 10" and 10⁸ PFU, and more preferably about 10⁷ PFU Generally, the effective amount of therapeutic composition compπses an amount in a range about from 10' to 10⁹ oπginal cancer cells per injection Preferably, the effective amount is between about 10⁶ and 10^s cells, and more preferably about 10 cancer cells The preferred number of antigen presenting cells (APC) in one dose of a vaccine is about 1 to 5 million cells. The ratio between cancer cells and plaque forming units (PFU) of recombinant vaccinia virus is selected from the range of about 1,000-1 cancer cells to about 0.001-1 of PFU. Preferably the ratio between cancer cells and PFU of recombinant vaccinia virus is about 10 to about 1. In turn, the ratio between cancer cells and antigen presenting cells is selected from the range of about 1,000-1 cancer cells to about 10-1 antigen presenting cells. The preferable ratio between cancer cells and antigen presenting cells is about 10 cancer cells to 1-5 APC It is preferable that the instant immunotherapeutic vaccine is administered subcutaneously or intradermally for a penod of time and in an amount necessary to provide the therapeutic effect Accordingly, preferred sites of the injection are on anteπor thighs, anterior upper arms, or the anteπor thorax The minimum duration time of the vaccine therapy is at least one day, preferably at least three months, more preferably at least one year or longer and even more preferably until disease remission or disease recurrence

Therapy can also continue after disease recurrence if considered beneficial to the host In this case, changing tumor antigens may be desired and is contemplated.

In preferred embodiments of the invention, DC/M-rIL-2VV-CS can be injected intradermally or subcutaneously into sites near to regional lymph node groups. Each injection can be equally divided among at least 4 to 6 injection sites - at least 2 to 4 above the waist and at least 2 below the waist near inguinal nodes In a preferred embodiment, rIL-2VV is injected first, and the DC/M-MS is injected about 30 min. later at approximately the same sites Other routes of administration are envisioned and can include continuous (such as intravenous dπp), intramuscular, transdermal (which may include a penetration enhancement agent), sustained release by encapsulating into delivery vehicles such as posomes

Preferably, immunization with a composition of the invention is performed using multiple injections administered over a time course which is selected to maximize an immune response. In a preferred embodiment, melanoma patients receive six biweekly injections for 12 weeks, then every three months for 2 years or until cancer recurrence However, any suitable immunization regimen can be used One of ordinary skill can modify methods of administration within the teachings of the specification to provide numerous routes without rendeπng the composition of the present invention unusable or compromising its therapeutic value.

The DC/M obtained are used in DC/M-MS preparation and also for in vitro studies to determine immune activation signs.

Biopsies can be taken for determination of IL-2 production or production of any other immunostimulatory molecule by methods known in the art

The following Examples are presented to illustrate the present invention. These examples are not intended in any way to otherwise limit the scope of the disclosure

EXAMPLES

EXAMPLE I Preparation and testing of a prototype CVACII vaccine in a muπne model.

Cell lines

Munne colon cancer cell line CC-36, human fibroblast cell line MRC5, monkey kidney cell line VERO, NK sensitive cell line YAC-1 , muπne antι-CD4 antibody secreting cell line GK1.5, munne antι-CD8 secreting cell line TD3210-2 43, IL-2 dependent cell line CTLL, thymidine kmase negative cell line 143B were obtained from ATCC and maintained in either EMEM or RPMI media with appropπate nutnents. Mice

Balb/C mice of 4-6 weeks old were used in all the therapeutic cancer vaccine experiments, and for the isolation of dendritic cells, and for the preparation of anti-CD4 or anti-CD8 monoclonal antibody-containing ascites.

Vaccinia virus (VV)

A recombinant vaccinia virus (rVV) was prepared with a complete Escherichia coli β-galactosidase gene (lacZ) and another rVV having the human IL-2 gene (rIL-2VV) inserted into the same locus as the lacZ gene of rVV. These viruses were expanded in either MRC5 or VERO cells and quantified by a plaque-forming assay using VERO cells.

The method of preparation of rIL-2VV is described by Flexner et al. (Flexner C, Hugin A, Moss B. Prevention of vaccinia virus infection in immunodeficient mice by vector- directed IL-2 expression, Nature 330:259-262, 1987)

Vaccinia colon sonicate (rIL-2VV-CS) preparation

Vaccinia colon sonicate using rIL-2VV is prepared by a modified method as described. (Sivanandham, et al., Cancer Immunol. Immunother. 38:259-264, 1994). Briefly, CC-36 cells were co-cultured for 24 hours with rIL-2VVat a ratio of one cell to one PFU of virus. Vaccinia virus-infected CC-36 cells were then collected, separated by centrifugation, and broken by sonication. Cell nuclei were removed from the sonicate by centrifugation at 800 φm for 10 minutes. Vaccinia virus and tumor cell debris were isolated from the cell/virus co-culture supernatant by centrifugation at 100,000 x g for 1 hour and mixed with the cell sonicate. The resultant suspension was exposed to short wave UV for 1 hour to inactivate virus and then aliquoted and stored at -70°C until use. Aliquots contained about 10⁶ cell equivalents in 0.2 ml of saline (0.9% sodium chloride)

Analysis of the production of IL-2 from the rIL-2VV

IL-2 was measured in culture supernatants of rIL-2VV-infected CC-36 cells and in sera of mice injected with rIL-2VV using commercial ELISA kits and by a suitable bioassay. The kinetics of the production of IL-2 were tested both in vitro and in vivo procedures. Preparation of spleen-derived dendπtic/monocytic cells

Dendπtic and monocytic (DC/M) cells were isolated from spleens from the same mouse strain The spleens were minced on a wire mesh and all dissociated cells were collected in Hank's balanced salt solution (HBSS) These cells were overlaid on Ficoll- Hypaque and centπfuged at 400 g for 30 minutes, harvested from the Ficoll-Hypaque interface, washed twice in HBSS, counted in trypan blue, and then resuspended in complete RPMI medium The cells were allowed to adhere to the plastic surface of a tissue culture dish for 1 -2 hours Non-adherent cells were removed by 4 washes with medium The adherent cells contained predominantly macrophages/monocytes and dendπtic cells Cells were kept overnight in RPMI 1640 complete medium The following day, floating cells were removed, washed with HBSS and kept in complete RPMI medium containing 2000 units of muπne GM-CSF and 1 ,000 units of muπne IL-4 These cytokines favor predominantly the growth of DC However, some monocytic cells are also found in the culture After cultuπng for five days, DC/M cells were harvested, washed twice with PBS and then pulsed with rIL-2VV-CS as follows One million DC/M were incubated in 5 ml of FBS free RPMI medium with the contents of one vial (about 10⁶ cell equivalents) of rIL-2VV-CS Thus, pulsing involved a mixture containing approximately one DC/M cell to one cell equivalent of rIL-2VV-CS The mixture was incubated overnight at 37 °C with rocking The next day, pulsed DC/M cells were spun down and washed with HBSS twice

