Title:  Composition comprising tumor cells and extracts and method of using thereof

United States Patent:  6,458,369

Issued:  October 1, 2002

Inventors:  Berd; David (Wyncote, PA)

Assignee:  Thomas Jefferson University (Philadelphia, PA)

Appl. No.:  304859

Filed:  May 4, 1999

The present invention is directed to compositions containing hapten-modified tumor cells and extracts and methods of treating cancer by administering a therapeutically effective amount of a composition containing a tumor cell or tumor cell extract to a subject in need of such treatment. The tumor cells and extracts of the invention and compositions thereof are capable of eliciting T lymphocytes that have a property of infiltrating a mammalian tumor, eliciting an inflammatory immune response to a mammalian tumor, eliciting a delayed-type hypersensitivity response to a mammalian tumor and/or stimulating T lymphocytes in vitro. The invention also relates to an effective vaccination schedule useful for inducing an antitumor response in a mammalian patient suffering from cancer by inducing at least one of the following: tumor necrosis, tumor regression, tumor inflammation, tumor infiltration by activated T lymphocytes, delayed-type hypersensitivity response, and prolongation of patient survival.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to cancer immunotherapy. A tumor composition and methods of treating cancer are included in the scope of the invention. The invention further relates to a method of inducing an antitumor response according to a weekly vaccination schedule. For purposes of the present invention an antitumor response is at least one of the following: tumor necrosis, tumor regression, tumor inflammation, tumor infiltration by activated T lymphocytes, delayed-type hypersensitivity response, and prolongation of patient survival. The tumor cells and extracts of the invention and compositions thereof are capable of eliciting T lymphocytes that have a property of infiltrating a mammalian tumor, eliciting an inflammatory immune response to a mammalian tumor, eliciting a delayed-type hypersensitivity response to a mammalian tumor and/or stimulating T lymphocytes in vitro.

An anti-tumor response resulting from the treatment according to the present invention may be a partial or a complete regression of the metastatic tumor or a stable disease. A "complete" regression indicates about 100% regression for a period of at least one month, more preferably for a period of at least three months. A "partial" regression indicates more than about 50% regression for a period of at least one month, more preferably for a period of at least three months. A "stable" disease indicates a condition in which there is no significant growth of the tumor after the vaccine treatment. Another anti-tumor response that may be observed upon following the treatment of the invention is prolongation of survival.

Any malignant tumor may be treated according to the present invention including metastatic and primary cancers and solid and non-solid tumors. Solid tumor include carcinomas, and non-solid tumors include hematologic malignancies. Carcinomas include and are not limited to adenocarcinomas and epithelial carcinomas. Hematologic malignancies include leukemias, lymphomas, and multiple myelomas. The following are non-limiting examples of the cancers treatable with isolated modified tumor cell membranes according to the methods of the present invention: ovarian, including advanced ovarian, leukemia, including and not limited to acute myelogenous leukemia, colon, including colon metastasized to liver, rectal, colorectal, melanoma, breast, lung, kidney, and prostate cancers. The ovarian cancers may be adenocarcinomas or epithelial carcinomas. Colon and prostate cancers are adenocarcinomas. Leukemias may originate from myeloid bone marrow or lymph nodes. Leukemias may be acute, exhibited by maturation arrest at a primitive stage of development, and chronic, exhibited by excess accrual of mature lymphoid or myeloid cells. Stage I, II, III, or IV cancer may be treated according to the present invention, preferably stages III and IV, even more preferably stage III. Any mammal, preferably a human, may be treated according to the present invention.

The compositions of the present invention are prepared from a tumor cell or tumor cell extract. A tumor cell may 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 ultimately lead to cervical cancer, and dysplastic nevi which are abnormal skin cells which lead to melanoma. The tumor cells and extracts preferably originate from the type of cancer which is to be treated. For example, a melanoma cell or cell extract is used to treat melanoma type cancer. The tumor cells and extracts may be, and are not limited to, autologous and allogenic cells dissociated from biopsy specimens or tissue culture, as well as stem cells and extracts from these sources. In one preferred embodiment, the cells and extracts are autologous. However, any non-allogeneic cell, including tumor cells produced in culture from autologous cells isolated from the patient's tumor, may be used. Tumor cells need not be completely (i.e., 100%) genetically identical to either the tumor cell or the non-tumor, somatic cell of the treated patient. Genetic identity of the MHC molecules between the tumor cell and the patient is generally sufficient. Additionally, there may be genetic identity between a particular antigen on the melanoma cell and an antigen present on the patient's tumor cells. Genetic identity may be determined according to the methods known in the art. For purposes of the present invention, a tumor cell that has been genetically altered (using for example recombinant DNA technology) to become genetically identical with respect to, for example, the particular MHC molecules of the patient and/or the particular antigen on the patient's cancer cells is within the meaning of "non-allogeneic" and within the scope of the present invention. Such cells may also be referred to as "MHC-identical" or "MHC-compatible."

