Checkpoint inhibitor loaded activated dendritic cells

Abstract

The invention relates to dendritic cells loaded with nucleic acid sequences expressing checkpoint inhibitors and optionally other molecules, pharmaceutical compositions comprising such dendritic cells, and medical use of such dendritic cells and compositions.

Description

[0001] Transimmune AG

[0002] Yale University Emory University

[0003] Checkpoint inhibitor loaded activated dendritic cells

[0004] FIELD OF THE INVENTION

[0005] The invention relates to activated dendritic cells loaded with nucleic acid sequences expressing checkpoint inhibitors and other molecules, pharmaceutical compositions comprising such activated dendritic cells, and medical uses of activated dendritic cells or the pharmaceutical composition comprising the same.

[0006] BACKGROUND OF THE INVENTION

[0007] Immunotherapy can be employed for the treatment of various human diseases, such as infections, degenerative conditions, and cancer. In cancer, immunotherapy sometimes involves stimulating the patient’s own immune system to attack cancer cells or other cellular components of the tumor.

[0008] A hallmark of the tumor microenvironment is the exhaustion and functional impairment of T cells. There is extensive evidence that CD8+T cells become unresponsive to cancer cells in the course of tumorigenesis, a process called T cell dysfunction. Dysfunctional T cells commonly express high levels of inhibitory receptors, while failing to produce effective immune responses.

[0009] Dendritic cells (DCs) have the ability to take up, process, and present antigens on their cell surface to T-cells and B-cells. As DCs thereby activate naive, effector, and memory immune cells, they are a promising therapeutic agent against diseases such as cancer or infectious diseases. The potent activation of de novo T-cell and B-cell

[0010] DB:SUL T08741ANM - 2 - responses also suggests dendritic cells as promising agents for prophylactic purposes.

[0011] Ex vivo activated dendritic cells, also called physiological dendritic cells (phDCs), are dendritic cells generated through applying shear stress on a blood sample, e.g., by running a blood sample through a flow chamber (such as a blood bag or an electrophoresis chamber). In one embodiment, a patient's blood sample is applied to an extracorporeal photopheresis (ECP) device. The process results in activated dendritic cells with improved clinical effectiveness compared to monocyte-derived, cytokine-differentiated DCs.

[0012] Eliciting or enhancing cancer-specific T lymphocytes by vaccinating the patient against tumor-associated antigens (TAAs) represents an attractive means of treating a subject by immunotherapy. One type of cancer vaccine involves the use of dendritic cells (DCs). DCs are a family of immune cells endowed with the ability to capture and present TAAs to T lymphocytes through a variety of mechanisms, priming potent effector responses against the tumor. DCs are also capable of migration between lymphoid and non-lymphoid tissues and modulating cytokine and chemokine gradients to control inflammation and lymphocyte homing. However, improving the efficacy of DCs for therapeutic use has been challenging.

[0013] OBJECTIVES AND SUMMARY OF THE INVENTION

[0014] The inventors surprisingly found that ex vivo activated dendritic cells expressing at least one checkpoint inhibitor exhibited significant anti-tumor activity in vivo when administered alone. Furthermore, the checkpoint inhibitor-loaded activated dendritic cells can augment standard of care therapy. Ex vivo activated dendritic cells as described herein provide a means to improve the efficacy of DC vaccines. T08741ANM - 3 -

[0015] Hence, one objective of the present invention is to provide an ex vivo activated dendritic cell, wherein the activated dendritic cell comprises at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor. The activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor is hereinafter used interchangeably with the term activated dendritic cell of the invention.

[0016] Another objective of the present invention is to provide an ex vivo activated dendritic cell, wherein the activated dendritic cell comprises at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and further comprises at least one exogenous nucleic acid sequence encoding an immune modulator and / or at least one exogenous nucleic acid sequence encoding an antigenic peptide.

[0017] A further objective of the invention is to provide a pharmaceutical composition comprising the activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, and a pharmaceutically acceptable carrier. Such a pharmaceutical composition is hereinafter used interchangeably with the term pharmaceutical composition of the invention.

[0018] Another objective of the invention is to provide a pharmaceutical composition comprising a first activated dendritic cell which comprises at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and a second activated dendritic cell which comprises at least one exogenous nucleic acid sequence encoding a cytokine. Another objective of the invention is to provide a pharmaceutical composition comprising a first activated dendritic cell which comprises at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and a second activated dendritic cell which comprises at least one exogenous nucleic acid sequence encoding an antigenic peptide. Another objective of the invention is to provide a pharmaceutical composition comprising a first activated dendritic cell which T08741ANM - 4 - comprises at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, a second activated dendritic cell which comprises at least one exogenous nucleic acid sequence encoding a cytokine, and a third activated dendritic cell which comprises at least one exogenous nucleic acid sequence encoding an antigenic peptide.

[0019] Another objective of the invention is to provide the activated dendritic cell or the pharmaceutical composition for use in therapy.

[0020] The activated dendritic cell or the pharmaceutical composition of the invention are capable of efficiently taking up and presenting tumor-associated antigens and induce strong and broad T-cell responses against multiple tumor-associated antigens, resulting in an effective anti-tumor response, thereby making them attractive therapeutics against a broad range of cancers.

[0021] Another objective of the invention is to provide a method for preparing the ex vivo activated dendritic cell, the method comprising the step of combining the activated dendritic cell with at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor.

[0022] A further objective of the invention is to provide an ex vivo activated dendritic cell obtainable by the methods provided herein.

[0023] Finally, it is an objective of the invention to provide a kit comprising a plurality of ex vivo activated dendritic cells and LNPs comprising at least one mRNA encoding a checkpoint inhibitor.

[0024] These and other objectives as they will become apparent from the ensuing description hereinafter are solved by the subject matter of the independent claims. Some of the preferred embodiments of the present invention form the subject matter of the dependent claims. Yet other embodiments of the present invention may be taken from T08741ANM - 5 - the ensuing description.

[0025] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

[0026] The present invention is described with respect to particular embodiments below and with reference to certain figures, but the invention is not limited thereto but only by the claims.

[0027] The present invention is based to some extent on data and experiments presented hereinafter, which led to the insight that dendritic cells loaded with checkpoint inhibitors can be used effectively for the prevention or treatment of diseases such as cancer. As key prerequisites for obtaining activated DCs loaded with a nucleic acid sequence encoding a checkpoint inhibitor, activated dendritic cells need to be (i) obtained and (ii) equipped with a specific nucleic acid sequence. As regards (i), so- called activated dendritic cells (previously also known as physiologic dendritic cells or phDCs) can be used. Activated dendritic cells can inter alia be produced ex vivo or in vitro by applying a shear force to monocytes (e.g. from a blood sample), see e.g. Ventura et al., 2018 “Extracorporeal Photochemotherapy Drives Monocyte-to- Dendritic Cell Maturation to Induce Anticancer Immunity”, WO 2014 / 106629, WO 2016 / 001405 or Hanlon et al., 2020 “Rapid Production of Physiologic Dendritic Cells (phDCs) for Immunotherapy”). Activated dendritic cells can be characterized by marker expression including markers such as HLA-DR, CD83, CD86, ICAM and / or PLAUR and / or no increased expression of GILZ (see, e.g., Ventura et al., 2018 “Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity” or WO 2014 / 106629). As the monocytes do not need to be subjected to cytokines to induce differentiation into activated dendritic cells, it is assumed that activated dendritic cells which are obtained by subjecting monocytes to a physical force more closely resemble the properties of naturally-occurring dendritic cells. T08741ANM - 6 -

[0028] As described previously (Ventura et al., 2018 “Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity” or WO 2014 / 106629), a physical force to induce differentiation of monocytes into activated dendritic cells can be applied by passing monocytes through a flow chamber which can take the form of, e.g., a plate or, e.g., a bag (e.g. flexible bag, such as a plastic bag). Such a bag is described, e.g., in Buechler et al., 2004 “Generation of Dendritic Cells Using Cell Culture Bags - Description of a Method and Review of Literature”. It is assumed that such passing through, e.g., a plate or placing in, e.g., a flexible bag or a combination thereof (e.g., a hybrid of a bag and a chamber as disclosed herein), exposes the monocytes to shear stress, inducing differentiation into dendritic cells.

[0029] If platelets are present, the maturation process can be improved. As mentioned, the monocytes can be matured into activated dendritic cells using this method without the need for adding cytokine cocktails. Cytokine-derived dendritic cells are not only expensive to generate but have been proven to be clinically disappointing.

[0030] As regards (ii), the inventors found that activated dendritic cells are capable of internalizing mRNA-containing LNP and expressing encoded proteins, such as checkpoint inhibitors. The resulting activated dendritic cells of the invention are capable of eliciting a disease-preventing immune response by their unique ability of supporting effector T cells in the immunosuppressant tumor microenvironment.

[0031] First aspect: Ex vivo activated dendritic cell expressing checkpoint inhibitors

[0032] In a first aspect, the present invention relates to an ex vivo activated dendritic cell, wherein the activated dendritic cell comprises at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor.

[0033] In one embodiment, the checkpoint inhibitor is capable of specifically binding to an immune checkpoint molecule, optionally wherein the immune checkpoint molecule is at least partially on a cell surface. In one embodiment, the immune checkpoint T08741ANM - 7 - molecule is PD-1, PD-L1 and / or CTLA-4. In one embodiment, the immune checkpoint molecule is PD-1. In another embodiment, the immune checkpoint molecule is PD- Ll. In another embodiment, the immune checkpoint molecule is CTLA-4. Additional embodiments could include, but are not limited to, other clinically-relevant CPI such as TIM-3, LAG-3 and TIGIT or additional checkpoint inhibitors which enter the commercial market concurrent with this application.

[0034] In one embodiment, the checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof. In one embodiment, the checkpoint inhibitor comprises an antibody or an antigen-binding fragment thereof. The antibody or an antigen-binding fragment thereof is capable of binding to PD-1, PD-L1 or CTLA-4 or a combination thereof. In some embodiments, the antibody is a monoclonal antibody or an antigen-binding fragment thereof. In one embodiment, the antibody is a multispecific antibody or an antigen-binding fragment thereof, preferably a bispecific antibody or an antigen-binding fragment thereof.

[0035] Hence, in a preferred embodiment, the checkpoint inhibitor is selected from an anti- PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD- L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof. In a preferred embodiment, the checkpoint inhibitor is an anti -PD-1 antibody or a PD-1 binding fragment thereof. In a further preferred embodiment, the checkpoint inhibitor is an anti-PD-Ll antibody or a PD-L1 binding fragment thereof. In a further preferred embodiment, the checkpoint inhibitor is an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof.

[0036] In one embodiment, the ex vivo activated dendritic cell comprises two exogenous nucleic acid sequences encoding two checkpoint inhibitors, e.g. an anti -PD-1 antibody or a PD-1 binding fragment thereof, and an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof. So-called dual checkpoint inhibitor loaded activated dendritic cells are believed to have increased anti-tumor specificity and efficacy. In T08741ANM - 8 - some embodiments, the dual checkpoint inhibitor loaded activated dendritic cells comprise an anti-PDL-1 antibody or a PDL-1 binding fragment thereof, and an anti- CTLA-4 antibody or a CTLA-4 binding fragment thereof. The two checkpoint inhibitors may be encoded on the same nucleic acid molecule, i.e. the first and second exogenous nucleic acid molecules may be covalently linked, e.g., be on the same construct or RNA molecule. In a preferred embodiment, the two checkpoint inhibitors are co-expressed in the same activated dendritic cell and anchored or bound to the cell surface of the activated dendritic cell. Hence, in some embodiments, the two checkpoint inhibitors, e.g. an anti-PD-1 antibody and an anti-CTLA-4 antibody, are anchored to the cell surface of the activated dendritic cell via an GPI anchor.

[0037] The treatment with the activated dendritic cells of the invention are highly efficacious in reducing tumor burden, tumor volume, extending the life span and reducing the side effects associated with traditional antibody treatment (e.g., a systemic administration via injection) of the checkpoint inhibitors. Treatment using CTLA-4 antibody by injection is tolerated poorly and associated with numerous negative side effects. Surprisingly, CTLA-4 antibody administered with activated DCs (either secreted or anchored to the surface of the activated DC) avoids or reduces the side effects that have been thought to be inherent ot the CTLA-4 antibodies. The present invention thus provides anti-tumor efficacy while improving the tolerance of the checkpoit inhibitor in the subject.

[0038] The checkpoint inhibitor may be either secreted by the activated dendritic cell or may be attached or anchored to the cell surface of the activated dendritic cell. For the purpose of anchoring to the cell surface, the nucleic acid sequence may comprise a nucleic acid sequence encoding a GPI anchor. Thus, the encoded checkpoint inhibitor may comprise a GPI anchor.

[0039] In one embodiment, the exogenous nucleic acid sequence comprises RNA or DNA. In one embodiment, the exogenous nucleic acid sequence comprises RNA and the RNA comprises mRNA. T08741ANM - 9 -

[0040] In one embodiment, the RNA (e.g. mRNA) comprises at least one chemical modification. Thus, the mRNA encoding the checkpoint inhibitor comprises modified nucleotides.

[0041] If more than one RNA (e.g. mRNA) is used, each RNA (e..g mRNA) independently comprises at least one chemical modification (i.e. the chemical modification from one RNA to another can be different). For the purposes of the present disclosure, chemical modification means that one of the four naturally-occuring standard nucleosides which occur in RNA (adenosine (A), guanosine (G), uridine (U), and cytidine (C)) are replaced by modified forms thereof wherein the modification affects the base moiety within the nucleoside. Modified nucleosides can be naturally-occurring or non- naturally-occurring modified nucleosides. Naturally-occurring modified nucleosides are preferred.

[0042] In one embodiment, the chemical modification is a substitution of one or more nucleosides of the RNA (e.g. mRNA) by one or more modified nucleosides.

[0043] Naturally occurring modified nucleosides comprise 1 -methyladenosine (nriA), N6- methyladenosine (m6A), 2'-O-methyladenosine (Am), 5-methylcytidine (m5C), 2'-O- methylcytidine (Cm), 2-thiocytidine (s2C), N4-acetylcytidine (ac4C), 5-formylcytidine (FC), 2'-O-methylguanosine (Gm), inosine (I), pseudouridine ( ), 5-methyluridine (m5U), 2'-O-m ethyluridine (Um). 1 -methylpseudouridine (ml'P), 2-thiouridine (s2U), 4-thiouridine (s4U), 5-methoxyuridine (mo5U) and 3 -methyluridine (m3U).

[0044] In one embodiment, the RNA (e.g. mRNA) comprises structural elements comprising a 5' untranslated region (UTR), a 3' UTR, a 5' cap and / or a poly(A) tail. In one embodiment, the RNA (e.g. mRNA) contains all of these elements. In one embodiment, the 5' cap is a Capl structure or a m7GpppG cap. Preferably, the 5 ’cap is a Capl structure.

[0045] In one embodiment, the checkpoint inhibitor is exogenous to the activated dendritic cells. The term "exogenous" as used herein, in relation to a specific cell type, e.g., T08741ANM - 10 - activated dendritic cells, can refer to a nucleic acid sequence, e.g. mRNA, or a protein, that originates from a source other than the specified cell type, e.g., the activated dendritic cell. A heterologous mRNA or exogenous mRNA to activated dendritic cells thus refers to an mRNA that is not endogenous to the activated dendritic cell, but has been introduced to the activated dendritic cell, e.g. by transfection or other means.

[0046] In one embodiment, the at least one exogenous nucleic acid sequence (e.g., mRNA) is comprised in a nanoparticle. Nanoparticles comprise lipid nanoparticles, poly(amine- co-ester) particles (PACE), poly-beta-amino-ester particles, PACE polyplex particles, lipoplexes and poly(N,N-cystaminebis(acrylamide)-co-4-amino-l-butanol) (pABOL) particles. In one embodiment, the nanoparticle is a lipid nanoparticle (LNP).

[0047] In one more preferred embodiment, the at least one exogenous nucleic acid (e.g. mRNA) is comprised in a lipid nanoparticle (LNP). A lipid nanoparticle can comprise a cationic lipid, a PEG-modified lipid, a cholesterol, a DSPE-PEG-maleimide, DSPN- PEG-azide and / or a non-cationic lipid. In one embodiment, a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a cholesterol, and / or a non-cationic lipid. In one embodiment, the lipid nanoparticle comprises a cationic lipid, a PEG- modified lipid, a cholesterol and a non-cationic lipid. Cationic lipids comprise cKK- E12, cKK-E14, LP01, SM102, Lipid 5, etc.. In one embodiment, the cationic lipid is a cKK-E12 lipid. In one embodiment, the cationic lipid is a SM102 lipid. In one embodiment, the cationic lipid is a MC3 lipid (DLin-MC3-DMA). In one embodiment, the lipid nanoparticle comprises cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE.

