WO2007130050A1 - Hybrid cells for treating early and late stage-ned melanoma - Google Patents

Abstract

The present invention relates to cancer treatment compositions and methods for a specific cancer patient population. In particular, the application describes methods of treating a patient with NED cancer, such as a stage IV-NED cancer, with a hybrid cells preparation.

Description

HYBRID CELLS FOR TREATING EARLY AND LATE STAGE-NED MELANOMA

BACKGROUND OF THE INVENTION

The incidence of malignant melanoma has dramatically increased over the past few decades, and indications are that the incidence of this deadly disease will continue to rise 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 variety of skin 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 responsiveness to immunomodulators such as interleukins and interferons. (Mukherji B and Chakraborty N G., Immunobiology and immunotherapy of melanoma, Curr. Opin. Oncol., 7:175-184, 1995).

Indeed, recent advances in molecular immunology now make immunotherapy a truly viable option for the treatment of patients with cancer and metastatic disease. The past decade has seen the approval and introduction of several immunotherapeutic strategies for wide ranging use against several metastatic cancers (Parkinson et al. , in CANCER MEDICINE, 4^th ed., pp. 1213-1226 (Holland et al., eds. 1997)). Perhaps the best known strategies include IL-2 therapy (Philip et al., Seminars in Oncology. 24(1 Suppl 4): S32-8, 1997 Feb.) and tumor vaccines targeted against melanoma (Smith et al., Int J Dermatol 1999; 38(7): 490- 508.) While these strategies are efficacious against some tumors, their potency is limited because they only enhance the already enfeebled ability of tumor cells to present their "foreign" epitopes to CD8 T-cells, and to generate thereby a tumor-specific cytotoxic T lympocyte (CTL) response.

Autologous whole tumor cell-based vaccines were first used for immunotherapy of melanoma. Such whole tumor cell-based vaccines are advantageous, because they contain large numbers of antigens, which eliminates the need for targeting the immune response against one antigen at a time. This is important because currently there is little ability to identify specific tumor-associated antigens (TAA) that are useful to induce immune system- mediated tumor regression (Boon et ah, Immunol Today 1997; 18:267-268). To date, however, autologous whole tumor cell-based vaccines alone have shown only some isolated or marginal successes. Smith et ah, supra. As seen below, the marginal success of whole tumor cell-based vaccines likely results from tumor cell mutations that impair their ability to act as antigen presenting cells ("APCs").

Evidence from many tumor immunology laboratories demonstrates that tumor cells persist in part because they have selected a mutation which partially or completely destroys their ability to act as APCs in the process of cytotoxic T lymphocyte CTL generation. (Stockert et al., J. Exp. Med. 1998; 187: 1349-1354; Sahin et α/,, Proc. Natl Acad. ScI USA 1995; 92:11810-11813; Gabrilovich et α/., JVαtwre Med. 1996; 2:1096-1103; Ishida et α/., J. Immunol. 1998; 161:4842-4851.) These observations spurred development of strategies that attempt to replace the tumor cell as the APC, rather than trying to boost the tumor's enfeebled antigen presenting process. The best candidate for such a replacement is the dendritic cell ("DC"). DCs are "professional" antigen presenting cells that play a vital role in stimulating immune responses. DCs not only can activate naive CD4+ T helper cells but also stimulate unprimed CD8+ cytotoxic T lymphocytes. (Steinman, R. M. Annu. Rev. Immunol. 1991; 9, 271-296; Macatonia, et al., J. Exp. Med. 1988; 169, 1255-1264; Mehta-Damaniet α/., J. Immunol. 1994;153, 996-1003; Porgador et ah, J. Exp. Med. 1995;182, 255-260.) Because of these characteristics, DCs have been widely studied as antigen presenting cells for cancer immunotherapy. DCs can be loaded with tumor antigens by pulsing with whole tumor antigens or tumor antigen peptides. (Young et ah, J. Exp. Med. 1996;183, 7-11; Mayordoma et ah, Nat. Med. 1995;1, 1297-1302; Bakkar et α/., Cancer Res. 1995; 55, 5330- 5334; Flamand et ah, Ew. J. Immunol. 1994; 24, 605-610; Gong et ah, Gene Ther. 1997; 4, 1023-1028; Song et ah, J. Exp. Med. 1997;186, 1247-1256; Specht et a J. Exp. Med. 1997;186, 1213-1256.)

Peptide- or tumor lysate-pulsed dendritic cells have been used, for example, to vaccinate melanoma patients. (Rosenberg et ah, Nature Med 1998; 4: 321-327; Wallack et ah, Cancer 1995; 75:34-42; Bystryn, Rec. Results Cancer Res. 1995; 139:337-348; Mitchell et ah, Semin. Oncol. 1998; 25: 623-635; Morton et ah, Ann. N. Y. Acad. ScI 1993; 690:120- 134; Berd et ah, Semin Oncol. 1998; 25:646-653; Berd et ah, J. CHn. Oncol. 1997; 15:2359- 2370.) DCs loaded with tumor antigens are able to induce both cellular and humoral, antigen- specific, anti-tumor immune responses. (Shurin, M. R. Cancer Immunol. Immunother. 1996; 43, 158-164.) This approach, however, is limited to application against tumors expressing known tumor antigens. See, Haigh et al, Oncology 1999; 13, 1561-1573. It is worthless for those tumors with no identified tumor antigen, like primary tumors from patients, which constitute most real-world situations. Obviously alternative strategies are needed.

An additional problem with antigen pulsing techniques is that the antigen presenting system of an APC works more effectively and efficiently when the protein/antigen is synthesized inside the cell rather than outside the cell, a substantial drawback to using antigen-pulsed cells. In an effort to avoid this problem, a number of laboratories have attempted to use gene therapy to introduce specific tumor antigens into dendritic cells. (Gong et al, 1997, Gene Ther. 4, 1023-28; Song et al, 1997, J Exp. Med. 186: 1247-56; and Specht et al, 1997, supra.) However, this gene therapy approach is also fraught with many disadvantages including: 1) the limited ability to identify all of the important specific tumor antigens, 2) the limited ability to map the genes of the specific tumor antigens, 3) only one or a small number of the known tumor antigen genes can be introduced into the dendritic cell, and 4) the process is time-consuming and cumbersome.

On the other hand, fusions between DCs and tumor cells represents an alternative way to produce effective tumor antigen presenting cells by presenting the immune cells with all possible tumor antigens. (Gong et al., Nat. Med. 1997; 3: 558-561; Wang et al, J. Immunol. 1998;161, 5516-5524; Lespagnard et al, Int. J. Cancer 1998; 76, 250-258; Rowse et al, Cancer Res. 1998; 58, 315-321.) DCs have been fused with tumor cells and the fused cells efficiently presented tumor antigens to the immune cells and stimulated a specific anti-tumor immune responses. (Gong et al; Wang et al; Lespagnard et al, all supra) These fusion schemes, however, rely on selectable markers (gene products which render the cell resistant to specific cell toxins or allow them to grow under certain metabolic conditions) in each of the DCs and the tumor cells to isolate the resultant hybrid. The rare cell fusion products are selected by long-term culture in the presence of both cell toxins where only the fusion product, containing both selectable markers, can survive. Since the introduction and selection schemes using markers requires culture and multiple cell division, they cannot be applied to dendritic cells because DCs are terminally differentiated, non- dividing cells. Thus, it is no surprise that the previous fusion work relied on well-defined tumor cell lines, bearing such a marker, and DC- and tumor-specific conjugated antibodies, which limits the usefulness of this strategy in cancer treatment.

U.S. Patent No. 6,849,451 addresses the need for cancer treatment compositions and methods. In particular, the '451 patent discloses compositions that comprise tumor cell- 5 antigen presenting cell fusions that were prepared in the absence of antibiotic and metabolic selection. Consequently, the resulting hybrid cell has unique properties, such as maintained antigen diversity, that make it particularly suitable for treating different cancers. The '451 patent, however, does not describe the unexpected results conferred by treating a specific population of patients with the hybrid cells.

