MRNA vaccines

Abstract

The present invention generally relates to a combination of an mRNA molecule encoding a functional immunostimulatory protein and a CTLA4 pathway inhibitor. In particular, it relates to a combination of one or more mRNA molecules encoding at least one functional immunostimulatory protein selected from a list including CD40L, CD70 and caTLR4, and a CTLA4 pathway inhibitor, optionally also in the form of an mRNA molecule. The present invention also relates to a vaccine comprising such a combination, and the use of the combination and vaccine of the present invention in human or veterinary medicine, in particular in the prevention and / or treatment of cell proliferative diseases.

Description

Field of the Invention

[0001] The present invention generally relates to a combination of an mRNA molecule encoding a functional immunostimulatory protein and a CTLA4 pathway inhibitor. In particular, it relates to a combination of one or more mRNA molecules encoding at least one functional immunostimulatory protein selected from a list including CD40L, CD70 and caTLR4, and a CTLA4 pathway inhibitor, optionally also in the form of an mRNA molecule. The present invention also relates to a vaccine comprising such a combination, and the use of the combination and vaccine of the present invention in human or veterinary medicine, in particular in the prevention and / or treatment of cell proliferative diseases. Background of the Invention

[0003] Inducing potent cytolytic CD8 T cell responses that can recognize and kill cancer cells constitutes a key goal of any therapeutic cancer vaccine. The ability of vaccines to elicit such cytolytic T cell responses depends largely on early interactions between the vaccine and dendritic cells (DCs), which are the most potent antigen presenting cells and initiators of T cell immunity. In contrast to protein-based vaccines, mRNA vaccines are able to express mRNA-encoded antigens in the cytoplasm of DCs, which is the natural pathway for antigen processing and antigen presentation to CD8 T cells.

[0004] Furthermore, in vitro transcribed mRNA—produced by viral polymerases such as T7—partially resembles viral RNA and is therefore recognized by innate immune sensors, conferring inherent adjuvant properties to mRNA (Kariko et al., 2005; Yoneyama et al., 2010). Nevertheless, activation of DCs by IVT mRNA is suboptimal and can be further enhanced by co-delivery of TriMix mRNA, a mixture of three mRNAs encoding the immunostimulatory proteins CD40L, CD70, and caTLR4 (Bonehill et al., 2008; Van Lint et al., 2012; Van Lint et al., 2016). In preclinical models, the addition of TriMix mRNA to mRNA encoding tumor antigens has been shown to strongly enhance the magnitude of T cell responses and their antitumor potency and is currently being explored in clinical studies.

[0005] Anti-CTLA4 antibodies can interfere with tumor immunosuppression and restore the anti-tumor efficacy of pre-existing effector T cells, among which the anti-CTLA-4 blocking antibody ipilimumab is the first immune checkpoint inhibitor approved for the treatment of cancer patients (Seidel et al., 2018). The interaction of CTLA-4 with CD80 and CD86 present on the surface of antigen-presenting cells provides inhibitory signals for T cells during the initial priming period. In addition, CTLA-4 is highly expressed on the surface of regulatory T cells.

[0006] mRNA vaccines have the ability to prime / expand antitumor T cells against mRNA-encoded tumor-associated antigens. Nevertheless, the therapeutic efficacy of vaccine-primed T cells is often hampered by the strong immunosuppressive microenvironment present at the tumor site. Since CTLA-4 antibodies are able to block co-inhibitory signals during T cell priming and can interfere with the immunosuppressive function of regulatory T cells, we evaluated whether the combination of TriMix-based mRNA vaccination with anti-CTLA4 antibodies would increase antitumor efficacy.

[0007] We now unexpectedly found that, although anti-CTLA4 monotherapy had no therapeutic effect, the combination of Trimix-based mRNA vaccination with anti-CTLA4 therapy showed excellent antitumor efficacy, which was even better than Trimix monotherapy. SUMMARY OF THE INVENTION

[0009] The present invention is defined by the following numbered statements:

[0010] 1. A combination comprising:

[0011] - one or more mRNA molecules encoding at least one functional immunostimulatory protein selected from the list comprising CD40L, CD70 and caTLR4; and

[0012] -CTLA4 pathway inhibitors, which prevent or block CTLA4-initiated signaling.

