NOVEL IMMUNOTHERAPEUTIC TREATMENTS FOR TUMOURS

Abstract

The present invention provides a method for the treatment of a tumour in a subject, said method comprising administering (i) an autologous immature dendritic cell, or a precursor thereof, and (ii) an immune cell checkpoint inhibitor to said subject subsequent to the at least partial ablation of said tumour in said subject, wherein the administration of said autologous immature dendritic cells, or a precursor thereof, and said immune cell checkpoint inhibitor is local to the site of the ablated tumour or part thereof. The invention further provides a product containing an autologous immature dendritic cell, or a precursor thereof, and an immune cell checkpoint inhibitor as a combined preparation for separate, simultaneous or sequential use in a method for the treatment of a tumour in a subject, said method comprising administering (i) the autologous immature dendritic cell, or a precursor thereof, and (ii) the immune cell checkpoint inhibitor to said subject subsequent to the at least partial ablation of said tumour in said subject, wherein the administration of said autologous immature dendritic cells, or a precursor thereof, and said immune cell checkpoint inhibitor is local to the site of the ablated tumour or part thereof.

Claims

1. A method for the treatment of a tumour in a subject, said method comprising administering (i) an autologous immature dendritic cell, or a precursor thereof, and (ii) an immune cell checkpoint inhibitor to said subject subsequent to the at least partial ablation of said tumour in said subject, wherein the administration of said autologous immature dendritic cells, or a precursor thereof, and said immune cell checkpoint inhibitor is local to the site of the ablated tumour or part thereof.

2.-3. (canceled)

4. A product containing an autologous immature dendritic cell, or a precursor thereof, and an immune cell checkpoint inhibitor as a combined preparation for separate, simultaneous or sequential use in a method for the treatment of a tumour in a subject, said method comprising administering (i) the autologous immature dendritic cell, or a precursor thereof, and (ii) the immune cell checkpoint inhibitor to said subject subsequent to the at least partial ablation of said tumour in said subject, wherein the administration of said autologous immature dendritic cells, or a precursor thereof, and said immune cell checkpoint inhibitor is local to the site of the ablated tumour or part thereof.

5. The method of claim 1, wherein said immune cell checkpoint inhibitor interferes with the interaction between at least one immune checkpoint ligand and receptor thereof.

6. The method of claim 1, wherein said immune cell checkpoint inhibitor is an affinity macromolecule reagent which binds to at least one immune checkpoint ligand and/or receptor thereof and the binding of said reagent to its target binding site on said immune checkpoint ligand and/or receptor thereof interferes with the interaction between the receptor and the ligand.

7. The method of claim 1, wherein said immune cell checkpoint or immune checkpoint ligand and/or receptor thereof is selected from (i) CTLA4 and CD80 or CD86, (ii) PD1 and PDL1 or PDL2, (iii) BTLA and HVEM, (iv) KIR and MHC class I or II, (v) LAG3 and MHC class I or II, (vi) TIM3 and galectin 9, (vii) A2aR and adenosine, (viii) B7-H3, (ix) B7-H4, and (x) 2B4

8. (canceled)

9. The method of claim 5, wherein said immune cell checkpoint inhibitor comprises a portion of the amino acid sequence of the checkpoint ligand and/or receptor thereof.

10. The method of claim 5, wherein said immune cell checkpoint inhibitor is an antibody, a phage display antibody or an antibody mimetic.

11. The method of claim 10, wherein said antibody, phage display antibody or antibody mimetic binds at least one immune checkpoint ligand and/or receptor thereof selected from CTLA4, CD80, CD86, PD1, PDL1, PDL2, LAG3, B7-H3, B7-H4, or TIM3.

12. The method of claim 1, wherein said immune cell checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, CT-011, AMP-224, MDX-1105, RG7446, BMS-936559, MGA271, atezolizumab, avelumab and durvalumab.

