WO2020187975A1

WO2020187975A1 - Patient selection for treatment with dendritic cell vaccination

Info

Publication number: WO2020187975A1
Application number: PCT/EP2020/057415
Authority: WO
Inventors: Jirina Bartunková; Ladislav PECEN; Hariz Iskandar Bin HASSAN
Original assignee: Sotio AS
Current assignee: Sotio AS
Priority date: 2019-03-19
Filing date: 2020-03-18
Publication date: 2020-09-24
Anticipated expiration: 2021-09-19

Abstract

The present invention relates to 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 by determining in a blood sample from the patient the amount of NK cells and comparing it to a threshold level.

Description

Patient selection for treatment with dendritic cell vaccination

The immune system is a powerful tool that, if better harnessed, could enhance the efficacy of cytotoxic agents and improve outcomes for cancer sufferers. Vaccination is hoped to be an effective method of harnessing the immune system to eliminate cancer cells. Its activity is mostly dependent on antigen-specific CD8⁺ T cells that generate cytotoxic T lymphocytes (CTLs) to reject cancer (Palucka and Banchereau 2013). The immune system components necessary for the induction of such CD8⁺ T cells include the presentation of antigens by appropriate antigen-presenting cells (APCs). Dendritic cells (DCs) are the most efficient APCs, and are an essential component of vaccination through their capacity to capture, process, and present antigens to T cells (Banchereau and Steinman 1998). Multiple cancer vaccination strategies based on DCs have been developed (Galluzzi et al. 2012). One strategy is to culture patient-derived (autologous) monocytes with specific cytokine combinations, load them with tumor-associated antigen (TAAs) ex-vivo in the presence of adjuvants to promote DC maturation and eventually re-administer them into the patient. Other strategies are to deliver TAAs to DCs in vivo or approaches based on DC-derived exosomes (Galluzzi et al. 2012). These strategies have been tested in multiple clinical trials, ranging over a large variety of cancers (Galluzzi et al. 2012; Vacchelli et al. 2013; Bloy et al. 2014; Garg et al. 2017). A newly developed DC vaccine, based on dendritic cell obtained from patient-derived (autologous) monocytes loaded with tumor cell undergoing immunogenic cell death (ICD) and matured with Toll-like receptor (TLR) agonists binding TLR3 or TLR4 has been shown to be more beneficial for the overall survival (OS) and progression free survival (PFS) of ovarian cancer patients than standard of care chemotherapy alone (Cibula et al. 2018; Rob et al. 2018).

Efficacy of such a DC vaccine treatment may further be improved by identifying, prior to treatment, patients who would benefit from such treatment. Since blood tests are routinely used in health care to determine physiological and biochemical states of patients, and are often preceding the initiation of many existing cancer treatments, such blood tests may thus also be an appropriate pre-treatment evaluation of patients eligible for a DC vaccine treatment. The inventors have now surprisingly identified blood biomarkers, namely levels of NK cells and levels of platelets in blood, enabling to identify, prior to treatment, patients suffering from ovarian cancer who would benefit the most from such a DC vaccine treatment. Definitions

“Dendritic cell vaccine” or“DC vaccine” refers to human DCs for therapeutic use that may be administered, being prepared without an antigen source or with an antigen source. Preferably, the DCs have been prepared by loading the DCs with an antigen sourced from tumor associated peptide(s), whole antigens from DNA or RNA, whole antigen-protein, idiotype protein, tumor lysate, whole tumor cells or viral vector-delivered whole antigen and subsequently optionally matured with toll-like receptor agonists. More preferably, the DCs have been loaded with whole tumor cells undergoing immunogenic cell death as described for example by Fucikova et al. (2014). Further, the loaded DCs may be further matured. Maturation occurs e.g. by culturing the loaded DCs in the presence of maturation factors such as Toll-like receptor agonists (e.g., poly[I:C] or LPS).

“Toll-like receptor agonists” refers to molecules binding toll-like receptors (TLR), e.g. lipopolysaccharides binding to TLR4, double-stranded RNA or polyinosinic:polycytidylic acid (poly[I:C]) binding to TLR3. Further, TLR1, TLR2, TLR5 and TLR8 agonists are suitable for maturation. TLR expression and function in DCs is reviewed by Schreibelt et al. (2010), see table 1 for monocyte derived DCs (moDC).

“Chemotherapy” is a treatment that uses drugs or agents to stop the growth of cancer cells, either by killing the cells (cytotoxic agents) or by stopping them from dividing (cytostatic agents). Chemotherapy drugs may include alkylating agents or alkylating-like agents such as carboplatin or cisplatin, antimetabolites such as gemcitabine, pemetrexed, methotrexate, anti-tumor antibiotics such as doxorubicin, topoisomerase inhibitors such as topotecan, irinotecan or etoposide, mitotic inhibitors such as docetaxel, paclitaxel, vinblastine, or vinorelbine. Further examples of chemotherapy drugs are provided by the American Cancer Society and can be found here:

Cancer Society - How Chemotherapy Drugs Work' 2016). Such drugs may typically be small molecule drugs (organic compounds of low molecular weight, e.g. < 900 Dalton). Chemotherapy may also include other treatments such as hormones and hormone analogous, small molecules drugs (e.g. kinase inhibitors, PARP inhibitors), vaccines other than DC vaccines, or biologies (e.g. antibodies), or combinations thereof. Chemotherapy may be given orally, by injection, or infusion, or on the skin, depending on the type and stage of the cancer being treated. It may be given alone or with other treatments, such as surgery, radiation therapy.

The term chemotherapy also covers“neoadjuvant chemotherapy”, which is a chemotherapy treatment given as a first step to shrink a tumor before the main treatment, which is usually surgery.

The term chemotherapy also covers“adjuvant chemotherapy”, which is a chemotherapy treatment given after the primary treatment (for example surgery or radiation) to treat residual tumor and/or to prevent or treat metastasis. In the context of the present invention, dendritic cell vaccines are considered not to be adjuvant chemotherapy.

“First-line therapy” is a first treatment given for a disease. It is often part of a standard set of treatments, such as surgery followed by chemotherapy and radiation. When used by itself, first- line therapy is typically the one accepted as the best treatment for a disease. If it does not cure the disease or if it causes severe side effects, other treatment options may be added or used instead. The first-line therapy can also be called induction therapy, primary therapy, or primary treatment.

“First-line chemotherapy” is a therapy with at least one chemotherapeutic agent as part of a first- line therapy. It is understood that the term“first-line chemotherapy” does not comprise a parallel use of a dendritic cell vaccine therapy.

“Second-line chemotherapy” is a chemotherapy that is given when the patient does not respond to the initial treatment (first-line therapy), or if the first-line therapy stops being effective.“Third-line chemotherapy” is the chemotherapy that is given when both initial treatment (first-line therapy) and subsequent treatment (second-line therapy) were not effective or stopped being effective.

A treated cancer may“relapse” or be“recurrent” when the cancer or signs and symptoms of the cancer return after a period of improvement.

“Overall survival” (OS) is the length of time from either the date of diagnosis, date of randomization or the start of treatment for a disease, such as cancer, that patients diagnosed with the disease are still alive. In a clinical trial, measuring the overall survival is one way to measure the efficacy of a new treatment. Randomization into a clinical trial is the process by which subjects are assigned by chance to separate groups that compare different treatments or other interventions. Randomization gives each participant an equal chance of being assigned to any of the groups.

