MXPA97009733A - Extract of tumor cell modified with hapten and methods for the treatment or classification decan - Google Patents

Abstract

A covedosa composition comprising a tumor cell depleted with hapten prepared from cancer cells, or membranes or cancer cell peptides obtained therefrom is described. Also disclosed are methods for the treatment of cancer comprising administering therapeutically effective amounts of cyclophosphamide and extracts of hapten-modified tumor cell, with or without adjuvants and cytokines. Also described is a method for the classification of cytokine production through tumors in order to determine the efficacy of the treatment, wherein the cytokine-specific nucleic acids from a sample of patient tissue are amplified in order to produce a signal. Detect

Description

EXTRACT OF TUMOR CELL MODIFIED WITH HAPTEN AND METHODS FOR THE TREATMENT OR CLASSIFICATION OF CANCER REFERENCE TO RELATED REQUESTS This application is a continuation in part of the application Series No. 08 / 203,004, filed on February 28, 1994, which is a continuation in part of the application Series No. 07 / 985,334, filed on December 4, 1992, now US patent No. 5,290,551, which is a continuation in part of the application Series No. 07 / 520,649, filed May 8, 1990, now abandoned.

REFERENCE TO GOVERNMENT CONCESSIONS The invention described herein was made in the course of work under a concession or award of an NIH Cancer Research grant, grant no. CA39248. The government of the United States may have certain rights in this invention. Some of this invention was described in a Description Document filed with the Patent and Trademark Office on April 18, 1990.

BACKGROUND OF THE INVENTION It was presented as a theory in the 1960s that tumor cells carry specific antigens (TSA), which are not present in normal cells and that the immune response to these antigens can allow an individual to reject a tumor. It was then suggested that the immune response to TSA can be increased by introducing new determinants into the cells. Mitchison, Transplant. Proc., 1970, 2, 92. Said "auxiliary determinant", which may be a hapten, a protein, a viral coating antigen, a transplantation antigen, or a xenogeneic cell antigen, may be introduced into a population of tumor cells. Then, an individual who is expected to tolerate the growth of unmodified tumor cells could be injected. Clinically, the hope was that an immunological reaction could occur against the auxiliary determinants, as a consequence of which the reaction towards the accompanying TSAs increases, and the tumor cells are destroyed which could otherwise be tolerated. Mitchison, supra, also suggests several modes of action of the auxiliary determinants, including, 1) that the unmodified cells are merely attenuated, in the sense that their growth rate is reduced or their susceptibility to immune attack is increased; 2) that the auxiliary determinants merely provide attack points and thus allow the modified cells to be annihilated by a non-directed immune response against TSA; 3) that the auxiliary determinants have an auxiliary action such as binding to an antibody or promoting the localization of the cells in the correct part of the body for immunization, in particular, in lymph nodes. Fujiwara et al., J. Immunol. , 1984a, 132, 1571, showed that tumor cells conjugated to the hapten, trinitrophenyl (TN P), could induce systemic immunity against unmodified tumor cells in a murine system, provided that the mice were first sensitized to the hapten in the absence of cells T hapten-specific suppressors. The spleen cells of the mice treated completely and specifically prevented the development of tumors in untreated recipient animals. Flood et al., J. Immunol, 1987, 138, 3573 showed that mice immunized with a "regressor" tumor induced with ultraviolet light, conjugated with TN P, were able to reject a tumor "progressor" conjugated with TN P which was another non-immune way. In addition, these mice were subsequently resistant to attack with unconjugated "progressor" tumor. In another experimental system, Fujiwara et al., J. Immunol. 1984b, 133, 510, demonstrated that mice sensitized to trinitrochlorobenzene (TNCB) after pretreatment with cyclophosphamide could be cured of large tumors (10 mm) through in situ hatenization of tumor cells; Subsequently, these animals were specifically resistant to attack with unconjugated tumor cells. Recently, the existence of T cells has been demonstrated, which react in crossing with non-modified tissues Weitzien and co-workers have shown that T-cell clones restricted by MHC class I generated from mice immunized with syngeneic lymphocytes modified with TN P respond to "auto" "Peptides modified with TN P, associated with M HC. Otmann, B., and others, J. Immunol. , 1992, 148, 1445. Furthermore, it has been established that immunization of mice with lymphocytes modified with TN P results in the development of splenic T cells that exhibit proliferative and cytotoxic responses secondary to TNP-modified cells, in vitro. Shearer, G M. Eur. J. Immunol. , 1974, 4, 527. The potential of lymphocytes produced by immunization with autologous cells modified with DN P or TN P to respond to systemic pus erythematosus, can develop and continue after the absence withdrawal of the offensive drug. This could imply the final generation of T lymphocytes that react in crossing with unmodified tissues. Others have shown that membranes or peptides of cells, and in one case a peptide of a virus, can produce a cell lymphocyte response, in vitro. Heike, M. , and others, J. immunotherapy, 1994, 75: 165-174, describe a method to stimulate mouse and human cytotoxic anti-tumor T lymphocytes (CTL) with plasma membrane preparations. This reference identifies differences between the reports of CTL responses and associates these differences with differences in immunogenicity of the tumors used and the mode of immunization, for example. Others report that the recognition of human melanoma cells by cytotoxic T cells is mediated by a number of peptides, Slingluff, C, et al., J. Immunology 1993, 150: 2955-2963; Wolfel, T., Int. J. Cancer 1994 57: 413-418; and Castelli, C, et al., J. Exp. Med. 1995 181: 363-368, the descriptions of each of these are incorporated herein by reference in their entirety. Each of these references indicates that there are a number of epitopes defined by T cells, which are unique to a given tumor. None of these references discloses a tumor cell extract, which is obtained through the hapten modification of a tumor cell. The common denominator of these experiments is hapten sensitization in a medium where the suppressor cells are not induced. Spleen cells from mice sensitized with TNCB, pretreated with cyclophosphamide exhibited radioresistant "amplified auxiliary function", that is, they specifically increased the in vitro generation of anti-TNP cytotoxicity. Furthermore, once these adjuvants have been activated through in vitro exposure to autologous TNP-conjugated lymphocytes, they could also increase cytotoxicity to unrelated antigens as well, including tumor antigens (Fujiwara et al., 1984b). Flood et al., (1987), supra, showed that this amplified auxiliary activity was mediated through T cells with the Lyt "1+, Lyt" 2", L3T4 +, l" J + phenotype, and suggests that these cells were contrasuppressor cells, a new class of immuno-regulatory T cell.

Immunotherapy of patients with melanoma has shown that the administration of cyclophosphamide, at a high dose (1000 mg / M2) or at a low dose (300 mg / M2), three days before sensitization with primary antigen key lymphocyte hemocyanin markedly increases the acquisition of delayed-type hypersensitivity to that of the antigen (Berd et al., Cancer Res. 1982, 42, 4862; Cancer Res., 1984a, 44, 1275). Pre-treatment with cyclophosphamide at a low dose allows patients with metastatic melanoma to develop a delayed-type hypersensitivity to autologous melanoma cells in response to injection with autologous melanoma vaccines (Berd, et al., Cancer Res., 1986, 46, 2572). The combination of cyclophosphamide at a low dose and vaccine can produce a clinically important regression of metastatic tumor (Berd et al. (1986), supra; Cancer Invest. 1988a, 6, 335). The administration of cyclophosphamide results in the reduction of non-specific peripheral blood lymphocyte T suppressor function (Berd et al., Cancer Res. 1984b, 44, 5439; Cancer Res., 1987, 47, 3317), possibly depleting the cells T inducing suppressors of CD4 +, CD45RA + (Berd et al., Cancer Res, 1988b, 48, 1671). The effects against tumors of this immunotherapy regimen appear to be limited by the excessively long interval between the initiation of vaccine administration and the development of delayed-type hypersensitivity to tumor cells (Berd et al., Proc. Amer. Assoc. Cancer Res. ., 1988c, 29, 408 (# 1626)). Therefore, there is a need to increase the therapeutic efficiency of said vaccine to make it more immunogenic. Most immunologists now agree that T lymphocyte, white blood cells responsible for tumor immunity, infiltration to the tumor mass, is a prerequisite for the destruction of the tumor through the immune system. Consequently, a good object of attention has been focused on what has become known as "TIL" therapy, initiated by Dr. Stephen Rosenberg at NCI. Dr. Rosenberg and others have extracted from human cancer metastases, some T lymphocytes that are naturally present and greatly expand their numbers by culturing them in vitro with Interleukin 2 (IL2), a growth factor for T lymphocytes. Topalian et al., J. Clin. Oncol. 1988, 6, 839. However, this therapy has not been very effective, since the injected T cells are limited in their ability to "stay" at the tumor site. The ability of high concentrations of IL2 to induce lymphocytes to become non-specifically cytotoxic killing cells has been exploited therapeutically in a number of studies (Lotze et al., J. Biol. Response, 1982, 3, 475; West et al., New Engl. J. Med., 1987, 316, 898). However, this aspect has been limited by the severe toxicity of high dose intravenous IL2. Less attention has been paid to the observation that much lower concentrations of IL2 can act as an immunological aid by inducing the expansion of antigen-activated T cells (Talmadge et al., Cancer Res., 1987, 47, 5725; Meuer et al., Lancet , 1989, 1, 15). Therefore, there is a need to understand and try to exploit the use of IL2 as an immunological aid. It is believed that human melanomas express unique surface antigens recognizable by T lymphocytes. Oíd, L. J., Cancer Res., 1981, 41, 361; Van der Bruggen, P., et al., Science, 1991, 254, 1643; Mukherji, B., et al., J. Immunol., 1986, 136, 1888; and Anichini, A., et al., J. Immunol., 1989, 142, 362. However, the immunoteraphatic aspects for updating have been limited by the difficulty of inducing an effective T cell-mediated response to said antigens in vivo. There are several proposed models to explain what appears to be tolerance to antigens associated with human tumors. These include: 1) tumor suppressor-specific antigen cells that reduce incipient tumor responses. Mukherji, and others, supra; Berendt, M. J. and R. J. North, J. Exp. Med., 1980, 151, 69. 2) Failure of human tumor cells to produce T helper cells or provide co-stimulatory signals to those T cells.

Fearon, E. R., et al., Cell, 1990, 60, 397; Towsend, S.E. and J.P. Allison, Science, 1993, 259, 368; and 3) Reduced surface expression of major histocompatibility products in tumor cells, which limit their recognition by T cells. Ruiter, DJ Seminars in Cancer Biology, 1991, 2, 35. None of these hypotheses has yet been corroborated in a clinical system. The objective of effective immunotherapy for tumors is the development of a productive systemic T cell mediated by specific immunity in the tumor. The specific immunity in the tumor could act both as the primary tumor site as well as in clearing small metastatic foci in distant sites. The generation of T cell immunity has been shown as a highly regulated response that requires cell-cell interaction and the production of a number of cytokines. Then, studies in a number of human and murine systems have shown that T cell responses can be separated into two categories called Type I and II (Mossman, et al., J. Immunol, 1986) 136: 2348). Type I responses are required for the development of delayed-type hypersensitivity (DTH), are associated with macrophage activation and interferon-gamma production (IFN?), And have been shown to be associated with the resolution of leprosy. in humans (Yamamura M., et al., Science, 1991, 254: 277-279) and murine leishmaniasis (Scott, P., et al., Immunological Review 1989 112: 161-182). Type II responses are associated with the production of IL4 and IL10, mainly support antibody responses, and are associated with the progressive forms of leprosy (Yamamura, M., et al., Supra) and leishmaniasis (Yamamura et al., Supra; and Scott, P., and others, supra). In addition to the development of DTH, Type I responses are expected to improve the generation of specific CTL in tumors via the up-regulation of MHC and antigens associated with tumors, as well as the presentation of improved antigen secondary to the production of IFNα. located. More recently, Type I and II responses have shown a crossover regulation: IFN? inhibits Type II responses, while IL4 and IL10 inhibit Type I (Scott, J. Immunol., 1991 147: 3149-3155); and Firentino et al., J. Immunol. 1991 146: 3444-3451). In the leismania system, the modulation of cytokines at the site of injury allows the conversion of a Type II response to a Type I response, and, consequently, a progressive infection change to the eradication of the disease (Scott, 1991, supra). Pisa and others, Proc. Nati Acad. Sci. USA, 1992, 89: 7708-7712 detected IL10 mRNA in ovarian carcinoma biopsies, but not in ovarian carcinoma cell lines; concluded that the source of IL10 was tumor infiltration lymphocytes. Gast et al., Int. J. Cancer 1991 55-96-101, found that 16/48 tumor cell lines released IL10 into the culture supernatant; only 3/8 melanoma cell lines were positive. Finally, Chen et al., Int. J. Cancer, 1994, 56: 755-760 recently reported that 6/9 cell lines derived from metastatic melanomas expressed IL10 mRNA. However, the present invention is the first known report to the inventors of mRNA for IL10 in metastatic melanoma biopsies. It is not known if these observations are applicable to the human-host tumor relationship, that is, if the pattern of cytokine production by T cells infiltrating tumors is an indicator of the effectiveness of the immune response. Patients with metastatic melanoma treated with a DNP-modified, autologous vaccine developed inflammatory responses at tumor sites, Berd et al., 1991, supra. Histologically, these inflamed lesions are characterized by infiltration of the T cell, which is sometimes associated with the destruction of the tumor cell. The present invention finds that tumors of patients treated with the DNP vaccine contain Type I T lymphocytes, which are not detectable in tumors excised prior to administration of the vaccine.