13. The method of claim 1, wherein said autologous DC or precursor thereof expresses no more than low levels of each of CD40, CD80, CD86, and HLA-DR

14. The method of claim 1, wherein said DC precursor is selected from a monocyte, a macrophage and DC progenitor, a common DC progenitor, a common myeloid progenitor; a granulocyte and macrophage progenitor; and a haematopoietic stem cell.

15. The method of claim 1, wherein said method further comprises prior to administration of the DCs or precursor thereof: (a) leukapheresis of the subject to isolate leukocytes or mononuclear cells from the blood of the subject and/or to isolate monocytes from the blood of the subject, (b) optionally, isolation of monocytes from the isolated leukocytes or mononuclear cells of (a), and (c) optionally, culture of said monocytes under conditions which permit differentiation of DCs therefrom.

16. The method of claim 1, wherein said method comprises a step prior to the administration of the immune cell checkpoint inhibitor and the autologous immature DCs or precursor thereof of at least partial tumour ablation.

17. The method of claim 1, wherein said at least partial ablation of the tumour is by cryoablation, hydrothermal ablation, ionising radiation ablation, radioablation, ultrasound ablation, laser ablation, microwave ablation or electroablation.

18. The method of claim 17, wherein said cryoablation comprises a plurality of freeze/thaw cycles of freezing to less than or equal to about ?40? C. and warming to about 37? C.

19. The method of claim 1, wherein said tumour is a colorectal tumour, prostate tumour, testicular tumour, skin tumour, breast tumour, kidney tumour, ovarian tumour, stomach tumour, intestinal tumour, liver tumour, pancreatic tumour, lung tumour, oesophageal tumour, oral tumour, throat tumour, brain tumour, adrenal tumour, thyroid tumour, uterine tumour or haematological tumour.

20. (canceled)

21. The method of claim 20, wherein said tumour is prostate cancer.

22. The method of claim 1, wherein a further pharmaceutical useful in the treatment of neoplastic disease is administered together with the immune cell checkpoint inhibitor and the autologous immature DCs or precursor thereof.

23. (canceled)

24. The method of claim 22, wherein said further pharmaceutical useful in the treatment of neoplastic disease is selected from a cytotoxic chemotherapy agent, an anti-hormonal agent, an angiogenesis inhibitor, an anti-cancer monoclonal antibody, a radioimmunotherapeutic, a cancer treatment vaccine, an immunostimulatory agent or an immunosuppressant.

25. The method of claim 24, wherein said further pharmaceutical useful in the treatment of neoplastic disease is cyclophosphamide or a prodrug or metabolite thereof.

Description

[0168] The invention will be further described with reference to the following non-limiting Examples, in which

[0169] FIG. 1 shows microscopic (A) and flow cytometric (B) analysis of DC cytotoxicity in the presence of 0.5 mg/ml ipilimumab or DMSO control for 24 hours.

EXAMPLES

Example 1Dendritic Cell Production

[0170] Following completion of leukapheresis of the patient's whole blood, the resultant cell product will contain an estimated 5?10.sup.10 leukocytes in an estimated volume of 400 ml plasma. The leukapheresis product will be further enriched for monocyte content by processing on the ELUTRA? Cell Separation System following established and certified routines. ELUTRA? processing will result in the separation of the lymphocyte fraction from the mononuclear cell collection obtained by leukapheresis. The post-ELUTRA? product will thus be comprised predominantly of a CD14+ monocytic cell population, which will serve as the precursor of autologous induced immature dendritic cells (DCs).

[0171] DCs will then be differentiated from peripheral blood CD14+ monocytes collected during leukapheresis in a four-day ex vivo closed culture system in the presence of two cytokines: Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF at 50 ng/ml and IL4 at 20 ng/ml).

[0172] Following three days of culture, the cells will be harvested, washed, and cryo-preserved in vials at a concentration of 5?10 total cells/vial suspended in 1.0 ml of human-injectable (USP) cryopreservative media. A number of the vials will be thawed and the cells subjected to a panel of release testing regiments including identity and viability assays, a 14 day USP sterility test, mycoplasma test and endotoxin test. Immunophenotyping will then be used to assess the contents of induced immature dendritic cells, including markers CD14, CD80, CD83, CD86, CD40, CCR7, HLA-DR, CD3, CD19, CD16+CD56, CD66b. Induced immature dendritic cells are characterized by absence of CD14, not more than low expression of CD80, CD83, CD86, low expression of CD40, HLA-DR and no expression of CD3, CD19, CD16+CD56, CD66b.

