PATIENT SELECTION FOR TREATMENT WITH DENDRITIC CELL VACCINATION

Abstract

The invention relates to a dendritic cell (DC) vaccine for use in a method of treating cancer in a patient, wherein the patient is selected for treatment with said DC vaccine. The selection inter alia comprises determining in a tumor sample from the patient the amount of CD8+ T-cells and/or the tumor mutation burden (TMB) and comparing the determined amount to a predetermined threshold level. The selection allows identifying patients that particularly benefit from DC treatment.

Claims

1. A dendritic cell vaccine for use in a method of treating a tumor in a patient, wherein the patient is selected for treatment with said dendritic cell vaccine, the selection comprising determining that the tumor is a cold tumor.

2. A dendritic cell vaccine for use in a method of treating a tumor in a patient, wherein the patient is selected for treatment with said dendritic cell vaccine, the selection comprising determining that the tumor exhibits a low amount or the absence of infiltrating CD8.sup.+ T-cells.

3. The dendritic cell vaccine for use of claim 1 or claim 2, wherein the selection comprises determining in a tumor sample from the patient the amount of CD8.sup.+ T-cells and comparing the determined amount to a predetermined threshold level, optionally wherein the amount of CD8.sup.+ T-cells is determined from at least one section of the tumor sample, the section having a thickness between 2 and 4 ?m.

4. The dendritic cell vaccine for use of claim 3, wherein the predetermined threshold level is between about 30-40 CD8.sup.+ T-cells/mm.sup.2, preferably about 40 CD8.sup.+ T-cells/mm.sup.2, more preferably about 35 CD8.sup.+ T-cells/mm.sup.2 and most preferably about 30 CD8.sup.+ T-cells/mm.sup.2, optionally wherein the patient is selected for treatment in case the patient has an amount of CD8.sup.+ T-cells/mm.sup.2 equal to or below the predetermined threshold level.

5. The dendritic cell vaccine for use of any one of claims 1 to 4, wherein the selection comprises determining in a tumor sample from the patient the tumor mutation burden (TMB) and comparing the determined TMB to a predetermined threshold level, optionally wherein the predetermined threshold level is about 2.5 mut/Mb, preferably about 2.3 mut/Mb.

6. The dendritic cell vaccine for use of claim 5, wherein the patient is selected for treatment in case the patient has a TMB equal to or below the predetermined threshold level.

7. A dendritic cell vaccine for use in a method of treating cancer in a patient, wherein the method comprises a step of determining in a tumor sample of the patient, whether or not the patient has (i) an amount of CD8.sup.+ T-cells/mm.sup.2 that is equal to or below a predetermined threshold level of between about 30-40 CD8.sup.+ T-cells/mm.sup.2, preferably about 40 CD8.sup.+ T-cells/mm.sup.2, more preferably about 35 CD8.sup.+ T-cells/mm.sup.2 and most preferably about 30 CD8.sup.+ T-cells/mm.sup.2, and/or (ii) a TMB that is equal to or below a predetermined threshold level of about 2.5 mut/Mb, preferably about 2.3 mut/Mb.

8. The dendritic cell vaccine for use of any one of claims 1 to 7, wherein the cancer is ovarian, lung or prostate cancer, preferably ovarian cancer, more preferably newly diagnosed ovarian cancer.

9. The dendritic cell vaccine for use of any one of claims 1 to 8, wherein the tumor sample is obtained prior to the treatment of the patient with the dendritic cell vaccine.

10. The dendritic cell vaccine for use of claim 9, wherein the tumor sample is obtained prior to the treatment of the patient with chemotherapy.

11. The dendritic cell vaccine for use of any one of claims 3 to 10, wherein the amount of CD8.sup.+ T-cells in the tumor section is measured by immunostaining, preferably immunohistochemistry or immunofluorescence and/or wherein the TMB in the tumor sample is measured by sequencing, preferably next generation sequencing.

12. The dendritic cell vaccine for use of any one of claims 1 to 11, wherein the dendritic cells are derived from monocytes that are autologous to the patient to be treated and wherein the monocytes are obtained by leukapheresis.

13. The dendritic cell vaccine for use of any one of claims 1 to 12, wherein the dendritic cell vaccine is prepared by loading immature dendritic cells in a first step with tumor cells undergoing immunogenic cell death and thereafter maturing the loaded dendritic cells with Toll-like receptor 3 agonists or Toll-like receptor 4 agonists.

14. The dendritic cell vaccine for use of any one of claims 1 to 13, wherein the dendritic cell vaccine is administered to a patient in combination with a further treatment modality selected from the group consisting of chemotherapy, targeted therapy, and biologics.

15. The dendritic cell vaccine for use of any one of claims 1 to 14, wherein the dendritic cell vaccine is administered to a patient a. in parallel to chemotherapy with at least one chemotherapeutic agent, or b. sequentially to chemotherapy after completion of chemotherapy with at least one chemotherapeutic agent.

Description

DESCRIPTION OF THE DRAWINGS

[0089] FIG. 1: Schematic diagram of the clinical study on patients with newly diagnosed ovarian cancer, comparing the standard of care chemotherapy treatment to the DC vaccine treatment administered in parallel or sequentially to chemotherapy. DCVAC OvCa stands for a dendritic cell vaccine wherein dendritic cells have been loaded with ovarian cancer cells undergoing immunogenic cell death and matured by a Toll-like receptor ligand. The treatment period 1 corresponds to the period of chemotherapy, also including DC vaccine administration for treatment Group A. The treatment period 2 corresponds to the period when the DC vaccine was administered after completion of the chemotherapy.