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. Extracts may also be first isolated from tumor cells and then hapten modified.

For purposes of the present invention, peptides are compounds of two or more amino acids including proteins. Peptides will preferably be of low molecular weight, of about 1,000 kD to about 10,000 kD, more preferably about 1,000 to about 5,000, which are isolated from a haptenized tumor cell and which stimulate T cell lymphocytes to produce gamma interferon. T cells are lymphocytes which mediate two types of immunologic functions, effector and regulatory, secrete proteins (lymphokines), and kill other cells (cytotoxicity). Effector functions include reactivity such as delayed hypersensitivity, allograft rejection, tumor immunity, and graft-versus-host reactivity. Lymphokine production and cytotoxicity are demonstrated by T cell effector functions. Regulatory functions of T cells are represented by their ability to amplify cell-mediated cytotoxicity by other T cells and immunoglobulin production by B cells. The regulatory functions also require production of lymphokines. T cells produce gamma interferon (IFN.gamma.) in response to an inducing stimulus including and not limited to mitogens, antigens, or lectins. The peptide may preferably be about 8 to about 20 amino acids, in addition the peptide is preferably haptenized. Peptides may be isolated front the cell surface, cell interior, or any combination of the two locations. The extract may be particular to type of cancer cell (versus normal cell). The peptide of the present invention includes and is not limited to a peptide which binds to the major histocompatibility complex, a cell surface-associated protein, a protein encoded by cancer oncogenes or mutated anti-oncogenes.

The cancer cell membrane of the present invention may be all or part of a membrane from 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 may be isolated then haptenized, alternatively, a cancer cell may be haptenized and the membrane subsequently isolated therefrom.

The cell extracts are able to stimulate T cells. Stimulation for purposes of the present invention refers to proliferation of T cells as well as production of cytokines by T cells, in response to the cell extract. Membranes and proteins isolated from hapten modified tumor cells and proteins each independently have the ability to stimulate T cells. Proliferation of T cells may be observed by uptake by T cells of modified nucleic acids, such as and not limited to 3H thymidine, ¹²⁵ IUDR (iododeoxyuridine); and dyes such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) which stains live cells. In addition, production of cytokines such as and not limited to IFN.gamma., tumor necrosis factor (TNF), and IL-2. Production of cytokines is preferably in an amount of greater than 15 picograms/ml, more preferably about 20 to about 30 picograms/ml, even more preferably about 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 may be live cells. In one preferred embodiment, the tumor cells and extracts of the present invention are incapable of growing in the body of the patient after injection. Method of preventing cells from growing are known to those of skill in the art. For example, tumor cells may be irradiated prior to use. In one embodiment, tumor cells or extracts are irradiated at about 2500 cGy to prevent the cells from growing after injection.

The compositions of the invention may be employed in the method of the invention singly or in combination with other compounds, including and not limited to other compositions of the invention. Accordingly, cancer cells and cancer cell extracts may be used alone or coadministered. For purposes of the present invention, co-administration includes administration together and consecutively. In addition, the cancer cell membrane may be co-administered with the peptide. Further, the cancer cells and/or extracts may be co-administered with other compounds including and not limited to cytokines such as interleukin2, 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 treatments including and not limited to chemotherapy, radiation, antibodies, oligonucleotide sequences, and antisense oligonucleotide sequences.

The compositions of the invention may be administered in a mixture with a pharmaceutically-acceptable carrier, selected with regard to the intended route of administration and the standard pharmaceutical practice. Dosages may be set with regard to weight, and clinical condition of the patient. The proportional ratio of active ingredient to carrier naturally depend on the chemical nature, solubility, and stability of the compositions, as well as the dosage contemplated. Amounts of the tumor cells and extracts of the invention to be used depend on such factors as the affinity of the compound for cancerous cells, the amount of cancerous cells present and the solubility of the composition.

The composition of the present invention may be mixed with an immunological adjuvant and/or a pharmaceutically acceptable carrier. Any known aqueous vehicle useful in drug delivery, such as and not limited to saline, may be used in accordance with the present invention as a carrier. In addition, any adjuvant known to skilled artisans may be useful in the delivery of the present invention. The adjuvant has the property of augmenting an immune response to the tumor cell preparations of the present invention. Representative examples of adjuvants are BCG, or the synthetic adjuvant, QS-21 comprising a homogeneous saponin purified from the bark of Quillaja saponaria, Corynebacterium parvum (McCune et al., Cancer 1979 43:1619), saponins in general, detoxified endotoxin and cytokines such as interleukin-2, interleukin-4, gamma interferon (IFN-.gamma.), interleukin-12, interleukin-15, GM-CSF and combinations thereof.

