Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to the field of mRNA formulations, and in particular provides a combination of one or more mRNA molecules encoding the functional immunostimulatory proteins CD40L, CD70 and caTLR4; and one or more mRNA molecules encoding an antigen.
• the combinations of the present invention are in particular characterized in that the mRNA molecules comprise a 5' CAP-1 structure and may further contain one or more modified nucleosides, such as pseudouridines.
• the present invention also provides compositions comprising said combinations and uses thereof, in particular in vaccination, and treatment of cell proliferative disorders.
• T cell responses elicited by mRNA vaccines are governed by the complex interplay between route of administration, delivery vehicle, and the intrinsic properties of the mRNA.
• the route of administration will determine the biodistribution of mRNA at the organ and cellular level.
• intravenous immunization with mRNA complexed into nanocarriers has emerged as a particularly powerful approach to elicit high magnitude/high quality T cell responses, yet immunogenicity critically depends on the physicochemical properties of the delivery vehicle.
• the mRNA format itself is likely to have a huge impact on immunogenicity.
• in vitro transcribed (IVT) RNA can indeed be recognized by various RNA sensors. Whereas triggering of these RNA sensors elicits the necessary cytokine responses to support T cell differentiation, it also elicits antiviral signatures aimed at degrading mRNA and shutting down translation arrest. Hence, in case of mRNA vaccines, innate activation needs to be tightly balanced to enable adequate expression of the mRNA encoded antigen.
• ARCA-Capped, non-modified mRNA 2 CleanCapped, non-modified mRNA 3 CleanCapped, modified mRNA.
• ARCA-Capped mRNA possesses a so-called 5'Cap-O structure, which stabilizes the mRNA and drives translation initiation, but is recognized by several RNA sensors that drive RNA degradation and translation arrest.
• Clean-Capped mRNA instead possesses a Cap-1 structure, which renders mRNA invisible for several RNA sensors (MDA-5, RIG-I and IFIT-1 ).
• Eukaryotic mRNAs typically have various nucleoside modifications (eg pseudo-uridine, 5 methylcytidine, N1 methylspeudo-uridine) build-in that lower their interaction with Toll-like Receptors (TLRs) 3, 7 and 8.
• TLRs Toll-like Receptors
• T cell response In terms of magnitude of T cell response, no significant differences were observed between the three different mRNA formats in the absence of TriMix (antigen only). Nonetheless, upon addition of TriMix, strong differences in T cell responses emerged between mRNA formats. Whereas TriMix did not improve the magnitude of the T cell response in case of ARCA-Capped, unmodified mRNA, TriMix did strongly enhance the magnitude of the T cell response in case of CleanCapped, unmodified and CleanCapped modified mRNA. Overall, T cell responses were most elevated in case of immunization with E7: TriMix at a ratio of 5:5 and in the Cleancap non- modified and CleanCapped modified mRNA formats, with as high as 80% of all circulating CD8 T cells being specific for E7.
• the present invention provides a combination comprising:
• CD70 and caTLR4 CD70 and caTLR4;
• mRNA molecules wherein at least one of said mRNA molecules is characterized in having a 5' CAP-1 structure.
• At least 2, at least 3, or all of said mRNA molecules have a 5' CAP-1 structure.
• said mRNA molecules further comprise at least one modified nucleoside, such as selected from the list comprising pseudouridine, 5-methoxy-uridine, 5- methyl-cytidine, 2-thio-uridine, and N6-methyladenosine.
• said modified nucleoside may be a pseudouridine selected from the list comprising: 4-thio-pseudouridine, 2-thio-pseudouridine, 1-carboxymethyl- pseudouridine, 1 -propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, N1-methyl- pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1 - deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio- dihydropseudouridine, 4-methoxy- pseudouridine, and 4-methoxy-2-thio-pseudouridine.
• said at least one modified nucleoside is N1 -methyl-pseudouridine.
• uridines in said mRNA molecules is replaced by N1-methyl-pseudouridine.