“Overall survival rate” is the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with a disease, randomized or started treatment for a disease, such as cancer. The overall survival rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment.

“Standard of care” (SOC) is a treatment that is accepted by medical experts as a proper treatment for a certain type of disease and that is widely used by healthcare professionals. It can also be called best practice, standard medical care, and standard therapy.

A“blood test” helps doctors to check for certain diseases and conditions. The Complete Blood Count (CBC) is the most common blood test. This test measures the count of erythrocyte (or red blood cells), of leucocytes (or white blood cells), of platelets, hemoglobin, hematocrit and mean corpuscular volume. Methods to practice blood tests can be found in guides or textbooks for use in diagnostic hematology laboratories (Bain 2015) (see especially Chapter 2“Performing a blood count”, p 17- 66).

The“platelet count” in blood can be measured during the blood test. Platelets (or thrombocytes) can be counted in a hemocytometer using either diluted whole blood or platelets-rich plasma prepared by sedimentation or centrifugation. In this method, platelets are visualized by light or phase-contrast microscopy. Platelets can also be counted by semi-automated or fully automated methods using flow cytometry based on impedance counters. The most accurate way to perform a platelet count require fully automated blood cell counters, where platelets are either counted using impedance, light-scattering or optical fluorescence technology. When direct current (DC) impedance is used to count platelets, it can be combined with hydrodynamic focusing. DC impedance technology is based on the principle that an electrical field, created between two electrodes of opposite charge, can be used to count and determine the size of cells. Blood cells are poor conductors of electricity. The diluent (reagent) in which they are suspended during counting is an isotonic solution which is a good conductor of electricity. Consequently, when the cells suspended in the diluent pass through an aperture (a narrowing) between the electrodes, each individual cell will momentarily increase the impedance (resistance) of the electrical path between the electrodes. Each cell generates an electrical pulse in proportion to its size (volume). Cell/particle counting can be optimized by hydrostatic focusing, where by means of a sheath flow, the cells or particles are separated and aligned upon entering the flow cell or detection unit to effectively prevent coinciding or recirculation of cells/particles (Sun and Morgan 2010; Briggs, Harrison, and Machin 2007). When fluorescence technologies are used, fluorochrome-labelled monoclonal antibodies raised against a platelet glycoprotein or against proteins expressed by platelets such as CD41, CD42a or CD61, can be incorporated so that platelets are reliably distinguished from other small particles. Alternatively, platelets can also be counted by fluorescence technologies using fluorescent dyes selectively labelling platelets, such as the PLT-F dye (Wada et al. 2015). Automated blood cell counters have been optimized to count platelets using PLT-F or similar dyes (Wada et al. 2015; Lim et al. 2018; Bain 2015). The average range of platelets count in healthy human patients is 150-400 x 10⁹/1.

A“natural killer cells or NK cells count” in blood can be measured from blood samples. NK cells are a type of cytotoxic lymphocytes critical to the innate immune system. The population of NK cells is heterogeneous. Using flow cytometry, NK cells can be sorted and counted by flow cytometry as cells being CD45⁺CD56^{~(or bn}s^ht)CD16 and CD45^~CD56^{-(or dim)}CD16⁺ (Caligiuri 2008). The average count of NK cells in healthy human patients is about 250 cells/mΐ of blood (absolute count) or about 12% of all blood lymphocytes (relative count) (Apoil et al. 2017; Pascal et al. 2004).

When the term“about” is used herein in relation to numerical values, it is understood as being ±5%, preferably ±2%, of the numerical value.

Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used. Description of the invention

In a first aspect, the invention relates to 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 by determining in a blood sample from the patient the amount of NK cells and comparing it to a threshold level. Determining the amount of NK cells may be part of the routine blood test during physical examination prior to treatment or prescription of the dendritic cell vaccine. NK cells are understood to be CD45⁺ CD56^{+ (or bn}s^ht) CD 16 and CD45⁺ CD56 ^{(or dim)} CD16⁺ and may be counted by flow cytometry or other means known to a person skilled in the art. Blood tests a routinely performed by specialized hematology laboratories according to guidelines. Blood sampling may be performed by trained laboratory personnel at the practicing doctor’s office. The blood sample may be obtained the most easily from a vein in the antecubital fossa using needles and either syringes or evacuated tubes (such as a vacutainer tubes). Blood collection tube may additionally contain additives such as anticoagulants (EDTA, sodium citrate, or heparin) or gels to separate blood cells from plasma (Bain 2015) (see Chapter 1“Blood sampling and blood film preparation and examination”, p 1-16).

The inventors have now surprisingly found that tumor patients can retrospectively be stratified with respect to their levels of NK cells in blood, and the resulting two groups had a significant difference in survival when treated with a dendritic cell vaccine (see Example 4. Clinical data in first recurrence of ovarian cancer).

In another aspect, the invention relates to 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 by determining in a blood sample from the patient the count of platelets and comparing it to a threshold level. Determining the count of platelets in a blood sample may be part of the routine blood test during physical examination prior to treatment or prescription of the dendritic cell vaccine. Multiple methods have been developed to count platelets (Bain 2015) (see p. 22-23). Platelets may be measured by flow cytometry using DC impedance method with hydrodynamic focusing (Briggs, Harrison, and Machin 2007; Sun and Morgan 2010). Platelets may also be accurately measured by fluorescent flow cytometry as CD41⁺, CD42a⁺ or CD61⁺ cells, or using fluorescent dyes selectively labelling platelets, such as the PLT-F dye (Wada et al. 2015). The platelets count is usually part of a complete blood count (CBC) and can be measured using automated hematology analyzer in specialized hematology laboratories according to guidelines.

Similarly as compared to NK cell, the inventors have surprisingly found that tumor patients can retrospectively be stratified with respect to their levels of platelets in blood, and the resulting two groups had a significant difference in survival when treated with a dendritic cell vaccine (see Example 4. Clinical data in first recurrence of ovarian cancer).

In yet another aspect, the invention relates to 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 by determining in a blood sample from the patient the amount of NK cells and comparing it to a threshold level and by determining in a blood sample from the patient the count of platelets and comparing it to a threshold level. Blood from the same sample may be used to determine the amount of NK cells and the count of platelets. Surprisingly, both NK cell levels and platelet levels were correlating together with the survival of the treated tumor patients (see Example 4. Clinical data in first recurrence of ovarian cancer).

In one embodiment, the amount of NK cells measured for the patient selection may be expressed as the relative level of NK cells of total lymphocytes. The total amount of lymphocytes may be determined from a blood sample by fluorescent flow cytometry as the count of all CD45⁺ blood cells in the blood sample. The relative level of NK cells of total lymphocytes is the percentage of CD45⁺ CD56⁺ <^{or bri}s^ht> CD 16 and CD45⁺ CD56 <^{or dim)} CD 16⁺ cells among all CD45⁺ cells. The amount of NK cells measured for the patient selection may be expressed as the absolute count of NK cells per microliter (mΐ) of blood. Such measurement may also require measurements by fluorescent flow cytometry.

In another embodiment, the relative level of NK cells of total lymphocytes measured for the selection of patient to be treated with a DC vaccine is compared to a threshold level between 1 1% and 16% of total lymphocytes. In a preferred embodiment, the relative level of NK cells of total lymphocytes measured for the selection of patient to be treated with a DC vaccine is compared to a threshold level between 12% and 15% of total lymphocytes. In yet a more preferred embodiment, the relative level of NK cells of total lymphocytes measured for the selection of patient to be treated with a DC vaccine is compared to a threshold level of 13% of total lymphocytes.