Expeπmental design

Solid tumors were induced in mice by injecting 10⁴ CC-36 cells in 100-200 μl of HBSS at the πght flank region, and the efficacy of the rIL-2VV +DC/M-rIL-2VV-CS combination therapy was studied Eight groups of 10 mice were used m this expeπment

Group I was treated with rIL-2VV + DC/M-IL-2VV-CS (CVACII), Group II was treated with a control VV+DC/M-VV-CS, Group III was treated with DC/M-rIL-2VV-CS, Group IV was treated with DC/M-VV-CS, Group V was treated with DC/M-CC-36 lysate (l e , crude cell lysate as used in the pπor art methods), Group VI was treated with rIL-2VV, Group VII was treated with a non-recombmant VV control, and Group VIII had no treatment These groups, as treated with respective vaccines are shown m Table 1

In the first and second groups, the administration was earned out in two steps. First, one million PFU of viable rIL-2VV or control "wild-type" VV in 100 μl was injected subcutaneously in the left flank region Thirty minutes later, DC/M-rIL-2VV-CS or DC/M-VV-CS in 200 μl was injected at the same site. Mice received injections on day 4, 10, and 17 after the tumor transplantation

To determine the efficacy of vaccine preparations, the mice were observed for tumor incidence Tumor incidence and diameter was measured at 2-3 days interval to assess tumor growth The effect of the treatments on survival was also determined

Analysis of the induction of tumor specific immunity

Sera and lymphocytes from mice of different treatment groups were collected and used in the suitable in vitro analysis to demonstrate the induction of antι-CC-36 immune responses Cellular assays were performed using peπpheral blood lymphocytes (PBL) or splenic lymphocytes of mice from the different groups Lymphocytes were prepared from peπpheral blood or spleen by standard methodology Cytolytic assays

CC-36 specific cytolytic activity of PBL or splenic lymphocytes was measured using a standard chromium release assay. Freshly prepared lymphocytes were co-cultured with irradiated rIL2-VV-CS pulsed DC/M for 5 days to 2 weeks in RPMI medium containing 10 units of interleukin-2. CC-36 or YAC-1 target cells were harvested, washed and incubated with 100 μCi sodium chromate/10⁶ cells (^{5 l}Cr, Amersham), in 0.5 ml volume for 60 minutes at 37 °C. After washing with RPMI 1640 four times, radiolabelled target cells were added at 10⁴/ml in 100 μl volume to conical microtiter plates (Costar). Effector lymphocyte to target tumor cell ratios (E:T) were in a range of 100:1 - 10:1. E:T were used in quadruplicate wells. Plates were spun at 250 g for 5 minutes and incubated at

37°C in a humidified incubator containing 5% CO₂. After four hours, the supernatants from the wells were harvested on fiber filters utilizing a Skatron Collection System and counted for one minute on a gamma counter. To determine maximum ⁵¹Cr release, 0.1 ml of 1% sodium dodecyl sulfate solution was added to appropriate wells. Counts per minute (cpm) were used to calculate percent release (%R) according to the formula:

experimental release - spontaneous release %R = X 100 maximum release - spontaneous release

CTL precursor frequency (CTLp) assay

Fresh lymphocytes were isolated from mice from the above treatment groups. 2-10 x 10^"1 lymphocytes in 50 μl of medium containing 50 units/ml of recombinant IL-2 were added to each well with 10³ CC-36 sonicate-pulsed DC/M. At least 50 replicates were used in this test. The co-cultures are kept for 21 days in a humidified CO₂ incubator. On day 6, 12, and 18, 50 μl of fresh medium was added to each well. On day 21, about 10³ YAC-1 cells were added followed by 10^{3 51}Cr-labeled CC-36 cells. After four hours, supernatants were harvested and counted using a gamma counter. Positive wells were those showing greater than 10% cytotoxicity in comparison to the control wells.

ELISA measuring the induction of antibody to CC-36 tumor ELISA plates were coated with 10 μg/well of CC-36 tumor cell sonicate in bicarbonate buffer (pH 9 4) Unbound sites capable of non-specific binding were blocked with 1% human serum albumin and incubated overnight at 4°C 100 u\ of diluted serum from immunized mice were added per well and incubated at 4°C overnight Assays included a negative control using normal mouse serum Plates were washed three times with PBS 100 μl of a 1 1000 dilution of biotinylated goat anti-mouse second antibody (Sigma, St Louis, MO) was added to each well After incubation at 37°C for 1 hour, plates were washed three times with PBS and incubated with 100 μl of avidin-alkahne phosphatase conjugate (Sigma, St Louis, MO) (1 1000 dilution) for 30 mm The plates were washed three times with PBS and 100 μl of p-mtrophenyl phosphate (PNPP, Sigma,

St Louis. MO) substrate was added After 30 minutes, the reaction was stopped by addition of 50 μl of 3 M sodium hydroxide per well OD ₀₆ values obtained from the ELISA assay indicated the titer of anti-tumor antibodies

Statistical analysis

A statistical prediction was used to find the total number of animals (10) required per treatment group to produce a 30-40%) difference in treatment arm in favor of the new cancer vaccine treatment with an 85% confidence interval Student's T test and Wilcoxon survival analysis or log-rank survival analysis was used to determine the difference between groups of vaccines

Results

Four separate expenments were performed in this muπne model study Figure 2 shows the efficacy of the vaπous vaccine preparations on incidence of tumors of 5±2 mm mean diameter dunng the 30 days following tumor implantation Only one mouse in the

CVACII vaccine group (group I) developed tumor (10%) In other groups, the tumor incidence observed during the 30-day penod was 50-100% Thus, tumor incidence was significantly reduced in group I as compared to other groups (p<0 05) Group V represents mice which were treated with a typical DC-based vaccme In addition, a survival expeπment was performed using the same treatment groups and the same tumor induction protocol An autopsy was performed on each mouse to identify the cause of death. The results are represented in the Figure 3 In this survival study, 90% of mice that received rIL-2VV + DC/M-rIL-2VV-CS (CVACII) treatment survived beyond 100 days following tumor transplantation The survival rate for mice in the other groups was 0-60% Thus, when compared with other treatments the rIL-2VV + DC/M-rIL-2VV-CS treatment produced a significantly better survival rate (p<0.05) The results shown in Figures 2 and

3, indicate that rIL-2VV + DC/M-rIL-2VV-CS therapy induces a significant and durable anti-tumor response.

