23 February 2018

Powering and arming to combat glioblastoma

Cancer Clinical

Glioblastoma is a highly aggressive, incurable cancer with uniformly poor outcomes for patients diagnosed with primary tumours. The median overall survival rate is one to two years with the five-year survival rate less than 10%.

Affected patients suffer devastating impacts on their quality of life, with symptoms including headaches, nausea and vomiting, seizures and changes in sensation and personality.

Available therapies, including surgery, chemotherapy, whole-brain radiation, and anti-angiogenic drugs only interrupt disease progression temporarily, and do so at a tremendous cost in the form of major toxicities and side effects.

Current therapies aim to slow disease progression and relieve symptoms, but have only a modest impact on the course of disease progression, and nearly all patients will relapse.

The high unmet need for effective therapies drives a strong pharmaceutical pipeline, with several therapies currently in late-stage clinical development.

Most drugs, however, try to directly kill the tumour cells and this imposes a large selection pressure on the heterogeneous tumour cell population by favouring those cells able to resist the treatment and relapse.

Cancer immunotherapies 

Immunotherapy has become an emerging area of research for this cancer due to the challenges faced by traditional drug therapy and targets.

More recently, several immunotherapeutic approaches aimed at promoting a direct anti-tumour immunity have shown promising results in the treatment of some advanced solid tumours.2

However, a major hurdle to the efficacy of anti-cancer therapy is the immunosuppressive tumour microenvironment, which counteracts effective and long-lasting anti-tumour responses.

Tumour-derived factors lead to the expansion and recruitment of immunosuppressive myeloid cells, including tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), that keep T cells at bay and protect tumours from the effector arm of the immune system.

Equally important is the level of intrinsic immunogenicity of different tumour types, with brain tumours considered to be of low immunogenicity3 and therefore poorly attackable by and/or visible to the immune system. Immunogenicity of tumour cells plays an important role in determining the magnitude and efficacy of anti-tumour immune responses.

Indeed, the development of effector CD8+ T cells capable of recognising and killing tumour cells is dependent on their stimulation by fully activated dendritic cells that present tumour antigens acquired by dying tumour cells to the naïve T cells to activate them.

Poorly immunogenic tumours typically have a low mutational and therefore antigen load, which is often combined with a lack of an efficient antigen presentation system, and/or poor T cell infiltration.

Thus, approaches aiming at reprogramming the tumour microenvironment towards a less immunosuppressive state, combined with strategies enhancing tumour immunogenicity, could promote the generation and deployment of immune effector responses that are crucial to achieve clinically relevant anti-tumour immune responses.

Evidence suggests that the central nervous system can develop and recruit tumour- specific immune cells and could, therefore, be a target for cancer immunotherapies.

However, immunotherapies failed to show reproducible clinical responses in glioblastoma because of its poor immunogenicity and the presence of a highly immunosuppressive microenvironment.3

To overcome these limitations, our research is focusing on developing clinically implementable strategies aimed at addressing both local immunosuppression and low tumour immunogenicity.

With the long-term goal of designing new combination therapies for the treatment of glioblastoma, the main aim of our current research is to provide preclinical data supporting the feasibility, safety and therapeutic efficacy of two novel strategies to induce powerful immune responses to combat this malignancy.

Approaches aimed at promoting direct anti-tumour immunity have shown promising results

Reversing local immunosuppression

As a strategy aimed at reversing the immunosuppressive microenvironment, we exploited the tumour-homing ability of a subpopulation of monocytes to turn them into an efficient vehicle for the tumour-targeted delivery of a potent immune-stimulatory molecule: interferon-alpha (IFN?).

Previous studies from our team showed that this cell- and gene-based delivery therapy strongly inhibited primary breast cancer tumours and lung metastases. Most importantly, it did so by inducing the recruitment and activation of immune cells as shown in our Cancer Cell and Science Translational Medicine papers, with no evidence of toxicity.4,5

Relevant to brain tumours, we also demonstrated its anti-tumour efficacy in a preclinical model of human glioblastoma, and proved the ability of our genetically engineered monocytes to deliver IFN? to human brain tumours.4

This work was started in the laboratory of Professor Luigi Naldini at the Telethon Institute for Gene Therapy in Milan, Italy. After relocating to the University of Queensland in Brisbane to set up an independent research group, I am working with my collaborators on further developing this therapy by investigating its efficacy in combination with additional therapies on several types of tumours.

