Means and methods for active cellular immunotherapy of cancer by using tumor cells killed by high hydrostatic pressure and dendritic cells

Abstract

Disclosed are pharmaceutical compositions for inducing an immune response against tumor cells comprising tumor cells which are made apoptotic by treatment with high hydrostatic pressure and dendritic cells, and methods for producing such compositions.

Claims

1. A method for treating cancer in a patient in need thereof comprising: (i) obtaining a monocyte from the patient, (ii) culturing the monocyte in the presence of GM-CSF and IL-4 to obtain an immature dendritic cell, (iii) obtaining a tumor cell from the patient or from one or more tumor cell lines, (iv) inducing apoptosis in the tumor cell by applying high hydrostatic pressure of 100 MPa to 300 MPa for 10 min to 2 h, (v) loading in vitro the immature dendritic cell obtained in step (ii) with the apoptotic tumor cell obtained in step (iv) to obtain a loaded dendritic cell, wherein the immature dendritic cell is combined with the apoptotic tumor cell at a ratio between about 1:1 to about 10:1, (vi) further maturing the loaded dendritic cell obtained in step (v) by treating the loaded dendritic cell with Poly I:C or LPS in vitro to obtain a loaded mature dendritic cell, and (vii) administering a therapeutically effective amount of the mature loaded dendritic cell obtained in step (vi) to the patient.

2. The method according to claim 1, wherein the tumor cells are rendered apoptotic with high hydrostatic pressure (HHP) ranging from 200 MPa to 300 MPa.

3. The method according to claim 1, wherein the tumor cells are rendered apoptotic with high hydrostatic pressure (HHP) applied to said tumor cells for 10 minutes to 1 hour.

4. The method according to claim 1, wherein the apoptotic tumor cells are not necrotic.

5. The method according to claim 1, wherein the tumor cells are derived from tumor cell lines.

6. The method according to claim 1, wherein the tumor cells are derived from a tumor isolated from the patient to be treated.

7. The method according to claim 1, wherein said mature dendritic cells express elevated levels of co-stimulatory and maturation-associated molecules as compared to dendritic cells cultured with tumor cells killed by UV irradiation.

8. The method according to claim 7, wherein said co-stimulatory and maturation-associated molecules are CD86 and/or HLA-DR.

9. The method according to claim 1, wherein said mature dendritic cells induce lower numbers of regulatory T-cells and higher numbers of tumor antigen-specific T-cells as compared to dendritic cells pulsed with tumor cells treated with UV irradiation.

10. The method according to claim 1, wherein the immature dendritic cell to tumor cell ratio ranges from 4:1 to 6:1.

11. The method according to claim 1, wherein the mature loaded dendritic cell is formulated for intravenous (IV), intradermal, subcutaneous or intralymphatic administration to said patient.

12. The method according to claim 1, wherein the mature loaded dendritic cells are capable of maintaining a low level of Forkhead Box P3 (FoxP3) positive T regulatory cells compared to dendritic cells loaded with tumor cells treated with UV irradiation.

13. The method according to claim 1, wherein the tumor cells are ovarian cancer cells and the cancer to be treated is ovarian cancer.

14. The method of claim 1, wherein the matured loaded dendritic cell has an increased expression of activation markers CD86 and/or HLA-DR compared to mature dendritic cells loaded with tumor cells killed with UV irradiation.

15. The method of claim 1, wherein the monocyte is obtained by leukapharesis.

16. The method of claim 1, which further comprises confirming by a clonogenic assay that the apoptotic tumor cell obtained in step (iv) is incapable of growing and forming a metastatic tumor after administration to the patient.

17. The method of claim 1, wherein the loaded matured dendritic cell obtained in step (vi) induces CD4 and CD8-mediated tumor specific T cell responses while inducing fewer regulatory T-cells than dendritic cells loaded with tumor cells treated with UV irradiation.

18. The method of claim 1, wherein the high hydrostatic pressure is applied at a temperature of about 21 C.

19. The method according to claim 1, wherein the tumor cells are prostate cancer cells and the cancer to be treated is prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1

(2) The schematic drawing shows how a pharmaceutical composition of the present invention can be obtained. Tumor cells obtained either from the patient or from cell lines are treated with high pressure whereby the cells become apoptotic.

