“Now this is not the end. It is not even the beginning of the end. But it is, perhaps, the end of the beginning.”
– Winston Churchill, aft er Britain’s fi rst victorious battle of World War II
Cancer immunotherapies have provided several success stories during the last years. While BCG and interferon-alpha used to be the only immunotherapies for decades, now the range is increasing. Checkpoint inhibitors and T-cell therapies have shown their capability in a few cancer types and new indications are being approved. In China, an oncolytic virus (H101) has been approved years ago, while latest reports indicate that in the near future also the fi rst western approval is expected with the T-Vec oncolytic virus (Voted 22-1 in favor of approval by the FDA 29.4.2015, (Robert Hans Ingemar Andtbacka 2013)). Th e limitations of these therapies at the moment seem to be the cost, while the effi cacy in the responding patients is oft en dramatic and long lived. At the moment, it seems almost inevitable that in the following years many, if not all, cancers shall have their own form of immunotherapy (Homet Moreno and Ribas 2015).
Th ese might fi nally even take the place of traditional treatments which cause more side-eff ects (especially chemotherapy). Also treatments at early stage cancer are considered. Th e biggest obstacle for cancer therapy, however, seems to be the same old: who to treat, when to treat and how to treat. Hopefully better imaging, sequencing and immunological knowledge will tackle these questions.
In study I, we showed that cloning of a functional oncolytic adenovirus of other serotype than 5 is possible. We show that the Ad3-hTERT-E1A is eff ective in cell lines representing seven major cancer types, while low toxicity was seen in cell lines representing normal cells. In vivo, serotype 3 seemed to be at least as potent as the serotype 5 based oncolytic control viruses in three independent human cancer models. As the serotype is diff erent and the receptor for serotype 3 adenovirus is known to be highly present in advanced cancer, Ad3-hTERT-E1A is a promising new oncolytic virus.
In study II, we fi rst continued the work started in study I. We performed biodistribution and toxicity experiments with the Ad3-hTERT-E1A and found that in mice higher amounts of cytokines were released but less liver damage was induced compared to the serotype 5 or 5/3 control viruses. Next, the results of the fi rst 25 patients treated with this virus were published. Th e safety profi le of the virus was good, resembling mild adverse events seen with serotype 5 viruses.
Patients that had been previously treated with a serotype 5 based virus showed deeper and longer lasting lymphocytopenia than patients that received Ad3-hTERT-E1A as the fi rst treatment. We also noted frequent alterations in the T-cells collected from blood, indicating immunological activity. Interestingly, patients that were pretreated with serotype 5 viruses showed re-emerging of this virus (or its DNA) to blood, and vice versa, indicating that perhaps the old virus is lurking passively inside malignant cells. Signs of effi cacy was seen in many patients, and decrease in tumors markers was observed in 11/15 (73%) patients. Four of these responding patients were treated only intravenously, indicating that at least the fi rst oncolytic virus of a certain serotype could be given by intravenous route only. Interestingly, good results were seen with breast cancer patients, especially the ones on trastuzumab, indicating that the synergy suggested in preclinical
models might be true. As the safety and effi cacy seemes good, further testing or development of armed serotype 3 viruses seems rational.
During studies I-II, we noted that an oncolytic adenovirus based on serotype 3 seems to have many benefi ts compared to the serotype 5 (or some other coxsackie-adenovirus receptor binding adenovirus), which has been the most used adenovirus on the fi eld. While the main receptor for serotype 3 (DSG-2) is widely present in advanced cancer, it seems to have other potentially important aspects, such as the suggested opening of the tight junction, production of dodecahedral particles (PtDds), transforming the cells to an epithelial-to-mesenchymal transition (EMT), binding to the CD80/CD86 present at antigen-presenting cells, and might thus have a role in activating T-cells.
In study III, we wanted to study if magnetic resonance imaging (MRI) and/or spectroscopy (MRS) could be used in the evaluation of oncolytic immunotherapy, as little previous data existed. Currently used methods are proven to be unreliable due to infl ammation and initial tumor swelling. A GM-CSF armed and a non-armed control adenovirus was used. We show that in T2-weighed MRI a hypodense core area develops in the responding tumors in an immunocompetent Syrian hamster carcinoma model. Th is core area is consistent with coagulative necrosis. Similar fi ndings were observed in a neuroblastoma patient who responded to the treatment. We found that MRS of choline, taurine and unsaturated fatty acids can be useful indicators of response at early days aft er treatment. A high amount of calprotectin positive infl ammatory cells and T-cells were found surrounding necrotic areas, suggesting a possible link between necrosis, oncolysis and/or immune response in the GM-CSF treated cells. MRI and MRS could thus be used in the estimation of effi cacy in oncolytic immunotherapy at early time points. Patients benefi tting from the therapy could be selected from nonresponding ones treatable with other modalities.