In the case where the cells and cell extracts are irradiated and haptenized, the cells may be conjugated to a hapten and then irradiated. Alternatively, the cells may be irradiated then conjugated to a hapten. In either case, the extracts are subsequently purified and then may also be irradiated and/or haptenized. To irradiate and haptenize the extracts, either method may be performed first, followed by the other method.

Alternatively, the tumor cells or tumor cell extracts may be added to antigen presenting cells. The cancer cell extract may be used to treat cancer together with another cell type, an antigen presenting cell, selected from the group consisting of autologous cultured macrophages and autologous cultured dendritic cells. Macrophages are any large ameboid mononuclear cell, regardless of origin, such as and not limited to histiocytes and monocytes, which phagocytose, i.e. engulf 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 more efficient antigen presenting cells than macrophages. They are potent stimulators of T cells and may be isolated from a variety of body organs and tissues including and not limited to blood, skin (where dendritic cells are referred to as Langerhans cells), lymphoid tissues.

The antigen presenting cells with peptide or membrane bound thereto, for example, may be used to immunize patients. The patient's blood is obtained and macrophages or dendritic cells are extracted therefrom. High concentrations of the peptide (about 1 ng/ml to about 1 .mu.g/ml, preferably about 10 ng/ml to about 100 ng/ml), or membrane (about 10⁵ to about 10⁷ cell equivalents (c.e.), cell equivalents are in relation to the number of starting cells, i.e., the amount of cell extract obtained from the indicated number of cells) are incubated with the cells overnight or for about 8 hours. In the case of incubating with membranes, the membranes are phagocytized by the macrophages or dendritic cells. The macrophages or dendritic cells which have phagocytized the membranes are used to immunize the patient, Grabbe, S., et al., Immunology Today 1995 16:117-121, the disclosure of which is incorporated herein by reference in its entirety.

The vaccine composition of the invention may contain, for example, at least 10⁴ tumor cells or c.e. of tumor cell extract (e.g. isolated membrane or peptide) per dose, preferably at least 10⁵ cells/c.e. extract, and most preferably at least 10⁶ cells/c.e. extract. A dose is that amount of the vaccine composition that is administered in a single administration. In one embodiment, the vaccine composition contains from about 10⁵ to about 2.5.times.10⁷ cells/c.e. extract per dose, more preferably about 5.times.10⁶ cells/c.e. In one preferred embodiment, the vaccine composition contains a maximum of 7.5.times.10⁶ cells/c.e. extract. The amount of the tumor cells and tumor cell membranes of the invention to be used generally depends on such factors as the affinity of the compound for cancerous cells, the amount of cancerous cells present and the solubility of the composition. Dosages may be set with regard to weight, and clinical condition of the patient.

The vaccine composition of the invention may be packaged in a dosage form suitable for intradermal, intravenous, intraperitoneal, intramuscular, and subcutaneous administration. Alternatively, the dosage form may contain the preparations of the invention (e.g. tumor cells, membranes, peptides) to be reconstituted at the time of the administration with, for example, a suitable diluent.

The tumor cells, tumor cell extracts and compositions thereof of the invention may be administered by any suitable route, including inoculation and injection, for example, intradermal, intravenous, intraperitoneal, intramuscular, and subcutaneous. There may be multiple sites of administration per each vaccine treatment. For example, the vaccine composition may be administered by intradermal injection into at least two, and preferably three, contiguous sites per administration. In one embodiment of the invention, the vaccine composition is administered on the upper arms or legs.

Prior to administration of the vaccine composition of the invention, the subject may be immunized to the hapten which is to be used to modify tumor cells and membranes by applying it to the skin. For example, dinitrofluorobenzene (DNFB) may be used. In one embodiment of the invention, the patient is not immunized to a hapten prior to vaccine administration. Subsequently (about two weeks later, for example), the subject may be injected with a tumor cell or extract composition. The composition may be administered (such as by reinjection) for a total of at least three and preferably at least six treatments. In one embodiment, the total number of administrations (including the initial administration) may be eight, and in another embodiment may be ten. The vaccination schedule may be designed by the attending physician to suit the particular subject's condition. The vaccine injections may be administered, for example, every 4 weeks, preferably every 2 weeks, and most preferably every week. In one preferred embodiment, the vaccine is injected every week for a total of six treatments. Haptenized and non-haptenized vaccine may be alternated. In one preferred embodiment, all vaccines contain hapten modified tumor cells or extracts. A booster vaccine may be administered. Preferably, one or two booster vaccines are administered. The booster vaccine may be administered, for example, after about six months or about one year after the initial administration.