(3) Dendritic cells are isolated via leukapheresis, Immature dendritic cells and apoptotic tumor cells are combined whereby mature dendritic cells are produced which can be used as vaccine,

(4) FIG. 2

(5) High hydrostatic pressure induces the expression of heat shock proteins on human tumor cells. The summary of a total of 5 experiments is shown. *P value for comparison with irradiated tumor cells, P<0.05. The time dependent expression of the markers HSP70, HSP90 and calreticulin on two tumor cell lines (OV90 and LNCap) caused by different treatments is shown.

(6) FIG. 3

(7) High hydrostatic pressure induces the release of HMGB1 (high-mobility group protein B1) from treated tumor cells (OV90 and LNCap). HMGB1 is a cytokine mediator of inflammation. The summary of a total of 5 experiments is shown. *P value for comparison with irradiated tumor cells, P<0.05. FIG. 3 shows that concerning the time dependent release of HMBG1 the HHP treatment is much more effective than other conventional treatments.

(8) FIG. 4

(9) The kinetics of phagocytosis of high hydrostatic pressure treated tumor cells by immature DCs. Summary of 5 independent experiments and representative results are shown. In the experiment either OV90 or LNCap tumor cells were used. HHP treatment is compared with UV treatment at 0 C. and 37 C.

(10) FIG. 5

(11) The phenotype of dendritic cells based on the markers OD86 and HLA-DR after interaction with high hydrostatic pressure-killed tumor cells (OV90 and LNCap) is shown. Day 5 immature DCs were cultured for 24 h with tumor cells killed by HHP or irradiation. After 24 h, the expression of maturation associated molecules on DCs was analyzed by flow cytometry. LPS was used as control. The mean fluorescence intensity (MFI) are shown. *P value for comparison with irradiated tumor cell-loaded DCs, P<0.05.

(12) FIG. 6

(13) The induction of tumor-specific T cells by dendritic cells loaded with hydrostatic pressure killed tumor cells (LNCap and OV90) is compared with dendritic cells loaded with tumor cells killed by UV irradiation. The data show a summary of five independent experiments. *P value for comparison with irradiated tumor cells, P<0.05.

(14) FIG. 7

(15) FIG. 7 demonstrates the superiority of the treatment of tumor cells with high hydrostatic pressure (HHP) compared with tumor cells killed by UV irradiation (UV irr). The tests have been performed with prostate cancer cell line (LNCap) and with ovarian cancer cell line (OV90). Controls have been performed with dendritic cells alone and cells stimulated with Poly I:C.

(16) The results summarized in FIG. 7 show the induction of prostate specific antigen (PSA)-specific T cells by dendritic cells loaded with high hydrostatic pressure killed tumor cells (LNCap and OV90, respectively). A comparison was made between high hydrostatic pressure killed tumor cells alone and dendritic cells loaded with tumor cells killed by UV irradiation. The data presented in FIG. 7 show a summary of five independent experiments. *P value for comparison with irradiated tumor cells, P<0.05. FIG. 7 summarizes the results obtained in example 7.

(17) FIG. 8

(18) The induction of regulatory T cells by high hydrostatic pressure killed tumor cells is compared with the induction of Tregs by UV irradiated tumor cells. The data show a summary of five independent experiments.

(19) The experiments summarized in FIG. 8 show that the teaching of the present invention can be applied to different types of tumors. The upper part of FIG. 8 shows the experiments performed with ovarian cancer cells (OV90). The lower part shows the experiments performed with prostate cancer cell line (LNCap). In the experiments the concentration of Fox P3 (Forkhead Box P3) has been determined in order to further differentiate the regulatory T cells (Tregs). The experiments show that tumor cells treated according to the invention with HHP do induce lower numbers of regulatory T cells than UV irradiated tumor cells.

(20) FIG. 9

(21) The results of an in vivo study are shown wherein patients were treated with a tumor vaccination as disclosed herein. All patients had radical prostatectomy or radiotherapy. As relevant parameter the PSA doubling time has been determined. According to Antonarakis et al., BJU Int. 2011, 108(3), p. 378-385, the PSA doubling time is the strongest determinant of metastatic free survival time and overall survival time of patients with prostate specific antigen (PSA)-recurrent prostate cancer. PSA doubling time means the time difference wherein the PSA value is doubled. The higher the PSA doubling time is, the better the survival prospect for the treated patient is. By applying the tumor vaccination of the present invention the PSA doubling time could be substantially prolonged.