[0090] FIG. 2: Analysis of progression-free survival (PFS) in the clinical study for the mITT population with newly diagnosed ovarian cancer stratified for the relative count of CD8.sup.+ T cells/mm.sup.2 in a tumor tissue section. A) patients with CD8.sup.+ T cells/mm.sup.2?30; B) patients with CD8.sup.+ T cells/mm.sup.2>30.

[0091] FIG. 3: Analysis of overall survival (OS) in the clinical study for the mITT population with newly diagnosed ovarian cancer stratified for the relative count of CD8.sup.+ T cells/mm.sup.2 in a tumor tissue section. A) patients with CD8.sup.+ T cells/mm.sup.2?30; B) patients with CD8.sup.+ T cells/mm.sup.2>30.

[0092] FIG. 4: Representative immunohistochemistry microscopy images of ovarian cancer tumor tissue samples showing infiltrated CD8.sup.+ T-cells. Top panel: tumor sample with an amount of CD8.sup.+ T-cells/mm.sup.2 equal or below 30. The black arrow points to stained CD8.sup.+ T-cells. Bottom panel: tumor sample with an amount of CD8.sup.+ T-cells/mm.sup.2 above 30. The black dots are stained CD8.sup.+ T-cells.

[0093] FIG. 5: Distribution of amount of CD8.sup.+ T-cells/mm.sup.2 among the mITT population (shows the percent of patient samples containing a specific amount of CD8.sup.+ T-cells/mm.sup.2). [0094] A. Distribution among all the tested samples. [0095] B. Distribution among samples having less than 200 CD8.sup.+ T-cells/mm.sup.2. [0096] C. Distribution among samples of amount of CD8.sup.+ T-cells/mm.sup.2 for patients treated with DCVAC in parallel to chemotherapy (top), with DCVAC sequentially to chemotherapy (medium) and with standard of care (SoC) alone (bottom). [0097] D. Distribution among samples having less than 200 CD8.sup.+ T-cells/mm.sup.2 for patients treated patients with DCVAC in parallel to chemotherapy (top), with DCVAC sequentially to chemotherapy (medium) and with standard of care (SoC) alone (bottom).

[0098] FIG. 6: Correlation between the amount of CD8+ T-cells/mm.sup.2 in ovarian tumor tissue sections and the measured TMB in tumor samples. Upper panel: average amount of CD8+ T-cells/mm.sup.2 in tumor tissue sections from tumor samples with a TMB?2.3 mut/Mb (TMB.sup.Lo) or a TMB>2.3 mut/Mb (TMB.sup.Hi); lower panel: average TMB in tumor samples where CD8.sup.+ T cells/mm.sup.2?30 in the tumor tissue section (CD8 Low) or CD8.sup.+ T cells/mm.sup.2>30 (CD8 High).

[0099] FIG. 7: Analysis of progression-free survival (PFS) in the clinical study for the population with newly diagnosed ovarian cancer stratified for the TMB in the tumor sample. Upper panel: patients with TMB?2.3 mut/Mb; Lower panel: patients with TMB>2.3 mut/Mb.

[0100] FIG. 8: Analysis of overall survival (OS) in the clinical study for the population with newly diagnosed ovarian cancer stratified for the TMB in the tumor sample. Upper panel: patients with TMB?2.3 mut/Mb; Lower panel: patients with TMB>2.3 mut/Mb.

THE INVENTION IS ALSO DESCRIBED BY THE FOLLOWING EMBODIMENTS

[0101] 1. A dendritic cell vaccine for use in a method of treating cancer in a patient, wherein the patient is selected for treatment with said dendritic cell vaccine, the selection comprising determining in a tumor sample from the patient the tumor mutation burden (TMB) and comparing the determined TMB to a predetermined threshold level. [0102] 2. The dendritic cell vaccine for use of embodiment 1, wherein the predetermined threshold level is about 2.5 mut/Mb, preferably about 2.3 mut/Mb. [0103] 3. The dendritic cell vaccine of for use of embodiment 2, wherein the patient is selected for treatment in case the patient has a TMB equal to or below the predetermined threshold level. [0104] 4. A dendritic cell vaccine for use in a method of treating cancer in a patient, wherein the method comprises a step of determining in a tumor sample of the patient, whether or not the patient has a TMB that is equal to or below a predetermined threshold level of about 2.5 mut/Mb, preferably about 2.3 mut/Mb. [0105] 5. The dendritic cell vaccine for use of any one of embodiments 1 to 4, wherein the cancer is ovarian, lung or prostate cancer, preferably ovarian cancer, more preferably newly diagnosed ovarian cancer. [0106] 6. The dendritic cell vaccine for use of any one of embodiments 1 to 5, wherein the tumor sample is obtained prior to the treatment of the patient with the dendritic cell vaccine. [0107] 7. The dendritic cell vaccine for use of embodiment 6, wherein the tumor sample is obtained prior to the treatment of the patient with chemotherapy. [0108] 8. The dendritic cell vaccine for use of any one of embodiments 1 to 7, wherein the TMB in the tumor sample is measured by sequencing, preferably next generation sequencing. [0109] 9. The dendritic cell vaccine for use of any one of embodiments 1 to 8, wherein the dendritic cells are derived from monocytes that are autologous to the patient to be treated and wherein the monocytes are obtained by leukapheresis. [0110] 10. The dendritic cell vaccine for use of any one of embodiments 1 to 9, wherein the dendritic cell vaccine is prepared by loading immature dendritic cells in a first step with tumor cells undergoing immunogenic cell death and thereafter maturing the loaded dendritic cells with Toll-like receptor 3 agonists or Toll-like receptor 4 agonists. [0111] 11. The dendritic cell vaccine for use of any one of embodiments 1 to 10, wherein the dendritic cell vaccine is administered to a patient in combination with a further treatment modality selected from the group consisting of chemotherapy, targeted therapy, and biologics. [0112] 12. The dendritic cell vaccine for use of any one of embodiments 1 to 11, wherein the dendritic cell vaccine is administered to a patient [0113] a. in parallel to chemotherapy with at least one chemotherapeutic agent, or [0114] b. sequentially to chemotherapy after completion of chemotherapy with at least one chemotherapeutic agent.