In yet another embodiment, the absolute count of NK cells measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level between 150 and 250 cells/mΐ of blood. In a preferred embodiment, the absolute count of NK cells measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level between 160 and 200 cells/mΐ of blood. In yet a more preferred embodiment, the absolute count of NK cells measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level of about 177 cells/mΐ of blood. The inventors stratified the patients the patients into two groups using the median number of NK cells of this cohort, being 177 cells/mΐ of blood thereby defining two separate group of patients with a significantly different survival outcome of the DC vaccination.

In another aspect of the invention, the platelet count measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level between 250 x 10⁹ and 310 x 10⁹ platelets/1. In a preferred aspect, the platelet count measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level between 260 x 10⁹ and 300 x 10⁹ platelets/1. In yet a more preferred aspect, the platelet count measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level of about 279 x 10⁹ platelets/1.

In another aspect of the invention, the platelet count measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level between 270 x 10⁹ and 330 x 10⁹ platelets/1. In a preferred aspect, the platelet count measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level between 280 x 10⁹ and 310 x 10⁹ platelets/1. In yet a more preferred aspect, the platelet count measured for the selection of patients to be treated with a DC vaccine is compared to a threshold level of about 300 x 10⁹ platelets/1.

This finding was surprising, as high platelet counts have been associated with a rather bad prognosis for tumor patients in general. Thrombocytosis, a disorder in which the body produces too many platelets, has been shown to correlate with poor prognosis in patients with gastric, pancreatic, colorectal, ovarian, and endometrial cancer (Suzuki et al. 2004; Long et al. 2016; Pietrzyk et al. 2016; Ekici et al. 2016; Qiu et al. 2012; Yin et al. 2018).

In another embodiment, the invention relates to 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 if the amount of NK cells and/or the amount of platelets is equal or above the threshold level. Surprisingly, patients selected with a NK cells level equal or above the threshold level for a treatment with the DC vaccine in combination with standard of care chemotherapy will have a higher chance of survival to cancer than patients with a NK cells level below this threshold. Likewise, patients selected with a platelet count equal or above the threshold for a treatment with the DC vaccine in combination with standard of care chemotherapy will have a higher chance of survival to cancer than patients with a platelet count below this threshold.

The level of NK cells have been previously described as prognostic factors, but mainly based on tumor infiltrating NK cells (Lundgren et al. 2016; Takanami, Takeuchi, and Giga 2001; Drakes and Stiff 2018) and more rarely based on the level of NK cells in circulating blood (Kondo et al. 2003; Terme et al. 2016). However, such prognostic factor based on the level of NK cells was never described in relation to a DC vaccination alone or DC vaccination in combination with chemotherapy. Typically, the artisan would expect that rather CD8⁺ cytotoxic T cells are important to mount an effective immune response in a vaccination approach. However, the presence of absence of CD8⁺ cytotoxic T cells did not correlate with the outcome of the study.

In another embodiment, the invention relates to a dendritic cells vaccine for use in a method of treating cancer in a patient, wherein the treatment comprises a step of determining in a blood sample of the patient whether or not the patient has a relative level of NK cells that is equal or above said threshold level. The invention also relates to a dendritic cells vaccine for use in a method of treating cancer in a patient wherein the treatment comprises a step of determining in a blood sample of the patient whether or not the patient has an absolute count of NK cells that is equal or above said threshold level. Further, the invention relates to a dendritic cells vaccine for use in a method of treating cancer in a patient wherein the treatment comprises a step of determining in a blood sample of the patient whether the patient has a level of platelets that is equal or above said threshold level.

In another embodiment, the selected patients are treated for cancer with a DC vaccine and standard of care chemotherapy, preferably wherein the cancer to be treated is ovarian, lung or prostate cancer. Ovarian cancer includes epithelial ovarian, fallopian tube, or primary peritoneal cancer and may be stage I to IV according to the International Federation of Gynecology and Obstetrics (FIGO). In one embodiment, the cancer to be treated is prostate cancer. Prostate cancer includes acinar adenocarcinoma, ductal adenocarcinoma, transitional cell cancer, squamous cell cancer and small cell cancer and may be graded using the Gleason scoring system. In one embodiment, the cancer to be treated is lung cancer. Lung cancer is preferentially non-small cell lung cancer and includes lung adenocarcinoma, squamous cell lung carcinoma and large-cell lung carcinoma. In a preferred embodiment, the cancer to be treated is ovarian cancer. In another preferred embodiment, the cancer is recurrent. The cancer is recurrent when a previous chemotherapy treatment failed to cure the patient from cancer. A previous chemotherapy may have failed to cure the patient because the patient relapsed after completion or termination of the previous chemotherapy or because the patient was refractory to the chemotherapy. Preferably, during a second line chemotherapy setting or later chemotherapy settings, the first DC vaccine dose administration starts after the 2^nd cycle of chemotherapy and continues after completion/termination of the chemotherapy. In a particularly preferred embodiment, the cancer is recurrent advanced ovarian cancer or relapsed platinum- sensitive epithelial ovarian carcinoma. Different types of DC vaccines are well known in the art and can be divided into different groups according to the method how the DCs are loaded or pulsed with tumor antigens (Tumis and Rooney 2010; Elster, Krishnadas, and Lucas 2016), incorporated herein by reference. Accordingly, the DC vaccine for use in the treatment of cancer administered to a patient in parallel to chemotherapy is a DC vaccine loaded ex-vivo with an antigen source, which is preferably selected from tumor associated peptide(s), whole antigens from DNA or RNA, whole antigen-protein, idiotype protein, tumor lysate, whole tumor cells or viral vector-delivered whole antigen. The DC vaccine may optionally be matured with toll-like receptor (TLR) agonists (e.g. poly[I:C] or LPS). Preferentially, the DC vaccine is matured with the TLR3 agonist poly[I:C]. The tumor lysate may be autologous or allogeneic/heterologous. Likewise, whole tumor cells may be autologous or allogeneic/heterologous. Whole tumor cells and tumor lysate may originate from one or multiple tumor cell lines, and may be selected for a specific presence of antigens matching the patient’s tumor to be treated. The source of antigens may be selected and DCs may be loaded by techniques known to one of skill in the art (Tumis and Rooney 2010; Elster, Krishnadas, and Lucas 2016).

In a preferred embodiment, the whole tumor cells loaded upon the DC vaccine are allogeneic to the patient. Whereas autologous tumor cells purportedly have a better match with the patient’s tumor antigens, in practice it is highly complicated to manufacture a DC vaccine from autologous tumor biopsies. Therefore, it is preferred to use allogeneic tumor cells, e.g. tumor cell lines, which have an overlap of expressed tumor antigens with the typical tumor antigens of the tumor disease to be treated. The DC vaccine may be formulated for intravenous, intradermal or subcutaneous administrations, preferably for subcutaneous administration. The DC vaccine can be formulated for example for infusion or bolus injection, and may be administered together with an adjuvant (Elster, Krishnadas, and Lucas 2016). Administration can be systemic, loco-regional or local. When a dose is injected, the dose can be administered in one or multiple injections, preferably in two injections. Preferably, the DC vaccine may be administered to the patient subcutaneously into lymph-node regions close to the region where the cancer to be treated is developing. Various delivery systems are known and can be used to deliver the DC vaccine. The mode of administration may be left to the discretion of the practitioner.