[0173] Upon meeting the criteria for release, cryovials containing preserved autologous immature DCs will be stored in the gaseous phase of a dedicated liquid nitrogen tank at the Clinical Trial Unit until the patient is ready for intratumoural administration in combination with the cryotherapy procedure. This administration is described in Examples 2 and 3.

Example 2Cryoablation of the Tumour

[0174] Cryoablation of prostate tumours is currently an accepted, definitive treatment for primary prostate cancer (Long et al., 2001, Urology 57:518-523.). All subjects will be administered an intravenous dose of ciprofloxacin (in D5W (5% dextrose in water), 400 mg/200 ml) beginning one hour prior to the cryoablation procedure. Upon discharge, all subjects will continue ciprofloxacin therapy, 500 mg b.i.d. per orum for a period of eight days. Cryoablative temperatures, which have been histologically demonstrated to be ?60? C. when a single freeze is employed, and ?40? C. when a double freeze/thaw technique is used (Larson et al., 2000, Urology 55:547-552).

[0175] Subjects will be treated under spinal or general anesthesia (according to subject preference) and sedation. Dependent upon the volume of the prostate gland to be treated, between six and eight sharpened 2.4 mm?15 cm insulated cryoprobes (Endocare, Inc., Irvine, Calif.) will be placed into the prostate under transrectal ultrasound guidance (Bahn et al., 2002, Urology 60:3-11). Placement of the probes by the operator will be such that the three-dimensional ice-ball formed by the confluence of the contributing cryoprobes will contain the prostate tumour, but not extend to adjacent anatomy. Seminal vesicles will not be treated.

[0176] The prostate will then be subjected to cryoablative temperatures at less than or equal to ?40? C. followed by warming to 37? C. Freezing and warming will be accomplished by the delivery of pressurized argon and helium gases, respectively, through the same cryoprobe. At the time of the placement of the cryoprobes into the prostate under ultrasound guidance, thermocouples will be guided to the following positions: right and left neurovascular bundles laterally; external sphincter inferiorly, apex; and posterior prostate at the prostato-rectal interface. Placing of the thermocouples, which supply real-time data to the operator via the cryoablation control unit, allows for objective data to be collected related to actual intraprostatic temperatures achieved. By providing real-time feedback to the operating console during the freezing process, the thermocouples also assist in preventing the operator from freezing through structuressuch as the anterior rectal wallduring cryoablation of the prostate. A warming catheter circulating irrigant maintained at 40? C. will protect the urethral lining from destructive temperatures. Once core body temperature is achieved as demonstrated by thermocouple temperature reading, a second freeze/thaw cycle will be undertaken, thus accomplishing a double freeze/thaw cryotreatment of the prostate. Cryoablation will end with the attainment of 37? C. in the previously frozen prostate following the second freeze/thaw cycle. At that time, the autologous dendritic cell injection(s) will be performed as described in Example 3.

Example 3Autologous Dendritic Cell Administration

[0177] An individualized Treatment Plan will be prepared for each subject. The Treatment Plan will envision the prostate in four quadrants from a coronal perspective: right superior (RS), right inferior (RI), left superior (LS), and left inferior (LI). Using the RS/RI/LS/LI quadrant system and most recently obtained prostatic biopsy, the investigator will grade each quadrant on a scale of: ? No cancer present; + Low volume of cancer in the quadrant; (<50% index tumour volume) ++ Intermediate volume of cancer in the quadrant; (=50% index tumour volume) +++ High volume of cancer in the quadrant. (Index tumour location).