The mechanism of induction of anti-tumor response by rIL-2VV + DC/M-rIL-2W-CS therapy was evaluated in vivo using mice depleted in either CD4^* helper T cells or CD8 cytolytic T cells (Fig 4) and in vitro using PBL from mice treated with rIL2-VV + DC/M-rIL-2VV-CS (Fig. 5) On day 2 following tumor induction, mice in the group II were injected with 0 5 ml of antι-CD4 antibody-containing ascites and mice in the group III were injected with 0 5 ml of antι-CD8 antibody-containing ascites. Mice in group I, II and III were then treated with rIL-2VV + DC/M-rIL-2VV-CS on day 4, 1 1 and 18 Mice in group IV received no treatment following tumor induction. The incidence of tumor in each group was checked every 2-3 days and recorded. Groups I and II showed similar protection against tumor development. This protection is significantly higher than the tumor protection in groups III and IV Mice depleted of CD4^T helper T cells showed a level of protection against tumor similar to that of CD47CD8^* mice treated with rIL-2VV + rIL-2VV-CS However, mice depleted of CD8^~ cytolytic T cells did not show any protection against tumor development in response to the IL-2VV+DC^/M-rIL- 2VV-CS therapy (Fig 4) These results indicate that the anti-tumor response induced by the rIL-2VV + DC/M-rIL-2VV-CS is due to the induction of CD8^{^} cytolytic T cells

Analysis of PBL from mice treated with rIL-2VV + DC/M-rIL-2VV-CS therapy indicated a significantly higher cytotoxicity against CC-36 tumor (Fig. 5) Mice (n = 20 per group) were treated with either CVACII or DC/M-CS (standard type DC vaccme, control) on day 1, 7 and 14 PBL were collected and stimulated with CVACII or control for 5 days in RPMI complete medium containing 10 units of rIL-2. PBL were then tested for cytotoxicity in a Cr release assay using CC-36 (Fig. 5, top panel) and YAC-1 (bottom panel) cells as targets at vaπous effector to target ratios PBLs from mice immunized with

CVACII demonstrated greater cytotoxicity against CC-36 cells as compared with PBLs from mice immunized with the control vaccine These results and the in vivo experiments support the involvement of CD8 positive cytolytic T cells in the anti-tumor response induced by CVACII therapy

EXAMPLE 2

Preparation and clinical testing of CVACII melanoma vaccine in humans

Pieparation and administration of CVACII

Preparation and administration of CVACII to cancer patients involves the following steps^* Preparation of clinical grade recombmant vaccinia virus encoding human ιnterleukιn-2 (rIL-2VV), Preparation of clinical grade melanoma cell lines deπved from humans with metastatic melanoma, Incubation of rIL-2VV with melanoma cells for a sufficient penod of time; Disruption of the melanoma cells by sonication and isolation of vaccinia virus, enucleated cytosol and cell membranes, Irradiation of the melanoma sonicate (MS) by UV m order to inactivate the virus; Preparation of dendπtic/monocytic cells (DC/M) preferably from patient's own blood; Pulsing DC/M with melanoma sonicate to obtain a DC/M-MS preparation, Vaccinating a patient, first with rIL-2VV and then with the DC/M-MS preparation, preferably subcutaneously and preferably at the sites near regional lymph nodes

Method of administration

In the first step of a two step procedure, live rIL-2VV (10⁷ PFU) was injected subcutaneously or intradermally into at least 4-6 sites near the regional lymph node groups These sites were located on antenor thigh, upper arm, or antenor thorax About thirty minutes later, DC/M-MS was injected at substantially the same sites as the initial rIL-2VV injection The vaccme was administered once every two weeks for three months and thereafter once every three months for one or two years or until recurrence or progression of disease

Preparation of recombinant vaccinia virus encoding IL-2

Recombinant vaccinia virus encoding IL-2 (rVV-IL-2) was prepared using a clinical grade Wyeth strain vaccinia virus (a strain of virus similar to one used in the first generation VMO vaccine) and recombinant human IL-2 gene (pTCGF-1 1, No. 39673, ATCC. Manassas, VA) by an established molecular methodology known in the art. The method of constructing a rVV is described -in detail elsewhere (Sivanandham, M., Scoggin, D.S., Tanaka, N., Levi, M., Wallack, M.K., Therapeutic effect of a vaccinia colon oncolysate prepared with interleukin-2 gene encoded vaccinia virus studied in a syngeneic CC-36 murine colon hepatic metastasis model, Cancer Immunol. Immunother. 38:259-264, 1994; Lee, S.S., Eisenlohr, L.C., McCue, P.A., Mastrangelo, M.J., Lattime, E.C., Vaccinia virus vector mediated cytokine gene transfer for in vivo tumor immunotherapy, Proc. Am. Assoc. Can. Res. 1035:514, 1994). Briefly, a human cDNA clone specific for the IL-2 gene was isolated from a plasmid DNA encoding IL-2 using proper restπction enzymes. This IL-2 gene was blunt ended using a filling reaction and ligated into the Smal restriction site in the tK gene segment of the plasmid pSC65.

The pSC65 plasmid containing the IL-2 gene and the Wyeth strain vaccinia vaccine virus (also containing tK gene in the non-essential region) was allowed to undergo homologous recombination in CV-1 cells as follows. First, a CV-1 cell monolayer was infected with the vaccinia vaccine virus (one cell to one PFU of virus) for 2 hours. The plasmid DNA encoding IL-2 was then transduced into these cells using a calcium chloride method. Five (5) ml of culture medium was added to the CV-1 cell culture followed by incubation for 3 hours. The medium was removed and 5 ml of fresh medium was added.

The cell culture was incubated for 48 h in a CO, incubator. CV-1 cells from the culture were scraped and collected with the medium. Virus was released from the cells by 3 cycles of freezing and thawing followed by sonication for 1 min in a bath sonicator. The recombinant viral clones were selected for growth on tK-143B cells in the presence of 5-bromo-deoxyuridine (BUDr) and 5-bromo-chloro-3-indoyl-beta-D galactoside (X-gal).

Blue plaques were selected and purified by at least 3 more cycles of plaque isolation. The rIL-2VV thus obtained was tested in mice for toxicity and in vitro for the absence of bacteria, fungi, and mycoplasma. The presence of vaccinia virus in the rIL-2VV preparation was confirmed by an antibody neutralization assay using anti-vaccinia polyclonal antibody. Tests were also carried out to rule out contamination with human infectious viruses such as HIV, HPV, and HBV. A seed lot was established with 10⁹ plaque forming units of rIL-2VV

Melanoma cell lines

Melanoma cells were deπved from five established melanoma cell lines Mel-2, Mel-3, Mel-4, Mel-6 and Mel-9 These cells were oπginally denved from patients with metastatic melanoma These cells express at least two HLA class I-A antigens These cells also express a vanety of melanoma antigens The melanoma cell lines have been characterized for the expression of melanoma antigens that induce melanoma specific antibodies and cytolytic T cells The cells contained characteπstic melanoma cellular components such as melanosomes and pre-melanosomes that could be detected microscopically Cells were free of contaminants such as bacteria, mycoplasma, viruses, and other biohazardous agents A seed lot was established for each melanoma cell line One vial from this seed lot was used for the preparation of one batch of vaccine

