Design and delivery strategies of alphavirus replicon-based cervical cancer vaccines
van de Wall, Marie-Nicole Stephanie
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2018
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
van de Wall, M-N. S. (2018). Design and delivery strategies of alphavirus replicon-based cervical cancer vaccines. Rijksuniversiteit Groningen.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
replicon-based cervical cancer vaccines
Microbiology of the University Medical Center Groningen (UMCG) within the Groningen University Institute for Drug Exploration (GUIDE), research programme Microbes in Health and Disease (MHD).
The work in this thesis was supported by GUIDE, the Dutch Cancer Society, the National Cancer Control Program, the European Fund for Regional Development and the Jan Kornelis de Kock foundation (Groningen).
The printing of this thesis was financially supported by:
ISBN: 978-94-034-0489-9(printed version)
ISBN: 978-94-034-0488-2(electronic version)
Cover and layout design: Iliana Boshoven-Gkini | www.AgileColor.com Printed by: Ridderprint | www.ridderprint.nl
Copyright © 2018 by Stephanie van de Wall. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without permission of the author and when appropriate, the publisher holding the copyrights of the published articles.
replicon-based cervical cancer vaccines
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken
en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op
woensdag 28 maart 2018 om 12.45 uur
Prof. dr. C.A.H.H. Daemen Prof. dr. H.W. Nijman Beoordelingscommissie Prof. dr. A.G.J. van der Zee Prof. dr. A.L.W. Huckriede Prof. dr. T.D. de Gruijl
Georgia Koutsoumpli José Alberto Aguilar Briseño
Chapter 1
General Introduction and outline 9
Chapter 2
Development and preclinical evaluation of an alphavirus therapeutic cancer vaccine against cervical cancer
Manuscript in preparation
23
Chapter 3
Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens
Submitted
41
Chapter 4
HPV-Specific Immunotherapy: Key Role for Immunomodulators
Anti-Cancer Agents Med Chem. 2014 Feb;14(2):265-79.
61
Chapter 5
An immunotherapeutic design approach an alphavirus-based immunotherapeutic vaccine, PD-1 blockade and sunitinib
Study in progress
95
Chapter 6
Tattoo Delivery of a Semliki Forest Virus-Based Vaccine Encoding Human Papillomavirus E6 and E7
Vaccines. 2015 Mar;3(2):221-38.
The prognostic benefit of CD27+ tumor-infiltrating lymphocytes in cervical cancer: implications for treatment regimen
Manuscript in preparation
Chapter 8
CD103+ tumor-infiltrating lymphocytes are tumor-reactive intraepithelial CD8+ T cells associated with prognostic benefit and therapy response in cervical cancer
Oncoimmunology. 2017 Jul 24;6(9)e1338230.
153
Chapter 9
Noninvasive monitoring of cancer therapy-induced activated T cells using [18F]
FB-IL-2 PET imaging
Oncoimmunology. 2016 Nov 18;6(1):e1248014.
183
Chapter 10
Summarizing Discussion and Future Perspectives 201
Addendum
Nederlandse Samenvatting 219
Curriculum Vitae 225
General introduction and outline
Chapter 1
1
Scope of the Thesis
The work presented in this thesis focuses on immunotherapeutic Semliki Forest virus (SFV) replicon vaccines. This platform is used for the treatment of human papillomavirus (HPV) 16-induced neoplasia. The aim is to optimize this platform for current and future use in the clinic by evaluating rational strategies based on vaccine design and delivery. The replicon vaccines in this thesis were designed as recombinant viral particles or naked DNA.
The recombinant SFV (rSFV) viral particle vaccine encoding for a fusion protein of E6 and E7 of HPV16 is currently being evaluated in a phase I clinical trial as Vvax001. We describe the production and preclinical evaluation of Vvax001 according to GMP standards. Alternative design and delivery strategies to those currently used in the clinical trial were explored which include the development of a HPV16 DNA replicon platform (DREP), combination strategies with other immunomodulatory treatments and intradermal delivery methods. Furthermore, we assessed relevant biomarkers for evaluating responses to treatment regimens in the clinic.
In this first chapter, we will introduce HPV and the associated (pre)-malignant lesions. Next, the developed HPV replicon vaccines will be described followed by design and delivery approaches to potentially improve vaccine immunogenicity. Lastly, markers to predict and monitor immune response to novel therapeutics will be discussed.
Human Papillomavirus and Cervical Cancer
Human papillomavirus (HPV), belonging to the family of Papovaviridae, is a double-stranded DNA virus synthesizing six early protein (E1, E2, E4, E5, E6 and E7) and two late capsid proteins (L1 and L2) upon infection in epithelial cells.1,2 Despite the existence of
more than 200 identified genotypes, only a few are known to cause cancer. These are categorized in the high-risk group.2 HPV infection is associated with nearly all cases of
cervical cancer, the fourth most common cancer type among women worldwide.3,4 The
virus has also been implicated as a causal agent of other cancers including penile, vaginal, vulvar, anal and oropharyngeal.5-7
High risk types 16 and 18 are the most common and are associated with approximately 70% of cervical cancer cases.8 Most sexually active women will be infected
with HPV at some point in their life yet many will remain asymptomatic as the virus is cleared by the immune system.9 If the infection were to persist, individuals are at risk
of developing low to high-grade cervical intraepithelial neoplasia (CIN) and eventually cervical carcinoma.10 Malignant transformation of epithelial cells is accomplished through
integration of HPV viral DNA genome into the host genome with the disruption of genes including E2, a negative regulator for the HPV oncoproteins E6 and E7.11 High expression
of these oncoproteins in transformed cells is essential for the induction and maintenance of cellular transformation.12 They interfere with the normal function of tumor suppressor
1
proteins by binding to p53 and retinoblastoma protein (pRb), respectively and inhibiting apoptosis in the infected cell.13
As HPV is the causal factor of HPV-associated malignancies, opportunities arise for effective tumor control. Successful prophylactic HPV vaccines have been developed at preventing infections through neutralizing antibodies against viral surface proteins.14 Yet
these vaccines are not effective at treatment and clearance of established HPV- associated lesions. The standard treatment for cervical cancer, depending on the stage of the disease, is surgery alone or the combination with (chemo)radiotherapy.15-17 As healthy tissues may
also be affected by the conventional treatment, side effects may ensue such as infertility.18
Alphavirus Replicons for HPV Immunotherapy
Immunotherapy is a novel and promising strategy for the specific treatment of CIN lesions and cervical cancer.19 As E6 and E7 are necessary for malignant transformation, these
proteins represent attractive targets for therapeutic HPV vaccination. Several therapeutic vaccines have been developed which include synthetic peptides, recombinant proteins, nucleic acids, autologous cell (dendritic cell, tumor cell or adoptive T-cell therapy) and bacterial or recombinant viral vectors.20 Recombinant viral vector vaccination is attractive
above all others as it offers high infection efficiency and antigen expression due to the nature of the vector. The associated potent antigen-specific responses are ones that develop towards a natural virus infection. For the treatment of HPV-associated (pre) malignant lesions, adenovirus, alphavirus, vaccinia virus and fowlpox virus have all been investigated.21
Replicon viral vector system
Alphaviruses belong to the Togaviridae family of small, enveloped, and positive-stranded RNA viruses.22 Among those identified, Semliki Forest virus, Sindbis virus and Venezuelan
equine encephalitis virus have been used as delivery vehicles for vaccination against a range of infectious diseases and cancer.23-27 In our studies we constructed alphavirus