[0048] Second aspect: Ex vivo activated dendritic cell expressing checkpoint inhibitors and one or two further nucleic acid sequences

[0049] In a second aspect, the invention relates to an ex vivo activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least one further exogenous nucleic acid sequence encoding an immune modulator. Hence, in some embodiments, the ex vivo activated dendritic cell comprises T08741ANM - 11 - at least two exogenous nucleic acid sequences, a first exogenous nucleic acid sequence encoding a checkpoint inhibitor as described above, and a second exogenous nucleic acid sequence encoding an immune modulator. In one embodiment, the activated dendritic cell is capable of expressing and / or secreting the immune modulator.

[0050] In one embodiment, the immune modulator is a cytokine. In one embodiment, the immune modulator is a cytokine selected from the group consisting of interleukin- lb (IL- lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL- 18), interleukin-23 (IL-23), interleukin-36y (fL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof. In a preferred embodiment, the cytokine is interleukin- 12 (IL- 12).

[0051] In one embodiment, the cytokine is a chemokine or a chemokine receptor. In one embodiment, the cytokine is a chemokine. In one embodiment, the cytokine is a chemokine receptor, such as CCR7 or XCR1. In one embodiment, the invention provides an ex vivo activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, and at least one further exogenous nucleic acid sequence encoding at least one chemokine or chemokine receptor. The expression of the chemokine receptor on the activated dendritic cell improves the recruitment of the activated dendritic cell to tumor cells and / or tumor-associated lymphoid organs expressing the corresponding chemokine.

[0052] In another embodiment of the second aspect, the invention relates to an ex vivo activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, and at least one further exogenous nucleic acid sequence encoding at least one antigenic peptide. T08741ANM - 12 -

[0053] In another embodiment of the second aspect, the invention relates to an ex vivo activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, at least one further exogenous nucleic acid sequence encoding an immune modulator, and at least one further exogenous nucleic acid sequence encoding at least one antigenic peptide.

[0054] Hence, in some embodiments, the ex vivo activated dendritic cell comprises at least three exogenous nucleic acid sequences, a first exogenous nucleic acid sequence encoding a checkpoint inhibitor as described above, a second exogenous nucleic acid sequence encoding an immune modulator as described above, and a third exogenous nucleic acid sequence encoding at least one antigenic peptide.

[0055] In some embodiments, the first exogenous nucleic acid sequence and the second exogenous nucleic acid sequence encoding an immune modulator as described above are comprised in the same nucleic acid molecule, e.g. on the mRNA molecule. In another embodiment, the first exogenous nucleic acid sequence and the third exogenous nucleic acid sequence encoding at least one antigenic peptide are located on the same nucleic acid molecule, e.g. on the mRNA molecule. In some embodiments, the first exogenous nucleic acid sequence, the second exogenous nucleic acid sequence and the third exogenous nucleic acid sequence are comprised on the same nucleic acid molecule, e.g. the same mRNA molecule.

[0056] The exogenous nucleic acid molecules (such as the first, second and / or third exogenous nucleic acid molecule) may be loaded in the same LNP. The exogenous nucleic acid molecules (such as the first, second and / or third exogenous nucleic acid molecule) may be loaded into separate LNPs. If separate LNPs are used, the activated dendritic cells may be co-incubated with a mixture of the LNPs, e.g. with an 1 :1 ratio of LNPs comprising the first, second, and / or third exogenous nucleic acid sequence.

[0057] In one embodiment, the at least one antigenic peptide is a tumor-associated peptide or protein, a viral antigenic protein, a bacterial antigenic protein, or a fungal antigenic T08741ANM - 13 - protein. In one embodiment, the at least one antigenic peptide is a tumor-associated antigen (TAA), such as a tumor-associated peptide or protein, and wherein the tumor- associated peptide or protein is a tumor-specific peptide or protein, preferably wherein the tumor-specific peptide or protein comprises at least one tumor-specific neoantigen. The identification of and isolation or generation of a tumor-specific neoantigen is routine for the skilled person (see e.g. Lang F, Schrbrs B, Lower M, Tiireci O, Sahin U. Identification of neoantigens for individualized therapeutic cancer vaccines. Nat Rev Drug Discov. 2022 Apr;21(4):261-282. doi: 10.1038 / s41573-021- 00387-y; Xie et al., Neoantigens: promising targets for cancer therapy, 2023, Sig Transduct Target Ther , 9 (2023). https: / / doi.org / 10.1038 / s41392-022-01270-x). In one particular embodiment, the at least one tumor-specific neo-antigen comprises a sequence of Table. 1. It will be routine for the skilled person in the art to use tumorspecific antigens that are specific to the patient to be treated. Hence, in some embodiments, the tumor-specific or tumor-associated antigen will be first identified and isolated from the patient, and subsequently the nucleic acid sequence encoding for the tumor-specific or tumor-associated antigen will be loaded into LNPs and combined with the activated dendritic cell.

[0058] Third aspect: Pharmaceutical composition comprising the activated dendritic cell and a pharmaceutically acceptable carrier

[0059] In a third aspect, the invention relates to a pharmaceutical composition comprising the activated dendritic cell described herein and a pharmaceutically acceptable carrier.

[0060] In one embodiment, the pharmaceutical composition comprises the activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor.

[0061] In another embodiment, the pharmaceutical composition comprises the activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a T08741ANM - 14 - checkpoint inhibitor and at least one further exogenous nucleic acid sequence as described above.

[0062] In one embodiment, the pharmaceutical composition further comprises an immune modulator. The immune modulator may be comprised in the pharmaceutical compositing in addition to the activated dendritic cell.

[0063] The immune modulator may be expressed by the same activated dendritic cell that comprises the at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor. Alternatively, or in addition, the immune modulator may be expressed by a second activated dendritic cell. In one embodiment, the second activated dendritic cell is capable of expressing and / or secreting the immune modulator.

[0064] Hence, in one embodiment, the pharmaceutical composition comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding an immune modulator.

[0065] In one embodiment, the immune modulator is a cytokine.

[0066] In one embodiment, the cytokine is capable of inducing CD8+ T cells.

[0067] In one embodiment, the immune modulator is selected from a cytokine of the group consisting of interleukin- lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL-15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL- 18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof.

[0068] In a preferred embodiment, the cytokine is interleukin- 12 (IL-12).

[0069] In another embodiment, the cytokine is a chemokine. In one embodiment, the cytokine is a chemokine receptor, such as CCR7 or XCR1. In one embodiment, the invention provides an ex vivo activated dendritic cell comprising at least one T08741ANM - 15 - exogenous nucleic acid sequence encoding a checkpoint inhibitor, and at least one further exogenous nucleic acid sequence encoding at least one chemokine or chemokine receptor. The expression of the chemokine receptor on the activated dendritic cell improves the recruitment of the activated dendritic cell to tumor cells and / or tumor-associated lymphoid organs expressing the corresponding chemokine.

[0070] In another embodiment, the pharmaceutical composition comprises the first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide.

[0071] In one embodiment, the at least one antigenic peptide is a tumor-associated peptide or protein, a viral antigenic protein, a bacterial antigenic protein, or a fungal antigenic protein. In one embodiment, the at least one antigenic peptide is a tumor- associated antigen (TAA), such as a tumor-associated peptide or protein, and wherein the tumor-associated peptide or protein is a tumor-specific peptide or protein, preferably wherein the tumor-specific peptide or protein comprises at least one tumor-specific neo-antigen. The identification of and isolation or generation of a tumor-specific neo-antigen is routine for the skilled person (see e.g. Lang F, Schrbrs B, Lower M, Tiireci O, Sahin U. Identification of neoantigens for individualized therapeutic cancer vaccines. Nat Rev Drug Discov. 2022 Apr;21(4):261-282. doi: 10.1038 / s41573-021-00387-y; Xie et al., Neoantigens: promising targets for cancer therapy, 2023, Sig Transduct Target Ther 8, 9 (2023). https: / / doi.org / 10.1038 / s41392-022-01270-x Error! Hyperlink reference not valid.; Li, J., Xiao, Z., Wang, D. et al. The screening, identification, design and clinical application of tumor-specific neoantigens for TCR-T cells. Mol Cancer 22, 141 (2023). https: / / doi.Org / 10. l 186 / sl2943-023-01844-5). In one particular embodiment, the at least one tumor-specific neo-antigen comprises a sequence of Table 1 .It will be routine for the skilled person in the art to use tumor-specific T08741ANM - 16 - antigens that are specific to the patient to be treated. Hence, in some embodiments, the tumor-specific or tumor-associated antigen will be first identified and isolated from the patient, and subsequently the nucleic acid sequence encoding for the tumorspecific or tumor-associated antigen will be loaded into LNPs and combined with the activated dendritic cell.

[0072] In another embodiment, the pharmaceutical composition comprises the first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide; and further comprises an immune modulator.

[0073] In another embodiment, the pharmaceutical composition a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least two additional activated dendritic cells comprising different exogenous nucleic acid sequences. Hence, in one embodiment, the pharmaceutical composition a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor; at least one second activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding an immune modulator; and at least one third activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding at least one antigenic peptide or tumor- associated antigen (TAA).

[0074] In all embodiments listed above, the pharmaceutical composition of the invention optionally comprises a pharmaceutically acceptable carrier and / or diluent. Additionally, the pharmaceutical composition can comprise adjuvants and / or immuno-modulators to boost the activity of the pharmaceutical composition and the subject’s response. Such adjuvants and / or immuno-modulators are understood by those skilled in the art, and are readily described in available published literature. T08741ANM - 17 -

[0075] Fourth aspect: The ex vivo activated dendritic cell or the pharmaceutical composition for use in therapy

[0076] In a further aspect, the invention relates to the ex vivo activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor or the pharmaceutical composition comprising the same for use in therapy.

[0077] Specifically, the invention relates to the ex vivo activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor or the pharmaceutical composition comprising the same for use in a method of treating cancer in a subject in need thereof.

[0078] In one embodiment, the method comprises administering a therapeutically effective amount of the activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor or the pharmaceutical composition of the invention to the subject. In one embodiment, the treatment prolongs the median survival of the subject compared to a subject having cancer and being treated with an activated dendritic cell or a checkpoint inhibitor alone. In one embodiment, the activated dendritic cell of the invention inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces tumor growth rate, eradicates the tumor or tumor cells, and / or prolongs the median survival period relative to treatment with an activated dendritic cell or a checkpoint inhibitor alone.

[0079] In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the activated dendritic cells of the invention.

[0080] In one embodiment, the cancer is a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or a germ cell tumor. In one embodiment, the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis T08741ANM - 18 - fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, and pancreatic cancer .

[0081] In one embodiment, the activated dendritic cell or the pharmaceutical composition of the invention is administered locally or systemically to the subject.

[0082] In some embodiments, the method further comprises administration of a further checkpoint inhibitor.

[0083] In one aspect of the invention, the invention relates to a pharmaceutical composition comprising a first activated dendritic cell of the invention and a second and / or a third activated dendritic cell, as described above, for use in a method of treating cancer.

[0084] In one embodiment, the first activated dendritic cell of the invention is administered separately, simultaneously or sequentially from the second and / or the third activated dendritic cell. In one embodiment, the first activated dendritic cell of the invention, the second activated dendritic cell and / or the third activated dendritic cell are administered in a pre-defined order. In one embodiment, the first activated dendritic cell of the invention, the second and / or the third activated dendritic cell are administered repeatedly to the subject. In one embodiment, the first activated dendritic cell of the invention, the second and / or the third activated dendritic cell are administered at least twice, 3-times, 4-times, 5-times, 6-times or more to the subject. T08741ANM - 19 -

[0085] In one embodiment, the combined treatment with the first activated dendritic cell of the invention in conjunction with the second and / or the third activated dendritic cell prolongs the median survival of the treated subject compared to a subject being treated with any one of the first activated dendritic cell of the invention, the pharmaceutical composition, the second or the third activated dendritic cell alone. In one embodiment, the combined treatment with the first activated dendritic cell of the invention with the second and / or the third activated dendritic cell inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces tumor growth rate, eradicates the tumor or tumor cells, and / or prolongs the median survival period relative to treatment with the activated dendritic cell of the invention, the pharmaceutical composition of the invention, the second or the third activated dendritic cell alone.

[0086] In another aspect of the invention, the invention relates to the activated dendritic cell of the invention comprising at least one exogenous mRNA encoding a checkpoint inhibitor, at least one further exogenous mRNA encoding an immune modulator, and / or at least one further exogenous mRNA encoding at least one antigenic peptide; for use in treatment of cancer.

[0087] In one embodiment, the treatment with the activated dendritic cell of the invention co-expressing and / or secreting a checkpoint inhibitor with one or both of an immune modulator and / or at least one antigenic peptide prolongs the median survival of the treated subject compared to a subject being treated with any one of the first activated dendritic cell of the invention, the pharmaceutical composition of the invention, the second or the third activated dendritic cell alone.

[0088] In another embodiment, the treatment with the activated dendritic cell of the invention co-expressing and / or secreting a checkpoint inhibitor with one or both of an immune modulator and / or at least one antigenic peptide inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces tumor growth rate, eradicates the tumor or tumor cells, and / or prolongs the median T08741ANM - 20 - survival period relative to treatment with the activated dendritic cell of the invention, the pharmaceutical composition of the invention, the second and / or the third activated dendritic cell alone.

[0089] Fifth aspect: Method for preparing the ex vivo activated dendritic cell

[0090] In one aspect, the invention provides a method for preparing the ex vivo activated dendritic cell of the invention, the method comprising the step of combining an activated dendritic cell with at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor.

[0091] The activated dendritic cell (also known as physiologic dendritic cell or PhDC) may be obtained by methods known in the art, e.g. Hanlon et al, 2020 (“Rapid Production of Physiologic Dendritic Cells (phDCs) for Immunotherapy”), Ventura et al., 2018 “Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity”, WO 2014 / 106629, WO 2016 / 001405, WO2014 / 106629 Al, W02014 / 106631 Al, W02016 / 001405 Al, and WO20 17 / 005700 Al, each of which is incorporated herein by reference in its entirety. Thus, the activate dendritic cell is obtained by a cytokine-independent method.

[0092] In one embodiment, in order to obtain the activated dendritic of the invention, i.e. the activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, the activated dendritic cell is incubated with the at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor. Incubation can be performed under standard conditions for culturing of human cells, e.g. at 37° C and 5% CO2 in standard mediums such as in RPMI-1640 medium, plasma or saline. In one embodiment, the incubation is performed in plasma, e.g. human plasma. In another embodiment, the incubation is performed in saline. Alternatively or additionally, an incubation step can also be inserted after generation of activated dendritic cells from monocytes. T08741ANM - 21 -

[0093] The incubation can be performed for 0.1 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h or 24 h. In another embodiment, the incubation step can be performed for at least 0.1 h, at least 0.5 h, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 8h, at least 12 h or at least 20h. In one embodiment, incubation is performed for 4 h. In one embodiment, incubation is performed for 5 h. In one embodiment, incubation is performed for 6 h. In one embodiment, incubation is performed for 12 h. In one embodiment, incubation is performed for 20 h. In one embodiment, incubation is performed for 6 to 20 hours. In one embodiment, incubation is performed for 8 to 20 hours. In one embodiment, incubation is performed overnight, such as about 16 h, about 18 h, about 20 h, or about 24 h. In general, activated dendritic cells have a higher transfection capability as compared to other DCs. The incubation step increases transfection efficiency. The incubation step further increases the degree of maturation of the monocytes to activated dendritic cells, wherein a shorter incubation period (e.g. 4 h, 6 hr or 8 h) is associated with less mature or immature dendritic cells, and a longer incubation period (e.g. at least 12 h, 14h, 16h, 20 h or 24 h) is associated with more mature activated dendritic cells.

[0094] The at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor may be incubated with the activated dendritic cell in any suitable form, including but not limited to nanoparticles, lipid nanoparticles (LNPs), polymeric nanoparticles, lipidpolymer hybrid nanoparticles, synthetic lipopolymer hybrid nanoparticles, proteins or peptides-mediated nanoparticles. Any delivery mode may be used to deliver the exogenous nucleic acid sequence into the activated dendritic cell. Exemplary delivery platforms disclosed e.g. in Zhong et al. (mRNA delivery in cancer immunotherapy. Acta Pharm Sin B. 2023 Apr;13(4): 1348-1357. doi: 10.1016 / j.apsb.2023.03.001) are incorporated herein and may be used to deliver the exogenous nucleic acid sequence into the activated dendritic cell.

