A New Strategy of Cancer Immunotherapy Combining Hyperthermia/Oncolytic Virus Pretreatment with Specific Autologous Anti-Tumor Vaccination – A Review

Special Issue - Cancer Vaccines

Austin Oncol Case Rep. 2017; 2(1): 1006.

A New Strategy of Cancer Immunotherapy Combining Hyperthermia/Oncolytic Virus Pretreatment with Specific Autologous Anti-Tumor Vaccination – A Review

Schirrmacher V*, Lorenzen D, Van Gool SW and Stuecker W

Immunological and Oncological Center Cologne (IOZK), Germany

*Corresponding author: Schirrmacher Volker, Immunological and Oncological Center Cologne (IOZK), Cologne, Germany

Received: February 02, 2017; Accepted: February 14, 2017; Published: February 17, 2017


This review describes and explains a specific immunotherapy strategy that has been developed at the Immunological and Oncological Center Cologne in Germany. The strategy is based on many years of basic and translational research. It is a highly individualized approach to activate and target the patient’s immune system against the patient’s own tumor. In a first step, the patient’s immune system is conditioned by pretreatment with oncolytic virus in combination with hyperthermia. The second step consists of active-specific autologous anti-tumor vaccination. The vaccine, VOL-DC, consists of autologous Dendritic Cells (DCs) which are loaded with Viral Oncolysate (VOL) from the patient’s tumor cells. VOL transfers to the DCs information about tumor-associated antigens from the patient’s tumor and Pathogen-Associated Molecular Patterns (PAMPs, danger signals) from the virus. The product VOL-DC, which involves Newcastle Disease Virus, has been approved in Germany as an Advanced Therapeutic Medicinal Product for individual treatment by the institution which received this permit. This review includes promising results from case-series studies of glioblastoma patients. It also discusses future strategies for treatment of late-stage disease including combination of this immunotherapy with immune checkpoint blocking antibodies and/or with costimulatory bispecific antibodies.

Keywords: Cancer vaccine; Dendritic cell; Oncolytic virus; Newcastle disease virus; Immunogenic cell death; Memory T cell


APC: Antigen-Presenting Cell; ATMP: Advanced Therapeutic Medicinal Product; ATV-NDV: Autologous Tumor cell Vaccine modified by infection with NDV; BM: Bone Marrow; Ca: Carcinoma; COMP: Committee for Orphan Medical Products; CRC: Colorectal Carcinoma; CTL: Cytotoxic T Lymphocyte; DAMP: Damage- Associated Molecular Pattern; DC: Dendritic Cell; DFS: Disease- Free Survival; ELISPOT: Enzyme-Linked Immuno-Spot; EMA: European Medicines Agency; ER: Endoplasmic Reticulum; GBM: Glioblastoma Multiforme; GMP: Good Manufacturing Practice; HLA: Human Lymphocyte Antigen; ICD: Immunogenic Cell Death; IFN: Interferon; IFNRA: α chain of the type I Interferon Receptor; IOZK: Immunological and Oncological Center Cologne; IPS: IFN-ß Promoter Stimulator; IRF: Interferon Regulatory Factor; ISG: IFNStimulated Gene; ISRE: IFN-Stimulated Response Element; mEHT: modulated Electrohyperthermia; MHC: Major Histocompatibility Complex; MTC: Memory T Cell; NDV: Newcastle Disease Virus; NK cell: Natural Killer cell; OS: Overall Survival; OV: Oncolytic Virus; PAMP: Pathogen-Associated Molecular Pattern; PFS: Progression-Free Survival; RIG: Retinoic acid Inducible Gene; STAT: Signal Transducers and Activators of Transcription; TAA: Tumor Associated Antigen; TIL: Tumor-Infiltrating Lymphocyte; TLR: Toll- Like Receptor; TF: Transcription Factor; Th: T-helper cell; Treg: T regulatory cell; VOL: Viral Oncolysate; VOL-DC: Dendritic Cell pulsed with VOL


Immunotherapy of cancer holds promise for cancer patients as a new biological treatment procedure. In contrast to surgery, chemoand radiotherapy - present-day standard therapies - which focus only on the tumor, immunotherapy is concerned with both, the tumor and the tumor-bearing host. Tumor-host interactions are particularly relevant in the fight against metastatic disease. Because the immune system is systemic it has the potential to protect against systemic disease such as metastasis.