COMPENDIUM OF THE INVENTION The present invention is directed to a treatment for cancer. Compositions and methods for the treatment of cancer are included within the scope of the present invention. The compositions of the present invention include a composition prepared from a tumor cell or tumor cell extract. The methods of the present invention are directed to the treatment of cancer, comprising administering a therapeutically effective amount of a composition comprising a tumor cell or a tumor cell extract.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the kinetics of the development of DTH to autologous PBL modified with DNP and melanoma cells. Patients with metastatic melanoma treated with the DNP vaccine were serially tested on the skin with PBL (LY) melanoma cells modified with DNP or modified with DNP, disassociated from a metastatic mass. Each bar indicates the average DTH response for the group of patients at each time point; the error bars = normal error. By day 119, only responses to PBL were measured. Sample sizes: Days 0, 14, 63, N = 84; day 119, N = 57; day 175, N = 42; day 231, N = 35. Figure 2 shows the antibody response to DNP. The serum obtained at various time points was tested for antibody (total immunoglobulin) to DNP using ELISA. The titration was defined as: (OD sample peak) X (reciprocal of the dilution that gave an OD equal to half of the peak OD of the positive control). Figure 3 is a graph of proliferative response of PBL to autologous lymphocytes modified with DNP. The PBLs were obtained from four patients who received the DNP vaccine at the peak of their responses to DTH. The cells were tested for their proliferation capacity to autologous PBL modified with DNP (autol LY-DNP) with unmodified autologous PBL (autol LY) as a control. The cultures were pulsed with 125IIUDR on day 6. Figure 4 shows the kinetics of the proliferative response to lymphocytes modified with DNP. A patient was serially taken PBL from DM2, while receiving the DNP vaccine. These were cryopreserved and then all samples were tested simultaneously for the proliferative response to autologous PBLs modified with DNP (autol LY-DNP). Pulses with 12 SIUDR were applied to the cultures on day 6. Figure 5 represents the specific character of the proliferative responses to the cells modified with DNP. PBL from two patients were tested for the proliferative response to autologous PBL, either unmodified (autol LY), modified with DNP (autol LY-DNP), or modified with TNP (autol LY-TNP), and to autologous melanoma cells cultivated, either unmodified (MEL) or modified with DNP (MEL-DNP). The cultures were pulsed with 125 IUDR on day 6. Figure 6 is a specific character analysis of expanded T cells. PBL of DM2 from one patient was expanded in IL2 and repeatedly stimulated with DNP-modified B lymphoblastoid cells. These were tested for the proliferative response to autologous PBL, either unmodified (autol LY), modified with DNP (autol LY-DNP), or modified with TNP (autol LY-TNP); autologous melanoma cells cultured, either unmodified (MEL) or modified with DNP (MELDNP); and allogeneic PLBs (Alio LY). The cultures were pulsed with 125IUDR on day 6. Figure 7 exhibits CD8 + and CD4 + T cell responses to autologous cells modified with DNP. Expanded T cells were separated in populations rich in CD8 or CD4 through positive separation. Then, they were tested for the proliferative response to autologous PBL, either unmodified (autol LY), modified with DNP (autol LY-DNP), and to cultured autologous melanoma cells, either unmodified (MEL) or modified with DNP (MEL-DNP). The cultures were pulsed with 125 IUDR on day 6. Figure 8 shows cytokine production through DNP-reactive T cells. The T-cell line reactive to DNP ("Genitora"), and three subcultures (2F8, 1D7, 1C2), obtained by plating at limiting dilution, were cultured with T lymphoblastoid cells modified with autologous DNP for 18 hours; the supernatants were collected and analyzed for gamma interferon (IFN) and IL4. Figure 9 shows blocking of the T cell response through the anti-MHC class I monoclonal antibody. Expanded CD8 + T cells were stimulated with DNP-modified B lymphoblastoid cells and the cultures were analyzed for gamma interferon after 18 hours. The stimulator cells were pre-incubated with any of the following: no antibody (none), non-specific mouse IgG (non-specific), monoclonal antibody W6 / 32 (class I), or monoclonal antibody L243 (class II).

Figure 10 shows the MHC restriction of T-cell response. Expanded CD + 8 T cells (HLA-AI, A2, B8, Bw6) were tested for proliferation capacity in response to autologous PBLs modified with DNP and Allogenic PBL modified with DNP from four other patients. Three of the allogeneic stimulators were compared with one or more sites of class I as shown, and the fourth was completely decoupled (A24, A26, B44, B63). The cultures were pulsed with 125 IUDR on day 6. Figure 11 shows cytotoxicity graphs of DNP reactive T cells. Melanoma cells, decoupled from class I either autologous or allogeneic (alio), were used as targets in a 51C 6-hour trial. The effector cells were T cells reactive to DNP, CD8 +, expanded. Figure 11A - the target cells were haptenized with various concentrations of DNBS or TNBS. The effector: target cell ratio was 20: 1. Figure 11B - Target cells haptenized with 2.5 mg / ml of DNBS or TNBS were mixed with effector cells to a series of effector: target ratios (E: T). Figure 12 shows a graph of percentage of tumor-free patients in the months following surgery treated with DNP vaccine and non-haptenized control vaccine. Figure 13 shows the HPLC fractions, which were emptied into five groups of 10 fractions each. Peptides derived from dinitrophenyl-modified cells (DNP-MEL) or dinitrophenyl-modified B cells (DNP-LY) were stimulators in void 2. Figure 14 shows an inflamed subcutaneous melanoma nodule from a patient immunized with DNP vaccine and expresses mRNA for IFN? and IL10. Figure 14-1 shows mRNA for cytokines determined by RT-PCR (lane 1 = size marker, 2 = β-actin, 3 = IFN ?, 4 = IL4; 5 = IL10); Figure 14-2 is an H & E stained section of the subcutaneous lesion (10OX). Figure 15 exhibits a lymph node metastasis from a non-immunized patient expressing mRNA for IL10, but not IFN ?. Figure 15-1 shows mRNA for cytokines determined by RT-PCR (lane 1 = size marker, 2 = β-actin, 3 = IFNα, 4 = IL4, 5 = IL10); Figure 15-2 is an H & E stained section of the lymph node metastasis (40OX). Figure 16 is a gel of IL10 mRNA expressed in melanoma metastasis. The mRNA for cytokines was determined through RT-PCR (lane 1 = size labeling, C = control of cDNA IL10, 1.7 = patient samples). Figure 17 is an IL10 mRNA gel expressed by melanoma cells. Figure 17 shows IL10 mRNA expression through RT-PCR of a representative tumor biopsy and cell line drift (lane 1 = size marker, 2 = IL10 cDNA, 3 = tumor biopsy, 4 = tumor line ). Figure 18 is an in situ RT-PCR of a paraffin section of a non-inflamed melanoma biopsy (A = 100x, B = 400x).

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to cancer immunotherapy. A composition and novel methods for tumor for the treatment of cancer are included in the scope of the invention. The present invention is directed to the use in the treatment of cancer, including metastatic and primary cancers. The cancers treatable with the present invention include the following non-limiting examples: melanoma, breast, lung, colon, kidney and prostate. Mammals, particularly humans, that have metastatic cancer can be treated with the compositions and methods of the present invention. The compositions of the present invention are prepared from a tumor cell or a tumor cell extract. A tumor cell can be a malignant or pre-malignant cell of any type of cancer. In accordance with the present invention, pre-malignant refers to any abnormal cell suggestive of a cancer cell, which is not yet a cancer cell; such as and not limited to dysplastic changes in cervical cells, which eventually lead to cervical cancer, and nevus dysplastic, which are abnormal cells in the skin, which lead to melanoma. The tumor cells and extracts preferably originate from the type of cancer, which is going to be treated. The tumor cells and extracts may be, but are not limited to, autologous and allogeneic cells dissociated from biopsy specimens or tissue culture, as well as stem cells and extracts from these sources. Preferably, the cells and extracts are autologous. The tumor cell extracts of the present invention may be a peptide isolated from a hapten-modified cancer cell or a cell membrane isolated from a hapten-modified cancer cell. For the purposes of the present invention, the peptides are compounds of two or more amino acids and include proteins. The peptides will preferably be of a molecular weight, from about 1,000 kD to about 10,000 kD, most preferably from about 1,000 to 5,000, which are isolated from a hardened tumor cell and which stimulate cell lymphocytes. T to produce gamma interferon. T cells are lymphocytes, which mediate two types of immunological, effector and regulatory functions, secrete proteins (lymphokines), and annihilate other cells (cytotoxicity). Effector functions include reactivity such as delayed hypersensitivity, allograft rejection, tumor immunity, and graft-host reactivity. Lymphokine production and cytotoxicity are demonstrated through T cell effector functions. The regulatory functions of T cells are represented by their ability to amplify cell-mediated cytotoxicity through other T cells and immunoglobulin production through of B cells. Regulatory functions also require the production of lymphokines. T cells produce gamma interferon (I FN?) In response to an induction stimulus including but not limited to mitogens, antigens or lectins. The peptide may be preferably from about 8 to about 20 amino acids, in addition the peptide is preferably haptenized. The peptides can be isolated from the surface of the cell, inside the cell or any combination of the two locations. The extract may be particular to the type of cancer cell (against normal cell). The peptide of the present invention includes and is not limited to a peptide which binds to the highest histocompatibility complex, a cell surface associated protein, a protein encoded by cancerous oncogenes or mutated anti-oncogenes. The cancer cell membrane of the present invention may be all or part of a membrane of a membrane isolated from a haptenized cancer cell. In accordance with the definition of cancer cell membrane as set forth for the present invention, a cancer cell membrane can be isolated, then haptenized, alternatively, a cancer cell can be haptenized and the membrane subsequently isolated from it. The cell extracts are capable of stimulating T cells. The stimulation for the purposes of the present invention relates to the proliferation of T cells, as well as the production of cytokines through T cells, in response to the cell extract. The membranes and proteins isolated from tumor cells modified with hapten and proteins, each independently have the ability to stimulate T cells. The proliferation of T cells can be observed through the consumption by T cells of modified nucleic acids, as such and not of limited to 3H thymidine, 125IUDR (iododeoxyuridine); and dyes such as 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), which stains living cells. In addition, the production of cytokines such as and are not limited to IFN?, tumor necrosis factor (TNF), and IL-2. The production of cytokines is preferably in an amount greater than 15 picograms / ml, most preferably around 20 s 30 picograms / ml, most preferably around 50 picograms / ml. Preferably, the tumor cell extract comprises cellular materials which are unique, or substantially specific to, a particular type of cancer. The tumor cells of the present invention can be living cells. The tumor cells and extracts of the present invention can be irradiated before use. The tumor cells or extracts are irradiated at approximately 2500 cGy to prevent the cells from growing after the injection. The compositions of the invention can be used in the method of the invention individually or in combination with other compounds, including and not limited to other compositions of the invention. Accordingly, cancer cells and cancer cell extracts can be used alone or co-administered. For purposes of the present invention, co-administration includes administration together and consecutively. In addition, the cancer cell membrane can be co-administered with the peptide. In addition, cancer cells and / or extracts may be co-administered with other compounds including and not limited to cytokines such as interleukin-2, interleukin-4, gamma interferon, interleukin-12, GM-CSF. The tumor cells and extracts of the invention may also be used in conjunction with other cancer treatment including but not limited to chemotherapy, radiation, antibodies, oligonucleotide sequences, and antisense oligonucleotide sequences. The compositions of the invention can be administered in a mixture with a pharmaceutically acceptable carrier, selected with respect to the intended route of administration and normal pharmaceutical practice. The doses can be set with respect to the weight and the clinical condition of the patient. The proportional ratio of the active ingredient to vehicle naturally depends on the chemical nature, solubility and stability of the compositions, as well as the dose contemplated. The amounts of tumor cells and extracts of the invention that will be used depend on factors such as the affinity of the compound for cancer cells, the amount of cancer cells present and the solubility of the composition. The compounds of the present invention can be administered by any suitable route, including inoculation and injection, for example, intradermal, intravenous, intramuscular and subcutaneous.