[0013] 2. A combination as defined in statement 1, wherein the one or more mRNA molecules encode all functional immunostimulatory proteins selected from the list comprising CD40L, CD70 and caTLR4.

[0014] 3. A combination as defined in statement 1, wherein the CTLA4 pathway inhibitor is in the form of mRNA encoding the CTLA4 pathway inhibitor.

[0015] 4. A combination as defined in statement 1, wherein the CTLA4 pathway inhibitor is an antagonistic antibody, nanobody or derivative thereof against CTLA4.

[0016] 5. A combination as defined in statement 4, wherein the antagonist antibody against CTLA4 is selected from the list comprising ipilimumab and tremelimumab.

[0017] 6. A combination as defined in any one of statements 1 to 5, further comprising one or more mRNA molecules encoding a tumor-specific antigen.

[0018] 7. A combination as defined in any one of statements 1 to 6, wherein the one or more mRNA molecules are formulated for parenteral administration; more particularly for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

[0019] 8. A combination as defined in any one of statements 1 to 7, wherein the mRNA molecule is contained in a nanoparticle.

[0020] 9. A combination as defined in any one of statements 1 to 8, wherein the nanoparticles are selected from the list comprising lipid nanoparticles and polymer nanoparticles.

[0021] 10. A combination as defined in any one of clauses 1 to 7, wherein the mRNA molecule is formulated for intranodal or intratumoral administration and is in the form of a naked mRNA molecule in a suitable injection buffer, such as Ringer's lactate buffer.

[0022] 11. A combination as defined in any one of statements 1 to 10, wherein the CTLA4 pathway inhibitor is formulated for parenteral administration; more particularly for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

[0023] 12. A vaccine comprising a combination as defined in any one of sentences 1-11.

[0024] 13. A combination as defined in any one of clauses 1 to 11 or a vaccine as defined in clause 12, for use in human or veterinary medicine.

[0025] 14. A combination as defined in any one of clauses 1 to 11 or a vaccine as defined in clause 12 for use in the prevention and / or treatment of a cell proliferative disease.

[0026] 15. A combination as defined in any one of clauses 1 to 11 or a vaccine as defined in clause 12 for use in eliciting an immune response against a tumor in a subject.

[0027] Thus, in a first aspect, the present invention provides a combination comprising:

[0028] - one or more mRNA molecules encoding at least one functional immunostimulatory protein selected from the list comprising CD40L, CD70 and caTLR4; and

[0029] -CTLA4 pathway inhibitors, which prevent or block CTLA4-initiated signaling.

[0030] In a specific embodiment of the invention, the one or more mRNA molecules encode all functional immunostimulatory proteins selected from the list comprising CD40L, CD70 and caTLR4.

[0031] In another embodiment of the present invention, the CTLA4 pathway inhibitor is in the form of mRNA encoding the CTLA4 pathway inhibitor. Alternatively, the CTLA4 pathway inhibitor is an antagonist antibody, nanobody or derivative thereof against CTLA4; more specifically, the antagonist antibody against CTLA4 is selected from the list including ipilimumab and tremelimumab.

[0032] In a specific embodiment, the combination of the present invention may further comprise one or more mRNA molecules encoding tumor-specific antigens.

[0033] In yet another specific embodiment of the invention, the one or more mRNA molecules are formulated for parenteral administration; more particularly for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

[0034] In a particularly preferred embodiment, the combination or mRNA molecule of the invention is formulated in the form of nanoparticles, for example in the form of lipid nanoparticles or polymer nanoparticles.

[0035] In yet another specific embodiment of the present invention, the 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 Ringer's lactate buffer.

[0036] The invention also provides a combination as defined herein, wherein the CTLA4 pathway inhibitor is formulated for parenteral administration; more particularly for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

[0037] In a further aspect, the invention provides a vaccine comprising a combination as defined herein.

[0038] In a specific embodiment, the invention provides a combination or vaccine as defined herein for use in human or veterinary medicine; more particularly for use in the prevention and / or treatment of a cell proliferative disease; for example for eliciting an immune response against a tumor in a subject.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] With specific reference now to the accompanying drawings, it should be emphasized that the details shown are merely examples and are only for the purpose of illustrative discussion of different embodiments of the present invention. They appear to provide what is considered to be the most useful and easiest description of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show more detailed structural details of the present invention than is necessary for a basic understanding of the present invention. The description in conjunction with the accompanying drawings makes it clear to those skilled in the art how several forms of the present invention can be implemented in practice.