In another aspect of the invention, the blood to be analyzed for patient selection is obtained prior to the treatment of the patient with the dendritic cell vaccine and an optional concomitant chemotherapy. The blood sample may be obtained within one week, two weeks, three weeks, four weeks or within one month or two month of the start of the treatment.

The optional concomitant chemotherapy may be the standard of care chemotherapy. The chemotherapeutic agent or agents to be used may be determined by the qualified practitioner, and may be part of the standard of care recommended for the type of cancer to be treated. Depending on the standard of care, the chemotherapeutic agent/s may be administered in one or multiple cycles. The duration, frequency and number of cycles will depend on the cancer to be treated and on the agent/s used. The chemotherapeutic agent/s may all be given on a single day, several consecutive days, or continuously, to an outpatient or to an inpatient. Treatment could last minutes, hours, or days, depending on the specific protocol. Chemotherapy may be repeated weekly, bi weekly, or monthly. Usually, a cycle is defined in monthly intervals. For example, two bi-weekly chemotherapy sessions followed by a recovery period may be classified as one cycle. In most cases, the total number of cycles - or the length of chemotherapy from start to finish - has been determined by research and clinical trials. As long as there are detectable signs of cancer, the length of chemotherapy treatment will depend upon the response of the cancer to therapy. If the cancer disappears completely (complete response), chemotherapy may continue for 1-2 cycles beyond this observation to maximize the chance of having attacked all microscopic, i.e. undetectable, tumors. If the cancer shrinks but does not disappear (partial response), chemotherapy may continue as long as it is tolerated and the cancer does not grow (progression). In one embodiment, the level of NK cells in blood is measured by flow cytometry/FACS. NK cells are understood to be CD45⁺ CD56^{+ (}°^rbnght) CD16 and CD45⁺ CD56 ⁽°^{r dim)} CD16⁺ cells, and further CD3 cells. Any fluorophore-coupled antibodies against CD45, CD3, CD16 and CD56 may be used to measure the amount of NK cells by flow cytometry. FACS is the same technique as flow cytometry and stands for fluorescence-activated cell sorting. Flow cytometry may be performed as described in Adan A. et al. (2017) and Jevremovic & Olteanu (2019). Blood samples for flow cytometry analysis may be prepared according to McCoy (2001).

In another embodiment, the level of platelets in blood is measured by flow cytometry. Using flow cytometry, platelets may be measured by DC impedance method with hydrodynamic focusing (Briggs, Harrison, and Machin 2007). Platelets may also be measured by fluorescent flow cytometry. For fluorescent flow cytometry, platelets may be stained with fluorochrome-labelled monoclonal antibodies raised against a platelet glycoprotein or against proteins expressed by platelets such as GPIIb/IIIa, CD41, CD42a or CD61. When flow cytometry is performed using antibodies to stain platelets, it may be performed as described in Karolczak et al. (2019). Alternatively, platelets can also be labelled with fluorescent dyes selectively labelling platelets, such as the PLT-F dye (Wada et al. 2015). Automated blood cell counters have been optimized to count by flow cytometry platelets using PLT-F or similar dyes (Wada et al. 2015; Lim et al. 2018; Bain 2015).

In yet another aspect of the invention, the levels of NK cells and platelets is determined in blood obtained from the patient prior to leukapheresis. The blood sample may be obtained prior to prescription of the DC vaccine treatment. Leukapheresis will only be performed once the DC vaccine treatment is prescribed. Leukapheresis is a laboratory procedure in which leukocyte are separated from a sample of blood. Therefore, an NK cells or platelet count may not be accurate, if performed after leukapheresis. In another aspect of the invention, the DCs may be derived from monocytes that are autologous to the patient being treated. As used herein, the term“monocytes” refers to leukocytes circulating in the blood characterized by a bean-shaped nucleus and by the absence of granules. Monocytes can give rise to dendritic cells. The monocytes can be isolated from a patient’s blood by any technique known to one of skill in the art, the preferred method being leukapheresis. Leukapheresis allows to collect monocytes that are autologous to the patient being treated, to be used for the preparation of the DC vaccine. Leukapheresis may be performed by any technique known to one of skill in the art. Typically, dendritic cells are derived from monocytes obtained by leukapheresis prior to chemotherapy, which is combined with the DC vaccination. As many chemotherapies induce neutropenia, a leukapheresis after initiation of chemotherapy may lead to less viable cells and therefore to a lower quantity and/or quality of the DC vaccine.

In one embodiment, the immature dendritic cells derived from monocytes are in a first step loaded with tumor cells undergoing immunogenic cell death (ICD). ICD, a specific type of apoptosis, may be characterized by expression of immunogenic molecules on the cell surface such as HSP70, HSP90 and calreticulin and the release of late apoptotic markers HMGB 1 and ATP and thus increases the uptake of these cells by DCs, resulting in loaded DCs presenting the multiple tumor antigens. In a preferred embodiment of the invention, the antigen source is whole tumor cells and wherein preferably the tumor cells were killed by high hydrostatic pressure (HHP), e.g. as described in WO 2013/004708 and WO 2015/097037, incorporated herein by reference (see examples 1 to 4 of WO 2013/004708 and examples 2 and 3 of WO 2015/097037). In brief, whole tumor cells from cell lines or from the patient are treated by HHP between 200 and 300 MPa for 10 min to 2 hours. Such a treatment will induce ICD. Other methods to introduce ICD are the treatment with anthracyclines (Casares et al. 2005) or heat - preferably severe heat above 45°C (Adkins et al. 2017). Prior to being loaded on DCs, the apoptotic tumor cells may be cryopreserved (see WO 2015/097037).

In one embodiment, the whole tumor cells loaded upon the DC vaccine are allogeneic to the patient. Whereas autologous tumor cells purportedly have a better match with the patient’s tumor antigens, in practice it is highly complicated to manufacture a DC vaccine from autologous tumor biopsies. Therefore, it is preferred to use allogeneic tumor cells, e.g. tumor cell lines, which have an overlap of expressed tumor antigens with the typical tumor antigens of the tumor disease to be treated.

In yet another embodiment, following leukapheresis, the collected autologous monocytes are cultivated in the presence of cytokines to obtain immature DCs. Preferred cytokines are GM-CSF and IL-4. The DC vaccine is obtained by loading the immature DCs with tumor cells undergoing ICD, preferably from HHP treated allogeneic tumor cell lines of the same tumor origin as the one to be treated. In another embodiment, loaded DCs are further matured with TLR agonists. TLR agonists may be TLR 3 or TLR4 agonists, preferably the TLR3 agonist poly[I:C]. Resulting DC vaccines may be fractioned and stored in individual doses of approximately lxlO⁷ DCs per dose.

Mature DCs generated as described herein may be characterized as displaying significantly higher expression of maturation markers, such as CD80, CD83, HLA-DR and CD86, than immature DCs and DCs loaded with tumor cells killed by other modalities, such as UV irradiation, as shown by Hradilova et al. and Fucikova et al. (Hradilova et al. 2017; Fucikova et al. 2014). Furthermore, Fucikova et al. and Hradilova et al. also have shown that DCs generated as described herein induced a greater number of tumor-specific CD4⁺ and CD8⁺ IFN-y-producing T cells and decreases the number of CD4⁺CD25⁺Foxp3⁺T regulatory cells compared to DCs pulsed with UV-B light- exposed cells.