The compositions of the present invention can be administered alone or will generally be administered in admixture with a selected pharmaceutical carrier with respect to the intended route of administration and normal pharmaceutical practice. The composition of the present invention is a therapeutically effective amount of a composition selected from the group consisting of living tumor cells, tumor cell extracts, such as a peptide and a cancer cell membrane, and a mixture of tumor cells and one or more Tumor cell extracts. The composition can be mixed with an immunological aid and / or a pharmaceutically acceptable carrier. Any known aqueous vehicle useful in the drug delivery, such as and not being limited to saline, may be used in accordance with the present invention as a carrier. In addition, any auxiliary known to those skilled in the art may be useful in the provision of the present invention. Assistants include and are not limited to Bacillus Calmette-Guerin (BCG); cytokines, such as and not being limited to interleukin-12, interleukin-2; and synthetic auxiliaries such as and not limited to QS-21, (Cambridge Biotech, Worcester, MA) described by Livingston et al., Vaccine 1994, 12: 1275, the disclosure of which is incorporated herein by reference in its entirety. In the case where cells and cell extracts are irradiated and haptenized, the cells can be conjugated to a hapten and then irradiated. Alternatively, the cells can then be conjugated to a hapten. In any case, the extracts are subsequently purified and can then also be irradiated and / or haptenized. To irradiate and haptenize the extracts, any method can be performed first, followed by the other method. Alternatively, tumor cells or tumor cell extracts can be added to the antigen presenting cells. The cancer cell extract can be used to treat cancer together with another type of cell, a cell that has an antigen, selected from the group consisting of autologous cultured macrophages and autologous cultured dendritic cells. Macrophages are any large mononuclear amoebic mononuclear cell, regardless of origin, such as and not limited to histiocytes and monocytes, phagocytose, that is, encompass and destroy, other cells, dead tissue, degenerated cells and the like. Macrophages are antigen presenting cells, which present antigens, including tumor antigens, to cells including T cells. Dendritic cells are also antigen presenting cells and appear to be closely related to macrophages, however, dendritic cells are cells that present antigen more efficient than macrophages. They are potent stimulants of T cells and can be isolated from a variety of organs and tissues of the body including and not limited to blood, skin (where dendritic cells are termed as Langerhans cells), lymphoid tissues. Patients can be immunized to the dinitrifeni lo chem (DNP) through the application of dinitrofluorobenzene (DN FB) to the skin. Two weeks later, patients were injected with a vaccine, which may include irradiated cells hardened to DNP. The vaccine was reinjected every 4 weeks for a total of eight treatments. The drug cyclophosphamide (CY) can be administered 3 days before the administration of each vaccine to increase the immune response to tumor cells. A non-haptenized form of the vaccine can be similarly administered. The vaccine of the present invention can be haptenized or non-haptenized. The haptenized, or chemically linked form of the vaccine may include a haptenized tumor cell to di nitrophenyl (DN P), for example. Other haptens include and are not limited to trinitrophenyl (TN P) and N-iodoacetyl-N '- (5-sulfonic 1 -naphthyl) ethylene diamine (A ED). A vaccine from tumor cell extracts can be similarly haptenized. In the case of haptenized cancer cell extracts, the extracts, a peptide, and a cancer cell membrane, are isolated from the haptenized cancer cells. The present invention also contemplates a non-haptenized vaccine of tumor cells and / or cell extracts. In the methods of the present invention, a method for the treatment of a patient suspected of having cancer, comprises administering a pharmaceutically acceptable amount of cyclophosphamide, a pharmaceutically acceptable amount of a composition selected from the group consisting of liver tumor cells. , tumor cell extracts, and a mixture of tumor cells and tumor cell extracts. When the composition is a cancer cell extract, the extract can be a peptide or a membrane isolated from a haptenized cancer cell. The composition can be mixed with an immunological aid and / or a pharmaceutically acceptable carrier. The haptenized vaccine may optionally be followed by the administration of a pharmaceutically acceptable amount of a non-haptenized vaccine. A non-haptenized vaccine can also be administered according to the methods of the present invention. The vaccine of the present invention may comprise tumor cells and / or tumor cell extracts. Tumor cells for use in the present invention can be prepared as follows. Tumor mass is processed, as described by Berd et al. (1986), supra, incorporated herein by reference in its entirety. The cells are extracted through enzymatic dissociation with collagenase and DNAse by mechanical dissociation, frozen in a controlled-regime freezer, and stored in liquid nitrogen until needed. On the day that a patient is going to be tested or treated on the skin, the cells are thawed, washed and irradiated at approximately 2500 R. They were washed again and then suspended in a Hanks balanced salt solution without phenol red. The conjugation of the cells prepared with DN P was carried out by the method of Miller and Claman, J. Immumol. , 1976, 1 15, 1519, incorporated herein by reference in its entirety, which implies a 30 minute incubation of your cells with DN FB under sterile conditions, followed by washing with sterile saline. The cancer cells of a patient can be conjugated to a hapten by isolating the membranes and modifying the membranes or conjugating the cells to a hapten without first isolating the membrane. A cancer cell membrane can be prepared by isolating the membranes of the unmodified preparation from a patient's cancer cells. The cells were suspended in approximately five volumes of approximately 30 mM sodium bicarbonate pH regulator with approximately 1 μM of phenyl methyl sulfonyl fluoride and interrupted with a glass homogenizer. The residual intact cells and the nuclei are removed by centrifugation at approximately 1000 g. The membranes were pelleted by centrifugation at 100,000 g for 90 minutes. The membranes were resuspended in approximately 8% sucrose and frozen at approximately -80 ° C until needed. To a membrane suspension (approximately 5,000,000 cell equivalents / ml), approximately 0.5 ml of 1 mg / ml of initofluorobenzene (DN FB) were added for approximately 30 minutes. Similarly, other haptens such as and not limited to trinitriphenyl and N-iodoacetyl-N '- (5-sulfonic-1-naphthyl) ethylenediamine can be used. The excess DNP was removed by dialyzing the membranes against approximately 0.15 M PBS for approximately three days. The membranes were pelleted. Alternatively, the cancer cell extract, the peptide or the membrane, can be prepared by modifying cancer cells from a patient with a hapten such as dinitrophenyl and then preparing the membranes therein. A patient's cancer cells are obtained during the biopsy and freezing until needed. Approximately 100 mg of DN FB (Sigma Chemical Co., St. Louis, MO) were dissolved in approximately 0.5 ml of 70% ethanol. Approximately 99.5 ml of PBS was added. The concentration of DN FB should be approximately 152 mg / 0.1 ml. The solution was stirred overnight in a water bath at 37 ° C. The storage life of the solution is approximately 4 weeks. The cells were thawed and the pellet was resuspended in 5X cells / ml in a Hanks balanced salt solution. Approximately 0. 1 ml of the DN FB solution was added to each ml of cells and incubated for approximately 30 minutes at room temperature. Similably, other haptens may be used such as but not limited to trinitrophenyl and N-iodoaceti-N '-) 5 sulfonic 1 -naphthyl) ethylenediamine. the cells were then washed twice in a Hanks balanced salt solution. Cells were suspended in approximately five volumes of approximately 30 mM sodium bicarbonate pH buffer with approximately 1 mM phenyl methyl sulfonyl fluoride and were disrupted with a glass homogenizer. The intact cells and residual nuclei were removed by centrifugation at approximately 1000 g. The membranes were pelleted by centrifugation at 100,000 g for 90 minutes. The membranes were resuspended in approximately 8% sucrose and frozen at approximately -80 ° C until needed. Of the cells modified with DNP, the peptide can be extracted, part of which is modified with DNP as a result of the modification of the cells. Extraction techniques, known to those skilled in the art, can be followed through antigen assays to isolate effective antigens for patient treatment. Methods of isolation of cell extracts are readily known to those skilled in the art. In summary, cancerous cells are isolated from one another and cultured in vitro. The dinitrophenyl is added to the cells grown according to the method established above. The peptides are isolated from the cells according to an established technique of Rotzschke et al., Nature, 1990, 348, 252, the disclosure of which is hereby incorporated by reference in its entirety. The cells are treated with a weak acid. Then, they are centrifuged and the supernatants are preserved. The fractions containing small peptides are obtained through H PLC, concentrated and frozen. The fractions are classified for immunological activity allowing them to bind to autologous B infoblastoid cells, which are then tested for the ability to stimulate melanoma-specific T lymphocytes.

The cells are treated with a weak acid, such as but not limited to trifluoroacetic acid (TFA). The cells are then centrifuged and the supernatant is preserved. Compounds having a molecular weight greater than 5,000 were removed from the supernatant through gel filtration (G25 Sepharose, Pharmacia). The remainder of the supernatant was separated on a reverse phase HPLC column (Superpac Pep S, Pharmacia LKB) in 0.1% trifluoroacetic acid (TFA) using a gradient of an increasing concentration of acetonitrile; flow rate = 1 ml / min., fraction size = 1 ml. Fractions containing small peptides are obtained by HPLC according to the method of Sambrook et al., Molecular Cloning: A Laboratory Manual, 2a. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), were concentrated and frozen. The fractions were classified for immunological activity by allowing them to bind to autologous B lymphoblastoid cells, which were then tested for the ability to stimulate melanoma-specific T lymphocytes. The Epstein barr virus (EBV, ATCC CRL-1612, transformed B95 EBV leukocytes, cotton top marmoset, Saguinus oedipus) was added to B lymphoblastoid cells in the culture. The B lymphoblastoid cells are transformed to a B cell tumor of the patient's own lymphocytes. Melanoma was cultured from a metastasis in fetal calf serum RPMI 1640 + 10% or 10% human serum drained, the non-adhered cells were washed with RPMI medium. When the cells are confluent, they are separated with 0.1% EDTA and divided into two flasks. This procedure continues when the confluent cells are continuously divided. To test the production of interferon gamma through T cells, lymphocytes were obtained from a patient's blood. The patient's own tumor cells, which have been modified with a hapten, such as DN P, were mixed with the lymphocytes to stimulate the T cells. Every seven days, interleukin-2 was added. The T cells expanded by dividing as described above. The T cells were then further stimulated through hapten-modified cells. An enriched population of T cells resulted, which are responsible for the hapten-modified cells. Human cancer vaccines have been developed and tested through a number of workers. Although they may sometimes induce weak immunity to a patient's cancer, they rarely cause tumor regression. The development of inflammatory responses in metastatic tumors was surprisingly found with the DN P vaccine of the present invention. The tumor reddens, warms and softens. Finally, in some cases, the tumor returns to the degree that the tumor disappears, at first sight and microscopically.

Microscopically, the infiltration of T lymphocytes towards the tumor was observed. Therefore, this aspect, which increases the inflammatory response and the number and capacity of lymphocytes entering the tumor, is a significant advance in the art. The effectiveness of the vaccine can be improved by adding several biological response modifiers. These agents work directly or indirectly stimulating the immune response. The biological response modifiers of the present invention include and are not limited to interleukin-12 and gamma-interferon. In this modality, a I L12 will be given following the injection of each vaccine. The administration of I L 12 to patients with inflammatory response is thought to cause the T lymphocytes, within the tumor mass; they proliferate and become more active. Increased T cell numbers and functional capacity leads to immunological destruction of the tumors. The doses for I L12 will be prepared in accordance with the dosage indications stated above.

Patients with metastatic melanoma were tested using an immunotherapeutic regimen with the following components: 1) vaccine consisting of autologous tumor cells conjugated to DN P; and 2) pre-treatment with cyclophosphamide at low dose. Patients were evaluated to determine if tumor regression occurred, to verify inflammatory responses, and to measure delayed-type hypersensitivity to autologous melanoma cells, autologous lymphocytes conjugated with DN FB (the form of DN P used for the sensitization of skin), DN P, diluent (Hanks solution), purified protein derivative (PP D), and call antigens (cand ida, trichophyton and mumps). Patients who are considered to be derived benefit (clinical or immunological) from therapy continue in the immunotherapy regimen. Subsequent vaccines may be given without cyclophosphamide. In a similar experiment, interleukin 2 linked to polyethylene glycol was found to be ineffective. In another embodiment, a vaccine comprising chemical extracts of cancer cells conjugated to a hapten and mixed with an immunological aid, such as bacillus Calmette-Guerin, BCG, was used. In the present invention, biopsies of human melanoma metastases were examined for the expression of cytokine mRNA using RT-PCR. It was found that mRNA for IFN? in DNP post-vaccine, it inflamed the metastasis, but only sparingly in pre-treatment metastasis, even those containing large numbers of residual lymph node lymphocytes. In addition, the Type II cytokine, IL10, was found in almost all melanoma metastases and appears to be produced by the same melanoma cells. Patients with metastatic melanoma treated with a vaccine modified with DNP, autologous, develop inflammatory responses in tumor sites. Histologically, these inflamed lesions are characterized by T cell infiltration, which is sometimes associated with the destruction of the tumor cell. In the present invention, biopsy specimens from 8 subcutaneous metastases that had developed inflammation after treatment with the vaccine were tested for mRNA expression for IFN ?, II4, TNF, and IL10. Biopsies after the vaccine, inflamed contained mRNA for I FN? (5/8), IL4 (4/8) or both (3/8), and for TN F (4-7). In contrast, mRNA of I FNα was detected. in only 1/17 and AR Nm of TN F in 2/1 control specimens (pre-treatment lymph node metastasis or non-inflamed subcutaneous metastasis). The mRNA for I L10, a cytokine with anti-inflammatory properties, was detected in 24/24 melanoma metastases and was independent of the lymphoid content; PCR in situ confirmed that melanoma cells were the major source. These findings provide a new parameter through which the effects of cancer treatment are measured. The present invention is directed to analyze biopsies of recently obtained metastatic melanoma for the presence of cytoqine mRNA, which correlates with a reproductive immune response at the tumor site. The expression of A RN m of I FN? or I L4 is characteristic of the metastasis of melanoma that has developed an inflammatory response after the administration of autologous vaccine modified with DN P. On the one hand, mRNA expression of I L10 is dependent on an inflammatory response and seen in almost all melanoma biopsy specimens. The examination of the cell lines derived from melanoma biopsies as well as the analysis of PC R in situ, showed that the source of I L10 is the same melanoma cells instead of the associated lymphocytes. Perhaps the most important finding of this work is a negative one: RNA m for I FN? and I L4 is generally not found in melanoma metastases from untreated patients, nor in metastatic masses that contain large numbers of lymph node lymphocytes. This provides a low background activity of in situ cytokine production against which melanoma tissues compete, whose T cell population has been altered by immunotherapy. further, this accentuates an important biological point: the T cells extracted from melanoma nodal metastasis probably represent the residues of the original lymph node population instead of the lymphocytes that have actually infiltrated the tumor as a result of their recognition of melanoma antigens . Since they are not activated by the antigen, they have not received any stimulus to produce IFN? or IL4. In contrast, biopsy specimens obtained following administration of the dNP vaccine typically expressed mRNA for IFN ?. However, the inflammatory responses induced by the DNP vaccine can not be characterized as Type I, since some of these also showed IL4. Given the sensitivity of OCR-based mRNA analysis, this pattern can be produced by a small focus of T cells that produce IL4 in half of the T cell infiltrate that is predominantly of IFN? Production. On the other hand, the presence of IFNα mRNA and IL4 may mean the presence of T cells that produce tato cytokines, so-called THs cells (Lee et al., Eur. J. Immunol., 1992, 22: 1455_1459). The resolution of this emission will require analyzes that allow the correlation of mRNA expression with morphology, such as OCR in situ. Whatever the outcome, these findings suggest that measurement of intra-tumor cytokine production may be an important parameter to measure in patients undergoing immunotherapy. The present invention strongly suggests that the source of MRNA I L 10 is the same melanoma cells, instead of the associated lymphocytes. Strong IL10 mRNA bands were detected in 24/15 biopsies, and their expression was independent of the number of associated lymphocytes or the presence of inflammation induced by the DNP vaccine. In addition, in situ PCR clearly showed IL10 mRNA within melanoma cells. The cell lines derived from the biopsy material expressed IL10 mRNA and produced IL10, as measured by ELISA. The physiological importance of IL10 production in melanoma tissues is unclear. It is known that IL10 is an anti-inflammatory cytokine with the ability to inhibit T cell proliferation and IL2 production (Jinquan, T. et al, J. Immunol., 1993, 151: 4545-4551) delayed the type of hypersensitivity (Lee, supra), probably by reducing the co-stimulating function of the macrophage. In this way, IL10 can suppress the activation and proliferation of melanoma-reactive T cells that have infiltrated the tumor site. However, IL10 has recently been shown to be a chemoattractant for CD8 + T cells (Jinquan, supra); this property represents the predominance of CD8 + cells in lymphoid infiltrates induced by the DNP vaccine. In any case, the modulation of I L10 production at the tumor site can have important consequences for the tumor-host relationship. The scope of the present invention also includes a method for the classification of cytokine production through a tumor to determine the efficacy of an autologous, irradiated hapten-conjugated cell composition in a patient suspected of having cancer, said method comprises administering said hapten-conjugated composition to said patient; obtaining a sample comprising nucleic acids from a sample of the tissue of a patient; amplifying the nucleic acids for a cytokine or amplifying a signal generated through the hybridization of a specific probe to a cytokine-specific nucleic acid in said tissue sample; and detecting the presence of the amplified nucleic acids or the amplified signal, wherein the presence of amplified nucleic acids or the signal indica cancer, wherein the presence of amplified nucleic acids or amplified signal of the tissue sample of said patient indicates the efficacy of a conjugate composition with hapten. The tissue sample can be a malignant or pre-malignant tumor, a melanoma tumor, for example, or a subcutaneous inflammatory metastatic melanoma, for example. In addition, a tissue sample may be a sample of solid or fluid tissue such as and not being limited to all or part of a tumor, saliva, sputum, mucus, bone marrow, serum, blood, urine, lymph, or a tear of a patient with suspected cancer.