The Invention is Further Described by the Following Embodiments

[0115] 1. A dendritic cell vaccine for use in a method of treating cancer in a patient, wherein the patient is selected for treatment with said dendritic cell vaccine, the selection comprising determining in a tumor sample from the patient the amount of CD8.sup.+ T-cells and comparing the determined amount to a predetermined threshold level. [0116] 2. The dendritic cell vaccine for use of embodiment 1, wherein the amount of CD8.sup.+ T-cells is determined from at least one section of the tumor sample, the section having a thickness between 2 and 4 ?m. [0117] 3. The dendritic cell vaccine for use of embodiment 1 or 2, wherein the predetermined threshold level is between about 30-40 CD8.sup.+ T-cells/mm.sup.2, preferably about 40 CD8.sup.+ T-cells/mm.sup.2, more preferably about 35 CD8.sup.+ T-cells/mm.sup.2 and most preferably about 30 CD8.sup.+ T-cells/mm.sup.2. [0118] 4. The dendritic cell vaccine for use of embodiment 3, wherein the patient is selected for treatment in case the patient has an amount of CD8.sup.+ T-cells/mm.sup.2 equal to or below the predetermined threshold level. [0119] 5. A dendritic cell vaccine for use in a method of treating cancer in a patient, wherein the method comprises a step of determining in a tumor sample of the patient, whether or not the patient has an amount of CD8.sup.+ T-cells/mm.sup.2 that is equal to or below a predetermined threshold level of between about 30-40 CD8.sup.+ T-cells/mm.sup.2, preferably about 40 CD8.sup.+ T-cells/mm.sup.2, more preferably about 35 CD8.sup.+ T-cells/mm.sup.2 and most preferably about 30 CD8.sup.+ T-cells/mm.sup.2. [0120] 6. The dendritic cell vaccine for use of any one of embodiments 1 to 5, wherein the cancer is ovarian, lung or prostate cancer, preferably ovarian cancer, more preferably newly diagnosed ovarian cancer. [0121] 7. The dendritic cell vaccine for use of any one of embodiments 1 to 6, wherein the tumor sample is obtained prior to the treatment of the patient with the dendritic cell vaccine. [0122] 8. The dendritic cell vaccine for use of embodiment 7, wherein the tumor sample is obtained prior to the treatment of the patient with chemotherapy. [0123] 9. The dendritic cell vaccine for use of any one of embodiments 1 to 8, wherein the amount of CD8.sup.+ T-cells in the tumor section is measured by immunostaining, preferably immunohistochemistry or immunofluorescence. [0124] 10. The dendritic cell vaccine for use of any one of embodiments 1 to 9, wherein the dendritic cells are derived from monocytes that are autologous to the patient to be treated and wherein the monocytes are obtained by leukapheresis. [0125] 11. The dendritic cell vaccine for use of any one of embodiments 1 to 10, wherein the dendritic cell vaccine is prepared by loading immature dendritic cells in a first step with tumor cells undergoing immunogenic cell death and thereafter maturing the loaded dendritic cells with Toll-like receptor 3 agonists or Toll-like receptor 4 agonists. [0126] 12. The dendritic cell vaccine for use of any one of embodiments 1 to 11, wherein the dendritic cell vaccine is administered to a patient in combination with a further treatment modality selected from the group consisting of chemotherapy, targeted therapy, and biologics. [0127] 13. The dendritic cell vaccine for use of any one of embodiments 1 to 12, wherein the dendritic cell vaccine is administered to a patient [0128] a. in parallel to chemotherapy with at least one chemotherapeutic agent, or [0129] b. sequentially to chemotherapy after completion of chemotherapy with at least one chemotherapeutic agent.