[0041] Figure 1 : Tumor growth curves of individual mice after TC-1 bearing mice were treated with PBS, anti-CTLA4, E7-TriMix mRNA or a combination of E7-TriMix mRNA and anti-CTLA-4, respectively. CR = Complete responder, which means the number of mice free of tumor on day 90 after tumor inoculation.

[0042] Figure 2 : Average tumor growth curves of TC-1-inoculated mice treated with PBS, anti-CTLA4, E7+TriMix mRNA, and E7+TriMix mRNA+anti-CTLA4. **p=0.0012, two-way ANOVA with Bonferroni's multiple comparison test. DETAILED DESCRIPTION OF THE INVENTION

[0044] As has been discussed in detail herein above, the present invention relates to a combination comprising:

[0045] - one or more mRNA molecules encoding at least one functional immunostimulatory protein selected from the list comprising CD40L, CD70 and caTLR4; and

[0046] -CTLA4 pathway inhibitors, which prevent or block CTLA4-initiated signaling.

[0047] In the present invention, the term "TriMix" represents a mixture of mRNA molecules encoding CD40L, CD70 and caTLR4 immunostimulatory proteins. The combined use of CD40L and caTLR4 produces mature, cytokine / chemokine-secreting DCs, as shown by the ligation of CD40 and TLR4 by the addition of soluble CD40L and LPS. The introduction of CD70 into DCs stimulates CD27 by inhibiting activated T cell apoptosis and supporting T cell proliferation. +Naive T cells provide a co-stimulatory signal. Other Toll-like receptors (TLRs) can be used as alternatives to caTLR4. For each TLR, a constitutively active form is known and may be introduced into DCs to elicit a host immune response. However, in our opinion, caTLR4 is the most potent activating molecule and is therefore the preferred activating molecule.

[0048] The mRNA or DNA used or mentioned herein can be naked mRNA or DNA, or protected mRNA or DNA. The protection of DNA or mRNA increases its stability while retaining the ability to use mRNA or DNA for vaccination purposes. Non-limiting examples of protecting mRNA and DNA can be: liposome encapsulation, protamine protection, lipid complex of (cationic) lipid, lipid, cation or polycation composition, mannosylated lipid complex, bubble liposome formation, polyethyleneimine (PEI) protection, liposome loading microvesicle protection, etc. In a specific embodiment, mRNA or DNA molecules as defined herein are isolated mRNA or DNA molecules, specifically, they are preferably not a part of cells (e.g., dendritic cells). The present invention is particularly intended for in vivo applications, including directly using isolated mRNA or DNA molecules, contrary to the in vitro method including using dendritic cells transfected with such mRNA or DNA molecules.

[0049] While the present invention is particularly suitable for use in conjunction with tumor-specific antigens, it may also be suitable for use in conjunction with other types of target-specific antigens.

[0050] The term "target" used throughout the specification is not limited to the specific examples that may be described herein. Any infectious agent such as a virus, bacteria or fungus can be targeted. In addition, any tumor or cancer cell can be targeted. The term "target-specific antigen" used throughout the specification is not limited to the specific examples that may be described herein. It is clear to those skilled in the art that the present invention is related to the induction of immune stimulation in APCs, and has nothing to do with the target-specific antigen presented. The antigen to be presented will depend on the target type of immune response that one intends to induce in the subject. Typical examples of target-specific antigens are expressed or secreted markers that are specific to tumors, bacteria and fungal cells or specific viral proteins or viral structures. Without wishing to limit the scope of protection of the present invention, some examples of possible markers are listed below.

[0051] The terms "vegetation", "cancer" and / or "tumor" used throughout the specification are not intended to be limited to the types of cancer or tumors that may have been exemplified. Therefore, the term encompasses all proliferative diseases, such as neoplasms, dysplasia, premalignant lesions or precancerous lesions, abnormal cell growth, benign tumors, malignant tumors, cancers or metastases, wherein the cancer may be selected from the following group: leukemia, non-small cell lung, 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, gastric cancer, duodenal cancer, esophageal cancer, thyroid cancer, blood cancer and lymphoma. The specific antigen of cancer can be, for example, MelanA / MART1, cancer germline antigen, gp100, tyrosinase, CEA, PSA, Her-2 / neu, survivin, telomerase.

