WO2002053176A2

WO2002053176A2 - An autologous anti-cancer vaccine

Info

Publication number: WO2002053176A2
Application number: PCT/IL2002/000012
Authority: WO
Inventors: Michal Lotem; Tamar Peretz; Eitan Shiloni; Olga Drize; Shoshana Frankenburg
Original assignee: Hadasit Medical Research Services And Development Ltd.
Priority date: 2001-01-08
Filing date: 2002-01-06
Publication date: 2002-07-11
Also published as: AU2002219485A1; WO2002053176A3; IL140796A0

Abstract

The invention relates to a pharmaceutical composition for inducing an immune response directed against malignancies in a mammalian subject. The composition comprises as active ingredients autologous antigen presenting cell (APC) which were loaded with mixture of at least two allogenic tumor cell line lysates. Optionally, the composition of the invention may further comprise an IL-2 adjuvant. The invention further relates to a method for conferring immunity against a malignancy, in a mammalian subject in need, by administering an autologous antigen presenting cells (APC) loaded with a mixture of allogeneic cell lysates mixture, or a composition comprising the same to said subject. The invention further relates to the use of these loaded APC cells in the preparation of pharmaceutical composition for the treatment of malignant disorders, particularly, melanoma.

Description

AN AUTOLOGOUS ANTI-CANCER VACCINE

Field of the Invention

The invention relates to compositions for inducing mammalian immune response against malignancies. More particularly, the invention provides a composition and method for inducing the production of specific both helper and cytotoxic T lymphocytes directed against malignancies such as melanoma.

Background of the Invention

General

Melanomas are aggressive, frequently metastatic tumors derived from either melanocytes or melanocyte related nevus cells ["Cellular and Molecular Immunology" (eds) Abas A.K., et al., Saunders Company, Philadelphia: 340-341 (1991)]. Melanomas make up approximately three percent of all skin cancers and the worldwide increase in melanoma is unsurpassed by any other neoplasm with the exception of lung cancer in women. Even when melanoma is apparently localized to the skin, up to 30% of the patients will develop systemic metastasis and the majority will die. Patients with metastatic (stage IV) malignant melanoma have a median survival of approximately one year. The incidence of advanced melanoma, the deadliest form of skin cancer, is increasing. Classic modalities of treating melanoma include surgery, irradiation and chemotherapy. Chemotherapy, in addition to its toxic effects, is usually ineffective against this type of cancer. In recent years, several medical research centers have implemented clinical trials using novel vaccines based upon dendritic cells (DC).

Vaccines are best known for preventing disease; however, cancer vaccines are specifically designed to stimulate the immune system to recognize and attack cancer cells. Cancer cells mask themselves in ways that make them undetectable to the immune system T-cells. Dendritic cells are a blood component whose role is to recruit and stimulate T-cells to detect and destroy cancer cells.

DC vaccines

Recently, new approaches to immunotherapy have used antigen-loaded autologous dendritic cells (DC) as means of inducing specific T-cell responses. DC, very potent antigen presenting cells (APC), are grown ex υivo from peripheral blood monocytes in the presence of cytokines, loaded with antigen, and re-injected into the patient. Malignant melanoma has provided an appropriate setting for pursuit of these studies, with the goal of generating strong and long-lasting tumor-specific T-cell immunity. Several melanoma associated antigens (MAA) have been defined and characterized and, when associated with particular HLA class I molecules, have been recognized by cytotoxic T lymphocytes (CTL) of melanoma patients. For these reasons, peptide-based vaccination protocols, such as those using autologous DC loaded with a single defined peptide for the treatment of malignant melanoma, are ongoing in several institutions. One example is described in US Patent No. 6,080,399, disclosing a peptide- based vaccination protocol that includes loading of APC with an antigenic peptide or a recombinant peptide derived from at least one antigen such as MAGE-3.

There is increasing consensus among researchers that dendritic cells provide strong stimuli for the generation of immunity, and that dendritic cell therapy has great potential and should be pursued aggressively [Carson et al., Program and abstracts of the American Society of Clinical Oncology 36^th Annual Meeting; May 20-23, 2000; New Orleans, Louisiana. Oral presentation (2000)]. However, the immunity obtained may be shortlived, and there is no consensus as to the identity and format of the antigens, type of adjuvants or other enhancers that should be used, and optimal mode of immunization. Clearly, there is a need for the development of new approaches to DC immunotherapy. A number of melanoma antigens capable of activating tumor-specific cytotoxic T-cell (CTL) responses have been identified. These include the melanoma tumor-specific MAGE-1 and -3, the melanocytic lineage-specific tyrosinase, gplOO, melan-A/MART-1, and gp75. These antigens have been loaded on DC and evaluated for efficacy in melanoma patients. In the pioneer work of Nestle et al. [Nature Medicine 4:328-332 (1998)], and in the study of Mackensen et al. [Int. J. Cancer 86:385-92 (2000)], different mixtures of peptides were used depending upon the HLA type of the patient. Thurner et al. [J. Immunol. Methods 223:1-15 (1999)] pulsed DC from HLA-A1 patients with MAGE-3A1, and Lotze et al. [Cancer J. Sci. Am. 2000 Feb, 6 Suppl. l:S61-6] treated HLA-A2 melanoma patients with a mixture of appropriate peptides. Although the treatment elicited specific immune responses to the peptides, clinical response was only observed in a relatively small number of patients.

Use of tumor lysates as a source of antisen for DC loading An approach different from peptide-DC vaccination is to use total tumor cell lysates for antigen loading. Crude cell lysates may have advantages over peptides in presenting important but undefined tumor epitopes to T- cells, and provide a wide variety of antigens that DC can internalize and present via both class I and class II molecules. While peptides undergo exogenous presentation to CD8+ cells, proteins are presented on APC after uptake and intracellular processing. Bone marrow-derived DC were shown to present exogenous antigens constitutively on their MHC class I molecules following in vitro exposure to soluble intact protein [Paglia et al., J. Exp. Med. 183:317-22 (1996)], due to their constitutive macropinocytosis ability. Thus, antigens presented after protein uptake by APC are independent of HLA class I restriction, since the peptide is complexed to the class I molecule of the APC. DC pulsed with tumor cell lysate therefore have the potential of synergistically stimulating different clones of both helper and cytotoxic T-cells, independently of HLA class I restriction. In r

addition, when using lysates, immunogenicity is not dependent on the stability of a given HLA/peptide complex.

Based on the fact that cell lysates are independent of HLA restriction, it is an object of the present invention to prepare a standard lysate which may be used for all patients. This approach has several major benefits:

(1) A large number of antigens, derived from several different melanoma lines, will enhance the chance of an effective T cell response in more patients.

(2) A lysate mixture which does not have to be custom-made is easier to use and cheaper to prepare. Such vaccine can be standardized and used for all patients, irrespective of their HLA class, thus enabling "immunotherapy for all".

(3) A standardized lysate can be used to treat patients when autologous tumor is not available (e.g. for the vaccination of high risk patients).

Interleukin (ID-2 as an adjuvant

A critical point in the therapeutic efficacy of tumor vaccines is the need to boost the induced immune response. IL-2 is a natural choice as a vaccine adjuvant. It promotes the proliferation and differentiation of T and B cells and enhances cytolytic activity. When given systemically, IL-2 induces clonal expansion of T cell subpopulations both in the tumor and in peritumoral tissues. Administration of this cytokine alone or in conjunction with tumor infiltrating lymphocytes (TILs) has resulted in several objective melanoma regressions [Rosenberg et al., Ann. Surg. 210:474-85 (1989)]. When given as a monotherapy, the major disadvantage of IL-2 is the requirement for high doses, which are associated with life threatening toxicity. Rosenberg et al. [Nat. Med. 4:321-7 (1998)] reported tumor regression in 40% of patients treated with a melanoma peptide derived from the gp-100 antigen and systemic administration of high-dose IL-2. This pioneering study strongly supports the assumption that IL-2 enhances the therapeutic effect of anti cancer vaccines. However, the adjuvant effect of IL-2 should be pursued at much lower doses compared to those given by Rosenberg. This has already been demonstrated in anti-viral vaccines: Anti-rabies and herpes simplex virus vaccines showed significant enhancement of both humoral and specific cell mediated cytotoxicity when co- administered with non-toxic doses of IL-2.