Results of recent clinical studies involving novel immunotherapy strategies such as immune checkpoint blockade and adoptive T-cell transfer approaches have clearly established immunotherapy as an important modality for the treatment of cancer [1]. Immunotherapy can complement the traditional approaches of standard therapy or small molecule targeted therapy or it can be employed as monotherapy. To date, immunotherapy has been shown to be capable of inducing durable clinical benefit, although only in a fraction of the patients. The challenge, therefore, is how to increase the fraction of responding patients. It is generally accepted that the ultimate goal and read-out of immunotherapy is the increase in median Overall Survival (OS) and particularly in the percentage of patients with longterm survival. There is no doubt that this necessitates new strategies for personalization and combinatorial approaches [2,3].

A personalized immunotherapy approach is what the Immunological and Oncological Center (IOZK) in Cologne, Germany, is aiming at. It has developed a treatment technology involving the patient’s tumor cells, the patient’s Dendritic Cells (DCs) and an Oncolytic Virus (OV) [4,5] to produce a vaccine called VOLDC. VOL stands for Viral Oncolysate and the virus used is the bird virus Newcastle Disease Virus (NDV). Treatment of cancer patients by NDV oncolysate vaccines without DCs, performed in the 1970s, had already given interesting results [6,7] but for decades thereafter there had been only little interest in OVs. A revival of interest occurred luckily in the 2000s with more knowledge about OVs and new technologies, such as DC culture [8].

VOL-DC is a DC1 polarized vaccine which presents Tumor- Associated Antigens (TAAs) from the patient’s tumor to the patient’s T cells and activates the latter to TAA-specific T helper cells and Cytotoxic T Lymphocytes (CTLs). After several such vaccinations, the patient’s immune system is enriched with TAA-specific Memory T Cells (MTCs) which help to protect against possibly disseminated tumor cells and micro-metastases. It is this immunological memory which distinguishes immunotherapy from conventional therapies and provides a basis for improvements in long-term survival.

VOL-DC is a licensed Advanced Therapeutic Medicinal Product (ATMP) in Northern-Westfalia, Germany, and applied to patients at IOZK on a compassionate use basis.

Immune modulation by hyperthermia/oncolytic virus pretreatment

Active specific immunization of cancer patients requires an immune system which is competent and not dysregulated. Therefore, before patients at IOZK receive vaccinations, their immune system is assessed in depth. Immune modulation/conditioning of the immune system 1 week before vaccination is done by modulated Electrohyperthermia (mEHT) combined with systemic (i.v.) application of NDV [5].

Modulated electrohyperthermia: Hyperthermia with a tissue temperature of 38,5 to 40,5°C can activate the immune system [9,10]. mEHT is combined with systemic oncolytic NDV virotherapy based on the observations that mEHT can enhance virus tumor targeting [10] and virus replication [11]. mEHT is applied either locally or systemically, depending on the clinical situation. Viral infection of tumor cells and hyperthermia with a radiofrequency of 13 MHz cause an Endoplasmic Reticulum (ER) stress response, modify the surface properties of tumor cells and induce Immunogenic tumor Cell Death (ICD) mechanisms [4,5,12].

Systemic NDV application: It is the high safety profile which allows the avian paramyxovirus NDV to be applied to cancer patients [5]. The reported side effects after experience with clinical application for 50 years [7] are grade 1 and 2. Special characteristics of NDV include:

i) Lack of gene exchange via recombination,

ii) Lack of interaction with host cell DNA,

iii) Viral replication in the cytoplasm of cells being independent of host cell proliferation and

iv) Tumor selectivity of virus replication and of host cell lysis [5,7].

Identified mechanisms that can explain tumor selectivity of virus replication and oncolysis in tumor cells of non-permissive hosts such as man involve:

i) Defects in activation of anti-viral signaling pathways,

ii) Defects in type I IFN signaling pathways and

iii) Defects in apoptotic pathways [5].

Systemic NDV application has the following positive effects:

i) Induction of type I Interferons (IFNs) [13] which inhibit secretion of Th2 cytokines (IL-4 and IL-5), stimulate Th1 cells and counter-act Treg cells,

ii) Induction of ICD in virus-infected tumor cells [4,5,7,14] and

iii) Priming of NDV Viral Oncolysate (VOL) - reactive T cells which can be monitored by an in vitro ELISPOT assay [15].

Immune modulation prior to specific vaccination has a preconditioning effect on the patient’s immune system. The induction of VOL-reactive T cells will create systemic recall responses at the site of VOL-DC vaccination and thereby enhance DC migration to the draining lymph nodes with improved antitumor efficacy [16].