In another aspect of the invention, the dendritic cell vaccine is administered to a patient in combination with a different treatment modality selected from the group of chemotherapy, targeted therapy and biologies. In the context of the present invention, the term chemotherapy is understood to relate to the class of compounds used as cytostatic or cytotoxic agents. In a preferred embodiment, the at least one chemotherapeutic agent is selected from carboplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, pegylated liposomal doxorubicin, etoposide, topotecan, irinotecan, olaparib, rucaparib, trabectedin, niraparib, mitoxantrone, cabazitaxel, vinorelbine, pemetrexed, vinblastine, and albumin-bound paclitaxel. The specific regimen of chemotherapy may be dictated by the established standard of care of the specific cancer to be treated, and may be at the discretion of the practitioner. In a preferred embodiment, cyclophosphamide is not a chemotherapeutic agent. In particular, a low dose cyclophosphamide is not a chemotherapeutic agent according to the present invention; it is used to reduce the function of regulatory T cells (Berd and Mastrangelo 1987; Fucikova et al. 2017) and has been used prior to DC vaccination in the prior art (Dong et al. 2016). Chemotherapy may include drugs prescribed as maintenance therapies or prescribed until progression of the cancer such as hormonal therapies (e.g. enzalutamide, abiraterone, tamoxifen, LHRH agonists and antagonists), targeted therapies (e.g. erlotinib, afatinib, gefitinib, crizotinib, alectinib, ceritinib, dabrafenib, trametinib and osimertinib) and biologies (e.g. antibodies as for example bevacizumab, pembrolizumab, nivolumab). For the avoidance of doubt, chemotherapy in the meaning of this invention does not include drugs prescribed to treat co- morbidities, to avoid or treat side-effects or help the patient to recover from side-effects (e.g. erythropoietin).

The first dose of DC vaccine is administered in parallel to each chemotherapy cycle, and may be further administered after completion/termination of the chemotherapy. The first DC vaccine administration may start with the first cycle of chemotherapy, with the second chemotherapy cycle, with the third chemotherapy cycle, or with later chemotherapy cycles. Preferentially, the first DC vaccine dose administration starts after the 2^nd cycle of chemotherapy and continues after completion/termination of the chemotherapy. In a preferred embodiment, the patient received at least 6 cycles of chemotherapy.

In another embodiment, DC vaccine is administered to a patient in parallel to chemotherapy with at least one chemotherapeutic agent. In a preferred embodiment, the invention relates to a DC vaccine treatment administered to a patient in parallel to chemotherapy, where a previous chemotherapy treatment failed to cure the patient from cancer. A previous chemotherapy may have failed to cure the patient because the patient relapsed after completion or termination of the previous chemotherapy or because the patient was refractory to the chemotherapy. Preferably, during a second line chemotherapy setting or later chemotherapy settings, the first DC vaccine dose administration starts after the 2^nd cycle of chemotherapy and continues after completion/termination of the chemotherapy.

In yet another aspect of the invention, the dendritic cell vaccine is administered to a patient after completion of chemotherapy with at least one chemotherapeutic agent. The first dose of the DC vaccine is administered to the patient preferably within two months after completion of the last cycle of the chemotherapy, preferably within one month after completion of the last cycle of the chemotherapy, more preferably within two weeks after completion of the last cycle of the chemotherapy, most preferably immediately after the completion of the last cycle of the chemotherapy. The timing between completion of the last cycle of chemotherapy and administration of the DC vaccine may depend on the chemotherapeutic agent/s, on the cancer to be treated, on the reaction of the patient regarding toxicity of the chemotherapeutic agent/s. In a preferred embodiment, the patient received at least 3 cycles of chemotherapy and preferably at least 6 cycles of chemotherapy. Alternatively, the first dose of the DC vaccine is administered to the patient within two months after administration of the last dose of the chemotherapy, preferably within one month after administration of the last dose of the chemotherapy, more preferably within two weeks after administration of the last dose of the chemotherapy. The last dose may be given within the last cycle of chemotherapy, i.e. before completion of the last chemotherapy cycle.

The invention is also described by the following items:

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 by determining in a blood sample from the patient the amount of NK cells and comparing it to a threshold level.

2. 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 by determining in a blood sample from the patient the count of platelets and comparing it to a threshold level.

3. 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 by determining in a blood sample from the patient the amount of NK cells and comparing it to a threshold level and by determining in a blood sample from the patient the count of platelets and comparing it to a threshold level.

4. The dendritic cell vaccine for use of item 1 or item 3, wherein the threshold level is a relative level of NK cells of the total lymphocytes.

5. The dendritic cell vaccine for use of item 1 or item 3, wherein the threshold level is an absolute count of NK cells.

6. The dendritic cell vaccine for use of item 4, wherein the threshold level is a relative NK cell level between 11% and 16% of total lymphocytes, between 12% and 15% of total lymphocytes and preferably of 13% of total lymphocytes.

7. The dendritic cell vaccine for use of item 5, wherein the threshold level is an absolute count of NK cells between 150 and 250 cells/mΐ of blood, between 160 and 200 cells/mΐ of blood and preferably of 177 cells/mΐ of blood.

8. The dendritic cell vaccine for use of item 2 or item 3, wherein the threshold level is a count of platelets between 250 x 10⁹ and 310 x 10⁹ platelets/1, between 260 x 10⁹ and 300 x 10⁹ platelets/1 and preferably of 279 x 10⁹ platelets/1. 9. The dendritic cell vaccine of items 1 to 8, wherein the patient is selected for treatment ifthe amount of NK cells and/or the amount of platelets is equal or above the threshold level.

10. The dendritic cells vaccine for use of items 1 to 9, wherein the treatment comprises a step of determining in a blood sample of the patient,

a. whether or not the patient has a relative level of NK cells that is equal or above said threshold level, and/or

b. whether or not the patient has an absolute count of NK cells that is equal or above said threshold level, and/or

c. whether the patient has a level of platelets that is equal or above said threshold level.

11. The dendritic cell vaccine for use of items 1 to 10, wherein the cancer is ovarian, lung or prostate cancer, preferably ovarian cancer.

12. The dendritic cell vaccine for use of items 1 to 11 wherein the cancer is recurrent.

13. The dendritic cell vaccine for use of items 1 and 12, wherein the blood sample is obtained prior to the treatment of the patient with the dendritic cell vaccine and an optional concomitant chemotherapy.

14. The dendritic cell vaccine for use of items 1 and 13, wherein the levels of CD16⁺CD56⁺ NK cells and platelets are determined in blood obtained from the patient prior to leukapheresis.

15. The dendritic cell vaccine for use of items 1, 3-7 and 9-14, wherein the level of NK cells in blood is measured by flow cytometry/FACS.

16. The dendritic cell vaccine for use of items 1, 2 and 8-14, wherein the level of platelets in blood is measured by flow cytometry.

17. The dendritic cell vaccine for use of items 1 to 16, wherein the dendritic cells are derived from monocytes that are autologous to the patient to be treated.

18. The dendritic cell vaccine for use of items 1 to 17, wherein the monocytes are obtained by leukapheresis.

19. The dendritic cell vaccine for use of items 1 to 18, wherein immature dendritic cells are in a first step loaded with tumor cells undergoing immunogenic cell death.