[0095] In a preferred embodiment, the exogenous nucleic acid sequence encoding a checkpoint inhibitor, an immune modulator, or an antigenic peptide is comprised in a LNP. T08741ANM - 22 -

[0096] In a preferred embodiment, the at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor is comprised in LNPs, and these LNPs are used for the incubation step with the activated dendritic cells.

[0097] The LNPs comprising the exogenous nucleic acid may be added to the blood sample before, during or after the blood sample is exposed to shear stress suitable to activate the monocytes (non-limiting examples of such suitable shear stress include a blood bag, flow chamber or transimmunization chamber). The blood sample may be whole blood, an apheresis product, isolated monocytes or a monocyte concentrate. In a preferred embodiment, the blood sample comprises concentrated monocytes. In another preferred embodiment, the blood sample is whole blood. Hence, in one embodiment, the LNPs are mixed with the blood sample (e.g., concentrated monocytes, apheresis product or whole blood) before the start of the activation procedure, and hence prior to monocyte processing and activation to generate activated dendritic cells. In one embodiment, the LNPs are added to the blood sample (e.g., concentrated monocytes, apheresis product or whole blood) before monocyte activation. In another embodiment, the LNPs are added to the blood sample (e.g., concentrated monocytes, apheresis product or whole blood) during monocyte activation. In a further embodiment, the LNPs are added to blood sample (e.g., concentrated monocytes, apheresis product or whole blood) or activated dendritic cells after monocyte activation.

[0098] In one embodiment, the incubation time period is measured from the point of addition of LNPs comprising the exogenous nucleic acid to the blood sample (e.g., concentrated monocytes, apheresis product or whole blood). Hence, in one embodiment, the incubation time is measured from the start of the monocyte activation procedure or beginning of adding the blood sample comprising the monocyte to the flow chamber. Hence in one embodiment, the incubation time is measured from the start of addition of the blood sample to the activation device, such as the transimmunization chamber, the blood bag or the flow chamber. In one embodiment, the LNPs are added to the cells prior to or at the beginning of the activation procedure T08741ANM - 23 -

[0099] (i.e. the administration of the blood sample to the activation device, e.g., transimmunization chamber, the blood bag or the flow chamber). The incubation can be brief (such as O.lh, 0.5 h, 0.5-1 h, 1-1.5 h, 1.5-2 h) or longer. Should the LNPs be added to the monocytes during the activation procedure, the incubation time is calculated from the time point of the LNPs and monocytes coming into contact. In another embodiment, if the LNPs are added to the blood sample after the activation procedure, the incubation time is measured from the moment of adding the LNPs.

[0100] In one embodiment, the incubation with the activated dendritic cells and LNPs comprising the at least one exogenous nucleic acid sequence (e.g. mRNA comprised in LNPs) encoding a checkpoint inhibitor is performed for about 4 hours. In another embodiment, the incubation is performed for about 4-6 h, 6-8 h, 8-10 h, 10-12 h, 12- 14 h, 14-16 h, 16-18 h, 18-20 h, 20-22 h, 22-24 h or longer than 24 h. Preferably, the incubation is performed for less than 8 h or less than 6 h or 4 h to obtain immature activated dendritic cells that comprise the LNP comprising the at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor. In another preferred embodiment, the incubation is performed for longer than 16 h, such as for about 18- 20 h to obtain mature activated dendritic cells that comprise the LNP comprising the at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor.

[0101] Monocytes may be obtained by any suitable means, e.g., from a blood sample obtained from a donor or a fraction thereof. The blood sample or fraction thereof may be, e.g., a buffy coat including white blood cells and platelets. In some embodiments, platelets are present in residual or small quantities. Alternatively, the blood sample or fraction thereof may be isolated peripheral blood mononuclear cells (PMBC). In one embodiment, the monocytes are autologous.

[0102] In one embodiment, the exogenous nucleic acid sequence is mRNA and comprised in a lipid nanoparticle (LNP). Hence, in one embodiment, the activated dendritic cell is incubated with the at least one LNP comprising at least one exogenous mRNA T08741ANM - 24 - encoding a checkpoint inhibitor. In one embodiment, the activated dendritic cell is incubated with the at least one LNP comprising at least one exogenous mRNA encoding a checkpoint inhibitor for a time period sufficient for the activated dendritic cells to take up the LNP. In one embodiment, the time period is at least 0.1 h, at least 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h or 24 h. In one embodiment, the incubation is performed for about 4-6 h, 6-8 h, 8-10 h, 10-12 h, 12-14 h, 14-16 h, 16-18 h, 18-20 h, 20-22 h, 22-24 h or longer than 24 h. Preferably, the incubation is performed for less than 8 h or less than 6 h or 4 h to obtain immature activated dendritic cells that comprise the LNP comprising the mRNA encoding a checkpoint inhibitor. In another preferred embodiment, the incubation is performed for longer than 16 h, such as for about 18-20 h to obtain mature activated dendritic cells that comprise the LNP comprising the mRNA encoding a checkpoint inhibitor.

[0103] In one embodiment, the ex vivo activated dendritic cell is not incubated with an exogenous checkpoint inhibitor molecule.

[0104] In one embodiment, the method for preparing the ex vivo activated dendritic cell of the invention comprises subjecting monocytes (e.g. obtained from a donor) to a physical force. The physical force can be applied to the monocytes by passing said monocytes through a flow chamber.

[0105] Hence, in one embodiment, the method for preparing the ex vivo activated dendritic cell of the invention comprises subjecting monocytes (e.g. obtained from a donor) to a physical force in order to obtain ex vivo activated dendritic cell; and incubating the activated dendritic cell with the at least one LNP comprising at least one exogenous mRNA encoding a checkpoint inhibitor.

[0106] The flow chamber can be a plate, a bag or a flow chamber of a device, e.g. such as a large-scale activation device, e.g., a clinical device (e.g., a THERAKOS® CELLEX® device; Combination of Terumo’s Spectra Optia with UVA PIT; Apheresis device Amicus Blue from Fresenius Kabi; Single-Needle Option for the Amicus® T08741ANM - 25 -

[0107] Extracorporeal Photopheresis Protocol), or a miniaturized device, e.g., a Transimmunization plate as described in WO2017 / 005700 Al, or a combination thereof (e.g. a hybrid of a bag and a chamber as disclosed herein). In one embodiment of the present invention, the method for preparing the ex vivo activated dendritic cell comprises plate-passage of monocytes using a flow chamber to activate the monocytes comprised in the blood sample to generate activated dendritic cells. Thus, in one embodiment, the flow chamber is a plate. Monocytes (e.g. obtained from a donor) are passed through a plate such that the monocytes are exposed to shear forces. Preferably, platelets are present in the plate which can be either derived from the donor' s blood sample or a fraction thereof or provided separately. Additionally or alternatively, plasma components can be present in the plate which can be either derived from the donor' s blood sample or a fraction thereof or provided separately. The process of preparing activated dendritic cells (previously also known as physiologic dendritic cells)has been described previously in, for example, Hanlon et al, 2020 (“Rapid Production of Physiologic Dendritic Cells (phDCs) for Immunotherapy”). In one embodiment, all method steps are carried out in vitro.

[0108] In another embodiment, the flow chamber is a bag. Optionally, the bag is a flexible bag or a plastic bag. In one embodiment, the flow chamber is a flexible bag. In one embodiment, the bag is a plastic bag. In one embodiment, the material of the flow chamber (e.g. plate, flexible bag or plastic bag) is plastic. In one embodiment, the material of the flow-chamber is non-plastic such as glass, ceramic or silicone. If plastic materials are considered, one may use acrylics, polycarbonate, polyetherimide, polysulfone, polyphenyl sulfone, styrenes, polyurethane, polyethylene, teflon or any other appropriate medical grade plastic. In a preferred embodiment, the flow chamber is made from acrylic plastic, preferably polymethyl methacrylate (PMMA) and / or polystyrol (PS). If a bag (e.g. flexible bag) is considered, the material may be plastic, rubber or silicone. In a preferred embodiment, the material is plastic. Plastic materials comprise polyolefin, polyethylene, fluoropolymer, polyvinyl chloride, ethylene-vinyl T08741ANM - 26 - acetate-copolymer, ethylene vinyl alcohol, polyvinylidene fluoride, and / or other plastic comprising materials approved for medical use.

[0109] In some embodiments, the flow chamber comprises or consists of a plate. The plate can be made of various materials, including but not limited to a plastic material. In one embodiment, the material of the plate is plastic. In one embodiment, the material of the plate is non-plastic such as glass, ceramic or silicone. Non limiting examples of materials for the plate comprise acrylics, polycarbonate, polyetherimide, polysulfone, polyphenylsulfone, styrenes, polyurethane, polyethylene, teflon or any other appropriate medical grade plastic. The plate may be rigid or flexible. In some embodiment, the material of the plate may comprise or consist of plastic, rubber or silicone. In some embodiments, the plate is elastic, i.e. is made of an elastic material. The elastic material may comprise cyclic olefin copolymer (COC), polyolefin, polyethylene, fluoropolymer, polyvinyl chloride, ethylene-vinyl acetate-copolymer, ethylene vinyl alcohol, polyvinylidene fluoride, polydimethylsiloxane (PDMS), dimethicone, and / or other plastic comprising materials approved for medical use. In one embodiment, the plate is made of PDMS, e.g., PDMS RTV-615 or PDMS Sylgard In another preferred embodiment of the present invention, the plate is made from an acrylic plastic.

[0110] In some embodiments, the flow chamber is a hybrid flow chamber. The hybrid flow chamber may comprise a chamber and a bag, a chamber and a plate, or a bag and a plate. The individual components of the hybrid flow chamber, i.e. the bag, plate or chamber, are as defined herein.

[0111] In one embodiment, human AB serum, autologous (e.g. human) serum, autologous (e.g. human) plasma, allogeneic (e.g. human) serum, allogeneic (e.g. human) plasma, mouse serum, mouse plasma or FBS is added additionally to the composition. In a preferred embodiment, human serum or plasma is used. In one embodiment, autologous (e.g. human) serum, autologous (e.g. human) plasma, allogeneic (e.g. human) serum, allogeneic (e.g. human) plasma, mouse serum, mouse plasma or FBS is added additionally to the composition. In one embodiment, mouse serum, mouse T08741ANM - 27 - plasma or FBS is added additionally to the composition. Alternatively, the flow chamber, bag, plate or hybrid flow chamber can be coated with human AB serum, autologous (e.g. human) serum, autologous (e.g. human) plasma, allogeneic (e.g. human) serum, allogeneic (e.g. human) plasma, mouse serum, mouse plasma or FBS before the monocytes are added. In one embodiment, the flow chamber, bag, plate or hybrid flow chamber is coated with autologous (e.g. human) serum, autologous (e.g. human) plasma, allogeneic (e.g. human) serum, allogeneic (e.g. human) plasma, mouse serum, mouse plasma or FBS. In another embodiment, the flow chamber, bag, plate or hybrid flow chamber is coated with mouse serum, mouse plasma or FBS.

[0112] Sixth aspect: ex vivo activated dendritic cell obtainable by the methods provided herein

[0113] In one further aspect, the invention provides an ex vivo activated dendritic cell of the invention obtainable by the methods described herein.

[0114] In one embodiment, the first ex vivo activated dendritic cell of the invention is obtainable by combining a monocyte with at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor; and subjecting the monocyte and the at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor to a physical force.

[0115] In one embodiment, the second ex vivo activated dendritic cell of the invention is obtainable by combining a monocyte with at least one exogenous nucleic acid sequence encoding an immune modulator; and subjecting the monocyte and the at least one exogenous nucleic acid sequence encoding an immune modulator to a physical force. T08741ANM - 28 -

[0116] In one embodiment, the third ex vivo activated dendritic cell of the invention is obtainable by combining a monocyte with at least one exogenous nucleic acid sequence encoding at least one antigenic peptide; and subjecting the monocyte and the at least one exogenous nucleic acid sequence encoding at least one antigenic peptide to a physical force.

[0117] In one embodiment, the first ex vivo activated dendritic cell of the invention is obtainable by combining a monocyte with at least one lipid nanoparticle (LNP) comprising an exogenous mRNA encoding a checkpoint inhibitor; and subjecting the monocyte and the at least one LNP to a physical force.

[0118] In some embodiments, the invention relates to an ex vivo activated dendritic cell comprising an exogenous nucleic acid sequence expressing a checkpoint inhibitor and at least one further exogenous nucleic acid sequence. Hence, in one embodiment, the ex vivo activated dendritic cell is obtainable by combining a monocyte with at least one lipid nanoparticle (LNP) comprising an exogenous mRNA encoding a checkpoint inhibitor, and at least one LNP comprising an exogenous mRNA encoding an immune modulator; and subjecting the monocyte and the LNPs to a physical force.

[0119] In another embodiment, the ex vivo activated dendritic cell of the invention is obtainable by combining a monocyte with at least one lipid nanoparticle (LNP) comprising an exogenous mRNA encoding a checkpoint inhibitor, and at least one LNP comprising an exogenous mRNA encoding at least one antigenic peptide; and subjecting the monocyte and the LNPs to a physical force. T08741ANM - 29 -

[0120] In another embodiment, the ex vivo activated dendritic cell of the invention is obtainable by combining a monocyte with at least one lipid nanoparticle (LNP) comprising an exogenous mRNA encoding a checkpoint inhibitor, at least one LNP comprising an exogenous mRNA encoding an immune modulator, and at least one LNP comprising an exogenous mRNA encoding at least one antigenic peptide; and subjecting the monocyte and the LNPs to a physical force.

[0121] In one embodiment, the ex vivo activated dendritic cell of the invention is obtainable by a method comprising combining the activated dendritic cell with at least one exogenous mRNA encoding a checkpoint inhibitor. In one embodiment, the method comprises incubating a LNP comprising the at least one exogenous mRNA encoding a checkpoint inhibitor with an activated dendritic cells for a time period sufficient for the activated dendritic cells to take up the LNP. In one embodiment, incubating the LNP with an activated dendritic cell means adding the LNP to the activated dendritic cell. In one embodiment, the time period sufficient for the activated dendritic cells to take up the LNP is at least 0.1 h.

[0122] A physical force (e.g. shear force) can be applied by passing monocytes through a flow chamber which can take the form of, e.g., a plate or, e.g., a bag (e.g. flexible bag or plastic bag) or a combination thereof (e.g. hybrid of a plate and bag). The at least one mRNA can be comprised in nanoparticles, lipid nanoparticles in particular. All embodiments, e.g. as regards the at least one nucleic acid sequence, the encoded proteins, nanoparticles (in particular lipid nanoparticles), or incubation methods, as disclosed herein, apply mutatis mutandis.

[0123] Seventh aspect: A kit comprising a plurality of ex vivo activated dendritic cells and LNPs comprising at least one mRNA encoding a checkpoint inhibitor T08741ANM - 30 -

[0124] In another aspect, the present invention relates to a kit comprising a plurality of ex vivo activated dendritic cells and at least one exogenous nucleic acid sequence (e.g. mRNA), which encodes for a checkpoint inhibitor.

[0125] The kit may further comprise nanoparticles (e.g. lipid nanoparticles). In case the kit comprises nanoparticles, the nanoparticles comprise the exogenous nucleic acid sequence (e.g. mRNA), which encodes for the checkpoint inhibitor. As regards the activated dendritic cells, nanoparticles (e.g. lipid nanoparticles), exogenous nucleic acid sequence (e.g. mRNA) and checkpoint inhibitor, the respective embodiments described herein apply mutatis mutandis.

[0126] In one embodiment, the kit further comprises at least one exogenous mRNA encoding a cytokine. In one embodiment, the kit further comprises at least one exogenous mRNA encoding at least one antigenic peptide.

[0127] In one embodiment, the at least one exogenous mRNA encoding a cytokine and the at least one exogenous mRNA encoding at least one antigenic peptide are either

[0128] (i) expressed by the same activated dendritic cell as the at least one mRNA encoding a checkpoint inhibitor, or

[0129] (ii) each expressed by a different activated dendritic cell.

[0130] Hence, in one embodiment, the invention provides a kit comprising a plurality of ex vivo activated dendritic cells and at least one exogenous mRNA encoding a checkpoint inhibitor; and at least one exogenous mRNA encoding a cytokine and at least one exogenous mRNA encoding at least one antigenic peptide, wherein the exogenous mRNAs are either

[0131] (i) expressed by the same activated dendritic cell, or

[0132] (ii) each expressed by a different activated dendritic cell. T08741ANM - 31 -

[0133] In one embodiment, the kit further comprises at least one LNPS comprising the at least one exogenous mRNA encoding for the cytokine, the immune modulator (e.g. a checkpoint inhibitor) and / or the antigenic peptide..