Concept of the DC vaccine VOL-DC

Tumorlysate-pulsed DCs as APCs for TAAs: Human TAAs were first described in the early 1990s. It formed the basis for a new era of molecularly defined tumor immunology. In the following years, numerous TAAs, either uniquely expressed or more common, have been characterized and respective HLA-restricted epitopes or neo-epitopes that are capable of triggering CD8+ and CD4+ T cell responses have been identified.

With the introduction of dendritic cell culture and the demonstration of DC’s T cell stimulatory capacity [8], methods have been developed to load DCs with TAAs or tumorlysate [17] and to use them as professional Antigen-Presenting Cells (APCs) for the de novo induction of TAA-specific T cell responses in cancer patients.

We decided to use autologous tumorlysate instead of defined TAAs to produce APCs based on the following findings:

1. Upon loading with crude tumor lysate proteins, human DCs cross-present CD8+ T cell epitopes in the context of MHC class I and present CD4+ T cell epitopes in the context of MHC class II. This was shown to be an active metabolic process leading to functional DCs able to induce TAA-specific CD8+ CTL activity [18].

2. Using a short-term IFN- γ ELISPOT assay, cancer-reactive MTCs from patient’s Bone Marrow (BM) were analyzed by stimulation with tumorlysate-pulsed DCs. Altogether, T cells reactive against the entirety of TAAs could be detected in about 40% of primary operated breast cancer patients. These existed at frequencies of 1:200 to 1:10,000 of total T cells and included CD8+ CTL and CD4+ Th cells [19].

3. The memory T-cell repertoire of the BM of primary operated breast cancer patients was analyzed in more detail and compared to that of healthy female donors. This was done by tumorlysate-pulsed DCs and DCs pulsed with HLA-A2-peptides from 10 different breast TAAs and a variety of normal breast tissue-associated antigens. The T-cell repertoire was highly polyvalent and exhibited pronounced inter-individual differences in the pattern of TAAs recognized by each patient [20].

4. In comparison to reactivity towards TAAs, reactivity to normal breast tissue-associated antigens was much lower. Healthy individuals also contained TAA-reactive T cells but this repertoire was more restricted and the frequencies were in the same low range as T cells reacting to normal breast tissue-associated antigens [20]. Since there was hardly a repertoire of MTCs reacting to normal tissue antigens, it does not seem necessary to purify single TAAs from tumor cells for vaccination purposes to avoid reactivity to tumor-derived normal tissue antigens.

Taken together, the analysis revealed that the BM appears as a lymphoid organ particularly involved in the induction and/or maintenance of natural T cell immunity against tumor antigens [19]. The spontaneous generation of cancer-reactive MTCs appears as a physiological process, also independent of TAA purification. The analyses involving tumorlysate-pulsed DCs revealed

i) That tumorlysate can serve as a source of TAAs,

ii) That tumorlysate-pulsed DCs as APCs do not induce autoimmune reactivity and

iii) That tumorlysate makes it unnecessary to purify TAAs.

Tumorlysate obtained from heat-stressed tumor cells was found to generate more potent DC vaccines than other antigen-loading strategies [21].

Viral oncolysate for DC programming, polarization and TAA information transfer: To create an effective DC vaccine it is important to avoid that the antigen-loaded cells induce tolerance instead of an immune responses. DCs are not only activators but also modulators of immune responses. Depending on the context in which DCs interact with antigens they will affect T helper precursor cells in different ways, for instance to differentiate into Th1, Th2, Th17 or Treg.

To avoid a wrong polarization of T helper cells we decided to introduce so called “danger signals” into the DC vaccine by virus infection. Foreign viral non-capped RNA in the cytoplasm of a cell infected by an RNA virus is a Pathogen-Associated Molecular Pattern (PAMP) that stimulates innate immunity [5]. Thus, VOL-DCs consist of three components: autologous tumor cells as a source of tumorcharacteristic TAAs, patient-derived DCs as professional APCs and the oncolytic virus NDV do deliver PAMPs. In addition, the emission of Damage-Associated Molecular Patterns (DAMPs) following ICD favors the establishment of a productive interface with the immune system [5,22]. Further features of NDV relate to the immune response:

i) Up-regulation of MHC I,

ii) Activation of NK cells,

iii) Activation of monocytes,

iv) Activation of DCs,

v) Costimulation of CD4+ and CD8+ T-cells [5].

This results in the elicitation of tumor-targeting immune responses associated with the potential to eliminate residual, treatment-resistant cancer cells and to establish tumor-reactive immunological memory.