10 In particular, the present application describes a cancer treatment method for a certain class of patients that have had an area of cancer removed and are considered NED, i.e., to have no evidence of disease. Since NED patients still have a risk of cancer recurrence, either near the site of original cancer or at a distant site in the body, it is still necessary for such patients to be closely monitored and in some instances, undergo additional cancer therapy to

15 minimize that risk. See, for example, Rivera et ah, The Breast Journal, 8:2-9 (2002), which describes chemotherapy after local treatment of stage IV-no evidence of disease (NED) breast cancer. The stage of cancer at the time of cancer removal is correlative of cancer recurrence and survival rate. Thus, a patient with stage IV- NED cancer has a lower expected survival than a patient with stage I-NED cancer.

20 Accordingly, there is a need in the art for improved treatment options for NED cancer patients, including late stage cancer patients. The present invention satisfies that need.

SUMMARY OF THE INVENTION

It is an object of the invention to provide solutions to the aforementioned deficiencies in the art. Further to this objective, the invention provides a method for treating a cancer

25 patient who has no evidence of disease (NED) comprising administering to a patient a composition that comprises a hybrid of a primary tumor cell and a dendritic cell, wherein the composition is substantially free of non-hybrid cells. The patient to be treated may have stage I, stage II, stage III, or stage IV cancer (staged according to the AJCC 2002 staging). Exemplary types of cancer to be treated include, but are not limited to, renal, ovarian, or

30 melanoma. The treatment method may also include adjunct treatment with a cytokine or lymphokine, such as interleukin-2.

IΛ/ΔCU Λ CVOOQO Λ In one embodiment, the patient has no evidence of disease following surgery, radiation, and/or chemotherapy. If the patient has had chemotherapy, the patient can have been treated with an agent such as, but not limited to, nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogues, pyrimidine analogues, purine analogs, platinum complexes, adrenocortical suppressants, adrenocorticosteroids, progestins, estrogens, antiestrogens and androgens, cyclophosphamide, chlorambucil, methotrexate, fluorouracil, cytarabine, thioguanine, vinblastine, vincristine, doxorubicin, daunorubicin, mitomycin, cisplatin, hydroxyurea, prednisone, hydroxyprogesterone caproate, medroxyprogesterone, megestrol acetate, diethyl stilbestrol, ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone, or any combination thereof.

Also described herein is a method for increasing survival of a late stage cancer patient that has no evidence of disease, comprising administering to a patient a composition that comprises a hybrid of a primary tumor cell and a dendritic cell, wherein the composition is substantially free of non-hybrid cells. In one aspect of the invention, the cancer patient has renal cancer, ovarian cancer, or melanoma. Additionally, the cancer patient may have stage I, stage II, stage III, or stage IV-NED cancer, such as stage IV-NED melanoma.

Both the foregoing general description and the following brief description of the drawings and the detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that hybrid cells according to the invention, between dendritic cells and cancer cells are capable of generating a tumor-specific cytotoxic T cell response, which is relevant to in vivo immunotherapy.

Figure 2A shows FAC S -detection of antigen-presentation markers on control dendritic cells. A normal distribution is shown.

Figure 2B shows FACS-detection of antigen-presentation markers on dendritic/tumor cell hybrids. A normal distribution is shown, as compared to the control dendritic cells, meaning that the hybrid cells retain all of the markers necessary for antigen presentation. Figure 3 describes the AJCC 2002 revised melanoma staging classification, reproduced from the Cleveland Clinic Foundation website (www.clevelandclinicmeded.com).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors unexpectedly discovered that hybrid cells are exceptionally useful for treating a cancer patient, even a late stage cancer patient, who has no clinical evidence of disease (NED). Although the NED diagnosis is given when a cancer has been removed, such as through surgery and/or radiation and/or chemotherapy, NED patients are still at a risk for cancer recurrence since it is believed that cancer cells still exist in the body even though they are not clinically detectable. Thus, the present invention is directed to the surprising discovery that combination therapy with cancer removal (e.g., tumor resection) and hybrid cells treatment can decrease risk of cancer recurrence and increase patient survival rate.

METHOD OF PREPARING HYBRID CELLS

The hybrid cells of the present invention can be prepared by a rapid, simple to use method that is applicable to fully differentiated, non-dividing cells. The method comprises contacting at least two different cells ("reactant cells") under conditions that promote cell fusion, and then purifying the resultant hybrid ("product cell") without antibiotic or metabolic selection. In general, the purification is accomplished in a relatively short period of time, for example, in less than about 24 to 48 hours, after exposure to conditions that promote cell fusion.

In an exemplary embodiment, the method is accomplished with the aid of at least two different dyes, which can be fluorescent dyes. Thus, the method may entail separately contacting, each with a different dye, the two cell types to be fused. This pre-fusion labeling marks each cell with a different dye, and permits discrimination among each fusion parent cell and the hybrid fusion product: the reactant cells (e.g., tumor cell and dendritic cell) each are stained with one dye, and the product cells are stained with both. This way, the hybrid fusion product may be separated from the reactant cells, for example, by fluorescence activated cell sorting (FACS), magnetic cell sorting, and other cell sorting techniques that do not employ antibiotic or metabolic selection as the means for sorting. The resulting hybrid cell retains the antigen diversity of the tumor reactant cell. Dyes useful according to the invention have the characteristic of associating with a cell for a time sufficient to detect them in such association. In addition, useful dyes do not substantially diminish cell viability, with greater than about 50% cell viability being preferred. Typically, they are fluorescent dyes. One useful class of dyes comprises the so- called "cyanine" dyes. Cyanine dyes come in a variety of types that fluoresce at different wavelengths such that they can be individually or jointly detected when associated with a cell. Some exemplary cyanine dyes are found in Horan et al, U.S. Pat. Nos. 4,783,401 (1998), 4,762,701 (1988) and 4,859,584 (1989), the structures of which are hereby specifically incorporated by reference. Two particularly useful cyanine dyes are PKH26-GL and PHK2-GL (Sigma Chemical

Co.). These dyes are preferred because they have been widely studied and used. For instance, they have been used in animal studies in vivo for cell trafficking studies. Horan et al, Nature 1989; 340, 167-168; Horan et al, Methods Cell Biol. 1990; 33, 469-490; Michelson et al, Proc. Natl Acad. Sci. USA 1996; 93, 11877-11882. In laboratory animals these dyes have been shown not to affect cell growth or function and not to migrate from the cells stained with these dyes to other cells (Horan et al., 1989). Thus, these dyes have low toxicity, which is a desirable quality for in vivo applications.

Dyes employed in vivo in accordance with the present invention should be free of endotoxin, as measured, for example, by the Limulus amaebocyte (LAL) assay. Typically, when the measured endotoxin level is less than about 1 ng/μg dye, and preferably less than about 0.1 ng/μg dye, the dye is considered "endotoxin-free."

More generally, the dyes are essentially pyrogen-free, whether pyrogenicity is contributed by endotoxin or other pyrogens. Thus, a dye is considered "essentially pyrogen free" when the final formulation of hybrid cells labeled with the dye (in a form to be injected into a subject, for example) yields less than about 1 endotoxin unit (EU)/dose, but preferably less than about 0.1 EU/dose, and most preferably less than about 0.05 EU/dose. Toxicity thresholds are informed by the fact that most in vivo methods contemplated herein result in less than about 10^"8g of these dyes, in association with cells, being introduced into a patient when undertaking the inventive methods of treatment. Conventional cyanine dye labeling methodologies require the presence of cellular stabilizers (osmolarity regulating agents), like sugars (e.g., glucose or mannitol), amino acids and/or certain Goods buffers. See, for example, Horan et al., U.S. Pat. No. 4,783,401 (1998). The inventors discovered that dimethyl sulfoxide (DMSO) can substitute for such stabilizers. In particular, DMSO diluted in a standard culture medium may be used as a solvent for cyanine dyes, and it promotes efficient and stable uptake of dye without substantial loss of cell viability. A generally useful range of DMSO concentration is from about 10 to about 50%, but a preferred range is from about 20 to about 40%. The invention therefore also contemplates methods of labeling cells, and corresponding kits, with cyanine dyes using DMSO in place of the conventional stabilizers.