(22) *P value for comparison with irradiated tumor cells, P<0.05.

(23) FIG. 10

(24) FIG. 10 is a Kaplan-Meier survival curve of patients at a late stage of prostate cancer which were treated according to the present invention.

(25) In the Kaplan-Meier survival curve each death of a patient causes a drop of the percent survival starting from 100% to lower values. The Halabi nomogram is the normally expected reduction of survivors whereby the medium survival time is 12 months.

(26) The active cancer immunotherapy using the cancer vaccine as described herein results in a prolongation of the medium survival time to 23 months.

(27) The present invention is further illustrated by the following examples which are, however, not limiting:

EXAMPLES

Example 1

(28) Expression of Immunogenic Cell Death Markers hsp70, hsp90 and Calreticulin by Human Cancer Cell Lines and Human Primary Tumor Cells After the Treatment with High Hydrostatic Pressure

(29) Leukemic, ovarian and prostate cancer cell lines and primary tumor cells were treated for 10 min with high hydrostatic pressure (HHP, 200 MPa) at 21 degrees centigrade's and the expression of the known immunogenic cell death markers hsp70, hsp90 and calreticulin was monitored at 6, 12 and 24 h. Significant expression of calreticulin, hsp70 and hsp90 was detected 6, 12 and 24 h after HHP treatment for all tested tumor models. The expression of immunogenic molecules was significantly higher than the expression induced by anthracyclins, the only known inducers of immunogenic cell death (FIG. 2). Increased expression of calreticulin and heat shock proteins after HHP treatment was accompanied by their translocation to the cell surface. HHP treatment also induced a rapid and substantial release of HMGB1, a soluble marker of immunogenic cell death. Release of HMGB1 was much higher than in the case of UV irradiation or anthracyclines. (FIG. 3).

(30) Maximal release of HMGB1 nuclear protein was detected 48 h after the induction of tumor cell death.

Example 2

(31) Treatment of Tumor Cells by High Hydrostatic Pressure Increases Their Phagocytosis by Antigen Presenting Cells

(32) In view of the established role of calreticulin as an eat me signal, the rate of phagocytosis of tumor cells killed by high hydrostatic pressure by dendritic cells (DCs) was investigated, the most efficient antigen presenting cells that are crucial for the initiation of an immune response. High hydrostatic pressure treated tumor cells were phagocytosed at faster rate and to a higher extent than the tumor cells killed by other modalities, such as anthracyclines or UV irradiation. After 12 h, the extent of phagocytosis of leukemic cells treated with HHP was 4-fold higher than of cells killed by UV irradiation.

(33) Similarly, the kinetics of phagocytosis of high hydrostatic pressure treated tumor cells by immature DC was investigated. Summary of 5 independent experiments and representative results are shown. In the experiment either OV90 or LNCap tumor cells were used. HHP treatment is compared with UV treatment at 0 C. and 37 C. (FIGS. 4a and 4b).

Example 3

(34) Phagocytosis of High Hydrostatic Pressure-Treated Tumor Cells Induces the Maturation of DCs

(35) The ability of DCs to activate the immune response depends on their activation status and the expression of costimulatory molecules. In normal circumstances the most efficient maturation of DCs is induced by molecules derived from pathogens, such as lipopolysacharide (LPS) from Gram negative bacteria. Only activated (mature) DCs that express high levels of costimulatory molecules can initiate the immune response. We analyzed the phenotype of DCs that phagocytosed tumor cells killed by the HHP. The interaction of DCs with HHP-treated tumor cells induced the upregulation of costimulatory molecules (CD86, CD83) and maturation associated molecules (HLA-DR) to a similar extent as activation by LPS (FIG. 5). Thus tumor cells killed by HHP can induce DCs maturation comparable to pathogen derived LPS.

Example 4

(36) DCs Presenting High Hydrostatic Pressure Treated Tumor Cells Induce Tumor-Specific T Cells and Induce Low Numbers of Inhibitory Regulatory T Cells

(37) To investigate whether tumor cells treated with HHP and expressing immunogenic cell death markers induce anti-tumor immunity, we evaluated the ability of tumor cell-loaded DCs to activate tumor cell-specific T cell responses. Tumor cells killed by HHP were cocultured with immature DCs with or without subsequent maturation with LPS. These DCs were then used as stimulators of autologous T cells, and the frequency of IFN--producing T cells was analyzed one week later after restimulation with tumor cell-loaded DCs. DCs pulsed with HHP killed tumor cells induced a greater number of tumor-specific IFN--producing T cells in comparison with DCs pulsed with irradiated cells, even in the absence of additional maturation stimulus (LPS).