replicons based on SFV as immunotherapy for HPV-induced (pre)-malignant lesions. The expression vectors is generated from SFV by replacing the genes encoding for the structural proteins with a transgene encoding for a fusion protein of E6 and E7.28 On the
same reading frame upstream of the transgene, are genes encoding for nonstructural proteins of SFV (nsPs or replicase) and the resulting RNA is termed a replicon due to its self-replicating property. Recombinant viral particles are produced upon cotransfection of cells with the replicase RNA and helper RNA encoding for the structural proteins. Only the replicase RNA together with E6,7 is packaged due to the absence of a packaging signal
1
of the two RNAs could lead to formation of replication-competent virus. To decrease the probability of recombination, the structural genes encoding for the spike and capsid proteins are placed on two separate helper RNA constructs as a so-called ‘split’ helper system.29
Upon infection of cells, the RNA enters the cytoplasm and the viral replicase is first translated. The self-amplifying nature of the replicon driving the replication and amplification of RNA as well as transcription of subgenomic RNA on which the E6,7 fusion protein is encoded. As a result, a large amount of antigen is produced. This production is also enhanced eight-fold due to the 5’ end of the capsid gene containing a translational enhancer encoding the first 34 amino acids of the capsid.30–32 In order to obtain translation
of E6,7 that is not attached to this capsid fragment, a 17 amino acid sequence of the 2A autoprotease of foot-and-mouth disease virus was inserted in frame directly after the capsid translational enhancer.29,33 The broad range of host cells that are infected
express high levels of transgene expression and undergo apoptosis. As the replicon RNA is amplified to a great extent, mimicking viral infection with the production of ssRNA and dsRNA intermediates, activation of pathogen recognition receptors (PRRs) leads to production of type I IFN.34,35 Apoptotic bodies are taken up by nearby antigen presenting
cells (APCs) that cross-present antigen for CD8+ and CD4+ T cell priming. For treatment of HPV-induced (pre)malignant lesions, we evaluated the immunogenicity of rSFVeE6,7 in preclinical studies which demonstrate potent E7-specific cellular responses as well as the ability to overcome immune tolerance.36,37 These responses translated to effective
anti-tumor responses with eradication of established HPV-transformed anti-tumors.38 Due to these
findings, we are currently conducting a phase I clinical trial with GMP-grade rSFVeE6,7. DNA replicon vector system
Replicon vectors can also be delivered as naked DNA (DREP). DREP has a CMV promoter, having replaced the SP6 RNA polymerase promoter of rSFV, and when first transfected in a cell, is transported to the cell nucleus for transcription into replicon RNA.39 Once the
replicon RNA is transported to the cytoplasm, a sequence of events occuring thereafter is the same between all replicon vector systems with translation of the replicase, amplification of the subgenomic RNA and extensive antigen production (Figure 1.1).39
So far, there are no DNA vaccines approved for human use. DNA vaccines are poorly translatable to the clinic as they are characterized by low immunogenicity in higher ordered species.40 Compared to these conventional DNA vectors (e.g. pVAX), DREP is superior
in immunogenicity due to the intrinsic immunostimulatory properties of replicon RNA, increasing gene expression and eliciting stronger immune responses (Figure 1). The dose-sparing effect can be further improved through electroporation-assisted delivery further increasing antigen-specific immune responses.25,41,42 DREP has been used as a delivery
1
models. DREP candidates, based on Sindbis virus or Venezuelan encephalitis virus, have been tested in three cancer models: melanoma, breast cancer and prostate cancer.43-46 To
date, no preclinical study for cancer immunotherapy study has focused on the SFV DNA platform, let alone for treatment of HPV-induced (pre)-malignant lesions. In this thesis, we developed a DREP vaccine encoding for a fusion protein of E6 and E7 (DREPeE6,7) and assessed it’s vaccine potency. Due to safety concerns in the clinic regarding the chromosomal integration of foreign DNA, a gene encoding a shuffled version of the E7 protein was also incorporated in DREP.47,48
transcription nuclear export translation of SFV replicase (-)RNA Translation of subgenomic RNA E6,7
RNA replicase E6,7 protein 26S CMV SFV replicon: DREPeE6,7 E6,7 protein Conven1onal DNA: pVAXeE6,7 CMV nuclear export transcription (+) RNA transfection infection SFV replicon: rSFVeE6,7 mRNA mRNA translation
negative strand synthesis transfection
(+)RNA
Figure 1.1. Expression of antigen by SFV replicons compared to conventional vaccine. Cellular delivery of DNA vectors (e.g. pVAXeE6,7 or DREPeE6,7) results in transcription in the nucleus and subsequently export of RNA to the cytoplasm. Recombinant viral vectors (e.g. rSFVeE6,7) directly introduce replicon RNA in the cytoplasm. For SFV replicon vaccines, the RNA in the cytoplasm first translates the SFV replicase which catalyzes the amplification of replicon RNA. Production of large amounts of subgenomic RNA by the replicase results in a large amount of transgene expression.
1
Improving Vaccine-induced Immunity
SFV replicon vaccination has immense potential for clinical use in cancer therapeutics due to induction of robust effector CD4+ and CD8+ effector and memory responses. Two
clinical trials thus far demonstrating potent clinical responses towards HPV-induced lesions fuels the optimism that vaccination with SFV replicons may be a viable treatment option in the near future.49,50 Yet, in order to guarantee the likelihood of success in the
clinic, cancer vaccination may be complimented with strategies to further enhance immunogenicity. For improvement in vaccine immunogenicity, one may consider combining immunotherapy with approaches that include immunomodulators (e.g. adjuvants) or target immunosuppression in the tumor microenvironment. Another way to potentially improve immunity is through the route of administration as well as device-assisted delivery to enhance antigen availability for efficient priming of T cells. These strategies are further described below.
Targeting immunosuppression
The efficacy of immunotherapy is hampered by the accumulation of immunosuppressive mechanisms in the cancer microenvironment. One of these mechanisms is the infiltration and activation of immunosuppressive cells, namely regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). The removal of Tregs can be accomplished using cyclophosphamide or anti-CD25 antibodies whereas sunitinib has been used to deplete MDSCs.51-55 We have previously reported the combination of using depleting agents
of Tregs and MDSCs with rSFV immunization in a preclinical model of cervical cancer. Interestingly, the therapeutic efficacy of rSFV immunization was not enhanced with Treg depletion, whereas MDSC depletion led to a synergistic effect.56,57 The enhanced tumor
control with dual treatment of rSFV immunization and MDSC depletion is in part due to the significant increase in the levels of antigen-specific T cells infiltrating the tumor.57
Another strategy for combating immunosuppression involves the use of antagonists of immune checkpoint proteins such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) or programmed cell death protein 1 (PD1)-PD1 ligand 1 (PDL1) pathways. The recent successes of these blocking antibodies as immunotherapy has been proven with durable clinical responses in a number of different cancer types.58-60 The success of treatment with
checkpoint blockade inhibitors is dependent on the a high mutational burden as well as the presence of tumor-infiltrating lymphocytes (TIL).61-63 For rational vaccine design, it is
therefore of interest to combine checkpoint blockade inhibition with immunotherapy as activated antigen-specific T cells have been found to predominantly express PD-1.64 The
ligand for PD-1, PD-L1, is expressed on tumors cells as well as various immune subsets that include MDSCs. It would be of interest to assess the possible synergistic effect of targeting both immunosuppressive mechanisms.