Once the reactant cells are labeled, they are put into contact with one another, under conditions that promote fusion. Such fusion-promoting conditions are well known to the artisan, and typically involve the addition of an agent that promotes cell fusion. These agents are thought to work by a molecular crowding mechanism to concentrate cells to an extent that they are in close enough proximity to cause fusion of cell membranes. While the invention contemplates any agent that meets these characteristics, exemplary useful agents are polymeric compounds, such as polyethylene glycols. An effective amount of such an agent generally will be from about 20% to about 80% (w/v). A preferred range is from about 40% to about 60%, with about 50% being more preferred.

After hybrid cell formation, it is usually beneficial to isolate them from the un-fused reactant cells. In the case of cellular vaccines, for example, this purification substantially increases the potency. Purification may be accomplished by conventional FACS methodologies and the like. The method explicitly contemplates hybrid cells of higher order, which are fusions between more than two cells. In each case, all that is needed is an additional dye that can serve as a marker for selection of the higher-order hybrid. For example, three different reactant cells labeled with three different dyes are used to form a "tribred," and so on. Thus, as used herein, the term "hybrid cell" contemplates fusions between two or more reactant cells.

HYBRID CELL PREPARATIONS

In one embodiment, the inventive hybrid cell preparation comprises a primary tumor cell and an antigen presenting cell (APC) as reactants. Such hybrids may be used as cellular vaccines to induce an immune response against a tumor. The tumor cell may be of any type, including breast, renal, prostate, ovarian, skin, lung, and colon cancer, and the like. In a preferred embodiment, the cancer to be treated is melanoma. The APC preferably is a professional APC, like a macrophage or a dendritic cell. Due to their superior antigen presentation capabilities, dendritic cells are more preferred. Both syngeneic and allogeneic fusions are contemplated as the inventors have discovered using a mouse model that both work equally well. Ultimately, the antigen diversity of the starting tumor cell population is maintained in the resultant hybrid cell population. The inventive hybrid cell preparation may be made using a combination of dyes, as detailed above. Thus, the inventive hybrid cell may be labeled with at least two different dyes. These dyes are preferably fluorescent and, again, cyanine dyes are favored. Alternatively, hybrid cells may be prepared, for example, using cell surface markers differentially expressed on the reactant cells and corresponding antibodies to them. The antibodies may be used to pan sequentially for each marker.

METHODS OF TREATMENT

Multiple factors determine the survival rate of a patient with malignant melanoma, including tumor size, depth of invasion, tumor thickness, and tumor stage at diagnosis. While some factors can positively influence a patient's prognosis (e.g., smaller tumor diameter (less than 2 cm) and tumor thickness (less than 2 mm), and an early tumor stage at diagnosis), overall survival rate nevertheless is low.

For example, according to the AJCC 2002 revised melanoma staging, stage FV melanoma with distant skin, subcutaneous, or nodal metastasis with normal LDH (any TanyNMla), the 1 year, 5 year, and 10 year overall survival rate is 59%, 19% and 16%, respectively. Similarly, stage IV melanoma (AJCC) with lung metastasis with normal LDH (any TanyNBlb) has a 1 year, 5 year, and 10 year overall survival rate of 57%, 7%, and 3%, respectively. Stage IV melanoma (AJCC) with all other visceral metastasis with normal LDH or any distant metastasis with increased LDH (any TanyNMlc) has a 41%, 9%, and 6% overall survival rate for 1 year, 5 years, and 10 years, respectively. See, Figure 3, which describes the AJCC 2002 revised melanoma staging classification, reproduced from the

Cleveland Clinic Foundation website, which was adapted from Balch et ah, Final Version of the American Joint Committee on Cancer Staging System for Cutaneous Melanoma., J. Clin. Oncol, 19:3635-3548 (2001). The most recent AJCC staging classification for various types of cancers can be found in Greene et al, (Eds), AJCC CANCER STAGING MANUAL, 6^th Edition. Springer Publishers (2002).

As provided above, the present inventors surprisingly discovered that hybrid cells are exceptionally useful in treating a cancer patient, even a late stage cancer patient, who has no clinical evidence of disease. In this patient population, cancer removal (e.g., tumor resection) followed by hybrid cells treatment can decrease the risk of cancer recurrence and increase patient survival rate. This method improves the likelihood of a favorable long-term prognosis in patients with stage I-NED, stage II-NED, stage III-NED, and even stage IV-NED cancer (stages identified according to the 2002 AJCC classification). The cancer can be any solid tumor cancer, including but not limited to renal cancer, ovarian cancer, lung cancer, breast cancer, prostate cancer, colon cancer, or skin cancer, such as melanoma.

In one aspect of the invention, the patient has stage I cancer (e.g., stage IA or IB), such as stage I melanoma (according the AJCC 2002 classification). In another embodiment, the patient has stage II cancer (e.g., stage HA, HB, or IIC), stage III cancer (e.g., IDA, IIIB, or IIIC), or stage IV cancer, according to the AJCC 2002 classification. Preferably, the hybrid cells of the invention are administered to a patient who has no clinical evidence of disease (NED) as a result of, for example, surgery (including surgical resection of tumor, as well as other destructive methods such as freezing, burning, laser removal, curettage, etc.) and/or radiation therapy and/or chemotherapy (including antimetabolites, alkylating agents, immunomodulatory agents, various natural products (e.g., vinca alkaloids, epipodophyllotoxins, antibiotics, and amino acid-depleting enzymes), antibodies, hormones and hormone antagonists), regardless of the stage of cancer. In other words, prior to treatment with hybrid cells, the patient's tumor has been surgically resected or somehow removed/destroyed, at least entirely or substantially, so as to be considered NED by one of skill in the art.

A method of treatment according to the present invention comprises administering to a NED cancer patient a hybrid between a first reactant cell and a second reactant cell, typically an antigen-presenting cell. The first reactant cell is one against which an immune response is sought, such as a primary tumor cell. For example, the treatment method involves administering a hybrid cell prepared by fusing a first reactant cell isolated from a patient, such as a neoplastic cell, with an antigen presenting cell, such as a dendritic cell. Prior to fusion, both first and second reactant cells are labeled with different dyes. Following cell fusion, the resulting hybrid cell is isolated and then administered subcutaneously, preferably near a lymph node, to a patient in an acceptable excipient, such phosphate buffered saline (PBS). The neoplastic cell is irradiated to avoid administration of a viable cancer cell. Preferably, the tumor reactant cells are lethally irradiated prior to fusion. This step kills the cell, but does not prevent efficient presentation of the tumor antigen(s) by the resultant hybrid cell. Both syngeneic and allogeneic fusions are contemplated.

The cancer treatment may also be augmented, moreover, by using additional antineoplastic agents in conjunction with the hybrid cells. One class of such agents is immunomodulators. These include cytokines and lymphokines, especially interleukin-2 (IL-2) and IL-2 derivatives, like aldesleukin (Proleukin, Chiron Corp.). The use of IL-2 is preferred because it should further enhance the immune response generated by the hybrid cell. As used herein, "interleukin-2" is used generically to refer to the native molecules and any derivatives or analogs that retain essential interleukin-2 activity, like promoting T cell growth. Other lymphokines and cytokines may also be used as an adjunct to treatment. Examples include interferon gamma (IFN-γ), granulocyte macrophage colony simulating factor (GM- CSF), and the like.