Nucleic acids, such as DNA (including cDNA) and RNA (including mRNA), were obtained from the tissue sample of a patient. Preferably, RNA was obtained from a tissue sample. Total RNA extracted through any method known in the art, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989), incorporated herein by reference in its whole. Nucleic acid extraction is followed by amplification thereof by any technique known in the art. The amplification step induces the use of at least one primer sequence, which is complementary to a portion of a specific cytokine sequence. Specific cytokine sequences are defined for the purposes of the present invention to include (and are not limited to) all or parts of the sequences, which encode IFN ?, TBF, I L-2, IL-12 and I L- 13 Generally, the primer sequence is from about 21 nucleotides to about 33 nucleotides, preferably about 21 nucleotides, about 31 nucleotides, 32 nucleotides and about 33 nucleotides in length. Initiator sequences useful in the amplification methods include, and are not limited to, β-actin, SEQ ID NOS: 1 and 2; IFN ?, SE ID NOS: 3 and 4; IL4, SEQ ID NOS: 5 and 6; IL10, SEQ ID NOS: 7 and 8; and TNF, SEQ ID NOS: 9 and 10. When a method depends on an amplification template using a pair of primers, a primer of the pair can comprise oligonucleotide, which are complementary to the nucleic acid sequences, which encode protein specific to cytokine. The one primer of the pair can be selected from the group consisting of SEQ ID NOS: 1 A 10. Alternatively, each of the two oligonucleotides in the primer pair can be specific to a nucleic acid sequence, which encodes a cytokine. The primers can be designed to be complementary to separate regions of a cytokine sequence, for example. By "dividing regions" is meant that a first primer is complementary to a 3 'region of a cytokine sequence and a second primer is complementary to a 5' region of a cytokine sequence. Preferably, the primers are complementary to separate, separate regions and are not complementary to each other. The primers of SEQ ID NOS: 1-19 are merely illustrative of the primers that may be useful in the present invention. When an amplification method includes the use of two primers, such as the polymerase chain reaction, the first primer can be selected from the group consisting of SEQUENCE ID NOS: 1, 3, 5, 7 and 9, and the second primer can be selected from the group consisting of SEQUENCE ID NOS: 2, 4, 6, 8, and 10. Any primer pairs, which transcribe nucleic acids from each other and which are specific for cytokines, can be used in accordance with the methods of the present invention. Preferably, the total extraction of AR N is carried out. As used herein, the term "amplification" refers to template-dependent procedures and vector-mediated propagation, which results in an increase in the concentration of a specific nucleic acid molecule relative to the initial concentration, or in an increase in the concentration of a detectable signal. As used herein, the term "template dependent process" is intended to refer to a method that involves the template-dependent extension of an initiator molecule. The term "template-dependent process" refers to the synthesis of RNA nucleic acid or a DNA molecule, wherein the sequence of the newly synthesized structure of the nucleic acid is dictated by the well-known rules of the complementary base pairs (see, for example, Watson, JD and others, in: Molecular Biology of the Gen, 4th ed., W. A. Benjamin Inc., Menlo Park, Calif. (1987) incorporated herein by reference in its entirety). Typically, methodologies mediated by the vector involve introducing the nucleic acid fragment to a DNA or RNA vector, clonal amplification of the vector, and recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.A. Patent No. 4,237,224), Maníatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, each i Incorporated here by reference in its entirety. A number of template dependent procedures are available to amplify the target sequences of interest in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR), which is described in detail in the U.S. Patents. 4,683,195; 4,683, 202 and 4,800,159, and in Innis et al., PCR Protocols, Academic Press, Inc., San Diego CA., 1990, each of which is incorporated herein by reference in its entirety. In summary, in the PCR, two primer sequences were prepared, which are complementary to regions in complementary complementary structures of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture together with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By increasing and reducing the temperature of the reaction mixture, the extended primers will dissociate from the target to form the reaction products, the excess of initiators will bind to the target and the reaction products and the procedure is repeated. Preferably, a reverse transcriptase PCR amplification procedure can be performed in order to quantify the amount of amplified mRNA. The methodologies of the polymerase chain reaction are well known in the art.

Another method for amplification is the ligase chain reaction (referred to as LCR), described in EPA No. 320,308, incorporated herein by reference in its entirety. In the LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will be joined to complementary complementary structures of the target, so that they are supported. In the presence of a ligase, the two pairs of probes will be linked together to form an individual unit. By cyclizing the temperature, as in PCR, the bound units are dissociated from the target and then serve as "target sequences" for the ligation of excess probe pairs. Patent of E.U.A. No. 4,883,750, incorporated herein by reference in its entirety, discloses an alternative method of amplification similar to CSF for joining probe pairs to a target sequence. Qbeta replicasa, described in the PCT application no.

PCT / US87 / 00880, incorporated herein by reference in its entirety, may also be used as another method of amplification in the present invention. In this method, a RNA replication sequence, which has a region complementary to that of a target, is added to a sample in the presence of an RNA polymerase. the polymerase will copy the sequence of replication, which can then be detected. An isothermal amplification method, where endonuclease and restriction ligases are used to obtain the amplification of target molecules containing the nucleotide 5 '- [alpha-thio] triphosphates in a chain of a restriction site (Walker, GT et al. Proc. Nati, Acad. Sci. (USA) 192, 89: 392-396, incorporated herein by reference in its entirety], may also be useful in the amplification of nucleic acids in the present invention. SDA) is another method for carrying out the isothermal amplification of nucleic acids, which involves multiple turns of displacement and chain synthesis, ie, slot translation.A similar method, called Repair Chain Reaction (RCR) is another method of amplification, which may be useful in the present invention and involves the reinforcement of several probes through a target region for amplification, followed by a repeat reaction. aration where only two of the four bases are present. The other two bases can be added as biotinylated derivatives to facilitate detection. A similar aspect is used in SDA. Specific cytokine sequence can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having a 3 'and 5' sequence of the specific DNA without cytokine and the cytokine specific RNA mediated sequence is hybridized to DNA, which is present in a sample. After hybridization, the reaction is treated with RNasH, and the products of the probe identified as distinct products that generate a signal, which are released after digestion. The original template is reinforced to another cyclization probe and the reaction is repeated. In this manner, CPR involves amplifying a signal generated by the hybridization of a probe to a cytokine-specific nucleic acid.

Still other amplification methods described in GB application No. 2 202 328, and PCT application No. PCT / US89 / 01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the first application, "modified" primers are used in a PCR as template-dependent synthesis and enzyme. The primers can be modified by labeling with a capture portion ('e.g., biotin) and / or a detector portion (e.g., enzyme). In the last application, an excess of labeled probes was added to a sample. In the presence of the target sequence, the probe binds and cleaves catalytically. After cleavage, the target sequence is released intact to be joined by the excess probe. The excision of the labeled probe signals the presence of the target sequence. Other methods of nucleic acid amplification include transcription based amplification (TAS) systems (KWoh D., and others, Proc. Nati Acad. Sci. (E.U.A.) 1989, 86: 1173, Gingeras T. R. and others, PCT application WO 88/1031, incorporated herein by reference in its entirety), including amplification based on nucleic acid sequence (NASBA) and 3SR. In NASBA, nucleic acids can be prepared for amplification through normal extraction of phenol / chloroform, thermal denaturation of a clinical sample, treatment with pH regulator and mini spin columns for DNA and RNA isolation or chloride extraction of RNA guanidinium. These amplification techniques involve reinforcing an initiator, which has specific prostate sequences. Following the polymerization, the DNA / RNA hybrids were digested with RNas H, while the double-stranded structure DNA molecules are again denatured with heat. In any case the DNA of individual chain structure is made of completely double-structure chain by the addition of a second prostate-specific initiator, followed by polymerization. DNA molecules of double-stranded structure are then multiplied transcribed by a polymerase such as T7 or SP. In an isothermal cyclic reaction, the RNAs are reverse transcribed to double-stranded chain DNA, and transcribed once more with polymerase such as T7 or SP6. The resulting products, either truncated or complete, indicate specific sequences of prostate cancer. Davey C. et al., European Patent Application No. 32,822, incorporated herein by reference in its entirety, disclose a nucleic acid amplification method that involves cyclically synthesizing single chain structure ("ssRNA") RNA, ssDNA, and DNA of double-stranded structure (DNA ds), which can be used according to the present invention. The ssRNA is a first template for a primer priming oligonucleotide, which is elongated through reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the duplex of DNA: RNA through the action of ribonuclease H (RNase H, a RNase specific for RNA in a duplex with either DNA or RNA). The resulting ssDNA is a second template for a second primer, which also includes the sequence of an RNA polymerase promoter (illustrated by T7 RNA polymerase) 5 'to its homology to its template. This initiator is then extended through DNA polymerase (illustrated by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a DNA molecule of double-stranded structure ("dsDNA"), which it has a sequence identical to that of the original RNA between the primers and also having, at one end, a promoter sequence. This promoter sequence can be used through the appropriate RNA polymerase to make many copies of DNA RNA. These copies can then re-enter the cycle that leads to very rapid amplification. With an appropriate choice of enzymes, this amplification can be done isothermally without the addition of enzymes in each cycle. Due to the cyclic nature of this procedure, the starting sequence can be chosen to be in the form of DNA or RNA. Miller, HI, et al., PCT application WO 89/06700, incorporated herein by reference in its entirety, discloses a nucleic acid sequence amplification scheme based on the hybridization of a promoter / primer sequence to a DNA structure of individual target chain ("ssDNA") followed by the transcription of many RNA copies of the sequence. This scheme is not cyclic, that is, no new templates are produced from the resulting RNA transcripts. Other amplification methods include "RACE" described by Frohman, M. TO . , in: PCR Protocols: AGuide to Methods and Applications 1990, Academic Press, N.Y. ) and "One-sided PCR" (Ohara, O. et al., Proc. Nati. Acad. Sci. (E. U.A.) 1989, 86: 5673-5677), all references are incorporated herein by reference in their entirety. Methods based on the ligation of two (or more) oligonucleotide in the presence of nucleic acid having the resulting "di-oligonucleotide" sequence, thus amplifying the di-oligonucleotide (Wu, DY et al., Genomics 1989, 4: 560 , incorporated herein by reference in its entirety), may also be used in the amplification step of the present invention. After amplification, the presence or absence of the amplification product can be detected. The amplified product can be sequenced by any method known in the art, including and not limited to the method of Maxam and Gilbert, see Sambrook, supra. The sequenced amplified product can then be compared to the results obtained with the divided tissue before treatment with the vaccine. Tissue samples obtained prior to treatment with the vaccine should be free of cytokine sequences, particularly I FN ?, TN F, IL2, I L12 and I L13. The nucleic acids can be fragmented into variable sizes of discrete fragments. For example, DNA fragments can be separated according to molecular weight by methods such as and not being limited to electrophoresis through an agarose matrix. The gels are then analyzed through Southern hybridization. Briefly, the DNA in the gel is transferred to a hybridization substrate or matrix such as and not limited to a sheet of nitrocellulose and a nylon membrane. A labeled probe is applied to the matrix under selected hybridization conditions for the purpose of hybridization with the complementary DNA located on the matrix. The probe can be a length capable of forming a stable duplex. The probe may have a size scale of about 200 about 10,000 nucleotides in length, preferably about 200 nucleotides in length. Imbalances such as, but not limited to, sequence with similar hydrophobicity and hydrophilicity, will be well known to those skilled in the art once armed with the present disclosure. Various labels for visualization or detection are known to those skilled in the art, such as but not limited to fluorescent staining, staining with ethidium bromide, for example, avidin / biotin radioactive labeling such as 32P labeling, and the like. Preferably, the product, such as the PCR product, can be operated on an agarose gel and visualized using a stain such as lithium bromide. See Sambrook et al., Supra. The matrix can then be analyzed by autoradiography to locate the particular fragments, which hybridize to the probe. A diagnostic kit for classifying the efficacy of an irradiated, autologous, hapten-conjugated cell composition comprising one or more vessels, a pair of primers, wherein one of the primers within said pair is complementary to a specific cytokine sequence. , wherein said initiator is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and means for visualizing amplified DNA; said equipment is useful for determining the effectiveness of the composition. The invention is further characterized through the following real examples 1-8 and 11 and prophetic example 9-10 and 12-14, which represent illustrations only and are not intended to limit the present invention to these specific embodiments.

EXAMPLE 1 64 patients with metastatic melanoma were treated using a melanoma vaccine, prepared according to the methods established above, preceded by a low dose of cyclophosphamide (CY) and verified for immunological effect and activity against tumor. On day 0, patients were given cyclophosphamide 300 mg / M2 i.v. Three days later, they were injected intradermally with a vaccine consisting of 10 x 106 to 25 x 106 of irradiated, cryopreserved, autologous tumor cells (2500 R) mixed with BDG; the tumor cells were obtained through the dissociation of enzymatic metastatic masses (collagenase and DNAse). This sequence of treatment was repeated every 28 days for 8 treatments. The toxicity of the therapy was limited to a local inflammatory response at the injection site and moderate nausea and vomiting followed by the administration of cyclophosphamide. There were 40 evaluable patients with measurable metastasis; 5 had answers, 4 complete and 1 partial. The average duration of response was 10 months (7-84 + months). A patient continues in remission at 11 years, regression occurred only in the skin and nodal metastasis, but also in lung and liver metastasis, in 6 additional patients, a response was observed against the tumor that seemed peculiar to this vaccine therapy , that is, the regression of metastatic lesions that appeared after immunotherapy was initiated. In 3 patients, this "delayed" regression occurred in two or more tumors. Delayed-type hypersensitivity (DTH) was detected in mechanically dissociated, autologous melanoma cells in only 16% of patients before treatment, as compared to 46%, 56% and 73% of patients on days 49 , 161 and 217, respectively. Increases in delayed-type hypersensitivity after immunotherapy were statistically significant through a non-independent t-test; day 0 vs. day 49, p < 0.001; day 0 vs. days 161, p < 0.001; day 0 vs. day 217, p < 0.021 Of all, 26/43 patients (61%) exhibited a positive delayed-type hypersensitivity response (> 5 mm) to the autologous melanoma cells at some point in time. Patients also developed a strong delayed-type hypersensitivity to the enzymes used to prepare the tumor cell suspensions: of 24 patients tested for delayed-type hypersensitivity with a mixture of collagenase and DNAD (each at 1 μg / ml) after two treatments with the vaccine, 21 (88%) had answers > 5 mm The responses against tumors to the vaccine were strongly associated with delayed-type hypersensitivity to mechanically dissociated, autologous melanoma cells, as indicated by three observations: 1) 8/10 patients who exhibited tumor regression had delayed-type hypersensitivity; 2) in post-surgical auxiliary patients, there was a significant correlation between the intensity of delayed-type hypersensitivity to autologous melanoma cells and the time of tumor recurrence (r = 0.680, p <0.001); 3) nine patients who developed delayed-type hypersensitivity to autologous melanoma cells in their original vaccine ("old" tumor) developed new metastasis ("new" tumor) that did not produce delayed-type hypersensitivity or produced a much smaller response. The patients were compared with their previous condition before treatment with the vaccine. Patients treated before the study with the vaccine were removed from the treatment one to two months before the start of the study with the vaccine. Therefore, patients were not treated at the beginning of the study with the vaccine. In three cases regression tumors could be removed for a histological examination; such tumors were characterized by an intense infiltration of lymphocytes. In contrast, tumors excised from these patients prior to immunotherapy consisted of homogenous masses of malignant cells without significant lymphocytic infiltration. This study shows that the use of cyclophosphamide allows the development of an immune response to antigens associated with melanoma in patients who have cancer.