1
Intradermal delivery methods
One of the attractive sites for vaccine delivery is the skin due to the presence of APCs for effective T cell priming and induction of tumor-infiltrating lymphocytes (TIL). The intradermal route has been shown to be superior to that of conventional routes such as intramuscular or subcutaneous injection.65 Clinical trials targeting influenza and rabies
vaccination have shown equal immune responses upon immunizing with 10-20% lower antigen dose.66,67 Studies utilizing hypodermic needles for intradermal injection require
considerable expertise. Hence, there is a need for devices to provide accurate and precise administration.68 These devices include microneedles arrays, liquid jet injectors, gene gun,
electroporation and tattooing.69 Electroporation is one of the most promising approaches
for delivery of naked DNA and is currently being used in the clinic, accompanying intramuscular injection in recent HPV phase I and II trials.49,70,71 Yet, intradermal injection
followed by electroporation, despite showing dose-sparing effects in preclinical studies, is still at an early stage for clinical application.42,72 A device that has been utilized for delivery
of conventional DNA vectors encoding HPV antigens is tattoo injection that elicits higher cellular responses compared to intramuscular injection.73,74 Whether this device can also
enhance immunity of other vaccine platforms is still under question.75
Markers to Predict and Monitor Responses to Immunotherapy
As clinical responses are generally observed in a minority of patients receiving treatment, there is a dire need to identify which patients are likely to respond towards novel treatments. Biomarkers cannot only guide patient selection, but also optimize treatment regimens for combination approaches with newly introduced therapies. The engagement of T cells with most immunotherapies is an essential process for the selection of biomarkers. Yet, the tumor microenvironment comprises of an intricate network of multiple cell subsets of the tumor, stroma and vasculature. The cellular interactions as a multi-step process contributes to tumor progression due to the presence of immune evasion mechanisms in the different compartments.76,77 A strong lymphocytic infiltration has been reported to be
associated with a beneficial clinical outcome for many different tumor types such as head and neck cancer, breast cancer, lung cancer and melanoma.7879 The immune biomarkers
have been explored in cervical cancer include CD3+ T cells CD8+ T cells, PD-L1, PD-1 and FoxP3+ Tregs.80-84 Yet, from these studies, it is challenging to determine which biomarker
is of prognostic and predictive value given that clinical responses vary dependent on the stage and treatment type. Effectively monitoring of antitumor immune responses elicited by immunotherapeutics in the clinic may also be useful as a biomarker in predicting clinic benefit. This can be accomplished using noninvasive tools such as positron emission
1
Outline of this Thesis
Chapter 2 describes the evaluation of a GMP-grade recombinant Semliki Forest virus replicon vaccine, Vvax001, for clinical application for the treatment of HPV-induced (pre) malignant lesions. In this study, the development from bench to bedside is presented along with pre-clinical results relating to toxicity and HPV immunogenicity.
Chapter 3 describes the production and therapeutic evaluation of a DNA replicon (DREP) platform based on Semliki Forest virus for the treatment of HPV-induced (pre)malignant lesions. DREP, encoding for a fusion protein of E6 and E7 (DREPeE6,7) was evaluated for immunogenicity and anti-tumor responses compared to a conventional DNA vaccine, pVAXeE6,7. We also assessed the impact of dose on the anti-tumor response of DREP. Furthermore, we evaluated a DREP vaccine encoding for a shuffled version of the E7 protein to overcome any safety concern in the clinic. Gene-shuffling results in loss of oncogenic potential of the E7 protein leaving putative T cell epitopes unaffected. The immunogenicity and anti-tumor response of the shuffled version of E7 was evaluated. To test whether the potency of DREP can be further improved, we included a carrier-protein providing CD4+ T cell help, improvement of antigen stability and alteration of subcellular localization of the antigen for evaluation of immunogenicity and anti-tumor responses. Chapter 4 provides a detailed review on the pre-clinical and clinical studies of immunotherapeutic strategies against HPV as part of combination strategies with immunomodulators. Immunomodulators include toll-like receptor adjuvants, cytokines and costimulatory molecules and strategies to target immunosuppression.
Chapter 5 provides immunotherapeutic design approaches with targeting PD-1, sunitinib and SFVeE6,7 immunization. We evaluated the expression levels of PD-1 and PD-L1 as well as TIL infiltration in tumors of mice immunized with SFVeE6,7 and SFVeE6,7 with PD-1 blockade. Subsequently, the anti-tumor efficacy of dual and triple treatment approaches was evaluated.
Chapter 6 describes the delivery of rSFVeE6,7 via tattoo injection with a head-to-head comparison with intramuscular injection for HPV-specific immunogenicity and anti-tumor responses. This is the first study to evaluated the administration of replicon particles with skin tattooing.
Chapter 7 analyses the prognostic and predictive value of tumor-infiltrating lymphocytes expressing CD27+, CD8+ and FoxP3+ in tissue from a cohort of cervical cancer patients. Two patient cohorts were analyzed separately according to the primary treatment. The relations to disease-free and disease-specific survival were assessed for each TIL subset.
1
Chapter 8 analyses the prognostic value of CD103+ TL in tissue from a cohort of cervical cancer patients. The localization of CD103+ cells was determined in epithelial and stromal compartments by immunofluorescence. In a preclinical tumor model of cervical cancer, the value of CD103 as an immunotherapeutic response biomarker was assessed.
Chapter 9 describes using positron emission tomography (PET) as a tool for monitoring activated T cells upon rSFVeE6,7 immunization and low-dose local tumor irradiation. Activated T cells were targeting using the radiotracer N-(4-[18F]flyorobenzoyl)interleukin-2
as a biomarker in tumor-bearing mice. We quantified the uptake of tracer in tumors and various non-target organs.
Chapter 10 presents a summarizing discussion of this thesis and perspectives on future research.
1
References
1. Kranjec, C. & Doorbar, J. Human papillomavirus infection and induction of neoplasia: a matter of fitness.
Curr. Opin. Virol. 20, 129–136 (2016).
2. Egawa, N., Egawa, K., Griffin, H. & Doorbar, J. Human Papillomaviruses; Epithelial Tropisms, and the
Development of Neoplasia. Viruses 7, 3863–3890 (2015).
3. Wakeham, K. & Kavanagh, K. The Burden of HPV-Associated Anogenital Cancers. Curr. Oncol. Rep. 16, 402
(2014).
4. Parkin, D. M. & Bray, F. Chapter 2: The burden of HPV-related cancers. Vaccine 24, S11–S25 (2006).
5. Stratton, K. L. & Culkin, D. J. A Contemporary Review of HPV and Penile Cancer. Oncology (Williston Park).
30, 245–9 (2016).
6. Mehanna, H. et al. Prevalence of human papillomavirus in oropharyngeal and nonoropharyngeal head and
neck cancer-systematic review and meta-analysis of trends by time and region. Head Neck 35, 747– 755 (2013).
7. Forman, D. et al. Global Burden of Human Papillomavirus and Related Diseases. Vaccine 30, F12–F23 (2012).
8. Schiffman, M., Castle, P. E., Jeronimo, J., Rodriguez, A. C. & Wacholder, S. Human papillomavirus and cervical
cancer. Lancet 370, 890–907 (2007).
9. de Jong, A. Human Papillomavirus Type 16-Positive Cervical Cancer Is Associated with Impaired CD4+ T-
Cell Immunity against Early Antigens E2 and E6. Cancer Res. 64, 5449–5455 (2004).
10. Snijders, P. J., Steenbergen, R. D., Heideman, D. A. & Meijer, C. J. HPV-mediated cervical carcinogenesis: concepts and clinical implications. J. Pathol. 208, 152–164 (2006).
11. Grabowska, A. K. The Invisible Enemy – How Human Papillomaviruses Avoid Recognition and Clearance by the Host Immune System. Open Virol. J. 6, 249–256 (2012).
12. Moody, C. A. & Laimins, L. A. Human papillomavirus oncoproteins: pathways to transformation. Nat. Rev.
Cancer 10, 550–560 (2010).
13. Narisawa-Saito, M. & Kiyono, T. Basic mechanisms of high-risk human papillomavirus-induced carcinogenesis: Roles of E6 and E7 proteins. Cancer Sci. 98, 1505–1511 (2007).
14. Roden, R. & Wu, T. Preventative and therapeutic vaccines for cervical cancer. Expert Rev. Vaccines 2, 495–516 (2003).
15. Tangjitgamol, S. et al. Adjuvant chemotherapy after concurrent chemoradiation for locally advanced cervical cancer. in Cochrane Database of Systematic Reviews (ed. Tangjitgamol, S.) (John Wiley & Sons, Ltd, 2014).
16. Eifel, P. J. Pelvic Irradiation With Concurrent Chemotherapy Versus Pelvic and Para-Aortic Irradiation for High-Risk Cervical Cancer: An Update of Radiation Therapy Oncology Group Trial (RTOG) 90-01. J. Clin.
Oncol. 22, 872–880 (2004).