[0134] FIGURE LEGENDS

[0135] Figure 1 Murine activated dendritic cells (aDCs) specifically internalize and express Spike protein encoded by mRNA-containing LNPs. A, B, FACS plot showing spike protein positivity in CDl lb positive aDCs without LNP transfection (A) and with LNP transfection (B). C, D, Confocal microscopy analysis using same sample showing spike protein expression in CD1 lb+Ly6G- aDCs (projected Z-stack images). E. Z-plane slice image of the LNP transfected aDC.

[0136] Figure 2 A, C57BL / 6 mice were administered i.v. treatment with aDCs transduced with OVA mRNA LNP, or treated with OVA mRNA LNP via i.m. route (no aDCs), 2 days post EG7-0VA subcutaneous tumor implant (3xl06cells per mouse). Approximately lOng of LNP were used to transduce the aDCs (per mouse), and same amount was injected via i.m. per mouse (0.5ug / kg of LNP, dose equivalent to COVID-19 Pfizer mRNA LNP vaccine in current human vaccine setting). Treatments were given twice per week, with each mouse receiving a total of 5 treatments. Tumors were visible and palpable (>50mm3) at the time of treatment initiation. B, EG7-0VA tumor growth was monitored throughout the experiment.

[0137] Figure 3 A, aDCfova] or IMfova] mice were given five total treatments at lOng [ova] per mouse, starting two days following EG7-0VA subcutaneous tumor inoculation with 3*106cells per mouse (same experiment as Fig. T08741ANM - 32 -

[0138] 3). B-C, tumor growth was monitored for the duration of the experiment, with cumulative tumor growth per experimental group (B) and individual mouse tumor growth curves (C) represented. D-E, CD8+ splenic T cells were isolated from treated mice on Day 30 and immediately placed into an 18hr IFN-g Elispot assay at l*105cells per well in the presence or absence of lOug / mL SIINFEKL peptide. Representative Elispot well images (D) and IFNg spot quantitation (E) are provided for each experimental group. *[ova] = LNP-mRNA encoding ovalbumin.

[0139] Figure 4 A, aDCfova] or IMfova] mice were given five total treatments at lOng [ova] per mouse, starting two days following EG7-OVA subcutaneous tumor inoculation with 3*106cells per mouse (same experiment as Fig. 3). Splenocytes from the treated mice were harvested at the end of the 28-day observation period for T cell analysis and characterization by flow cytometry of A, antigen (SIINFEKL)-specific CD8 T cells via dextramer analysis; Tern (effector) / Tcm (central memory) type phenotype analysis via CD44 / CD62L expression; detection of stemlike T cell via IL7Ra / SCA-l expression, and B, T cell exhaustion marker evaluation via PD-1 expression. *[ova] = LNP-mRNA encoding ovalbumin.

[0140] Figure 5 aDCfova] or IM[ova] mice were vaccinated (prime / boost) on days -7 and 0, respectively. Undifferentiated splenocytes were harvested post vaccination and immediately placed in an 18hr IFN-g Elispot without added antigenic stimulation (A, B and C). To identify cells responsible for splenic IFN-g, isolated CD8+ orNKl.U- splenocytes were collected post vaccination from aDCfova] mice and assessed for IFN-g spots (D). *[ova] = LNP-mRNA encoding ovalbumin.

[0141] Figure 6 A, C57BL / 6 mice were administered i.v. treatment with aDCs transduced with lug / mL OVA mRNA LNP, or treated i.v. with soluble T08741ANM - 33 -

[0142] OVA protein (50ug / mouse, no aDCs), on days -14 and -7 prior to EG7- OVA subcutaneous tumor implant (IxlO6cells per mouse). B, EG7- OVA tumor growth was monitored throughout the experiment.

[0143] Figure 7 Mouse aDCs were transduced with OVA mRNA LNP (CKK) at 1 ug / ml, and stained for intracellular OVA protein after over-night culture. aDCs transduced with SP LNP (1 ug / ml) placed as control. (n=3) OVA detection with Rockland OVA ab (FITC conjugated).

[0144] Figure 8 Mouse aDCs were incubated overnight with OVA in various antigen form; soluble, expressed in tumor cells or OVA encoding mRNA containing LNPs. Cells were then harvested, and stained for surface CD 11b, Ly6G and 25. DI (ab against SIINFEKL bound MHC I in H- 2Kb strain). Ly6G positive cells were selected out for neutrophil exclusion. FACs plot showing 25. DI positivity on CD1 lb+ aDCs.

[0145] Figure 9 aDCs (PP PBMCs from B6 mice) were transduced with OVA mRNA LNPs (1 ug / ml) in standard overnight culture protocol. Cells were harvested, washed and fixed (intraprep kit) at various time points, and stained for surface CD1 lb and SIINFEKL-MHC I complex (25. DI ab). 25.D1 expression level show in CDl lb+ cells at various time points shown in FACs plot (upper panel). Percentage of 25. DI positive cells and MFI level of 25. DI in CD1 lb+ subset shown in bar graph (below).

[0146] Figure 10 Enriched aDCs (monocytes purified from plate passed PBMCs from mice) or BMDC (cytokine induced DC from bone marrow of mice) were incubated over-night with / without LNPs containing OVA mRNA 0.1 ug / ml in standard overnight protocol. Cells were harvested and stained for surface CD 11b and 25. DI (ab against SIINFEKL bound MHC I in H-2Kb strain) for aDC samples, and CDl lc and 25. DI for BMDC sample. FACs plot showing 25. DI positivity on CD1 lb+ aDCs and CDl lc+ BMDCs.

[0147] Figure 11 Mouse aDCs were transduced with either SIINFEKL peptide mRNA T08741ANM - 34 -

[0148] LNP or OVA protein mRNA LNP. After over-night incubation, cells were harvested and stained with 25. DI ab for detection of SIINFEKL MHC I complex signal. FACS plot showing 25. DI signal on CD1 lb+ aDC subset.

[0149] Figure 12 aDCs from mouse were pulsed with OVA mRNA LNPs at differing concentration, then cultured with OT1 T cells. Proliferation of the OT1 CD8 T cells (CFSE labeled) was evaluated as shown by reduction in CFSE signal.

[0150] Figure 13 aDCs from mouse (B6) were pulsed with 1 ug / ml OVA mRNA, then cultured with 100K OT1 T cells at differing aDC number (96 well U bottom plate). Proliferation of the OT1 CD8 T cells (CFSE labeled) were evaluated as readout.

[0151] Figure 14 C57BL / 6 mice were administered i.v. treatment with aDCs transduced with OVA mRNA LNP* or injected with with mock (SP) mRNA LNP* or OVA mRNA LNP via i.m.* route (no aDC), 4 days post EG7 tumor implant (3x106 cells per mouse). Treatments were given twice per week (Tx on day 4, 8, 11, 15, 18 and 25). Tumors were visible and palpable (>50mm3) at the time of treatment initiation. N=10 for each groups.

[0152] Figure 15 (a) Splenocytes from mice in experiment 21-1008 (Therapeutic exp,

[0153] HB) were analyzed for SIINFEKL tetramer positivity at the end of the in vivo tumor monitoring period (day 32). H-2Kb OVA SIINFEKL tetramer positivity on CD8+ subset shown in FACs plot. Splenocytes from 10 mice pooled prior to analysis. Last treatment given 1 week prior to analysis, (b) T cells isolated from the splenocytes of the three experimental groups were transferred (15M T cells / mouse) into EG7 tumor bearing mice, and tumor development was monitored over the course of 19 days.

[0154] Figure 16: Spike protein ELISpot detection of SARS-CoV-2 reactive T cells. A: T08741ANM - 35 -

[0155] Representative image of an aDC ELISpot using PBMC isolated from a single human donor, 4 weeks post SARS-CoV2 infection. [Spike] indicates aDC transduced with LNP containing mRNA encoding Spike protein. Positive control wells are treated with an overlapping pool of Spike Class I & II peptides (Miltenyi Biotec, PepTivatorSARS-CoV-2 Prot-S Complete). Negative control wells are treated with 62.5- 250ng / well of cKK-E12 based LNP containing an irrelevant (eg Nanoluciferase) protein mRNA, and untreated control (No Ag). B: An example of an aDC ELISpot dose response using PBMC from a single human donor, 4 weeks post SARS-CoV2 infection. IFNy release increased to levels above 150 SFU / million when LNP [Spike] antigen was added at 62.5ng / well.

[0156] Figure 17: Spike protein ELISpot detection of SARS-CoV-2 reactive T cells. A: Quantification of aDC ELISpot against SARS-CoV-2 Spike antigen of 18 previously vaccinated human donors, separated in previously infected with SARS-CoV-2 (left, inf.) or not (right, uninf.). B: 11 previously vaccinated and convalescent human donors were screened in aDC ELISpot against SARS-CoV-2 Spike antigen. Dotted line represents the standard threshold cutoff for positive response (50 SFU / Million cells). C: Left: Representative images of Elispot. Right: Quantification of aDC ELISpot against SARS-CoV-2 Spike antigen of vaccinated and convalescent donors after either CD8 or CD4 T cell depletion showing % of total response. D: Quantification of IFNy release using aDC ELISpot assay using PBMC isolated from a single human donor prior to, and six weeks post, SARS-CoV2 infection.

[0157] Figure 18: Systemic activated dendritic cell vaccination in the MC38 colon carcinoma mouse model. A: Schematic overview of experimental design. Treatment groups are untreated control, commercially available anti-PD-1 antibody by BioXcell (control, aPDl (BioXcell)), aDCs T08741ANM - 36 - loaded with LNP containing mRNA encoding secreted anti-PD-1 antibody (aDC[sec-aPDl]), or aDCs loaded with LNP containing mRNA encoding anchored anti-PD-1 antibody (aDC[anc-aPDl]). B: Tumor volume over time. Treatment groups correspond to lines as indicated by symbols. C: Survival plot for three treatment groups and control, as indicated. Re-challenge at indicated time-points at days 80 and / or 120 after the original tumor challenge was with a similar additional inoculum of the same tumor cell line. D: Tumor volume graphs of individual animals for each treatment group as indicated. CR: complete response.

[0158] Figure 19: Systemic activated dendritic cell treatment in the YUMMER1.7 mouse model. A: Schematic overview of experimental design. Treatment groups are untreated control, commercially available anti-PD-1 antibody by BioXcell (control, aPDl (BioXcell)), aDCs loaded with LNP containing mRNA encoding secreted anti-PD-1 antibody (aDC[sec-aPDl]), or aDCs loaded with LNP containing mRNA encoding anchored anti-PD-1 antibody (aDC[anc-aPDl]). B: Tumor volume over time. Treatment groups correspond to lines as indicated by symbols. C: Survival plot for treatment groups and control, as indicated. Re-challenge at indicated time-points at day 80 after the original tumor challenge was with a similar additional inoculum of the same tumor cell line. D: Tumor volume graphs of individual animals for each treatment group as indicated. CR: complete response.

[0159] Figure 20: Systemic activated dendritic cell treatment using anti-PDl antibody construct mRNA and IL-12 mRNA-containing LNP. A: Schematic overview of experimental design. Treatment groups are untreated control, aDCs loaded with LNP containing mRNA encoding anchored anti-PD-1 antibody (aDC[anc-aPDl]), aDCs loaded with LNP containing mRNA encoding NeoStrings (aDCfNeoStrings]) and aDCs T08741ANM - 37 - loaded with LNP containing mRNA encoding murine IL- 12 (aDCfmlL- 12]). Systemic injections (ie., Strings and anti-PD-1) began on Day+4, for a total of 6 treatments (see schematic of Fig. 20 A). mIL-12 injection #1 given concurrently w / tumor inoculum on DayO, followed #2 injection given on Day+4 i.t.

[0160] Figure 21 : Systemic activated dendritic cell treatment using anti-PDl antibody construct mRNA and IL-12 mRNA-containing LNP. A: Tumor volume over time. Treatment groups correspond to lines as indicated by symbols. B: Survival plot for treatment groups and control, as indicated. Re-challenge at indicated time-points at day 80 after the original tumor challenge was with a similar additional inoculum of the same tumor cell line. C: Tumor volume graphs of individual animals for each treatment group as indicated. CR: complete response.

[0161] Figure 22: Systemic activated dendritic cell treatment using anti-CTLA-4 antibody mRNA and anti-PDl antibody mRNA-containing LNP. A: Schematic overview of experimental design. Activated dendritic cells are transduced with surface-anchored or secreted anti-CTLA4 antibody construct (500ug / mouse of cKK-E12 based LNP), and injected into the flank intratumorally / peritum orally, at ~5xl05cells / mouse (indicated as “Dendritic Cell Treatments”). Treatment groups are untreated control, aDCs loaded with LNP containing mRNA encoding anchored anti- CTLA-4- 1 antibody and mRNA encoding NeoStrings (aDCfanc- aCTLA4 + strings]), aDCs loaded with LNP containing mRNA encoding secreted anti-CTLA-4- 1 antibody and mRNA encoding NeoStrings (aDC[sec-aCTLA4 + strings]), control treated with standard soluble anti-PD-1 antibody, aDCs loaded with LNP containing mRNA encoding anchored anti-CTLA-4- 1 antibody and mRNA encoding NeoStrings and additionally treated with standard soluble anti-PD-1 antibody (aDC[anc-aCTLA4 + strings] + aPDl), aDCs T08741ANM - 38 - loaded with LNP containing mRNA encoding secreted anti-CTLA-4-1 antibody and mRNA encoding NeoStrings and additionally treated with standard soluble anti-PD-1 antibody (aDC[sec-aCTLA4 + strings] + aaPDl), B: Tumor volume over time. Treatment groups correspond to lines as indicated by symbols. C: Tumor volume graphs of individual animals for each treatment group as indicated. CR: complete response.

[0162] Figure 23: Modulation of Type-1 diabetes onset in non-obese diabetic (NOD) mouse model. A: Diabetes onset in NOD mouse model by age. B: Survival plot showing diabetes free % of mice when treated with anti- PD-1 antibody compared to control, X axis shows days since treatment. C: Survival plot showing diabetes free % of mice comparing female and male mice treated with anti-PD-1 antibody at day 0. D: Survival plot showing diabetes free % of mice comparing mice treated with anti- PD-1 antibody at day 0, mice where either 3-5 weeks old at day 0 (top line) or 8-12 days old at day 0 (bottom line).

[0163] Figure 24: Modulation of Type-1 diabetes onset in non-obese diabetic (NOD) mouse model. A: Survival plot showing % of Diabetes free mice. NOD mice (male, 7wk) were treated with standard anti-PD-1 antibody (group B, aPDl Bioxcell, lOmg / kg) or either an anti-PDl -antibody loaded activated dendritic cell (group C, aDCfaPDl]) or an activated dendritic cell loaded with an anti-PD-1 antibody and anti-CTLA-4 antibody (group D, [aPDl] / CTLA4], anchored version, 150ng was administered per animal on day 0). Untreated animals served as control (group A, no Tx). B: Survival plot showing % of Diabetes free mice. Treatment groups were the same as above with the addition of group E, an anti- PDl -antibody loaded activated dendritic cell plus addition of an anchored CTLA4 antibody from Bioxcell (group E, aDC[aPDl]+aCTLA4ab bioxell). T08741ANM - 39 -

[0164] Figure 25: EliSpot assay showing IFNy release of mock transfected (aDC alone) or aDCs that were loaded with IL-12 mRNA LNPs. A: Representative images of EliSpot assay. B: Quantification of IFNy spot forming units (SFU) per million cells.

[0165] Figure 26: Systemic activated dendritic cell treatment using IL-12 mRNA- containing LNP. A: Schematic overview of experimental design. Treatment groups are unloaded aDCs (aDCfmock]) and aDCs loaded with LNPs containing mRNA encoding IL-12 (aDC[IL-12]). The treatment was administered 6 times (6X). B: Graph showing mean tumor volume over time starting from DO for both treatment groups according to symbols as shown. C: Survival plot showing probability of survival over time for both groups, the line showing extended survival is the group aDC [IL- 12],

[0166] Figure 27: Systemic activated dendritic cell treatment using IL-12 mRNA- containing LNP and CpG co-stimulation. A: Schematic overview of experimental design. Treatment groups are unloaded aDCs (aDCfmock]), aDCs loaded with LNPs containing mRNA encoding IL- 12 (aDC[IL-12]) treated once, aDCs loaded with the same LNPs containing mRNA encoding IL- 12 but being administered twice, once at DO and once at D4 (aDC[IL-12] / 2X), and a group receiving aDCs loaded with the same LNPs containing mRNA encoding IL- 12 and further receiving co-stimulation with CpG. All treatments are administered at DO. B: Graph showing mean tumor volume over time starting from DO for both treatment groups according to symbols as shown. C: Survival plot showing probability of survival over time for both groups, the lines correspond to, from left to right: aDC empty (mock), untreated, aDC[IL-12], aDC[IL-12] / 2X, aDC[IL-12] / CpG.