20. The dendritic cell vaccine for use of items 1 to 19, wherein the loaded dendritic cells are thereafter matured with Toll-like receptor 3 agonists or Toll-like receptor 4 agonists. 21. The dendritic cell vaccine for use of items 1 to 20, wherein the dendritic cell vaccine is administered to a patient in combination with a different treatment modality selected from the group of chemotherapy, targeted therapy, and biologies.

22. The dendritic cell vaccine for use of items 1 to 21, wherein the dendritic cell vaccine is administered to a patient in parallel to chemotherapy with at least one chemotherapeutic agent.

23. The dendritic cell vaccine for use of items 1 to 22, wherein the dendritic cell vaccine is administered to a patient after completion of chemotherapy with at least one chemotherapeutic agent.

Description of the drawings

Figure 1: Schematic diagram of the clinical study on patients with 1^st recurrence of ovarian cancer, comparing the standard of care chemotherapy treatment to the DC vaccine treatment administered in parallel 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. A day 1/8 regimen means that gemcitabine is given on day 1 and 8 of each cycle and carboplatin is given on the first day of every cycle. The length of a cycle is 21 days.

Figure 2: Analysis of OS in the clinical study for the ITT population with 1^st recurrence of ovarian cancer.“Standard of care” stands for chemotherapy treatment alone.

Figure 3: Analysis of OS in the clinical study for the ITT population with 1^st recurrence of ovarian cancer stratified for the absolute count of NK cells. A) CD 16⁺ CD56⁺ NK cell count < 13% of total lymphocytes in blood; B) CD16⁺ CD56⁺ NK cell count > 13% of total lymphocytes in blood.

Figure 4: Analysis of OS in the clinical study for the ITT population with 1^st recurrence of ovarian cancer stratified for the relative amount of NK cells. A) CD16⁺ CD56⁺ NK cell count < 177 cells/mΐ of blood; B) CD16⁺ CD56⁺ NK cell count > 177 cells/mΐ of blood. Figure 5: Analysis of OS in the clinical study for the ITT population with 1^st recurrence of ovarian cancer stratified for the level of platelets. A) Platelet count < 279 x 10⁹/1; B) Platelet count > 279 x 10⁹/1.

Figure 6: Analysis of OS in the clinical study for the ITT population with 1^st recurrence of ovarian cancer stratified for the level of platelets. A) 140 x 10⁹/1 < platelet count < 300 x 10⁹/1; B) Platelet count > 300 x 10⁹/1.

Figure 7: Graph representing the distribution of absolute NK cells counts over the patient population treated with DCVAC (left graph) and patient population treated with standard of care (right graph). X axis: count of NK cells/mΐ of blood; Y axis: frequency of patients. Figure 8: Graph representing the distribution of platelet counts over the patient population treated with DCVAC (left graph) and patient population treated with standard of care (right graph). X axis: platelet count in 10⁹ platelets/1 of blood; Y axis: frequency of patients.

Examples

Example 1. DC vaccine

The DC vaccine consisted of autologous DCs loaded ex vivo with killed ovarian cancer cells 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.

The final product was cryopreserved in doses of approximately lxlO⁷ 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. Measurement of platelets counts and NK cell counts in blood

Platelets count in a blood sample

Venous blood samples of patients to be screened have been collected in BD Vacutainer® EDTA tubes (BC Biosciences, Heidelberg, DE) according to approved protocol and stored at 2 to 8°C until analysis. Prior to analysis, samples were brought back to room temperature.

Platelets count was measured by flow cytometry with a Sysmex XN1000 hematology analyzer (Sysmex Europe GmbH, Norderstedt, DE) according to the manufacturer’s instruction ("Automated Hematology Analyzer XN series (XN-1000) Instructions for Use" 2014).

In brief, the platelets (as well as the erythrocytes) were analyzed using the DC impedance method with hydrodynamic focusing. In this method, the electrical signals of the cells are analyzed as they pass through an aperture in the device. From these primary signals, the device can identify the cell type by determination of the particle size.

The device automatically evaluates the particle distribution in a range from 2-6 fL (low discriminator) to 12-30 fL (high discriminator). For comparison, the size limits (discriminators) for erythrocytes are 25-75 fL and 200-250 fL, respectively. If any irregularities were detected (e.g. abnormal distribution width, multiple peaks), the measurement would have to be repeated using an optical readout (same method as for reticulocytes) according to the programming of the device. The platelet count is given in number of cells x 10⁹/l.

NK cells count in a blood sample

Venous blood samples of patients to be screened have been collected in BD Vacutainer® EDTA tubes (BC Biosciences, Heidelberg, DE) according to approved protocol. NK cells count was measured by flow cytometry/FACS using a Coulter Epics XL flow cytometer (Beckman Coulter, Krefeld, DE).

In brief, a fraction of the blood sample was treated for red blood lysis with a red blood lysis buffer (RBC Lysis Buffer (10X), Beckman Coulter, Krefeld) according to the manufacturer’s instructions. After lysis the cells were stained with CD3, CD 16 and CD56 (anti-human FITC CD3, clone UCHT1, Beckman Coulter; anti-human CD16 PE clone 3G8, Beckman Coulter; anti-human CD56PE clone N901, Beckman Coulter). The lymphocytes were live gated during acquisition using the side and forward scatter dot plot display. The NK cell population was further identified and differentiated into cytotoxic NK cells (CD3 , CD16⁺, CD56⁺) on the basis of the expression of CD56 and CD16. The count of NK cells is expressed either as absolute count of cell/ mΐ of blood or as relative count of % of NK cell/total lymphocytes.

Example 3. Standard therapy after first recurrence of platinum-sensitive ovarian cancer

The European Society of Medical Oncology (ESMO) established guidelines (Ledermann et al. 2013) of treatment options for therapy after first recurrence of platinum-sensitive ovarian cancer. It also included recently authorized maintenance treatments.

• Second-line therapy: taxane in combination with a platinum compound (platinum doublet) is considered valuable, with other agent combinations tested in clinical trials, including gemcitabine, trabectedin and pegylated liposomal doxorubicin. The choice should be based on the toxicity profile and convenience of administration.

• Targeted therapy: bevacizumab in combination with carboplatin and gemcitabine has been approved and is recommended in platinum-sensitive relapsed disease in patients not previously treated with bevacizumab.

• Maintenance treatments after second-line chemotherapy: olaparib, a PARP inhibitor, is indicated as monotherapy for the maintenance treatment of adult patients with platinum sensitive relapsed BRCA-mutated (germline and/or somatic) high-grade serous epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in response (complete response or partial response) to platinum-based chemotherapy. Niraparib, another PRAP inhibitor, is indicated for the maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in complete or partial response to platinum-based chemotherapy.

Despite great efforts to devise an optimal therapeutic approach to overcome the relapse following chemotherapy, only a limited number of treatment options remain available to patients with relapsed disease and the majority of such patients eventually die. Patients with disease recurrence are in high need of novel therapeutic options, such as a DC vaccine, which could improve their life expectancy and quality of life.

Example 4. Clinical data in first recurrence of ovarian cancer

The study was a randomized, open-label, parallel group, multicenter, phase II clinical trial evaluating the effect of addition of a DC vaccine to standard of care chemotherapy (carboplatin and gemcitabine) in women with relapsed platinum-sensitive epithelial ovarian cancer. The aim of this study was to explore the efficacy and safety of a DC vaccine administered in parallel to chemotherapy, as an add-on to standard of care chemotherapy with carboplatin and gemcitabine as compared to chemotherapy alone.