17. Rosa, D. D., Medeiros, L. R., Edelweiss, M. I., Pohlmann, P. R. & Stein, A. T. Adjuvant platinum-based chemotherapy for early stage cervical cancer. in Cochrane Database of Systematic Reviews (ed. Rosa, D. D.) (John Wiley & Sons, Ltd, 2012).
18. Herzog, T. J., Huh, W. K., Downs, L. S., Smith, J. S. & Monk, B. J. Initial lessons learned in HPV vaccination.
Gynecol. Oncol. 109, S4–S11 (2008).
19. Lee, S.-J., Yang, A., Wu, T.-C. & Hung, C.-F. Immunotherapy for human papillomavirus-associated disease and cervical cancer: review of clinical and translational research. J. Gynecol. Oncol. 27, (2016).
20. Van de Wall, S., Nijman, H. W. & Daemen, T. HPV-specific immunotherapy: key role for immunomodulators.
Anticancer. Agents Med. Chem. 14, 265–79 (2014).
21. Skeate, J. G., Woodham, A. W., Einstein, M. H., Da Silva, D. M. & Kast, W. M. Current therapeutic vaccination and immunotherapy strategies for HPV-related diseases. Hum. Vaccin. Immunother. 12, 1418–1429 (2016). 22. Jose, J., Snyder, J. E. & Kuhn, R. J. A structural and functional perspective of alphavirus replication and
assembly. Future Microbiol. 4, 837–56 (2009).
23. Ljungberg, K. & Liljeström, P. Self-replicating alphavirus RNA vaccines. Expert Rev. Vaccines 14, 177–94 (2015).
1
24. Berglund, P., Fleeton, M. N., Smerdou, C. & Liljeström, P. Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice. Vaccine 17, 497–507 (1999).
25. Knudsen, M. L. et al. Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine. J. Virol. 86, 4082–90 (2012).
26. Hallengard, D. et al. Novel Attenuated Chikungunya Vaccine Candidates Elicit Protective Immunity in C57BL/6 mice. J. Virol. 88, 2858–2866 (2014).
27. Ip, P. P. et al. Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses. Mol. Ther. 22, 881–90 (2014).
28. Quetglas, J. I. et al. Alphavirus vectors for cancer therapy. Virus Res. 153, 179–196 (2010).
29. Smerdou, C. & Liljeström, P. Two-helper RNA system for production of recombinant Semliki forest virus particles. J. Virol. 73, 1092–8 (1999).
30. Frolov, I. & Schlesinger, S. Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation. J. Virol. 70, 1182–90 (1996).
31. Frolov, I. & Schlesinger, S. Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon. J. Virol. 68, 8111–7 (1994).
32. Sjöberg, E. M., Suomalainen, M. & Garoff, H. A significantly improved Semliki Forest virus expression system based on translation enhancer segments from the viral capsid gene. Biotechnology. (N. Y). 12, 1127–31 (1994).
33. Liljeström, P. & Garoff, H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology. (N. Y). 9, 1356–61 (1991).
34. Huckriede, A. et al. Induction of cytotoxic T lymphocyte activity by immunization with recombinant Semliki Forest virus: indications for cross-priming. Vaccine 22, 1104–1113 (2004).
35. Barry, G. et al. Semliki Forest Virus-Induced Endoplasmic Reticulum Stress Accelerates Apoptotic Death of Mammalian Cells. J. Virol. 84, 7369–7377 (2010).
36. Daemen, T. et al. Superior therapeutic efficacy of alphavirus-mediated immunization against human papilloma virus type 16 antigens in a murine tumour model: Effects of the route of immunization. Antivir.
Ther. 9, 733–742 (2004).
37. Riezebos-Brilman, A. et al. Induction of human papilloma virus E6/E7-specific cytotoxic T-lymphocyte activity in immune-tolerant, E6/E7-transgenic mice. Gene Ther. 12, 1410–4 (2005).
38. Daemen, T. et al. Eradication of established HPV16-transformed tumours after immunisation with recombinant Semliki Forest virus expressing a fusion protein of E6 and E7. Vaccine 21, 1082–8 (2003). 39. Lundstrom, K. Replicon RNA Viral Vectors as Vaccines. Vaccines 4, 39 (2016).
40. Kutzler, M. A. & Weiner, D. B. DNA vaccines: ready for prime time? Nat. Rev. Genet. 9, 776–88 (2008). 41. Knudsen, M. L. et al. Kinetic and phenotypic analysis of CD8+ T cell responses after priming with alphavirus
replicons and homologous or heterologous booster immunizations. J. Virol. 88, 12438–51 (2014). 42. Johansson, D. X., Ljungberg, K., Kakoulidou, M. & Liljeström, P. Intradermal electroporation of naked
replicon RNA elicits strong immune responses. PLoS One 7, e29732 (2012).
43. Yamanaka, R. & Xanthopoulos, K. G. Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18. DNA Cell Biol. 24, 317– 24 (2005).
44. Lachman, L. B. et al. DNA vaccination against neu reduces breast cancer incidence and metastasis in mice.
Cancer Gene Ther. 8, 259–68 (2001).
45. Wang, X. et al. Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice. Breast Cancer Res. 7, R580-8 (2005).
46. Garcia-Hernandez, M. de la L., Gray, A., Hubby, B., Klinger, O. J. & Kast, W. M. Prostate stem cell antigen vaccination induces a long-term protective immune response against prostate cancer in the absence of
1
48. Osen, W. et al. A DNA vaccine based on a shuffled E7 oncogene of the human papillomavirus type 16 (HPV 16) induces E7-specific cytotoxic T cells but lacks transforming activity. Vaccine 19, 4276–4286 (2001). 49. Kim, T. J. et al. Clearance of persistent HPV infection and cervical lesion by therapeutic DNA vaccine in CIN3
patients. Nat. Commun. 5, 5317 (2014).
50. Kenter, G. G. et al. Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia. N. Engl.
J. Med. 361, 1838–1847 (2009).
51. Le, D. T. & Jaffee, E. M. Regulatory T-cell Modulation Using Cyclophosphamide in Vaccine Approaches: A Current Perspective. Cancer Res. 72, 3439–3444 (2012).
52. Poehlein, C. H., Haley, D. P., Walker, E. B. & Fox, B. A. Depletion of tumor-induced Treg prior to reconstitution rescues enhanced priming of tumor-specific, therapeutic effector T cells in lymphopenic hosts. Eur. J.
Immunol. 39, 3121–3133 (2009).
53. Ko, J. S. et al. Direct and Differential Suppression of Myeloid-Derived Suppressor Cell Subsets by Sunitinib Is Compartmentally Constrained. Cancer Res. 70, 3526–3536 (2010).
54. Draghiciu, O., Lubbers, J., Nijman, H. W. & Daemen, T. Myeloid derived suppressor cells—An overview of combat strategies to increase immunotherapy efficacy. Oncoimmunology 4, e954829 (2015).
55. Ozao-choy, J. et al. The Novel Role of Tyrosine Kinase Inhibitor in the Reversal of Immune Suppression and Modulation of Tumor Microenvironment for Immune-Based Cancer Therapies The Novel Role of Tyrosine Kinase Inhibitor in the Reversal of Immune Suppression and Modulation o. Cancer Res. 69, 2514–2522 (2009).
56. Walczak, M. et al. Role of regulatory T-cells in immunization strategies involving a recombinant alphavirus vector system. Antivir. Ther. 16, 207–18 (2011).
57. Draghiciu, O., Nijman, H. W., Hoogeboom, B. N., Meijerhof, T. & Daemen, T. Sunitinib depletes myeloid- derived suppressor cells and synergizes with a cancer vaccine to enhance antigen-specific immune responses and tumor eradication. Oncoimmunology 4, e989764 (2015).
58. Topalian, S. L., Drake, C. G. & Pardoll, D. M. Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy. Cancer Cell 27, 450–461 (2015).
59. Parry, R. V. et al. CTLA-4 and PD-1 Receptors Inhibit T-Cell Activation by Distinct Mechanisms. Mol. Cell. Biol. 25, 9543–9553 (2005).