EXAMPLE 2 Patients with metastatic melanoma were sensitized to DNP through topical application in the upper arm with 1% dinitrochlorobenzene (DNCB) or dinitrofluorobenzene (DCFB). Two weeks later, they were injected with a vaccine consisting of 10 x 106 to 25 x 106 of irradiated, autologous melanoma cells conjugated to DNP and mixed with BCG. Cyclophosphamide 300 mg / M 2 i.v. 3 days before DNCB (or DNFB) or vaccine. Of 4 evaluable patients, 3 developed an inflammatory response in tumor masses after 2 treatments with vaccine (8 weeks).

Patient # 1 developed erythema and swelling in the dermal metastasis > 50 large (1-3 cm) in his leg and lower abdomen, followed by ulceration and drainage of necrotic material, and something began to return. The biopsy showed infiltration with CD4 + CD8 + T lymphocytes. Patient # 2 developed erythema and swelling in the skin of his lower abdomen and large modal masses (8 cm) of intersecting cover. They did not return, but they changed in stone consistency to variable. Patient # 3 exhibited moderate erythema in 3 subcutaneous metastases covering the skin. The 3 patients developed delayed-type hypersensitivity for autologous lymphocytes conjugated with both DNCB and DNP. Patients were compared with their previous condition before treatment with the vaccine. Patients treated before the study with the vaccine were removed from the treatment one or two months before the start of the study with the vaccine. Therefore, patients were not treated at the beginning of the study with the vaccine.

EXAMPLE 3 patients (including 3 patients of Example 2) were treated with metastatic melanoma using a novel form of immunotherapy, ie, tumor cell vaccine conjugated to DN P. Patients were sensitized to DNP through topical application to upper arm with 5% dichlorobenzene. Then, every 4 weeks they received cyclophosphamide 300 mg / M2 followed by 3 days through an injection of 10 x 106 to 25 x 106 of irradiated, autologous melanoma cells conjugated to DNP. The patients received 6-8 treatments. The majority of patients (92%) developed delayed-type hypersensitivity (DTH) to autologous lymphocytes conjugated with DNP or tumor cells (mean DTH = 17 mm). The vaccine induced an inflammatory response in subcutaneous and nodal metastases in 11/15 patients, consisting of erythema, swelling, fever, and irritation around the tumor masses, and, in one case, purulent drainage. The biopsies showed infiltration with lymphocytes, which, through immunopathological and flow cytometric analysis, were mainly CD3 +, CD4-, CD8 +, HLA-DR + T cells. The melanoma cells in these tissues strongly expressed ICAM-1, serving as an adhesion molecule for T cells. In this way, the DNP vaccine appears to induce a degree of immunity against melanoma not seen with previously tested immunotherapy. The patients were compared with their condition prior to treatment with the vaccine. Patients treated before the study with the vaccine were removed from the treatment one to two months before the start of the study with the vaccine. Therefore, the patients were not treated at the beginning of the study with the vaccine.

EXAMPLE 4 this example examined the therapeutic effects of the DNP vaccine in patients with surgically resected metastases and no clinical evidence of metastatic disease. 47 patients were sensitized to the hapten, DNFB) dinitrofluorobenzene) Then, they were treated by intradermal injection of irradiated, autologous melanoma cells conjugated to DNP. Additional vaccine injections were administered for 28 days for a total of 8 treatments. All patients were periodically tested for delayed-type hypersensitivity, DTH, exposed to autologous melanoma cells, autologous lymphocytes conjugated with DPN, microbial antigens. An in vitro study was carried out, with cryopreserved lymphocytes extracted from metastatic tumors and / or separated from the peripheral blood. The graph in Figure 12 compares the percentage of tumor-free patients in the months following surgery treated with DNP vaccine and non-haptenized control vaccine. The study examined the therapeutic effects of the DNP vaccine in patients with surgically resected metastases and no clinical evidence of metastasis disease. All patients were sensitized to the hapten, DNFB (dinitrofluorobenzene). Afterwards, they were treated by injection of irradiated, autologous melanoma cells conjugated to DNP. Vaccine injections were administered for 28 days for a total of 8 treatments. All patients were periodically tested for delayed-type hypersensitivity, DTH, responses to autologous melanoma cells, autologous lymphocytes conjugated in DNP and microbial antigens. In vitro studies were performed with cryopreserved lymphocytes extracted from metastatic and / or peripheral blood tumors.

MELANOMA THERAPY WITH DNP VACCINE CY (cyclophosphamide) = 300 mg / M i.v. bolus only before the first two vaccines. Vaccine = 5 x 106 to 20 x 106 irradiated melanoma cells, autologous mixed with BCG Sens. DNFB = 1.0 mg in 0.1 ml of acetone-corn oil applied to the ventral upper arm Attack DNFB = 200 μg in 0.1 ml of acetone-oil of corn applied to the forearm Apply Skin Tests = autologous melanoma cells, peripheral blood lymphocytes (PBL), peripheral blood lymphocytes conjugated to DNP (PBL-DNP), purified protein derivative (PPD) (skin test for tuberculosis ), microbial call antigens * Day 0: PBL, PBL-DNP only Read Skin Tests = mean hardening diameter Obtain PBL = 100 heparinized blood ce Labs Routine = blood count (CBC), differential blood count (Dif) ), platelet count (platelets), SMA-12 (routine laboratory test panel, BUN blood urea nitrogen).

Sensitization to DNP Patients were initially sensitized to DNP as follows: On day 17, cyclophosphamide, 300 mg / M2, was administered as a rapid infusion i.v. Three days later, on days -14 and -15, patients were sensitized with DNFB (dinitrofluorobenzene): 1 mg of DNFB dissolved in acetone-corn oil and applied topically in a volume of 0.1 ml within the confines of a steel ring with a diameter of 2 cm. Two weeks later, patients were tested for DNP reactivity through topical application of 200 μg of DNFB and intradermal injection of autologous PBL conjugated with DNP. Cyclophosphamide was reconstituted in sterile water and the appropriate dose was administered through rapid i.v. infusion.

Preparation of the Vaccine - Tumor masses were processed. The cells were extracted through enzymatic dissociation with collagenase and DNAse and through mechanical dissociation, frozen in a controlled-regime freezer, and stored in liquid nitrogen until needed. On the day a patient was tested, the cells were thawed, washed and irradiated at 2500 R. They were then washed again and suspended in a balanced salt solution of Hanks without phenol red. The conjugation of melanoma cells with DNP was performed.

This involved a 30 minute incubation of tumor cells with dinitrofluorobenzene (DNFB) under sterile conditions, followed by washing with sterile saline. The vaccine consists of 5-20 x 106 liver tumor cells suspended in 0.2 ml of Hanks solution. When BCG was added, it consisted of 0.1 ml of a 1:10 dilution of Tice BCG. Each vaccine treatment consisted of three injections in contiguous sites on the upper arms or legs, excluding ipsilateral limbs to a lymph node dissection. Procedure Study - On day 0, patients received cyclophosphamide, 300 mg / M2, as a rapid infusion i.v. Three days later, on day +3, they were injected intradermally with the autologous melanoma vaccine. Additional vaccine injections were administered every four hours for a total of 8 treatments. Cyclophosphamide was only given before the first two injections. All vaccines were conjugated with DNP and mixed with Bacillus Calmette-Guerin (BCG). BCG is the Tice strain (subcepa of the Pasteur Institute strain) obtained from Organon Teknika Corporation, Durham, NC. The dried frozen material was reconstituted with 1 ml of sterile water and diluted with 1: 10 in pH regulated saline with phosphate, pH 7.2; then 0.1 ml was extracted, mixed with the vaccine and injected. All vaccines were injected to the same site (upper arm or leg).

Immunological Evaluation - The skin test was performed by intradermal injection of 0.1 ml of the test material on the forearm, and delayed-type hypersensitivity was analyzed at 48 hours by measuring the mean hardening diameter. The positive reactions were photographed. The following materials were tested: 1) 1 x 106 autologous melanoma cells irradiated; 2) 3 x 106 of autologous peripheral blood lymphocytes, both unconjugated and conjugated to DNP; 3) Hanks solution; 4) intermediate resistance to PPD; and 5) microbial call antigens - Candida, trichophyton and mumps. Also, the contact sensitivity to DNFB was tested by applying 200 μg to the forearm skin and examining the area for a hardening circle at 48 hours. All patients were collected blood for separation and cryopreservation of lymphocytes and serum each time the test was performed on the skin (see Table 1 for blood collection schedule). Periodically, they were treated for: 1) proliferative and cytotoxic response to autologous melanoma cells; and 2) proliferative response to autologous lymphocytes conjugated to DN P.

Duration of the Study 1) The patients were treated with eight courses of vaccine, which required approximately 8 months. Afterwards, the treatment was stopped. These patients will be checked until at least 5 years have passed since their initial surgery. 2) Patients, who developed regional recurrence or distant metastasis before the end of the 8 treatments, were removed from the study and treated as clinically indicated (chemotherapy or surgery). The control group consisted of 22 patients with metastatic melanoma at regional lymph nodes. They underwent surgical resection of their disease, at that time they had no clinical evidence of metastatic melanoma. Later, they received the treatment with the autologous melanoma vaccine, not hapten raised. First, they were given cyclophosphamide, 300 mg / M2. Three days later, they were injected intradermally with the vaccine, which consisted of 10 x 10 6 to 25 x 10 6 autologous, irradiated melanoma cells mixed with BCG. Treatment with the cyclophosphamide vaccine was repeated every 28 days. A total of eight treatments were given. The patients were clinically evaluated every two months. Only 20% of control patients were released from cancer at two years. In contrast, patients treated with the DNP vaccine of the invention had a significantly higher cancer-free survival, as stated above. Patients who received the haptenized vaccine all had metastatic melanoma at regional lymph nodes, but there was no evidence of distant metastasis. Patients in this condition were treated routinely through surgical resection of the diseased nodes. Surgical resection freed them clinically from the disease, but presented an 80-85% chance of developing metastatic melanoma within two years. The patients in the control group were in the same condition in order to be able to compare with the haptenized vaccine group. In this way, the control group also consisted of patients with metastatic melanoma at the regional nodes, but there was no evidence of distant metastasis, who underwent surgical resection of the diseased nodes. When the treatment was started with the non-haptenized vaccine, the control patients were clinically liberated from the disease, but as previously observed, 80% developed distant metastasis. Patients with surgically incurable melanoma were not selected as controls, since such patients have a cure rate approaching zero, and an even shorter survival than patients with metastasis of the resection node. Furthermore, it is not possible in such patients to measure disease-free survival, a parameter that was dramatically prolonged by the vaccine of the present invention. A statistical analysis of the data was made as follows: Kaplan-Meir graphs of disease-free survival and total survival were constructed. The difference between patients with DNP vaccine and control patients was analyzed through the Mantel registry category test, these are normal statistical methods to analyze such data. The difference was highly significant with p < .01. Subsequently, 17 additional patients were treated according to the protocol outlined above (the size of the control group was not increased for the reasons stated above). The results maintained a statistically significant difference in disease-free survival and total survival.

EXAMPLE 5 The administration of a melanoma vaccine conjugated with dinitrophenyl (DNP), autologous induces T cell infiltration of metastatic tumors, and prolongs the survival of patients who underwent lymphadenotocmia for voluminous regional metastasis. These effects appear to be due to specific T cells. of melanoma, its generation is contingent on T cells with specific character for melanoma cells modified with DNP (DNP-MEL).

Clinical Protocol All patients had metastatic melanoma and underwent immunotherapy with DNP conjugate tumor vaccine, autologous, as previously described in Berd, D., and Oros, Cancer Res., 1991, 51, 273 I, incorporated herein by reference In its whole. The informed consent of the patients was obtained. Patients were pretreated with cyclophosphamide, 300 mg / M2, see Berd et al. (1986) supra, and three days later they were synthesized to DNFB through the topical application of 0.1 ml of a solution of DNFB in acetone-corn oil. Two consecutive days. Two weeks later, the patients were again given cyclophosphamide, followed 3 days after injection of the melanoma vaccine conjugated with DNP. The DNP vaccine was repeated every 28 days. Cyclophosphamide was given before the first two cycles. The vaccine consisted of 10 to 106 - 25 x 106 melanoma cells conjugated with DNP, irradiated (2500 R), cryopreserved autologous, conjugated to DNP mixed with BCG. All tumor preparations contained lymphocytes, which were lymph node tissue residues infiltrated in the tumor. Serum and PBL were collected at the following time points: day 0 (before sensitization), day 14 (2 weeks after sensitization with DNFB), day 63 (after two vaccines), day 119 (after 4 vaccines) ), day 175 (after 6 vaccines), and day 231 (after 8 vaccines).

Cellular Reagents PBL was separated by density gradient centrifugation over Ficoll metriozoate. These were suspended in a freezing medium (RPMI-1640 (Mediatech, Washington DC) + 1% human albumin + dimethyl sulfoxide) were frozen in a controlled regime freezer, and stored in liquid nitrogen. HLA typing of the PBL was performed through the Thomas Jefferson University Hospital Clinical Laboratory. The melanoma cells were enzymatically extracted from the metastatic masses according to the method of Berd, D, et al., (1986), supra, incorporated herein by reference in its entirety, and cryoconserved. The cell lines were derived from these suspensions and were maintained in RPMI-1640 with 10% fetal bovine serum. The melanoma cell lines of the patients used in this study were distinguished through MHC class I differences determined by cytometric analysis with a panel of monoclonal antibodies obtained from the American Type Culture Collection: (HB82 = HB122 = HLA-A3, HB164 = HLA-A11.24).