NDV viral oncolysate (VOL) from autologous tumor cells is used to load patient-derived immature DCs. The material is taken up by macropinocytosis and processed along endogenous professional antigen-presentation pathways for generation of HLA-class II peptide complexes and along cross-presentation pathways to generate HLAclass I peptide complexes. VOL also contains viable NDV.

With the viral oncolysate the DCs receive information about the patient’s own tumor, in particular about its individually specific and common TAAs. The rationale for this autologous approach has been summarized [23]. New findings about the individuality of genetic and immunobiological changes in human tumor cells support this rationale. For instance, a dominance of mutation-derived unique tumor neo-antigens was described in an analysis of 598 human colorectal carcinomas indicating that cancer vaccination requires individualized strategies [24].

Importance of NDV-induced signaling through RIG-I and type I interferon receptor: The bird virus NDV induces in DCs and other normal cells from non-permissive hosts such as man a strong type I interferon response. This response which occurs within 18 hrs after infection consists of an early phase and a late phase [25].

The early phase is initiated through the recognition of viral RNA by two types of pathogen recognition receptors:

i) Endosomal Toll-Like Receptors (TLRs), particularly TLR3,7 and 8 and

ii) Cytoplasmic Retinoic acid Inducible Gene I (RIG-I) receptors. RIG-I binds specifically to RNA containing 5`-phosphate, such as viral RNA. Mammalian RNA is either capped or contains base modifications. RIG-I triggering involves the upregulation of RIG-I RNA copies. Once activated, RIG-I binds to the adaptor protein IFN-ß Promoter Stimulator-1 (IPS-1) which, after a further signaling cascade, activates the IFN regulatory factor IRF-3. This Transcription Factor (TF) is then phosphorylated, translocates to the nucleus and induces the IFN response [25].

During the late phase of the IFN response, the type I IFN molecules secreted during the early phase (IFN- α and IFN-ß) interact with the cell surface expressed α-chain of the type I Interferon Receptor (IFNRA). This initiates an amplification loop of the IFN response, which involves Signal Transducers and Activators of Transcription (STAT) proteins and the IFN regulatory factor IRF-7. Upon ligand binding, receptor associated Tyk2 and JAK1 become activated by transphosphorylation. These in turn phosphorylate STAT1 and STAT2. These STAT proteins then heterodimerize and form a complex with the IFN regulatory factor IRF-9 known as ISGF3. It translocates to the nucleus to bind to the IFN-Stimulated Response Element (ISRE) and directs the expression of IFN-Stimulated Genes (ISGs) that create the antiviral state in the target cells which blocks viral replication [25].

The effect of NDV on human DCs has been studied in a very sophisticated way using technologies developed by systems biology [26]. NDV was considered a prototype avian virus to study an uninhibited cellular response to virus infection. The analysis revealed that the genetic program underlying the anti-viral cell-state transition during the first 18 h post-infection can be explained by a single convergent regulatory network involving 24 critical TFs. These factors regulated 779 of the 1351 up-regulated genes. Many of these genes were associated with polarization of the DCs towards DC1 and for induction of Th1 T-cell mediated immune responses.

Such programming and polarization of human DCs by NDV to DC1 was confirmed in functional studies [27].

Signaling through RIG-I and IFNRA plays an important role for immune activation by the avian virus NDV in man. They also play an important role for immune evasion by the primate virus Ebola. The latter does not induce type I IFN responses in man as it has viral proteins that specifically and strongly interfere with RIG-I and IFNRA signaling [28].

Preparation of VOL-DCs and functional tests

Preparation: In 2015, IOZK succeeded in producing NDV from cell culture at high titers and with high purity according to GMP guidelines. This is worldwide achieved for the first time. Also, the methodology to produce VOL-DC has meanwhile been established and quality certified as an ATMP at the IOZK in Germany.

Figure 1 illustrates the main features for the preparation of the three-component vaccine, exemplified for Glio Blastoma Multiforme (GBM). Following consent between the patient, the operating institution and IOZK, a sample of freshly operated tumor is received in IOZKs GMP facility for further processing via cell separation, cell culture, cell characterization and virus infection. The viral oncolysate is freeze-thawed to become devoid of viable tumor cells and then co-incubated with immature DCs from a short-term culture of a sample of the patient’s white blood cells. After a further maturation step, the vaccine VOL-DC is ready for intradermal application to the patient. The differentiation process from adherent monocytes (CD14++,CD86+,CD209-,CD83-) via semiadherent immature DCs (CD14+,CD86+,CD209++,CD83-) to floating mature DCs (CD14- ,CD86++,CD209+,CD83++) is followed by flow cytometry.