Type I interferons (IFNs) are pleiotropic cytokines involved in innate and adaptive immunity that have been shown to promote anti-tumour immune responses. Moreover, IFNs increase natural killer cell cytotoxicity and enhance natural killer cell survival and expansion IFNs also increase the expression of tumour antigens on neoplastic cells, rendering them more immunogenic.7

The broad biological activities of type I IFNs provided the rationale for testing administration of exogenous recombinant IFN? as an anticancer treatment, which proved effective against several solid and hematological malignancies.

However, clinical use of IFN? has since declined because of the substantial toxicity associated with systemic administration and the limited efficacy at the maximal tolerated doses, thus calling for safer and more effective delivery strategies.8

We addressed this issue by developing a cell- and gene-based IFN? delivery platform.

How does it work?

The immune system has the great advantage of being transplantable.

Haematopoietic stem cells can be isolated and genetically modified using appropriate transfer vectors to introduce a transgene of interest (in our case the gene encoding for IFN?).

After reintroducing the genetically engineered haematopoietic stem cells into the autologous host, they engraft and differentiate into all the different haematopoietic cell lineages.9 By carefully choosing the genetic switch (promoter) to turn-on the expression of the transgene we can decide in which of the stem cell’s progeny we will have the transgene expressed (IFN? production).

We knew that a specific subpopulation of monocytes, were preferentially recruited to tumours in mice and humans.10,11 We therefore decided to exploit their tumour-homing capability to turn them into efficient IFN? delivery vehicles.

Using transfer vectors, we introduced an IFN? transgene into murine haematopoietic stem cells, and upon transplantation we showed selective activation of IFN? expression in their tumour associated Tie2-expressing macrophages These macrophages represent an advantageous cellular vehicle because they are recruited to the tumour, they go where the drug needs to be delivered.

These two aspects, tumour tropism together with low systemic versus high local delivery of IFN?, have been fundamental to achieving good efficacy with low toxicity. Moreover, moving towards a possible clinical implementation of our strategy, we also developed a human IFN? delivery platform and a preclinical model in human haematochimeric mice in which we demonstrated the efficacy of our strategy on human breast cancer tumours in the presence of a functional human haematopoietic system.5,12

Adoptive transfer of mature monocytes. Evidence for safe and effective haematopoietic stem cell gene transfer by vectors in clinical trials of gene therapy for inherited monogenic diseases were recently reported by our collaborator Professor Naldini in his Science, Nature and Lancet papers9, 13-15.

Feasibility and long-term safety issues may, however, exist when proposing transplantation of genetically engineered autologous haematopoietic stem cells. Therefore, we are also exploring the possibility of transferring autologous mature monocytes with the IFN?-delivery platform.

Given the multiple immunostimulatory activities of type I IFNs and the ability of our IFN-delivery platform to reverse the immunosuppressive tumour microenvironment in breast cancer,4,5,12 we expect this strategy to also modulate the immune infiltrate in glioblastoma, thus rendering the microenvironment of this specific type of tumour more permissive for further immune stimulation.

Reversal of an immunosuppressive microenvironment is an absolute requirement for the onset of an effective and long-lasting anti-tumour immune response, however, it is not sufficient.

Equally important for the establishment of efficient anti-tumour immunosurveillance, is the level of tumour cell immunogenicity. Based on the observation that antigen availability is generally poor in glioblastoma, dendritic cell- based vaccination approaches have been attempted to overcome this limitation.

