Self-amplifying RNA virus vectors: clinical applications in cancer drug delivery

KEYWORDS:

• Self-amplifying RNA virus
• RNA replicon
• cancer therapy
• vaccines

1. Introduction

Self-amplifying RNA viruses share the common features of single-stranded RNA surrounded by a capsid structure and a protein envelope [1]. However, alphaviruses and flaviviruses possess a genome of positive polarity, while measles viruses (MV) and rhabdoviruses contain a negative-sense RNA genome. Moreover, all self-amplifying RNA viruses carry in their genome the nonstructural genes (RNA replicon) responsible for extreme RNA replication in infected host cells, providing the potential for high-level expression of recombinant proteins from engineered expression vectors [2]. Expression vectors have been developed for transfection of mammalian and non-mammalian cell lines, transfection of primary cells such as neuronal cells, and delivery in vivo [3]. Alphaviruses have demonstrated great flexibility as expression vectors can be delivered as recombinant viral particles (in the form of replication-proficient, replication-deficient, and liposomal nanoparticles), RNA replicons, and DNA/RNA layered plasmids. In the context of therapeutic and prophylactic applications, self-amplifying RNA virus vectors have been utilized for vaccine development and gene therapy in various areas such as infectious diseases and different cancers. In many cases, immunization with self-amplifying RNA virus vectors has provided protection against challenges with lethal doses of infectious agents and tumor cells in several animal models [1]. Moreover, tumor regression and prolonged survival have been established in rodents after administration of self-amplifying RNA viral vectors. In this Editorial the focus will be on cancer therapy. It is important to point out that both prophylactic and therapeutic approaches are presented.

2. Preclinical applications for cancer therapy

A large number of preclinical studies have been conducted with self-amplifying RNA virus vectors in animal tumor models as listed by examples in Table 1. For example, an MV vector was constructed with the CD46 and the signaling lymphocyte activation molecule (SLAM) incorporated in the hemagglutinin (HA) protein in combination with the display of a single-chain antibody against the epidermal growth factor receptor (EGFR) at the HA C-terminus. It was shown that intratumoral MV particle administration generated potential antitumor activity in glioblastoma cell lines and therapeutic efficacy by providing tumor regression and significantly prolonged survival in mice with glioblastoma xenografts [4]. Neuronal targeting of micro-RNA miRT124 sequences introduced into an oncolytic Semliki Forest virus (SFV) strain demonstrated virus replication in tumors, therapeutic activity by significant inhibition of tumor growth, and improved survival of C57BL/6 mice carrying CT-2A orthotopic gliomas after a single intraperitoneal injection [5]. In the context of RNA-based delivery, a single intramuscular administration of 0.1 µg SFV-LacZ RNA elicited antigen-specific antibody and CD8⁺ T cell responses and protected immunized mice from challenges with colon CT26.CL25 tumor cells [6]. In addition to prophylactic activity, therapeutic immunization demonstrated prolonged survival of mice with established tumors. Related to breast cancer, immunization of mice with dendritic cells (DCs) transduced with Venezuelan equine encephalitis virus (VEE) replicon particles expressing a truncated form of the neu oncogene generated robust neu-specific CD8⁺ T cell and anti-neu IgG responses and therapeutic regression of large established tumors [7].

Table 1. Examples of pre-clinical studies using self-amplifying RNA viral vectors.

Cancer	Target/Antigen	Vector	Response	Ref
Brain	SLAM, EGFR miRT124	MV particles SFV VLPs	Tumor regression Tumor growth inhibition, prolonged survival	[45]
Breast	Truncated neu	VEE VLPs	Tumor regression	[7]
Cervical	HPV16 E7 HPV E6,7	VEE VLPs SFV VLPs + I + Sun	Tumor prevention Tumor-free mice	[89]
Colon	LacZ	SFV RNA	Protection against tumor challenges	[6]
Melanoma	VEGFR2-IL-12 + Surv-βhCG	SFV DNA	Prolonged survival	[10]
Prostate	VSV	VSV-GP	Prolonged survival	[11]

Abbreviation: EGFR, epidermal growth factor receptor; HPV, human papilloma virus; I, irradiation; IL-12, Interleukin-12; MV, measles virus; SFV, Semliki Forest virus; SLAM, signaling lymphocyte activation molecule; Sun, sunitib; Surv, survivin; VEE, Venezuelan equine encephalitis virus; VEGFR2, vascular endothelial growth factor receptor-2; VLPs, virus-like particles; VSV, vesicular stomatitis virus

