The increase of titles related to RNA-based therapeutics and vaccines within the CIMT poster session compared to previous years, highlighted the increased focus on RNA approaches. Therefore, some details will be given in this second CIMT Blog (for more highlights of the CIMT meeting please read our previous blog CIMT 2016 "Mechanisms of efficacy in cancer immunotherapy").
Messenger RNA (mRNA) is a carrier for genetic information that specifies the amino acid sequence of the encoded protein. It is a nontoxic molecule that allows transient protein expression of a desired protein product in virtually every transducable cell type. Since RNases – enzymes that rapidly degrade RNA – can be found almost everywhere, for many years mRNAs were believed to be too unstable and that it would be too much effort to handle mRNA as an efficient therapeutic.
Recently, researchers succeeded in the replacement of several of the limiting mRNAs structures by more stabilizing ones and consequently artificial transcribed mRNAs could be produced in vitro. But how are mRNAs produced? Nowadays the in vitro synthesis of single-stranded RNA is a routine laboratory procedure: a DNA template is used by a specialized enzyme to synthesize a complementary RNA strand. The in vitro transcribed-RNA (IVT-RNA) mimics the function of naturally occurring mRNAs and if administered to cells it can be shown that mRNA is translated into the encoded protein. The protein synthesis takes place within the cytoplasm of cells that incorporate the artificial mRNA, making use of the cell’s own protein synthesis machinery. Thus actually mRNA allows patients to produce their own tailored drug product.
This simple in vitro transcribed RNA format has many advantages over for example traditional plasmid and viral-based approaches. Using traditional and simple DNA engineering methods, the in vitro mRNA today is easily customizable, moreover allowing the design of patient personalized mRNAs. This mRNA production is effortlessly scalable which offers great flexibility with respect to the manufacturing processes. The IVT-RNA application can be a fast and affordable alternative to the complex, laborious and time-consuming procedure of GMP protein production that is associated with the protein delivery process, making the time and cost of production for IVT-RNA far lower. A further advantage is that with the aim to be translated into a protein, cytosolic active mRNA-based therapeutics do not need to pass the nuclear membrane such as DNA and thus mRNA carries little to no risk of genomic integration. Hence, mRNA is considered to be a safe vector with a superior safety profile compared to DNA.
However, one of the biggest challenges of this relative new field of mRNA remains the tactics to deliver and target the mRNA to specific desired places or cell types within the body. Two CIMT posters from Heleen Dewitte and Rein Verbeke from the university of Ghent for examples addressed this field of research presenting new techniques using mRNA lipoplexes or mRNA sonoporation. mRNA delivery to immune cells has been done in various ways. The remarkable amount of papers found for the last five years within this relatively new class of therapies mirrors the research interest and needs within this area.
All mRNA delivery approaches aim for a protective immune response. Once administered the antigens encoding mRNA is translated and the resulting antigens are presented to host antigen-presenting cells (APC) in order to stimulate an adaptive immune response (shown in Figure 1). Unlike peptide-based vaccines, mRNA-based vaccines are not restricted by the patient’s HLA type. They are recognized by the immune system in an MHC class-independent manner. Remarkable results for this kind of immune response either in dendritic cells (DCs) or T cells were shown in various posters. For instance, Iris Bigalke from the Department of Cellular Therapy from Oslo University hospital describes that five acute myeloid leukemia (AML) patients have received four times mRNA based vaccination targeting the two antigens Wilm’s tumor-1 (WT-1) and preferentially expressed antigen in melanoma (PRAME). Maturated DCs were electroporated with the mRNA for both targets and given to patients. The vaccination was well tolerated with patients showing strong local delayed hypersensitivity test (DTH) responses in week 6 and antigen specific immune responses could be detected in patients by CD8 responses against WT-1. Lukasz Bialkowski from the “Vrije Universiteit Brussel” reported on successful E7-TriMix mRNA-vaccine induced CD8 T-cell responses that inhibited growth of mucosal located tumor lesions (i.e. subcutaneously, in the lungs and in the genital tract) in cervical cancer mouse models. In addition, it could be shown in mice that cisplatin treatment combined with mRNA immunization can reverse a suppressive tumor microenvironment and allow for antigen specific T cells migrating to and infiltrating the tumor tissues. His colleague Kevin Van der Jeught presented the delivery of intravenously administered antigen encoding mRNAs, which were packed into a lipoplex RNAiMAX to protect it from RNases, resulting in rapid translation in splenic DCs of mice and a consequent strong activation of Tcells with antigen specific lysis.