60. Victor, C. T.-S. et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature (2015).
61. Ji, R.-R. et al. An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer
Immunol. Immunother. 61, 1019–31 (2012).
62. Rizvi, N. A. et al. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science (80-. ). 348, 124–128 (2015).
63. Hamid, O. et al. A prospective phase II trial exploring the association between tumor microenvironment biomarkers and clinical activity of ipilimumab in advanced melanoma. J. Transl. Med. 9, 204 (2011). 64. Gubin, M. M. et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.
Nature 515, 577–581 (2014).
65. Kis, E. E., Winter, G. & Myschik, J. Devices for intradermal vaccination. Vaccine 30, 523–538 (2012).
66. Chen, W. & Gluud, C. Vaccines for preventing hepatitis B in health-care workers. in Cochrane Database of
Systematic Reviews (ed. Chen, W.) (John Wiley & Sons, Ltd, 2005).
67. Sangaré, L., Manhart, L., Zehrung, D. & Wang, C. C. Intradermal hepatitis B vaccination: A systematic review and meta-analysis. Vaccine 27, 1777–1786 (2009).
68. Kim, Y. C., Jarrahian, C., Zehrung, D., Mitragotri, S. & Prausnitz, M. R. Delivery Systems for Intradermal Vaccination. in 77–112 (2011).
69. Chen, D., Bowersock, T., Weeratna, R. & Yeoh, T. Current opportunities and challenges in intradermal vaccination. Ther. Deliv. 6, 1101–1108 (2015).
70. Trimble, C. L. et al. A phase I trial of a human papillomavirus DNA vaccine for HPV16+ cervical intraepithelial neoplasia 2/3. Clin. Cancer Res. 15, 361–7 (2009).
1
71. Trimble, C. L. et al. Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial. Lancet (London, England) (2015).
72. Geall, A. J. et al. Nonviral delivery of self-amplifying RNA vaccines. Proc. Natl. Acad. Sci. U. S. A. 109, 14604–9 (2012).
73. Oosterhuis, K., van den Berg, J. H., Schumacher, T. N. & Haanen, J. B. A. G. DNA Vaccines and Intradermal Vaccination by DNA Tattooing. in 221–250 (2010).
74. Oosterhuis, K., Aleyd, E., Vrijland, K., Schumacher, T. N. & Haanen, J. B. Rational Design of DNA Vaccines for the Induction of Human Papillomavirus Type 16 E6- and E7-Specific Cytotoxic T-Cell Responses. Hum.
Gene Ther. 1312, 121031083217002 (2012).
75. van de Wall, S. et al. Tattoo Delivery of a Semliki Forest Virus-Based Vaccine Encoding Human Papillomavirus E6 and E7. Vaccines 3, 221–238 (2015).
76. Galon, J., Angell, H., Bedognetti, D. & Marincola, F. The Continuum of Cancer Immunosurveillance: Prognostic, Predictive, and Mechanistic Signatures. Immunity 39, 11–26 (2013).
77. Angell, H. & Galon, J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer. Curr. Opin. Immunol. 25, 261–267 (2013).
78. Galon, J. et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313, 1960–4 (2006).
79. Ascierto, P. A. et al. The additional facet of immunoscore: immunoprofiling as a possible predictive tool for cancer treatment. J. Transl. Med. 11, 54 (2013).
80. Piersma, S. J. et al. High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer. Cancer Res. 67, 354–61 (2007).
81. Karim, R. et al. Tumor-expressed B7-H1 and B7-DC in relation to PD-1+ T-cell infiltration and survival of patients with cervical carcinoma. Clin. Cancer Res. 15, 6341–7 (2009).
82. Shah, W. et al. A reversed CD4/CD8 ratio of tumor-infiltrating lymphocytes and a high percentage of CD4+FOXP3+ regulatory T cells are significantly associated with clinical outcome in squamous cell carcinoma of the cervix. Cell. Mol. Immunol. 8, 59–66 (2010).
83. Nedergaard, B. S., Ladekarl, M., Thomsen, H. F., Nyengaard, J. R. & Nielsen, K. Low density of CD3+, CD4+ and CD8+ cells is associated with increased risk of relapse in squamous cell cervical cancer. Br. J. Cancer 97, 1135–8 (2007).
84. Punt, S. et al. FoxP3(+) and IL-17(+) cells are correlated with improved prognosis in cervical adenocarcinoma.
Cancer Immunol. Immunother. 745–753 (2015).
85. Tavare, R. et al. An effective immuno-PET imaging method to monitor CD8-dependent responses to immunotherapy. Cancer Res. (2015).
86. McCracken, M. N. et al. Noninvasive detection of tumor-infiltrating T cells by PET reporter imaging. J. Clin.
Development and preclinical evaluation
of an alphavirus therapeutic cancer vaccine
against cervical cancer
Chapter 2
Stephanie van de Wall1, Annemarie Boerma1, Joke Regts,
Baukje-Nynke Hoogeboom1, Hans W. Nijman3, Jan C. Wilschut,
Jolande Schoemaker4, Janneke J.M. Meulenberg4, Derk P. Allersma2,
Jos W.G. Kosterink2, Coba van Zanten2, Annelies Jorritsma-Smit2
and Toos Daemen1
1Department of Medical Microbiology, Tumor Virology and
Cancer Immunotherapy, University of Groningen, University Medical Center Groningen, The Netherlands
2Department of Clinical Pharmacy and Pharmacology,
University of Groningen, University Medical Center Groningen, The Netherlands
3Department of Obstetrics & Gynecology, University of Groningen,
University Medical Center Groningen, The Netherlands
2
Abstract
Currently, a multitude of immunotherapeutic strategies are being explored for clinical translation for the treatment of patients with primary or metastatic tumours. We developed a strategy against (pre)malignant cervical lesions based on recombinant Semliki Forest virus (rSFV)encoding a fusion protein of E6 and E7 from HPV type 16 (SFVeE6,7). For scalability of production of SFVeE6,7, an efficient production method was established for the clinical batch of SFVeE6,7, termed Vvax001. Vvax001 was tested for toxicity in mice and no adverse effects were observed for any of the evaluated parameters. Furthermore, robust antitumor immunity was induced in mice with eradication of established tumors. These studies warrant clinical testing of Vvax001 in patients with (pre)malignant cervical lesions or cervical cancer.
2
Introduction
Cervical cancer is the second most common cancer diagnosed in women worldwide with an estimated half a million cases per annum.1 All cases are primarily attributed to infection
with high- risk human papillomavirus (HPV).2 The three prophylactic vaccines currently on
the market, Gardasil, Gardasil-9 (Merck) and Cervarix (GlaxoSmithKline), protect against HPV types that account for approximately 70% or 90% of cervical cancer cases.3 As these
vaccines are unable to clear existing HPV infections, therapeutic strategies to treat cases of established infections and developed (pre)malignant cervical lesions are warranted.4
As HPV E6 and E7 are primary viral factors that are involved in cellular transformation and cervical cancer development, these oncoproteins serve as potential targets for therapeutic immunization.5 Various immunotherapeutic strategies are currently being
developed to induce HPV- specific T cell-mediated immune responses for clearance of (pre) malignant cervical lesions.6 The strategy we have employed is a recombinant Semliki Forest
virus (rSFV) replicon system to produce SFV particles encoding for a fusion protein of E6 and E7 from HPV type 16 (SFVeE6,7). In preclinical studies we demonstrated that SFVeE6,7 can induce potent HPV-specific cellular responses concomitant with eradication of established tumors transformed with HPV.7-9 Furthermore, SFVeE6,7 immunization elicited strong
antitumor responses in immune-tolerant HPV-transgenic mice.10 In the present study,
we describe the studies conducted with SFVeE6,7 before technology transfer to a good manufacturing practice (GMP) setting. The production process was optimized with regard to safety, scalability and translation to GMP. Furthermore, the ultimate clinical vaccine, termed Vvax001, was preclinically tested for toxicity, biodistribution and antitumor efficacy.