• the present invention further provides a pharmaceutical composition comprising the combination as defined herein and at least one pharmaceutically acceptable agent.
• the combination as defined herein is formulated in nanoparticles, such as lipid nanoparticles or polymeric nanoparticles.
• composition as defined herein is formulated for intravenous, intranodal, intratumoral, subcutaneous, intradermal or intramuscular formulation.
• composition according to this invention may be in the form of a vaccine.
• said antigen is a cancer antigen.
• the present invention further provides a combination, or composition as defined herein for use in human or veterinary medicine; more in particular for use in vaccination and/or for use in the treatment of cell proliferative disorders.
• the present invention also provides a method for treating a cell proliferative disorder, said method comprising: administering to a subject in need thereof, a combination or composition as defined herein.
• FIG. 2 Kinetics of inflammatory responses to CleanCap/modified mRNA LNPs.
• E7 and TriMix mRNA were encapsulated at the indicated ratio's into LNPs and administrated intravenously at a total mRNA dose of 10 pg. All mRNA contained a CleanCap and a 100% uridine substitution by Nl methylpseudo-uridine. Serum cytokines were measured at 6 hours and 24 hours post injection.
• Figure 3 A) Impact of mRNA format on the magnitude of the E7-specific T cell response upon immunization with E7/TriMix mRNA LNPs at E7/TriMix ratios of 10:0 (no TriMix) and 5:5.
• Two-way ANOVA analysis followed by Tukey's multiple comparisons test ns nonsignificant; * p ⁇ 0,05 ** p ⁇ 0,01 ***p ⁇ 0,001.
• Figure 4 Magnitude of the IFN-y T cell response as assessed by ELISPOT after the 3th immunization with E7 E7/TriMix mRNA LNPs at E7/TriMix ratios of 10:0 (no TriMix) and 5:5.
• mRNA was either ARCA capped without nucleoside modifications (ARCA non-mod), CleanCapped without nucleoside modifications (CleanCap/non-mod) or CleanCap and containing Nl methylpseudo-uridine (CleanCap/mod).
• Figure 5 A) Weight of mice expressed as % of initial body weight upon injection of mice with E7 mRNA LNPs with the indicated mRNA formats. LNPs were intravenously injected at days 0, 7 and 14. B) Weight of mice expressed as % of initial body weight upon injection of mice with LNPs at the indicated E7/TriMix mRNA LNPs for respectively ARCA, non-mod mRNA, CleanCap, non-mod mRNA and CleanCap, mod mRNA.
• Figure 6 ALT/AST levels in serum of mice after the third injection with PBS or the indicated mRNA LNPs.
• Two-way ANOVA analysis followed by Tukey's multiple comparisons test ns non-significant; * p ⁇ 0,05 ** p ⁇ 0,01 ***p ⁇ 0,001.
• the present invention provides a combination comprising:
• CD70 and caTLR4 CD70 and caTLR4;
• mRNA molecules wherein at least one of said mRNA molecules is characterized in having a 5' CAP-1 structure.
• TriMix stands for a mixture of mRNA molecules encoding CD40L, CD70 and caTLR4 immunostimulatory proteins.
• the use of the combination of CD40L and caTLR4 generates mature, cytokine/chemokine secreting DCs, as has been shown for CD40 and TLR4 ligation through addition of soluble CD40L and LPS.
• the introduction of CD70 into the DCs provides a co-stimulatory signal to CD27 + naive T-cells by inhibiting activated T- cell apoptosis and by supporting T-cell proliferation.
• TLR Toll-Like Receptors
• a constitutive active form is known, and could possibly be introduced into the DCs in order to elicit a host immune response. In our view however, caTLR4 is the most potent activating molecule and is therefore preferred.
• the mRNA or DNA used or mentioned herein can either be naked mRNA or DNA, or protected mRNA or DNA. Protection of DNA or mRNA increases its stability, yet preserving the ability to use the mRNA or DNA for vaccination purposes.