(38) Additionally, the frequency of regulatory T cells (Tregs) induced in DC and T cell cocultures was also tested. Induction of Tregs is undesirable in the case of tumor immunotherapy as Tregs inhibit the immune response directed against the tumor. DCs pulsed with tumor cells killed by HHP had a lower capacity to expand regulatory T cells when compared with both immature DCs and LPS-activated DCs (FIG. 8). The FoxP3 surface marker is specific for regulatory T cells.

Example 5

(39) Active cellular immunotherapy can be administered as a single treatment modality in the case of minimal residual disease after primary treatment of the tumor by surgery or radiotherapy. In prostate cancer it may concern patients with signs of biochemical relapse (increasing levels of prostate-specific-antigen PSA in the peripheral blood measured by ultrasensitive method),

(40) The best results of the present invention can be obtained when the primary tumor is removed from the patient by surgery. The pharmaceutical composition as described in the present application can be produced from the tumor cells which have been isolated from the tumor tissue or from tumor cell lines.

(41) A patient (68 years old) suffering from prostate cancer was diagnosed at an early stage of the tumor development. Tumor was removed but few months after the surgery rising levels of PSA were detected. The patient thus underwent leukapheresis and immature dendritic cells were differentiated from isolated monocytes. Tumor cells from the prostate cancer cell line were rendered apoptotic treatment with high hydrostatic pressure as described herein and the apoptotic tumor cells were brought into contact with the immature dendritic cells in order to prepare the vaccine composition.

(42) The pharmaceutical composition was divided into aliquots that were frozen in the liquid nitrogen until use. The first application of the tumor vaccination occurred 4 weeks after the detection of the biochemical relapse of the prostate cancer. Booster applications followed every four weeks for a period of one year.

(43) Vaccination induced an immune response against the small number of surviving tumor cells that has lead to a substantial slowing down of regrowth of tumor cells and resulted in the prolongation of the survival of the patient.

Example 6

(44) In advanced cancer patients, active cellular immunotherapy should be combined with chemotherapy (i.e. docetaxel in prostate cancer) according to the concept of chemoimmunotherapy.

(45) A patient (76 years old) suffering from advanced prostate cancer was treated according to the present invention. The usual chemotherapy was combined with the active cellular immunotherapy as disclosed herein. The patient has been treated at the age of 65 years with prostate tumor. After removal of the tumor by surgery and hormone treatment the level of PSA (prostate specific antigen) was kept at a low level showing that the prostate cancer cells did not grow. After 12 months of hormone therapy metastatic prostate cancer developed at several positions in the body (in particular in the bones) and the tumor became hormone refractory. The patient was approved for the treatment of hormone refractory prostate cancer with docetaxel in combination with active cellular immunotherapy based on dendritic cells.

(46) Before the chemotherapy started, immature dendritic cells were generated from monocytes obtained during leukapheresis. Tumor cells from prostate cancer cell lines were treated with hydrostatic pressure for 30 minutes at a pressure of 210 MPa at 21 C. 10.sup.9 tumor cells treated according to the present invention were used to pulse 10.sup.9 immature dendritic cells and aliquots of the mature dendritic cells which have been pulsed before with those tumor cells were deep-frozen in liquid nitrogen and used for later applications.

(47) Active cancer immunotherapy was administered every 4-6 weeks in alternate cycles with standard chemotherapy by docetaxel and alone (after the end of docetaxel treatment) for a period of one year. Combined chemoimmunotherapy led to the stabilization of the disease, decrease in the intensity of bone marrow metastases and longer than expected survival. Patient currently survives for over three years, compared to the expected survival of 6 months at the beginning of the therapy.

Example 7

(48) In Vitro Experiment Showing the Superiority of HHP Killed Tumor Cells Versus UV Killed Tumor Cells

(49) In the in vitro experiments the ability of immature dendritic cells, poly I:C activated mature dendritic cells, and dendritic cells loaded with tumor cells which were either HHP treated or UV irradiated was checked with regard to their ability of induce tumor specific immunity. Tumor specific immunity was measured as percent tumor specific T cell lymphocytes.