The Invention is Additionally Described by the Following Embodiments

[0130] 1. A dendritic cell vaccine for use in a method of treating a tumor in a patient, wherein the patient is selected for treatment with said dendritic cell vaccine, the selection comprising determining that the tumor is a cold tumor. [0131] 2. A dendritic cell vaccine for use in a method of treating a tumor in a patient, wherein the patient is selected for treatment with said dendritic cell vaccine, the selection comprising determining that the tumor exhibits a low amount or the absence of infiltrating CD8.sup.+ T-cells. [0132] 3. The dendritic cell vaccine for use of embodiment 1 or embodiment 2, wherein the selection comprises determining in a tumor sample from the patient the amount of CD8.sup.+ T-cells and comparing the determined amount to a predetermined threshold level. [0133] 4. The dendritic cell vaccine for use of embodiment 3, wherein the amount of CD8.sup.+ T-cells is determined from at least one section of the tumor sample, the section having a thickness between 2 and 4 ?m. [0134] 5. The dendritic cell vaccine for use of embodiment 3 or 4, wherein the predetermined threshold level is between about 30-40 CD8.sup.+ T-cells/mm.sup.2, preferably about 40 CD8.sup.+ T-cells/mm.sup.2, more preferably about 35 CD8.sup.+ T-cells/mm.sup.2 and most preferably about 30 CD8.sup.+ T-cells/mm.sup.2. [0135] 6. The dendritic cell vaccine for use of embodiment 5, wherein the patient is selected for treatment in case the patient has an amount of CD8.sup.+ T-cells/mm.sup.2 equal to or below the predetermined threshold level. [0136] 7. The dendritic cell vaccine for use of any one of embodiments 1 to 5, wherein the selection comprises determining in a tumor sample from the patient the tumor mutation burden (TMB) and comparing the determined TMB to a predetermined threshold level. [0137] 8. The dendritic cell vaccine for use of embodiment 7, wherein the predetermined threshold level is about 2.5 mut/Mb, preferably about 2.3 mut/Mb. [0138] 9. The dendritic cell vaccine for use of embodiment 8, wherein the patient is selected for treatment in case the patient has a TMB equal to or below the predetermined threshold level. [0139] 10. A dendritic cell vaccine for use in a method of treating cancer in a patient, wherein the method comprises a step of determining in a tumor sample of the patient, whether or not the patient has [0140] (i) an amount of CD8.sup.+ T-cells/mm.sup.2 that is equal to or below a predetermined threshold level of between about 30-40 CD8.sup.+ T-cells/mm.sup.2, preferably about 40 CD8.sup.+ T-cells/mm.sup.2, more preferably about 35 CD8.sup.+ T-cells/mm.sup.2 and most preferably about 30 CD8.sup.+ T-cells/mm.sup.2, and/or [0141] (ii) a TMB that is equal to or below a predetermined threshold level of about 2.5 mut/Mb, preferably about 2.3 mut/Mb. [0142] 11. The dendritic cell vaccine for use of any one of embodiments 1 to 10, wherein the cancer is ovarian, lung or prostate cancer, preferably ovarian cancer, more preferably newly diagnosed ovarian cancer. [0143] 12. The dendritic cell vaccine for use of any one of embodiments 1 to 11, wherein the tumor sample is obtained prior to the treatment of the patient with the dendritic cell vaccine. [0144] 13. The dendritic cell vaccine for use of embodiment 12, wherein the tumor sample is obtained prior to the treatment of the patient with chemotherapy. [0145] 14. The dendritic cell vaccine for use of any one of embodiments 3 to 13, wherein the amount of CD8.sup.+ T-cells in the tumor section is measured by immunostaining, preferably immunohistochemistry or immunofluorescence and/or wherein the TMB in the tumor sample is measured by sequencing, preferably next generation sequencing. [0146] 15. The dendritic cell vaccine for use of any one of embodiments 1 to 14, wherein the dendritic cells are derived from monocytes that are autologous to the patient to be treated and wherein the monocytes are obtained by leukapheresis. [0147] 16. The dendritic cell vaccine for use of any one of embodiments 1 to 15, wherein the dendritic cell vaccine is prepared by loading immature dendritic cells in a first step with tumor cells undergoing immunogenic cell death and thereafter maturing the loaded dendritic cells with Toll-like receptor 3 agonists or Toll-like receptor 4 agonists. [0148] 17. The dendritic cell vaccine for use of any one of embodiments 1 to 16, wherein the dendritic cell vaccine is administered to a patient in combination with a further treatment modality selected from the group consisting of chemotherapy, targeted therapy, and biologics. [0149] 18. The dendritic cell vaccine for use of any one of embodiments 1 to 17, wherein the dendritic cell vaccine is administered to a patient [0150] a. in parallel to chemotherapy with at least one chemotherapeutic agent, or [0151] b. sequentially to chemotherapy after completion of chemotherapy with at least one chemotherapeutic agent.

EXAMPLES

Example 1. DC Vaccine (DCVAC)

[0152] The DC vaccine consisted of autologous DCs loaded ex vivo with ovarian cancer cells which were killed by immunogenic cell death and matured by a Toll-like receptor 3 (TLR-3) ligand. DCs were derived from autologous monocytes that were obtained by leukapheresis. Monocytes isolated from the leukapheresis product were cultured in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4 to obtain immature DCs. Immature DCs were loaded with cells of the ovarian cancer cell lines OV-90 and SK-OV-3 (in a ratio of 2:1). Before being added to the DC culture, OV-90 and SK-OV-3 cells were treated with high hydrostatic pressure (HHP) (as described in WO 2013/004708, examples 1-4), which induces immunogenic cell death (Fucikova et al. 2014). The tumor cell-loaded DCs were matured by polyinosinic:polycytidylic acid (poly[I:C]), a TLR-3 ligand.