Conjugation of Hapten The PBL were modified with DNP through a 30 minute incubation with DNFB or aqueous DNBS, according to the methods of Miller, SD and HN Claman, J. Immunol., 1976, 117, 1519 and Geczy, AF and A. Baumgarten, Immunology, 1970, 19, 189, (incorporated herein by reference in its entirety) respectively; the two methods produced equivalent results. For controls of specific character, the cells were modified with TNP through incubation with TNBS, or with oxazolone, according to the methods of Fujiwara, H. et al., J. Immunol., 1980, 124, 863 and Boerrigter, GH and RJ Scheper, J. Invest. Dermatol., 1987, 88, 3, (incorporated herein by reference in its entirety), respectively. Conjugation with hapten was repeated with melanoma cells.

Delayed Type Hypersensitivity (DTH) Cryopreserved PBLs were thawed and resuspended in Hanks' balanced salt solution. The cells were divided into three groups: unmodified, conjugated to DNP, and conjugated to TNP. After washing, 1 x 106 melanoma cells or 3 x 106 PBL were suspended in 0.1 ml of a solution of Hanks and injected intradermally into the forearm. The DTH was determined at 48 hours by measuring the mean hardening diameter. The DTH analysis was reported with melanoma cells. All patients developed DTH to autologous PBL modified with DNP (Figure 1). The DTH responses were evident two weeks after the topical application of DNFB (day 14), and then remained stable throughout the entire period of the monthly administration of the vaccine. Autologous melanoma cell suspensions conjugated with DNP produced a stronger DTH than DN P-PBL (S E medium: PBL = 13.3 mm + 1.3 mm, melanoma cells = 21.9 mm + 3.6 mm); p < 0.01). The DTH was specific for the "same" modified with DNP, since autologous PBL conjugated to TN P did not produce any response in 50 tested patients.

Anti-DNP Antibody ELISA was developed through the coating of microtiter wells with PBL conjugated with DN P. This method was found preferable to coat plates with albumin conjugated with DN P, since it resulted in readings of lower background with serum from patients pre-immunized. PBL conjugates with DN P (5 x 10 5 in 0.1 ml) were added to each well of a 96-well flat bottom plate. The cells were fixed to the plate by drying followed by a 5 minute exposure to 100% methanol. Then, the plates were washed five times with saline regulated at pH with phosphate + 0.05% Tween-20. Serial dilutions of test sera were added to the wells and the plate was placed in a humidified chamber at 37 ° C for 1 hour. After incubation, the plate was washed five times, and then goat anti-human immunoglobulin conjugated with peroxidase (Cappel Laboratories, Malvern, PA) was added at a predetermined optimal dilution. For the detection of IgG or IgM antibody, goat anti-human IgG or IgM conjugated with peroxidase, respectively, were used. After one hour of incubation at 37 ° C, the plate was washed five times and 0.1 ml of substrate (O-phenylenediamine, Sigma Chemical Co., St. Louis, MO) was added to each well followed by 50 μl of 0.12% of hydrogen peroxide. The plate was read on an ELISA plate reader. The assay was validated using a murine anti-DNP monoclonal antibody (clone SPE-7; Sigma ImmunoChemicals) and an anti-mouse immunoglobulin antibody conjugated with peroxidase as the second step reagent. Subsequently, the positive control consisted of a serum sample from a patient who had received multiple injections of the DNP vaccine. Titration of the anti-DNP antibody from each serum sample was defined as follows: (OD little sample) X (reciprocal of the dilution having an OD equal to half the OD of a positive control peak) Butler, JE, Methods Enzymol., 1981, 73, 482. The anti-DNP antibody was developed in 24 of the 27 patients tested (Figures 2). In contrast to DTH, the antibody was not induced by the topical application of DNFB (day 14). In 19 patients, titrations were increased above pre-immunization levels after two intradermal injections of melanoma cells conjugated with DNP (day 63); in 5 patients, significant titers were found only after 4 to 6 vaccines. In all patients, the IgG antibody was detected: only in three patients was the anti-DNP IgM found. The anti-DNP antibody was made to cross-react with TNP, showed that the lymphocyte colonies were classified to see their ability to proliferate in response to DNP-modified B lymphoblastoid cells. Positive cavities expanded in IL2 and were re-stimulated with autologous DNP-conjugated B lymphoblastoid cells every 14 days.

Lymphoproliferative responses - PBLs were tested as response cells. They were suspended in a lymphocyte culture medium (RPMI-1640, 10% human AB + serum emptying, insulin-transferase-selenite medium supplement (Sigma Chemical Co.), 2 mM glutamine, 1% non-essential amino acids, 25 mM pH buffer from HEPES, penicillin + streptomycin) and added to round bottom microtiter plates, from 96 wells to 1 x 105 cells / well. Stimulant cells included: 1) autologous or allogeneic PBL, 2) autologous or allogeneic B lymphoblastoid lines made through transfection with Epstein-Barr virus, 3) cultured, autologous melanoma cells; were inactivated through irradiation (5000 R). In most of the experiments, the response: stimulant ratio was 1: 1. The plates were incubated in a CO2 incubator at 37 ° C for 5 days; then pulses were applied to the cavities with IUDR marked with 1 5l (ICN Radiochemical, Costa Mesa, CA) for 6 hours, were harvested with an automatic harvesting device, and counted in a gamma counter. The cavity mean was calculated in triplicate. T cells were also tested for a lymphoproliferative response according to the above methods. The PBL, obtained and cryopreserved from four patients at the time of the maximum DTH reactivity to autologous cells modified with DNP, were thawed and tested in vitro for proliferative responses. The PBLs of the four patients proliferated after stimulation with cells modified with DNP (Figure 3). The kinetics of the development of the proliferative response in one of the patients (DM2) is shown in Figure 4. The single application of DNFB (day 14) did not result in detectable numbers of response cells in circulation. The reactive OBL were detected after two injections of DNP vaccine (day 63) and continued to be detected throughout the 8-month period of treatment with the vaccine. The proliferative response to the cells modified with DNP was specific, since neither the unconjugated PBL nor the PBL modified with TNP produced responses (Figure 5). PBLs after the vaccine also proliferated actively when stimulated with a DNP-modified melanoma cell line derived from autologous tumor tissue. When stimulated with allogeneic lymphocytes, the PBLs exhibited the expected mixed lymphocyte reaction, which was three to five times greater than the DNP responses. The circulating T lymphocytes of one of these patients (DM2) were expanded in vitro through culture in IL2 and repeated restimulation with DNP-modified B lymphoblastoid cells. After four weeks of expansion, the T cells were 70% CD3 +, CD8 + and 30% CD3 +, CD4 +. They proliferated when stimulated through lymphoblastoid B cells of cultured melanoma, modified with DNP, but not through non-conjugated autologous cells (Figure 6). These cells were separated through positive separation to populations rich in CD8- and CD4- rich, which were 98% pure as determined through flow cytometric analysis. As shown in Figure 7, cells enriched with both CD4- and CD8- exhibited a proliferative response to autologous B lymphoblastoid cells modified with DNP. However, only CD8 + t cells responded to autologous melanoma cells modified with DNP. This result could have been due to the low constitutive expression (<; 5%) of MHC class II through the melanoma cell line. Expanded T cells were tested for the ability to produce cytokines when stimulated with DNP-modified B lymphoblastoid cells. As shown in figure 8 these produced gamma interferon but not IL4. To determine if both CD4 + and CD8 + T cells were involved in the cytokine response, sublines, which were obtained by plating T cells at a limiting dilution, were analyzed. Each of these cultures was homogenous with respect to the expression of CD4 or CD8. Three of these sublines (two of CD4 +, one of CD8 +) were tested for the cytokine response to lymphoblastoid B cells modified with DN P. All three produced interferon gamma, while none produced I L4 (Figure 8).

Cytokine Production - T cells were added to round bottom microtiter plates at 1 x 10 5 cells / well. An equal number of stimulants (autologous B lymphoblastoid cells modified with DNP) was added, and the supernatants were collected after 18 hours of incubation. Commercially available ELISA kits were used to measure the interferon range (Endoge, Boston, MA, sensitivity = 5 pg / ml) and I L4 (R &D Systems, Minneapolis, MN; sensitivity = 3 pg / ml). To determine the MHC dependence of the response, stimulating cells were pre-incubated with monoclonal antibodies to class I MHC (W6 / 32) or class II and MHC (L243) at a concentration of 10 μg / ml during a hour before adding the response cells. No non-specific mouse immunoglobulin at the same concentration was tested as a negative control. The CD8 + T cells reactive with DN P obtained by separation from the bulky population were able to be maintained in a long-term culture (> 3 months) in a medium containing I L2 through repeated stimulation with modified autologous B lymphoblastoid B cells. with DN P; retained the stable phenotype, CD3 +, CD8 +. Two lines of evidence confirmed that their response was class I M HC restricted; 1) the production of gamma interferon was blocked through pre-incubation of the stimulating cells with a class I framework antibody, but not through the anti-class II antibody (Figure 9), 2) T cells were able to respond to alloagénic DNP-modified stimulants that were combined in one or both HLA-A sites, but not to stimulants that were decoupled from HLA-A. As shown in Figure 10, T cells were proliferated by stimulation with autologous PBLs modified with DNP (HLA-A1, A2, B8 +, Bw6) and with PBL modified with DNP that expressed A1 or A2, or both; no response occurred through allogeneic stimulants modified with DNP that were negative to A1 and A2.

Cytotoxicity - Melanoma targets were labeled for two hours with 51 Cr (Amersham Corp. Arlington Heihts, IL), and 2500 cells were added to round-bottom microtiter cavities. Then effector cells were added to obtain a series of E: T ratios. After 6 hours of incubation at 37 ° C, the supernatants were removed and counted in a gamma counter. Lysis was defined as: ([CPM prußba - CPM eSponténeo] / [CPM tota, - Spontaneous CPM]) * 100. The cytotoxicity of the CD8 + T cell line was tested in a 51Cr release assay with autologous melanoma cells as objectives. To minimize the spontaneous release of s1Cr, modification by DNP was achieved with DNBE instead of DNFB. T cells used autologous melanoma cells modified with DNP, but not allogenic melanoma cells (uncoupled class I) (Figures 11a, 11b). There was a direct relationship between the susceptibility to lysis and the degree of modification through DNP, as determined by the concentration of DNBS used. Neither the autologous nor the allogeneic targets modified with TPN were lysed.

EXAMPLE 6 Clinical data were collected to suggest that an autologous DNP-conjugated melanoma vaccine prolongs disease-free survival (DFS) and total survival (TS) in melanoma patients with bulky regional lymph node metastases but with resection. Forty-seven patients underwent lymphadenoctomy with resection of metastatic masses. The tumor cells were enzymatically dissociated from these tissues and were cryopreserved. The vaccines consisted of 10 x 106 to 20 x 106 irradiated melanoma cells (2500 cGy), conjugated to DNP and mixed with BCG. They were injected i d. every 28 days for a total of 8 treatments. Cyclophosphamide, 300 mg / M2, i.v. 3 days before the first 2 vaccines. The DFS and TS of these patients were compared with those of 22 melanoma patients with resection nodal metastases previously treated with a non-conjugated vaccine, see Example 4. Of the 36 patients with stage 3 melanoma (palpable mass in a lymph node region), 22 are free of diseases with up to a mean of 33 months. The Kaplan-Meir analysis projects a 3-year DFS and TS of 59% and 71%, respectively. In contrast, the DFS and TS of stage 3, patients treated with the unconjugated vaccine was 22% and 27%, respectively (p = 0.01, record category test). Of the 11 patients in stage 4 (palpable mass in two lymph node regions), 5 are NED (no evidence of disease) with an average of up to 41 months. For patients in both stages 3 and 4, the highest relapse rate was in the first 6 months, a time when melanoma immunity could not yet be established. This experiment will be followed by an accelerated schedule of immunizations to reduce the early relapse regimen and improve overall clinical production. Patients were compared with their condition before treatment with the vaccine. Patients treated before the vaccine study were removed from the treatment, one or two months before the start of the vaccine study. Therefore, the patients were not treated at the beginning of the vaccine study. EXAMPLE 7 Materials and Methods Melanoma tissue and Human cell lines - The tissue was obtained from patients with metastatic melanoma before entering the vaccine program and at post-vaccine time points. The clinical protocol for the administration of the DNP vaccine was performed according to Berd, D., et al., Cancer Res. 1991 51: 2731-2734. After surgery, the tumor specimen was transported to the laboratory, the tumor tissue was isolated from the surrounding face and connective tissue, and the tumor pieces, measuring 2-4 mm3, were frozen in liquid nitrogen. The melanoma cell lines of the same specimens were derived from enzyme digestions (DNAase and collagenase) of the tumor and propagated as described by Berd, D., et al., Cancer Res., 1986, 46: 2572-2577.

RNA isolation and Amplification via RT-PCR - Total RNA was extracted from frozen tissues by grinding in guanidinium isocyanate, followed by isolation using a CsCl gradient as described by Lattime, E. C, et al., J. Immunol., 1988, 144: 3422-3428. To minimize the loss of RNA in the tissue, 15 μg of E. coli ribosomal RNA (Sigma Chemical Corp., St. Louis, MO) was added to each sample. The isolated RNA was resuspended in diethyl pyrocarbonate treated with (DEPC-tx) (Sigma) deionized water. The cDNA synthesis was performed using 10 μg of total RNA, random Primer (Gibco BRL, Gaithersburg, MD), and pH regulator RT in water DEPC-tx. This was incubated at 65 ° C for 10 minutes and then placed at 4 ° C. To these, 10 mM of DTT (Gibco BRL), 0.5 mM of each of dATP, dCTP, dTTP, dTTP, (Gibco BRL), and 500 U of MMLV-RT (Gibco BRL) were added to obtain a volume of final reaction of 50 μl. The samples were incubated at 37 ° C for 1 hour, then heated at 95 ° C for 5 minutes. For amplification through PCR, 5 μ of each cDNA was then added to the MicroAmp reaction tubes (Perkin Elmer, Norwalk, CT) containing a PCR reaction regulator, 0.2 mM each of dATP, dCTP , dTTP, dTTP, 1.25 U of AmpliTaq DNA polymerase (Perkin Elmer), MGCI2, concentrations determined to be optimal for each initiator pair (final concentrations of 1.5-6.0 mM), and 0.5 mM of each of the appropriate pairs in the final volume of 50 μl. The primer pairs in this study included β-actin, TNF-α, IL-4, IFN ?, South San Francisco, CA) and IL10 (Clonatech, Palo Alto, CA). Β-actin served as a standard for the comparison of relative mRNA expression between samples, as well as a control for RT and PCR reactions. The PCR samples were amplified using a GeneAmp System 9600 Perkin Elmer thermocycler). Each sample was denatured at 94 ° C for 37 sec., Reinforced at 55 ° C for 45 sec., And extended at 72 ° C for 60 sec., For 39 cycles, followed by an extension of 10 minutes at 72 ° C. C. PCR products and size markers (Novagen, Madison, Wl) were separated on 0.2% agarose gel (FMC BioProudcts, Rockland, ME). The gel was stained with ethidium bromide, visualized and photographed under UV illumination. Electrophoresis of the PCR products revealed a band corresponding to the fragment size predicted for each group of primers. Non-reverse transcribed RNA was subjected to amplification via PCR as a control for contamination of genomic DNA. It was found that cell suspensions dissociated with enzyme, cryopreserved from melanoma tissues are not suitable for RNA analysis. These samples usually expressed mRNA for all tested cytokines, probably a result of activation through the dissociation procedure.