A total of 71 patients were centrally randomized in a ratio of 1 : 1 to treatment group A (39 patients) to receive the DC vaccine in parallel with standard of care chemotherapy or to treatment group B (32 patients) to receive standard of care chemotherapy alone. The DC vaccine was administered to the patients in treatment group A in up to 10 doses. A total of 6, 8, or 10 cycles of standard of care chemotherapy were to be completed by patients in both treatment groups as per investigators’ decision (Figure 1).

The intent-to-treat (ITT) population included all randomized patients regardless of whether they received treatment or not; however, patients randomized to treatment group A had to receive at least 1 dose of DC vaccine to be included into the ITT population (32 patients in treatment group A (parallel DC vaccine) and 32 patients in treatment group B (standard of care).

Analysis of OS

The difference in OS in favor of treatment group A was close to statistical significance in the ITT population (HR = 0.38, p = 0.0032, Table 1, Figure 2) meaning that the risk of death was reduced by 62% in the ITT population, compared to standard of care. Statistical significance has not been reached, likely due to the relatively small size of the study.

Stratification for NK cells counts

OS was analyzed upon stratification of the patients based on their count of NK cells (see Figure 7 for the distribution of NK cell counts over the treated population). When the relative count of CD16⁺ CD56⁺ NK cells threshold value was 13% of total lymphocytes in blood, the difference in favor of patients within treatment group A having a CD16⁺ CD56⁺ NK cell count > 13% of total lymphocytes in blood was significant in the ITT population (HR = 0.189, p = 0.0018) while no difference was noted between the two treatment groups within patients having a CD16⁺ CD56⁺ NK cell count < 13% of total lymphocytes in blood (HR = 0.508, p = 0.1951) (Table 1, Figure 3). This means that the risk of death was reduced by 81.1% in patients treated by DCVAC having a CD16⁺ CD56⁺ NK cell count > 13% of total lymphocytes in blood compared to the same group of patients treated by standard of care.

When the absolute count of CD16⁺ CD56⁺ NK cells threshold value was 177 cells/mΐ of blood, the difference in favor of patients within treatment group A having a CD16⁺ CD56⁺ NK cell count > 177 cells/mΐ of blood was significant in the ITT population (HR = 0.306, p = 0.0082) while no difference was noted between the two treatment groups within patients having a CD16⁺ CD56⁺ NK cell count < 177 cells/ mΐ of blood (HR = 0.887, p = 0.8149) (Table 1, Figure 4). This means that the risk of death was reduced by 69.4% in patients treated by DCVAC having a CD16⁺ CD56⁺ NK cell count > 177 cells/mΐ of blood compared to the same group of patients treated by standard of care.

Stratification for platelet counts

OS was analyzed upon stratification of the patients based on their platelet count (see Figure 8 for the distribution of platelet counts over the treated population). When a median threshold value of 279 x 10⁹ platelets/1 of patient’s blood was chosen, the difference in favor of patients within treatment group A having a platelet count > 279 x 10⁹ platelets/1 was significant in the ITT population (HR = 0.175, p = 0.0005) while no difference was noted between the two treatment groups within patients having a platelet count < 279 x 10⁹ platelets/1 (HR = 1.025, p = 0.9617) (Table 1, Figure 5). This means that the risk of death was reduced by 82.5% in patients treated by DCVAC having a platelet count > 279 x 10⁹ platelets/1 compared to the same group of patients treated by standard of care.

When the platelet count threshold value was 300 x 10⁹ platelets/1, the difference in favor of patients within treatment group A with a count above this threshold 1 was significant in the ITT population (HR = 0.161, p = 0.0027) while no difference was noted between the two treatment groups within patients having a platelet count between 140 and 300 x 10⁹ platelets/1 (HR = 0.669, p = 0.3635) (Table 1, Figure 6). This means that the risk of death was reduced by 83.9% in patients treated by DCVAC having a platelet count > 300 x 10⁹ platelets/1 compared to the same group of patients treated by standard of care.

Table 1 : Benefit of DCVAC/OvCa on patient survival

References

Adan, A._; G. Alizada, Y. Kiraz, Y. Baran, and A. Nalbant. 2017. 'Flow cytometry: basic principles and applications', Crit Rev Biotechnol, 37: 163-76.

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: el311433.

'American Cancer Society - How Chemotherapy Drugs Work'. 2016. American Cancer Society, Accessed

Apoil, P. A., B. Puissant-Lubrano, N. Congy-Jolivet, M. Peres, J. Tkaczuk, F. Roubinet, and A. Blancher. 2017.

'Reference values for T, B and N K human lymphocyte subpopulations in adults', Data Brief, 12: 400-04.

"Automated Hematology Analyzer XN series (XN-1000) Instructions for Use." In. 2014. Sysmex Corporation Kobe Japan.

Bain, Barbara J. 2015. Blood Cells: A Practical Guide, Fifth Edition (John Wiley & Sons Ltd: Chichester, West Sussex, UK).

Banchereau, J., and R. M. Steinman. 1998. 'Dendritic cells and the control of immunity', Nature, 392: 245- 52.

Berd, D., and M. J. Mastrangelo. 1987. 'Effect of low dose cyclophosphamide on the immune system of cancer patients: reduction of T-suppressor function without depletion of the CD8+ subset', Cancer Res, 47: 3317-21.

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.

Briggs, C., P. Harrison, and S. J. Machin. 2007. 'Continuing developments with the automated platelet count', Int J Lab Hematol, 29: 77-91.

Caligiuri, M. A. 2008. 'Human natural killer cells', Blood, 112: 461-9.

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.

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 lst-line platinum (Pt)-based ct: Primary analysis of a phase 2, open-label, randomized, multicenter trial', Journal of Clinical Oncology, 36: el7515- el5.

Dong, W., R. Wei, H. Shen, Y. Ni, L. Meng, and J. Du. 2016. 'Combination of DC Vaccine and Conventional Chemotherapeutics', Anticancer Agents Med Chem, 16: 558-67.

Drakes, M. L., and P. J. Stiff. 2018. 'Regulation of Ovarian Cancer Prognosis by Immune Cells in the Tumor Microenvironment¹, Cancers (Basel), 10.

Ekici, H., E. Malatyalioglu, A. Kokcu, E. Kurtoglu, M. Tosun, and H. Celik. 2016. 'Do Leukocyte and Platelet Counts Have Benefit for Preoperative Evaluation of Endometrial Cancer?', Asian Pac J Cancer Prev, 16: 5305-10.

Elster, J. D., D. K. Krishnadas, and K. G. Lucas. 2016. 'Dendritic cell vaccines: A review of recent developments and their potential pediatric application¹, Hum Vaccin Immunother, 12: 2232-9. 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.

Fucikova, J., M. Podrazil, L. Jarolim, P. Bilkova, M. Hensler, E. Becht, Z. Gasova, J. Klouckova, J. Kayserova, R. Horvath, A. Fialova, K. Vavrova, K. Sochorova, D. Rozkova, R. Spisek, and J. Bartunkova. 2017. 'Phase I/ll trial of dendritic cell-based active cellular immunotherapy with DCVAC/PCa in patients with rising PSA after primary prostatectomy or salvage radiotherapy for the treatment of prostate cancer', Cancer Immunol Immunother.