[0052] In a preferred embodiment of the vaccine of the present invention, the mRNA or DNA molecule encodes CD40L and CD70 immunostimulatory proteins. In a particularly preferred embodiment of the vaccine of the present invention, the mRNA or DNA molecule encodes CD40L, CD70 and caTLR4 immunostimulatory proteins.

[0053] The mRNA or DNA molecule encoding the immunostimulatory protein can be part of a single mRNA or DNA molecule. Preferably, the single mRNA or DNA molecule is capable of expressing two or more proteins simultaneously. In one embodiment, the mRNA or DNA molecule encoding the immunostimulatory protein is separated in a single mRNA or DNA molecule by an internal ribosome entry site (IRES) or a self-cleaving 2a peptide coding sequence.

[0054] In a specific embodiment, one or more of the mRNA molecules of the present invention may further comprise a translation enhancer and / or a nuclear retention element. Suitable translation enhancers and nuclear retention elements are those described in WO2015071295.

[0055] Cytotoxic T lymphocyte antigen 4 (CTLA-4) is primarily expressed in the intracellular compartment of T cells. Upon activation of naive T cells, CTLA-4 is transported to the cell surface and concentrated at the immune synapse, where it competes with CD28 for CD80 / CD86 and downregulates TCR signaling.

[0056] As used herein, the term "CTLA4 pathway inhibitor" includes any compound that prevents or blocks CTLA4-initiated signal transduction. Therefore, it can directly or indirectly affect the regulation of CTLA4 by reducing, for example, the expression of the CTLA4 receptor (i.e., transcription and / or translation) or its natural ligands B7-1 (CD80) and B7-2 (CD86). Without being limited thereto, such inhibitors include siRNA, antisense molecules, proteins, peptides, small molecules, antibodies, nanobodies, and derivatives of any of these. In a specific embodiment, the CTLA4 inhibitor can also be provided in the form of mRNA encoding the inhibitor, such as mRNA encoding an anti-CTLA4 antibody.

[0057] Preferred anti-CTLA4 antibodies are human antibodies that specifically bind to human CTLA4. Human antibodies offer significant advantages in the therapeutic methods of the invention because they are expected to minimize the immunogenicity and allergic reactions associated with the use of non-human antibodies in human patients.

[0058] Exemplary human anti-CTLA4 antibodies are described in detail, for example, in WO 00 / 37504. Such antibodies include, but are not limited to, 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, ticilimumab, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1, as well as tremelimumab and ipilimumab. In another embodiment, the antibody is selected from an antibody having the full length, variable region, or CDR amino acid sequence of the heavy and light chains of the antibodies defined above.

[0059] In other embodiments of the present invention, the antibody inhibits the binding of CTLA4 to B7-1, B7-2 or both. Preferably, the antibody can inhibit the binding to B7-1 with an IC50 of about 100nM or less, more preferably, about 10nM or less, such as about 5nM or less, more preferably, about 2nM or less, or even more preferably, such as about 1nM or less. Similarly, the antibody can inhibit the binding to B7-2 with an IC50 of about 100nM or less, more preferably, 10nM or less, such as even more preferably, about 5nM or less, more preferably, about 2nM or less, or even more preferably, about 1nM or less.

[0060] Although the anti-CTLA4 antibodies discussed previously herein may be preferred, based on the disclosure provided herein, one skilled in the art will appreciate that the present invention encompasses a wide variety of anti-CTLA4 antibodies and is not limited to these specific antibodies. More particularly, although human antibodies are preferred, the present invention is by no means limited to human antibodies; on the contrary, the present invention encompasses useful antibodies regardless of species origin, and includes chimeric humanized and / or primatized antibodies, etc.