Materials and Methods
Plasmid construction
Three plasmids were constructed for the production of Vvax001, using pSP6-SFV4: 1) pSFV3eE6,7 encoding the replicase and E6,7 fusion protein, 2) pSFV-helper-C-S219A encoding the capsid protein and 3) pSFV-helper-S2 encoding the spike proteins. pSP6-SFV4 was constructed as previously described by P.Liljestrom and H. Garoff with the replacement of a full-length cDNA clone of SFV4 in a plasmid containing the SP6 RNA polymerase promoter to allow for in vitro transcription of full-length and infectious RNA transcripts.11 The 26S subgenomic promoter in pSP6-SFV4 was replaced with a polylinker
sequence for insertional cloning of cDNA sequences under the 26S promoter resulting in the so-called pSFV3 vector. This pSFV3 vector was used as backbone for the insertion of HPV16 E6 and E7 fusion gene in the polylinker region. The E6 and E7 genes were obtained
2
and the stop codon of E6 changed from TAA to GAA. In front of the E6,7 fusion protein, a translational enhancer (“e”) derived from the SFV capsid protein is encoded. A sequence encoding for the autoprotease 2A of foot-and-mouth disease virus is inserted directly behind the enhancer for cleavage of the enhancer from the E6,7 fusion protein.7 The
pSP6-SFV4 was also modified for the helper plasmids to encode the SFV capsid protein on one plasmid and the envelope genes p62 on the other.13 pSP6-SFV4, pSFV-helper-C-S219A and
pSFV-helper-S2 were kind gifts of P.Liljestrom and C.Smerdou. High Quality grade plasmid DNA was manufactured by PlasmidFactory (Bielefeld, Germany) and genetic stability was assessed by sequence analysis. Maps of the plasmids are provided in Figure 2.1.
Cell lines
Vero cells (green monkey kidney cells) were inlicensed from Intravacc (Bilthoven, The Netherlands). Ampules of a fully characterized and QC tested Vero cell line Master Working Cell Bank were obtained from Intravacc. Baby hamster kidney cells (BHK-21) were purchased in 1996 from the American Type Culture Collection (# CCL-10). The TC-1 cell line, a kind gift from Prof. C. Melief (Leiden University Medical Center (LUMC), Leiden, The Netherlands), was generated from C57BL/6 primary lung epithelial cells with a retroviral vector and expressing human papillomavirus 16 (HPV16) E6E7. The C3 cell line, received in 1998 from Dr Mariet Feltkamp and Prof. dr. Jan ter Schegget (LUMC), expresses the complete genome of HPV16. The cell lines were tested for mycoplasma before freezing and authenticated by morphology and growth characteristics. The cell lines were cultured as described before.9 The growth kinetics of the cell lines were recorded and validated at
least twice per week.
Pre-GMP development of Vvax001
The development of Vvax001 included different conditions tested at laboratory scale for toxicity, scalability and translation to GMP for production of the clinical batch. The development process included a switch from a one helper system to a two helper system. The one helper system (development batch) was produced on BHK-21 cells and purified by sucrose gradient whereas the two helper system was selected for production of the clinical batch by electroporation of Vero cells and purification using chromatography. The conditions tested during electroporation included different voltages, cell number, pulse length, number of pulses and total amount of RNA. Other conditions tested for large-scale production of Vvax001 included the fetal bovine serum (FBS) percentage and number of cells plated after electroporation.
2
pSFV-Helper-S2 7543 bp Subgenomic promoter SpeI FMDV 2A Enhancer A pSFV-helper C-S219A 5504 bp SpeI B C pSFV3eE6,7 11,981 bp FMDV 2 A Enhancer SpeI2
Production of development batch Vvax001
The efficacy of clinical batch produced at the GMP unit (two helper system) was compared to that of the development batch (one helper system). The production, purification and titer determination of the development batch were performed as previously described.7,14
In brief, for the development batch, BHK-21 cells were co-electroporated with RNA derived from two plasmids; one encoding the replicase and E6,7 fusion protein and one encoding for both the capsid and envelope spike proteins (helper RNA). The recombinant SFV replicon particles produced by the transfected cells were purified on a discontinuous sucrose density gradient and further titrated using an infectivity assay: serial dilutions of recombinant SFV were added to BHK-21 cells and a polyclonal rabbit anti-replicase (nsP3) antibody [a kind gift from Dr. T. Ahola (Biocentre Viiki, Helsinki, Finland)], was used to stain infected cells by immunohistochemistry. Before use, the SFV particles of the one helper system were activated with α-chymotrypsin (Sigma Chemical Co., St. Louis, MO, USA) to cleave the mutated p62 spike protein after which the enzyme was inactivated with the addition of aprotinin (Sigma).
Toxicology and Biodistribution
The toxicity study was performed with a nonclinical batch of Vvax001 produced at the Unit Biotech & ATMPs using the two-helper system in Vero cells essentially using the same process as the clinical batch. Four groups of 10 female C57BL/6 mice were immunized intramuscularly on four occasions (day 1, 15, 29 and 43) at 2-week intervals and monitored for 13 weeks (day 90) in a toxicity study performed by Huntingdon Life Sciences (Cambridgeshire, UK). Group 1 received the vehicle (formulation buffer), group 2 received 5 x 105 Vvax001 infectious particles (IP), group 3 received 5 x 107 Vvax001 IP and group 4
received 5 x 108 Vvax001 IP. Group 4 mice were sacrificed on day 16 or 17. Seven mice of
Group 1 were also analyzed at an early time point (day 15). The other mice of Group 1, 2, and 3 were sacrificed at day 22, 50 and 90 to investigate the kinetics and/or resolution of potential adverse effects. The parameters that were determined in the study included HPV16-E749-57 dextramer (Immudex, Copenhagen, Denmark) staining analysis, cytokine analysis, anti-viral antibody response, clinical condition, body weight, food consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, organ weight, gross pathology and histopathology investigations. The tissue distribution and persistence of Vvax001 was evaluated upon administration of a single intra-muscular injection of 5 x 108
IP of Vvax001 to female C57/BL6 mice. The control group received vehicle (formulation buffer). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis was performed on a panel of 14 selected tissues on Day 7 (6 days after the Drug Product was administered) on Day 1, 10, 28, 49 and 91 to assess the persistence of the Vvax001 RNA in these tissues.
2
Tumor inoculation
Specified pathogen-free female C57BL/6 mice were used between the age of 8 and 10 weeks. Mice were purchased from Harlan CPB (Zeist, the Netherlands) and kept according to institute’s guidelines. Mice were maintained at 12h day/night regime and fed standard laboratory chow. The Institutional Animal Care and Use Committee (IACUC) approved all experiments.
C57BL/6 mice were implanted subcutaneously (s.c.) in the neck with 2x104 TC-1 tumor
cells suspended in 0.2 mL Hank’s Balanced Salt Solution (Invitrogen). After inoculation, the mice were randomly divided among 3 different groups to ensure equal tumor size variations for all mice. Control mice were injected intramuscularly (i.m.) with PBS. The tumor volumes were assessed by measurement using a caliper twice per week using the following formula: length x width2 x 0.7854 for cylindrical tumors and diameter3 x
0.5236 for spherical tumors. If the tumor volume exceeded 1000 mm3 or if the tumor grew
through the skin, the mice were sacrificed. Immunizations with Vvax001
For comparison of immune responses elicited by the development (one helper) and the (two helper) clinical batch of Vvax001, mice were immunized at a prime-boost interval of 2 weeks with 50 µL administered i.m. (25 µL/hind leg muscle) of 5 x 106 Vvax001 IP. For
determining the anti-tumor efficacy of Vvax001, 7 mice were immunized 3 times at a one-week interval with the same dosage starting day 7 after tumor inoculation. For negative controls, 5 mice were injected i.m. with PBS of same volume.