The drug cyclophosphamide (CY) may be administered several days (e.g. 3 days) prior to each vaccine administration to augment the immune response to the tumor cells. In one preferred embodiment, CY is administered only prior to the first vaccine injection.

The vaccine of the present invention may be haptenized or non-haptenized. The haptenized, or chemically-linked, form of the vaccine may include a tumor cell haptenized to dinitrophenyl (DNP) for example. Other haptens include and are not limited to trinitrophenyl, N-iodoacetyl-N'-(5-sulfonic 1-naphthyl)ethylene diamine, trinitrobenzenesulfonic acid, fluorescein isothiocyanate, arsenic acid benzene isothiocyanate, trinitrobenzenesulfonic acid, sulfanilic acid, arsanilic acid, dinitrobenzene-S-mustard. Combinations of hapten may also be used. A vaccine of tumor cell extracts may similarly be haptenized. In the case of haptenized cancer cell extracts, the extracts, a peptide, and a cancer cell membrane, are isolated from haptenized cancer cells. The present invention also contemplates a non-haptenized vaccine of tumor cells and/or cell extracts.

In one embodiment of the present invention, a method of treating a patient suspected of having cancer, may comprise administering a pharmaceutically acceptable amount of cyclophosphamide, and a pharmaceutically acceptable amount of a composition selected from the group consisting of live tumor cells, tumor cell extracts, and a mixture of tumor cells and tumor cell extracts. Where the composition is a cancer cell extract, the extract may be a peptide or a membrane isolated from a haptenized cancer cell. The composition may be mixed with an immunological adjuvant and/or a pharmaceutically acceptable carrier. The haptenized vaccine may optionally be followed by administration of a pharmaceutically acceptable amount of a non-haptenized vaccine. A nonhaptenized vaccine may also be administered in accordance with the methods of the present invention.

In another embodiment of the invention, the composition of the invention is administered every week for a period of at least six weeks, and the first administration is preceded by a pharmaceutically acceptable amount of cyclophosphamide. Preferably, the composition may contain a maximum of about 7.5.times.10⁶ cells or c.e. extract. The patient need not be immunized to hapten prior to vaccine administration.

The vaccine composition of the present invention may comprise tumor cells and/or tumor cell extracts. The tumor cells for use in the present invention may be prepared as follows. Tumor masses are processed as described by Berd et al. (1986), supra, incorporated herein by reference in its entirety. The cells are extracted by enzymatic dissociation with collagenase and DNAse by mechanical dissociation, frozen in a controlled rate freezer, and stored in liquid nitrogen until needed. On the day that a patient is to be skin tested or treated, the cells are thawed, washed, and irradiated to about 2500 R. They are washed again and then suspended in Hanks balanced salt solution without phenol red. Conjugation of the prepared cells with DNP is performed by the method of Miller and Claman, J. Immunol., 1976, 117, 1519, incorporated herein by reference in its entirety, which involves a 30 minute incubation of tumor cells with DNFB under sterile conditions, followed by washing with sterile saline.

Cancer cells of a patient may be conjugated to a hapten by isolating the membranes and modifying the membranes or by conjugating the cells to a hapten without first isolating the membranes.

A cancer cell membrane may be prepared by isolating membranes from non-modified preparation of cancer cells of a patient. Cells are suspended in about five volumes of about 30 mM sodium bicarbonate buffer with about 1 mM phenyl methyl sulfonyl fluoride and disrupted with a glass homogenizer. Residual intact cells and nuclei are removed by centrifugation at about 1000 g. The membranes are pelleted by centrifugation at 100,000 g for 90 minutes. The membranes are resuspended in about 8% sucrose and frozen at about -80^oC. until needed. To a suspension of membranes (about 5,000,000 cell equivalents/ml), about 0.5 ml of 1 mg/ml dinitrofluorobenzene (DNFB) is added for about 30 minutes. Similarly, other haptens such as and not limited to trinitrophenyl and N-iodoacetyl-N,-(S sulfonic 1-naphtyl)ethylene diamine may be used. Excess DNP is removed by dialyzing the membranes against about 0.15 M PBS for about three days. The membranes are pelleted.