One method to avoid the systemic toxicity of IL-2 is to genetically modify tumor cells to produce IL-2. Osanto et al. [Hum. Gene. Ther. 11: 739-50 (2000)] used an IL-2 producing allogeneic melanoma cell line with two of 33 patients attaining tumor regressions and seven patients attaining stable disease. Sobol et al. [Clin. Cancer Res. 9:2359-65 (1999)] showed a five-fold increase in tumor specific CTL numbers from peripheral blood of colorectal carcinoma patients vaccinated with an autologous tumor and with genetically modified IL-2 producing fibroblasts. In fact, gene-modification leads to a local, sustained effect, due to IL-2 release.

So far, the enhancing effect of IL-2 on DC vaccines has been evaluated in murine studies only. Shimizu et al. [Proc. Natl. Acad. Sci. USA 96:2268-73 (1999); Cancer J. Sci. Am. Suppl. 1:567-75 (2000)] demonstrated significant cure rates of pulmonary metastases of weakly immunogenic sarcoma and melanoma, when using lysate-pulsed DC combined with systemic administration of non-toxic doses of IL-2.

Liposomal cytokines

The same effect as with genetically modified cells can be achieved more easily, using the depot effect of liposomal formulations. Liposomes are bi- layer vesicles (with a diameter of 30 nm-10 μm), formed by amphipathic lipids, generally phospholipids, entrapping a water-soluble phase. They serve as drug carriers when a drug is dissolved in the water phase, and allow for a local protracted release of the drug. Liposomes have very low or no toxicity, are not immunogenic, and can be safely administered to humans. Liposome-encapsulated IL-2 was already evaluated as a vaccine adjuvant [Adler et al, Cancer Biotherapy 10:193-206 (1995)]. They demonstrated 6 out of 10 objective responses to allogeneic human liposomal melanoma vaccine combined with regional IL-2, as compared to 0/5 without the liposomal IL-2. Interestingly, low dose liposomal IL-2 without vaccine also resulted in several clinical responses, attesting to its independent therapeutic role. This effect was repeated in a murine model of renal cell carcinoma [Adler, et al., (1995) ibid; Krup et al., J. Immunother. 6:525-38 (1999)] and it was shown in the murine B16 melanoma model that the enhancement is mainly due to a depot effect, since it cannot be attained with the soluble form of IL-2 [Van Slooten et al., Int. J. Pharm. 183:33-6 (1999)].

E. Kedar and co-workers have gained much experience using liposomal formulations of IL-2, Tumor Necrosis Factor-α (TNF) and GM-CSF. [Kedar et al., In: "The Biotherapy of Cancers: From Immuno-therapy to Gene Therapy" (S. Chouaib, ed), INSERM, Paris, pp 333-62 (1998]. These liposomal cytokine formulations exhibited in mice greater immunomodulatory and therapeutic effects, and lower toxicity, as compared with soluble cytokines. In addition, lower doses and less frequent administration was required.

Based on recent developments in DC growth in vitro, in accordance with the present invention these potent antigen presenting cells are loaded with a mixture of antigen-rich melanoma lysates, with the object of preparing a vaccine which does not have major histocompatibility complex restrictions. Moreover, the effect of the vaccine of the invention can be enhanced by the concomitant administration of liposomal IL-2. This low toxicity formulation allows for a sustained effect of IL-2 at the site of vaccination. Clearly, an effective universal vaccine, which can be used for all patients, would represent a significant advance in the treatment of this fatal disease. These and other objects of the invention will be elaborated on as the description proceeds.

Summary of the Invention

The first aspect of the present invention relates to a pharmaceutical composition for inducing an immune response directed against malignancy in a mammalian subject. The composition comprises as active ingredient autologous antigen presenting cell (APC) loaded with mixture of at least two tumor cell line lysates. The composition of the invention may optionally further comprise an IL-2 adjuvant and optionally further pharmaceutically acceptable carrier, diluent, excipient and/or additive.

A preferred embodiment relates to the composition of the invention for inducing immune response against carcinomas, lymphomas, melanomas and sarcomas, and most preferably, melanoma.

In a specifically preferred embodiment the composition of the invention is intended to induce an immune response in humans.

In a further embodiment the composition of the invention comprises APC that are autologous dendritic cells (DC). After being loaded with lysates mixture, the DC are subjected to maturation by treatment with any one of tumor necrosis factor (TNF), TNF+ prostaglandin E2 (PG) and polyribocytidylic acid (poly (1:C)), preferably by treatment with TNF+PG.

In a specifically preferred embodiment the composition of the invention employs APC loaded with the cell-lysate mixture, that express peptides derived from said cell-lysate mixture in context of both MHC Class I and Class II molecules. This composition is intended to induce an immune response that results in the production of both helper and cytotoxic T lymphocytes specific for different antigens that are present in the lysate mixture and are associated with the malignancy. Further particular embodiments relate to the composition of the invention in which the said tumor cell line lysate is prepared from allogeneic tumor cell line selected from the group consisting of M-10, M-12, M-16 and M-24 deposited under DSM Accession Nos. ACC2481, 2482, 2483 and 2484, respectively, or any cell lines derived therefrom. More preferably, the composition of the invention employs mixture of tumor cell line lysates comprising at least two allogeneic tumor cell line lysates.

In a specifically preferred embodiment those tumor cell lines are allogeneic melanoma cell lines that express at least one of the melanoma associated antigens (MAA) tyrosinase, gp-100, MAGE-3 and MART-1. More particularly, this mixture of tumor cell line lysates is prepared from at least two of M-10, M-12, M-16 and M-24 deposited under DSM Accession No. ACC2481, 2482, 2483 and 2484, respectively, or any cell lines derived therefrom.

In the composition of the invention the APC may be loaded with cell lysate mixture at a concentration of from 70 to 200 μg/ml. Preferably, the APC are pulsed/loaded with cell lysate mixture at a concentration of 120 μg/ml. Further, the composition of the invention utilizes a liposomal IL-2 dose of 6xlOHo 6xl0⁶ U.

In a further aspect, the present invention relates to carcinoma, lymphoma, sarcoma and melanoma cell lines selected from the group consisting of M- 10, M-12, M-16 and M-24 deposited under DSM Accession No. ACC2481, 2482, 2483 and 2484, respectively, and any cell lines derived therefrom. Lysates of cells and cell lines of the invention are also contemplated. The invention further relates to the use of the cell lines of the invention, as well as cell lysates derived therefrom, in the preparation of pharmaceutical composition for the treatment of melanoma. In a further aspect the present invention relates to a method for conferring immunity against a malignancy in a mammalian subject, comprising the steps of: obtaining autologous antigen presenting cells (APC) from the subject; loading the APC with a mixture of allogeneic tumor cell line lysates; subjecting the loaded autologous APC to maturation; and administering to the subject an autologous APC loaded with mixture of at least two tumor cell line lysates or composition comprising the same. The loaded APC or composition comprising the same are administrated at an amount sufficient to induce an immune response against said malignancy in the subject. These compositions may optionally further comprise an IL-2 adjuvant and optionally further pharmaceutically acceptable carrier, diluent, excipient and/or additive.

The method of the invention is particularly suitable for conferring immunity against carcinomas, lymphomas, melanomas and sarcomas, preferably melanoma, in humans.

The method of the present invention preferably employs compositions comprising as APC autologous dendritic cells (DC). After being pulsed/loaded with the cell lysates of the invention, the DC are matured by treatment with tumor necrosis factor (TNF), TNF + prostaglandin E2 (PG) and polyribocytidylic acid (poly (1:C)), preferably, with TNF+PG.

Still preferably, the method of the invention employs compositions comprising APC loaded with the cell-lysate mixture that express peptides derived from said cell-lysate mixture in a context of both MHC Class I and Class II molecules. Thus, the method of the invention may confer an immune response that results in the production of both helper and cytotoxic T lymphocytes specific for different antigens that are present in the cell lysate and are associated with the malignancy. The method of the invention preferably employs compositions comprising cells loaded with tumor cell line lysates prepared from allogeneic tumor cell lines selected from the group consisting of M-10, M-12, M-16 and M-24 deposited under DSM Accession No. ACC2481, 2482, 2483 and 2484, respectively, and any cell lines derived therefrom. More specifically, the mixture of tumor cell line lysates comprises at least two allogeneic tumor cell line lysates. Preferred tumor cell lines are allogeneic melanoma cell lines, particularly those expressing at least one of the melanoma- associated antigens (MAA) tyrosinase, gp-100, MAGE-3 and MART-1. More particularly, the mixture of tumor cell line lysates is prepared from at least two of the melanoma cell lines M-24, M-16, M-12 and M-10 or any cell lines derived therefrom.