(50) Dendritic cells with HHP killed tumor cells were directly compared with HHP killed tumor cells alone and dendritic cells loaded with tumor cells killed by UV irradiation. The results of the experiments are shown in FIG. 7.

(51) In order to test the capacity to induce tumor-specific T cells unpulsed or loaded with tumor cells dendritic cells were added to autologous T cells at a ratio of 1:10 on days 0 and 7 of culture. 25-50 international units/mL of IL2 (PeproTech) were added on days 2 and 7 to the culture. The cultures were tested for the presence of tumor specific T cells 7-9 days after the last stimulation with DCs. The induction of tumor-reactive, interferon (IFN)--producing T cells of prostate specific antigen (PSA) reactive T cells by tumor-loaded DCs was determined by flow cytometry. The T cells were stained with anti-human CD8/IFN-.

(52) The induction of prostate specific antigen (PSA)-specific T cells by dendritic cells loaded with high hydrostatic pressure killed tumor cells (LNCap) is compared with high hydrostatic pressure killed tumor cells alone and with dendritic cells loaded with tumor cells killed by UV irradiation.

(53) The results of the experiments are shown in FIG. 7. The upper part of FIG. 7 shows that DCs loaded with HHP killed tumor cells can induce tumor specific T cells even in the absence of a maturation signal. DCs loaded with tumor cells killed by UV treatment or HHP killed tumor cells alone do not induce tumor immunity. It is surprising that only HHP treated tumor cells (according to the invention) and immature dendritic cells can induce tumor specific immune response whereas this result cannot be obtained by UV treated tumor cells and immature dendritic cells. Without wishing to be bound to a theory it seems that only the HHP treated tumor cells can together with immature dendritic cells induce the tumor specific T cell immune response. The HHP treated tumor cells seem to act as a kind of activator of the immature dendritic cells whereas UV treated tumor cells do not have this effect.

(54) The lower part of FIG. 7 shows that when Poly I:C treatment is applied the treated HHP tumor cells can better induce specific T cell lymphocytes than tumor cells irradiated with UV.

Example 8

(55) In Vivo Data Obtained with the Tumor Vaccination According to the Present Invention

(56) Dendritic cells were obtained from a cohort of patients similar to those as described above. The dendritic cells were pulsed with killed tumor cells as described above and the tumor vaccination was administered repeatedly in up to 12 doses in 4-6 weeks intervals to patients with a biochemical relapse of the prostate cancer after radical prostatectomy or radiotherapy. The progression of the disease in each single patient has been evaluated by the PSA doubling time. Under PSA doubling time the time period is understood which is required for the PSA value to double. PSA doubling time has been shown as the strongest and most reliable determinant of the overall survival and metastatic free survival in men with prostate cancer. Short PSA doubling time correlates with a shortened survival and with shortened time to metastasis appearance (Antonarakis et al., BJU Int., 2012, 108(3); pp 378-385.

(57) As shown in FIG. 9 the continuous administration of the tumor vaccination according to the present invention in patients with biochemical relapse of the prostate cancer after radical prostatectomy or radiotherapy leads to a significant prolongation of the PSA doubling time. It has been found that by using the tumor vaccination as disclosed herein mean PSA doubling time increases from 5 months before the initiation of cancer immunotherapy to 30 months after 12 months of immunotherapy. This represents a significant benefit to patients with the biochemical relapse of the prostate cancer.

Example 9

(58) Clinical Trial with Patients in Late Stage of Prostate Cancer

(59) In this clinical trial dendritic cells were pulsed with killed tumor cells as described herein. The tumor vaccination was administered repeatedly to patients at a later stage of the prostate cancer. Said patients suffered from castration resistant metastatic prostate cancer. In those patients cancer immunotherapy was administered in alternate dosing schedule with docetaxel chemotherapy.

(60) The survival of the treated cohort was compared to the historical cohort or to the survival estimated by Halabi nomogram. It has been shown that the continuous administration of active cancer immunotherapy significantly prolongs the survival time of treated patients (median survival of 23 months) compared with the cohort of the historical controls based on the expected survival calculated by Halabi nomogram (13 months).

(61) This experiment proves that the tumor vaccination of the present invention substantially extends the survival time of patients which are in a late state of prostate cancer. The average survival expectation of such patients is 13 months without treatment compared to 23 months after treatment with tumor vaccination according to the present invention. This represents a substantial improvement for such patients which are extremely difficult to medicate successfully.