Preparation of melanoma sonicate (MS) containing recombinant vaccinia virus

(rIL-2VV-MS)

A single cell suspension of melanoma cells was exposed to rIL-2VV -preparation at a ratio of about 10 cells to about 1 PFU of the rIL-2VV The ratio can vary and can be anywhere between about 1000-0 001 cells to about 1 PFU of virus After incubation for 24 hrs (viral replication is not yet substantially cytolytic and substantially all cells remain intact) in a CO, incubator, the melanoma cells were separated by spinning at approximately 1 ,200 φm for 10 mm in a refngerated centnfuge The supernatant (SI) was saved and the cell pellet (PI) was reconstituted in PBS The cells were disrupted by sonication using a probe sonicator (1,500 Watts, Heat System XL seπes) for approximately 1 mm The sonication was repeated about three times or until all cells were broken but nuclei were still intact (as monitored under microscope) The mixture of broken cells was then spun at approximately 800 φm for 10 mm and the resulting melanoma sonicate (MS) containing vaccinia virus particles, enucleated cytosol and cell membranes was saved (the pellet containing mostly nuclei was discarded) The supernatant SI was centnfuged at approximately 100,000 g for 30 min using an ultracentrifuge to pellet the virus from the supernatant. The pellet was combined with the nuclei-free melanoma cell sonicate (MS) to form the rIL-2VV-MS preparation. Each melanoma cell line was processed separately in the same manner. The rIL-2VV-MS from each melanoma cell line was then placed in a Petri dish about 10 cm from a germicidal UV lamp (short-wave UV, 256 nm, 1.5-1.75 microwatt/cm²) for about 1 hour to inactivate the vaccinia virus. Melanoma sonicates from all five melanoma cell lines were mixed in an equal cell number ratio. One unit of rIL-2VV-MS contained the equivalent of about 10⁷ original melanoma cells and 10⁶ PFU of rIL-2VV. The rIL-2VV-MS preparations were dispensed in 1 ml vials with saline (0.9% sodium chloride solution) and stored at -70°C until needed.

Dendritic/monocytic cells (DC/M)

100 ml of peripheral blood was collected from the patient and 50 ml was reserved for HLA analysis. Dendritic cells were isolated from the remaining 50 ml as follows. Peripheral blood lymphocytes (PBLs) were isolated from blood using lymphocyte separating medium (Ficoll-Hypaque or Lymphocyte Separation Medium (LSM), Litton).

The blood was collected in sterile heparinized tubes, diluted in Hank's balanced salt solution (HBSS), overlaid on Ficoll-Hypaque, and centrifuged at 400 g for 30 minutes. Cells at the LSM-aqueous interface were then harvested, washed twice in HBSS, and counted in trypan blue to assess the viability. PBLs were reconstituted at 10⁶ cell/ml in RPMI- 1640 complete medium with 10% FBS and 1 mM glutamine, transferred into a T75 tissue culture flask and incubated at 37 °C for 2 hrs in a CO, incubator. The flask was then washed at least 4 times with complete RPMI medium to remove non-adherent cells. After the final wash 35 ml RPMI-1640 complete medium was added to the flask followed by incubation overnight (approximately 18 hours). The next day, floating, non-adherent cells (dendritic/monocytic cells) were washed with medium and transferred to a new flask with

RPMI-1640 complete medium supplemented with 2,000 unit/ml IL-4 and 2,000 unit/ml GM-CSF and incubated for 18-24 hours in a CO₂ incubator. (Increased DC yield can be obtained with the addition of FLT-3 ligand, which is an in vivo dendritic-cell growth factor.) The yield of DC/M was measured and the cells further cultured for about 5 days in a CO, incubator. Usually 1-5 x 10⁶ viable DC/M cells were obtained after 5 days of culture. (The culture period can be longer to obtain larger numbers of cells.) The day prior to the DC/M-MS administration, cultured DC/M were harvested from the flask, washed once with PBS and mixed with 1 ml unit of rIL-2VV-MS in 5 ml of AIM V (serum-free) medium containing 2,000 unit/ml IL-4 and 2,000 unit/ml GM-CSF The passage through serum-free medium was intended to eliminate the toxic effects associated with administration to a subject of a preparation containing bovine serum The mixture was incubated in a glass vial at 37 T overnight (approximately 18 hours) to allow time for processing and presentation of tumor and vaccinia virus antigens The minimal time for antigen presentation is about six hours The next day, the resulting DC/M-MS were spun down and the supernatant was discarded (an aliquot is saved for endotoxin testing) The DC/M-MS was reconstituted in 1 ml of rIL-2VV-MS and divided into 5-6 a quots for administration to the subject

Monitoπng the Induction of Immune Responses

Indications of induction of anti-melanoma immunity in patients treated with the melanoma vaccine are useful in determining the potency of the vaccme These indications are especially valuable in early stages of vaccine therapy when end-point clinical results are not yet available Induction of anti-melanoma immunity was analyzed by determming the delayed type hypersensitivity (DTH) response against melanoma antigens pnor to and three months after the melanoma vaccine treatment In addition, serum and peπpheral blood lymphocytes were obtained pnor to vaccine injection and one month after the vaccme injection to test the induction of anti-melanoma immunity by cytotoxicity assay, CTL precursor frequency (CTLp) assay and phenotypic analysis of lymphocytes

Delayed type hypersensitivity (DTH) test This test was performed to evaluate the immune response against melanoma antigens If a patient was positive to HLA Al then melanoma peptide antigen MAGE-1 loaded-DC/M was used. If a patient was positive for HLA A2 then MAGE-3, MART-1 or gplOO melanoma antigen-loaded DC/M was used MAGE-1 peptide has amino acid sequence NH,-EADPTGHSY-COOH, MAGE-3 peptide has amino acid sequence NH₂-EVDPIGHLY-COOH, and MART- 1 peptide has ammo acid sequence

NH,-AAGIGILTV-COOH. The peptides were obtained from a commercial source The puπty of this peptide was >90% and showed a single peak in the mass spectrum analysis These peptides were reconstituted in pure water at 1 mg/ml and steπhzed by filtration through a 0.2 μm steπle filter Reconstituted peptides were ahquoted at 1 ml per vial and frozen at -70°C Ten million DC/M in 1 ml of medium containing 50-200 μg of peptide were incubated at 37 °C for 4 hours. The peptide loaded DC/M were washed two times with medium and then kept in 100 μl of PBS for injection to assess DTH response, in vitro cytotoxicity assay, proliferation, and ELISPOT assay.

For DTH test, the melanoma peptide antigen-pulsed autologous DC/M (10^{5 6}) were injected subcutaneously at the deltoid region After 48 hours, induration was measured and photographed The patient also received plain DC M as a control m the other arm. If a DTH site showed a response, the site was biopsied and analyzed for the presence of CTLp by CTLp frequency assay and for the presence of cytotoxic lymphocytes by the ⁵¹Cr release assay. In addition, immunohistology was performed to analyze the phenotype of tumor-infiltrating lymphocytes. DTH test was performed pnor to the initiation of CVACII therapy and one month after initiation of the therapy.