[0178] The number of DCs to be delivered to each of the quadrants will be proportionate to the relative estimated volume of disease residing there. For instance, in the case that the RS quadrant is graded as (+++), and the LS quadrant is graded as (++), with the other two quadrants not exhibiting disease, 60% (?) of the cell volume will be administered to the RS quadrant and 40% (?) of the cell volume will be administered to the LS quadrant (e.g. 2.5?10.sup.7 dose in 1 ml; 0.6 ml injected to RS and 0.4 ml to LS quadrant, respectively). This proportional approach will hold for all cell doses delivered.

[0179] Frozen DC prepared in Example 1 will be placed into a 37? C. water bath for thawing. Thawing will be noted as the disappearance of ice crystals from the interior of the cryovial while in the water bath. Once thawed the vial contents will be drawn into a sterile 1 ml tuberculin syringe fitted with an 18 gauge, 1.5 inch drawing-up needle. Once the vial contents have been drawn into the syringe, the operator remove the drawing-up needle, and attach to the syringe an 18 gauge, 20 cm Chiba? biopsy needle (Cook Medical, Inc., Bloomington, Ind.). Referring to the Treatment Plan, the operator will insert the needle into the prostate gland under ultrasound guidance until the base of the prostate is reached by the needle tip. Using the quadrant system described he/she will begin with the RS quadrant (if appropriate) and move clockwise to all involved quadrants. Following localization of the needle tip to the prostate base as confirmed by ultrasound imaging, he/she will depress the plunger and slowly withdraw the syringe/needle complex, depositing cells along the length of the quadrant. In the event that a large lesion is noted at diagnosis in a particular location within the quadrantfor instance, where cancer has been noted at the base, but not the mid-gland or apex, he/she may elect to preferentially inject cells at that location. Approximately 0.3 ml of cell suspension will remain in the needle following the injection. Leaving the needle in place, the physician-operator will remove the now-empty syringe used for cell administration and replace with a syringe containing saline solution. He/she will flush the needle by injecting 0.3 ml of saline solution into the needle, thereby ensuring the administration of the entire dosage of DCs. Each 1.0 ml vial of dendritic cells with be injected using a 1.0 ml tuberculin syringe. This process will be repeated in accordance with the Treatment Plan, until all cell vials have been deposited in the thawed, post-cryoablation prostate.

Example 4Local (Intratumoural) and Systemic (i.v.) Administration of Immune Cell Checkpoint Inhibitors

[0180] The immune checkpoint inhibitor ipilimumab (Yervoy?)) is a fully human anti-CTLA-4 monoclonal antibody (IgG1?) produced in Chinese hamster ovary cells by recombinant DNA technology. Each ml of concentrate contains 5 mg ipilimumab. The solution is clear to slightly opalescent, colourless to pale yellow liquid that may contain light (few) particulates and has a pH of 7.0 and an osmolarity of 260-300 mOsm/kg. Each ml of concentrate contains 0.1 mmol sodium, which is 2.3 mg sodium. One 10 ml vial contains 50 mg of ipilimumab. One 40 ml vial contains 200 mg ipilimumab.

[0181] An individualized Treatment Plan will be prepared for each subject in order to supplement the cryoimmunotherapy procedure of Examples 1 to 3 with the immune checkpoint inhibitor ipilimumab. Several patients will be administered ipilimumab at 0.3 mg/kg bodyweight locally and several patients will be administered ipilimumab at 0.6 mg/kg bodyweight locally. The maximum administered dose will be limited to 50 mg irrespective of bodyweight.

[0182] The autologous dendritic cell administration will first be performed as described in Example 3. Immediately upon completion of the intratumoral administration of autologous dendritic cells one vial of 10 ml ipilimumab 5 mg/ml (Yervoy?) will be presented to the urologist by the assistant nurse and the required volume, restricted to maximum 50 mg (10 ml) will be drawn into a sterile 10 ml Braun Omnifix syringe using a BD Microlance 18 gauge, 1.5 inch drawing needle. Once the vial contents have been drawn into the syringe, the operator will remove the drawing-up needle, and attach to the syringe an 18 gauge, 20 cm Chiba? biopsy needle (Cook Medical, Inc., Bloomington, Ind.). Referring to the Treatment Plan, the operator will insert the needle into the ablated prostate gland under ultrasound guidance until the base of the prostate is reached by the needle tip. Thereafter the volume of ipilimumab in the syringe will be distributed slowly (about 1 ml per minute) into the cryoablated area following the same injection routes as used for injection of autologous dendritic cells. It is acceptable if not the entire volume is deposited into the cryoablated area, but additionally into neighbouring non-necrotic tissue.