[0153] The final product was cryopreserved in doses of approximately 1?10.sup.7 DCs per vial in 1 ml of CryoStor CS10 freezing medium containing 10% dimethyl sulfoxide. DC vaccine aliquots were transported to the study sites on dry ice at a temperature below ?50? C. Each DC vaccine dose was then thawed and diluted in saline to a final volume of 5 ml. The diluted dose was administered to the patient subcutaneously in two applications: one into the inguinal area and one into the contralateral axillary area (2.5 ml to each of the application sites).

Example 2: Preparation of Tumor Tissue and Cell Quantification

Immunohistochemistry

[0154] Immunostaining with antibody specific for CD8 was performed according to conventional protocols as published previously (Fucikova et al. 2019; Goc et al. 2014; Truxova et al. 2018). Briefly, tumor specimens were fixed in neutral buffered 10% formalin solution for 24 hours and embedded in paraffin as per standard procedures. The standard sample size was 0.5 cm?1 cm?1 cm. Tissue sections of 3 ?m of thickness where cut on a microtome. Tissue sections were deparaffinized, followed by antigen retrieval with Target Retrieval Solution (Leica) in TRIS EDTA at pH 8.0 in a heated water bath (98? C., 30 min). Endogenous peroxidase and alkaline phosphatase were blocked with 3% H.sub.2O.sub.2 and levamisole, respectively for 15 min. Thereafter, sections were treated with protein block (DAKO) for 15 min and incubated with the primary anti-CD8 antibody (clone SP16) (Abcam ab1010500), followed by the revelation of enzymatic activity (EnVision+ Sytem-HRP Labelled Polymer anti-rabbit, DAKO). Sections were counterstained with hematoxylin (DAKO) for 30 sec. Images of whole tumor sections were acquired using a Leica Aperio AT2 scanner (Leica).

Immunofluorescence

[0155] Tumor specimens were fixed in neutral buffered 10% formalin solution and embedded in paraffin as per standard procedures. Immunostaining with the primary anti-CD8 antibody (clone SP16) (Abcam ab1010500) was performed according to conventional protocols. Briefly, tissue sections were deparaffinized and rehydrated descending alcohol series (100, 96, 70, and 50%), followed by antigen retrieval with Target Retrieval Solution (Leica) in EDTA pH 8.0 in preheated water bath (97? C., 30 min). Sections were allowed to cool down to RT for 30 min. Sections were then treated with Signal enhancer (Fisher Thermoscientific) for 30 min and blocking buffer for 60 min. The anti-CD8 antibody was applied for 2 hours at RT. Thereafter, slides were incubated with appropriate fluorophore-labelled secondary antibodies for 1 hour at RT. Finally, sections were treated with TrueBlack? Lipofuscin Autofluorescence Quencher (Biotium) for 30 seconds and mounted with ProLong Gold antifade reagent containing DAPI (Thermo Fisher Scientific). The specificity of the staining was determined using appropriate isotype controls. Images of whole tumor sections were acquired using a Leica Aperio AT2 scanner (Leica).

Cell Quantification

[0156] Infiltration of tumor nests by CD8.sup.+ T cells was quantified in whole tumor sections with Calopix software (Tribvn) as published previously (Goc et al. 2014; Fucikova et al. 2019). Data are reported as absolute number of positive cells/mm.sup.2.

[0157] FIG. 4 shows representative immunohistochemistry microscopy images for a patient with a low amount of CD8.sup.+ T-cells/mm.sup.2 in the tumor section (4A) and with higher amount of CD8.sup.+ T-cells/mm.sup.2 in the tumor section (4B).

Example 3. Clinical Data of DCVAC Treatment in Combination with Chemotherapy in Women with Newly Diagnosed Epithelial Ovarian Carcinoma

[0158] The clinical study (Clinical trial information: NCT02107937, see https://www.clinicaltrials.gov/ct2/show/NCT02107937) was conducted as an open label, multicenter, three-arm phase II clinical trial in women with newly diagnosed epithelial ovarian carcinoma, who have undergone debulking surgery. The aim of this study was to evaluate the efficacy and safety of the DC vaccine (DCVAC) administered in parallel to chemotherapy as an add-on to standard of care chemotherapy with carboplatin and paclitaxel or sequentially after standard of care chemotherapy with carboplatin and paclitaxel compared to chemotherapy alone.

[0159] A total of 99 patients were centrally randomized in a ratio of 1:1:1 to treatment groups [0160] A (34 patients) to receive the DC vaccine in parallel with standard of care chemotherapy, [0161] B (34 patients) to receive the DC vaccine sequentially after standard of care chemotherapy, or [0162] C (31 patients) to receive standard of care chemotherapy alone.

[0163] The DC vaccine was administered subcutaneously (SC) to patients in treatment groups A and B in up to 10 doses. The planned number of chemotherapy cycles was 6 in all treatment groups (FIG. 1).