Lymphocytes from blood (PBL) and lymphocytes from DTH sites

Cellular assays such as CTL assay, and CTLp assay were performed using patient's penpheral blood lymphocytes (PBL) as effector cells. PBL were harvested from blood as descπbed above. Infiltrating lymphocytes at the DTH sites were isolated from biopsies taken at the peptide pulsed DC/M-injected sites Biopsies were also taken at the control DC M-injected sites. Isolation of lymphocytes from the biopsy tissue was performed by collagenase treatment. Tissue was minced and kept in 10 ml of RPMI medium containing collagenase-DNase (1 4 mg of collagenase type IV and 1 0 mg of DNase per ml) for 3-4 hours at 37°C. Cells were washed 3 times with medium and kept overnight in a CO₂ incubator The next day, non-adheπng lymphocytes were separated from adhered fibroblast cells and cultured in the presence of IL-2 containing medium and peptide-pulsed DC/M for 1-2 months. These lymphocytes were phenotyped and tested for peptide specific CTL and CTLp.

Cytolytic assay MAGE- 1 , MAGE-3 , MART- 1 /Melan A, or gplOO peptide-specific cytolytic activity of PBL was measured using a standard chromium release assay Freshly prepared PBL or frozen PBL from several time intervals were tested in the same assay Melanoma cell lines positive to both HLA-A1 and -MAGE-1 (if patient is Al positive) or to both HLA-A2 and MAGE-3, MART-1/Melan A, or gplOO (if patient is A2 positive) and autologous DC/M-pulsed with the above peptide antigens were used as stimulators in this assay. Patient's PBL stimulated with melanoma antigen were cultured in IL-2 RPMI medium for at least 3 weeks and used in this assay Target cells in the assay routinely included two melanoma cell lines, normal skin fibroblasts pulsed with appropπate peptide, and erythroleukemia line K-562. For the assay, target cells were harvested, washed and incubated with 100 Ci sodium chromate/10⁶ cells, in 0 5 ml volume for 60 minutes at 37°C After washing with RPMI 1640 four times, radiolabelled target cells were added at 10⁴/ml in 100 μl volume to conical-bottomed microtiter plates (Costar). Effector to target ratios (E.T) 40.1 - 5.1 were used in quadruplicate wells. Plates were spun at 250 g for 5 minutes and incubated at 37 °C in a humidified incubator containing 5% CO,. To determine maximum release, 0.1 ml of 1 % sodium dodecyl sulfate solution was added to the appropπate wells. At the end of four hours the supernatants from the wells were harvested on fiber filters and then counted for one minute on a gamma counter. Counts per minute (cpm) were used to calculate percent release (%R) according to the formula as disclosed in Example 1

ELISPOT assay may be used to quantify the CTLp specific to a melanoma peptide antigen

This assay can be performed according to a published method but with slight modification (Miyahira, Y. et al., Quantification of antigen specific CD8+ T cells using an ELISPOT assay, J. Immunol. Meth. 181 :45-54, 1995). Wells in a 96 well plate are coated with 10 μg/ml mouse anti-human IFN-γ m 75 μl of PBS and incubated overnight at room temperature under a laminar flow hood. The plate is washed three times with culture medium (Complete RPMI-1640). The last wash is performed with medium. PBL, in the amounts of 10⁴, 5xl0⁴, 10\ or 5xl0⁶ PBL (10⁶ for fresh PBL) in 50 μl of medium are added in each well At least 25 replicates are kept for each dilution. Peptide pulsed DC/M at a ratio to PBL of 1 : 1 are added into each well and incubated 24-48 h in a CO₂ incubator. Wells are washed with PBS containing 0.05% Tween 20 Fifty (50) μl (5 μg/ml) of biotinylated mouse anti-interferon-gamma (with different clone) is added per well and incubated overnight at 4°C The next day, wells are washed three times with PBS-Tween 20 and 50 μl of avidin-peroxidase is added (as per the manufacturer's instruction) The plate is incubated at 37 °C for 1 h and washed two times with PBS-Tween and one time with PBS (no Tween-PBS) Fifty (50) μl of Tπs-HCl (50 mM and pH 7 5) containing 3,3' diaminobenzidine-tetra-hydrochlonde dehydrate (DAM) (1 mg/ml) and 5 μl/ml of 30% hydrogen peroxide is added and allowed to develop color. Black green spots in each well are counted using a simple microscope The number of spots for each dilution is calculated and compared

Immunophenotypmg

Penpheral blood lymphocytes and DTH-mfiltratmg lymphocytes were phenotyped for CD4, CD8, CD 16 and CD25 using direct or indirect immuno fluorescence or immunohistologic methods. Appropnate monoclonal antibodies to CD4, CD8, CD 16 and

CD25 were purchased from commercial sources DC M were phenotyped for the expression of CD80, CD85, CD1 lb and la antigens

Expression of IL-2 at the Vaccine Injection Site Punch biopsies were taken from the vaccine-injected and control vaccine-mjected sites At least one vaccme biopsy and one control vaccine biopsy was taken at each time point m the injection schedule and tested for the presence of IL-2 For immunohistology, frozen biopsy tissue was used for preparation of 6 μm tissue sections on treated glass slides which were then air dπed for 30 mm at room temperature Tissue was fixed with Situfix (Kreatech Diagnostics) and then washed twice with phosphate-buffered saline

(PBS) A drop of appropπately diluted biotinylated antibody to human IL-2 (5 μg/ml concentration) was added and incubated m a humid chamber at room temperature for 30 minutes to 1 hour The slide was washed three times with PBS and then incubated with appropπately diluted avidm peroxidase for 30 min The slide was washed twice with PBS and then treated with a drop of diaminobenzidine (1 3 mM DAB in PBS containing 0.02%o hydrogen peroxide) for 20-30 minutes The slide was washed with PBS and dehydrated with alcohol and Xylene, and mounted with DPX. Brown staining indicates the presence of IL-2.

Statistical analysis All immunological tests compared induction of immunity in the pre- and post- treatment samples. In these analyses, an ANOVA statistical method was used to identify changes due to the vaccine therapy.

Results The patient received six biweekly subcutaneous injections of CVACII over three months (induction phase). Clinically, no major side-effects were observed except for mild fever, headache, and inflammation and swelling at the vaccine sites. Post-vaccination, peripheral mononuclear cells contained an increased number of tumor-reactive proliferative and cytolytic cells. In general, the patient had no DTH response prior to the melanoma vaccine therapy. However, DTH response was apparent when tested after the completion of the induction phase.