A specific embodiment relates to the method of the invention employing a composition comprising APC loaded with cell lysate mixture at a concentration of from 70 to 200μg/ml, and preferably 120 μg/ml. The composition comprises APC in an amount from 10⁶ to 5xl0⁶ cells, and liposomal IL-2 dose of 6 xlO⁵ to 6 xlO⁶ U.

The invention further provides for a method for the treatment of a malignant disorder in a mammalian subject in need. This method comprises the steps of: (a) obtaining autologous antigen presenting cells (APC) from said subject; (b) loading said APC with a mixture of allogeneic tumor cell line lysates; (c) subjecting said loaded autologous APC to maturation; and (d) administering to said subject an autologous APC loaded with mixture of at least two tumor cell line lysates or composition comprising the same. The loaded APC or the composition are administered according to the method of the invention in an amount sufficient to induce in the treated, subject an immune response against the malignant disorder. The present invention further relates to the use of an autologous antigen presenting cell (APC) loaded with a mixture of allogeneic tumor cell line lysates, in the preparation of the pharmaceutical composition of the invention, for the treatment of a malignant disorder, more particularly, for the treatment of melanoma. According to a specific embodiment, the composition comprises APC in an amount of from about 10⁶ to about 5xl0⁶ cells. This composition preferably further comprised liposomal IL-2. According to a particular embodiment, the composition applied by the method of the invention comprises liposomal IL-2 dose of about 6 xlO⁵ to about 6 xlO⁶ U.

The invention will be described in more detail on hand of the following drawings.

Brief Description of the Figures

Figure 1: IFN-γ secretion measured of DC induced CD8⁺ and CD8- lymphocytes

The figure shows IFN-γ secretion measured by ELISPOT of CD8⁺ and CD8- lymphocytes from 19 vaccinated patients in response to lysate mixture- loaded autologous DC. The mixture consisted of M-10, M-12, M-16, and M- 24 lysates (total protein 120 μg/ml). Lymphocytes from all patients were tested during the first stimulation. Each set of bars represents the response of one patient. Abbreviations: ce.=cells, Indiv. Patie.= individual patients, sp.= spots.

Figure 2: Comparison of IFN-γ secretion

The figure shows comparison of IFN-γ secretion measured by ELISPOT of CD8⁺ and CD8- lymphocytes from vaccinated patients in response to allogeneic melanoma lysate mixture-loaded autologous DC and autologous lysate-loaded DC. DC were loaded with 120μg/ml autologous lysate (empty bars), or 120 μg/ml lysate mixture, containing 30 μg/ml of each lysate (full bars). Lymphocytes from all patients were tested during the first stimulation. Each set of bars represents the response of one patient. Abbreviations: ce.=cells.

Detailed Description of the Invention

Conventional chemotherapy aims at controlling the growth of cancer such as melanoma by targeting rapidly growing cells. However, this function is not specific, as many normal cells, like those of the bone marrow and the intestinal epithelium, also have a basal level of proliferation. Therefore, many normal cells of the body also are susceptible to the toxic effects of chemotherapy, and conventional chemotherapy may have a substantial negative impact on the patient.

Immunotherapy is a specific protocol and is therefore attractive. If antigens were expressed on the tumor cells that were not expressed by normal cells of the host, then specific cytolytic T lymphocytes could theoretically be activated to selectively kill the tumor cells while sparing the normal tissue of the patient. To this end, considerable effort has been made in the last decade to identify such tumor specific antigens, which may serve as targets for specific tumor cell killing.

Immune recognition of these antigens occurs via specific CD8+ CTL (cytotoxic T lymphocytes) that interact with antigenic peptides bound to a groove in MHC Class I (HLA) molecules. MHC Class II-binding epitopes recognized by CD4+ T cells have also been described. Under optimal circumstances, initiation of an immune response is triggered by peptide presented by the MHC complexes expressed by host APC, and additionally requires multiple cofactors provided by APC. After initial activation, CTL induced by APC interactions are thought to migrate throughout the host, recognize the same MHC/peptide complex in the tumor cells, and be triggered to kill them. This antigen-specific cytolysis is mediated largely via induction of apoptosis.

Most studies done so far have used either very small melanoma-derived components (proteins or peptides), or an extract of the tumor from the patient himself (autologous extracts) to pulse APCs such as dendritic cells. This approach limits the number of patients that can be treated, since such components (antigens) can be used only for a certain genetic type, and often, sufficient tumor from the patient is not available.

The present invention describes a novel approach of dendritic cell vaccination, using a mixture of melanoma cell extracts obtained from several patients as a standard antigen for pulsing dendritic cells, thus, exposing the immune system to a wide spectrum of melanoma antigens, without limiting the prospective use to patients of a specific genetic type. The immune response may be amplified and the effect of the vaccine enhanced by using adjuvants, and particularly liposomal IL-2, that is much less toxic and more efficient than the regular soluble form of IL-2.

Thus, in a first aspect the present invention relates to a pharmaceutical composition for inducing an immune response in a mammalian subject, directed against malignancies. The composition comprises as active ingredients autologous antigen presenting cell (APC) which were loaded with mixture of at least two tumor cell line lysates. Optionally, the composition of the invention may further comprise an IL-2 adjuvant and optionally further pharmaceutically acceptable carrier, diluent, excipient and/or additive. Compositions containing an IL-2 adjuvant are preferred compositions of the invention.

The composition of the invention is particularly directed at inducing immune response against carcinomas, lymphomas, melanomas and sarcomas. For example, prostate, ovary, kidney, lung, brain, breast, colon, bone, skin, testes and uterus cancer may be treated, and most preferably, melanoma.

The term melanoma includes, but is not limited to, melanoma, metastatic melanoma, melanoma derived from either melanocytes or melanocyte- related nevus cells, melanocarcinoma, melanoepithelioma, melanosarcoma, melanoma in situ, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginoous melanoma, invasive melanoma or familial atypical mole and melanoma (FAM-M) syndrome. Such melanomas may be caused by chromosomal abnormalities, degenerative growth and developmental disorders, mitogenic agents, ultraviolet radiation (UV), viral infections, inappropriate tissue gene expression, alterations in gene expression, or carcinogenic agents. The aforementioned melanomas can be treated by the method and the composition described in the present invention.

The compositions of the invention are particularly intended for the induction of immune response in a mammalian subject, preferably, in humans, but other mammals including, but not limited to, monkeys, equines, cattle, canines, felines, mice, rats, pigs, horses, sheep and goats may be treated.

The tumor cell line lysate is preferably prepared from allogeneic tumor cell lines selected from the group consisting of M-10, M-12, M-16 and M-24 deposited under DSM Accession Nos. ACC2481, 2482, 2483 and 2484, respectively, or any cell lines derived therefrom. More preferably, the composition of the invention employs mixture of tumor cell line lysates comprising at least two allogeneic tumor cell line lysates, particularly cell lines selected from the group consisting of M-10, M-12, M-16 and M-24 deposited under DSM Accession Nos. ACC2481, 2482, 2483 and 2484 respectively, or any cell lines derived therefrom. By "cell lines derived therefrom" is meant "variants" or "sub-clones" of the cell line. A "variant" of such cell line is meant to refer to a naturally occurring cell line by different passages or different treatments. These variants and sub-clones are functionally similar to the original cell line.

By "functional" is meant having same biological function, for example, having identical ability to induce an immune response directed against malignancy, and expressing essentially the same tumor specific antigens.

In a specifically preferred embodiment the tumor cell lines are allogeneic melanoma cell lines that express at least one of the melanoma associated antigens (MAA) tyrosinase, gp-100, MAGE-3 and MART-1. More particularly, this mixture of tumor cell line lysates is prepared from at least two of M-10, M-12, M-16 and M-24 deposited under DSM Accession No. ACC2481, 2482, 2483 and 2484, respectively, or any cell lines derived therefrom.

Several cell types appear to be capable of serving as APC, including dendritic cells (DC), activated B cells, and activated macrophages. In accordance with the invention the APCs are preferably autologous cells and in some illustrative embodiments the antigen-presenting cell may be a dendritic cell (DC). It is understood that one of skill in the art will recognize that other antigen presenting cells may be useful in the invention, such as B cells activated by lipopolysaccharide, whole spleen cells, peripheral blood macrophages, fibroblasts or non-fractionated peripheral blood mononuclear cells (PBMC). Therefore, the invention is not limited to the exemplary cell types which are specifically mentioned and exemplified herein.