Alternatively, the cancer cell extract, the peptide or the membrane, may be prepared by modifying cancer cells of a patient with a hapten such as dinitrophenyl and then preparing membranes therefrom. Cancer cells of a patient are obtained during biopsy and frozen until needed. About 100 mg of DNFB (Sigma Chemical Co., St. Louis, Mo.) was dissolved in about 0.5 ml of 70% ethanol. About 99.5 ml of PBS was added. DNFB concentration should be about 152 mg/0.1 ml. The solution was stirred overnight in a 37^o C. water bath. The shelf life of the solution is about 4 weeks. The cells were thawed and the pellet was resuspended in 5.times.cells/ml in Hanks balanced salt solution. About 0.1 ml DNFB solution was added to each ml of cells and incubated for about 30 minutes at room temperature. Similarly, other haptens such as and not limited to trinitrophenyl, N-iodoacetyl-N'-(5-sulfonic 1-naphthyl)ethylene diamine, trinitrobenzenesulfonic acid, fluorescein isothiocyanate, arsenic acid benzene isothiocyanate, trinitrobenzenesulfonic acid, sulfanilic acid, arsanilic acid, dinitrobenzene-S-mustard and combinations thereof may be used. The cells were then washed twice in Hanks balanced salt solution. Cells are suspended in about five volumes of about 30 mM sodium bicarbonate buffer with about 1 mM phenyl methyl sulfonyl fluoride and disrupted with a glass homogenizer. Residual intact cells and nuclei are removed by centrifugation at about 1000 g. The membranes are pelleted by centrifugation at 100,000 g for 90 minutes. The membranes are resuspended in about 8% sucrose and frozen at about -80^oC. until needed.

From the DNP modified cells, peptide may be extracted, some of which are DNP modified as a result of modifying the cells. Protein extraction techniques, known to those of skill in the art, may be followed by antigen assays to isolate antigen(s) effective for patient treatment. The methods of isolating cell extracts are readily known to those skilled in the art. Briefly, cancerous cells are isolated from a tumor and cultured in vitro. Dinitrophenyl is added to the cultured cells in accordance with the method set forth above. Peptides are isolated from 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 saved. Fractions containing small peptides are obtained by HPLC, concentrated, and frozen. The fractions are screened for immunological activity by allowing them to bind to autologous B lymphoblastoid cells which are then tested for ability to stimulate melanoma-specific T lymphocytes.

The cells are treated with a weak acid, such as and not limited to trifluoroacetic acid MA). The cells are then centrifuged and the supernatant is saved. Compounds having a molecular weight greater than 5,000 were removed from the supernatant by gel filtration (G25 Sepharose, Pharmacia). The remainder of the supernatant is separated on a reversedphase HPLC column (Superpac Pep S, Pharmacia LKB) in 0.1% trifluoroacetic acid (TFA) using a gradient of increasing acetonitrile concentration; 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, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), concentrated, and frozen. The fractions are screened for immunological activity by allowing them to bind to autologous B lymphoblastoid cells which are then tested for ability to stimulate tumor- (e.g. melanoma) specific T lymphocytes.

Epstein barr virus (EBV, ATCC CRL-1612, B95-8 EBV transformed leukocytes, cotton top marmoset, Saguinus oedipus) is added to B lymphoblastoid cells in culture. The B lymphoblastoid cells are transformed into a B cell tumor from the patient's own lymphocytes. Melanoma from a metastasis is cultured in RPMI 1640+10% fetal calf serum or 10% pooled human serum. The non-adhered cells are washed off with RPMI medium. When the cells are confluent, they are detached with 0.1% EDTA and split into two flasks. This process continues where the confluent cells are continuously split. To test for gamma interferon production by T cells, lymphocytes from a patient's blood are obtained. The patient's own tumor cells, which have been modified with a hapten, such as DNP, are mixed with the lymphocytes to stimulate the T cells. Every seven days, interleukin-2 is added. The T cells are expanded by splitting as disclosed above. The T cells are then restimulated by the hapten modified cells. An enriched population of T cells result which are responsive to the hapten modified cells.

Human cancer vaccines have been developed and tested by a number of workers. Although they can 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 DNP-vaccine of the present invention. The tumor becomes reddened, warm and tender. Ultimately, in some cases, the tumor regresses to the extent that the tumor disappears, to the naked eye and microscopically. Microscopically, infiltration of T lymphocytes--into the tumor mass is observed. Therefore, this approach, 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 may be improved by adding various biological response modifiers. These agents work by directly or indirectly stimulating the immune response. Biological response modifiers of the present invention include and are not limited to interleukin-12 and gamma interferon. In this embodiment, IL12 will be given following the each vaccine injection. Administration of IL12 to patients with inflammatory responses is believed to cause the T lymphocytes within the tumor mass to proliferate and become more active. The increased T cell numbers and functional capacity leads to immunological destruction of the tumors. Dosages for IL12 will be prepared in accordance with the dosage indications set forth above.