Upon treatment of the patient with the hybrid cells of the present invention, the patient can have a mixed response (e.g., one or more tumor site(s) responds to treatment by, for example, decreasing in size, but one or more other tumor site(s) does not respond to treatment and progresses, as determined by, for example, an increase in size), but preferably has a partial response (e.g., one or more tumor site(s) responds to treatment by decreasing in size, for example, but one or more other tumor site(s) does not respond to treatment) or complete response (e.g., all tumor site(s) respond to treatment). Although the patient may have a "mixed response," the treatment method may nevertheless still be considered effective. A treatment regimen is considered "effective" if it can increase a patient's overall survival beyond the patient's expected age of survival, given the stage and type of cancer immediately prior to treatment with the hybrid cells of the present invention. For example, the hybrid cells of the present invention are useful in increasing survival of a patient with NED cancer by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 1 year, at least about 1.5 years, at least about 2 years, at least about 2.5 years, at least about 3 years, at least about 3.5 years, at least about 4 years, at least about 4.5 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, etc. The foregoing detailed description and the following examples are offered for illustrative purposes and are not meant to be limiting. The artisan will recognize that there are additional embodiments that fall within the invention, but are not described with particularity. All references identified herein, including U.S. patents, are hereby expressly incorporated by reference.

Examples

Example 1: Animal Studies

This example demonstrates the preparation of certain hybrids between cancer cells and dendritic cells, called dendritomas. These hybrids were used as a cellular vaccine to prevent cancer in a murine metastatic cancer model system.

To prepare dendritic cells from bone marrow, the appropriate number of female C57BL/6J mice to support later Dendritoma injections (two mice for every one mouse to be injected) were sacrificed. The femur and tibia of both hind legs were removed from each mouse. Bone marrow was flushed out of the bones using a syringe containing RPMI 1640 with 25 mM Hepes (Gibco BRL). The media containing the bone marrow was filtered through a 40 μm cell strainer into a 50 ml conical centrifuge tube. The bone marrow cells were pelleted by centrifugation at 1500 rpm for five minutes. After removing the supernatant, the tube was gently tapped to loosen the cell pellet. Red blood cell lysis was achieved by adding 5 ml/mouse of ACK Lysing Solution (0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.3) and incubating at room temperature for five minutes. The cells were pelleted by centrifugation at 1500 rpm for five minutes. The supernatant was removed, and the cells were gently resuspended in 10 ml/mouse of complete DC media (RPMI 1640, 10% fetal bovine serum (FBS), 100 μg/ml gentamicin, 10 ng/ml GM-CSF, 10 ng/ml IL-4). The cells were plated into two wells/mouse of a six well tissue culture plate. After incubating the cultures overnight at 37° C, 5% CO₂, the floating cells were removed from each culture. Adherent cells were washed twice with IX Phosphate Buffered Saline (PBS). Each well of cells was fed 5 ml of complete DC media. The cultures were incubated for 48 hours at 37° C, 5% CO₂. The dendritic cells were harvested from the 6 well plate by removing the supernatant containing the cells to a 15 ml conical centrifuge tube. Each well was washed twice with 3 ml of IX PBS. The cells were lightly trypsinized by adding 1 ml of 0.25% Trypsin/EDTA (Gibco BRL) to each well. After rocking the plate to cover the entire surface, the trypsin solution was quickly removed from the plate. The plate was lightly tapped to remove any loosely attaching cells. These cells were resuspended in 2 ml of complete DC media and added to the 15 ml tube. The cells were pelleted by centrifugation at 1500 rpm for five minutes. After resuspending the cells in 10 ml of complete DC media, a cell count was taken.

B 16F0 murine melanoma cells were obtained from the ATCC (CRL-6322) and cultured using standard tissue culture techniques. When the cells were ready for use, they were trypsinized using 0.25% Trypsin/EDTA. After taking a cell count the number of cells needed for experimentation were pelleted by centrifugation at 1500 rpm for five minutes. The remaining cells were cultured for later use.

For general cell membrane labeling of murine dendritic cells and B 16F0 melanoma cells, a commercial fluorescent cell linker kit was used. The dendritic cells were labeled fluorescent green using Sigma stock number PKH2-GL; the B 16FO melanoma cells were labeled fluorescent red using Sigma stock number PKH26-GL. The staining procedure was performed at 25 ⁰C. The cells to be stained were washed with serum-free media. The cell suspension was centrifuged at 400 g for five minutes to obtain a loose pellet. Supernatant was removed leaving less than 25 μl of medium on the pellet. The pellet was resuspended by tapping the tube, and 1 ml of Diluent A or C for green or red staining respectively was added to resuspend the cells. Immediately prior to staining, 4 x 10^'6 molar dyes (2X) were prepared with Diluent A or C in polypropylene tubes. To minimize ethanol effects, the amount of dye added was less than 1% of the individual sample volume. The cells in the diluent were rapidly added into 1 ml of 2X dye. The cells and dye were immediately mixed by gentle pipetting. The mixture was then incubated at 25° C for five minutes. The staining process was stopped by adding an equal volume of FBS and incubating for one minute. The stained cells were diluted with equal volume of complete culture medium. Stained cells were removed from the staining solution by centrifuging at 400 g for 10 minutes. After a total of three washes, the cells were resuspended in complete medium at a proper concentration. Efficiency of staining was monitored by fluorescent microscopy.

Prior to the fusion process, the red fluorescently stained B 16F0 murine melanoma cells were irradiated with 5,000 rads. Murine dendritic cells and B 16F0 melanoma cells were fused together by mixing the two cell types at a 1 :1 ratio in a 50 ml conical centrifuge tube. The tube was filled with serum-free RPMI 1640 with 25 mM Hepes. The cell mixture was centrifuged at 1500 rpm for five minutes at room temperature. During the fusion process, all solutions as well as the tube in which the fusion was performed were kept at 37° C using double-beaker water baths. The supernatant from the mixed cell pellet was aspirated and discarded. Using a 1 ml serological pipet, 1 ml of prewarmed 50% PEG/DMSO (Sigma), which contained 50% PEG and 10% DMSO in PBS (Ca^+"1"- and Mg⁺⁺-free), was added to the mixed cell pellet drop-by-drop over one minute, stirring the cells with the pipet tip after each drop. The mixture was stirred for an additional minute with the pipet.

Using a clean 2 ml serological pipet, 2 ml of prewarmed serum free RPMI 1640 with 25 mM Hepes was added to the cell mixture drop-by-drop over two minutes, stirring after each drop. With a 10 ml serological pipet, 7 ml of prewarmed serum free RPMI 1640 with 25 mM Hepes was added drop-by-drop over two to three minutes. The cells were pelleted by centrifugation at 1500 rpm for five minutes at room temperature. The supernatant was discarded, and the tube was placed back into the beaker water bath. With a clean 10 ml serological pipet, the cell pellet was resuspended in 10 ml of complete DC media by forcefully discharging about 3 ml of media onto the pellet and then gently adding the remaining media. The resuspended cells were put into a T75 tissue culture flask. The Instant Dendritomas (fused dendritic cells with melanoma cells) were incubated overnight at 37° C, 5% CO₂. A drop of the cells was placed on a slide and evaluated by fluorescent microscopy to ensure the occurrence of fusion.

The Instant Dendritomas were removed from the tissue culture flask by saving the supernatant containing the cells as .well by lightly trypsinizing the adherent cells as previously described. The cells were pelleted by centrifugation at 1500 rpm for five minutes. The cell pellet was resuspended in 2 ml of IX PBS and put into a sterile, polystyrene, round bottom, 12 x 75mm Falcon tube. After centrifuging the cells at 1500 rpm for five minutes, they were resuspended in 1 ml of IX PBS. The Instant Dendritomas were sorted out based on dual green and red fluorescence using a FACS Caliber (Becton Dickinson), using standard methods.