[0183] Several other patients following cyroablation will receive one single intravenous administration of ipilimumab at a cumulative dose of 3 mg/kg bodyweight. The total dose will be diluted to 100 ml using sterile 0.9% NaCl and thereafter transferred to an empty PVC or non-PVC IV bag and infused over 90 minutes. A DEHP-free and latex-free IV set with a 0.2 micron in-line filter will be used.

Example 5Optional Concomitant Low-Dose Cyclophosphamide Therapy for Selective TReg Depletion

[0184] The following regimen for TReg depletive therapy will be undertaken if required.

[0185] At three days from cryoablation and DC administration 300 mg/m.sup.2 cyclophosphamide (Cy) will be administered by intravenous infusion (IV), (Berd et al., 1987, Cancer Research 47:3317-3321). Starting at week 2 from cryoablation and DC administration, 25 mg Cy, p.o., b.i.d. will be administered for 7 days on and then administration will cease for 7 days before restarting for 7 days. A further 7 days without Cy administration will follow. This 28 day cycle will be repeated 6 times. The effects of Cy will be monitored by measuring circulating levels of TReg cells (i.e. CD4+ CD25+ Foxp3+ T cells) in the subject's blood, as described extensively in in Ghiringhelli et al., 2007, Cancer Immunology, Immunotherapy 56:641-648).

Example 6Diagnostic Imaging

[0186] Disease progression and treatment effects will be assessed by in vivo radiology and nuclear medicine imaging, i.e. magnetic resonance imaging (MRI), contrast enhanced computer tomography (diagnostic CT), single photon emission computer tomographic (SPECT) and positron emission tomography (PET). The following imaging schedule will be performed in each patient: Inclusion/exclusion

TABLE-US-00001 Procedure Purpose Sequence/tracer Time Prostate MRI Staging of local disease T2W, DWI, DCE-MRI 40 min MRI columna/Pelvis Nodal/bone metastasis T1W/STIR 15 min SPECT Bone metastasis (Tc-99m-MD) 60 min PET-CT Metastasis/primary tumour FACBC/FDG 30 min (diagnostic CT) 22 weeks Prostate MRI Restaging of local disease T2W, DWI, DCE-MRI 40 min Verification of region treated MRI columna/Pelvis Nodal/bone metastasis T1W/STIR 15 min SPECT Bone metastasis (Tc-99m-MD) 60 min PET-CT Metastasis/primary tumour FACBC/FDG 30 min (diagnostic CT) 46 weeks Prostate MRI Local recurrence/restaging T2W, DWI, DCE 40 min MR columna/Pelvis Nodal/bone metastasis T1W/STIR 15 min SPECT Bone metastasis (Tc-99m-MD) 60 min PET-CT Metastasis/primary tumour FACBC/FDG 30 min (diagnostic CT)

[0187] Prostate MRI and MRI columna/pelvis is preferably done in same the imaging session, i.e. total scan time of about 60 minutes using a 1.5 Tesla MRI system. Prostate MRI is performed without use of endorectal coil to increase patient compliance and minimize total scan time.

[0188] SPECT and PET-CT will be performed on separate imaging days. The CT from the PET-CT examination will be used for RECIST evaluations.

[0189] PET: [18F] FACBC will be used as the preferred PET tracer, but if FACBC fails to identify the tumour/metastasis, e.g. in low differentiated tutors, imaging using [18F] FDG will be applied on a separate day.

[0190] No individual imaging session will exceed 75 minutes.