In study IV, a quadruple modifi ed CM-CSF producing oncolytic adenovirus was evaluated.
First the virus was shown to function as designed. Th e modifi cations made it prone in entering and replicating in cancer cells while replication in normal human cells was low. Th en we demonstrate that the virus produces functional GM-CSF. At the clinical part of the study, we evaluated the 13 fi rst patients treated with the virus. Treatments were found well tolerated and effi cacy was suggested. Signs of potential anti-tumor effi cacy (PET-CT and markers) was observed in 9/12 (75%) evaluable patients. Radiological disease control rate (stable metabolic disease or better) with PET-CT was 83% while the response rate (minor metabolic response or better) was 50%. Accumulation of immunological cells aft er treatment was seen in tumor biopsies. RNA expression analysis of these biopsies indicated immunological activity and metabolic changes aft er virus replication.
Conclusion from studies I-IV. A fully functional serotype 3 oncolytic adenovirus was constructed. Th e treatments showed promising safety and effi cacy data. Even though this virus was not armed with any immunomodulatory transgene, it seemed to be as good, or even better, as a GM-CSF armed Ad5/3 (serotype 5 virus with a serotype 3 fi ber knob) oncolytic virus.
A side by side comparison of two independent studies is hard, but if we hypothesize that the patients reported in study II and study IV were similar as well as all other major error factors, the non-armed serotype 3 virus seems promising. With serotype 3, 11/15 patients (73%) showed possible signs of effi cacy according to tumor markers while this was seen with 6/10 (60%) of the armed Ad5/3 virus. However, we must keep in mind that the number of patients is low for any comparison and there was signifi cant heterogeneity among the patients. Th e goal was to treat patients, not to evaluate effi cacy. Accordingly, no defi nite conclusions can be made of
effi cacy. Th is data is, however, provocative as the GM-CSF armed Ad5/3 construct seems to be one of the best oncolytic viruses we have used. Th erefore, the further development of a fully serotype 3 armed oncolytic virus is highly suggested. Preclinical work seems to propose that combining adenovirus serotype 3 with approved therapies, such as chemotherapy, trastuzumab or cetuximab (Beyer et al. 2011) (Beyer et al. 2012), may off er further potency. Results have been promising and adenovirus 3 dodecahedral particles are entering into clinical testing (Beyer et al.
2012). Other combinations that will probably be seen in the near future include combinations with checkpoint inhibition and T-cell based therapies (see table 11). Th ere even seems to be rationale for combining them all.
Table 11. Hypothetical new cancer treatment schema
Biopsy fi nding Problem Treatment Eff ect
High amount of
If patients is in a good general status, checkpoint combinations (CTLA-4 and PD-1 antibody) preferable in stead of monotherapy.
For non-necessary organs (e.g. prostate, breast, thyroid, ovary) targeting these cells with transfected T-cells might be an option. Feasibility of this approach is demonstrated with lymphoma/leukemia.
Similar therapy might be preferable if tumor presents cancer-testis antigens.
My favorite imaginary treatment design at the moment for advanced cancer:
Operation of the tumor and growing the tumor infi ltrating lymphocytes from the sample.
Priming the residual tumor with an immunostimulatory serotype 5 adenovirus and then boosting the treatment later with a serotype 3 adenovirus.
Th ese treatments would be combined with checkpoint inhibition and with trastuzumab and/or cetuximab when rational.
Finally returning the tumor infi ltrating lymphocytes to the patient.
ACKNOWLEDGEMENTS
Th is work was conducted during 2007-2015 in the Cancer Gene Th erapy Group (CGTG), University of Helsinki, Finland. CGTG was formerly part of the Molecular Cancer Biology Program in Biomedicum Helsinki and currently affi liated to Medicum, Haartman Institute. In brief, I am deeply grateful to all people working in these institutes and their help and assistance during these years. Without the cleaning man or the head of the department the journey would have been a lot harder, thank you all.
First I want to thank all the 290 patients that participated in the 821 ATAP treatments.
Th eir bravery and faith in these new treatments enabled us to gain experience and to develop the treatments further. While not all of the patients showed benefi t, I believe there were many that received advantage themselves.
A special thanks for the Dean of Faculty of Medicine, Professor Risto Renkonen for his continuous support over these years. I wish to thank also the head of Haartman Institute, Professor Tom Böhling and the current and former directors of Molecular Cancer Biology Program for the good research facilities.
I am grateful for professor Ruben Hernandez Alcoceba who accepted (in minutes aft er sending the email) the request to be my opponent. Professor Kari Airenne and adjunct professor Timo Muhonen are entitled to special recognition for their helpful comments during the pre-examination.
I am very grateful for my supervisor, group leader, professor and brother Akseli for giving me the opportunity to get acquainted into this truly fascinating world of gene therapy, rational cloning, viruses, immune cells, oncolysis, cancer, cancer treatments and oncolytic immunotherapy. I wish also to thank all former and present group members of the CGTG.