The sorted cells were pelleted by centrifugation at 2000 rpm for 30 minutes. After removing the supernatant, the cells were resuspended at a concentration of 50,000 cells/0.5 ml IX PBS. A drop of the cells was placed on a slide and evaluated by fluorescent microscopy to ensure the general purity of the sort. Three days prior to the fusion process, female C57BL/6J mice were challenged with 0.75 x 10⁶ B 16F0 melanoma cells in 0.4 ml IXPBS by intravenous injection. Once the Instant Dendritomas were pelleted and resuspended, each mouse was injected intravenously with 50,000 cells. This was followed by IL-2 treatment, which was administered intraperitoneally at 10,000 IU/day/mouse. The mice were monitored up to four weeks for pulmonary metastasis.

At the end of four weeks, the mice were sacrificed and the metastases were counted. Each of the four control animals, which were not treated with the Instant Dendritomas, had greater that 50 tumors. On the other hand, only one of the treated animals had measurable metastases. These data indicate that the hybrid cells are effective in treating cancer in a proven animal model system. The data are compiled in the following table.

Example 2: Human Studies

This example demonstrates that the inventive hybrid cells induce a cancer cell-specific cytotoxic T cell response.

To enhance the number of dendritic cells, which were isolated from peripheral blood, a panning technique using anti-CD 14 coated plates was utilized. Five hundred micrograms of anti-CD14 antibody was resuspended in 5 ml of IX PBS with BSA (700 μg of BSA per 100 μl of antibody). A 100 mm tissue culture plate was coated with 5 mis of the antibody. The plate was swirled and incubated for one hour at room temperature or overnight at 4° C. The antibody solution was removed, and the plate was washed five times with 5 mis of IX PBS.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by obtaining 50 ml of peripheral blood from the patient in preservative-free or sodium heparin tubes. The blood was diluted 1 : 1 with IX PBS. Eight ml of the diluted blood was layered over 4 ml of room temperature Ficoll-Paque Plus in 15 ml conical centrifuge tubes. The Ficoll gradients were centrifuged at 40Og at room temperature for 40 minutes. Using a Pasteur pipet, the PBMC layers were carefully removed from the Ficoll gradient and put into a clean 15 ml centrifuge tube. Four volumes of IX PBS were added to the tube and inverted several times to mix thoroughly. The PBMCs were centrifuged at lOOg at room temperature for 10 minutes. After removal of the supernatant, 10 ml of IX PBS was added to the cell pellet and inverted to mix. The PBMCs were pelleted by centrifugation at lOOg at room temperature for 10 minutes. The supernatant was removed, and the PBMCs were resuspended in 5 ml complete DC medium (RPMI 1640, 10% Human Serum, 800U/ml GMCSF, lOOOU/ml IL-4, lOOμg/ml gentamicin).

The resuspended PBMCs (up to 2 x 10⁸) were pipetted onto the anti-CD 14 coated plate and swirled to cover the entire plate. The plate was incubated at room temperature for 30 minutes. After gently swirling the plate again, the supernatant containing the non-adherent cells was removed. The adherent cells were washed by adding 10 ml of IX PBS and swirling. This wash was repeated for a total of four times, pipetting at the same place each time. Immediately after the washes, 10 mis of complete DC media was added to the plate. The culture was incubated at 37° C, 5% CO₂ for 5 to 10 days to generate dendritic cells.

A section of tumor that had been resected from a patient was received immediately after the surgery. The section of tumor was cut into several pieces and placed in a 50 ml conical centrifuge tube containing IX PBS. The tumor pieces were further cut into smaller pieces using sterile dissection scissors and placed into a T75 tissue culture flask. Ten ml of 0.25% Trypsin/EDTA was added to the flask, which was incubated at 37° C with rocking for one hour. Following incubation, 15 ml of complete tumor cell media (DMEM, 10% Human Serum, 200 μg/ml gentamicin) was added to the flask. The tumor pieces were removed and placed into a clean T75 flask, which was incubated overnight at 37° C, 5% CO₂.

The medium/trypsin mixture from the original flask contained some single tumor cells. These cells were filtered through a 40 μm cell strainer and pelleted by centrifugation at 1500 rpm for five minutes. The cells were resuspended in 10 ml of complete tumor cell media and put into a T75 tissue culture flask and incubated at 37° C, 5% CO₂. To the flask containing the tumor pieces, 20 ml of complete tumor cell media was added after the overnight incubation. Both flasks were carefully monitored for attaching cells. After several days, the tumor pieces were removed from the flask. The attached tumor cells were fed every three days and maintained for experimental use. They were subcultured using standard tissue culture techniques for adherent cells.

Cell membrane labeling of human dendritic cells and tumor cells were prepared as described in Example 1, above.

CD8+, cytotoxic T cells (CTLs) were prepared by the following method. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by obtaining 40 ml of peripheral blood from the patient in preservative-free or sodium heparin tubes and 10 ml in ACD tubes. The blood was diluted 1 :1 with IX PBS. Eight ml of the diluted blood was layered over 4 ml of room temperature Ficoll-Paque Plus in 15 ml conical centrifuge tubes. The Ficoll gradients were centrifuged at 40Og at room temperature for 40 minutes. Using a Pasteur pipet, the PBMC layers were carefully removed from the Ficoll gradients and put into a clean 15 ml centrifuge tube. Four volumes of IX PBS were added to the tube and inverted several times to mix thoroughly. The PBMCs were centrifuged at lOOg at room temperature for 10 minutes. After removal of the supernatant, 10 ml of IX PBS was added to the cells and inverted to mix. The PBMCs were pelleted by centrifugation at lOOg at room temperature for 10 minutes and resuspended in complete lymphocyte media (RPMI 1640, 10% FBS, 100 μg/ml gentamicin).

PBMCs were isolated from patients in preservative-free or sodium heparinized blood. They were subjected to the same panning technique as previously described except that anti- CD4 antibody was used to coat the plate. Prior to panning the PBMCs were enriched for T lymphocytes by passing them through a nylon wool column. This was done by packing 0.5 g of teased nylon wool into a 10 ml syringe which has a stopcock attached to the tip. The column was washed twice at 37° C with RPMI 1640 with 10% FBS. The stopcock was closed and incubated at 37° C for one hour. After draining the media from the column to the top of the wool, the PBMCs were added to the column (up to 2 x 10⁸ in 2 ml of media). The stopcock was opened the media was drained until the cell volume had entered the packed wool. After closing the stopcock additional media was added to cover the top of the wool. The column was incubated for one hour at 37° C. The nonadherent T cells were collected by two media washes. After this T cell enrichment, the T lymphocytes were panned using the anti-CD4 coated plate. T cells that were not bound by the CD4 antibody were recovered and assumed to be CD8+ cells (cytotoxic T lymphocytes). This was confirmed by FACS analysis. To have constant re-stimulators for tumor cell specific CTL' s, the PBMCs isolated from the ACD blood were immortalized by Epstein-Barr virus (EBV) transformation. This was accomplished by resuspending the PBMCs at a concentration of 1 x 10⁶ cells/ml complete lymphocyte media. To this, 1 ml of EBV supernatant and 0.2 ml of phytohemagglutinin were added. The cell mixture was cultured in a T25 tissue culture flask at 37° C₅ 5% CO₂.

The Instant Dendritomas obtained from the FACS sort were mixed with the enriched, panned CD8+ T lymphocytes in a 1:10 ratio. The CD8+ cells to be used were pelleted by centrifugation at 1500 rpm for five minutes and resuspended in 1 ml of medium containing RPMI 1640, 10% FBS, 1000 U/ml IL-6, 5 ng/ml IL-12, and 10 U/ml IL-2. This was added to the Instant Dendritomas plated after the sort. This culture was incubated at 37° C, 5% CO₂ for one week. During that week the cells were refed with the same media.

