Ex vivo, fast and efficient process to obtain activated antigen-presenting cells that are useful for therapies against cancer and immune system-related diseases

Abstract

The present invention relates to an ex vivo, fast and efficient process to obtain activated antigen-presenting cells that are useful for therapies against cancer and immune system-related diseases. At the same time, it is related to a cellular composition that contributes to stimulate the activated antigen-presenting cells to induce a specific immune response against tumors in patients with cancer or other pathologies involving immune responses.

1 . A method to generate vaccines to stimulate the immune system, wherein the peripheral blood mononuclear cells—preferably monocytes—are induced to differentiate into APCs, preferably DCs, by the stimulation of said monocytes in an in-vitro culture within less than 3 days from the start-up of culture, using cytokines, growth factors and/or a mixture of lysate or extracts of tumor cells, then separate said APCs and mixed with adjuvant. 
     
     
         2 . A method to generate vaccines according to  claim 1 , wherein the peripheral blood mononuclear cells—preferably monocytes—are induced to differentiate into APCs, preferably DCs, by the stimulation of those monocytes in in vitro culture with cytokines in a first stage and at least one growth factor, and with a growth factor and/or a mixture of lysate or extracts of tumor cells in a second stage, then separate said APCs and mixed with adjuvants; where said first and second stages of manufacturing take place within 48 hours from the start of culture and where each stage extends for about 24 hours. 
     
     
         3 . A method to generate vaccines according to  claim 1 , wherein the peripheral blood mononuclear cells are incubated at a concentration between 10 4  and 10 10  cells, preferably between 10 5  and 10 7  and ideally around 13×10 6  cells by ml in a culture medium free from bovine fetal serum. 
     
     
         4 . A method accordingly to  claim 1 , wherein the peripheral blood mononuclear cells were kept under culture in the presence of 100 to 800 U/mi, preferably between 400 and 600 and ideally around 500 U/ml of cytokines, like interleukins and preferably IL-4; and in the presence of 500 to 1,100, preferentially between 700 and 900 and ideally around 800 U/ml of at least one growth factor, most preferably GM-CFS, wherein the incubation has a minimum extension of at least 10 hours, preferably more than 18 hours and ideally for about 22 hours. 
     
     
         5 . A method accordingly to  claim 1 , wherein the mononuclear cells—after the incubation period described in  claim 3 —were incubated for at least further 10 hours, preferably over 18 hours, and ideally for about 24 hours, in medium alone or supplemented with a growth factor, preferably TNF-α, or supplemented with the mixture of tumor cells lysate described above or with both components at the same time; where the mixture of tumor cells lysate might be also combined with other pro-inflammatory cytokines such as IFN-γ, IL-6, IL-1β or such other factors as prostaglandin E2, CpG, thermal shock proteins, Toll-like receptor (TLR) ligands or other factors to activate DCs maturation. 
     
     
         6 . A method accordingly to  claim 1 , wherein the growth factors, such as TNF-α, is used at a concentration between 100 pg/ml to 100 ng/ml, preferably between 1 ng/ml to 50 ng/ml, more preferably between 2 ng/ml to 20 ng/ml and ideally around 10 ng/ml. 
     
     
         7 . A method to generate vaccines according to  claim 1  wherein the mixture of lysate of tumor cells used is made up by at least two cell lines obtained from tumors from metastatic tissue deriving from patients with cancer; preferably the tumor cells are selected from malign melanomas and correspond to three cell lines, preferably deriving from gland metastasis; optionally the lysate may be obtained from fresh tumor tissue deriving from patients with different types of cancer combined or not with lysate of allogeneic tumor cell lines of the same tumor type; lysate may be mixed or not in equal parts or used individually according to the tumor type to be treated, where they are used in the cultivation at a concentration between 1 μg/ml and 10 mg/ml, preferably 10 μg/ml to 1 mg/ml and ideally around 100 μg/ml. 
     
     
         8 . A method to generate vaccines according to  claim 1 , wherein said adjuvant may be KLW (hemocyanins deriving from the Keyhole limpet mollusk). 
     
     
         9 . A method to generate vaccines according to  claim 1 , where the adjuvant used is KLH (hemocyanin derived from the Keyhole limpet mollusk). 
     
     
         10 . A method to generate vaccines according to  claim 1 , wherein the lysate or the mixture of tumor cells lysate are obtained by incubating the cells between 15 minutes and 4 hours at temperature between 39 and 44° C. in a serum-free culture medium; then cells and/or tissues are lysated, to which effect they are subject to instantaneous freezing and thawing cycles, and then thawed to a temperature between 35 and 40° C.; later, the lysate or extract obtained is subject to ultrasound homogenization (30 to 40 KHz); finally each lysate or extract is irradiated to doses ranging between 40 and 120 Gy. 
     
     
         11 . A method to generate APCSs, preferably DCs, according to  claim 1 , wherein said method consists in inducing peripheral blood mononuclear cells—monocytes preferably—trough stimulation in an in vitro cultures in less than 3 days from culture start, where cytokines, growth factors and/or a mixture of lysate or extracts of tumor cells are used. 
     
     
         12 . An extract of tumor cells, wherein it is manufactured by the mixture of at least two cell lines derived from tumors from metastatic tissue of patients with cancer; preferably tumor cells are selected from malign melanomas and preferably corresponding to three cell lines, preferably derived from gland metastasis; optionally the lysate is made from fresh tumor tissue obtained from patients with different kinds of cancer combined or not with lysate of allogeneic tumor cell lines of the same tumor type. 
     
     
         13 . A pharmaceutical composition, wherein said composition comprises APCs, DCs preferably, with qualities of mature DCs developed in vitro in less than 3 days with the capacity to induce the immune response; and an adjuvant, preferably KLH (hemocyanin deriving from the Keyhole limpet mollusk).