[0167] Figure 28: Schematic overview of OT1 proliferation assay of Example 29. OT-1 cells are co-incubated with aDCs pulsed with soluble OVA (sOVA) T08741ANM - 40 - and loaded with LNPs containing mRNA encoding either anti-PD-1 antibody (LNP[a-PDl]), secreted anti-PD-1 antibody (LNPfsec a- PD1]) or Interleukin-12p70 (LNP[IL-12p70]). After incubation, the cells are analysed using FACS (fluorescence-activated cell sorting).

[0168] Figure 29: Antigen-specific CD8 T cell proliferation, as indicated by CFSE dilution in OT1 CD8+ T cells, assessed by flow cytometry (Cytoflex cytometer, analysis using Flow Jo vlO software). Legend underneath the plot indicates file name, event recorded and number of events recorded. Each LNPfmRNA] is used at three different concentrations (0.1, 1 or 5 ug / mL of cKK-E12 based LNP). Blank aDC (untransfected) and aDC pulsed with sOVA are used as controls. A: Representative flow cytometry plots of controls and cells treated with sOVA and LNP[anti-PD-l antibody], B: Representative flow cytometry plots of cells treated with sOVA and LNPfsecreted anti- PD-1 antibody], C: Representative flow cytometry plots of cells treated with sOVA and LNP[IL-12p70], D: Bar graph showing quantification of %proliferation of OT1 cells.

[0169] Figure 30: Schematic overview of aDC-TIL co-culture assay of Example 30.

[0170] Figure 31 : Representative flow cytometry plots. Legend underneath the plot indicates file name, event recorded and number of events recorded. A: Consecutive gating for selection of single cells (X: Forward Scatter Height (FSC-H), Y: Forward Scatter Area (FSC-A), live cells (Y: Zombie-UV die, binds to dead cells, and DAPI fluorescence intensity, X: FSC-A) and CD8+T-cells (Y: Fluorescence intensity detected using FITC (Fluorescein Isothiocyanate) conjugated to a marker detecting tumor-associated proteins or other specific surface proteins like GPR, X: Fluorescence intensity corresponding to CD8 expression, T08741ANM - 41 - measured using Pacific Blue dye) . B: Consecutive gating for selection for selection of Glycoprotein33 -specific T-cells (left panel, Y: Fluorescence intensity corresponding to GP33 tetramer, measured using ALLOPHYCOCYANIN, X: Fluorescence intensity corresponding to CD8 expression, measured using Pacific Blue dye) and selection of effector and memory T-cell subsets (right panel, Y: Fluorescence intensity corresponding to CD44 expression, measured using Alexa Fluor 680 dye, X: Fluorescence intensity corresponding to CD62L expression, measured using Brilliant Violet 711 dye). Lines are drawn into the plot to indicated selected cell populations.

[0171] Figure 32: Representative flow cytometry plots showing gating strategy to quantify Tet+cells. Y axis indicates fluorescence intensity corresponding to GP33 tetramer, measured using ALLOPHYCOCYANIN, X axis indicates fluorescence intensity corresponding to CD8 expression, measured using Pacific Blue dye. Legend underneath the plot indicates file name, event recorded and number of events recorded. A: Plots from culture without aDC (tumor / TIL only), left panel, and co-culture of TIL with aDC loaded with LNP[GP33], right panel. B: Plots from co-culture of TIL with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody], left panel, and with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody] plus treatment with recombinant IL-2.

[0172] Figure 33: Gating for memory and effector T-cell subpopulations. A: Representative flow cytometry plots showing gating strategy to quantify Tet+cells. Y: Fluorescence intensity corresponding to CD44 expression, measured using Alexa Fluor 680 dye, X: Fluorescence intensity corresponding to CD62L expression, measured using Brilliant Violet 711 dye. Legend underneath the plot indicates file T08741ANM - 42 - name, event recorded and number of events recorded. Plots from culture without aDC (tumor / TIL only), top left panel, co-culture of TIL with aDC loaded with LNP[GP33], top right panel, co-culture of TIL with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody], bottom left panel, and with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody] plus treatment with recombinant IL-2, bottom right panel. B: Pie charts showing quantification of memory 1 and 2 and effector 1 and 2 cell populations, showing changing proportions of the four cell populations under different conditions. Starting from the top of the circle (12 o’clock), going clockwise, in each graph, the first slice represents effector 1, the second slice effector 2, the third slice memory 1, and the last slice memory 2.

[0173] Figure 34: Bar plot showing quantification of memory 1 and 2 and effector 1 and 2 cell populations by experimental group (culture without aDC (tumor / TIL only), first bar; co-culture of TIL with aDC loaded with LNP[GP33], second bar; co-culture of TIL with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody], third bar; and with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody] plus treatment with recombinant IL-2, fourth bar.

[0174] Figure 35: IL7Ra and KLRG1 expression on whole memory subset cells. Representative flow cytometry plots showing gating strategy to quantify Tet+cells. Y: Fluorescence intensity corresponding to IL7Ra expression, measured using PerCP dye, X: Fluorescence intensity corresponding to KLRG1 expression, measured using Phycoerythrin dye. Legend underneath the plot indicates file name, event recorded and number of events recorded. From left to right, the panels show coculture of TIL with aDC loaded with LNP[GP33], co-culture of TIL with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody], T08741ANM - 43 - and with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody] plus treatment with recombinant IL-2.

[0175] Figure 36: Quantification of marker expression (IL7R, KLRG1, Tim3 and PD1) in memory sub-populations (selected population indicated in flow cytometry diagram on top left). For bar plots, X axis indicates gMFI (geometric mean fluorescence intensity). The bar plots show coculture of TIL with aDC loaded with LNP[GP33], first bar; co-culture of TIL with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody], second bar; and with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody] plus treatment with recombinant IL-2, third bar.

[0176] Figure 37: Quantification of marker expression (IL7R, KLRG1, Tim3 and PD1) in effector 1 sub-population(selected population indicated in flow cytometry diagram on top left). Bar plot X axis indicates gMFI (geometric mean fluorescence intensity). The bar plot on the top right shows PD1 expression in effector 1 cells of the indicated experimental groups. The bottom plot shows the expression of the indicated markers in the groups: culture without aDC (tumor / TIL only), first bar; co-culture of TIL with aDC loaded with LNP[GP33], second bar; co-culture of TIL with aDCs loaded with LNP[GP33] and LNPfanti- PD-1 antibody], third bar; and with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody] plus treatment with recombinant IL-2, fourth bar.

[0177] Figure 38: Quantification of marker expression (IL7R, KLRG1, Tim3 and PD1) in effector 2 sub-population(selected population indicated in flow cytometry diagram on top left). Bar plot X axis indicates gMFI (geometric mean fluorescence intensity). The bar plot on the top right T08741ANM - 44 - shows the expression of the indicated markers in the groups: culture without aDC (tumor / TIL only), first bar; co-culture of TIL with aDC loaded with LNP[GP33], second bar; co-culture of TIL with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody], third bar; and with aDCs loaded with LNP[GP33] and LNP[anti-PD-l antibody] plus treatment with recombinant IL-2, fourth bar. The three plots at the bottom show expression of the indicated marker in the indicated experimental groups.

[0178] DETAILED DESCRIPTION

[0179] I. The ex vivo activated dendritic cell loaded with a nucleic acid sequence

[0180] Activated dendritic cells are combined with at least one mRNA encoding, e.g., a checkpoint inhibitor. The process of introducing an exogenous mRNA encoding a protein of interest is also referred to as “loading”. Such loaded activated dendritic cells can elicit potent anti-tumor and / or immune responses. These activated dendritic cells and the disclosed embodiments thereof are also called “activated dendritic cells of the invention”.

[0181] Once activated dendritic cells have been obtained and combined with the at least one LNP comprising an exogenous mRNA, the mixture can for example be incubated under standard conditions. Culturing can be performed under standard conditions, e.g. at 37° C and 5% CO2in standard mediums for culturing of human cells such as in RPML1640 medium (obtainable e.g. from GIBCO), supplemented with 15% AB serum (obtainable from e.g. Gemini Bio-Products). An incubation step can be added after combining the activated dendritic cells with at least one LNP comprising an exogenous mRNA. Alternatively or additionally, an incubation step can also be inserted after generation of activated dendritic cells from monocytes. The incubation T08741ANM - 45 - step can be performed for 0.1 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 12 h, 20 h or 24 h. In another embodiment, the incubation step can be performed for at least 0.1 h, at least 0.5 h, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 12 h, at least 20 h or at least 24 h. It needs to be understood that all method steps are performed in vitro.

[0182] Checkpoint inhibitors

[0183] The invention relates to ex vivo activated dendritic cells comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor. Hence, the invention relates to an ex vivo activated dendritic cells capable of expressing and optionally secreting a checkpoint inhibitor.

[0184] Overexpression of immune checkpoints within the tumor microenvironment suppresses the ability of the immune system to attack tumor cells, enabling tumors to evade the immune system and grow. Signaling through immune checkpoints such as programmed death protein 1 (PD-1) and its ligand PD-L1 and cytotoxic T lymphocyte- associated protein 4 (CTLA4) on the exterior of T cells can stop their activation. Immune checkpoints also play a role in immune suppression via their expression on B cells and other immune response cells. Hence, immune checkpoint inhibitors, also termed “checkpoint inhibitors” herein, are a promising tool in cancer immunotherapy, as they can stop signaling through immune checkpoints decrease the suppression of the immune system.

[0185] Upon combination of activated dendritic cells with a checkpoint inhibitor, e.g. a LNP comprising an exogenous mRNA encoding the checkpoint inhibitor, activated dendritic cells are able to take up, process and express the checkpoint inhibitor. If the checkpoint inhibitor is secreted, for example a secreted antibody without an anchor, the checkpoint inhibitor is secreted from the activated dendritic cell. If the checkpoint inhibitor comprises a cell anchor, such as a GPI anchor, the checkpoint inhibitor is T08741ANM - 46 - tethered to the surface of the cell.

[0186] In one embodiment, the checkpoint inhibitor is a molecule that is capable of specifically binding to an immune checkpoint molecule. The immune checkpoint molecule may be at least partially on a cell surface.

[0187] In one embodiment, the checkpoint inhibitor is a small molecule compound. In another embodiment, the checkpoint inhibitor is an antibody or an antigen-binding fragment thereof, preferably a monoclonal antibody or an antigen-binding fragment thereof.

[0188] The antibody or an antigen-binding fragment thereof is capable of binding to PD-1, PD-L1 or CTLA-4 or a combination thereof. In some embodiments, the antibody is a monoclonal antibody or an antigen-binding fragment thereof. In one embodiment, the antibody is a multispecific antibody or an antigen-binding fragment thereof, preferably a bispecific antibody or an antigen-binding fragment thereof.

[0189] Antigen-binding fragments of a bispecific or multispecific antibody may be, for example, two or more single-chain variable fragments that are connected by a linker. In one embodiment, the bispecific antibody or antigen-binding fragment thereof is specific or anti -PD-1 and one further antigen. In one embodiment, the further antigen is a tumor antigen. In one embodiment, the bispecific antibody or antigen-binding fragment thereof is specific or anti -PD-1 and one further immune checkpoint molecule selected from PD-1L, CTLA-4, TIM-3, LAG-3 and TIGIT. In one preferred embodiment, the antibody is an anti-PD-l / anti-CTLA-4 bispecific antibody or a fragment thereof that binds to both antigens.

[0190] The checkpoint inhibitor as described herein may be an anti -PD-1 antibody, an anti- PD-L1 antibody, or an anti-CTLA4 antibody. These antibodies have in common that they block / antagonize cellular interactions that block or downregulate immune cells, especially T cells from killing cancer cells, accordingly these antibodies are all antagonistic antibodies. In one embodiment, the checkpoint inhibitor as described T08741ANM - 47 - herein is an anti-PD-1 antibody. Examples of anti-PD-1 antibodies are pembrolizumab, nivolumab, cemiplimab (REGN2810), BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100 and JSOO1. In one embodiment, the checkpoint inhibitor as described herein is an anti-PD-Ll antibody. Examples of anti-PD-Ll antibodies are avelumab, atezolizumab, durvalumab, KN035 and MGD013 (bispecific for PD-1 and LAG-3). In one embodiment, the checkpoint inhibitor as described herein is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies are ipilimumab and tremelimumab (ticilimumab).

[0191] Hence, in one embodiment, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, avelumab, atezolizumab, durvalumab, KN035 and MGD013, ipilimumab and tremelimumab. In a preferred embodiment, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab and ipilimumab. Other immune checkpoint molecules include T-cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3) and T cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif (ITIM) domain (TIGIT). Inhibitors targeting these immune checkpoint molecules are sometimes referred to as next generation immune checkpoint inhibitors.

[0192] Hence, in one embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-1L, CTLA-4, TIM-3, LAG-3 and TIGIT.

[0193] In one embodiment, the checkpoint inhibitor is selected from the group consisting of anti-PD-1 antibody or a PD-1 -binding fragment thereof, anti-PD-Ll antibody or a PD- L1 -binding fragment thereof, anti-CTLA4 antibody or a CTLA4-binding fragment thereof, anti-TIM-3 antibody or a TIM-3 binding fragment thereof, anti-LAG-3 antibody or a LAG-3 binding fragment thereof, and anti-TIGIT antibody or a TIGIT- binding fragment thereof. In one embodiment, the checkpoint inhibitor is anchored to the surface of the activated dendritic cell, and no further antigen (e.g., a tumor antigen) is provided. In another embodiment, the checkpoint inhibitor is secreted from the T08741ANM - 48 - activated dendritic cell, and no further antigen (e.g., a tumor antigen) is provided. In one embodiment, the checkpoint inhibitor is anchored to the surface of the activated dendritic cell, and at least one further antigen (e.g., a tumor antigen) is provided. In another embodiment, the checkpoint inhibitor is secreted from the activated dendritic cell, and at least one further antigen (e.g., a tumor antigen) is provided.

[0194] The checkpoint inhibitor may comprise a secretory signal peptide or an anchoring (cell surface expression) signal peptide.

[0195] A secretory signal peptide (sec) may be fused to the coding region preferably in a way that the sec is translated as N terminal tag. Sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins may be used as GS-linkers (glycine-serine-linkers). In other embodiments the RNA (e.g. mRNA) may have one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3’UTR. The AURES may be removed from the RNA (e.g. mRNA). Alternatively the AURES may remain in the RNA (e.g. mRNA).

[0196] Alternatively, an anchoring or tethering sequence (anc) may be fused to the coding region. The anchoring sequence targets the protein for lipid anchoring, resulting in a prenylated protein, fatty acid acylated protein or glycosylphosphatidylinositol (GPI)- linked protein. A protein may have multiple lipid groups covalently attached to it.

[0197] Prenylation includes the attachment of a farnesyl (15 carbon ) or geranylgeranyl (20 carbon) isoprenoid group by farnesytransferase or geranylgeranyltransferase to a cysteine residue of a C-terminal consensus sequence CaaX. In one embodiment, the checkpoint inhibitor comprises a CaaX sequence, wherein “a” is an aliphatic amino acid and X is either M, S, Q A, C, L, or E.

[0198] In one embodiment, the checkpoint inhibitor comprises an ER signaling sequence. In one embodiment, the checkpoint inhibitor comprises a GPI anchor. The GPI complex is attached to the checkpoint inhibitor’s C terminal carboxyl group via an amide linkage by the GPI-transamidase complex. The GPI anchor is assembled on a T08741ANM - 49 - phosphatidylinositol lipid in the endoplasmic reticulum by a series of enzymatic reactions and then is covalently attached to the carboxyl terminus.

[0199] Thus, in one embodiment, the checkpoint inhibitor is a secreted antibody or antigenbinding fragment thereof selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof. In one embodiment, the checkpoint inhibitor as described herein is a secreted anti-PD-1 antibody. In one embodiment, the checkpoint inhibitor as described herein is a secreted anti-PDL-1 antibody. In one embodiment, the checkpoint inhibitor as described herein is a secreted anti-CTLA-4 antibody.