• Non-limiting examples of protection of both mRNA and DNA can be: liposome-encapsulation, protamine-protection, (Cationic) Lipid Lipoplexation, lipidic, cationic or polycationic compositions, Mannosylated Lipoplexation, Bubble Liposomation, Polyethylenimine (PEI) protection, liposome-loaded microbubble protection etc..
• target-specific antigen used throughout the description is not limited to the specific examples that may be described herein. Any infectious agent such as a virus, a bacterium or a fungus may be targeted. In addition any tumor or cancer cell may be targeted.
• target-specific antigen used throughout the description is not limited to the specific examples that may be described herein. It will be clear to the skilled person that the invention is related to the induction of immunostimulation in APCs, regardless of the target-specific antigen that is presented.
• the antigen that is to be presented will depend on the type of target to which one intends to elicit an immune response in a subject.
• Typical examples of target-specific antigens are expressed or secreted markers that are specific to tumor, bacterial and fungal cells or to specific viral proteins or viral structures. Without wanting to limit the scope of protection of the invention, some examples of possible markers are listed below.
• neoplasms are not intended to be limited to the types of cancer or tumors that may have been exemplified.
• the term therefore encompasses all proliferative disorders such as neoplasma, dysplasia, premalignant or precancerous lesions, abnormal cell growths, benign tumors, malignant tumors, cancer or metastasis, wherein the cancer may be selected from the group of: leukemia, nonsmall cell lung cancer, small cell lung cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, glioma, colon cancer, bladder cancer, sarcoma, pancreatic cancer, colorectal cancer, head and neck cancer, liver cancer, bone cancer, bone marrow cancer, stomach cancer, duodenum cancer, oesophageal cancer, thyroid cancer, hematological cancer, and lymphoma.
• Specific antigens for cancer can e.g. be MelanA MART
• the mRNA or DNA molecule(s) encode(s) the CD40L and CD70 immunostimulatory proteins.
• the mRNA or DNA molecule(s) encode(s) CD40L, CD70, and caTLR4 immunostimulatory proteins.
• Said mRNA or DNA molecules encoding the immunostimulatory proteins can be part of a single mRNA or DNA molecule.
• said single mRNA or DNA molecule is capable of expressing the two or more proteins simultaneously.
• the mRNA or DNA molecules encoding the immunostimulatory proteins are separated in the single mRNA or DNA molecule by an internal ribosomal entry site (IRES) or a self-cleaving 2a peptide encoding sequence.
• IRES internal ribosomal entry site
• the mRNA used in the methods of the present invention has a 5' cap structure with a so-called CAP-1 structure (CleanCap), meaning that the 2' hydroxyl of the ribose in the penultimate nucleotide with respect to the cap nucleotide is methylated, such as illustrated below:
• two, three, four, ... or all of the used mRNA molecules of the present invention have a 5’ cap structure with a so-called CAP-1 structure.
• one or more of the mRNA molecules of the present invention may further comprise at least one modified nucleoside.
• two, three, four, ... or all of the used mRNA molecules of the present invention have at least one modified nucleoside.
• said mRNA molecules further comprise at least one modified nucleoside, such as selected from the list comprising pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine, 2-thio-uridine, and N6-methyladenosine.
• said at least one modified nucleoside may be a pseudouridine, such as selected from the list 4-thio-pseudouridine, 2-thio-pseudouridine, 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, N1 - methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1 -methyl-pseudouridine, 1-methyl- 1-deaza-pseudouridine, 2-thio-1 -methyl-1 -deaza-pseudouridine, dihydropseudouridine, 2-thio- dihydropseudouridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.
• said at least one modified nucleoside is N
• nucleoside modifications which are suitable for use within the context of the invention, include: pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5- carboxym ethyl - uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio- uridine, I- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1- methyl- pseudouridine, 2-thio- 1-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-1 -
• the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1 - methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-
• the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7- deaza-8- aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 - methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenos
• mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza- guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1 - methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8- oxo-guanosine, 7- methyl-8-oxo-guanosine, l-methyl-6-thio-guaiguanosine, and N2,N2-dimethyl-6-thio- guanosine.