[0164] The main population, modified intent-to-treat (mITT) population included all patients who were randomized and received at least 1 dose of chemotherapy in group C, or at least 1 dose of DC vaccine in groups A and B (mITT: 31 patients in treatment group A [parallel DC vaccine], 29 patients in treatment group B [sequential DC vaccine], and 30 patients in treatment group C [standard of care]).

Analysis of Subgroup of Patients with CD8.sup.+ Low Cells Counts

[0165] Both PFS and OS were analyzed on the subgroup of patients with low CD8.sup.+ cells counts (CD8.sup.+ cells counts ?30 cells/mm.sup.2) (see FIG. 5 on the distribution of CD8.sup.+ T-cells amounts in a tumor sample over the mITT population). When the count of CD8.sup.+ T-cells was at below or equal to 30 cells/mm.sup.2, in the mITT population, group A and B patient had a better PFS when compared to group C patients treated with the standard of care treatment (A vs C: HR=0.49, p=0.2219; B vs C: HR=0.25, p=0.0759). (Table 1, FIG. 2A). That difference in PFS was not seen for patients with count of CD8.sup.+ T-cells above 30 cells/mm.sup.2 in the tumor sample when comparing treatment groups A and B to C with standard of care in mITT population (A vs C: HR=1.37, p=0.5518; B vs C: HR=0.65, p=0.4908) (Table 1, FIG. 2B).

[0166] When the count of CD8.sup.+ T-cells was at below or equal to 30 cells/mm.sup.2, in the mITT population, group A and B patient had a better OS when compared to group C patients treated with the standard of care treatment (A vs C: HR=0.15, p=0.0131; B vs C: HR=0.15, p=0.0434) (Table 1, FIG. 3A). Again, that difference in OS was not seen for patients with count of CD8.sup.+ T-cells above 30 cells/mm.sup.2 in the tumor sample when comparing treatment groups A and B to C with standard of care in mITT population (A vs C: HR=1.41, p=0.5084; B vs C: HR=0.63, p=0.4516) (Table 1, FIG. 3B).

TABLE-US-00001 TABLE 1 Benefit of DCVAC/OvCa on patient progression-free survival and overall survival Treatment group A Treatment group B (parallel) over (sequential) over treatment group C (SoC) treatment group C (SoC) HR p-value HR p-value PFS on mITT population and 0.49 0.2219 0.25 0.0759 CD8.sup.+ count ?30 cells/mm.sup.2 PFS on mITT population and 1.37 0.5518 0.65 0.4908 CD8.sup.+ count >30 cells/mm.sup.2 OS on mITT population and 0.15 0.0131 0.15 0.0434 CD8.sup.+ count ?30 cells/mm.sup.2 OS on mITT population and 1.41 0.5084 0.63 0.4516 CD8.sup.+ count >30 cells/mm.sup.2 PFS: progression-free survival; OS: overall survival; SoC: standard of care; HR: hazard ratio

Example 4: Preparation of Tumor RNA and Quantification of the TMB

[0167] DNA/RNA Isolation from FFPE

[0168] RNA and DNA were isolated using AllPrep DNA/RNA FFPE Kit (Qiagen) according to the manufacturers' instructions. The RNA concentration and purity were determined using a NanoDrop 2000c (Thermo Scientific, Germany). Purified RNA samples were stored at ?80? C. until further use.

DNA Library

[0169] DNA libraries were prepared using the hybrid capture-based TruSight Oncology 500 Library Preparation Kit (Illumina, San Diego, CA, USA). TMB measurement and analyses were performed as described in the TruSight Oncology 500 Reference Guide (TruSight Oncology 500 Reference Guide 2020)

Example 5: TMB Status Positively Correlates with Anti-Tumor Immunity in Ovarian Carcinoma

[0170] Of the 90 patients enrolled and randomly assigned in the clinical study (Clinical trial information: NCT02107937, see https://www.clinicaltrials.gov/ct2/show/NCT02107937), 78 (86%) had tumor samples available to attempt the assessment of the tumor mutational burden (TMB) and had valid data for TMB-based efficacy analyses. Baseline characteristics of all randomly assigned patients and patients whose TMB could be evaluated were similar and balanced between the SOC treatment group and the DCVAC treatment group. The TrueSightOnco500 gene panel was used to compare the profile of somatic mutations and TMB in tumor samples from ovarian cancer patients involved in the study. A panel of 20 somatic mutations (TP53, BRCA1, BARD1, MDC1, PIK3CA, SDHA, TET1, ARID5B, KRAS, ZFHX3, ZNF703, RID1A, BOOR, BCORL1, FAWCD2, GNAS, MED12, NF1, SLX4 and SPTA1) was identified as occurring in patients and was well balanced between treatment groups. Similarly, a statistical difference between the TMB load in individual patients' treatment arms was not observed. The relationship between the TMB status and intratumoral abundance of CD8.sup.+ T-cells measured by immunofluorescence was analyzed. A significantly higher densities of CD8.sup.+ T-cells in tumor samples was observed in samples from TMB.sup.Hi patients, compared to TMB.sup.Lo patients (FIG. 6, upper panel). Likewise, patients with a low TMB had low CD8.sup.+ T-cells infiltration in tumor samples, compared to patients with high TMB (see FIG. 6 lower panel). Taken together, these findings indicate that a high TMB status in the tumor microenvironment of ovarian cancer were strongly associated with an immune infiltration and low TMB is associated with poor immune infiltration.