Histology and RT-PCR In-Situ - routine H & E staining of representative specimens was performed through the Department of Pahology. In situ RT-PCR was performed on paraffin sections, which were permeabilized using proteinase K, treated with reverse transcriptase and the resulting IL10 DNA was amplified using the same primers, as previously observed using the methodology according to Bagasra, OR ., and others, J. Immunol. Meth., 1993, 158: 131-145.

Cytokine mRNA in Inflammed Biopsies, Post-Vaccine - Since metastatic melanoma is characterized by a shortage of lymphocytic infiltration (Eider et al., "The surgical pathology of cutaneous malignant melanoma." In WH Clark, Jr., and others (Eds. ) Human Malignant Melanoma, page 100, New York; Gruñe and Stratton 1979), the administration of DNP vaccines induces the infiltration of T cells into metastatic masses (Berd et al., 1991 supra.) Eight (8) metastases were studied. subcutaneous (of 4 patients) who developed inflammation after treatment with the vaccine, and were compared with 3 subcutaneous metastases excised before the vaccine and 4 metastases after the vaccine that failed to develop an inflammatory response. The inflamed biopsies, subsequent to the vaccine, contained mRNA for IFN? (5/8), IL4 (4/8) or both (3/8). In contrast, neither the IFNα mRNA neither the IL4 mRNA was detected in the 7 control specimens. All but one of these 15 tissues expressed mRNA for IL10. Figure 14 shows the expression of cytokine mRNA for a subsequent biopsy of the infiltrated T-cell vaccine, representative together with the corresponding histology.

Lymph node metastasis - A group of lymph node metastasis biopsies was also studied. Histologically, these lesions are characterized by an abundance of lymphocytes (Figure 15B) that are thought to be lymphoid node lymphocyte residues infiltrated in the tumor (Cardi, et al., Cancer Res., 1989, 49: 6562-6565) . Of the 10 lymph node biopsies studied, only one expressed mRNA for IFNα; this specimen, and an additional specimen, contained mRNA for IL4. However, all 10 specimens contained mRNA for IL10. Figure 15 shows the expression of cytokine mRNA for a representative lymph node metastasis together with the corresponding histology.

Production of IL10 through Melanoma Metastasis and Cell Lines - As indicated above, IL10 mRNA expression was seen in 24/25 melanoma metastases (Figure 16). Since it was dependent on the expression of IFN? or of IL4 and did not correlate with T cell infiltration, melanoma cells, instead of lymphocytes, may be the source of IL10. Two aspects were used to test this hypothesis. First, the cell lines derived from two of the metastatic tumors described above were examined. As illustrated in Figure 17A, both the cell lines and the tissue from which they were derived expressed IL10 mRNA. Both cell lines produced IL10, as determined by a culture analysis of supernatants after 72 hours of incubation (IL10 concentrations: 760 pg / ml and 10 pg / ml respectively). Second, the expression of IL10 mRNA in a tissue section of a melanoma metastasis was studied using RT-PCR in situ. As can be seen in Figure 17B, IL10 mRNA is associated in the myeloma cells and not in elements without tumor.

TNF mRNA is expressed in melanoma metastasis - mRNA for TNF in human colon carcinoma biopsies using in situ hybridization (Naylos, MS, et al., Cancer Res., 1990 50: 4436-4440), and that resistance to TNF is associated with tumor growth in vivo (Lattime, E.C. and Stitman, O., J. Immuno., 1989, 143: 4317-4323). TNF mRNA was detected in 6/23 melanoma specimens. There was an association with the inflammation induced after the vaccine: 4/7 biopsies after the vaccine infiltrated in T cells were positive against 2/16 specimens before the vaccine or after the non-infiltrated vaccine.

EXAMPLE 8 This Example describes dinitrophenyl-modified tumor peptides for cancer immunotherapy. Epstein Barr virus (EBV) was added to B lymphoblastoid cells in culture. The B lymphoblastoid cells were transformed to a B-cell tumor of the patient's own lymphocytes. Melanoma was cultured from a metastasis in RPMI 1640 + 10% fetal calf serum or 10% human serum drained. The non-adherent cells were washed with a R PMI med. When the cells were confluent, they were separated with 0.1% EDTA and formed into passages in two flasks. This procedure continued for approximately 10 approximately 30 passages. To test the production for gamma-interferon through T cells, lymphocytes were obtained from the blood of a patient. Approximately 1,000,000 lymphocytes were mixed with autologous melanoma cells modified with DN P to stimulate T cells. Every seven days, 100 U / ml interleukin-2 was added. The T cells were expanded through passage as described above. The T cells were then further stimulated through autologous melanoma cells modified with DNP. A population enriched for T cells resulted, which were responsible for autologous melanoma cells modified with DNP. Stimulation was determined through the production amount of interferon gamma by T cells. Generally, gamma production interferon at more than 15 picograms / ml, was considered. Small peptides were extracted from 4 cell types, all generated from a single patient: 19 lymphoblastoid B cells, 2) B lymphoblastoid cells modified with (DNP), 3) cultured melanoma cells, 4) cultured melanoma cells modified with DNP. The cells were suspended in 0.1% trifluoroacetic acid, sound was applied and centrifuged at 100.00 xg for 90 minutes. The material in the supernatant was removed with a molecular weight of >; 10.00 through a Centricon 10 filter. The remaining material was separated on a reverse phase HPLC column. The individual fractions were collected, dried, resuspended in a culture medium, and added to autologous B lymphoblastoid cells, which were joined and presented to the peptides. These peptide-pulsed B cells were tested for their ability to stimulate a T lymphocyte cell line that was specifically sensitized to DNP-modified melanoma cells.

Initially, fractions of 50 HPLC (10 μl of each sample) were emptied into five groups of ten fractions of each. As shown in Figure 13, only the peptides derived from melanoma cells modified with DNP (DNP-MEL) or B cells modified with DNP (DNP-LY), were stimulants, and only deposit # 2 was positive. Each of the individual fractions of reservoir # 2 were analyzed by performing the stimulation test of the T cell with each fraction in reservoir 2; Activity was found only in fractions # 17 and # 18, and the DNP-MEL peptide stimulated production of interferon gamma twice more than DNP-LY. these results indicate that an individual HPLC fraction of low molecular weight peptide preparation contains the peptide or peptides responsible for the stimulation of sensitized T cells to DNP-modified melanoma cells.

EXAMPLE 9 This example will determine the inhibition of peptide stimulation through the anti-DNP antibody. The experiment will be identical to that described for Example 8 with one exception. After adding the peptide to the B lymphoblastoid cells, and just before adding the T response cells, variable concentrations (1-100 μg / ml) of the anti-DNP antibody will be added to different samples. The anti-DNP antibody can be obtained from ATCC, hybridoma # CRL-1968, or a similar antibody. If the stimulation is caused by peptides modified with DPN, the antibody will inhibit it. Fractions 17 and 18 are expected to be inhibited by the antibody.

EXAMPLE 10 It is expected that Example 10 determines whether the responding T cells are CD4 + or CD8 +. The experiment will be identical to that described for Example 8, with one exception. Responding T cells will be divided into subgroups before being added to the B lymphoblastoid cells pulsed by the peptide. This was achieved by mixing the T cells with magnetic beads coated with either anti-CD4 or anti-CD8 antibodies (commercially obtained from Immunotech, Inc., Westbrook, Maine). Then the pearls, and the cells that joined them, were removed with a magnet. The unbound cells were washed in the tissue culture medium (RPMI + 10% empty human serum), counted, and added to the microtitre cavities to measure stimulation.

EXAMPLE 11 Example 11 describes dinitrophenyl modified tumor membranes for cancer immunotherapy.

Melanoma cell membranes cultured from a patient were prepared according to the method of Heike et al., J. Immunol, 1994, 15: 165-174, the disclosure of which is incorporated herein by reference in its entirety. The melanoma cells were conjugated to dinitrophenyl (DNP) according to the methods of Miller and Claman, J. Immunology, 1976, the disclosure of which is incorporated herein by reference in its entirety. The cells were suspended in 5 volumes of 30 mM of sodium bicarbonate buffer with 1 mM of phenyl-methylsulfonyl fluoride and divided into a glass homogenizer. The residual intact cells were removed by centrifugation at 1000 g. Then, the membranes were pelleted by centrifugation at 100,000 g for 90 minutes. The membranes were suspended in 8% sucrose and frozen at -80 ° C until needed. Melanoma cells were similarly prepared for conjugation to dinitrophenyl. These melanoma cell membranes modified with DNP were tested for their ability to stimulate autologous T lymphocytes that have been sensitized to intact melanoma cells modified with DNP. This is done by incubating approximately 100,000 T lymphocytes / cavity with approximately 10,000 to approximately 100,000 membranes / cavity modified with equivalent cell DNF, and measuring gamma interferon production (greater than 15 picograms). This procedure was repeated by incubating the T lymphocytes with melanoma cells modified with DNP. The results revealed that the intact melanoma cells and the membranes derived from them were equally effective in stimulating T cells. This experiment, which was repeated several times (using the same patient sample) with similar results, indicates that the membranes of melanoma modified with DNP can substitute for intact melanoma cells modified with DNP to induce a T cell response.

EXAMPLE 12 Example 12 will determine whether the addition of autologous monocytes or dendritic cells increases the T cell response to tumor membranes. The autologous monocytes will be isolated as follows. Peripheral blood lymphocytes were separated from the peripheral blood through gradient centrifugation according to the methods of Boyum, A. Scand. J. CU. Lab. Invest., 21, 1968, Suppl. 7: 77-89, the description of which is hereby incorporated by reference in its entirety. They were suspended in a tissue culture medium (RPMI-1640 + 10% human serum drained) and added to plastic microtiter cavities for about two hours in order for the monocytes to adhere. Then, the non-adhered cells were washed with the culture medium. Various concentrations of GM-CSF (granulocyte macrophage colony stimulation factor, commercially obtained from Immunex, Seattle, WA) were added to stimulate the growth of monocytes. After approximately 2-3 weeks, monocytes, now considered macrophages, were removed from the plastic with 0.1% EDTA and added in graduated numbers (about 100 to 10,000 / well) to fresh microtiter cavities. The graduated numbers of membranes, prepared from autologous tumor cells modified with DN P (quantified as cell equivalents), will be added to the monocoated adherent macrophage layer. After about 6 to about 24, autologous T cells specific for DNA were added and incubated for an additional 24 hours. Then, the supernatants will be collected and tested for the production of cytokines such as, and not limited to, interferon range, I L2, tumor necrosis factor l; or for the proliferation or stimulation of T cells such as by 1 2 SI U DR, 3 H thymidine, or with dyes such as MTT. For example, 125I U DR will be added and the cells will be collected in an automatic harvesting device to test for T cell proliferation. The controls consisted of unstimulated T cells, and T cells stimulated with membranes in the absence of macrophages. The ability of autologous dendritic cells to improve the response to membranes will be tested in the same way. The dendritic cells were isolated from the peripheral blood mononuclear cells and grown in a tissue culture according to the method of O'Doherty, U. , and others. J. Exp. Med., 1993, 178: 10678-1078, the disclosure of which is hereby incorporated by reference in its entirety.

EXAMPLE 13 Example 13 is expected to determine whether patients who received the DNP-modified melanoma vaccine manifest delayed type hypersensitivity (DTH) to melanoma membranes modified with DNP. The subjects of the study are patients who received repeated doses of melanoma cell vaccine modified with DN P. The membranes were separated from melanoma cells modified with autologous DNP as described above. The graduated numbers of membranes (approximately 100 to approximately 10, 000 cell equivalents) were washed in PBS, resuspended in PBS, and injected intradermally in the forearm. The DTH was measured approximately 48 hours longer as the cutaneous hardening diameter. Controls consisted of autologous non-conjugated melanoma cell membranes and membranes prepared from autologous blood lymphocytes.