[0200] In another embodiment, the checkpoint inhibitor is an antibody or antigen-binding fragment thereof anchored to the cell surface selected from an anti-PD-1 antibody or a PD- 1 binding fragment thereof, an anti-PD-L 1 antibody or a PD-L 1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof. In one embodiment, the checkpoint inhibitor as described herein is an anchored anti-PD-1 antibody. In one embodiment, the checkpoint inhibitor as described herein is a anchored anti-PDL-1 antibody. In one embodiment, the checkpoint inhibitor as described herein is an anchored anti-CTLA-4 antibody.

[0201] In a preferred embodiment, the checkpoint inhibitor is selected from secreted anti-PD- 1 antibody or a PD-1 binding fragment thereof, anchored anti-PD-1 antibody or a PD-1 binding fragment thereof, secreted anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof and anchored anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof.

[0202] In one embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding a checkpoint inhibitor selected from the group consisting of anti-PD-1 antibody or a PD-1 -binding fragment thereof, anti-PD-Ll antibody or a T08741ANM - 50 -

[0203] PD-L1 -binding fragment thereof, anti-CTLA4 antibody or a CTLA4-binding fragment thereof, anti-TIM-3 antibody or a TIM-3 binding fragment thereof, anti-LAG-3 antibody or a LAG-3 binding fragment thereof, and anti-TIGIT antibody or a TIGIT- binding fragment thereof, and at least one further mRNA encoding a checkpoint inhibitor selected from the group consisting of anti-PD-1 antibody or a PD-l-binding fragment thereof, anti-PD-Ll antibody or a PD-L1 -binding fragment thereof, anti- CTLA4 antibody or a CTLA4-binding fragment thereof, anti-TIM-3 antibody or a TIM-3 binding fragment thereof, anti-LAG-3 antibody or a LAG-3 binding fragment thereof, and anti-TIGIT antibody or a TIGIT -binding fragment thereof; wherein the at least two mRNAs do not encode the same checkpoint inhibitor.

[0204] In one embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding an anti-PD-1 antibody or a PD-l-binding fragment thereof and comprises at least one mRNA encoding an anti-CTLA4 antibody or a CTLA4- binding fragment thereof. In another embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding an anti-PD-1 antibody or a PD-l-binding fragment thereof and further comprises at least one mRNA encoding an anti-TIM-3 antibody or a TIM-3 -binding fragment thereof. In another embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding an anti-PD-1 antibody or a PD-l-binding fragment thereof and further comprises at least one mRNA encoding an anti- LAG-3 antibody or a LAG-3-binding fragment thereof. In another embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding an anti-PD-1 antibody or a PD-l- binding fragment thereof and further comprises at least one mRNA encoding an anti- TIGIT antibody or a TIGIT-binding fragment thereof. In a further embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding an anti-PDL-1 antibody or a PDL-1 -binding fragment thereof and comprises at least one mRNA encoding an anti-CTLA4 antibody or a CTLA4-binding fragment thereof. In another embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding an anti-PDL-1 antibody or a PDL-1 -binding fragment T08741ANM - 51 - thereof and further comprises at least one mRNA encoding an anti-TIM-3 antibody or a TIM-3 -binding fragment thereof. In another embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding an anti-PDL-1 antibody or a PDL-1 -binding fragment thereof and further comprises at least one mRNA encoding an anti- LAG-3 antibody or a LAG-3 -binding fragment thereof. In another embodiment, the ex vivo activated dendritic cell of the invention comprises at least one mRNA encoding an anti-PDL-1 antibody or a PDL-1 -binding fragment thereof and further comprises at least one mRNA encoding an anti- TIGIT antibody or a TIGIT -binding fragment thereof.

[0205] Immune modulators

[0206] Some embodiments of the invention relate to an ex vivo activated dendritic cells comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and also comprising at least one further exogenous nucleic acid sequence encoding an immune modulator. Hence, the invention relates to an ex vivo activated dendritic cells capable of expressing and optionally secreting a checkpoint inhibitor and expressing and / or secreting an immune modulator. Furthermore, the invention also relates to pharmaceutical compositions comprising a first ex vivo activated dendritic cells comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and also comprising at least one second ex vivo activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding an immune modulator. The invention also relates to pharmaceutical compositions comprising a first ex vivo activated dendritic cells comprising at least one exogenous nucleic acid sequence (e.g. a mRNA) encoding a checkpoint inhibitor, said pharmaceutical composition further comprising an immune modulator. In some embodiments, the immune modulator or checkpoint inhibitor is cytoplasmic. In some embodiments, the immune modulator or checkpoint inhibitor is located on the cell surface of the activated dendritic cell, e.g. via a linker. T08741ANM - 52 -

[0207] In one embodiment, the immune modulator is selected from the group consisting of cytokines, co-stimulatory molecules (chemokines), growth factors or immunomodulatory imide drugs (IMiDs).

[0208] In one embodiment, the immune modulator is a cytokine. In one embodiment, the cytokine is selected from chemokines, interferons, interleukins, lymphokines and tumor necrosis factor.

[0209] In one embodiment, the cytokine is selected from the group consisting of interleukin- 1b (IL- lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL- 23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof. In a preferred embodiment, the cytokine is interleukin- 12 (IL-12).

[0210] Hence, in one embodiment, the ex vivo activated dendritic cell comprises at least two exogenous nucleic acid sequences, the first exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, and an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof, and the second exogenous nucleic acid sequence encoding a cytokine selected from the group consisting of of interleukin- lb (IL- lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL- 4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin- 21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL- 23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof. In a preferred embodiment, the cytokine is interleukin- 12 (IL-12). T08741ANM - 53 -

[0211] In a preferred embodiment, the at least two exogenous nucleic acid sequences encode anti-PD-1 antibody or a PD-1 binding fragment thereof and interleukin- 12 (IL-12).

[0212] In another embodiment, the ex vivo activated dendritic cell of the invention comprises, e.g. expresses, a co-stimulatory molecule. In one embodiment, the co-stimulatory molecule is a co-stimulatory receptor. In one embodiment, the co-stimulatory receptor is selected from the group consisting of ICOSL, CD70, CD30L, ICAM, PLAUR, CD40, CD80, CD86, OX40L, 4-1BBL, GITRL, and TIM-4.

[0213] Hence, in one embodiment, the ex vivo activated dendritic cell comprises at least two exogenous nucleic acid sequences, the first exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, and an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof, and the second exogenous nucleic acid sequence encoding an immune modulator selected from the group consisting of of interleukin- lb (IL- lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL- 4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin- 21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL- 23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa), ICOSL, CD70, CD30L, ICAM, PLAUR, CD40, CD80, CD86, OX40L, 4-1BBL, GITRL, and TIM-4, or a combination thereof. In one embodiment, the immune modulator is selected from a cytokine of the group consisting of interleukin-lb (IL- 1b), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL- 18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof.

[0214] In a preferred embodiment, the cytokine is interleukin- 12 (IL-12). T08741ANM - 54 -

[0215] In another embodiment, the cytokine is a chemokine. In one embodiment, the cytokine is a chemokine receptor, such as CCR7 or XCR1. In one embodiment, the invention provides an ex vivo activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, and at least one further exogenous nucleic acid sequence encoding at least one chemokine or chemokine receptor. The expression of the chemokine receptor on the activated dendritic cell improves the recruitment of the activated dendritic cell to tumor cells and / or tumor-associated lymphoid organs expressing the corresponding chemokine.

[0216] In another one embodiment, the ex vivo activated dendritic cell comprises at least two exogenous nucleic acid sequences, the first exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof, anti-TIM-3 antibody or a TIM-3 binding fragment thereof, anti-LAG-3 antibody or a LAG-3 binding fragment thereof, and anti-TIGIT antibody or a TIGIT- binding fragment thereof; and the second exogenous nucleic acid sequence encoding an immune modulator selected from the group consisting of interleukin- lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof.

[0217] In a preferred embodiment, the immune modulator is interleukin- 12 (IL-12).

[0218] In another embodiment, the immune modulator is a chemokine. In one embodiment, the immune modulator is a chemokine receptor, such as CCR7 or XCR1.

[0219] In another embodiment, the ex vivo activated dendritic cell comprises at least two exogenous nucleic acid sequences, the first exogenous nucleic acid sequence T08741ANM - 55 - encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, and an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof, and the second exogenous nucleic acid sequence encoding a at least one co-stimulatory receptor selected from the group consisting of ICOSL, CD70, CD30L, CD40, CD80, CD86, OX40L, 4-1BBL, GITRL, and TIM-4, or a combination thereof.

[0220] In a preferred embodiment, the ex vivo activated dendritic cell comprises at least two exogenous nucleic acid sequences, the first exogenous nucleic acid sequence encoding an anti-PD-1 antibody or a PD-1 binding fragment thereof and the second exogenous nucleic acid sequence CD80 or CD86.

[0221] In another one embodiment, the ex vivo activated dendritic cell comprises at least two exogenous nucleic acid sequences, the first exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof, anti-TIM-3 antibody or a TIM-3 binding fragment thereof, anti-LAG-3 antibody or a LAG-3 binding fragment thereof, and anti-TIGIT antibody or a TIGIT- binding fragment thereof; and the second exogenous nucleic acid sequence encoding a co-stimulatory receptor selected from the group consisting of ICOSL, CD70, CD30L, CD40, CD80, CD86, OX40L, 4-1BBL, GITRL, and TIM-4, or a combination thereof

[0222] Antigenic peptides or proteins

[0223] In one embodiment, the at least one antigenic peptide is a tumor-associated peptide or protein, a viral antigenic protein, a bacterial antigenic protein, or a fungal antigenic protein. In one embodiment, the at least one antigenic peptide is a tumor-associated peptide or protein, and wherein the tumor-associated peptide or protein is a tumor- T08741ANM - 56 - specific peptide or protein, preferably wherein the tumor-specific peptide or protein comprises at least one tumor-specific neo-antigen.

[0224] In one embodiment, the at least one tumor-specific neo-antigen is selected from the group consisting of Aatf, Cpnel, Dpagtl, Wbp7, Nlel, Irgq, Zbtb40, Cryl, pl5E, Gtf2i, Med 12, N4bp212 and Sydel, or a combination thereof. In one particular embodiment, the at least one tumor-specific neo-antigen comprises a sequence of Table 1 , or a combination thereof.

[0225] In a preferred embodiment, the tumor-associated or tumor specific antigen is identified from a human subject. In a more preferred embodiment, the tumor-associated or tumor specific antigen is a patient-specific neo-antigen, Oncoviral antigen (such as HPV E6ZE7 or Merkel Cell polyoma LTA), Tumor Associated Antigen (TAA) such as lineage-restricted Differentiation antigens (such as melanoma MART-1 or TRP2), cancer-testes antigen (such as MAGE or NY-ESO-1) or shared mutated antigen(such as p53 or Ras), or any combination thereof. The nucleic acid sequence encoding for said tumor-associated or tumor specific antigen may then be loaded into LNPs and combined with activated dendritic cells of the invention. In another embodiment, the activated dendritic cell of the invention is administered to the subject without a nucleic acid sequence encoding tumor-associated or tumor specific antigens. Without being bound by theory, it is believed that the activated dendritic cells of the invention are capable of processing and presenting tumor-associated or tumor specific antigen that are encountered in the body of the subject after administration, and thereby triggering an effective anti -turn or response in the subject.

[0226] In one embodiment, the antigenic peptide is a combination of tumor-specific neoantigens comprised in a lipid nanoparticle. Each tumor-specific neo-antigen is connected to the next by a linker. In one embodiment, the antigenic peptide comprises an LNP comprising a polyepitope construct, i.e. a construct comprising more than one epitope. T08741ANM - 57 -

[0227] In one embodiment, the polyepitope construct comprises the epitopes two or more of Aatf, Cpnel, Dpagtl, Wbp7, Nlel, Irgq, Zbtb40, Cryl, pl5E, Gtf2i, Medl2, N4bp2I2 and Sydel. In one embodiment, the polyepitope construct comprises the epitopes of two or more of Aatf, Cpnel, Dpagtl, Wbp7, Nlel, Irgq, Zbtb40, Cryl, pl5E, Gtf2i, Med 12, N4bp2I2 and Sydel each separated by linkers. In one embodiment, the polyepitope construct comprises at least two or more epitopes listed in Table 1

[0228] In a preferred embodiment, the polyepitope construct comprises the epitopes two or more human tumor-associated or tumor specific antigens, such as tumor neo-antigens. In one embodiment, polyepitope construct comprises tumor-associated or tumor specific antigens that are patient-specific neo-antigens, Oncoviral antigens (such as HPV E6ZE7 or Merkel Cell polyoma LTA), Tumor Associated Antigens (TAA) such as lineage-restricted Differentiation antigens (such as melanoma MART-1 or TRP2), cancer-testes antigens (such as MAGE or NY-ESO-1) or shared mutated antigen (such as p53 or Ras), or any combination thereof.

[0229] In one embodiment, the polyepitope construct comprises the epitopes two or more of Aatf, Cpnel, Dpagtl, Wbp7, Nlel, Irgq, Zbtb40, Cryl, and pl5E. In one embodiment, the poly epitope construct comprises the epitopes of two or more of Aatf, Cpnel, Dpagtl, Wbp7, Nlel, Irgq, Zbtb40, Cryl, and pl5E each separated by linkers. In one embodiment, the polyepitope construct comprises at least two or more epitopes of Table 1.

[0230] In one embodiment, the polyepitope construct comprises the epitopes Gtf2i, Medl2, N4bp2I2 and Sydel. In one embodiment, the polyepitope construct comprises the epitopes Gtf2i, Med 12, N4bp2I2 and Sydel each separated by linkers. In one embodiment, the polyepitope construct comprises the any of the epitopes of Table 1.

[0231] In one embodiment, the polyepitope construct comprises at least a sequence encoding for HPV E6ZE7. In one embodiment, the polyepitope construct comprises at least a sequence encoding for Merkel Cell polyoma LTA epitope. In one embodiment, the T08741ANM - 58 - polyepitope construct comprises at least a sequence encoding for a Tumor Associated Antigens (TAA). In one embodiment, the polyepitope construct comprises at least a sequence encoding for a lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2). In one embodiment, the poly epitope construct comprises at least a sequence encoding for MART-1. In one embodiment, the polyepitope construct comprises at least a sequence encoding for TRP2. In one embodiment, the polyepitope construct comprises at least a sequence encoding for cancer-testes antigens. In one embodiment, the polyepitope construct comprises at least a sequence encoding forMAGE. In one embodiment, the polyepitope construct comprises at least a sequence encoding for NY-ESO-1. In one embodiment, the polyepitope construct comprises at least a sequence encoding forp53. In one embodiment, the poly epitope construct comprises at least a sequence encoding for Ras. In one embodiment, the poly epitope construct comprises a plurality of sequences encoding for tumor neoantigens, wherein said sequences may be connected by a linker.

[0232] Hence, in one embodiment, the ex vivo activated dendritic cell comprises at least three exogenous nucleic acid sequences, the first exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, and an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; the second exogenous nucleic acid sequence encoding a cytokine selected from the group consisting of of interleukin- lb (IL- lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL- 4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin- 21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL- 23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof

[0233] ; and the third exogenous nucleic acid sequence encoding at least one antigenic peptide comprising at least one tumor-specific neo-antigen. T08741ANM - 59 -

[0234] In another embodiment, the ex vivo activated dendritic cell comprises at least three exogenous nucleic acid sequences, the first exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, and an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and the second exogenous nucleic acid sequence encoding at least one antigenic peptide comprising at least one tumor-specific neo-antigen.

[0235] In a preferred embodiment, the at least two exogenous nucleic acid sequences encode anti-PD-1 antibody or a PD-1 binding fragment thereof and at least one tumor-specific neo-antigen.

[0236] In a preferred embodiment, the at least three exogenous nucleic acid sequences encode anti-PD-1 antibody or a PD-1 binding fragment thereof; interleukin- 12 (IL- 12) and at least one tumor-specific neo-antigen. In one embodiment, the at least one tumorspecific neo-antigen is a patient-specific antigen. In one embodiment, the tumorspecific neo-antigen comprises one or more epitopes from HPV E6 / E7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patientspecific TAA), lineage-restricted Differentiation antigen (such as melanoma MART- 1 or TRP2), NY-ESO-1, p53 or Ras.

[0237] In another embodiment, the at least three exogenous nucleic acid sequences encode anti-PD-1 antibody or a PD-1 binding fragment thereof; interleukin- 12 (IL- 12) and the at least one tumor-specific neo-antigen.