Without Akseli´s driving force and the CGTG group’s team work, the accomplishments would not have been possible. During my time more than a hundred publications were published in highly ranked journals and also many patients were treated with the oncolytic viruses that were cloned and preclinically evaluated by the group. I saw the evolution from preclinical promising oncolytic agents to clinical treatments. Akseli and the CGTG made the leap from the bench to the bedside, something that is rarely accomplished with preclinical groups. I saw the encouraging responses and the attempts to fi nd money for clinical testing. Now the group has evolved back to the bench with the information gained from these treatments and is designing even more potent treatments. Hopefully someday these attempts will lead to late stage clinical trials so that the effi cacy of these promising treatments can be evaluated. Th is enables the possibility for global large scale patient benefi t.
I would like to thank Sergio, my hands on mentor, for introducing me to the cell culture and mouse work, and Sari for helping me out to plan and execute many of the experiments that were done and helping me for preparing the articles. I am grateful for the time Iulia helped me with the experiments and animal work. I would like to thank Gerd for passing on the information about the serotype 3 viruses; he was the one in our group who had tried to clone these viruses before me. For teaching of cloning, I would like to thank Eerika and our collaborators from Zurich and Seattle. Silvio kindly invited me to Zurich to learn new tricks with cloning, and Andre and Hongjie from Seattle were extremely helpful regarding the cloning of the armed viruses. Simona and Suvi, you have helped me fi nalize critical experiments. Saila, thank you especially for the help with the qPCR and Kikka for help with the ELISPOT, you both emit good atmosphere around you. Minna and Aila, thank you for the million little things you have done for me. Essential was
also the help and knowledge that more experienced researchers such as Iulia, Lotta, Tuuli, Kilian, Markus, Vince and Anna were able to give. Younger GCGT PhD students, Mikko, Siri, Riikka, Kristian, Noora, you are good company and seem enthusiastic, keep up the good work and spirit.
You are the heart of CGTG.
Th ank you for the fruitful collaboration in the MRIS project with the University of Eastern Finland. Riikka, Johanna and Olli, this would have not been possible without your welcoming attitude.
Th e scientifi c conferences we joined were memorable not only because of the scientifi c background and the many connections with collaborators we made but because we were able to project the work we are doing with work done by others. Some trips we made with friends from the lab were unforgettable, including kitesurfi ng in the Long Island, powder skiing in Utah, touring the Alps, road tripping Baja California or hanging out at diff erent summer cottages in Finland. Ilkka, Marko, Kilian, Petri, Joao, Iulia, Sari, Akseli, Anniina, Maria and others, thanks also for the time outside the lab.
Equally great were the many scientifi c and non-scientifi c interaction we had with diff erent groups in Meilahti. Especially regular fl oor ball with the Lauri Aaltonen group and beach volley with a heterogeneous team have oft en been good balance in the weekly cycle. Especial thanks to Alexandra, Heli, Erkka, Boris for keeping things active outside the lab.
Th ank you Ari Harjula and Maija Lappalainen for giving your time and advice during the yearly follow-up group meetings. Your comments were valuable and the meetings helped to concretely see the progress as sometimes it felt that no progress was made in ages. Special thanks also to Maija for helping with the validation of the Ad3-hTERT-E1A virus; electron microscopy, culture, serotyping and PCR. Special thanks for Ari Harjula and Mika Matikainen for accepting to become the reviewers of this thesis. Even greater thanks to Kari Airenne and Timo Muhonen who also accepted and were then appointed as reviewers by the Faculty.
I wish to thank the National Graduate School of Clinical Investigation for educational and fi nancial support during these years. In addition, I would like to thank the other fi nancial supporters including our CGTG and the following foundations: Duodecim, Biomedicum Helsinki, Irja Karvonen, Finnish Cancer Organisations, Sohlberg, Research on Viral Diseases, Maud Kuistila, Irja Karvonen, Eemil Aaltonen and Ida Montin.
I guess, I could not change my destiny. Th e genes had it encrypted or then it just was the environment. Having a research professor as an mother, as a father and nowadays also as a brother did not help. I feel I was newer pressurized or even lured, but sometime in mid-med school science started to interest me more and more. For few years I searched passively for something interesting and aft er graduating I ended up starting with the most interesting group I had come up with. Coincidence or not, it was the one of my brother. Although combining research with clinical work seems to be quite demanding, I have, however, enjoyed the experience and, if feasible, shall try to continue on this road. Th us, I would like to thank people involved in my upbringing, Inna, Ossi, Kari, Asta, Aksu, Liina. I don’t know how you did, it but I think you did it quite well. Finally, I want to thank my beloved Anni for understanding, caring and standing by my side through this sometimes stressful journey.
Last but not least I thank all my friends. Without you my life would be somewhat dull.
Helsinki, October 2015
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