Competence of immunity to common recall antigens was tested using the Multitest CMI (Pasteur-Merieux Connaught Laboratories, Swiftwater, PA) prior to vaccine treatment and two months after the initiation of vaccine treatment. The patient showed a reaction to at least one of the eight antigens in the Multitest prior to the treatment and exhibited a reaction to at least one of the eight recall antigens. Furthermore, the patient exhibited a vitiligo (discoloration of the skin) near the vaccine-injected sites, suggesting the induction of anti-melanocyte immune response.

Several in vitro assays were also performed using pre- and post-vaccination blood. Post-immune PBL showed an enhanced proliferation to melanoma antigens and increased anti-melanoma cytotoxicity (Figures 6 and 7 respectively). Figure 6 shows proliferation of pre-immune PBL and post-immune PBL or fractionated CD8⁺ T cells against autologous dendritic/monocytic cells pulsed with purified melanoma peptide antigen (MART-1 ), the patient's HLA-matched allogeneic melanoma cell lysate (Mel-9), melanoma cell lysate derived from melanoma cells that are not matched to the patient's HLA (Mel-2), or cell lysates from patient's own tumor cells. When compared with pre-immune PBL, post- immune PBL or CD8^* T-cells showed enhanced proliferative response to all sources of melanoma antigens Figure 7 shows cytotoxicity of pre- and post-immune PBL of a patient immunized with the CVACII. PBL were isolated from the blood and stimulated with melanoma antigen-pulsed DC/M for 1 -3 weeks in RPMI complete medium containing 10 units/ml of IL-2 When compared with pre-immune PBL, post-immune

PBL showed a higher lysis against the patient's HLA-matched melanoma cells (Mel-9) Although the post-immune PBL showed an increased lysis of melanoma cells that are not shaπng the patient's HLA (Mel-2), this increase was only moderate Post-immune PBL also showed an enhanced cytotoxicity against the NK target K562 The immune reaction was specific since the patient's response was more pronounced when HLA type was matched Several other assays such as ELISA to demonstrate the production of antibodies to melanoma antigens, lmmunophenotype of cells that infiltrated the DTH site and testing of the vaccine injected sites for IL-2 production were also performed. In addition, the induction of anti-melanoma immunity revealed an immune reaction against synthetic melanoma peptide antigens and the patient's own tumor cell antigen-pulsed dendntic cells.

Taken together these results indicated that CVACII vaccination induced positive immunological changes this advanced, terminally-ill patient These results demonstrate that immunization with CVACII confers cellular immunity and retards tumor growth, thus prolonging the survival of patients afflicted with melanoma. The cited references are mcoφorated by reference herein in their entirety The present invention is not to be limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention Vaπous modifications of the invention in addition to those shown and descπbed herein will become apparent to those skilled in the art from foregoing descπption and accompanying drawings. Such modifications are intended to fall withm the scope of the appended claims

Claims

What is claimed is:

1. A therapeutic composition comprising antigen presenting cells pulsed with a preparation comprising enucleated cytosol and cell membranes of cancer cells infected with a recombinant vaccinia virus encoding at least one immunostimulating molecule.

2. The composition of claim 1, wherein the enucleated cytosol is substantially free of nuclei.

3. The composition of claim 1, wherein the cell membranes comprise at least two HLA class I A antigens.

4. The composition of claim 1 , wherein the recombinant vaccinia virus is either live or inactivated.

5. The composition of claim 1. wherein the immunostimulating molecule is IL-2.

6. The composition of claim 1, wherein the immunostimulating molecules comprises FLT-3 ligand, FLT-3/FLK-2 ligand, GM-CSF, G-CSF, IL-3, IL-4, IL-6, IL-7, IL- 12, IL-15, IL-18, stem cell factor, an interferon, or a combination thereof.

7. The composition of claim 1 wherein the immunostimulating molecule compπses a melanoma immunogen selected from the group consisting of MAGE-1, MAGE-3, BAGE, GAGE, PRAME, NY-ESO-1 , tyrosinase. Melan-A, MART- 1 , gp 100, TRP- 1, TRP-2, MUM- 1 , CDK4, beta-catemn, gp 100in4, pi 5, N- acetylglucosaminyltransferase. B7-1 , TA-90. lysosome-associated membrane protein, melanocyte-stimulating hormone receptor, p90 calnexin, and a combination thereof.

8. The composition of claim 1 , wherein the antigen presenting cells are dendritic cells or monocytes.

9. The composition of claim 1, wherein the antigen presenting cells are dendritic cells and monocytes.

10. The composition of claim 1, wherein the antigen presenting cells are autologous cells.

1 1. The composition of claim 1, wherein the antigen presenting cells are HLA-matched to a host to be treated.

12. The composition of claim 1, wherein the cancer cells are melanoma cells. The composition of claim 12, wherein the melanoma cells comprise one or more cells from the group consisting of Mel-2, Mel-3, Mel-4, Mel-6, and Mel-9 The composition of claim 1, wherein the cancer cells are established cancer cell lines The composition of claim 1 , wherein the cancer cells are selected from the group consisting of fibrosarcoma, myxosarcoma, hposarcoma, chondrosarcoma, osteogemc sarcoma, chordoma, angiosarcoma, Kaposi's sarcoma, endothehosarcoma, lymphangiosarcoma. lymphangioendothehosarcoma, synovioma, mesothehoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma. colorectal carcinoma, pancreatic cancer, breast cancer, ovanan cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcmoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choπocarcmoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, g oma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pmealoma, hemangioblastoma. acoustic neuroma, oligodendroghoma, meningioma, neuroblastoma, retinoblastoma. myeloma, lymphoma, and leukemia cells The composition of claim 14, wherein the cancer cell lines are selected from the group of cancer cell lines consisting of fibrosarcoma. myxosarcoma, hposarcoma, chondrosarcoma, osteogemc sarcoma, chordoma, angiosarcoma, Kaposi's sarcoma, endothehosarcoma, lymphangiosarcoma, lymphangioendothehosarcoma, synovioma, mesothehoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma. colorectal carcinoma, pancreatic cancer, breast cancer, ovanan cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcmoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcmoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, ghoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroghoma, menmgioma, neuroblastoma, retinoblastoma, myeloma, lymphoma. and leukemia cell lines The composition of claim 1 , wherein the cancer cells are harvested from a host to be treated with the composition An immunotherapeutic vaccine compπsing

(a) a first part compnsing a first recombinant vaccinia virus encoding at least one first immunostimulating molecule, and