Example 6: Low Tumor Mutational Burden Status is Associated with Better Clinical Response to DCVAC Therapy

[0171] To assess the prognostic and predictive value of TMB in the tumor samples of ovarian patients involved in the clinical study NCT02107937, PFS and OS was evaluated upon median stratification. The median value calculated from the measured TMB on all patient tumor sample was equal to 2.3 mut/Mb. When the patients group was selected for having a TMB in the tumor sample below or equal to 2.3 mut/Mb, the patient group treated with DCVAC had a better PFS and OS compared to patients treated with standard of care only (PFS: FIG. 7, upper panel; OS: FIG. 8, upper panel). When the patients group was selected for having a TMB in the tumor sample above 2.3 mut/Mb, no significant difference in PFS and OS was observed between the patient group treated DCVAC and the patient group treated with standard of care alone (PFS: FIG. 7, lower panel; OS: FIG. 8, lower panel).

REFERENCES

[0172] Adkins, Irena, Lenka Sadilkova, Nada Hradilova, Jakub Tomala, Marek Kovar, and Radek Spisek. 2017. Severe, but not mild heat-shock treatment induces immunogenic cell death in cancer cells, Oncoimmunology, 6: e1311433. [0173] American Cancer SocietyHow Chemotherapy Drugs Work. 2016. American Cancer Society, Accessed 2018 Jan. 5. https://www.cancer.org/treatment/treatments-and-side-effects/treatment-types/chemotherapy/how-chemotherapy-drugs-work.html. [0174] Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity, Nature, 392: 245-52. [0175] Bloy, N., J. Pol, F. Aranda, A. Eggermont, I. Cremer, W. H. Fridman, J. Fucikova, J. Galon, E. Tartour, R. Spisek, M. V. Dhodapkar, L. Zitvogel, G. Kroemer, and L. Galluzzi. 2014. Trial watch: Dendritic cell-based anticancer therapy, Oncoimmunology, 3: e963424. [0176] Buttner, R., J. W. Longshore, F. Lopez-Rios, S. Merkelbach-Bruse, N. Normanno, E. Rouleau, and F. Penault-Llorca. 2019. Implementing TMB measurement in clinical practice: considerations on assay requirements, ESMO Open, 4: e000442. [0177] Casares, N., M. O. Pequignot, A. Tesniere, F. Ghiringhelli, S. Roux, N. Chaput, E. Schmitt, A. Hamai, S. Hervas-Stubbs, M. Obeid, F. Coutant, D. Metivier, E. Pichard, P. Aucouturier, G. Pierron, C. Garrido, L. Zitvogel, and G. Kroemer. 2005. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death, J Exp Med, 202: 1691-701. [0178] Cibula, D., P. Mallmann, P. Knapp, B. Melichar, J. Klat, L. Minar, Z. Novotny, P. Wimberger, A. Hein, R. Spisek, J. Bartunkova, L. Pecen, H. I. B. Hassan, L. Rob, and SOV02 Investigators. 2018. Dendritic cell vaccine (DCVAC) with chemotherapy (ct) in patients (pts) with recurrent epithelial ovarian carcinoma (EOC) after complete response (CR) to 1st-line platinum (Pt)-based ct: Primary analysis of a phase 2, open-label, randomized, multicenter trial, Journal of Clinical Oncology, 36: e17515-e15. [0179] Dey, Pranab. 2018. Basic and Advanced Laboratory Techniques in Histopathology and Cytology (Springer Nature Singapore: Singapore). [0180] Ellis, P. M. 2016. Anti-angiogenesis in Personalized Therapy of Lung Cancer, Adv Exp Med Blot, 893: 91-126. [0181] Elster, J. D., D. K. Krishnadas, and K. G. Lucas. 2016. Dendritic cell vaccines: A review of recent developments and their potential pediatric application, Hum Vaccin Immunother, 12: 2232-9. [0182] Fridman, W. H., L. Zitvogel, C. Sautes-Fridman, and G. Kroemer. 2017. The immune contexture in cancer prognosis and treatment, Nat Rev Clin Oncol, 14: 717-34. [0183] Fucikova, J., I. Moserova, I. Truxova, I. Hermanova, I. Vancurova, S. Partlova, A. Fialova, L. Sojka, P. F. Cartron, M. Houska, L. Rob, J. Bartunkova, and R. Spisek. 2014. High hydrostatic pressure induces immunogenic cell death in human tumor cells, Int J Cancer, 135: 1165-77. [0184] Fucikova, J., J. Rakova, M. Hensler, L. Kasikova, L. Belicova, K. Hladikova, I. Truxova, P. Skapa, J. Laco, L. Pecen, I. Praznovec, M. J. Halaska, T. Brtnicky, R. Kodet, A. Fialova, J. Pineau, A. Gey, E. Tartour, A. Ryska, L. Galluzzi, and R. Spisek. 2019. TIM-3 Dictates Functional Orientation of the Immune Infiltrate in Ovarian Cancer, Clin Cancer Res, 25: 4820-31. [0185] Galluzzi, L., L. Senovilla, E. Vacchelli, A. Eggermont, W. H. Fridman, J. Galon, C. Sautes-Fridman, E. Tartour, L. Zitvogel, and G. Kroemer. 