Besides encoding the information of the desired antigen, mRNA offers an additional benefit for immunotherapies. The innate immune system is naturally activated by foreign nucleic acids, thus mRNA can act as an adjuvant by providing costimulatory signals to the innate immune system, via toll-like receptors. Posters presented by Hadas Weinstein-Marom from the Sharett Institute of oncology in Jerusalem and Regina Heidenreich from CureVac AG demonstrated an induction of both the innate and cellular immune responses, immensely enhancing the anti-tumor effects. One of these immune-oncology therapies was described in the poster from Jan Dörrie (A phase I/II clinical trial on malignant melanoma with in vitro optimized mRNA-electroporated dendritic cells as therapeutic vaccine) where vaccination with in vitro transcribed mRNA molecules encoding tumor-associated antigens (TAAs) was shown to be an elegant approach to let patient’s cells produce its own desired drug and induce specific immune responses within the patient to the vaccination antigens.
The sum of these beneficial characteristics of IVT-RNA explains the growing interest in research and development of the latter. The RNA therapeutics market appears promising demonstrated by several collaborations between RNA- and pharmaceutical companies as well as by the emergence of early phase clinical trials initiated by RNA specialized companies such as CureVac or BioNTech. For the latter, in collaboration with TRON, Barbara Schrörs prepared a CIMT poster giving additional information regarding the IVAC (Individualized Vaccines Against Cancer) Mutanome approach, which is the first-in-human trial that introduces a fully personalized RNA vaccine for the treatment of malignant melanoma. In this work RNA and exome sequencing has been performed on tumor cell lines derived from one of the melanoma patients that received IVAC treatment. Aim was to show if a certain rare gene locus deletion could be driven by the obtained mRNA treatment. The tumor cell lines from melanoma patient PA018 was lacking the functional beta-2-microglobulin (B2M) locus, which represents a known tumor immune-escape mechanism. The researchers could demonstrate that this rare event – the loss of the B2M locus – already started before IVAC treatment and the increased infiltration of immune cells in the tumor tissues initiated by the IVAC mRNA treatment was not impaired. But it is stated that ongoing research on this issue shall reveal whether routine monitoring of B2M loss or equivalent events is advisable for all patients. This research is showing that mRNA treatment is just in its beginning and furthermore lots has to be learned from ongoing RNA treatments.
Together with the company Moderna, CureVac and BioNTech are three of the main players with a more advanced status of a mRNA therapeutic platform technology. Nevertheless, the CIMT congress shows that many biotech companies and academia increasingly entered the field, showing that pharmacologically optimized synthetic mRNAs have recently emerged as an attractive alternative for established therapeutic agents, hopefully holding potential for many more promising personalized immune-oncology therapies yet to come.
Selected CIMT RNA poster titles
- Bigalke; WT-1 and PRAME mRNA transfected TLR 7/8 polarized fast DCs can raise specificimmune responses in AML patients that correlate with clinical outcome
- Bialkowski; Intralymphatic mRNA vaccine induces CD8 T-cell responses that inhibit the growth of mucosally located tumors
- Dewitte; Theranostic mRNA-loaded microbubbles for ultrasound-assisted dendritic cell based cancer vaccination
- Dörrie; A phase I/II clinical trial on malignant melanoma with in vitro optimized mRNA-electroporated dendritic cells as therapeutic vaccine
- Heidenreich; RNAdjuvant®, a novel, highly-potent RNA-based adjuvant, combines strong immunostimulatory capacities with a favorable safety profile
- Jabulowsky; A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent cancer immunotherapy in patients
- Miller; IVAC® MUTANOME – a first-in-human phase I clinical trial targeting individual mutant neoantigens for the treatment of melanoma
- Ramachandran; Preclinical evaluation of triple microRNA-attenuated oncolytic Semliki forest virus in glioma and neuroblastoma
- Buonaguro; Effects of RNA-based RNAdjuvant® on PBMCs from liver cancer patients in an ex vivo model
- Van der Jeught; Type I IFN induced upon particle mediated intravenous delivery of antigen mRNA enhances specific immune responses
- Verbeke; Messenger RNA DOTAP-Cholesterol lipoplexes containing TLR agonists allow single step antigen-loading and maturation of dendritic cells
- Schrörs; Complex deletion event at B2M locus in a human melanoma patient treated with IVAC MUTANOME
- Weinstein-Marom; Enhancing the effector functions of T cells with a combination of new mRNA adjuvants for improving adoptive cell therapy