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.

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: el328341.

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.

Jevremovic, D., and H. Olteanu. 2019. 'Flow Cytometry Applications in the Diagnosis of T/N K-Cell Lymphoproliferative Disorders', Cytometry B Clin Cytom.

Karolczak, K., B. Soltysik, T. Kostka, P. J. Witas, and C. Watala. 2019. 'Platelet and Red Blood Cell Counts, as well as the Concentrations of Uric Acid, but Not Homocysteinaemia or Oxidative Stress, Contribute Mostly to Platelet Reactivity in Older Adults', Oxid Med Cell Longev, 2019: 9467562.

Kondo, E., K. Koda, N. Takiguchi, K. Oda, K. Seike, M. Ishizuka, and M. Miyazaki. 2003. 'Preoperative natural killer cell activity as a prognostic factor for distant metastasis following surgery for colon cancer', Dig Surg, 20: 445-51.

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.

Lim, Y. K., O. J. Kweon, J. H. Choi, W. Lee, and A. J. Park. 2018. 'Measurement uncertainty of platelet concentration using the Sysmex XN automated hematology analyzer', Scand J Clin Lab Invest, 78: 224-29.

Long, Y., T. Wang, Q. Gao, and C. Zhou. 2016. 'Prognostic significance of pretreatment elevated platelet count in patients with colorectal cancer: a meta-analysis', Oncotarget, 7: 81849-61.

Lundgren, Sebastian, Jacob Elebro, Margareta Heby, Bjorn Nodin, Jakob Eberhard, and Karin Jirstrom.

2016. 'Prognostic significance of tumor infiltrating natural killer cells in periampullary adenocarcinoma', Journal of Clinical Oncology, 34: 267-67.

McCoy, J. P., Jr. 2001. 'Handling, storage, and preparation of human blood cells', Curr Protoc Cytom, Chapter 5: Unit 5 1.

Palucka, K., and J. Banchereau. 2013. 'Dendritic-cell-based therapeutic cancer vaccines', Immunity, 39: 38- 48.

Pascal, V., N. Schleinitz, C. Brunet, S. Ravet, E. Bonnet, X. Lafarge, M. Touinssi, D. Reviron, J. F. Viallard, J.

F. Moreau, J. Dechanet-Merville, P. Blanco, J. R. Harle, J. Sampol, E. Vivier, F. Dignat-George, and P. Paul. 2004. 'Comparative analysis of N K cell subset distribution in normal and lymphoproliferative disease of granular lymphocyte conditions', Eur J Immunol, 34: 2930-40.

Pietrzyk, L., Z. Plewa, M. Denisow-Pietrzyk, R. Zebrowski, and K. Torres. 2016. 'Diagnostic Power of Blood Parameters as Screening Markers in Gastric Cancer Patients', Asian Pac J Cancer Prev, 17: 4433- 37.

Qiu, J., Y. Yu, Y. Fu, F. Ye, X. Xie, and W. Lu. 2012. 'Preoperative plasma fibrinogen, platelet count and prognosis in epithelial ovarian cancer', J Obstet Gynaecol Res, 38: 651-7. 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.

Schreibelt, G., J. Tel, K. H. Sliepen, D. Benitez-Ribas, C. G. Figdor, G. J. Adema, and I. J. de Vries. 2010. 'Tolllike receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy', Cancer Immunol Immunother, 59: 1573-82.

Sun, T., and H. Morgan. 2010. 'Single-cell microfluidic impedance cytometry: a review', Microfluid Nanofluid, 8: 423-43.

Suzuki, K., K. Aiura, M. Kitagou, S. Hoshimoto, S. Takahashi, M. Ueda, and M. Kitajima. 2004. 'Platelets counts closely correlate with the disease-free survival interval of pancreatic cancer patients', Hepatogastroenterology, 51: 847-53.

Takanami, I., K. Takeuchi, and M. Giga. 2001. 'The prognostic value of natural killer cell infiltration in resected pulmonary adenocarcinoma', J Thorac Cardiovasc Surg, 121: 1058-63.

Terme, Magali, Simon Pernot, Elie Marcheteau, Florence Castan, Olivier Bouche, Jaafar Bennouna, Eric Francois, Francois Ghiringhelli, Valerie Boige, Christelle De La Fouchardiere, Emmanuelle Samalin, Jean Baptiste Bachet, Christophe Borg, Trevor Stanbury, Sophie Gourgou, David Malka, and Julien Taieb. 2016. 'Peripheral natural killer cells are a prognostic factor in advanced oesogastric adenocarcinoma and are associated with intestinal types in the randomized trial PRODIGE17- ACCORD20 (UNICANCER Gl)¹, Journal of Clinical Oncology, 34: 4061-61.

Turnis, M. E., and C. M. Rooney. 2010. 'Enhancement of dendritic cells as vaccines for cancer', Immunotherapy, 2: 847-62.

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.

Wada, A., Y. Takagi, M. Kono, and T. Morikawa. 2015. 'Accuracy of a New Platelet Count System (PLT-F) Depends on the Staining Property of Its Reagents', PLoS One, 10: e0141311.

Yin, J. B., X. Wang, X. Zhang, L. Liu, and R. T. Wang. 2018. 'Mean platelet volume predicts survival in pancreatic cancer patients with synchronous liver metastases', Sci Rep, 8: 6014.

WO 2013/004708

WO 2015/097037

Claims

4. The dendritic cell vaccine for use of claim 1 or claim 3, wherein the threshold level is a relative level of NK cells of the total lymphocytes or an absolute count of NK cells.

5. The dendritic cell vaccine for use of claim 4, wherein the threshold level is a relative NK cell level between 11% and 16% of total lymphocytes, between 12% and 15% of total lymphocytes and preferably of 13% of total lymphocytes or an absolute count of NK cells between 150 and 250 cells/mΐ of blood, between 160 and 200 cells/mΐ of blood and preferably of 177 cells/mΐ of blood.

6. The dendritic cell vaccine for use of claim 2 or claim 3, wherein the threshold level is a count of platelets between 250 x 10⁹ and 310 x 10⁹ platelets/1, between 260 x 10⁹ and 300 x 10⁹ platelets/1 and preferably of 279 x 10⁹ platelets/1.

7. The dendritic cell vaccine of claims 1 to 6, wherein the patient is selected for treatment if the amount of NK cells and/or the amount of platelets is equal or above the threshold level.

8. The dendritic cells vaccine for use of claims 1 to 7, wherein the treatment comprises a step of determining in a blood sample of the patient,

9. The dendritic cell vaccine for use of claims 1 to 8, wherein the cancer is ovarian, lung or prostate cancer, preferably ovarian cancer and wherein the cancer is recurrent.

10. The dendritic cell vaccine for use of claims 1 and 9, wherein the blood sample is obtained prior to the treatment of the patient with the dendritic cell vaccine and an optional concomitant chemotherapy.

11. The dendritic cell vaccine for use of claims 1 and 10, wherein the levels of NK cells and platelets are determined in blood by flow cytometry/FACS and wherein the level of platelets in blood is measured by flow cytometry, and wherein the blood is obtained from the patient prior to leukapheresis.

12. The dendritic cell vaccine for use 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 claims 1 to 12, wherein immature dendritic cells are in a first step loaded with tumor cells undergoing immunogenic cell death and thereafter are matured with Toll-like receptor 3 agonists or Toll-like receptor 4 agonists.

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

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