[0238] In one embodiment, the activated dendritic cell comprises the at least three exogenous nucleic acid sequences at a molar ratio of about (l):(0. l-10):(0.1-10). In one embodiment, the activated dendritic cell comprises the at least three exogenous nucleic acid sequences at a molar ratio of l:(0.1-1): 1, 1 : 1 :(0.1-1), 1 :(0. l-l):(0.1-1), 1 : 1 : 1, 1 :(1- T08741ANM - 60 -

[0239] 10): 1, l:l:(l-10), or l :(l-10):(l-10). In a preferred embodiment, the activated dendritic cell comprises the at least three exogenous nucleic acid sequences at a molar ratio of about 1 : 1 : 1.

[0240] Hence, in one embodiment, the ex vivo activated dendritic cell of the invention comprises at least three LNPs, each comprising an exogenous nucleic acid sequence, the first exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, and an anti- CTLA-4 antibody or a CTLA-4 binding fragment thereof; the second exogenous nucleic acid sequence encoding a cytokine selected from the group consisting of of interleukin- lb (IL- lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL- 4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin- 21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL- 23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof; and the third exogenous nucleic acid sequence encoding at least one antigenic peptide comprising at least one tumor-specific neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0241] In another embodiment, the ex vivo activated dendritic cell of the invention comprises at least two LNPs, each comprising at least one exogenous nucleic acid sequence, the first LNP comprising an exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, and the second LNP comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide comprising at least one tumor-specific neo-antigen. T08741ANM - 61 -

[0242] In a preferred embodiment, the ex vivo activated dendritic cell of the invention comprises at least two LNPs, each comprising an exogenous nucleic acid sequence, wherein the two exogenous nucleic acid sequences encode anti-PD-1 antibody or a PD-1 binding fragment thereof and at least one tumor-specific neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras..

[0243] In a preferred embodiment, the ex vivo activated dendritic cell of the invention comprises at least three LNPs, each comprising at least one exogenous nucleic acid sequences, wherein the first LNP comprises at least one exogenous nucleic acid sequence encoding anti-PD-1 antibody or a PD-1 binding fragment thereof; the second LNP comprises at least one exogenous nucleic acid sequence encoding interleukin- 12 (IL-12) and the third LNP comprises at least one exogenous nucleic acid sequence encoding at least one tumor-specific neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patientspecific TAA), lineage-restricted Differentiation antigen (such as melanoma MART- 1 or TRP2), NY-ESO-1, p53 or Ras.

[0244] In another preferred embodiment, the ex vivo activated dendritic cell of the invention comprises at least three LNPs, each comprising at least one exogenous nucleic acid sequences, wherein the first LNP comprises at least one exogenous nucleic acid sequence encoding anti-PD-1 antibody or a PD-1 binding fragment thereof; the second LNP comprises at least one exogenous nucleic acid sequence encoding interleukin- 12 (IL-12) and the third LNP comprises at least one exogenous nucleic acid sequence T08741ANM - 62 - encoding at least one tumor-specific neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patientspecific TAA), lineage-restricted Differentiation antigen (such as melanoma MART- 1 or TRP2), NY-ESO-1, p53 or Ras.

[0245] In one embodiment, the activated dendritic cell of the invention comprises the at least three LNPs each comprising at least one exogenous nucleic acid sequence at a molar ratio of about (l):(0. l-10):(0.1-10). In one embodiment, the activated dendritic cell comprises the at least three LNPs each comprising at least one exogenous nucleic acid sequence at a molar ratio of l:(0.1-1): 1, 1 : 1 :(0.1-1), 1 :(0. l-l):(0.1-1), 1 : 1 : 1, l :(l-10):l, 1 : 1 :(l-10), or l :(l-10):(l-10). In a preferred embodiment, the activated dendritic cell comprises the at least three LNPs each comprising at least one exogenous nucleic acid sequence at a molar ratio of about 1 : 1 : 1.

[0246] The skilled person understands that term “at least one LNP comprising at least one exogenous nucleic acid sequence” also refers to a plurality of LNPs each comprising the same at least one exogenous nucleic acid sequence. In practice, a plurality of LNPs is used.

[0247] Furthermore, the skilled person understands that the term “at least three LNPs each comprising at least one exogenous nucleic acid sequence” also refers to three different pluralities of LNP, for example, three different mixtures of LNPs each comprising a different at least one exogenous nucleic acid sequence, which are combined to form a mixture of “at least three LNPs” or “at least three pluralities of LNPs”. Thus, to prepare such an activated dendritic cell, the cell is incubated with at least three different LNPs, or at least three different pluralities of LNPs.

[0248] In one embodiment, the ex vivo activated dendritic cell of the invention comprises at least three LNPs, each comprising an exogenous nucleic acid sequence, the first T08741ANM - 63 - exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti- PD-L1 antibody or a PD-L1 binding fragment thereof, and an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; the second exogenous nucleic acid sequence encoding an immune modulator selected from the group consisting of interleukin- lb (IL- lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL- 18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa), ICOSL ,CD70, CD30L, CD40, CD80, CD86, OX40L, 4-1BBL, GITRL, TIM-4 or a combination thereof; and the third exogenous nucleic acid sequence encoding at least one antigenic peptide comprising at least one tumor-specific neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6 / E7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0249] In one embodiment, the ex vivo activated dendritic cell of the invention comprises at least three LNPs, each comprising an exogenous nucleic acid sequence, the first exogenous nucleic acid sequence encoding a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti- PD-L1 antibody or a PD-L1 binding fragment thereof, an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof, anti-TIM-3 antibody or a TIM-3 binding fragment thereof, anti-LAG-3 antibody or a LAG-3 binding fragment thereof, and anti-TIGIT antibody or a TIGIT -binding fragment thereof; the second exogenous nucleic acid sequence encoding an immune modulator selected from the group consisting ofinterleukin-lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL- 4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin- 21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL- T08741ANM - 64 -

[0250] 23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) , ICOSL ,CD70, CD30L, CD40, CD80, CD86, OX40L, 4-1BBL, GITRL, TIM-4 or a combination thereof; and the third exogenous nucleic acid sequence encoding at least one antigenic peptide comprising at least one tumor-specific neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0251] In one embodiment, the activated dendritic cells described herein interacts with tumorspecific CD8+T cells and / or with tumor-specific CD4+T cells. In one embodiment, the activated dendritic cell of the invention interacts with tumor-specific CD8+T cells and with tumor-specific CD4+T cells at the same time, forming a three-cell-type cluster (triad).

[0252] In one embodiment, the activated dendritic cells described herein activate CD8+T cells. In one embodiment, the activated dendritic cells described herein activate antigen-reactive CD8+T cells.

[0253] In one embodiment, the activated dendritic cells described herein activate tumor- reactive CD8+T cells. In one embodiment, the activated dendritic cells described herein activate tumor-specific CD8+T cells.

[0254] In one embodiment, the activated dendritic cells described herein induce proliferation of CD8+T cells. In one embodiment, the activated dendritic cells described herein induce proliferation of antigen-reactive CD8+T cells.

[0255] In one embodiment, the activated dendritic cells described herein induce proliferation of tumor-reactive CD8+T cells. In one embodiment, the activated dendritic cells described herein induce proliferation of tumor-specific CD8+T cells.

[0256] In one embodiment, the activated dendritic cells comprising an mRNA encoding anti- T08741ANM - 65 -

[0257] PD-1 antibody described herein induce proliferation of CD8+T cells. In one embodiment, the activated dendritic cells comprising an mRNA encoding anti -PD-1 antibody described herein induce proliferation of antigen-specific CD8+T cells.

[0258] In one embodiment, the activated dendritic cells comprising an mRNA encoding anti- PD-1 antibody described herein induce proliferation of tumor-reactive CD8+T cells. In one embodiment, the activated dendritic cells described herein induce proliferation of tumor-specific CD8+T cells. The anti -PD-1 antibody may be an anchored or secreted anti -PD-1 antibody.

[0259] In one embodiment, the activated dendritic cells comprising an mRNA encoding IL- 12 described herein induce proliferation of CD8+T cells. In one embodiment, the activated dendritic cells comprising an mRNA encoding IL-12 described herein induce proliferation of antigen-specific CD8+T cells.

[0260] In one embodiment, the activated dendritic cells comprising an mRNA encoding IL- 12 described herein induce proliferation of tumor-reactive CD8+T cells. In one embodiment, the activated dendritic cells described herein induce proliferation of tumor-specific CD8+T cells.

[0261] In a particular embodiment, the activated dendritic cells described above are further stimulated by an antigen. In one embodiment, the antigen is the target of the induced or activated antigen-specific CD8+T cells. In one embodiment, the antigen is selected from the antigenic peptides or proteins described herein.

[0262] In one embodiment, the activated dendritic cells described herein interacts with tumorinfiltrating lymphocytes (TILs). TILs are primarily T-cells, that have migrated from the blood into a tumor to recognize and kill cancer cells.

[0263] In one embodiment, interaction of the activated dendritic cells described herein with TILs induces the generation of memory type T cells. In one embodiment, the memory type T cells can be identified by the expression of markers CD44+and CD62L+and / or T08741ANM - 66 -

[0264] IL7R.

[0265] In one embodiment, activated dendritic cells comprising at least one mRNA encoding an anti-PD-1 antibody induce increased production of CD44+ high, CD62L+ memory T cells. In one embodiment, activated dendritic cells comprising at least one mRNA encoding an anti-PD-1 antibody and are further treated with IL-2 induce increased production of CD44+ high, CD62L+ memory T cells. Increased production of CD44+ high, CD62L+ memory T cells can be seen in comparison to activated dendritic cells not comprising at least one mRNA encoding an anti-PD-1 antibody. Increased production of CD44+ high, CD62L+ memory T cells can also be seen in comparison to activated dendritic cells comprising at least one mRNA encoding glycoprotein 33. In one embodiment, activated dendritic cells comprising at least one mRNA encoding an anti-PD-1 antibody increases expression of IL7R in memory T cells.

[0266] In one embodiment, activated dendritic cells comprising at least one mRNA encoding an anti-PD-1 antibody induce decreased production of CD44+ low effector T cells. Decreased production of CD44+ low effector T cells can be seen in comparison to activated dendritic cells not comprising at least one mRNA encoding an anti-PD-1 antibody.

[0267] In one embodiment, interaction of the activated dendritic cells described herein with TILs induces phenotypic changes in effector T cells. In one embodiment, interaction of the activated dendritic cells described herein with TILs induces phenotypic changes in effector T cells, wherein the expression of markers IL7R and Tim3 and / or the expression of PD-1 in effector cells is reduced. In particular, interaction of activated dendritic cells comprising at least one mRNA encoding an anti-PD-1 antibody with TILs induces phenotypic changes in effector T cells, wherein the expression of PD-1 in effector cells is reduced.

[0268] Modifications ofRNA T08741ANM - 67 -

[0269] In one embodiment, the RNA (e.g. mRNA) is a modified RNA, in particular a stabilized mRNA. In some embodiments, the RNA (e.g. mRNA) may be modified for maximal efficacy, e.g. improved longevity in the cell, transcriptional efficacy, non- immunogenic properties (no toll-receptor induction etc.) and / or structural mRNA- stability.

[0270] In one embodiment, the RNA (e.g. mRNA) independently comprises at least one chemical modification. The chemical modification can e.g. be a modified nucleoside. In one embodiment, the chemical modification comprises a naturally occurring modified nucleoside. Naturally occurring modified nucleosides comprise 1- methyladenosine (nfA), N6-methyladenosine (m6A), 2'-O-methyladenosine (Am), 5- methylcytidine (m5C), 2'-O-methylcytidine (Cm), 2-thiocytidine (s2C), N4- acetylcytidine (ac4C), 5-formylcytidine (FC), 2'-O-methylguanosine (Gm), inosine (I), pseudouridine ( ), 5-methyluridine (m5U), 2'-O-methyluridine (Um). 1- methylpseudouridine (m I ), 2-thiouridine (s2U), 4-thiouridine (s4U), 5- methoxyuridine (mo5U), 3 -methyluridine (m3U). In one embodiment, the RNA (e.g. mRNA) comprises a modified nucleoside in place of at least one uridine. In one embodiment, the RNA comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleoside is independently selected from pseudouridine, Nl-methyl-pseudouridine, 5-methyl-uridine and Nl- ethylpseudouridine. In one embodiment, the modified nucleoside is a Nl- methylpseudouridine modification or a N1 -ethylpseudouridine modification. In one embodiment, the modified nucleoside is a N1 -methylpseudouridine. For instance, Nl- methyl-pseudouridine was found to be superior to several other nucleoside modifications and their combinations in terms of translation capacity. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine nucleosides in the mRNA are chemically modified.

[0271] Alternatively or additionally to a chemical modification, the sequence of the RNA, in particular mRNA, may be optimized. RNA sequence optimization inter alia comprises T08741ANM - 68 - codon-optimization, optimization of G / C content and optimization of structural elements (e.g. 5’ cap, 5’ UTR, 3’ UTR and poly(A)-tail).

[0272] In some embodiments, the amino acid sequence of the at least one checkpoint inhibitor, cytokine or antigenic peptide is encoded by a coding sequence which is codon- optimized and / or the G / C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and / or increased in the G / C content compared to the corresponding sequence regions of the wild type coding sequence. In one embodiment, the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence. In some embodiments, the G / C content of the coding region of the RNA is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild type RNA.

[0273] In some embodiments, the RNA (e.g. mRNA) may contain one or more optimized structural elements. Structural elements comprise a 5’ cap, 5’ UTR, 3’ UTR and poly(A)-tail. Thus, in one embodiment, the RNA (e.g. mRNA) comprises a 5' untranslated region (UTR), a 3' UTR, a 5' cap and / or a poly(A) tail. In one embodiment, the RNA (e.g. mRNA) contains all of these elements. In some embodiments, the RNA comprises a 5’-UTR and / or a 3’-UTR. In one embodiment, the RNA (e.g. mRNA) comprises a cap. In some embodiments, the RNA (e.g. mRNA) comprises a 3’-poly(A) sequence. In one embodiment, the cap is a Capl structure or a m7GpppG cap. In one embodiment, the sequence of the 5’ UTR and / or 3 ’UTR is optimized. In some embodiments, the mRNA comprises a 5' or 3' UTR that is a heterologous UTR. In some embodiments, the 5’ and / or 3 ’UTR sequences can be derived from mRNA which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA. For example, as 5’- UTR sequence, the 5 ’-UTR sequence of the human alpha-globin mRNA, optionally with an optimized ’Kozak sequence’ to increase translational efficiency may be used. Alternatively, the 5 ’UTR sequence of a human cytochrome mRNA may be used, e.g. T08741ANM - 69 - the human cytochrome b-245 alpha mRNA or cytochrome p4502El mRNA. As 3’- UTR sequence, a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) placed between the coding sequence and the poly(A)-tail to assure higher maximum protein levels and prolonged persistence of the mRNA may be used. Alternatively, the 3’-UTR may be two re-iterated 3’-UTRs of the human beta-globin mRNA.

[0274] In one embodiment, the poly-A sequence comprises at least 100 nucleotides. In one embodiment, the poly-A sequence comprises at least 150 nucleotides. In one embodiment, the poly-A sequence comprises at least 250 nucleotides. Furthermore, a poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues may be used. This poly(A)-tail sequence enhances RNA stability and translational efficiency . In one embodiment, a poly(A)-tail measures 300 to 800 nucleotides in length.

[0275] Lipid nanoparticles

[0276] The at least one exogenous nucleic acid sequence can be encapsulated in a lipid nanoparticle (LNP). For example, the exogenous nucleic acid sequence, e.g. an mRNA, can be encapsulated in a lipid nanoparticle for delivery to the activated dendritic cells of the invention. The encapsulated RNA (e.g. mRNA) is combined with the activated dendritic cells.

[0277] The LNP can comprise 3, 4 or 5 classes of lipids. LNP with three classes of lipids comprise: (1) an ionizable lipid, (2) a PEGylated lipid and (3) a cholesterol-based lipid. LNP with four classes of lipids comprise: (1) an ionizable lipid, (2) a PEGylated lipid,

[0278] (3) a cholesterol-based lipid and (4) a helper lipid. LNPs with five classes of lipids comprise: (1) an ionizable lipid, (2) a PEGylated lipid, (3) a cholesterol-based lipid,

[0279] (4) a helper lipid and (5) DSPE-PEG-maleimide or DSPN-PEG-azide. DSPE-PEG- T08741ANM - 70 - maleimide or DSPN-PEG-azide allow for adding ligands for targeted delivery.