(b) a second part compπsing antigen presenting cells pulsed with a preparation compnsing enucleated cytosol and cell membranes of cancer cells infected with a recombinant vaccinia virus encoding at least one second immunostimulating molecule The vaccine of claim 18, wherein the enucleated cytosol is substantially free of nuclei The vaccine of claim 18, wherein the cell membranes compπse at least two HLA class I A antigens The vaccine of claim 18, wherein the first recombinant vaccinia virus is a live virus The vaccine of claim 18, wherein the second recombinant vaccinia virus is either live or inactivated The vaccme of claim 18, wherein the first immunostimulating molecule is IL-2 The vaccine of claim 18, wherein the first immunostimulating molecule compπses FLT-3 ligand, FLT-3/FLK-2 ligand, GM-CSF, G-CSF, IL-3, IL-4, IL-6, IL-7, IL- 12, IL-15, IL-18, stem cell factor, an interferon, or a combination thereof The vaccine of claim 18, wherein the first immunostimulating molecule comprises a melanoma immunogen selected from the group consisting of MAGE-1, MAGE- 3, BAGE, GAGE, PRAME, NY-ESO-1 , tyrosinase, Melan-A, MART- 1 , gp 100, TRP- 1, TRP-2, MUM- 1, CDK4, beta-catenin, gp 100m4, pl5, N- acetylglucosaminyltransferase, B7-1, TA-90, lysosome-associated membrane protein, melanocyte-stimulatmg hormone receptor, p90 calnexm. and a combination thereof. 26 The vaccine of claim 18, wherein the second immunostimulating molecule is IL-2

27. The vaccine of claim 18, wherein the second immunostimulating molecule compnses FLT-3 ligand, FLT-3/FLK-2 ligand, GM-CSF, G-CSF, IL-3, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, stem cell factor, an interferon, or a combination thereof

28. The vaccme of claim 18. wherein the second immunostimulating molecule compπses a melanoma immunogen selected from the group consisting of MAGE- 1, MAGE-3, BAGE, GAGE, PRAME, NY-ESO-1, tyrosinase, Melan-A, MART- 1, gp 100. TRP- 1 , TRP-2, MUM- 1. CDK4, beta-catenm, gp 100m4, pi 5, N- acetylglucosaminyltransferase. B7-1 , TA-90, lysosome-associated membrane protein, melanocyte-stimulatmg hormone receptor, p90 calnexin, and a combination thereof.

29. The vaccme of claim 18, wherein the antigen presenting cells are dendntic cells or monocytes.

30. The vaccine of claim 18, wherein the antigen presenting cells are dendπtic cells and monocytes.

31. The vaccine of claim 18, wherein the antigen presenting cells are autologous cells.

32. The vaccine of claim 18, wherein the antigen presenting cells are HLA-matched to a host to be treated

33. The vaccine of claim 18, wherein the cancer cells are melanoma cells.

34 The vaccine of claim 33, wherein the melanoma cells compπse one or more cells from the group consisting of Mel-2, Mel-3, Mel-4, Mel-6, and Mel-9.

35. The vaccme of claim 18, wherein the cancer cells are established cancer cell lines.

36. The vaccme of claim 18, wherein the cancer cells are selected from the group consisting of fibrosarcoma, myxosarcoma, hposarcoma, chondrosarcoma, osteogemc sarcoma, chordoma, angiosarcoma, Kaposi's sarcoma, endothehosarcoma, lymphangiosarcoma, lymphangioendothehosarcoma, synovioma, mesothehoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovanan cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroghoma, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma, and leukemia cells.

37. The vaccine of claim 35, wherein the cancer cell lines are selected from the group of cancer cell lines consisting of fibrosarcoma, myxosarcoma, Hposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, Kaposi's sarcoma, endothehosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothehoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma. acoustic neuroma, oligodendroghoma, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma, and leukemia cell lines.

38. The vaccine of claim 18, wherein the cancer cells are harvested from a host to be treated with the composition.

39. A method for preparing an immunotherapeutic vaccine, which comprises:

(a) contacting cancer cells with a recombinant vaccinia virus encoding an immunostimulating molecule;

(b) disrupting the vaccinia virus-contacted cancer cells to obtain a preparation comprising enucleated cytosol, and cell membranes from said cancer cells, and (c) pulsing antigen presenting cells with the preparation The method of claim 39, wherein the contacting step is stopped before the cancer cells are lysed by the vaccinia virus The method of claim 39, wherein the contacting step lasts between about four hours and about thirty-six hours The method of claim 39, wherein the contacting step lasts about twenty-four hours The method of claim 39, wherein the preparation is substantially free of cancer cell nuclei The method of claim 39. wherein the disrupting step is earned out by physical or chemical means. The method of claim 39, wherein the disrupting step is earned out by sonication The method of claim 39, wherein the antigen presenting cells are obtained from peπpheral blood, spleen, skin, thymus, lymph nodes or bone marrow The method of claim 46, wherein the antigen presenting cells are HLA-matched to a host to be treated The method of claim 39, wherein the pulsing step is earned out for a time sufficient to allow the antigens in the preparation to be processed by and presented on the plasma membrane of the antigen presenting cells The method of claim 39, wherein the pulsing step is carried out for at least six hours. The method of claim 39, wherein the ratio between the cancer cells and plaque forming units (PFU) of the recombinant vaccinia virus is from about 1 million 1 to about 1.1. The method of claim 39, wherein the ratio between the cancer cells and PFU of the recombinant vaccinia virus is about 10 to 1 The method of claim 39, wherein the ratio between the cancer cells and the antigen presenting cells is about 1,000.1 to about 1.10 The method of claim 39, wherein the ratio between the cancer cells and the antigen presenting cells is from about 10 1 to about 2 1 54 A method for eliciting an anti-cancer immune response, which compπses administering a therapeutically-effective amount of a composition compπsing antigen presenting cells pulsed with a preparation comprising enucleated cytosol and cell membranes of cancer cells infected with a recombinant vaccinia virus encoding at least one immunostimulating molecule.

55. A method for eliciting an anti-cancer immune response in a subject, which compnses:

(a) admimstenng a first recombinant vaccinia virus encoding at least one first immunostimulating molecule; and

(b) admimstenng a composition comprising antigen presenting cells pulsed with a preparation compπsing enucleated cytosol and cell membranes of cancer cells infected with a second recombinant vaccinia virus encoding at least one second immunostimulating molecule.

56. The method of claim 56, wherein the amount of the first recombinant vaccinia virus is from about 10⁴ to about 10⁸ PFU.

57. The method of claim 56, wherein the amount of the first recombinant vaccinia virus is about 10⁷ PFU.

58. The method of claim 56, wherein the number of antigen presenting cells is from about 10⁵ to about 10⁷.

59 The method of claim 56, wherein the number of antigen presenting cells is from about 10⁶ to about 5 l0⁶ cells.

60. The method of claim 56, wherein the enucleated cytosol is substantially free of nuclei.

61. The method of claim 56, wherein the cell membranes comprise at least two HLA class I A antigens

62. The method of claim 56, wherein the first recombinant vaccinia virus is a live virus.

63 The method of claιm56, wherein the second recombinant vaccinia virus is either live or inactivated

64 The method of claim 56, wherein the first immunostimulating molecule is IL-2. 65. The method of claim 56, wherein the first immunostimulating molecule is selected from the group consisting of FLT-3 ligand, FLT-3/FLK-2 ligand, GM-CSF, G- CSF, IL-3, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, stem cell factor, an interferon, and a combination thereof.