Patients with metastatic melanoma were treated using an immunotherapy regimen with the following components: 1) vaccine consisting of autologous tumor cells conjugated to DNP; and 2) low dose cyclophosphamide pretreatment. Patients were evaluated to determine whether tumor regression had occurred, to monitor tumor inflammatory responses, and to measure delayed type hypersensitivity to autologous melanoma cells, DNFB (the form of DNP used for skin sensitization), DNP-conjugated autologous lymphocytes, diluent (Hanks solution), purified protein derivative (PPD), and recall antigens (candida, trichophyton, and mumps). Patients who are considered to be deriving benefit (clinical or immunological) from the therapy are continued in the immunotherapy regimen. Subsequent vaccines may be given without cyclophosphamide. In a similar experiment, Interleukin 2 linked to polyethylene glycol was found to not be effective.

In another embodiment, a vaccine comprising chemical extracts of cancer cells conjugated to a hapten and mixed with an immunological adjuvant, such as Bacillus Calmette-Guerin, BCG, is used.

In the present invention, biopsies from human melanoma metastases were examined for expression of cytokine mRNA using RT-PCR. mRNA for IFN.gamma. is found in post-DNP vaccine, inflamed metastases, but only rarely in pretreatment metastases, even those containing large numbers of residual lymph node lymphocytes. Moreover, the Type II cytokine, IL10, is found in almost all melanoma metastases and appears to be produced by the melanoma cells themselves.

Patients with metastatic melanoma treated with an autologous, DNP-modified vaccine develop inflammatory responses at tumor sites. Histologically, these inflamed lesions are characterized by T cell infiltration which is sometimes associated with tumor cell destruction. In the present invention, biopsy specimens of 8 subcutaneous metastases that had developed inflammation following vaccine treatment were tested for expression of mRNA for IFN.gamma., IL4, TNF, and IL10. Post-vaccine, inflamed biopsies contained mRNA for IFN.gamma. (5/8), IL4 (4/8) or both (3/8), and for TNF (4/7). In contrast, IFN.gamma. mRNA was detected in only 1/17 and TNF mRNA in 2/16 control specimens (pre-treatment lymph node metastases or non-inflamed subcutaneous metastases). mRNA for IL10, a cytokine with anti-inflammatory properties, was detected in 24/25 melanoma metastases and was independent of lymphoid content; in situ PCR confirmed that melanoma cells were the major source. These findings provide a new parameter by which to measure the effects of cancer immunotherapy.

The present invention is aimed at analyzing freshly obtained metastatic melanoma biopsies for the presence of cytokine mRNA which correlates with a productive immune response at the tumor site. The expression of IFN.gamma. or IL4 mRNA is characteristic of melanoma metastases that have developed an inflammatory response following administration of DNP-modified autologous vaccine. on the other hand, expression of IL10 mRNA is independent of an inflammatory response and seen in nearly all melanoma biopsy specimens. Examination of cell lines derived from melanoma biopsies as well as in situ PCR analysis demonstrated that the source of IL10 is melanoma cells themselves rather than the associated lymphocytes.

Perhaps the most important finding of this work is the negative one: mRNA for IFN.gamma. and IL4 generally is 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 to compete melanoma tissues whose T cell population has been altered by immunotherapy. Moreover, it underscores an important biological point: T cells extracted from melanoma nodal metastases probably represent the residua of the original lymph node population rather than lymphocytes that have actually infiltrated the tumor as a result of their recognition of melanoma antigens. Since they are not antigen-activated, they have received no stimulus to produce IFN.gamma. or IL4.

In contrast, biopsy specimens obtained following administration of DNP-vaccine typically expressed mRNA for IFN.gamma.. However, DNP-vaccine-induced inflammatory responses cannot be characterized as Type I since some of these samples contained IL4 as well. Given the sensitivity of PCR-based mRNA analysis, such a pattern could be produced by a small focus of IL4-producing T cells in the midst of a T cell infiltrate that is predominantly IFN.gamma.-producing. On the other hand, the presence of mRNA for IFN.gamma. and IL4 could signify the presence of T cells that produce both cytokines--so-called TH₀ cells (Lee et al, Eur. J. Immunol. 1992 22:1455-1459). Resolution of this issue will require analyses that allow correlation of mRNA expression with morphology, such as in situ PCR. Whatever the results, these findings suggest that intra-tumor cytokine production may be an important parameter to measure in patients undergoing immunotherapy.