CTL assay
TC-1 cells (as stimulators) were cultured with 50 U/ml of recombinant murine IFNγ (Peprotech, London, UK) for 48 hours (hr). Splenocytes as effector cells were isolated 11 days after the boost injection (day 14) and were co-cultured with irradiated TC-1 cells (100 grays) in a ratio of 25:1 at 5% CO2 in T25 culture flasks. After 5 days on co-culture, recombinant human IL-2 (4 U/ml) (Peprotech, London, UK) was added to the co-culture. C3 target cells were cultured 48 hr with 50 U/ml of recombinant murine IFNγ prior to harvesting the splenocytes. On day 7 the splenocytes were co- cultured with C3 cells (at different ratios) that had been labeled for 1 hr at 37°C with 51Cr (PerkinElmer, Groningen,
The Netherlands). After 4 hr of co-culture, 51Cr in the supernatant was measured with
RiaStar manual gamma counter (Packard, Meriden, CT). The percentage of cytotoxicity was calculated according to the formula: % specific release = ((experimental release- spontaneous release)/(maximal release-spontaneous release) count per minute x 100. The mean percentage is of samples analyzed in triplicate for each ratio tested.
2
Statistical analysis
Data is represented as the mean ± standard deviation (SD) and analyzed with GraphPad Prism software (La Jolle, CA). A student t-Test was performed to determine differences between two treatment groups. A log-rank (Mantel-Cox) test was used to determine the differences between two survival curves. P values of < 0.05 were considered as statistically significant.
Results
Vvax001 production at laboratory scale
Production of Vvax001 was initially optimized at the laboratory scale focusing on scalability and translation to GMP. The development work was performed at the Department of Medical Microbiology before technology transfer to Unit Biotech & ATMPs of the Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen for production of the engineering batch (nonclinical batch) of Vvax001 and GMP production of clinical grade Vvax001.
First, electroporation was optimized in Vero cells, the production cell line selected for production of clinical grade Vvax001. Initially, the conditions were tested using an SFV one helper system encoding for GFP with flow cytometry as a read-out method. Using the square wave protocol, the percentage of GFP-positive cells was assessed 18 hr after electroporation. The electroporation conditions tested include the voltage (110 vs. 160 V), pulse length (1, 2, 3, 5, 10, 15 vs. 20 ms) and number of pulses (1, 2, 3 vs. 4) (Figure 2.2A). A pulse interval of 0.1 s was used consistently throughout. The cell viability was also assessed directly after electroporation (Figure 2.2B). Using 4 pulses at 5 ms resulted in the highest percentage of GFP-positive cells, yet also resulted in a low viability (55%). The settings that generated the optimal combination of percentage of GFP-positive cells and viability were 160 V, 5 ms and 3 pulses (Figure 2.2A-B).
Subsequently, we tested whether these optimal electroporation conditions of Vero cells for the one helper system could also be applied to the production of SFV-GFP as a two helper system. The latter system was selected for production of clinical grade Vvax001in order to enhance safety of Vvax001 by encoding the capsid and envelope spike proteins on two separate plasmids (plasmid pSFV-helper-C-S219A and pSFV-helper-S2) for co-electroporation of three independent RNAs. Splitting the helper regions enhances the safety of Vvax001. The chance of recombination of three RNAs to produce replication competent virus is decreased to 10-12 compared to 10-6 for co- electroporation of two RNAs.13 Vero
cells were chosen for production of clinical grade VVax001 as it is a well-characterized cell line and used for the production of other commercial therapeutic products and vaccines, including Polio vaccine. Production in BHK-21 was compared to that in Vero cells with the
2
optimal temperature and time for harvesting virus after transfection determined. In BHK21, a higher number of IP was produced up to 48 hr after transfection with a substantially lower number at 72 hr after transfection (Figure 2.3A). In addition, at 48 hr after transfection, a higher number of IP is observed at 30oC compared to 37oC for both BHK-21 (Figure 2.3A)
and Vero cells (Figure 2.3B), despite the viral titer being 10x lower in Vero cells compared to BHK-21 cells. Hence, 30oC was used for all subsequent experiments. With a ratio of
1.5:1:1 for pSFVeGFP, pSFV-helper-C- S219A and pSFV-helper-S2-derived RNA, respectively, different electroporation conditions were tested for the two helper system in Vero cells (indicated in Figure 2.4). The best conditions tested initially were 110V, 5 ms and 4 pulses for harvest at 24 h or 160V, 3 ms and 2 pulses for harvest at 48 h (Figure 2.4A).
A B 1 2 3 4 1 2 3 4 0 20 40 60 80 5 ms 1 ms 2 ms 3 ms 10 ms 20 ms 15 ms 110 V 160 V Number of pulses % G FP + ce lls 1 2 3 4 1 2 3 4 0 20 40 60 80 100 5 ms 1 ms 2 ms 3 ms 10 ms 20 ms 15 ms 110 V 160 V Number of pulses % vi ab ilit y Lorem ipsum Lorem ipsum
Figure 2.2. Optimizing the upstream recombinant SFV production process in Vero cells using the SFVeGFP one
helper system. Vero cells were resuspended in 0.2 ml electroporation buffer at 2x106 cells per 0.2 cm cuvette
2
A B 24 48 72 0.0 0.5 1.0 1.5 30°C 37°CTime point of harvest (hr)
Vi ra l ti te r ( 10 9/m L) 24 48 0.0 0.5 1.0 1.5 2.0 2.5 30°C 37°C
Time point of harvest (hr)
Vi ra l ti te r ( 10 8/m L)
Figure 2.3. Optimizing the upstream recombinant SFV production process based on temperature using the SFVeGFP one helper system. BHK-21 cells were transfected using the exponential decay protocol at 420 V, 5 ms pulse length, 4 pulses and 0.1 pulse interval (A). Vero cells were transfected using the square save protocol at 110
V, 5 ms pulse length, 4 pulses and 0.1 pulse interval (B). Transfected cells were incubated at either 30 0C or 37 0C.
Supernatant was harvested 24, 48 or 72 hr later for titer determination.
As the viral titer with the above optimal conditions was still too low for optimal large-scale production, other electroporation conditions for production of SFV-GFP two helper viral particles in Vero cells were tested. The parameters tested included the total amount of RNA/electroporation, percentage of FBS during transfection, cell number/ electroporation at varying voltages and the number of cells plated after electroporation. Increasing the total amount of RNA had no effect on the number of IP/cell (data not shown) but as expected, increasing the number of cells/electroporation increased the number of IP/mL (Figure 2.4B). Furthermore, different percentages of FBS during transfection were tested. For GMP-compliant production of IP, it is desired to minimize the use of animal-derived materials in production to minimize the risk for Transmissible Spongiform Encephalopathy (TSE) and adventitious agents. Furthermore, as FBS proteins are process-related impurities, it is desired to keep their concentration as low as possible. No FBS versus 1, 2, 5 and 10% FBS was assessed. Adding no FBS to Vero cells in culture after electroporation resulted in a very low number of IP produced whereas 5% of FBS resulted in the highest number produced (Figure 2.4C). Other efforts to ensure optimized conditions to achieve high titers of recombinant virus included further increasing the number of cells, accommodated with a larger cuvette size (0.4 cm vs. 0.2 cm) while varying
2
the voltage. At 8x106 cells/electroporation, the conditions of 195V at 5 ms, 220 V at 3 ms
and 245V at 5 ms were tested and cells seeded at a density of 2 x 106 and 6 x 106 after
electroporation was also tested. 220V at 5 ms resulted in lower titer/ml and number of IP/ cell, compared to 195V at 5 ms (data not shown). 6x106 cells seeded in T25 flasks resulted
in a slightly higher number of IP/cell added from two 24 h incubation periods, yet a substantially higher titer/ml compared to 2x106 cells/plate (Figure 2.4D-E). Furthermore,
at 220 V at 5 ms the highest viral yields were obtained. A 24 48 72 0.0 0.5 1.0 1.5 2.0 2.5 160 V, 3 ms, 2 pulses 160 V, 3 ms, 3 pulses 160 V, 3 ms, 4 pulses 110 V, 5 ms, 4 pulses 110 V, 10 ms, 4 pulses n.d. n.d.