Preparation of cell lysates as well as loading or pulsing into dendritic cells may be performed in variety ways. Different procedures of lysate loading to DC may lead to enhancement of antigen presentation, or to the specific stimulation of a certain type of immune response. It is therefore appreciated that lysates as well as loading procedures may be performed in different ways.

After being loaded with lysates mixture, the DC of the invention are subjected to a maturation procedure by treatment with any one of tumor necrosis factor (TNF), TNF + prostaglandin E2 (PG) and polyribocytidylic acid (poly (1:C)), preferably by treatment with TNF+PG.

In a particular exemplary embodiment the peptides derived from the cell lysate mixture comprise one or more fragments of an antigen binding to MHC Class I or Class II molecules.

The loaded APC may present a peptide derived from the cell lysate mixture. Such peptide comprises an antigenic fragment capable of inducing an immune response that is characterized by the production of both helper and cytolytic T cells, which are directed against the malignancy.

In certain embodiments the APC are loaded with cell lysate mixture at a concentration of from 70 to 200 μg/ml. Preferably, the APC are pulsed/loaded with cell lysate mixture at a concentration of 120 μg/ml.

In yet other embodiment, the composition of the invention may comprise about 10⁶ to 5xl0⁶ APC per dose.

The compositions of the invention optionally further comprise as an active ingredient an IL-2 adjuvant.

It has also been found that administration of an immunomodulating drug, such as IL-2, further enhances the efficacy of the present invention. Administration of IL2 to patients with inflammatory responses may 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. It is to be appreciated that the use of other cytokines such as IL-12 as an adjuvant, is also contemplated within context of the present invention.

"IL-2" includes different species of such IL-2, for example, recombinant human IL-2, PEG-IL2 and so forth, preferably, liposomal IL-2.

Recently a new preparation of IL-2 has become available, which is covalently linked to polyethylene glycol (PEG). PEG-IL-2 has a much longer pharmacological half-life than unmodified IL-2, and the toxicity of weekly administration of PEG-IL-2 is milder. It was found that the administration of low dose IL-2 to patients whose tumor has become infiltrated with activated T cells results in expansion of those cells and more potent anti-tumor effects.

In a further embodiment the compositions comprises, liposomal IL-2 in a dose ranging from 6xl0⁵ to 6xl0⁶ U.

A second aspect of the present invention relates to any one of carcinoma cell line, lymphoma cell lines, sarcoma cell lines and melanoma cell lines. Specific cell lines are melanoma cell line selected from the group consisting of M-10, M-12, M-16 and M-24 deposited under DSM Accession Nos. ACC2481, 2482, 2483 and 2484. respectively, and any cell lines derived therefrom. The lysates of these cells and cell lines are also within ambit of the present invention.

Still further, the invention relates to the use of cell lines for the preparation of cell lysates mixtures for loading DC. The loaded DCs are than used for preparation of a pharmaceutical composition according to the invention. A third aspect of the present invention relates to a method for conferring immunity against a malignancy, in a mammalian subject in need, comprising the steps of:

(a) obtaining autologous antigen presenting cells (APC) from a mammal subject; (b) loading the APC obtained in step (a) with a mixture of allogeneic tumor cell line lysates; (c) subjecting the loaded autologous APC obtained in step (b) to maturation; and (d) administering to the subject an autologous APC loaded with mixture of at least two tumor cell line lysates or composition comprising the same. The loaded APC or the composition are administered in an amount sufficient to induce in said subject an immune response against said malignancy. The composition may optionally further comprise an IL-2 adjuvant and optionally further pharmaceutically acceptable carrier, diluent, excipient and additive.

In order to obtain APCs from a subject, particularly human patients, blood is drawn from the patient by cytopheresis, a procedure by which a large number of white cells are obtained, while other blood components are being simultaneously transferred back to the patient. The composition of the invention may be prepared from these cells and frozen in small aliquots.

As used herein, "an amount sufficient to induce effective immune response" means an amount necessary to achieve a selected result. For example, an effective amount of the composition of the invention will be conferring immunity against the treated malignant disorder.

The compositions of the present invention may be administered directly to the subject to be treated or it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration. Therapeutic formulations may be administered in any conventional dosage formulation. Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof.

Composition dosages may be any that induce an immune response. It is understood by the skilled artisan that the preferred dosage would be individualized to the patient following good laboratory practices and standard medical practices.

Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. While formulations include those suitable for oral, rectal, nasal, preferred formulations are intended for parenteral administration, including intramuscular, intravenous, intradermal and specifically subcutaneous administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy.

The compositions of the invention can be administered in a variety of ways. By way of non-limiting example, the composition may be delivered intravenously, or into a body cavity adjacent to the location of a solid tumor, such as the intraperitoneal cavity, or injected directly into or adjacent to a solid tumor. Intravenous administration, for example, is advantageous in the treatment of leukemias, lymphomas, and comparable malignancies of the lymphatic system.

As a preferred route the composition of the present invention may be administered via subcutaneous or intradermal injections in proximity to the tumor, via intralymphatic or intravenous injection.

The pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, gfycerol, propylene glycol, and liquid polyethylen glyol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of the sterile injectable solutions, the preferred method of preparation are vacuum-drying and freeze drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

Supplementary active ingredients can also be incorporated into the compositions.

Although it is not envisioned as a preferred route, the composition of the invention or its active ingredients, the IL-2 and the tumor cell lysate mixture pulsed DC, may also be orally administered, for example, with an inert diluent or with an assimilable carrier, or enclosed in hard or soft shell gelatin capsule, or compressed into tablets, or incorporated directly with the food of the diet.

In the present invention it is contemplated that systemic delivery of either or both cell lysate mixture pulsed DC and optionally IL-2 may be used. Thus, compositions comprising as active ingredient autologous antigen presenting cells (APC) pulsed/loaded with a mixture of at least two and preferably, four tumor cell line lysates may be used, as well as compositions comprising as active ingredient autologous antigen presenting cells (APC) pulsed or loaded with a mixture of at least two tumor cell line lysates in optional combination with an IL-2 adjuvant, preferably, liposomal IL-2.

The method of the invention is particularly useful in the treatment of carcinomas, lymphomas, melanomas and sarcomas, more preferably melanomas. The method of the invention may employ any of the compositions of the invention.

It is further contemplated that in practicing the invention one may wish to alter the DCs by ex vivo manipulation. In such ex vivo protocols, the biological sample, particularly a blood sample, may be drawn from the body of the human subject by methods known to the skilled artisan in the fields of oncology and surgery, and include sampling blood in well-known ways.

The method of the invention is intended to confer an immune response that results in the production of both helper and cytotoxic T lymphocytes specific for different antigens present in the employed lysate mixture and associated with the malignancy to be treated.

In a specific embodiment the method of the invention employed APC loaded with cell lysate mixture at a concentration of from 70 to 200 μg/ml, and preferably 120 μg/ml. The composition employed in the method of the invention may particularly comprise APC in an amount of from 10⁶ to 5xl0⁶ cells. Finally, the composition utilized in the method of the invention may optionally comprise IL-2 adjuvant, preferably liposomal IL-2, at a dose of 6x10⁵ to 6x10⁶ U.

The invention further provides for a method for the treatment of a malignant disorder in a mammalian subject in need. This method comprises the steps of: (a) obtaining autologous antigen presenting cells (APC) from said subject; (b) loading said APC with a mixture of allogeneic tumor cell line lysates; (c) subjecting said loaded autologous APC to maturation; and (d) administering to said subject an autologous APC loaded with mixture of at least two tumor cell line lysates or composition comprising the same. The loaded APC or the composition are administered according to the method of the invention in an amount sufficient to induce in the treated, subject an immune response against the malignant disorder.

The present invention further relates to the use of an autologous antigen presenting cell (APC) loaded with a mixture of allogeneic tumor cell line lysates, in the preparation of the pharmaceutical composition of the invention, for the treatment of a malignant disorder. More particularly, for the treatment of melanoma. According to a specific embodiment, the composition comprises APC in an amount from 10⁶-5xl0⁶ cells. This composition preferably further comprised liposomal IL-2. According to a particular embodiment, the composition applied by the method of the invention comprises liposomal IL-2 dose of 6 xlO⁵ to 6 xlO⁶ U. liposomal IL-2 dose of 6 xlO⁵ to 6 xlO⁶ U.