The present invention strongly suggest that the source of IL10 mRNA is the melanoma cells themselves, rather than the associated lymphocytes. Strong IL10 mRNA bands were detected in 24/25 biopsies, and its expression was independent of the number of associated lymphocytes or the presence of DNP-vaccine-induced inflammation. Moreover, in situ PCR clearly showed IL10 mRNA within melanoma cells. Cell lines derived from the biopsy material expressed IL10 mRNA and produced IL10 as measured by ELISA.

The physiologic significance of IL10 production in melanoma tissues is not clear. IL10 is known to be an antiinflammatory cytokine with ability to inhibit T cell proliferation and IL2 production (Jinquan, T., et al., J. Immunol. 1993 151:4545-4551) and delayed type hypersensitivity (Lee, supra), probably by reducing macro phage costimulatory function. Thus IL10 could suppress the activation and proliferation of melanoma-reactive T cells that have infiltrated the tumor site. However, IL10 recently has been shown to be a chemoattractant for CD8+ T cells (Jinquan, supra); this property could account for the predominance of CD8+ cells in DNP-vaccine-induced lymphoid infiltrates. In either case, modulation of IL10 production at the tumor site may have important consequences for the tumor-host relationship.

The scope of the present invention also includes a method of screening for cytokine production by a tumor to determine the efficacy of an autologous, irradiated hapten conjugated cell composition in a patient suspected of having cancer, said method comprising administering said hapten conjugated composition to said patient; obtaining a sample comprising nucleic acids from a patient tissue sample; amplifying nucleic acids specific for a cytokine or amplifying a signal generated by hybridization of a probe specific 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 amplified signal indicates cancer, wherein the presence of amplified nucleic acids or amplified signal from said patient tissue sample indicates efficacy of said hapten conjugated composition.

The tissue sample may be a malignant or premalignant tumor, a melanoma tumor for example, or a subcutaneous inflammatory metastatic melanoma, for example. In addition, a tissue sample may be a solid or liquid tissue sample such as and not limited to all or part of a tumor, saliva, sputum, mucus, bone marrow, serum, blood, urine, lymph, or a tear from a patient suspected of having cancer.

Nucleic acids, such as DNA (including cDNA) and RNA (including mRNA), are obtained from the patient tissue sample. Preferably RNA is obtained from a tissue sample. Total RNA is extracted by any method known in the art such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), incorporated herein by reference in its entirety.

Nucleic acid extraction is followed by amplification of the same by any technique known in the art. The amplification step includes the use of at least one primer sequence which is complementary to a portion of a cytokine specific sequence. Cytokine specific sequences are defined for purposes of the present invention to include (and are not limited to) all or part of sequences which encode IFN.gamma., TNF, IL-2, IL-12, and IL-13. Generally, the primer sequence is about 21 nucleotides to about 33 nucleotides, preferably about 21 nucleotides, about 31 nucleotides, 32 nucleotides, and about 33 nucleotides in length.

Primer sequences useful in the amplification methods include and are not limited to .beta. actin, SEQ ID NOS: 1 and 2; IFN.gamma., SEQ 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.

Where a template dependent process of amplification uses a pair of primers, one primer of the pair may comprise oligonucleotides which are complementary to nucleic acid sequences which encode cytokine specific proteins. The one primer of the pair may be selected from the group consisting of SEQ ID NOS: 1 to 10.

Alternatively, each of the two oligonucleotides in the primer pair may be specific to a nucleic acid sequence which encodes a cytokine. The primers may be designed to be complementary to separate regions of a cytokine sequence for example. By separate regions is meant that a first primer is complementary to a 31 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 distinct, separate regions and are not complementary to each other. The primers of SEQ ID NOS: 1-10 are merely exemplary of the primers which 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 may be selected from the group consisting of SEQUENCE ID NOS: 1, 3, 5, 7, and 9, and the second primer may be selected from the group consisting of SEQUENCE ID NOS: 2, 4, 6, 8, and 10. Any primer pairs which transcribe nucleic acids toward each other and which are specific for cytokines may be used in accordance with the methods of the present invention.

Total extraction of RNA is preferably carried out. As used herein, the term "amplification" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its 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 process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987) incorporated herein by reference in its entirety). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, each incorporated herein by reference in its entirety.