Cancer vaccines are perfectly equipped to enhance dendritic cell activation. However, cancer vaccines of high potency and antigen-specificity are not available yet.16 Developing new strategies that enhance dendritic cell activation, cross-presentation and survival is required to induce effective anti-tumour immune responses.17

A strategy largely adopted to pulse dendritic cells within cancer vaccines involves the use of tumour cell lysates which provide a broad spectrum of tumour-associated antigens, yielding promising, albeit still unsatisfactory, results.18

A possible strategy to improve current protocols may be the use of lysates obtained from tumour cells undergoing immunogenic cell death. The process of immunogenic cell death involves changes in the composition of the cell surface antigens as well as the release of soluble mediators. This may be useful to break tolerance and to allow refocusing of immune responses towards epitopes for which tolerance may not be established. This approach appears highly promising.19

Our collaborator Professor Riccardo Dolcetti has recently identified 9-cis-retinoic acid and IFN? (RA/IFN) combination as a novel and potent immunogenic cell death inducer able to enhance the tumour-specific killing activity of T-cells in mouse models of lymphoma20 and breast cancer.

Therefore, to address the low immunogenicity of glioblastoma, we will explore the ability of conventional and novel immunogenic cell death inducers to generate immunogenic tumour cell lysate for the development of more effective dendritic cell based vaccination protocols.

Significance of this research

Our research seeks to address the bleak outlook for glioblastoma patients by developing more effective therapies with the potential to deliver substantial improvement in survival and quality of life.

In addition to killing primary tumours, by establishing effective anti-tumour immunity, this class of therapy has the potential to prevent recurrence, which is what ultimately kills most glioblastoma patients.

Our research pioneers the use of two novel strategies to induce powerful immune responses to combat glioblastoma.

Importantly, both of these strategies are well differentiated from the immunotherapies that are currently being developed for glioblastoma. As a result, the strategies we are developing may provide treatment options for patients who fail to respond to other drugs.

Furthermore, our approaches are complementary to the major pipeline therapies and therefore have the potential to be used, eventually, in combination with other immunotherapy agents to induce synergistic effects and further improve treatment outcomes.

Finally, the new therapies we are developing have the potential to provide superior safety and side-effect profiles to existing treatments, thereby reducing the impact of glioblastoma therapy on patients’ quality of life and that of their families and carers.

If successful, our studies have the potential to deliver disease-modifying therapies for glioblastoma patients who currently lack satisfactory treatment options and/or to improve outcomes of treatment with standard and emerging therapies.

Dr Roberta Mazzieri is a senior research fellow and research group leader at The University of Queensland’s Diamantina Institute. Her studies focus on understanding the tumour microenvironment and developing strategies to interfere with the pro-tumoural activities of the different components of the tumour environment


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2. Galluzzi L, Buqué A, Kepp O, Zitvogel L, Kroemer G. Immunological Effects of Conventional Chemotherapy and Targeted Anticancer Agents. Cancer Cell. 2015 Dec 14;28(6):690-714. doi: 10.1016/j.ccell.2015.10.012.

3. Mira A. Patel & Drew M. Pardoll. Concepts of immunotherapy for glioma. J Neurooncol 2015 123:323–330.

4. Michele De Palma*, Roberta Mazzieri*, Letterio S. Politi, Ferdinando Pucci, Erika Zonari, Giovanni Sitia, Stefania Mazzoleni, Davide Moi, Mary Anna Venneri, Stefano Indraccolo, Andrea Falini, Luca G. Guidotti, Rossella Galli, and Luigi Naldini. Tumor-Targeted Interferon-a Delivery by Tie2-Expressing Monocytes Inhibits Tumor Growth and Metastasis. Cancer Cell 2008 14, 299–311. * These authors share first co-authorship.

5. Giulia Escobar, Davide Moi, Anna Ranghetti, Pinar Ozkal-Baydin, Mario Leonardo Squadrito, Anna Kajaste Rudnitski, Attilio Bondanza, Bernhard Gentner, Michele De Palma, Roberta Mazzieri*, Luigi Naldini*. Genetic Engineering of Hematopoiesis for Targeted IFN-a Delivery Inhibits Breast Cancer Progression. Sci Transl Med 2014 6, 217ra3. * These authors share senior co-authorship.

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