Time point of harvest (hr)
V ira l t ite r ( 10 7/m L) 2x106 6x106 0 10 20 30 1x RNA 2x RNA 3x RNA n.d. # of cells/electroporation B C 0 1 2 5 10 0 50 100 150 # of IP /c el l % serum 195 220 245 0 50 100 150 Volts (V) 195 220 245 0.0 0.5 1.0 1.5 2x106 6x106 D E V ira l t ite r ( 10 8/m L) 2x106 6x106 # of IP /c el l V ira l t ite r ( 10 7/m L) Volts (V)
Figure 2.4. Optimizing the upstream recombinant SFV production process using the SFVeGFP two helper system. Vero cells were transfected using the square save protocol with electroporation conditions tested for voltage, pulse length and number of pulses (A). Supernatant was harvested 24, 48 and 72 hr after transfection (A). The viral titers at 48 hr and 72 hr post electroporation represent the release of virus between 24-48 hr and 48-72 hr, respectively. As viral titers exponentially decrease at 72 hr, supernatant was collected at 24 and 48 hr for subsequent testing of other conditions. The viral titer or number of IP/cell indicated represents the additive result at 24 and 48 hr after electroporation. The electroporation conditions applied to Figure B-E include 5 ms, 4
2
To ensure that viral IP would be produced at a high titer with further upscaling, the cell number/electroporation and the number of cells plated out after electroporation were further increased. As it was evident that increasing the number of either parameter resulted in a higher titer, the subsequent conditions aimed to further optimize the number of IP produced per cell. Minimal differences in number of IP/cell were observed between 2 x 106
cells and 5.4-6 x 106 cells plated after electroporation at either 8x106 cells/electroporation
or 16 x 106 cells/electroporation (Figure 2.5A-B). Yet, at higher numbers of cells plated after
electroporation, 196V and 5ms resulted in a higher IP number/cell compared to 220V and 3 ms (Figure 2.5B). Although there was only a slight decrease in the number of viral IP/ cell with 22x106 cells plated in a T75 flask compared to 7.2-8x106 cells plated in a T25 flask
after electroporation, 22x106 cells plated after electroporation resulted in a factor 10 higher
concentration of viral IP (data not shown). Hence, this condition was considered optimal for large-scale production. The final optimized conditions for the production of Vvax001 include a temperature of 30oC, electroporation of in total 63 ug of RNA (ratio of RNA
transcribed from pSFV3eE6,7:pSFV-helper-S2:pSFV-helper-C-S219A is 11:5:4, respectively), to 14-18 x 106 cells in a 0.4 cm gap cuvette, 195V, 5ms, 4 pulses at 0.1s interval and plating
of 22 x 106 cells in a T75 flask after electroporation with harvest time of 24 and 48 hr.
195 V, 5 ms 220 V, 3 ms 0 50 100 150 200 2x106 5,4-6x106 # of IP /c el l 195 V, 5 ms 220 V, 3 ms 0 50 100 150 200 2x106 5,4-6x106 7,2-8x106 22x106 n.d. A B # of IP /c el l
Figure 2.5. Final testing of conditions for up-scaling of Vvax001 at 8x106 cells/electroporation (A) and 16x106/ electroporation (B). The most optimal electroporation conditions, as determined in previous experiments (195V at 5 ms or 220V at 3 ms), was used to test the effect of plating out different numbers of cells after electroporation
2
Preclinical toxicology and biodistribution study in mice
The toxicity study was performed using a nonclinical batch of Vvax001 produced at the Unit Biotech & ATMPs according to the same process as intended for production of clinical grade Vvax001. In a 13- week toxicity study in C57/BL6 female mice intramuscular injection of four consecutive doses of Vvax001 at 5 x 105 IP/animal and 5 x 107 IP/animal and a single
dose at 5x108 IP/animal was well tolerated. Findings were limited to transient clinical
signs and macroscopic and microscopic changes in the lumbar lymph nodes that were considered to be related to the expected stimulative effect of the vaccine on the immune system. Minor differences were observed in plasma blood chemistry, but these were of no toxicological significance. No effects on cytokine analysis (TNFα, IFNγ), bodyweight, food consumption, opththalmoscopy, hematology or organ weights. MHC Dextramer staining for HPV16-E749-57 positive cells confirmed the presence of E749-57 specific T-cells in all treated animals. The highest percentage was observed on day 22 and averaging at 1% for all mice immunized irrespective of dose. No Vvax001 related effects of toxicological significance or adverse events were observed in any of the treated animals. All immunized animals (groups 2 &3) exhibited a significant increase in anti-SFV antibodies at day 50 after immunization compared to pre-treatment. In a biodistribution/persistence study with a single administration of Vvax001 to female C57/BL6 mice at 5 x 108 IP/animal, the Vvax001
RNA expressing the tumor antigens E6 and E7 of HPV16 was persistent at quantifiable levels at the injection site up to day 10, but was below the limit of quantification (using a reverse transcriptase-quantitative PCR method) on Day 28, 49 and 91. Investigation of the tissue distribution showed that quantifiable levels of the Vvax001 RNA were detected also in the right inguinal lymph node for one animal on Day 7 and remained detectable in several animals until Day 10. All other tissues examined were below the limit of quantification. Altogether, it can be concluded that upon intramuscular injection the majority of Vvax001 RNA is found at the injection site, and Vvax001 RNA is rapidly cleared from all tissues, including the injected muscle.
HPV-specific anti-tumor immune responses of Vvax001 in mice
To compare the immunogenicity of the subsequently produced clinical batch of Vvax001 with a development batch, mice were immunized twice by i.m. injection with 5 x 106 IP
Vvax001 at a 2-week interval. The frequency of E7-specific CD8+ T cells was analyzed by HPV16-E749-57 dextramer analysis in splenocytes 7 days after co-culture with irradiated TC-1 cells demonstrating that CTL induced in vivo expanded to the same extend for both batches (Figure 2.6A). These CTLs exerted significantly high cytotoxic activity in vitro as demonstrated by the standard 51Cr-release assay with C3 cells as the target cells (Figure
2
tumor-free by day 89 after tumor inoculation indicating potent therapeutic antitumor responses for the viral vectors tested (Figure 2.6C).
30:1 10:1 3:1 1:1 0 20 40 60 80 100 PBS Development batch Clinical batch E:T ratio % C yt ot ox ic ity PBS Deve lopme nt ba tch Clini cal b atch 0 20 40 60 80 % E 7-sp ec ifi c C D 8 + T c el ls 0 20 40 60 80 0 200 400 600 800 1000 0/5
Days after tumor inoculation
Tu m or v ol um e (m m 3) 0 20 40 60 80 0 200 400 600 800 1000 6/7
Days after tumor inoculation # Tu m or v ol um e (m m 3) 0 20 40 60 80 0 200 400 600 800 1000 6/7
Days after tumor inoculation # Tu m or v ol um e (m m 3)
PBS Clinical batch Development batch
A
B
C
Figure 2.6. Vvax001 demonstrates high cytotoxicity and potent anti-tumor efficacy. Mice were immunized i.m.
with 5x106 IP of Vvax001 development batch or clinical batch and boosted 14 days later. Ten days after the
second immunization, mice were sacrificed and spleens were isolated for an in vitro 7-day restimulation. At the end of culture, splenocytes were analyzed for cytolytic activity in a CTL assay (A) and stained with E7-specific MHC class I dextramers and anti-CD8 antibodies for flow cytometric analysis of antigen-specific T cells (B). The mean ± SEM of each group is represented (A-B). For assessment of anti-tumor efficacy, mice were inoculated with
2 x 104 TC-1 cells on days 0 and immunized with 5x106 IP of Vvax001 development batch or clinical batch on days
7, 14 and 21 post tumor inoculation. The tumor growth was monitored for 89 days after inoculation. Mice were
sacrificed for ethical reasons once the tumor size reached 1000 mm3 or when the tumor grew through the skin
