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.

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.

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, gp100, TRP-1 and TRP-2; mutated or aberrantly expressed antigens such MUM-1, CDK4, beta-catenin, gp100-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 (MCIR), 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 A1. 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 period, to avoid virus-induced cell lysis, preferably 4-36 hrs, more preferably 18-30 hrs and even more preferably 24 hrs in a CO₂ incubator, the melanoma cells are separated from the culture supernatant. This can be accomplished, for example, by spinning at approximately 1,200 rpm for 10 min in a refrigerated centrifuge. 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 variety 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, various detergents, or any other methods known in the art. The preferred method is a high frequency vibration or sonication method using a probe sonicator. In preferred embodiments of the invention, cells are disrupted but nuclei remain substantially intact. 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 centrifugation at 800 rpm for 10 min. The remaining cellular material comprises 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 material 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. Prior 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 material recovered from the disrupted cells. In a preferred embodiment, contacting is for a period 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, C. M., 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 10⁵ and 10⁸ PFU, and more preferably about 10⁷ PFU. Generally, the effective amount of therapeutic composition comprises an amount in a range about from 10⁵ to 10⁹ original cancer cells per injection. Preferably, the effective amount is between about 10⁶ and 10⁸ cells, and more preferably about 10⁷ 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 period of time and in an amount necessary to provide the therapeutic effect. Accordingly, preferred sites of the injection are on anterior thighs, anterior upper arms, or the anterior 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 drip), intramuscular, transdermal (which may include a penetration enhancement agent), sustained release by encapsulating into delivery vehicles such as liposomes.

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 rendering 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.
 

Claim 1 of 38 Claims

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

(a) administering a first recombinant vaccinia virus encoding at least one first immunostimulating molecule, wherein the first immunostimulating molecule is IL-2; and

(b) administering a composition comprising antigen presenting cells, which are capable of inducing T cell activation, wherein the antigen presenting cells are dendritic cells and/or monocytes, and which are autologous or syngeneic, pulsed with a preparation comprising enucleated cytosol and cell membranes of cancer cells, which are derived from the subject or are the same cancer cell type as the patient-derived cancer cells, infected with a second recombinant vaccinia virus encoding at least one second immunostimulating molecule, wherein the second immunostimulating molecule is IL-2; and

(c) wherein administration of said first recombinant vaccinia virus and said composition is at or near lymph node(s); and wherein administration of said first recombinant vaccinia virus is approximately 30 minutes prior to said composition.
 

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