• nucleoside selected from the group consisting
• the mRNA molecules used in the present invention may contain one or more modified nucleotides, in particular embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of a particular type of nucleotides may be replaced by a modified one. It is also not excluded that different nucleotide modifications are included within the same mRNA molecule. In a very specific embodiment of the present invention, about 100% of uridines in said mRNA molecules is replaced by N1-methyl-pseudouridine.
• the present invention further provides a pharmaceutical composition comprising the combination as defined herein and at least one pharmaceutically acceptable agent.
• one or more of said mRNA molecules of the present invention may further contain a translation enhancer and/or a nuclear retention element. Suitable translation enhancers and nuclear retention elements are those described in WO2015071295.
• said one or more mRNA molecules are formulated for parenteral administration; more in particular for intravenous, intranodal, intratumoral, subcutaneous, intradermal or intramuscular formulation.
• said mRNA molecules are formulated for intranodal or intratumoral administration, and are in the form of naked mRNA molecules in a suitable injection buffer, such as a Ringer Lactate buffer.
• the present invention also provides a combination or composition as defined herein; wherein one or more of said mRNA molecules are encompassed in nanoparticles.
• nanoparticle refers to any particle having a diameter making the particle suitable for systemic, in particular intravenous administration, of, in particular, nucleic acids, typically having a diameter of less than 1000 nanometers (nm).
• the nanoparticles are selected from the list comprising: lipid nanoparticles and polymeric nanoparticles.
• a lipid nanoparticle is generally known as a nanosized particle composed of a combination of different lipids. While many different types of lipids may be included in such LNP, the LNP's of the present invention may for example be composed of a combination of an ionisable lipid, a phospholipid, a sterol and a PEG lipid.
• a polymeric nanoparticle can typically be a nanosphere or a nanocapsule.
• Two main strategies are used for the preparation of polymeric nanoparticles, i.e. the“top-down” approach and the “bottom-up” approach.
• the“top-down” approach a dispersion of preformed polymers produces polymeric nanoparticles
• the bottom-up approach polymerization of monomers leads to the formation of polymeric nanoparticles.
• top-down and bottom-up methods use synthetic polymers/monomers like poly(d, l-lactide-co-glycolide), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), and poly(isohexyl cyanoacrylate); stabilizers like poly(vinyl alcohol) and didecyldimethylammonium bromide; and organic solvents like dichloromethane and ethyl acetate, benzyl alcohol, cyclohexane, acetonitrile, acetone, and so on.
• Recently the scientific community has been trying to find alternatives for synthetic polymers by using natural polymers and synthesis methods with less toxic solvents.
• the present invention also provides the combinations and vaccines as defined herein for use in human or veterinary medicine, in particular for use in the treatment of cell proliferative disorders, more in particular for use in eliciting an immune response towards a tumor in a subject.
• the present invention provides a method for the treatment of a cell proliferative disorder comprising the steps of administering to a subject in need thereof a combination or vaccine of the present invention.
• compositions may also be of value in the veterinary field, which for the purposes herein not only includes the prevention and/or treatment of diseases in animals, but also - for economically important animals such as cattle, pigs, sheep, chicken, fish, etc. - enhancing the growth and/or weight of the animal and/or the amount and/or the quality of the meat or other products obtained from the animal.
• the invention will now be illustrated by means of the following synthetic and biological examples, which do not limit the scope of the invention in any way.
• E7 mRNA was prepared by eTheRNA by in vitro transcription (IVT) from the eTheRNA plasmid pEtherna-v2.
• IVT in vitro transcription
• the sequence encoding the HPV16-E7 protein was cloned in-frame between the signal sequence and the transmembrane and cytoplasmic regions of human DC-LAMP.
• This chimeric gene was cloned in the pEtherna-v2 plasmid (WO2015071295) that was enriched with a translation enhancer at the 5' end and an RNA stabilizing sequence at the 3' end.