66. The method of claim 18, wherein the first immunostimulating molecule comprises a melanoma immunogen selected from the group consisting of MAGE-1, MAGE- 3, BAGE, GAGE, PRAME, NY-ESO-1, tyrosinase, Melan-A, MART- 1, gp 100, TRP- 1, TRP-2, MUM- 1, CDK4, beta-catenin, gp 100in4, pi 5, N- acetylglucosaminyltransferase, B7-1, TA-90, lysosome-associated membrane protein, melanocyte-stimulating hormone receptor, p90 calnexin, and a combination thereof.

67. The method of claim 56, wherein the second immunostimulating molecule is IL-2.

68. The method of claim 56, wherein the second immunostimulating molecule is selected from the group consisting of FLT-3 ligand, FLT-3/FLK-2 ligand, GM- CSF, G-CSF, IL-3, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, stem cell factor, an interferon, and a combination thereof.

69. The method of claim 56, wherein the second immunostimulating molecule comprises a melanoma immunogen selected from the group consisting of MAGE- 1, MAGE-3, BAGE, GAGE, PRAME, NY-ESO-1, tyrosinase, Melan-A, MART- 1, gp 100, TRP- 1 , TRP-2, MUM- 1 , CDK4, beta-catenin, gp 100in4, pi 5, N- acetylglucosaminyltransferase, B7-1, TA-90, lysosome-associated membrane protein, melanocyte-stimulating hormone receptor, p90 calnexin, and a combination thereof.

70. The method of claim 56, wherein the antigen presenting cells are dendritic cells or monocytes.

71. The method of claim 56, wherein the antigen presenting cells are dendritic cells and monocytes.

72. The method of claim 56, wherein the antigen presenting cells are autologous cells.

73. The method of claim 56, wherein the antigen presenting cells are HLA-matched cells to the subject.

74. The method of claim 56, wherein the cancer cells are melanoma cells.

75. The method of claim 75, wherein the melanoma cells comprise one or more cells selected from the group consisting of Mel-2, Mel-3, Mel-4, Mel-6, and Mel-9.

76. The method of claim 56, wherein the cancer cells are established cancer cell lines.

77. The method of claim 56, wherein the cancer cells are from the subject.

78. A method of treating cancer in a subject, which comprises:

(a) administering a first live recombinant vaccinia virus encoding at least one first immunostimulating molecule; and

(b) administering an effective amount of a composition comprising antigen presenting cells pulsed with a preparation comprising enucleated cytosol and cell membranes of cancer cells infected with a second recombinant vaccinia virus encoding for at least one second immunostimulating molecule.

79. The method of claim 78, wherein the first live recombinant vaccinia virus encodes IL-2.

80. The method of claim 78, wherein about 10⁵ to about 10⁷ PFU of the first live recombinant vaccinia virus is provided.

81. The method of claim 78, wherein enucleated cytosol and cell membranes equivalent to about 10⁶ to about 10⁷ cancer cells are provided.

82. The method of claim 78, wherein at least one treatment is administered.

83. The method of claim 78, wherein parts said first recombinant vaccinia virus and said composition are injected subcutaneously in at least one site selected from the group consisting of an anterior thigh, an upper arm, or the anterior thorax.

84. The method of claim 78, wherein the at least one site is near regional lymph nodes.

85. The method of claim 78, wherein step (a) is carried out before step (b).

86. The method of claim 85, wherein steps (a) and (b) are carried out in substantially the same site.

87. The method of claim 78, wherein step (a) is carried out about thirty minutes prior to step (b).

88. The method of claim 78, wherein the cancer is a melanoma.

89. The method of claim 78, wherein the cancer cells are melanoma cells.

90. The method of claim 78, wherein the cancer is selected from the group consisting of fibrosarcoma, myxosarcoma, hposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, Kaposi's sarcoma, endothehosarcoma, lymphangiosarcoma, lymphangioendothehosarcoma, synovioma, mesothehoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroghoma, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma, and leukemia.

91. The method of claim 78, wherein the cancer cells are from cancers selected from the group consisting of fibrosarcoma, myxosarcoma, hposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, Kaposi's sarcoma, endothehosarcoma, lymphangiosarcoma, lymphangioendothehosarcoma, synovioma, mesothehoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroghoma, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma, and leukemia.

92. The method of claim 78, wherein the enucleated cytosol is substantially free of nuclei.

93. The method of claim 78, wherein the cell membranes comprise at least two HLA class I A antigens.

94. The method of claim 78, wherein the first recombinant vaccinia virus is either live or inactivated.

95. The method of claim 78, wherein the second recombinant vaccinia virus is either live or inactivated.

96. The method of claim 78, wherein the first immunostimulating molecule is IL-2.

97. The method of claim 78, wherein the first immunostimulating molecule is selected from the group consisting of FLT-3 ligand, FLT-3/FLK-2 ligand, GM-CSF, G- CSF, IL-3, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, stem cell factor, an interferon, and a combination thereof.

98. The method of claim 78, wherein the first immunostimulating molecule comprises a melanoma immunogen selected from the group consisting of MAGE-1, MAGE- 3, BAGE, GAGE, PRAME, NY-ESO-1, tyrosinase, Melan-A, MART- 1, gp 100, TRP- 1, TRP-2, MUM- 1, CDK4, beta-catenin, gp 100in4, pi 5, N- acetylglucosaminyltransferase, B7-1, TA-90, lysosome-associated membrane protein, melanocyte-stimulating hormone receptor, p90 calnexin, and a combination thereof.

99. The method of claim 78, wherein the second immunostimulating molecule is IL-2.

100. The method of claim 78, wherein the second immunostimulating molecule is selected from the group consisting of FLT-3 ligand, FLT-3/FLK-2 ligand, GM- CSF, G-CSF, IL-3, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, stem cell factor, an interferon, or a combination thereof.

101. The method of claim 78, wherein the second immunostimulating molecule comprises a melanoma immunogen selected from the group consisting of MAGE - 1, MAGE-3, BAGE, GAGE, PRAME, NY-ESO-1, tyrosinase, Melan-A, MART- 1, gp 100, TRP- 1, TRP-2, MUM- 1, CDK4, beta-catenin, gp 100in4, pi 5, N- acetylglucosaminyltransferase, B7-1, TA-90, lysosome-associated membrane protein, melanocyte-stimulating hormone receptor, p90 calnexin, and a combination thereof.

102. The method of claim 78, wherein the antigen presenting cells are dendritic cells or monocytes.

103. The method of claim 78, wherein the antigen presenting cells are dendritic cells and monocytes.

104. The method of claim 78, wherein the antigen presenting cells are autologous cells.

105. The method of claim 78, wherein the antigen presenting cells are HLA-matched to the subject.

106. The method of claim 78, wherein the cancer cells are from the subject.

107. The method of claim 78, wherein the subject is a human.