As used in the specifications and the appended claims and in accordance with long-standing patent Law practice, the singular forms "a" "an" and "the" generally mean "at least one", "one or more", and other plural references unless the context clearly dictates otherwise. Thus, for example "a cell", "a peptide" and "an adjuvant" include mixture of cells, one or more peptides and a plurality of adjuvants of the type described; and reference to "IL-2" includes different species of such IL-2, preferably but not limited to lyposomic IL-2.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The contents of all publications quoted to herein are fully incorporated by reference. The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Examples

Experimental procedures

General methods in molecular biology

A number of methods of the art of molecular biology are not detailed herein, as they are well known to the person of skill in the art. Such methods include PCR cloning, expression of cDNAs, analysis of recombinant proteins or peptides, transformation of bacterial and yeast cells, transfection of mammalian cells, and the like. Textbooks describing such methods are e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology ,by F. M. Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al. (eds.) 3rd ed. John Wiley & Sons; ISBN: 0471137812, 1995. These publications are incorporated herein in their entirety by reference. Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art.- See e.g., Current Protocols in Immunology, Coligan et al. (eds), John Wiley & Sons. Inc., New York, NY. Preparation of lysate-loaded dendritic cells

1. Peripheral Blood Dendritic Cell Isolation and Culture

Peripheral blood was drawn by either venous puncture or cytopheresis.

The method of choice, unless contraindicated is cytopheresis. Using this method, large numbers of mononuclear cells can be obtained, which can be frozen and used for the whole vaccination protocol.

Venous puncture: 60-80 ml blood is drawn 7-9 days before each vaccination, using preservative-free heparin.

Cytopheresis: The protocol used is as described by Thurner et al. [J.

Immunol. Methods 223:1-15 (1999)] for cytopheresis for clinical application. As cell separator, Cobe Spectra (Cobe BCT, Lakewood, CO, USA) is used, with the white blood cell set and the MNC program. As an anticoagulant, acid-citrate-dextrose (ACD-A) is used. The procedure takes about two hours, during which the patient sits on an armchair, with both his arms connected through peripheral veins to the cell separator. Side effects are unusual. Patients with low blood pressure or impaired cardiac function can suffer from a drop in blood pressure, and therefore are excluded from cytopheresis.

The cytopheresis product is filled into a 600 ml culture flask by using a perfusor syringe, then PBS containing 10% anticoagulant is added to a final volume of 480 ml. After dilution with PBS/10% ACD-A PBMC are isolated by centrifugation on Ficoll (density 1.077 g/ml) at 460xg and room temperature for 30 min (15 ml Ficoll are layered under 30 ml diluted cytopheresis product). Cells are then washed three times in PBS and re- suspended in culture medium. 2. Dendritic cell growth and loading with antigen

Ficoll- separated cells are re-suspended in culture medium, and incubated in T25 tissue culture flasks, 40-50xl0⁶ mononuclear cells per flask, in 5 ml medium. Non-adherent cells are discarded after 90 minutes, and after 5 days of growth in the presence of 1000 U/ml GM-CSF (Leucomax, Novartis) and 500 U/ml IL-4 (R&D Systems, MN, USA), the cells are washed, and re- suspended in complete medium without serum. IL-4 (500 U/ml), β-2 microglobulin (3 μg/ml, Sigma) and the lysate (30 μg/ml of each extract, i.e. 120 μg lysate/ml) are added. After 4 hours, 2% autologous serum, tumor necrosis factor-α (TNFα, lOOOU/ml, R&D) and prostaglandin E₂ (PG, Sigma, 1 μg/ml) are added for additional 48 hours of incubation.

3. Preparation of lysates from melanoma cell lines

Melanoma cell lines that grow in suspension are collected, and washed 3 times with saline (TevaMedical, for i.v. injection, pyrogen-free). Plastic- adherent melanoma cells are detached from culture flasks by brief (1 minute) treatment with a small volume of 0.5% trypsin/EDTA and immediate dilution in serum-containing medium. The cells are washed 3 times with normal saline, and suspended in saline at a concentration of 15- 20xl0⁶/ml. The tubes are frozen (liquid nitrogen) and thawed (37°C bath) consecutively as fast as possible at least six times and preparation is sonicated in a water bath for 5 minutes. Destruction of all the cells is confirmed by microscopical examination. The cell extract are centrifuged at 3,000xrpm for 15 minutes the supernatant collected, diluted 1:1 with saline and filtered through a 0.2 μm filter. After determination of protein content (Bradford method), the cell extract are frozen at -70°C.

4. Characterization of melanoma cell line lysates

Characterization of cell lysates: Cells from human metastatic melanoma biopsies were isolated, cultured and maintained in vitro under strict sterile conditions, and tested for mycoplasma, hepatitis B and C and HIV before harvesting for the extract preparation.

Selection of the melanoma lines used for the preparation of the lysate mixture was based on antigenic characterization of the melanoma cells, their ease of growth in vitro, and their ability to induce interferon γ (IFNγ) production and anti-melanoma T cell activity by lymphocytes in an ex-vivo co-culture of melanoma lysate-loaded autologous DC and autologous T lymphocytes. The lines used for lysate preparation were designated M-10, M-12, M-16, and M-24, and have been characterized for HLA A, B and C, and for melanoma antigens, as described below.

A pool containing equal amounts of protein from the four lysates is prepared, and used as the standard lysate mixture for DC loading, as described below.

5. Preparation of liposomal IL-2

Multilamellar liposomes (mean diameter 1-2 μm), composed of dimyristoyl phosphatidylcholine/dimyristoyl phosphatidylglycerol (mole ratio 9:1) encapsulating recombinant human IL-2 is prepared as described [Kedar, et al, J. Immunother. 16:47-59 (1994); J. Immunother. 23:131-45 (2000)]. Preparations are tested for sterility (for 14 days). Toxicity is tested in rabbits following subcutaneous administration. Encapsulation efficiency is tested by bioassay (80-90% encapsulation is expected).

Clinical protocol

The clinical protocol includes two-arm vaccination trial with melanoma antigen-pulsed autologous DC as follows:

Arm A: Melanoma lysate-pulsed dendritic cells.

Arm B: Melanoma lysate-pulsed dendritic cells plus local administration of liposomal IL-2 as an immuno adjuvant. 1. Patient Selection Inclusion criteria:

1. Measurable soft tissue or visceral metastases (AJCC stage IV).

2. Failure or unmaintained partial response (by local institution standard protocol of chemoimmunotherapy of metastatic melanoma).

3. Patients unwilling to receive the local institutional chemotherapeutic protocol.

4. Karnofsky performance status > 80 (Normal activity with effort).

5. He atocrit >25% and WBC >3000.

6. Positive cutaneous DTH to at least 1 of 5 recall antigens.

7. Informed consent of the patient.

Exclusion Criteria:

1. Administration of cytotoxic drugs or extensive radiotherapy within the preceding 1 month.

2. Brain metastases.

3. Concurrent malignancy (other than skin cancer, carcinoma in situ of cervix and early stage prostate cancer).

4. Active serious infection.

5. Patient's wish to withdraw from the study at any stage.

2. Patient randomization

Patients are randomized to one of the treatment groups and there is no stratification factors.

3. Brief outline of vaccination protocol

Arms A+B: Injection of melanoma lysate-loaded DC intradermally at 3 sites (lxl0⁶ per site) in close proximity to the regional lymph nodes. Arm B: Subcutaneous injection of liposomal IL-2 (6xl0⁵ IU) at the site of DC injection. The process is repeated weekly for one month and then at monthly intervals for 6 months. Responding patients are vaccinated 3 additional times at 3 month intervals.

4. Toxicity

There are no anticipated toxicities associated with the components of the vaccine. Liposomal IL-2 is administered at very low doses. Expected side effects include mild erythema and pain at injection site, and minor flu-like symptoms.

5. Clinical evaluation

Clinical efficacy is assessed by measurements of changes in diameter of radiographic or CT scan findings. Twenty to forty ml of blood are drawn before treatment, once in the course of the treatment, and after its completion. The blood is used for biochemistry, for measurement of antinuclear, anti-DNA, anti-microsomal and anti-thyroid antibodies and for immunological evaluation (see below).