• CD40L, caTLR4 and CD70 mRNA (TriMix components) were cloned in the pEtherna-v2 plasmid.
• dsRNA was removed by cellulose purification.
• Cellulose powder was purchased from Sigma and washed in 1xSTE (Sodium Chloride-Tris-EDTA) buffer with 16% ethanol.
• IVT mRNA in 1xSTE buffer with 16% ethanol was added to the washed cellulose pellet and shaken at room temperature for 20 minutes. This solution is then brought over a vacuum filter (Corning). The eluate contains the ssRNA fraction and was used for all experiments. mRNA quality was monitored by capillary gel electrophoresis (Agilent, Belgium).
• Lipid based nanoparticles are produced by microfluidic mixing of an mRNA solution in malic acid buffer (20mM malic acid (Sigma), 30mM NaCI (Sigma), pH3) and lipid solution in a 2: 1 volume ratio at a speed of 9mL/min using the NanoAssemblr Benchtop (Precision Nanosystems).
• the lipid solution contained a mixture of Coatsome-EC (NOF corporation), DOPE (Avanti), Cholesterol (Sigma) and DMG-PEG2000 (Sunbright GM-020, NOF corporation) in a molar ratio of 50/10/39.5/0.5 respectively.
• LNPs were dialysed against PBS (100 times more PBS volume than LNP volume) using slide-a-lyzer dialysis casettes (20K MWCO, 3mL, ThermoFisher). Size and polydispersity were measured by dynamic light scattering with a Zetasizer Nano (Malvern).
• Serum samples were diluted 3 times in universal assay buffer (included in ProcartaPlex kit) and incubated with fluorescently labelled beads for 120minutes. Read-out of Procartaplex assay was done on a MagPix instrument (Luminex).
• the assays were performed according to the kit protocols with only one deviation; only half of the prescribed volume of serum was added to the plates because of the limiting nature of this type of sample. Serum samples were diluted 5 times in sample diluent buffer included in the kits. Read-out of the ELISAs was done on a SpectraMax M3 plate reader (Molecular Devices). Flow cytometry Number of E7-specific T cells
• Table 1 List of antibodies used for flow cytometry analysis of number of E7-specific T cells.
• mice were euthanized and their spleens were harvested. Single cell suspensions of splenocytes were prepared and seeded into microtiter plates at 10000 cells/well, with or without peptide stimulation. 5ug/mL of E7 peptide was used for stimulation in the dedicated wells. As a positive control, T cells were stimulated with anti-CD3/anti-CD28 beads.
• mRNA LNPs were generated by microfluidic mixing on a NanoAssemblr (PNI).
• PNI NanoAssemblr
• an ethanolic lipid mix composed of SS-EC, DOPE, cholesterol and DMG-PEG2000 was mixed with an acidic solution of the mRNAs of interest as explained in detail in the materials and methods section.
• the molar % ratio for the constituent lipids is 50% CoatsomeSS-EC, 10% DOPE, 39,5% Cholesterol and 0,5% DMG-PEG2000.
• mRNA LNPs were subsequently dialyzed to PBS and size and polydispersity (PDI) of all mRNA LNPs were measured by Dynamic Light Scattering (DLS). As indicated in table 2, all mRNA LNPs were of similar size and PDI, regardless of mRNA format and E7: TriMix ratio.
• Table 2 Size and PDI (polydispersity) of lipid-based nanoparticles used for intravenous immunizations mRNA format determines inflammatory responses to E7/TriMix mRNA LNPs
• mRNAs were either co-transcriptionally capped with an ARCA-Cap or with a CleanCap. No nucleoside modifications were incorporated into the ARCA Cap mRNA (ARCA, non-mod mRNA). CleanCap mRNA either did not contain modified nucleosides (CleanCap, non-mod mRNA) or displayed a 100% substitution of uridine by Nl methylpseudo-uridine (CleanCap, mod mRNA). To address the impact of mRNA format and of TriMix on the inflammatory response, E7 mRNA was mixed with TriMix at the indicated ratio's and encapsulated into LNPs. C57BL/6 mice received a total dose of 10 pg mRNA administrated by the tail vein. Serum samples were collected at 6 hours and 24 hours post injection and analyzed for IFN-a, IFN-g, IL-6, MCP-1 , G- CSF and RANTES.