[0061] In one embodiment, the present disclosure provides a combined treatment method for an individual, comprising administering to the individual one or more immunostimulatory factors and an inhibitor of the CTLA4 pathway. The one or more immunostimulatory factors and the CTLA4 pathway inhibitor can be administered simultaneously or concurrently as a single composition or separate compositions, or the one or more immunostimulatory factors and the CTLA4 pathway inhibitor can be administered at different times.

[0062] Compositions of the invention typically comprise a CTLA4 pathway inhibitor in combination with one or more immunostimulatory factors.

[0063] The appropriate dosage of a CTLA4 pathway inhibitor will depend on many factors, including, for example, the age and weight of the individual, at least one precise condition to be treated, the severity of the condition, the nature of the composition, the route of administration, and combinations thereof. Ultimately, a person skilled in the art, such as a physician, veterinarian, scientist, and other medical and research professionals, can readily determine the appropriate dosage. For example, a person skilled in the art can start with a low dosage that can be increased until the desired therapeutic effect or result is achieved. Alternatively, a person skilled in the art can start with a high dosage that can be reduced until the minimum dosage required to achieve the desired therapeutic effect or result is achieved.

[0064] The invention also provides a combination as defined herein, wherein the mRNA molecule is contained in a nanoparticle.

[0065] As used herein, the term "nanoparticle" refers to any particle, particularly nucleic acids, having a diameter that makes the particle suitable for systemic administration, particularly intravenous administration, typically less than 1000 nanometers (nm).

[0066] In a particular embodiment of the present invention, the nanoparticles are selected from the list comprising lipid nanoparticles and polymer nanoparticles.

[0067] Lipid nanoparticles (LNP) are generally referred to as nanoscale particles composed of different lipid combinations. Although many different types of lipids can be included in such LNPs, the LNP of the present invention can, for example, be composed of a combination of ionizable lipids, phospholipids, sterols and PEG lipids.

[0068] Polymer nanoparticles can generally be nanospheres or nanocapsules. Two main strategies are used to prepare polymer nanoparticles, namely the "top-down" approach and the "bottom-up" approach. In the top-down approach, a dispersion of a preformed polymer produces polymer nanoparticles, while in the bottom-up approach, polymerization of monomers leads to the formation of polymer nanoparticles. Both the top-down and bottom-up approaches use synthetic polymers / monomers such as poly(d,l-lactide-co-glycolide), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), and poly(isohexyl cyanoacrylate); stabilizers such as poly(vinyl alcohol) and didecyldimethylammonium bromide; and organic solvents such as dichloromethane, ethyl acetate, benzyl alcohol, cyclohexane, acetonitrile, acetone, etc. Recently, the scientific community has been trying to find alternatives to synthetic polymers by using natural polymers and synthetic methods that use less toxic solvents.

[0069] The invention also provides compositions and vaccines as defined herein for use in human or veterinary medicine, particularly for use in the treatment of a cell proliferative disease, more particularly for use in eliciting an immune response against a tumour in a subject.

[0070] Finally, the present invention provides a method for treating a cell proliferative disease, comprising the step of administering the combination or vaccine of the present invention to a subject in need thereof.

[0071] The compositions may also be valuable in the veterinary field, which for purposes herein includes not only the prevention and / or treatment of animal disease, but also - in the case of economically important animals such as cattle, pigs, sheep, chickens, fish, etc. - improving the growth and / or weight of the animal and / or the quantity and / or quality of meat or other products obtained from the animal.

[0072] The invention will now be illustrated by the following synthetic and biological examples, which are not intended to limit the scope of the invention in any way. Example

[0073] Example 1: Antitumor efficacy of TRIMIX-based immunization as monotherapy or in combination with anti-CTLA4 antibody in TC-1 tumor-bearing mice

[0074] Materials and Methods

[0075] Synthesis of in vitro transcribed mRNA:

[0076] E7 mRNA and mouse caTLR4, mouse CD70 and mouse CD40L (TriMix) mRNA were synthesized from the corresponding linearized peTheRNA plasmids by in vitro transcription as described previously (EP3068888).

[0077] Mouse:

[0078] C57BL / 6 mice (female, 6 weeks old) were purchased from Janvier (Genest) and maintained under SPF (OncoDesign, Dijon) conditions according to FELASA guidelines. TC-1 cells were obtained from ATCC and cultured as described previously.