6. Immunological evaluation

(a) Delayed type hypersensitivity (DTH) is measured before the first vaccination, once in the course of treatment (after the 5^th vaccination), and after the treatment is completed, using the following sensitizers: DC with and without lysate, lysate alone, Melan-A (5 μl), tyrosinase (1.25 μl), gplOO (1.25 μl), and saline. Erythema and induration are measured 24 and 48 h after injection. The maximal area of induration serves as the primary endpoint of interest.

(b) Elicitation of a specific immune response is measured by IFN-γ production by specific CD8+ lymphocytes, using the ELISPOT assay, at 3 time points: before vaccination, once in the course of treatment (after the 5^th vaccination), and after treatment is completed. The cells are stimulated in the following way: mononuclear white blood cells from the patient are incubated for one week either with irradiated autologous melanoma cells or allogeneic lysate-loaded autologous DC. CD8+ cells then are separated using magnetic beads (Miltenyi, Biotec, Germany), and re-stimulated on IFN-γ-ELISPOT plates for further 48 hours, before development of spots (U-CyTech, the Netherlands). Each experiment includes a positive control, consisting of normal mononuclear cells (from a buffy coat frozen in small aliquots), stimulated for 48 hours with 100 U/ml IL-2.

(c) Staining and analysis of Ag-specific CTLs from peripheral blood. MHC-peptide tetrameric complexes are molecules designed in vitro to imitate a small but very specific antigenic fraction of tumor cells. Thus, CTLs can be recognized by tetramers in a very specific manner, depending upon the antigen for which the tetramer has been designed. Recombinant tetramers that are constructed as described below, with the relevant melanoma associated peptides and labeled with fluorochromes, are used for staining MNC after Ficoll separation. Double staining and FACS analysis of the CD8+ T cell sub-populations are performed. Results presented as percentage of Ag-specific CTLs from total CD8+ peripheral blood lymphocytes.

(d) Characterization of melanoma associated antigens.

Based on the inventors' previous results, biopsies from the patients are tested by RT-PCR for gplOO, MAGE3, MARTI and tyrosinase, in order to look for a possible correlation with disease outcome and immunological parameters such as DTH (delayed type hypersensitivity and in vitro immunological assays.

Data analysis

DTH and other in vitro immunological assays are expected in a higher percentage of treated patients. Thus, immunological evaluation provided with minimal resources a means to determine whether IL-2 has an effect in addition to the DC vaccine. Statistical analyses are performed using a nonparametric test, e.g. the Wilcoxon signed rank test. The SPSS program is used to run the analyses.

Example 1

Autologous melanoma cell vaccine as post-operative adjuvant treatment for high-risk melanoma patients (AJCC stage III and IV)

To evaluate the overall survival (OS) and disease free survival (DFS) of melanoma patients that were treated with an autologous melanoma cell vaccine, administered as a post-operative adjuvant, thirty patients were tested. This study group included patients with metastatic melanoma (14- AJCC stage III, 16-AJCC stage IV), with a median follow up of 25 months, and all patients were disease free after resection of their metastases. The treatment consisted of the administration of eight doses of a vaccine made of 10-25xl0⁶ autologous melanoma cells either released from the surgical specimen or grown in cell cultures. Tumor cells were conjugated with the hapten dinitrophenyl (DNP), mixed with Bacille Calmete Guerin (BCG) and irradiated with 110 Gy. Low dose cyclophosphamide, 300 mg/m², was administered before the first two doses of the vaccine [Berd et al., Cancer Res. 46:2572-7 (1986)].

The median actuarial OS for the whole group was 39 months with a median DFS of 12 months. Both DFS and OS were found to be correlated with intensity of evolving delayed type hypersensitivity (DTH) to subcutaneous injection of unmodified melanoma cells. Patients with a DTH reaction of 10 mm (strong DTH) had a mean DFS of 28 months and OS of 39 months (median OS has not been reached). In contrast, patients with a negative or weak DTH had a median DFS of 10 months and a median OS of 21 months (p=0.015 and 0.03). These differences were most prominent for AJCC stage IV patients, with a median DFS of 40 months for strong DTH compared to 6 months for negative/weak DTH (p=0.004). In conclusion, the adjuvant administration of autologous melanoma vaccine was associated with improved disease-free and overall survival in selected patients who successfully attained anti-melanoma reactivity as detected by positive DTH reactions to injected unmodified melanoma cells.

Example 2

Autologous melanoma cell vaccine alone or combined with systemic interleukin-2 (IL-2) for metastatic melanoma

Autologous melanoma cell vaccine has been administered to 15 patients with active metastatic melanoma. Survival data have not yet been summarized, but 7 of these patients developed strong DTH reaction and one of them achieved partial tumor regression. A pilot study on the use of autologous melanoma cell vaccine combined with intravenous, high dose interleukin-2 is being conducted. So far, 4 patients were treated. One achieved complete response, and 2 achieved partial responses.

Example 3

Human melanoma cell lines

As a corollary of the vaccination program, over 100 melanoma cell cultures were grown in vitro, as listed in Table 1. Fresh and sterile tumor specimens were procured. For large tumor bulks, cells were extracted by mechanic and by enzymatic dissociation with collagenase and DNAse (Sigma, St. Louis, Mo, USA), frozen in a controlled rate freezer and stored in liquid nitrogen in a medium containing 2.5% human albumin and 20% DMSO. When mechanical dissociation of the tumor was not possible, either because of small tumor size or dense connective tissue, tumor was cut to pieces of less than lmmc, put on a 60 mm² plastic dish (Nunc) and glued with drops of fetal calf serum (Gibco BRL). Twenty four hours later culture medium was added to the dish. When cell growth was demonstrated at the periphery of tumor pieces, passage into a culture bottle was performed. All cultured cells were stained with monoclonal antibodies S-100 and HMB-45 (Zymed Inc. San Fransisco, Ca. USA) to assure their melanocytic progeny.

Four melanoma-associated antigens were determined for 20 of the cell lines. These include tyrosinase, gp-100, MAGE-3 and MARTI. The antigens were determined using RT-PCR technique. Additionally, HLA class I and II typing was performed for several lines. Clinical information is available, concerning vaccination outcomes and course of disease (including radiosensitivity of tumor and response to chemotherapy in several cases).

Fifty cultures were maintained for over a year. Some melanoma cell lines developed by the inventors were already characterized by known melanoma-associated monoclonal antibodies (HMB-45, S-100) and by RT- PCR.

Table 1: List of tumor cell lines

Unless remarked otherwise - the origin is melanoma. Abbreviations: SC- subcutaneous; LN-lymph node Example 4

Characterization of tumor-associated antigens in melanoma cell lines derived from patients treated by autologous tumor vaccines

To investigate the possible correlation between expression of melanoma associated antigens (MAA), HLA-A2 molecules and prognosis of treated patients, identification and characterization of melanoma-associated antigens (MAA) was performed in melanoma patients undergoing autologous tumor vaccination.

The experiments were performed by RT-PCR and revealed a pattern of specific MAA (tyrosinase, gplOO, MAGE3, MARTI,) as well as the relevant class I MHC antigen (HLA-A2). In addition, delayed type hypersensitivity (DTH) was tested in order to evaluate the clinical response to vaccination.

The results hint to a correlation between the expression of class I MHC HLA-A2 molecules, the MAA tyrosinase and gplOO, and a better long-term prognosis in these patients.

Table 2

Characterization of melanoma lines

ND - not done.

HLA Class I was defined by serological tests, and tyrosinase, gp-100, MAGE-3, MART-1 were determined by RT-PCR. DTH-delayed hypersensitivity response of the patient from whom the melanoma was isolated. Example 5

DC growth and maturation

In vitro experiments were performed, to determine optimal conditions for DC preparation.

Comparison between DC grown from fresh heparinized peripheral blood and DC grown from buffy coat:

Blood was obtained either from volunteers (75 ml blood taken with preservative-free heparin) or from the Blood Bank of Hadassah Hospital (15 ml of buffy coat obtained from 450 ml of blood taken with acid-citrate- dextrose (ACD). The buffy coat was stored overnight at room temperature, and was used after verifying that it was hepatitis- and HIV-negative. Blood was separated on a Ficoll gradient and incubated at 37°C in RPMI medium containing 10 mM hepes, 1 mM non essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin and 2% autologous human serum (complete medium, CM). After 90 minutes, the nonadherent cells were discarded, and the adherent cells were further grown for 6 days in CM in the presence of 1000 U GM-CSF and 800 U IL-4, or GM-CSF alone. The cells, obtained from both sources, had the characteristics of immature DC, as determined by flow cytometry (1-3% CD14+, 17-19% CD83%), and by morphology (large cells with irregular outlines and few longer processes or veils, see also Thurner et al., 1999). Since many more cells are obtained from buffy coats, all further experiments with blood from healthy donors were performed with this source.