After one week, the primed CD8+ T cells (CTL' s) were restimulated with irradiated EBV-transformed lymphocytes that were pulsed with tumor lysate. Tumor cells, which had been previously cultured, were subjected to four freeze thaw cycles to lyse the cells. To obtain the lysate containing tumor antigens, the lysed cells were centrifuged at 600 g for ten minutes. The supernatant was collected and centrifuged at 13,000 g for one hour. The supernatant contain the lysate of tumor antigens was collected. To restimulate the CTL' s a viable cell count was taken using trypan blue exclusion. Once the viable cell number was determined the same number of EBV transformed lymphocytes were pulsed with the tumor lysate by incubating the lysate with the lymphocytes at 37° C, 5% CO₂ for one hour. The pulsed lymphocytes were irradiated with 5,000 rads and then mixed with the CTL' s in media containing RPMI 1640, 10% FBS, 10 U/ml IL-lα, 5 U/ml IL-2, 50 U/ml IL-4, 125 U/ml IL-6, and 30 U/ml IL-7. The culture was incubated at 37° C, 5% CO₂ and refed every two days. This re-stimulation was performed at 7 and 14 days after initial priming.

Each day the CTL' s were refed, the supernatant that was removed was stored at -20° C. When feasible, an Interferon-gamma (IFN-γ) assay was performed using an OptEIA Human IFN-γ Kit (PharMingen). The protocol was performed exactly according to the manufacturer's directions. The assay was read using a Benchmark Microplate Reader (BioRad).

To determine if the Instant Dendritomas stimulated a tumor cell specific CTL response, a CTL assay was performed using the cultured tumor cells as target cells. Fifty thousand tumor cells were harvested and pelleted in a 15 ml conical centrifuge tube by centrifugation at 200 g for five minutes. The supernatant was discarded leaving 0.1 ml of medium on the pellet. The cells were gently resuspended in the remaining medium. The tumor cells were then labeled with ⁵¹Cr by adding 0.1 ml of 1 mCi/ml ⁵¹Cr solution and 10 μl FBS and mixing gently. This mixture was incubated by loosening the cap of the tube and placing at 37° C, 5% CO₂ for one hour. After the incubation, the labeled tumor cells were washed twice with 14 ml of RPMI 1640 and resuspended at a concentration of 5 x 10⁴ cells/ml in complete lymphocyte media.

The CTL effector cells were plated in 4 wells of a round bottom 96 well tissue culture plate at concentrations that equaled 100:1, 30:1, 10:1, and 3:1 effector to target cell ratios. Five thousand labeled target cells were added to the wells containing the effector cells as well as two additional wells for natural and maximum release controls. The cells were mixed and centrifuged at 200 g for 30 seconds. The plate was then incubated at 37° C, 5% CO₂ for four hours. Thirty minutes prior to the end of the incubation, 0.1 ml of Triton X-IOO was added to the maximum release control well. At the end of the incubation, the cells were centrifuged in the plate at 200 g for five minutes. 0.1 ml of each supernatant was added to liquid scintillation counter vials containing 5 ml of scintillation cocktail. The amount of ⁵ Cr release was measured using a LS6500 Multi-purpose Scintillation Counter (Beckman).

The CTL assay results showed that as the ratio of hybrid cell-primed CTLs to tumor cells increased, the release of the isotope increased, indicating a positive correlation between the number of CTLs and tumor killing. Greater than 50% killing was observed at a 100:1 effectoπtarget ratio. On the other hand, there was no such correlation with control T cells that were not primed with the inventive hybrid cells. Even at a ratio of 100:1, the control T cells did not lyse more tumor cells than at lower ratios. These results demonstrated that the CTLs generated using our hybrid antigen presenting cells are fully functional and tumor cell specific. The results are depicted in Figure 1. Example 3; Dendritoma Characterization

This example provides further characterization of the reactant cells and the dendritomas described in Examples 1 and 2. Fluorescent microscopic analysis showed that 100% of the stained cells were successfully labeled. To test whether the dye can interstain between the two different type of cells, green DCs and red tumor cells were mixed together and incubated overnight. Fluorescent microscopic examination showed there was no interstaining. Immediate examination of the fusion product demonstrated that the green DCs and the red tumor cells were fused together and after an overnight recovery, the fused cells showed both colors. The double colored cells (approximately 10% of the total cells), instant dendritomas, were then purified by FACS sorting. More than 95% of the sorted cells were double colored fused cells.

Instant dendritomas express all the molecules necessary for antigen presentation. FACS analysis showed that instant dendritomas express the molecules required for antigen presentation, such as MHC class I and II and co-stimulating molecules CD80 (B7.1) and CD86 (B7.2). The data are depicted in Figure 2. "Isotype" is the negative control; HLA- A₅B₅C is MHC class I and HLA-DR is MHC class II. Under a microscope, moreover, instant dendritomas also have those dark granules that melanoma tumor cells have.

For Figure 2, human DCs from peripheral blood were stained with the green dye and tumor cells were stained with the red dye, respectively, and fused, using the above protocol. After overnight incubation, the cells were equally divided into 4 groups. They were then stained with Cy-Chrome conjugated antibodies by incubating the cells with the antibodies (1 million cells/microgram antibody, Becton/Dickinson) on ice for 30 min. The different groups were as follows: anti-human HLA-A₅B₅C [group I]; anti-human HLA-DR [group H]; anti- human CD80 [group III]; and anti-human CD86 [group IV]. The un-bound antibodies were removed by two washes and the cell pellet was re-suspended in 0.5 ml of staining buffer (PBS containing 0.1% BSA and 0.1% sodium azide). Three-color analysis was performed by FACS, using CellQuest software. Control human DCs were also stained with the same antibodies in the same way.

Example 4: Clinical Protocol

This example provides two exemplary clinical protocols for treating human cancer patients with the inventive hybrid cell preparations. This regimen is useful, for example, to treat melanoma patients with a dendritoma, prepared according to the inventive methodologies.

Protocol 1

Mature dendritic cells are generated from the patient's peripheral blood monocytes. 50 mis of peripheral blood are obtained from the patient in preservative free or sodium heparin tubes. Briefly, the blood are diluted 1:1 with IXPBS. Then, 8 mis of the diluted blood are layered over 4 mis of room temperature Ficoll-Paque Plus in a 15 ml centrifuge tube, and centrifuged at 4Og for 40 minutes. The PBMC layer is removed from the Ficoll gradient, and placed into a clean 15 ml centrifuge tube. 4 volumes of IX PBS are added and the tube is inverted to mix. The PBMCs are then centrifuged at lOOg at room temperature for 10 minutes. lOmls of IX PBS are added, and the cells are mixed by inverting the tube. The PBMCs are again centrifuged at lOOg at room temperature for 10 minutes. The PBMCs are resuspended in 5 mis complete DC medium (RPMI 1640 + 10% human serum + 800U/ml GMCSF + lOOOU/ml IL-4). Then the dendritic cells/precursors are panned using anti-CD 14 coated plates. 2X10 PBMCs are placed onto the anti-CD 14 coated plate and swirled. They are left to incubate at room temperature for 30 minutes. The non-adherent cells are then removed. 10 mis of IXPBS are added, the plate is swirled, and the PBS is removed. This PBS washing is repeated for a total of four times, pipetting at the same place each time. Afterwards, 10 mis of complete DC media is added to the plate. They are then incubated at 37° C, 5% CO2 for 5-10 days to generate dendritic cells.

A tumor section is obtained at the time of biopsy or excisional resection. The tumor cells are cultured using the following technique. After separating fat and necrotic tissue away from the tumor tissue (1-5 grams), the tumor are cut into small chunks and put into a T 75 flask. lOmls of 0.25% Trypsin-EDTA are added. This solution will rock for 1 hour at 37° C, and then 15 mis of complete media (DMEM + 10% human serum + gentamicin) are added. The chunks are then removed and put into a clean T75 flask. This flask is left at 37° C in 5% CO2 overnight. Then the cell suspension/typsin/complete media is centrifuged at lOOOg for 5 minutes. These cells are resuspended in 15 mis of complete media and cultured in a T75 flask at 37° C in 5% CO2 for 24 hours. After overnight incubation in the absence of media, 20 mis of complete media are added to the flask with chunks, and this solution is left for two days at 37⁰C in 5% CO2. The chunks are removed, and the adherent cells are cultured. The tumor cells used for dendritic fusion result from both cultures.