Example 7Cytotoxic Effects of Ipilimumab on Dendritic Cells

[0191] For generation of monocyte derived DC (moDC), 20 to 45 ml heparinized blood were collected from each participant and handled within 2 hours. Peripheral blood mononuclear cells were isolated by gradient centrifugation using Lymphoprep (Axis Shield PoC AS, Oslo, Norway) and monocytes isolated by plastic adherence in X-VIVO 20 medium (Lonza, Verviers, Belgium). Nonadherent cells were removed after 1 hour of incubation at 37? C. and 5% carbon dioxide. Remaining monocytes were cultured in RP10 medium (RPMI 1640 with Ultraglutamine (Bio Whittaker, Lonza), 10% FCS Gold (PAA, Pasching, Austria), 1% penicillin-streptomycin (Gibco, Invitrogen Corporation, Paisley, UK)) supplemented with 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; ImmunoTools GmbH, Friesoythe, Germany) and 20 ng/ml IL-4 (ImmunoTools GmbH). After 3 days in culture, maturation was induced in one part of the moDC by adding 0.1 ?g/ml TLR4 ligand lipopolysaccharide (LPS) (InvivoGen, San Diego, Calif., USA) for 24 hours. Ipilimumab up to 0.5 mg/ml or DMSO was added at the same time as LPS and at the same time to un-treated DCs. Following incubation for 24 hours the cells were harvested for Cytation 5? imaging system analyses and for flowcytometric analysis using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences Cat. No 556547) following the manufacturer's protocol, or for microscopic analysis.

[0192] Results are shown in FIG. 1. Microscopic analysis and flowcytometric apoptosis (Annexin V-FITC) or other cell death (propidium iodide) analysis of LPS induced mature DCs and immature DCs show that treatment with up to 0.5 mg/ml ipilimumab for 24 hours cause no detectable cytotoxic effects on DCs. The concentration of ipilimumab used in these studies is much higher than typical clinical doses. These results show, for the first time, that the use of immune cell checkpoint inhibitors together with DCs, e.g. during local co-administration to an at least ablated tumour, would not induce DC toxicity and thus limit the effectiveness of any anti-tumour response.

Example 8Clinical Treatment of Late Stage Prostate Cancer Patients in Accordance with the Invention

[0193] 14 late stage prostate cancer (metastatic castration resistant prostate cancer (CRPC)) patients were treated as described in Examples 1 to 4. Patients R01 to R09 received cryoablation and doses of DCs, but not ipilimumab. Patients R10 to R13 received highest dosing of DC and 0.3 mg/kg bodyweight of ipilimumab. Patient R14 received highest dosing of DC and 0.5 mg/kg bodyweight of ipilimumab. At predetermined time points circulating tumour cells (CTC) were enumerated according to the following protocol.

[0194] 7.5 ml peripheral whole blood was collected in CellSave tubes (Immunicon, Inc., Huntingdon Valley, Pa.). The semi-automated analysis was performed as described elsewhere. Blood samples were kept at room temperature for 572 hours before analysis using the CellSearch? assay (CellSearch? Epithelial Cell Kit/CellSpotter? Analyser, Menarini-Silicon Biosystems, San Diego, Calif., USA). The assay uses a ferrofluid coated with antibodies to epithelial cell adhesion molecule (EpCAM) to immunomagnetically separate cells of epithelial origin from blood, and fluorescent staining to differentiate between debris, hematopoietic cells, and epithelial-derived circulating tumour cells. CTCs quantified and characterized in this study were cells with a positive staining for keratins (K) and nuclear DAPI staining, but negative for the pan-leukocyte marker CD45. Results are shown in Table 1.

TABLE-US-00002 TABLE 1 CellSearch circulating tumor cell (CTC) enumeration in weeks pre- and post cryoimmunotherapy for patients R01 to R14 (per 7.5 ml peripheral blood sample) Pre- treatment Weeks post-treatment 1 2 2 6 10 14 22 30 38 46 52 72 R01 8 13 7 14 5 58 R02 0 0 0 0 0 0 0 0 0 0 0 R03 0 1 0 0 0 0 0 R04 0 0 0 0 0 0 0 R05 3 1 3 1 0 0 0 0 0 0 0 0 R07 0 1 0 0 0 1 0 0 0 R08 0 0 0 0 0 0 0 R09 2 31 67 137 6 3 4 9 40 R10 9 7 9 24 12 24 R11 0 0 0 R12 0 0 0 0 0 R13 6 14 8 18 2 R14 0 1

[0195] Adverse side effects were also monitored during treatment and follow up.