Virus-based cervical cancer has been a promising target for prophylactic vaccine development illustrated by immunization of mice with VEE particles expressing the human papilloma virus-16 (HPV16) E7 protein resulting in class I-restricted CD8⁺ T cell responses and prevention of tumor development [8]. Therapeutic elimination of established tumors was observed in 67% of tumor-bearing mice. Combination therapy with SFV-HPV E6,7, 40 mg/kg sunitib, and low-dose (14 Gy) irradiation dramatically changed the intratumoral compartment, resulting in enhanced immunotherapeutic antitumor activity, inhibition of tumor growth, and 100% tumor-free survival in mice with implanted xenografts [9]. In another combination therapy, co-immunization of two SFV DNA vectors expressing vascular endothelial growth factor-2 (VEGFR2) and interleukin-12 (IL-12), and survivin and β-hCG antigens, respectively, elicited humoral and cellular immune responses against VEGFR2, survivin, and β-HCG [10]. The combined DNA vaccine provided therapeutic activity by superior inhibition of tumor growth and prolonged survival in a B16 melanoma mouse model compared to immunization with individual SFV DNA vectors. Moreover, therapeutic efficacy was observed after intratumoral administration of an oncolytic pseudotyped vesicular stomatitis virus (VSV-GP), which generated long-term remission in murine prostate cancer models, also in subcutaneous tumors and bone metastases after intravenous administration [11]. Additional studies have confirmed therapeutic tumor regression in lung (MV particles and SFV VLPs), ovarian (oncolytic VSV particles and Sindbis virus VLPs), and pancreatic cancers [1].

3. Clinical applications for cancer therapy

Although not to the same extent as recorded for adenovirus vectors, self-amplifying RNA virus vectors have been subjected to clinical trials. Related to cancers several clinical trials have been conducted with self-amplifying RNA virus vectors (Table 2). For instance, VEE particles expressing the carcinoembryonic antigen (CEA) were intramuscularly injected four times at doses of 4 × 10⁷ to 4 × 10⁸ IU every 3 weeks in patients with advanced pancreatic cancer in a Phase I trial [12]. Clinically relevant CEA-specific T cell and antibody responses were elicited after repeated immunization. The antibody-dependent cellular toxicity against human colorectal cancer cells was mediated by CEA-specific antibodies and patients showed therapeutic relevance as prolonged overall survival. In the context of castration resistant metastatic prostate cancer (CRPC), five doses of either 0.9 × 10⁷ or 3.6 × 10⁷ IU of propagation-defective VEE particles expressing the prostate-specific membrane antigen (PSMA) were administered to patients with CRPC metastatic to bone in a Phase I trial [13]. Both doses were well tolerated, but only weak PSMA-specific signals were detected. As neither clinical benefit nor robust immune responses were achieved it is thought that the dosing was suboptimal. In a protocol for recurrent glioblastoma multiforme, patients were planned to be treated with MV particles expressing CEA at a starting dose of 1 × 10⁵ TCID₅₀ escalating to the maximum dose level of 2 × 10⁷ TCID₅₀ [14]. So far, three patients have been injected with 1 × 10⁵ TCID₅₀ and three other individuals received 1 × 10⁶ TCID₅₀ in the resection cavity, which generated no dose-limiting toxicity. MV-CEA was subjected to intraperitoneal injections in a Phase I trial in patients with advanced ovarian cancer at doses of 10³ to 10⁹ TCID₅₀, which confirmed no dose-limiting toxicity [15]. Stable disease was observed in 14 patients with a median duration of 88 days and a range of 55 to 277 days. The higher doses (10⁷–10⁹ TCID₅₀) provided stable disease in all patients, while stable disease was achieved in only 5 of 12 patients after vaccination with lower doses (10³–10⁶ TCID₅₀). Oncolytic MV particles expressing the human sodium iodide symporter (NIS) were subjected to a Phase I trial in patients with relapsed refractory myeloma [16]. As the maximum tolerated dose (MTD) was not achieved in a dose-escalation study (1 × 10⁶ to 1 × 10⁹ TCID₅₀), doses of 1 × 10¹⁰ and 1 × 10¹¹ TCID₅₀ were applied showing a complete response in one patient with the higher dose. The response persisted for nine months and the isolated occurring relapse was treated with irradiation, which kept the patient disease-free for an additional 19 months. In another patient, MV-NIS treatment resulted in subjective softening and shrinking of extramedullary plasmacytomas in the back and thighs. The MV Edmonston strain was subjected to a Phase I open-label, non-randomized dose-escalation study in patients with cutaneous T cell lymphoma [17]. Intratumoral administration in combination with subcutaneous injection of interferon-α (IFN-α) resulted in complete regression of CTCL lesions in one patient and in partial regression in other patients. In an approach of tumor targeting and protection against recognition by the host immune system, SFV particles expressing interleukin-12 (LipoVIL12) were encapsulated in liposomes [18]. Intravenous administration of LipoVIL12 in melanoma and kidney carcinoma patients showed up to 10-fold increase in IL-12 plasma levels in a Phase I trial after five days with an MTD of 3 × 10⁹ particles per m². The encapsulation enhanced tumor targeting, and repeated injections resulted in no toxicity related to the treatment.