A number of template dependent processes are available to amplify the target sequences of interest present in a sample. one of the best known amplification methods is the polymerase chain reaction (PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al., PCR Protocols, Academic Press, Inc., San Diego Calif., 1990, each of which is incorporated herein by reference in its entirety. Briefly, in PCR, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along 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 raising and lowering the temperature of the reaction mixture, the extended,primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated. Preferably a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (referred to as LCR), disclosed in EPA No. 320,308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750, incorporated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, incorporated herein by reference in its. entirety, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA 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 replicative sequence which can then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha -thio] triphosphates in one strand of a restriction site (Walker, G. T., et al., Proc. Natl. Acad, Sci. (U.S.A.) 1992, 89:392-396, incorporated herein by reference in its entirety), may also be useful in the amplification of nucleic acids in the present invention.

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e. nick translation. A similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA.

Cytokine specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having a 31 and 51 sequences of non-cytokine specific DNA and middle sequence of cytokine specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNaseH, and the products of the probe identified as distinctive products generating a signal which are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Thus, CPR involves amplifying a signal generated by hybridization of a probe to a cytokine specific nucleic acid.

Still other amplification methods described in GB Application No. 2 202 328, and in 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 former application, "modified" primers are used in a PCR like, template and enzyme dependent synthesis. The primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labelled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh D., et al., Proc. Natl. Acad. Sci. (U.S.A.) 1989, 86:1173, Gingeras T. R., et al., PCT Application WO 88/10315, incorporated herein by reference in their entirety), including nucleic acid sequence based amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has prostate specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second prostate specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate prostate cancer specific sequences.

Davey, C., et al., European Patent Application Publication No. 329,822, incorporated herein by reference in its entirety, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

Miller, H. I., et al., PCT Application WO 89/06700, incorporated herein by reference in its entirety, disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts. Other amplification methods include "race" disclosed by Frohman, M. A., In: PCR Protocols: A Guide to Methods and Applications 1990, Academic Press, N.Y.) and one-sided PCR11 (Ohara, O., et al., Proc. Natl. Acad. Sci. (U.S.A.) 1989, 86:5673-5677), all references herein incorporated by reference in their entirety.

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu, D. Y. 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.

Following amplification, the presence or absence of the amplification product may be detected. The amplified product may be sequenced by any method known in the art, including and not limited to the Maxam and Gilbert method, see Sambrook, supra. The sequenced amplified product may then be compared to results obtained with tissue excised prior to vaccine treatment. Tissue samples obtained prior to vaccine treatment should be free of cytokine sequences, particularly IFN.gamma., TNF, IL2, IL12, and IL13. The nucleic acids may be fragmented into varying sizes of discrete fragments. For example, DNA fragments may be separated according to molecular weight by methods such as and not limited to electrophoresis through an agarose gel matrix. The gels are then analyzed by Southern hybridization. Briefly, DNA in the gel is transferred to a hybridization substrate or matrix such as and not limited to a nitrocellulose sheet and a nylon membrane. A labelled probe is applied to the matrix under selected hybridization conditions so as to hybridize with complementary DNA localized on the matrix. The probe may be of a length capable of forming a stable duplex. The probe may have a size range of about 200 to about 10,000 nucleotides in length, preferably about 200 nucleotides in length. Mismatches such as and not limited to sequences with similar hydrophobicity and hydrophilicity, will be known to those of skill in the art once armed with the present disclosure. Various labels for visualization or detection are known to those of skill in the art, such as and not limited to fluorescent staining, ethidium bromide staining for example, avidin/biotin, radioactive labeling such as ³² P labeling, and the like. Preferably, the product, such as the PCR product, may be run on an agarose gel and visualized using a stain such as ethidium bromide. See Sambrook et al., supra. The matrix may then be analyzed by autoradiography to locate particular fragments which hybridize to the probe.

A diagnostic kit for screening for the efficacy of an autologous, irradiated, hapten conjugated cell composition comprising in one or more containers, a pair of primers, wherein one of the primers within said pair is complementary to a cytokine specific sequence, wherein said primer 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 a means for visualizing amplified DNA; said kit useful for determining the efficacy of said composition.

Claim 1 of 14 Claims

What is claimed is:

1. A method for inducing an anti-tumor response in a mammalian patient suffering from a tumor, which method comprises administering to said patient a composition comprising a tumor cell or tumor cell extract with an adjuvant, wherein the tumor cell or tumor cell extract is;

(i) conjugated to a hapten;

(ii) of the same tumor type as the patient's tumor;

(iii) not allogeneic to said patient; and

(iv) incapable of growing in the body of the patient after injection;

and repeating said administration at weekly intervals,

wherein a therapeutically effective amount of cyclophosphamide is administered only prior to the first administration of the composition, wherein the patient is not sensitized to the hapten prior to administration of the composition, and wherein the composition elicits an anti tumor response.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.