In order to allow ex vivo growth, cells require a source of protein. Fetal calf serum, the most common source of protein, is not recommended for human use, and causes non-specific stimulation of cells. Therefore several alternatives were evaluated, such as AIM-V serum-free medium (Gibco) and human serum. Best results were obtained with autologous plasma and autologous serum (obtained from plasma, by CaGb treatment for 30 min at room temperature). In one typical experiment, the yield of DC, as defined by morphology, for cells grown in 2% plasma or 2% serum, was 11% and 7.2% respectively.

Optimization of cytokine addition

DC were grown in GM-CSF and IL-4. Both cytokines were added on day 0 or day 1 ("day 0" is meant during culturing of the DC). IL-4 was added again on day 3 and on day 5 together with maturation factors (see below). Different concentrations of IL-4 and GM-CSF were tested. 500 U/ml IL-4 and 1000 U/ml GM-CSF gave optimal results, and this protocol was used in all further experiments.

Maturation of DC

After 5 or 6 days of incubation in the presence of GM-CSF and IL-4, maturation of DC was induced. Three different protocols for maturation were evaluated: tumor necrosis factor-α (TNF, 1000 U/ml), TNF + prostaglandin E₂ (PG, 1 μM) [Rieser et al, J. Exp. Med. 9:1603 (1997); Jonouleit et al, Eur. J. Immunol. 27:33135 (1997)], or poly rib oinosinic polyribocytidylic acid (poly (I:C), 12.5 μg/ml) [Cella et al, J. Exp. Med. 189:821 (1999); Verdijk et al, J. Immunol. 163:57 (1999)]. Maturation was less effective with TNF than after the addition of TNF+PG. Poly (I:C) seems to have a similar effect to TNF +PG. Morphologically, mature DC are large and mostly round cells with many motile veils.

Table 3 Maturation of DC

Functional parameters of maturation

To determine if functional differences are induced upon induction of maturation by the different protocols, mixed leukocyte reactions and cytokine production were measured. The experiments were performed in 96-well plates; each well contained 2xl0⁶ peripheral blood lymphocytes and allogeneic DC at a given ratio. Supernatants were collected after 48 hours (for the measurement of IFN-γ production), and the plates were incubated for 3 more days; ³H-thymidine was added for the last 18 hours, and the cells harvested for measurement of proliferation. DC derived from TNF+PG-treated cultures induced the strongest proliferation and IFN-γ production. Highest levels of IL-12 mRNA were also obtained after maturation with TNF+PG. Thus, this protocol was chosen for all further experiments.

Stability test

A significant characteristic of mature DC is their ability to maintain a stable phenotype in the absence of cytokines. DC matured with TNF+PG were washed after maturation on day 7, and maintained for 2 more days (until day 9) in the absence of cytokines. Percentages of CD83+, CD86+ and HLA-DR did not decrease, and were even somewhat increased. These results confirm that stable mature DC were obtained.

Freezing of DC

Freezing of DC will be required during clinical treatment, since in most cases it is expected that large numbers of cells will be obtained by cytopheresis, grown into DC, and loaded with melanoma antigen. Most cells will be frozen, and used later for all vaccinations of the given patient. It was therefore necessary to develop a freezing protocol using an appropriate source of protein. Freezing cells with 10% autologous or AB+ serum and 10% DMSO in CM gave a good recovery of DC, and cells retained morphology and phenotype of mature DC.

Lysate loading of DC

To test the efficacy of DC loading with lysates, blood samples from healthy donors and from high risk melanoma patients vaccinated with autologous melanoma cells, who had developed a strong DTH response were used. Immature DC obtained from the patients were loaded with lysates (prepared as described below), and allowed to mature for further 2 days in the presence of TNF+ PG.

DC loaded with lysate after maturation induced lower levels of IFN-γ production than DC loaded before maturation, suggesting that uptake of the lysate is required for presentation. Autologous lymphocytes were incubated with DC for 7 days, followed by restimulation. Lymphocyte response was measured by IFN-γ secretion (by ELISA or ELISPOT) and by cytotoxic activity.

DC were loaded with a series of lysates (70 μg/ml). There was variability in the response to DC loaded with the different lysates and, as expected, there was also variability between patients. However, several lysates seemed better stimulators than others. Four lysates, designated M-10, M- 12, M-16, M-24, that induced good IFN-γ production in several patients and healthy buffy coats, were mixed in equal amounts of protein. DC were loaded with different concentrations of the lysate mixture and the effect on activity determined after maturation. It was found that 30 μg/ml of each lysate (i.e., 120 μg/ml total) induced an optimal phenotype of mature DC, and stimulated autologous lymphocytes for highest IFN-γ production.

Autologous DC loaded with alogeneic cell lysates induce IFN-γ secretion by patient lymphocytes

IFN-γ secretion by lymphocytes from several patients, stimulated with autologous DC loaded with different lysates, was measured. The lymphocytes of the two patients tested so far with the lysate mixture- loaded DC produced high levels of IFN-γ: The fact that strong autologous induction of IFN-γ was observed (Pt-16 and M-16, Pt-77 and M-77, not shown), suggests that the response was due to specific melanoma stimulation.

Table 4

IFN-γ (pg/ml) after stimulation with DC+lysate - DC alone

Autologous DC loaded with alogeneic cell lysates induce IFN-γ secretion by patient lymphocytes - ELISPOT assay

Secretion of IFN-γ by CD8+ and CD8-lymphocytes in response to lysate mixture-loaded autologous DC was determined by ELISPOT. The assay was performed using U-CyTech (the Netherlands) reagents, and according to the manufacturer's suggested protocol, with slight variations.

CD8+ cells were >95% CD8+ and CD8- cells were about 75% CD4+, and 3% NK cells. Secretion of IFN-γ by CD8+ and CD8- lymphocytes from 19 patients and 5 healthy donors was measured during the first stimulation cycle in the presence of DC loaded with the lysate mixture. The results, summarized in Figure 1, show that the response of the patients varied; in 2 patients, only CD8+ cells responded, and in 7 patients only CD8- cells responded. In 9 patients, both cell populations responded to DC loaded with the lysate mixture, and one patient did not respond. Thus, in 58% of the patients (11/19) CD8+ cells responded to the lysate; this is in contrast to the cells from the healthy donors, where no CD8+ responded to the lysate. In two cases CD8- cells from healthy donors significantly responded to the lysate (not shown). Three patients were evaluated twice, several months apart. In two of these patients, the results at the two timepoints were similar; in one case, CD8+ cells converted to positive after 7 months.

Whenever autologous tumor was available, the IFN-γ response to DC loaded with autologous and allogeneic lysate was compared after one stimulation cycle in vitro (Figure 2). CD8⁺ cells from 4 out of 8 patients tested responded to the allogeneic lysate mixture, and 5 out of 8 responded to the autologous lysate; of these, 2 responded significantly to both types of lysate, and 2 responded only to the autologous lysate. CD8- lymphocytes from 5 out of 6 donors tested responded to the allogeneic lysates, whereas 2 out of 6 responded also to the autologous lysate.

Specific cytotoxicity of CTL's against target melanoma cells, induced by cell lysates loaded DC

Cytotoxicity was measured by lysis of ⁵¹Cr-labeled melanoma targets at an effector target ratio of 100:1. Specific lysis of autologous melanoma targets was low, and some lysis of allogeneic melanoma targets was also detectable, as shown in Table 5.

Table 5

Percent lysis of ⁵¹Cr-labeled melanoma targets

These results show that DC loaded with several melanoma lysates can present antigen to lymphocytes for IFN-γ production and for cytotoxic activity. Cells from several additional patients, loaded with the individual lysates, and with the mixture of lysates (M-10+12+16+24) are currently tested. Example 6

Generation of functional class I MHC-peptide complexes for the assessment of Ag-specific CTL in peripheral blood

Peptide-MHC Class I tetrameric complexes are a novel and unique tool to quantify specific CTLs generated against peptides. The human genes that encode the human MHC class I molecule HLA-A2, as well as human β-2 microglobulin that is an integral part of the MHC class I complex were cloned by the inventors. This is the most frequent HLA haplotype that is involved in the presentation of cancer-related peptides. The genes were cloned by RT-PCR, sequenced, and then subcloned into expression vectors to facilitate efficient production in E. coli. The expression system used is based on the T7 promoter - T7 RNA polymerase system; the components for generation of the complex, i.e. HLA-A2 and β-2 microglobulin, are expressed on two different plasmids. However, a construct in which the two components are expressed as a single polypeptide chain by connecting the HLA-A2 and β-2 microglobulin with a flexible linker was also developed. Refolding experiments of recombinant class I complexes with melanoma peptides have already been performed using established protocols of a redox-shuffling buffer system.