EXAMPLE 14 It is expected that Example 14 determines whether macrophages or dendritic cells process melanoma membranes and present them in an immunogenic form to T cells syngeneic to those of macrophages. The procedure will be similar to that described in Example 12, with the exception that the stimulation membranes were prepared from melanoma cells conjugated with allogenic DN P. The hypothesis is that the macrophages or dendritic cells of patient A can process in vitro the membranes obtained from the melanoma cells of patient B. This results in the stimulation of the T cells of patient A. This experiment may lead to a strategy for allogeneic immunization. Membranes modified with D N P prepared from a cell line of allogenic melanoma, or deposition of allogenic cell lines, can be processed through macrophages or dendritic cells, in vitro. Those cells could be used for immunization. The description of each patent, patent application and publication cited or described in this document is hereby incorporated by reference in its entirety. Various modifications of the invention in addition to that shown and described herein will be apparent to those skilled in the art from the foregoing description. Said modifications are also intended to fall within the scope of the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Berd, David; Eisenlohr, Lawrence; and Lattime, Edmund (ii) TITLE OF THE INVENTION: TUMOR CELL EXTRACT MODIFIED WITH HAPTEN AND METHODS FOR THE TREATMENT OR CLASSIFICATION OF CANCER (iii) SEQUENCE NUMBER: 10 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Woodcock Washburn Kurtz Mackiewicz & Norris (B) STREET: One Liberty Place - 46 h Floor (C) CITY: Philadelphia (D) STATE: PA (E) COUNTRY: USA (F) POSTAL CODE: 19103 (v) COMPUTER LEGIBLE FORM: (A) TYPE MEDIUM: disk, 3.5 in., 1.44 Mb STORAGE (B) COMPUTER: IBM PS / 2 (C) OPERATING SYSTEM: PC-DOS (D) SOFTWARE: WORDPERFECT 5.1 (vi) APPLICATION DATA: (A) APPLICATION NUMBER: unknown (B) SUBMISSION DATE: June 7, 1995 (C) CLASSIFICATION: Unknown (vii) DATA FROM THE PREVIOUS APPLICATION: (A) NO. APPLICATION: 08 / 203,004 (B) SUBMISSION DATE: February 28, 1994 (viii) AGENT INFORMATION: (A) NAME: Lori Y. Beardell (B) NO. REGISTRATION: 34,293 (C) REFERENCE / NO. PERMITTED: TJU-1582 (ix) TELECOMMUNICATION INFORMATION (A) PHONE: (215) 568-3100 (B) TELEFAX: (215) 569-3439 (2) INFORMATION FOR SEC ID NO: 1: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATGGATGATG ATACGCCGC G 21 (3) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 33 base pairs (B) TYPE: acid nucleic (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No (iv) ANTI-SENSE: yes (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ACTAGAAGCAT TTGCGGTGGA CGATGGAGGG GCC 13 (4) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: i ndividual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATGAAATATA CAAGTTATAT C 21 (5) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: acids nucleic (iii) HYPOTHETIC: No (iv) ANTI-SENSE: Yes (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 4 TTACTGGGAT GCTCTTCGAC CTCGAAACAG CAT 33 (6) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (i) ) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATGGGTCTCA CCTCCCAACT G 21 (7) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No (iv) ANTI-SENSE: Yes (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TCAGCTCGAA CACTTTGAAT ATTTCTCTCT CAT 33 (8) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (i) ) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AAGCTGAGAA CCAAGACCCA GACATCAAGG CG 32 (9) INFORMATION FOR SEQ ID NO: 8: (i) ) SEQUENCE CHARACTERISTICS (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No ( iv) ANTI-SENSE: Yes (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 8: AGCTATCCCA GAGCCCCAGA TCCGATTTTG G 31 (10) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 21 base pairs ( B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No (iv) ANT ISENANCE: No (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATGAGCACTG AAAGCATGAT C 21 (11) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acids (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TCACAGGGCA ATGATCCCAA AGT 23

Claims (2)

1. CLAIMS 1 .- A composition comprising a therapeutically effective amount of a hapten-modified tumor cell extract, said composition stimulates the T-cell lymphocytes and is useful for the treatment of cancer.
2. 2. The composition according to claim 1, wherein said tumor cell extract is selected from the group consisting of a hapten-modified cancer cell membrane, a low molecular weight peptide of a hapten-modified cancer cell, a antigen presenting a cell with a low molecular weight peptide of a hapten-modified cancer cell attached thereto, and an antigen presenting a cell with a hapten-modified cancer cell membrane attached thereto. 3 - The composition according to claim 1, wherein said tumor cell extract is a low molecular weight peptide of a cancer cell modified with dinitrophenyl. 4. The composition according to claim 1, wherein said extract is a membrane of a cancer cell modified with dinitrophenyl. 5. The composition according to claim 1, wherein said extract is an antigen that has a cell with a low molecular weight peptide of a cancer cell modified with hapten, bound thereto. 6. The composition according to claim 1, wherein said tumor cell extract is selected from the group consisting of an autologous cell and an allogeneic cell. 7. The composition according to claim 1, wherein the tumor is selected from the group consisting of melanoma, breast, lung, colon, breast, kidney and prostate. 8. The composition according to claim 1, wherein the melanoma tumor. 9. The composition according to claim 2, wherein the hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl and N-iodoacetyl-N '- (5-sulfonic-1-naphthyl) ethylenediamine. 10. The composition according to claim 9, wherein said hapten is dinitrophenyl. 11. The composition according to claim 1, wherein it also comprises an immunological aid. 12. The composition according to claim 11, wherein said immunological aid is Bacillus Calmatte-Guerin. 13. A composition comprising a therapeutically effective amount of a cancer cell membrane modified with dinitrophenyl, said composition stimulates T-cell lymphocytes and is useful for the treatment of cancer. 14. A composition comprising a therapeutically effective amount of a low molecular weight peptide of a cancer cell modified with dinitrophenyl, said composition stimulates T-cell lymphocytes and is useful for the treatment of cancer. 15. A method for the treatment of cancer comprising administering to a patient a therapeutically effective amount of cyclophosphamide; administering a therapeutically effective amount of a hapten-modified tumor cell extract, wherein the extract stimulates T-cell lymphocytes. 16. The method according to claim 1, wherein the tumor is selected from the group consisting of melanoma, lung, colon, breast, kidney and prostate. 17. - The method according to claim 1, useful for the treatment of cancer selected from the group consisting of melanoma, lung cancer, colon cancer, breast cancer, kidney cancer and prostate cancer. 18. The method according to claim 15, wherein the tumor cell extract is selected from the group consisting of a hapten-modified cancer cell membrane and a low molecular weight peptide of a hapten-modified cancer cell. The method according to claim 18, wherein the hapten is selected from the group consisting of di nitrophenol, tri-nitrophenyl and N-iodoacetyl-N '- (5-sulfonic-1-naphthyl) ethylenediamine. 20. - The method according to claim 18, wherein said hapten is dinitrophenyl. twenty-one . The method according to claim 1, wherein the tumor cell extract is a low molecular weight peptide of a cancer cell modified with dinitrophenyl. 22. The method according to claim 15, wherein the tumor cell extract is a membrane of a cancer cell modified with dinitrophenyl. 23. The method according to claim 15, wherein the tumor cell extract is selected from the group consisting of an autologous cell and an allogeneic cell. 24. The method according to claim 1, wherein said therapeutically effective amount of cyclophosphamide comprises administering a dose of approximately 300 mg / M2 of cyclophosphamide prior to administration of the composition. 25. The method according to claim 15, wherein the composition is mixed with an immunological aid prior to administration. 26. - The method according to claim 25, wherein the unological auxiliary is Bacillus Calmette-Guerin. 27. The method according to claim 15, further comprising sensitizing the patient with a therapeutically effective amount of 1-fluoro-2,4-dinitrobenzene before administering cyclophosphamide. 28 - A method for the treatment of human cancer which comprises administering to a patient a therapeutically effective amount of cyclophosphamide; administering a therapeutically effective amount of a tumor extract, which stimulates T cell lymphocytes, said composition is mixed with an immunological aid; and administering a therapeutically effective amount of a cytokine selected from the group consisting of interleukin-12, interluecin-2, and interleukin-13. The method according to claim 28, wherein the tumor cell extract is selected from the group which consists of a hapten-modified cancer cell membrane and a low molecular weight peptide of a hapten-modified cancer cell. 30. The method according to claim 29, wherein the hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl and N-iodoacetyl-N '- (5-sulfonic-1-naphthyl) ethylenediamine. 31. The method according to claim 29, wherein said hapten is dinitrophenyl. 32. The method according to claim 28, wherein the tumor cell extract is a low molecular weight peptide of a cancer cell modified with dinitrophenyl. 33. - The method according to claim 28, wherein the tumor cell extract is a membrane of a cancer cell modified with dinitrophenyl. 34 - The method according to claim 28, wherein the tumor cell extract is selected from the group consisting of an autologous cell and an allogeneic cell. The method according to claim 28, wherein said therapeutically effective amount of cyclophosphamide comprises administering a dose of approximately 300 mg / M of cyclophosphamide prior to administration of the composition. 36. The method according to claim 28, wherein the composition is mixed with an immunological aid prior to administration. 37. The method according to claim 28, wherein the immunological aid is Bacillus Calmette-Guerin. 38. The method according to claim 28, wherein the tumor is selected from the group consisting of melanoma, lung, colon, breast, kidney and prostate. 39.- The method according to claim 28, useful for the treatment of cancer selected from the group consisting of melanoma, lung cancer, colon cancer, breast cancer, kidney cancer and prostate cancer. 40. The method according to claim 28, further comprising sensitizing the patient with 1-fluoro-2,4-dinitrobenzene before administering cyclophosphamide. 41.- A method for the treatment of cancer comprising administering to a patient a therapeutically effective amount of cyclophosphamide; administering an effective amount of a tumor cell extract that stimulates T cell lymphocytes, said composition mixed with an immunological aid; administering a therapeutically effective amount of an irradiated, non-haptenized composition comprising a tumor cell extract. 42 - A method for the classification of cytokine production through tumors to determine the efficacy of an autologous, irradiated hapten-conjugated cell composition for alleviating cancer in a patient suspected of having cancer, said method comprises: administering said conjugated composition with hapten to the patient; obtaining a sample comprising nucleic acids from a tissue sample of the patient; amplifying the nucleic acids for a cytokine or amplifying a signal generated through the hybridization of a specific probe to a cytokine-specific nucleic acid in the tissue sample; and detecting the presence of the nucleic acids or the amplified signal, wherein the presence of the nucleic acids or the amplified signal of the tissue sample of a patient indicates cytokine production and determines the efficacy of said composition conjugated with hapten. 43. The method according to claim 42, wherein the hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl and N-iodoacetyl-N '- (5-sulfonic-1-naphthyl) ethylenediamine. 44. The method according to claim 42, wherein said tissue sample from the patient is a subcutaneous inflammation. Four. Five - . The method according to claim 42, wherein the amplification step comprises hybridization to a cytokine-specific nucleic acid of at least one oligonucleotide, which is complementary to a specific cytokine sequence. 46.- The method according to claim 42, wherein the nucleic acids specific for a cytokine comprise gamma interferon nucleic acids, tumor necrosis factor, interleukin 2, interleukin 12, and interleukin 13. 47.- The method according to Claim 42, wherein said amplification step comprises hybridization to a cytokine-specific nucleic acid with a pair of initiators, wherein an initiator within the pair is complementary to the specific cytokine sequence. 48. The method according to claim 42, wherein the amplification step comprises performing a method selected from the group consisting of polymerase chain reaction, ligase chain reaction, repair chain reaction, probe reaction cyclic, amplification based on nucleic acid sequence, chain shift amplification, and Qβ replicase replicase. 49. The method according to claim 42, wherein the step of amplification comprises carrying out a polymerase chain reaction, wherein said polymerase chain reaction comprises a first initiator and a second initiator, wherein the first primer is selected from the group consisting of SEQUENCE ID NOS: 1, 3, 5, 7 and 9, and said second primer is selected from the group consisting of SECU ENC IA ID NOS: 2, 4, 6, 8, and 1 0. 50. - The method according to claim 42, wherein said amplification step comprises performing the polymerase chain reaction, wherein said polymerase chain reaction comprises a first initiator and a second initiator, wherein the first initiator is SEQ. NO: 3 and the second initiator is SEQ ID NO: 4. 51.- The method according to claim 42, wherein said amplification step comprises performing the polymerase chain reaction, wherein said polymerase chain reaction comprises a pair of primers, wherein an initiator of said pair is complementary to the specific cytokine sequence. 52. The method according to claim 42, wherein the primer that is complementary to a specific cytokine sequence is selected from the group consisting of SEQUENCE ID NOS: 1 to 10. 53 - The method according to claim 47 , wherein the primer that is complementary to a specific cytokine sequence is selected from the group consisting of SEQ ID NOS: 1 to 10. The method according to claim 47, wherein the primer is complementary to a The cytokine specific sequence is selected from the group consisting of SEQ ID NO: 3. The method according to claim 42, wherein said tissue sample from the patient is a tissue selected from the group consisting of a tumor, saliva , sputum, mucus, bone marrow, serum, blood, urine, lymph and a tear. 56.- A diagnostic device for the classification of the effectiveness of an irradiated, autologous hapten-conjugated cell composition comprising, in one or more vessels, a pair of primers, wherein one of the primers within the pair is complementary to a cytokine-specific sequence, wherein said initiator is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO : 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and means for visualizing amplified DNA; said equipment useful for determining the effectiveness of said composition. 57. The diagnostic equipment according to claim 56, wherein said means for visualizing amplified DNA is selected from the group consisting of strain of ethidium bromide, 32P and biotin. 58. The method according to claim 42, wherein said tissue sample of the patient is a melanoma tissue. 59.- A method for the classification of cytokine production through tumors to determine the efficacy of an autologous irradiated hapten-conjugated cell composition in a patient suspected of having cancer, said method comprises: obtaining a sample of an RNA from a patient tumor sample; reverse transcribe said RNA to DNA; amplifying said DNA with the polymerase chain reaction using a pair of primers, which are complementary to separate regions of a cytokine sequence; and detecting the presence or absence of amplified DNA, wherein the presence of amplified DNA indicates cytokine production and determines the efficacy of said hapten-conjugated composition. The method according to claim 59, wherein the polymerase chain reaction is a polymerase chain reaction in situ. 61 - A composition comprising a human tumor cell modified with hapten. 62. A composition according to claim 61, wherein said tumor is selected from the group consisting of melanoma, lung, colon, breast, kidney and prostate. 63.- A composition according to claim 61, wherein the hapten is selected from the group consisting of d-nitrophenyl, trinitrophenyl and N-iodoacetyl-N '- (5-sulfonic-1-naphthyl) ethylenediamine. 64. - A composition according to claim 61, further comprising an auxiliary. 65.- A composition according to claim 64, wherein said auxiliary is selected from the group consisting of Bacillus Calmette-Guerin, cytokines and QS-21. 66. A composition according to claim 61, further comprising a vehicle. 67. - A composition according to claim 66, wherein the vehicle is selected from the group consisting of saline and culture medium. 68.- A composition comprising a human tumor cell modified with hapten, said hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl and N-iodoacetyl-N '- (5-sulfonic-1-naphthyl) ethylenediamine and an auxiliary selected from the group consists of Bacillus Calmette-Guerin, cytokines and QS-21. 69.- A composition comprising a tumor cell of human melanoma modified with hapten. 70.- A composition comprising a human lung tumor cell modified with hapten. 71 .- A composition comprising a human colon tumor cell modified with hapten. 72.- A composition comprising a human breast tumor cell modified with hapten. 73.- A composition comprising a tumor cell of human kidney modified with hapten. 74.- A composition comprising a human prostate tumor cell modified with hapten. 75.- A composition according to claims 69-74, wherein said hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl and N-iodoacetyl-N '- (5-sulfonic-1-naphthyl) ethylenediamine. 76.- A composition according to claim 75, wherein it also comprises a vehicle. 77. A composition according to claim 76, wherein said vehicle is selected from the group consisting of saline and a culture medium. 78. A composition according to claim 75, further comprising an auxiliary selected from the group consisting of Bacillus Calmette-Guerin, cytokines and QS-21. 79.- A composition according to claim 78, further comprising a vehicle. 80.- A composition according to claim 79, wherein said vehicle is selected from the group consisting of saline and culture medium.