The next step comprises the fusion of tumor cells and dendritic cells received from the patient. Hybrid formation by cell fusion became routine after the introduction of the use of polyethylene glycol as a fusing agent. The procedure outlined below is a variation of the one reported by Prado et aϊ., 1989 FEBS Lett., 259: 149-52., for the PEG-mediated fusion of somatic cells in monolayers. First, the tumor cells are exposed to a single dose of 5000 rads, sufficient to kill all of the cells. Then, the dendritic cells are stained green using the PKH2-GL fluorescent dye (Sigma), and the tumor cells are stained red using the PKH26 fluorescent dye (Sigma). The staining procedure is performed at 25 ⁰C, using a slight modification of the Sigma procedure. The cells to be stained are washed with serum-free media. The cell suspension is centrifuged at 400 g for five minutes to obtain a loose pellet, and the supernatant fraction is removed. The pellet is resuspended by tapping the centrifuge tube, and 1 ml of Diluent (20% DMSO in serum-free RPMI) is added to resuspend the cells. Immediately prior to staining, 4 x 10^" molar dyes (2X) were prepared with Diluent in polypropylene tubes. The cells in the diluent are rapidly added into 1 ml of 2X dye, and the mixture is immediately mixed by gentle pipetting. The mixture is then incubated at 25° C for five minutes. The staining process is stopped by adding an equal volume of 10% human serum, which may be the patient's own serum, and incubating for one minute. The stained cells are diluted with equal volume of complete culture medium. Stained cells are removed from the staining solution by centrifuging at 400 g for 10 minutes.

The green dendritic cells are mixed with the red tumor cells at a 1 : 1 ratio in a 50-ml conical centrifuge tube. The tube is filled with complete serum-free DMEM. The cell mixture is centrifuged for 5 minutes at 50Og. While the cells are being centrifuged, three 37° C double-beaker water baths are prepared in the laminar flow hood by placing a 400-ml beaker containing 100 ml of 37° C water into a 600-ml beaker containing 75 to 100 ml of 37° C water. Tubes of prewarmed 50% PEG solution and complete serum-free DMEM are placed into two of the 37° C water baths in the hood. Then, the supernatant from the cell mixture is aspirated and discarded. The cell fusion is performed at 37° C by placing the tube containing the mixed-cell pellet in on of the double-beaker water baths in the laminar flow hood. Then, 1 ml of prewarmed 50% PEG is added to the mixed-cell pellet drop-by-drop over one minute, stirring the cells with the pipette tip after each drop. The mixture is then stirred for an additional minute.

Using a clean pipette, 1 ml of pre- warmed RPMI + HEPES is added to the cell mixture drop-by-drop over one minute, stirring after each drop. This step is repeated once with an additional 1 ml of prewarmed RPMI + HEPES solution. With a 10-ml pipette, 7 ml of prewarmed RPMI +HEPES is added drop-by-drop over 2 to 3 minutes. This mixture is then centrifuged for five minutes at 500g. While the cells are in the centrifuge, the water baths are rewarmed to 37° C and placed in the hood. Prewarmed complete DC media is placed in the beaker water bath. Then the supernatant from the mixture is discarded; the tube is placed in the beaker water bath. With a pipette, 10 ml of prewarmed complete DC media are forcefully discharged onto the cell pellet and placed in a T75 flask. This is incubated overnight in a humidified 37° C, 5% CO2 incubator. The next day, the cells are analyzed on a FACS Caliber fluorescence activated cell sorter using the CELLQuest software (Becton/Dickenson), which will sort the fusion cells with both the green and red dye. These fusion cells, dendritomas, are then resuspended in 1 ml of NS (Normal Saline) and injected into the patient.

The vaccine will consist of 100,000 (or more) irradiated tumor cells fused to dentritic cells i.e. dendritomas. These dendritomas are resuspended in 1 ml of NS and injected IV into the patient.

Interleukin 2 (e.g., Aldesleukin) also may be given in a low-dose regimen. When used, IL-2 is administered by subcutaneous injection in a dosage of 18 million units daily for 5 days beginning on the day of vaccination.

Protocol 2

This protocol was used in the patient studies detailed in Example 6 below.

Fluorescent Dyes

The fluorescent dyes used in dendritoma generation were PKH26-GL and PKH2-GL from the Sigma Chemical Co. Less than 10¹⁴g of these dyes associated with the dendritoma vaccines were introduced into the patient.

Serum Preparation

For culture of the patient's dendritic cells and cryopreservation of tumor cells and dendritomas, the patient's own serum was used. Autologous serum was isolated using standard procedures from 200 mis of peripheral blood collected without anticoagulants. The serum was inactivated by incubating at 56 ⁰C for 30 minutes, aliquoted into 15 ml tubes marked with the patient's identifiers, and stored at -80 ⁰C. At the same time, a small amount of the serum sample was used to do QC testing including sterility, mycoplasma, and endotoxin. This autologous serum comprised 10% by volume of the culture media used for the culture of the patient's dendritic cells and the freezing medium for cryopreservation of tumor cells and/or dendritomas.

Dendritic cell generation

Dendritic cells were generated from the patient's peripheral blood monocytes. Three hundred milliliters of sodium heparinized peripheral blood were collected from the patient and diluted 1:1 with IX PBS. The diluted blood was evenly layered over 100 mis of room temperature Ficoll-Paque Plus in four 250 ml centrifuge tube bottles, and centrifuged at 1750 rpm for 20 minutes. The PBMC layers were removed from the Ficoll gradients, and placed into clean 250 ml centrifuge tubes. Four volumes of IXPBS were added and the tubes were inverted to mix. The PBMCs were then centrifuged at 1500 rpm at room temperature for 10 minutes. Ten milliliters of IXPBS were added, and the cells were mixed by inverting the tube. The PBMCs were again centrifuged at 1500 rpm at room temperature for 10 minutes. The PBMCs were resuspended at a concentration of 3 to 10 x 10⁶/ml in RPMI 1640/Hepes and placed onto 100 mm tissue culture plates and swirled. They were incubated at 37° C for 3 hours. The non-adherent cells were then removed and the adherent cells were washed once with RPMI 1640/Hepes. The cells were cultured in 10 mis of complete DC medium (RPMI 1640 + 10% autologous patient serum + 800U/ml GMCSF + 1 OOOU/ml IL-4 + 1 OOμg/ml gentamicin) in each plate for 8-10 days to generate dendritic cells with a fresh DC medium change every 3 days.

Tumor Cell Preparation from Surgically Excised Lesions

A tumor section was requested at the time of biopsy or excisional resection prior to the specimen being sent to pathology. The tumor cells were isolated using the following technique: After separating fat and necrotic tissue away from the tumor tissue, the tumor was cut into small pieces and put into a T75 flask. Twenty milliliters of digestion medium (RPMI 1640 + 1.5mg/ml Collagenase Type VIII + 26 μg/ml Pulmozyme) were added to the flask. The solution was rocked for 1-2 hours at 37° C. The cell suspension was then filtered through a 40 μm cell strainer (Falcon Cat# 2340). The cells were pelleted at 80Og for 5 minutes at room temperature. The cells were washed three times with 20 ml IX PBS and pelleted at 800 g for 5 minutes at room temperature. 15 ml of ACK Lysing Solution (Cambrex) was added to the cell pellet and incubated for 5 minutes at room temperature to lyse red blood cells. The cells were pelleted at 80Og for 5 minutes. The cells were then frozen for future dendritoma production and tumor cell skin test.

Dendritic Cell and Irradiated Tumor Cell Fusion

The next step in this protocol involves the fusion of tumor cells and dendritic cells received from the patient. Hybrid formation by cell fusion became routine after the introduction of the use of polyethylene glycol as a fusing agent. The procedure outlined below is a variation of the one reported by Prado et ah, FEBS Lett, 18;259(l):149-52 (1989) for the PEG-mediated fusion of somatic cells in monolayers.