• E7 mRNA was mixed with TriMix mRNA at the indicated ratios for the different mRNA formats.
• Mice were immunized at days 0, 7 and 14 with the respective mRNA LNPs at a total mRNA dose of 10 pg.
• PBMCs were collected and stained by flow cytometry to assess the percentages of E7-specific CD8 T cells.
• mice immunized with CleanCap/non-mod mRNA and CleanCap/mod mRNA showed significantly higher levels of E7- specific T cells compared to mice immunized with ARCA/non-mod mRNA ( Figure 3A).
• the impact of TriMix hence clearly dependent on the mRNA format, with TriMix showing no benefit in case of ARCA / non-mod mRNA yet showing a highly significant benefit in case of CleanCap/mod mRNA.
• TriMix did not augment the number of IFN- y producing splenocytes in case of ARCA, non-mod mRNA, yet TriMix did increase responses in case of CleanCap/non-mod mRNA and CleanCap/mod mRNAs ( Figure 4).
• Weight loss and ALT/AST levels were measured as surrogate markers for toxicity and liver damage. As can be appreciated from Figure 5, all LNPs induced transient weight loss of mice - most pronounced at 24hrs post injection - followed by rapid recovery. The mRNA format itself appeared to have little impact on the extent of weight loss, although ARCA-capped mRNA tended to evoke the highest weight loss compared to PBS injected mice. For none of the mRNA formats addressed, TriMix exacerbated weight loss.
• AST/ALT used as surrogate markers for liver damage.
• AST/ALT levels were assessed by ELISA at one week after the third immunization. For none of the treatment groups, ALT/AST levels were strongly elevated.
• E7/TriMix mRNA LNP treated mice no statistically significant increases in ALT nor AST levels compared to PBS mice were measured, regardless of the mRNA format. Surprisingly, mice treated with E7-only mRNA LNPs did show low but significant upregulation in AST levels to PBS ( Figure 6).
• ARCA replacement of ARCA by CleanCap resulted in a significant decrease in the systemic inflammatory response.
• the efficiency of co-transcriptional capping with ARCA is around 70%, leaving 30% of the generated mRNA with a 5' triphosphate end, which is sensed by RIG-1.
• ARCA instead of the natural methylated Cap1 structure, ARCA introduces a non-methylated “Cap-0” structure, which again triggers various RNA sensors that drive inflammation. CleanCap
• Cap-1 is incorporated with higher efficiency (> 90%) and does introduce the natural Cap1 structure, hence lowering innate recognition and inflammation.
• Substitution of uridine by N1 methyluridine further lowered serum titers of IFN-a, IFN-g and IL-6, in line with previous reports demonstrating that N1 methylpseudo-uridine substitution lowers TLR7 and RIG-I activation.
• TriMix exacerbated systemic inflammatory responses.
• TriMix In terms of magnitude and kinetics of the T cell response, no significant differences were observed between mRNA formats in the absence of TriMix. Yet, upon addition of TriMix, strong differences occurred between mRNA formats. In case of the high inflammatory ARCA, non-mod mRNA format, TriMix failed to further augment the magnitude of the T cell response. Nonetheless, in case of the low inflammatory mRNA formats CleanCap, non-mod and CleanCap, mod, TriMix exerted its immune-stimulatory functions and resulted in a strong increase in the magnitude of the E7-specific CD8 T cell response. Similar, TriMix mRNA also enhanced the numbers of IFN-y secreting T cells in case of immunization with CleanCap, non- mod and CleanCap, mod mRNA yet not with ARCA, non-mod mRNA LNPs.

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