Table 2. Examples of clinical trials using self-amplifying RNA viral vectors.

Cancer	Vector/Antigen	Phase	Response	Ref
CRPC	VEE-PSMA	Phase I	Neutralizing Abs, suboptimal dosing	[13]
CTCL	MV-EZ	Phase I	Regression of CTCL lesions	[17]
Glioblastoma	MV-CEA	Phase I	No dose-limiting toxicity	[14]
Kidney	LipoVIL12	Phase I	Tumor targeting, safe delivery	[18]
Melanoma	LipoVIL12	Phase I	Tumor targeting, safe delivery	[18]
Myeloma	MV-NIS	Phase I	Complete response in one patient	[16]
Ovarian	MV-CEA	Phase I	Stable disease with higher doses	[15]
Pancreatic	VEE-CEA	Phase I	CEA-specific Abs, prolonged survival	[12]

Abbreviation: Abs, antibodies; CEA, carcinoembryonic antigen; CRPC, castrate resistant metastatic prostate cancer; CTCL, cutaneous T cell lymphoma; LipoVIL12, liposome-encapsulated SFV-IL12 particles; MV, measles virus; NIS, sodium iodide symporter; PSMA, prostate specific membrane antigen; SFV, Semliki Forest virus; VEE, Venezuelan equine encephalitis virus

4. Expert opinion

Self-amplifying RNA virus vectors have been engineered for both prophylactic and therapeutic applications [1]. Prophylactic cancer prevention comprises primarily of targeting risk factors to avoid cancers such as carcinogens and tobacco smoke, which is outside the scope of application of viral vector-based vaccines. However, secondary cancer prevention aims at controling or reducing the growth of existing tumors and prevention of metastatic tumors [19]. Although promising results have also been obtained for targeting infectious diseases, the focus here is on cancer treatments. In this context, several studies in animal models have demonstrated efficient tumor regression, significant prolonged survival rates, and even cure. So far, the number of clinical trials has been fairly limited, and mainly Phase I trials have been conducted albeit showing good safety profiles. The therapeutic efficacy has not been outstanding, which may relate to the need of dose optimization and delivery modes (targeting DCs and encapsulation of viral particles). Although not targeting cancer, the encouraging results from a Phase III vaccination trial, which provided protection against EBOV challenges, bodes well for developing strategies for immunization against various cancers [20]. The flexibility of applying self-amplifying RNA viruses as recombinant particles, RNA replicons, plasmid DNA vectors, and LNPs is an additional asset. Moreover, due to the RNA replication mechanism the amounts of RNA and DNA used for immunizations can be reduced. For instance, comparison to synthetic mRNA, self-amplifying VEE RNA replicons expressing the influenza hemagglutinin (HA) required 1.25 µg (64-fold less material) to achieve protection of mice against influenza virus challenges [21]. Similarly, a 200-fold lower equimolar dose of 0.05 µg SFV DNA replicon-based cervical cancer vaccine was sufficient to generate complete tumor regression in 85% of immunized mice [22]. These findings make self-amplifying RNA viruses attractive as an alternative approach for future development of cancer drugs.

Declaration of interest

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

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Additional information

Funding

This paper was not funded.