The dendritic cells were stained green using the PKH2-GL fluorescent dye and the tumor cells were stained red using the PKH26-GL fluorescent dye as outlined by the protocol from the company. Before fusion, the red dye stained tumor cells were irradiated with a single dose of 5000 rads of γ-ray sufficient to render the cells' replication incompetent. The green dendritic cells were mixed with the red tumor cells at a 1 : 1 to 10: 1 ratios in a 50-ml conical centrifuge tube. The tube was filled with complete serum-free RPMI 1640 medium. The cell mixture was centrifuged for 5 minutes at 500g. While the cells are being centrifuged, three 37 ⁰C double-beaker water baths were prepared in the laminar flow hood by placing a 400-ml beaker containing 100 ml of 37 ⁰C water into a 600-ml beaker containing 75 to 100 ml of 37 ⁰C water. Tubes of prewarmed 50% PEG solution and complete serum-free RPMI 1640 medium were placed into two of the 37 ⁰C water baths in the hood. The supernatant from the cell mixture was aspirated and discarded. The cell fusion was performed at 37 ⁰C by placing the tube containing the mixed-cell pellet in one of the double- beaker water baths in the laminar flow hood. One milliliter of the prewarmed 50% PEG was added to the mixed-cell pellet drop-by-drop over one minute and the cells were then stirred with the pipette tip after each drop. The mixture was stirred for an additional minute. Using a clean pipette, one milliliter of prewarmed RPMI 1640/Hepes was added to the cell mixture drop-by-drop over one minute, stirring after each drop. This step was repeated once with an additional 1 ml of prewarmed RPMI 1640/Hepes solution. With a 10-ml pipette, 7 ml of prewarmed RPMI 1640/Hepes was added drop-by-drop over 2 to 3 minutes. This mixture was then centrifuged for five minutes at 50Og. While the cells are in the centrifuge, the water baths were rewarmed to 37° C and placed in the hood. Prewarmed complete DC medium containing 10% autologous serum was placed in the beaker water bath. After discarding the supernatant, the tube was placed in the beaker water bath and 10 ml of prewarmed complete DC medium containing 100 ng/ml TNF-α was forcefully discharged onto the cell pellet and placed in a T75 flask. This was incubated overnight in a humidified 37° C, 5% CO2 incubator.

The next day, the cells were analyzed and the dual colored hybrid cells (dendritomas) were sorted out by the FACS Vantage SE (Becton Dickinson). After sorting, the dendritomas were washed in 50 ml IX PBS and pelleted by centrifugation. The dendritomas were resuspended in 0.5-1.0ml NS medium in a sterile 50 ml conical tube. The dendritoma cells were then irradiated with 10,000 rads. A certified pharmacist working in a laminar flow biological safety cabinet transferred the 0.5-1.0 ml of dendritomas to a 3.0cc syringe and adjusted the final volume to 2-3 ml with NS medium in preparation for injection into the patient. The final formulation in 2-3 mis of NS contained 200,000 to 500,000 dendritoma cells. The dendritomas sorted out but not used for the vaccination were frozen in complete DC medium + 10% DMSO for future re-vaccination or re-evaluation.

Example 6. Clinical Trials

Patient 1 with stage IV melanoma (AJCC, 5^th Ed.) was rendered NED by left hemicolectomy. At time of study, patient 1 was given 3-6 months to live. Patient 1 received 5 courses of treatment with dendritoma cells according to the following vaccine protocol.

3, 6, 9 and 12 months from initial time of treatment with the dendritoma cells, Patient 1 was alive with a partial response. Patient 1 was classified as a complete responder and was alive 970 days from initial time of treatment.

Patient 2 with stage IV melanoma (AJCC, 6^th Ed.) was rendered NED by surgical removal of mass from lateral surface of right kidney. At time of study onset, patient 2 was expected to live for only 3-6 more months.

Patient 2 received 6 courses of treatment with dendritoma cells according to the following vaccine protocol.

Four vaccine skin test were negative and two were positive. Skin tests were done at least 3 months after administration of first vaccine.

At 3 months and 6 months post initial dose, Patient 2 was evaluated to determine disease status. Patient 2 was considered NED at both timepoints and was alive at day 878 post initial treatment.

Patient 3 with stage IV melanoma (AJCC, 6^th Ed.) was rendered NED by excision of nodule on left leg. At time of study, patient 3 was given 3-6 months to live.

Patient 3 received 3 courses of treatment with dendritoma cells according to the following vaccine protocol.

At 3 months and 6 months post initial dose, Patient 3 was evaluated to determine disease status. Patient 3 was considered to have stable disease and NED at both timepoints and while Patient 3 is currently classified as having progressive disease, Patient 3 was alive at day 606 post initial treatment.

Patient 4 with stage IV melanoma (AJCC, 6^th Ed.) was rendered NED by excision of metastatic lesion in the posterior superior aspect of the right lobe of the liver. At time of study, patient 4 was given 3-6 months to live.

Patient 4 received 6 courses of treatment with dendritoma cells according to the following vaccine protocol.

All five vaccine skin tests were negative. Skin tests were done at least 3 months after administration of first vaccine.

At 3 months and 6 months post initial dose, Patient 4 was evaluated to determine disease status. Patient 4 was considered as having stable disease and NED at both timepoints. Patient 4 was alive at day 578 post initial treatment. Patient 5 with stage IV melanoma (AJCC, 6^th Ed.) was rendered NED by surgical removal of nodal metastasis of melanoma in the right groin. At time of study, patient 5 was given 3-6 months to live.

Patient 5 received 2 courses of treatment with dendritoma cells according to the following vaccine protocol.

A vaccine skin test was not done.

Patient 5 was evaluated to determine disease status. Patient 5 was considered to have stable disease at 3 months post initial hybrid cells treatment. Patient 5 was alive at day 423 post initial treatment but with progressive disease.

It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modification and variations of the invention provided they come within the scope of the appended claims and their equivalents.

Claims

IN THE CLAIMS

1. A method for treating a cancer patient who has no evidence of disease (NED) comprising administering to a patient a composition comprising a hybrid of a primary tumor cell and a dendritic cell, wherein the composition is substantially free of non-hybrid cells.

2. The method of claim 1 , wherein the patient has stage I, stage II, or stage III cancer, with the staging defined according to the American Joint Committee on Cancer (AJCC) 2002 Staging System.

3. The method of claim 2, wherein the patient has stage IV cancer.

4. The method of claim 1 , wherein the patient has renal cancer, ovarian cancer, melanoma, or a combination thereof.

5. The method of claim 3, wherein the patient has melanoma.

6. The method of claim 1 , wherein the patient has no evidence of disease following surgery, radiation, chemotherapy, or a combination thereof.

7. The method of claim 6, wherein the patient has had chemotherapy with an agent selected from the group consisting of nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogues, pyrimidine analogues, purine analogs, platinum complexes, adrenocortical suppressants, adrenocorticosteroids, progestins, estrogens, antiestrogens and androgens, cyclophosphamide, chlorambucil, methotrexate, fluorouracil, cytarabine, thioguanine, vinblastine, vincristine, doxorubicin, daunorubicin, mitomycin, cisplatin, hydroxyurea, prednisone, hydroxyprogesterone caproate, medroxyprogesterone, megestrol acetate, diethyl stilbestrol, ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone, and combinations thereof.

8. A method for increasing survival of a late stage cancer patient that has no evidence of disease, comprising administering to a patient a composition comprising a hybrid of a primary tumor cell and a dendritic cell, wherein the composition is substantially free of non-hybrid cells.

9. The method of claim 6, wherein the cancer patient has melanoma.

10. The method of claim 9, wherein the cancer patient has stage IV-NED melanoma, with the staging defined according to the American Joint Committee on Cancer (AJCC) 2002 Staging System.