[0079] Antibody:

[0080] Anti-CTLA4 antibody was purchased from BioXCell (reference: BE0131; clone: ​​9H10; reactivity: mouse; isotype: hamster IgG1; storage condition: +4°C).

[0081] Tumor inoculation and treatment schedule:

[0082] On day 0, mice were subcutaneously inoculated with 1×10 6 TC-1 tumor cells in a volume of 200 μl PBS. Intrasegmental immunization with E7 / TriMix mRNA was performed on days 3, 8, and 13 after tumor inoculation. Mice received 10 μg E7 mRNA combined with 30 μg TriMix mRNA dissolved in 0.8× Ringer's lactate solution (20 μl). mRNA was injected into the inguinal lymph nodes. Anti-CTLA4 antibody (10 mg / kg / administration) was injected intraperitoneally on days 3, 6, 9, and 12 after tumor inoculation.

[0083] Intra-seasonal application

[0084] Before disinfection with 70% ethanol, mice were shaved in the inguinal region to remove hair. A small incision was made in the inguinal region to expose the inguinal lymph nodes. A total volume of 10 μl of mRNA solution was injected into the inguinal lymph nodes using a 0.3 ml 30G insulin needle (BD Biosciences, Ref 324826A). After injection, the skin was closed with a 4-0 crinerce suture. The same inguinal lymph nodes were injected for all three intranodal immunizations.

[0085] result:

[0086] like Figure 1 and 2 As detailed in , the anti-CTLA-4 monotherapy regimen did not provide any therapeutic benefit in the TC-1 model compared to control (PBS), with no complete responders in either group. On the other hand, intranodal immunization with E7 / TriMix showed a strong delay in TC-1 tumor growth and resulted in a limited proportion of complete responders (2 / 15).

[0087] More strikingly, the combination of anti-CTLA4 antibody and intranodal E7 / TriMix immunization showed superior anti-tumor efficacy and greatly increased the proportion of complete responders (9 / 15) compared with intranodal E7 / TriMix monotherapy (p=0.0012) and anti-CTLA4 monotherapy.

[0088] Therefore, these data clearly show the potential of the combination of Trimix and CTLA4 pathway inhibitory molecules in tumor therapy.

[0089] References

[0090] Bonehill A et al.Mol.Ther.2008;16:1170-80.

[0091] Kariko K et. Immunity 2005; 23,165-175.

[0092] Sahin et al.Nat.Discovery Rev.2014;13,759-780.

[0093] Seidel et al. Front Oncol.2018;8:86.

[0094] Van Lint S.et al.Cancer Res.2012;1;72(7):1661-71

[0095] Van Lint S.et al.Cancer Res Immunol.2016;4(2):146-156

[0096] Yoneyama,M.&Fujita,T.Rev.Med.Virol.2010;20,4-22

Claims

1. Use of a combination in the preparation of a medicament for preventing and / or treating HPV-related cancers, the combination comprising: -mRNA molecules encoding all functional immunostimulatory proteins in CD40L, CD70 and caTLR4; - mRNA molecules encoding E7 antigen; and - an antagonist antibody against CTLA4 selected from the list comprising ipilimumab and tremelimumab; wherein the combination does not comprise dendritic cells.

2. The use as defined in claim 1, wherein the antagonistic antibody against CTLA4 is ipilimumab.

3. The use as defined in claim 1, wherein the mRNA molecule is formulated for parenteral administration.

4. The use as defined in claim 1, wherein the mRNA molecule is formulated for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

5. The use as defined in claim 1, wherein the mRNA molecule is formulated in the form of nanoparticles.

6. The use as defined in claim 5, wherein the nanoparticles are selected from the list comprising lipid nanoparticles and polymer nanoparticles.

7. The use as defined in claim 1, wherein the mRNA molecule is formulated for intranodal or intratumoral administration and is in the form of a naked mRNA molecule in a suitable injection buffer.

8. The use as defined in claim 1, wherein the mRNA molecule is formulated for intranodal or intratumoral administration and is in the form of a naked mRNA molecule in Ringer's lactate buffer.

9. The use as defined in claim 1, wherein the CTLA4 pathway inhibitor is formulated for parenteral administration.

10. The use as defined in claim 1, wherein the CTLA4 pathway inhibitor is formulated for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

Images