After refolding, recombinant complexes were purified. Functional and structural analysis of the recombinant MHC-peptide complexes revealed that they are functional, tested on CTL clones with specificity for the appropriate peptide used to generate the MHC-peptide complex. These recombinant soluble single chain MHC-peptide tetramers are used with unique T-cell receptor like specificity to melanoma associated peptides, to analyze populations of lymphocytes of peripheral blood of vaccinated melanoma patients. Example 7

Liposomal IL-2 as vaccine adjuvant

It has been shown in studies in mice, that liposomal IL-2 proved a more potent vaccine adjuvant than soluble IL-2, when co-administered with influenza viral antigens [Babai, et al, Vaccine 17:1223-8 (1999) and Vaccine 17:239-50 (1999)] and tumor cell vaccines [Kedar et al, J. Immunother. 23:131-45 (2000)]. In a clinical trial in young (18-40 yr) healthy volunteers, a trivalent liposomal influenza vaccine containing IL-2 (6xl0⁵ to 6xl0⁶ U), administered once intramuscularly, proved to be significantly more effective than two standard commercial vaccines [Kedar, et al, in prep.]. The only side effect observed was mild local pain (that persisted for 1-3 days) in a higher proportion of patients that received the liposomal IL-2.

Claims

Claims:

1. A pharmaceutical composition for inducing an immune response directed against malignancy in a mammalian subject, comprising:

(a) autologous antigen presenting cells (APC) loaded with a mixture of at least two tumor cell line lysates;

(b) optionally an IL-2 adjuvant; and

(c) optionally further pharmaceutically acceptable carrier, diluent, excipient and additive.

2. The composition according to claim 1, wherein said malignancy is any one of carcinomas, lymphomas, melanomas and sarcomas.

3. The composition according to claim 2, wherein said malignancy is melanoma.

4. The composition according to claim 3, wherein said mammal is human.

5. The composition according to claim 1, wherein said APC are autologous dendritic cells (DC).

6. The composition according to claim 5, wherein said DC, after being loaded with said lysates, are subjected to maturation by treatment with any one of tumor necrosis factor (TNF), TNF+prostaglandin E2 (PG) and poly rib ocy tidy lie acid (poly (1:C)).

7. The composition according to claim 6, wherein said DC are matured by treatment with TNF+PG.

8. The composition according to any one of claims 1 to 7, wherein the

APC loaded with the tumor cell-lysate mixture express peptides derived from said cell-lysate mixture in context of both MHC Class I and Class II molecules.

9. The composition according to claim 8, wherein said immune response results in the production of both helper and cytotoxic T lymphocytes specific for different antigens associated with said malignancy present in said lysate mixture.

10. The composition according to claim 9, wherein said tumor cell line lysate is prepared from allogeneic tumor cell line.

11. The composition according to claim 10, wherein said cell line is a melanoma cell line selected from the group consisting of M-10, M- 12, M-16 and M-24 deposited under DSM Accession Nos. ACC2481, 2482, 2483 and 2484, respectively, or any cell lines derived therefrom.

12. The composition according to claim 1, wherein said mixture of tumor cell line lysates comprises at least two allogeneic tumor cell line lysates according to claim 11.

13. The composition according to claim 12, wherein said tumor cell lines are allogeneic melanoma cell lines expressing at least one of the melanoma- associated antigens (MAA) tyrosinase, gp-100, MAGE-3 and MART-1.

14. The composition according to any one of claims 1 to 13, wherein said APC are loaded with cell lysates mixture at a concentration of from 70 to 200 μg/ml.

15. The composition according to claim 14, wherein said APC are loaded with cell lysate mixture at a concentration of 120 μg/ml.

16. The composition according to claim 1, wherein said IL-2 is liposomal IL-2.

17. The composition according to claim 16, wherein said liposomal IL-2 is presented at 6xl0⁵ to 6xl0⁶ million U.

18. The composition according to any of the preceding claims for the treatment of a malignant disorder.

19. The composition according to claim 18, wherein said malignant disorder is melanoma.

20. A melanoma cell line selected from the group consisting of M-10, M- 12, M-16 and M-24 deposited under DSM Accession No. ACC2481, 2482, 2483 and 2484, respectively, and any cell line derived therefrom.

21. Use of an autolgous melanoma cell line according to claim 20 and cell lysates derived therefrom, in the preparation of pharmaceutical composition according to any one of claims 1 to 17, for the treatment of melanoma.

22. A method for conferring immunity against a malignancy in a mammalian subject, comprising the steps of:

(a) obtaining autologous antigen presenting cells (APC) from said subject;

(b) loading said APC with a mixture of allogeneic tumor cell line lysates;

(c) subjecting said loaded autologous APC to maturation; and

(d) administering to said subject the autologous APC loaded with mixture of at least two tumor cell line lysates or composition comprising the same, in an amount sufficient to induce in said subject an immune response against said malignancy, optionally together with an IL-2 adjuvant.

23. The method according to claim 22, wherein said malignancy is selected from the group consisting of carcinomas, lymphomas, melanomas and sarcomas.

24. The method according to claim 23, wherein said malignancy is melanoma.

25. The method according to claim 24, wherein said mammal is human.

26. The method according to claim 22, wherein said APC are autologous dendritic cells (DC).

27. The method according to claim 26, wherein said DC after being loaded with said cell lysates, are subjected to maturation by treatment with any one of tumor necrosis factor (TNF), TNF+prostaglandin E2 (PG) and polyribocytidylic acid (poly (1:C)).

28. The method according to claim 27, wherein said DC are matured by treatment with TNF+PG.

29. The method according to claim 28, wherein the APC loaded with the cell-lysate mixture express peptides derived from said cell- lysate mixture in a context of both MHC Class I and Class II molecules.

30. The method according to claim 29, wherein said immune response results in the production of both helper and cytotoxic T lymphocytes specific for different antigens associated with said malignancy, present in said lysate mixture.

31. The method according to claim 30, wherein said tumor cell line lysate is prepared from allogeneic tumor cell line selected from the group consisting of M-10, M-12, M-16 and M-24 deposited under DSM Accession Nos. ACC2481, 2482, 2483 and 2484, respectively, or any cell lines derived therefrom.

32. The method according to claim 22, wherein said mixture of tumor cell line lysates comprises at least two allogeneic tumor cell line lysates according to claim 31.

33. The method according to claim 31, wherein said tumor cell lines are allogeneic melanoma cell lines expressing at least one of the melanoma associated antigens (MAA) tyrosinase, gp-100, MAGE-3 and MART-1.

34. The method according to any one of claims 22 to 33, wherein said APC are loaded with cell lysate mixture at a concentration of from 70 to 200 μg/ml.

35. The method according to claim 32, wherein said APC are loaded with cell lysate mixture at a concentration of 120 μg/ml.

36. A method for the treatment of a malignant disorder in a mammalian subject in need, comprising the steps of:

(a) obtaining autologous antigen presenting cells (APC) from said subject;

(b) loading said APC with a mixture of allogeneic tumor cell line lysates;

(c) subjecting said loaded autologous APC to maturation; and (d) administering to said subject the autologous APC loaded with mixture of at least two tumor cell line lysates or composition comprising the same according to any one of claims 1 to 17, in an amount sufficient to induce in said subject an immune response against said malignant disorder.

37. Use of an autologous antigen presenting cell (APC) loaded with a mixture of allogeneic tumor cell line lysates, in the preparation of a pharmaceutical composition according to any one of claims 1 to 17, for the treatment of a malignant disorder.

38. The use according to claim 37, wherein said composition comprises APC in an amount from 10⁶-5xl0⁶ cells.

39. The use according to claim 38 wherein said composition comprises liposomal IL-2.

40. The use according to claim 39 wherein said composition comprises 6 xlO⁵ to 6 xlO⁶ U of liposomal IL-2.