[0196] Follow-up of patients R01-R09 previously treated with cryoablation of the prostate tumor followed by subsequent intratumoral DC injection showed remarkably few adverse events. Thus, it may be concluded that both cryoablation was safe without local surgical or freezing associated damage and no serious autoimmune adverse events are associated with DC administration.

[0197] According to published literature, systemic treatment with ipilimumab has been associated with high incidence of autoimmune adverse events, and the adverse events have been correlated to total injected dose per kg bodyweight. Serious adverse events include autoimmune colitis, hepatitis, uveitis, hypophysitis and difficult to treat skin disorders (Perez-De-Lis M, et al. 2017, Autoimmune diseases induced by biological agents. A review of 12,731 cases (BIOGEAS Registry), Expert Opin Drug Saf.; 16(11):1255-71; and Rijnders M, de Wit R, et al, 2017, Systematic Review of Immune Checkpoint Inhibition in Urological Cancers, Eur Urol.; 72(3):411-23). It is consequently strongly encouraging that no autoimmune adverse events have been observed during follow-up of patients treated with intratumoral injection of ipilimumab in addition to DC-mediated cryoimmunotherapy.

[0198] Neither was there any negative acute reactions observed as a consequence of ipilimumab and DC intratumoral injections.

[0199] Patients R10 to R14 also showed remarkably few surgical side effects, with no instances of infection being reported and low levels of fistula formation and similar local anatomical deformations being observed. It was not clear from the art whether or not tumour cryoablation and local administration of DCs followed by local administration of immune cell checkpoint inhibitors would cause significant surrounding tissue damage and thereby result in adverse surgical side effects. The follow-up results reveal that tumour ablation, e.g. by cryoablation, followed by local use of DCs together with immune cell checkpoint inhibitors is surprisingly well tolerated by patients and not associated with elevated infection rates, fistula formation and similar local anatomical deformations.

[0200] Based upon the CTC results shown in Table 1, the treatments of the invention can be considered to result in disease stabilisation in late stage prostate cancer patients, i.e. worsening of the disease is not observed over the follow-up period in any patient. This may be consider a very positive result in view of the lack of adverse effects seen in the treated patients as compared to those expected in the same patients treated systemically with ipilimumab alone.

[0201] It should also be noted that the patients used in this study are those with metastatic castration resistant prostate cancer (CRPC), i.e. very late stage cancer. The included patients have had their diagnosis of prostate cancer for extended periods. Patients were included only after their tumor progresses on standard androgen deprivation therapy (ADT). Patients both pre and post chemotherapy were included. The median overall survival of comparable patient groups according to international literature is 18 months even with docetaxel chemotherapy intervention (Schalken J, Fitzpatrick J M. Enzalutamide: Targeting the androgen signalling pathway in metastatic castration-resistant prostate cancer. BJU international. 2015). These patients are necessarily selected because ethical consent for such trials is only given for patients with very serious disease. It is therefore to be expected that efficacy results observed in this study will not be as apparent as would be expected in patients with earlier stage disease. Indeed, there is theoretical and clinical evidence to suggest that the curative potential of immunotherapy is stronger at an earlier stage and with lower total tumor load (Gulley J L, et al, 2011, Impact of tumour volume on the potential efficacy of therapeutic vaccines, Curr Oncol.; 18(3):e150-7). It therefore to be expected that the results observed in this trial will be replicated to a greater extent when repeated in patients with earlier stage disease.

[0202] Moreover, the stabilisation of disease in patients with such advanced prostate cancer without the adverse side effects associated with ipilimumab may be considered a particular success.