2012. Trial watch: Dendritic cell-based interventions for cancer therapy, Oncoimmunology, 1: 1111-34. [0186] Galon, Jerome, and Daniela Bruni. 2019. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies, Nature Reviews Drug Discovery, 18: 197-218. [0187] Garg, A. D., M. Vara Perez, M. Schaaf, P. Agostinis, L. Zitvogel, G. Kroemer, and L. Galluzzi. 2017. Trial watch: Dendritic cell-based anticancer immunotherapy, Oncoimmunology, 6: e1328341. [0188] Goc, J., C. Germain, T. K. Vo-Bourgais, A. Lupo, C. Klein, S. Knockaert, L. de Chaisemartin, H. Ouakrim, E. Becht, M. Alifano, P. Validire, R. Remark, S. A. Hammond, I. Cremer, D. Damotte, W. H. Fridman, C. Sautes-Fridman, and M. C. Dieu-Nosjean. 2014. Dendritic cells in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and license the positive prognostic value of infiltrating CD8+ T cells, Cancer Res, 74: 705-15. [0189] Head, S R. 2018. Next Generation Sequencing (Humana Press). [0190] Hradilova, N., L. Sadilkova, O. Palata, D. Mysikova, H. Mrazkova, R. Lischke, R. Spisek, and I. Adkins. 2017. Generation of dendritic cell-based vaccine using high hydrostatic pressure for non-small cell lung cancer immunotherapy, PLoS One, 12: e0171539. [0191] Ledermann, J. A., F. A. Raja, C. Fotopoulou, A. Gonzalez-Martin, N. Colombo, C. Sessa, and Esmo Guidelines Working Group. 2013. Newly diagnosed and relapsed epithelial ovarian carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up, Ann Oncol, 24 Suppl 6: vi24-32. [0192] Lisi, L., G. M. Ciotti, D. Braun, S. Kalinin, D. Curro, C. Dello Russo, A. Coli, A. Mangiola, C. Anile, D. L. Feinstein, and P. Navarra. 2017. Expression of iNOS, CD163 and ARG-1 taken as M1 and M2 markers of microglial polarization in human glioblastoma and the surrounding normal parenchyma, Neurosci Lett, 645: 106-12. [0193] Maleki Vareki, S. 2018. High and low mutational burden tumors versus immunologically hot and cold tumors and response to immune checkpoint inhibitors, J Immunother Cancer, 6: 157. [0194] Palucka, K., and J. Banchereau. 2013. Dendritic-cell-based therapeutic cancer vaccines, Immunity, 39: 38-48. [0195] Rob, L., P. Mallmann, P. Knapp, B. Melichar, J. Klat, L. Minar, Z. Novotny, J. Bartunkova, R. Spisek, L. Pecen, H. I. B. Hassan, D. Cibula, and SOV01 Investigators. 2018. Dendritic cell vaccine (DCVAC) with chemotherapy (ct) in patients (pts) with epithelial ovarian carcinoma (EOC) after primary debulking surgery (PDS): Interim analysis of a phase 2, open-label, randomized, multicenter trial, Journal of Clinical Oncology, 36: 5509-09. [0196] Sakarya, D. K.; Yetimalar, M. H. 2016. Is there a current change of maintenance treatment in ovarian cancer? An updated review of the literature, JBUON, 21: 290-300. [0197] Schreibelt, G., J. Tel, K. H. Sliepen, D. Benitez-Ribas, C. G. Figdor, G. J. Adema, and I. J. de Vries. 2010. Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy, Cancer Immunol Immunother, 59: 1573-82. [0198] Steele, K. E., T. H. Tan, R. Korn, K. Dacosta, C. Brown, M. Kuziora, J. Zimmermann, B. Laffin, M. Widmaier, L. Rognoni, R. Cardenes, K. Schneider, A. Boutrin, P. Martin, J. Zha, and T. Wiestler. 2018. Measuring multiple parameters of CD8+ tumor-infiltrating lymphocytes in human cancers by image analysis, J Immunother Cancer, 6: 20. [0199] TruSight Oncology 500 Reference Guide. In. 2020. edited by Inc. Illumina, 50. Illumina, Inc. [0200] Truxova, I., L. Kasikova, M. Hensler, P. Skapa, J. Laco, L. Pecen, L. Belicova, I. Praznovec, M. J. Halaska, T. Brtnicky, E. Salkova, L. Rob, R. Kodet, J. Goc, C. Sautes-Fridman, W. H. Fridman, A. Ryska, L. Galluzzi, R. Spisek, and J. Fucikova. 2018. Mature dendritic cells correlate with favorable immune infiltrate and improved prognosis in ovarian carcinoma patients, J Immunother Cancer, 6: 139. [0201] Turnis, M. E., and C. M. Rooney. 2010. Enhancement of dendritic cells as vaccines for cancer, Immunotherapy, 2: 847-62. [0202] Vacchelli, E., I. Vitale, A. Eggermont, W. H. Fridman, J. Fucikova, I. Cremer, J. Galon, E. Tartour, L. Zitvogel, G. Kroemer, and L. Galluzzi. 2013. Trial watch: Dendritic cell-based interventions for cancer therapy, Oncoimmunology, 2: e25771. [0203] WO 2013/004708 [0204] WO 2015/097037