[0280] (1) Ionizable lipids

[0281] An ionizable lipid facilitates mRNA encapsulation and may be a cationic lipid. A cationic lipid affords a positively charged environment at low pH to facilitate efficient encapsulation of the negatively charged mRNA. In one embodiment, the cationic lipid is cKK-E12 ((3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione); see Dong et al., PNAS (2014) 111(11):3955-60). Other cationic lipids that can be used include those described in Dong et al., 2014.

[0282] (2) PEG modified lipid

[0283] A PEG modified lipid component provides control over particle size and stability of the nanoparticle. The addition of such components may prevent complex aggregation and provide a means for increasing life-time and increasing the delivery of the LNPs to the activated dendritic cells.

[0284] Contemplated PEGylated lipids include, but are not limited to, a polyethylene glycol (PEG) chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 (for example, C8, CIO, C12, C14, C16 or C18) length, such as a derivatized ceramide (e.g., N-octanoyl-sphingosine-1- [succinyl(m ethoxypoly ethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipid is 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); l,2-distearoyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol (DSPE-PEG); l,2-dilauroyl-sn-glycero-3- phosphoethanolamine-poly ethylene glycol (DLPE-PEG); or 1,2-distearayl-rac- glycero-polyethelene glycol (DSG-PEG). In particularly exemplary embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In some embodiments, the PEG is PEG2000, also known as PEG-2K. In some embodiments, the PEG-modified lipid is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000 or C8 PEG2000.

[0285] (3) Cholesterol based lipid

[0286] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNPs comprise one or more cholesterol-based T08741ANM - 71 - lipids. Suitable cholesterol-based lipids include, for example: N,N-dimethyl-N- ethylcarboxamido-cholesterol, l,4-bis(3-N-oleylamino-propyl)piperazine , imidazole cholesterol ester, P-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol -based lipid used in the LNPs is cholesterol.

[0287] (4) Helper Lipids

[0288] A helper lipid enhances the structural stability of the LNP and helps the LNP in endosome escape. It improves uptake and release of the RNA (e.g. mRNA) load. The cross-presenting capacity of DCs can be limited by non-specific degradation during endosome maturation. Thus, LNPs providing better endosome escape may be useful in some embodiments. In one embodiment, the helper lipid is a non-cationic lipid. In some embodiments, the helper lipid is a zwitterionic lipid, which has fusogenic properties for enhancing uptake and release of the load. Examples of helper lipids are l,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero- 3 -phosphocholine (DSPC); l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2- dielaidoyl-sn-glycero-3 -phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-

[0289] Distearoylphosphatidylethanolamine (DSPE), and l,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE).

[0290] In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE. In some embodiments, the activated dendritic cells may be loaded with different antigenic peptides to give rise to multi-valent antigen-specific activated dendritic cells. For example, if the at least one antigenic peptide is encoded by an mRNA, encapsulated in a LNP, the LNP may carry mRNAs that encode more than one antigenic peptides or proteins , such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigenic peptides or proteins, from the same or different tumors.

[0291] Molar Ratios of the Lipid Components

[0292] Specific molar ratios of the above components may be important for the LNPs’ T08741ANM - 72 - effectiveness. The molar ratio of the cationic lipid, the PEGylated lipid, the cholesterol-based lipid, and the helper lipid is A: B: C: D, where A+B+C+D=100%. In some embodiments, the molar ratio of the cationic lipid in the LNPs relative to the total lipids (i.e., A) is 35-50%, optionally 35-45%. In some embodiments, the molar ratio of the PEGylated lipid component relative to the total lipids (i.e., B) is 0.25- 2.75%, optionally about 1.5%. In some embodiments, the molar ratio of the cholesterol-based lipid relative to the total lipids (i.e., C) is 20-46.5%. In some embodiments, the molar ratio of the cholesterol-based lipid relative to the total lipids (i.e., C) is about 46.5%. In another embodiment, the molar ratio of the cholesterol- based lipid relative to the total lipids (i.e., C) is about 38.5%. In some embodiments, the molar ratio of the helper lipid relative to the total lipids (i.e., D) is about 10-35%, such as 10-25%, or 16-35% (e.g., 16-32% such as 16%). In some embodiments, the molar ratio of the helper lipid relative to the total lipids (i.e., D) is about 10%. In another embodiment, the molar ratio of the helper lipid relative to the total lipids (i.e., D) is about 16%. In one embodiment, the ratio of the components is 35: 2.5: 46: 16 (A: B: C: D). In another embodiment, the ratio of the components is 50:38.5:1.5: 10 (A: B: C: D).

[0293] In one embodiment, the lipid nanoparticle comprises cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE. In one embodiment, the lipid nanoparticle comprises cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 35:46.5:2.5: 16.

[0294] In some embodiments, the lipid nanoparticle comprises SM102, cholesterol, DMG- PEG-2K, DSPC. In one embodiment, the lipid nanoparticle comprises SMI 02, cholesterol, DMG-PEG-2K, DSPC at a ratio of 50:38.5: 1.5: 10.

[0295] In some embodiments, the (PEGylated lipid+cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs contain a molar ratio of the cationic lipid to the helper lipid that is more than 1. To calculate the actual amount of each lipid to be put into an LNP formulation, the molar amount of the cationic lipid is first determined based on a desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the T08741ANM - 73 -

[0296] RNA (e.g. mRNA) to be transported by the LNP. Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the molar ratio selected. These molar amounts are then converted to weights using the molecular weight of each lipid.

[0297] The lipid nanoparticles comprising the mRNA can be provided frozen. For example, this may be useful if the activated dendritic cells and lipid nanoparticles comprising the mRNA are provided as a kit.

[0298] In one embodiment, the lipid nanoparticle comprises cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 30-40:41-51 : 1.0-4.0: 12-21. In one embodiment, the lipid nanoparticle comprises cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 33-37:44-48:2.0-3.0: 14-18. In one embodiment, the lipid nanoparticle comprises cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 34-36:45- 47:2.2-2.8: 15-17. In one embodiment, the lipid nanoparticle comprises cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 35:46.5:2.5: 16.

[0299] In one embodiment, the lipid nanoparticle comprises SM-102, cholesterol, C14-PEG 2000-PE and DOPE. In one embodiment, the lipid nanoparticle comprises SM-102, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 30-40:41-51 : 1.0-4.0: 12-21. In one embodiment, the lipid nanoparticle comprises SM-102, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 33-37:44-48:2.0-3.0: 14-18. In one embodiment, the lipid nanoparticle comprises SM102, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 34-36:45-47:2.2-2.8: 15-17. In one embodiment, the lipid nanoparticle comprises SM-102, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 35:46.5:2.5: 16. In some embodiments, the lipid nanoparticle comprises SM102, cholesterol, DMG-PEG-2K, DSPC. In one embodiment, the lipid nanoparticle comprises SM102, cholesterol at a ratio of 30-50:35-45: 1.0-4.0: 10-20, DMG-PEG- 2K, DSPC at a ratio of 50:38.5: 1.5: 10.

[0300] In one embodiment, the lipid nanoparticle comprises MC3, cholesterol, C14-PEG 2000-PE and DOPE. In one embodiment, the lipid nanoparticle comprises MC3, T08741ANM - 74 - cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 30-40:41-51 : 1.0-4.0: 12-21. In one embodiment, the lipid nanoparticle comprises MC3, cholesterol, C14-PEG 2000- PE and DOPE at a ratio of 33-37:44-48:2.0-3.0: 14-18. In one embodiment, the lipid nanoparticle comprises MC3, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 34-36:45-47:2.2-2.8: 15-17. In one embodiment, the lipid nanoparticle comprises MC3, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 35:46.5:2.5: 16.

[0301] In one embodiment, the lipid nanoparticle comprises C12-(2-3-2), DPPC, cholesterol and DMG-PEG-2K. In one embodiment, the lipid nanoparticle comprises C12-(2-3- 2), DPPC, cholesterol and DMG-PEG-2K at a ratio of 8:5.29:4.41 :0.88.

[0302] Size and amount of lipid nanoparticles

[0303] Suitable LNPs may be made in various sizes. In some embodiments, the majority of purified LNPs, i.e., greater than about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the LNPs, have a size of about 50 to 200 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., greater than 80 or 90%) of the purified lipid nanoparticles have a size of about 70 to 200 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, greater than about 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% of the LNPs in the present composition have a size ranging from about 85 to 100 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm) or about 50-70 nm (e.g., 55-65 nm). The smaller sizes are particular suitable for inhalative delivery via nebulization. In some embodiments, the activated dendritic cells are combined with at least about 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 10 pg or 20 pg of encapsulated RNA, particularly mRNA. In some embodiments, the activated dendritic cells are combined with about 10 pg of encapsulated RNA, particularly mRNA. In some embodiments, the activated dendritic cells are combined with about T08741ANM - 75 -

[0304] 20 pg of encapsulated RNA, particularly mRNA.

[0305] Lipoplexes

[0306] The at least one mRNA can be comprised in a lipoplex. Mixing of RNA (e.g. mRNA) and positive charged liposomes results in the formation of lipoplex particles by spontaneous self assembly. The liposomes typically contain at least two components: a cationic lipid and a neutral lipid. Lipoplexes have extensively be described in the art, see, e.g. Nanomedicine: Nanotechnology, Biology and Medicine, 2009. In one embodiment, the neutral lipid is a helper lipid as defined above. In an exemplary embodiment, the cationic lipid is DOTMA and the neutral lipid is DOPE.

[0307] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one neutral lipid is from about 10:0 to about 1 :9, about 4: 1 to about 1 :2, or about 3 : 1 to about 1 : 1. In specific embodiments, the molar ratio may be about 3:1, about 2.75: 1, about 2.5: 1, about 2.25: 1, about 2: 1, about 1.75: 1, about 1.5: 1, about 1.25: 1, or about 1 : 1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2: 1. RNA lipoplex particles described herein have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm or about 700 nm. In an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of T08741ANM - 76 - about 400 nm.

[0308] In one embodiment, the (mRNA) lipoplex particles comprise at least one cationic lipid and at least one neutral lipid. In one embodiment, the at least one cationic lipid comprises l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and / or

[0309] 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, the at least one neutral lipid comprises l,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol (Choi) and / or l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and the at least one neutral lipid comprises l,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE). In one embodiment, the lipoplex particles comprise

[0310] 1.2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and l,2-di-(9Z- octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). Spleen targeting RNA lipoplex particles are described in WO 2013 / 143683, herein incorporated by reference. It has been found that lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen- presenting cells, in particular dendritic cells.

[0311] II. The pharmaceutical composition

[0312] In a third aspect, the invention relates to a pharmaceutical composition comprising the activated dendritic cell described herein and a pharmaceutically acceptable carrier.

[0313] In one embodiment, the pharmaceutical composition of the invention comprises the activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor.

[0314] In another embodiment, the pharmaceutical composition of the invention comprises the activated dendritic cell comprising at least one exogenous nucleic acid sequence T08741ANM - 77 - encoding a checkpoint inhibitor and at least one further exogenous nucleic acid sequence as described above.

[0315] In one embodiment, the pharmaceutical composition of the invention further comprises an immune modulator. The immune modulator may be comprised in the pharmaceutical compositing in addition to the activated dendritic cell.

[0316] In one embodiment, the immune modulator is a cytokine.

[0317] In one embodiment, the cytokine is capable of inducing CD8+ T cells.

[0318] In one embodiment, the immune modulator is selected from a cytokine of the group consisting of interleukin- lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL-15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL- 18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof.

[0319] In a preferred embodiment, the cytokine is interleukin- 12 (IL-12).

[0320] Hence, in one embodiment, the pharmaceutical composition of the invention comprises the activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and a cytokine. In a preferred embodiment, the pharmaceutical composition of the invention comprises the activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and is interleukin- 12 (IL-12).

[0321] The immune modulator may be expressed by a second activated dendritic cell. In one embodiment, the second activated dendritic cell is capable of expressing and / or secreting the immune modulator.

[0322] Hence, in one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least one second activated T08741ANM - 78 - dendritic cell comprising an exogenous nucleic acid sequence encoding an immune modulator.

[0323] Hence, in one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding an immune modulator, wherein the immune modulator is selected from a cytokine of the group consisting of interleukin-lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL-15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL- 18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof. In a preferred embodiment, the cytokine is interleukin- 12 (IL-12).

[0324] In one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is selected from an anti -PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding an immune modulator, wherein the immune modulator is selected from a cytokine of the group consisting of of interleukin-lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL- 4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin- 21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof. In a preferred embodiment, the cytokine is interleukin- 12 (IL-12). In a preferred embodiment, the checkpoint inhibitor is T08741ANM - 79 - selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD- L1 antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and the cytokine is interleukin- 12 (IL-12).

[0325] In another embodiment, the pharmaceutical composition of the invention comprises the first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide.

[0326] In one embodiment, the at least one antigenic peptide is a tumor-associated peptide or protein, a viral antigenic protein, a bacterial antigenic protein, or a fungal antigenic protein. In one embodiment, the at least one antigenic peptide is a tumor- associated peptide or protein, and wherein the tumor-associated peptide or protein is a tumor-specific peptide or protein, preferably wherein the tumor-specific peptide or protein comprises at least one tumor-specific neo-antigen. In one embodiment, the least one tumor-specific neo-antigen is selected from the group consisting of Aatf, Cpnel, Dpagtl, Wbp7, Nlel, Irgq, Zbtb40, Cryl, pl5E, Gtf2i, Med 12, N4bp212 and Sydel, or a combination thereof. In one particular embodiment, the at least one tumor-specific neo-antigen is a patient-specific tumor-specific neo-antigen. In a preferred embodiment, the patient is a human subject. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6 / E7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0327] Hence, in one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic T08741ANM - 80 - acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide, wherein at least one antigenic peptide is a tumor- associated peptide or protein, and wherein the tumor-associated peptide or protein is a tumor-specific peptide or protein, preferably wherein the tumor-specific peptide or protein comprises at least one tumor-specific neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In a preferred embodiment, the patient is a human subject. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0328] In one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is selected from an anti -PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one tumor-specific neo-antigen. In a preferred embodiment, the checkpoint inhibitor is selected from an anti -PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and the at least one tumor-specific neo-antigen is a patient-sepcific tumor neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In a preferred embodiment, the patient is a human subject. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated T08741ANM - 81 -

[0329] Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0330] In one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one tumor-specific neo-antigen. In a preferred embodiment, the checkpoint inhibitor is selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and the least one tumor-specific neo-antigen is a patient-specific tumor neo-antigen.. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In a preferred embodiment, the patient is a human subject. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0331] In another embodiment, the pharmaceutical composition of the invention comprises the first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide; and further comprises an immune modulator. T08741ANM - 82 -

[0332] Hence, in one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD- L1 antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one tumor-specific neo-antigen; and further comprises a cytokine.

[0333] In one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one tumor-specific neo-antigen; and further comprises interleukin- 12 (IL-12). In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In a preferred embodiment, the patient is a human subject. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6 / E7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0334] In another embodiment, the pharmaceutical composition of the invention a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and at least two additional activated dendritic cells comprising different exogenous nucleic acid sequences. Hence, in one embodiment, the pharmaceutical composition of the invention comprises a first T08741ANM - 83 - activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor; at least one second activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding an immune modulator; and at least one third activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding at least one antigenic peptide.

[0335] Hence, in one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding an immune modulator, wherein the immune modulator is selected from a cytokine of the group consisting of of interleukin-lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL- 6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL-15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL- 18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof; and at least one third activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide, wherein at least one antigenic peptide is a tumor- associated peptide or protein, and wherein the tumor-associated peptide or protein is a tumor-specific peptide or protein, preferably wherein the tumor-specific peptide or protein comprises at least one tumor-specific neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In a preferred embodiment, the patient is a human subject. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras. T08741ANM - 84 -

[0336] In one embodiment, the pharmaceutical composition of the invention comprises a first activated dendritic cell comprising at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, wherein the checkpoint inhibitor is selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; and at least one second activated dendritic cell comprising an exogenous nucleic acid sequence encoding an immune modulator, wherein the immune modulator is selected from a cytokine of the group consisting of interleukin- 1b (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL- 23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof; and at least one third activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one tumor-specific neo-antigen. In a preferred embodiment, the checkpoint inhibitor is selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof; the cytokine is interleukin- 12 (IL-12); and the at least one tumorspecific neo-antigen is a patient-specific tumor neo-antigen. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In a preferred embodiment, the patient is a human subject. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0337] In another preferred embodiment, the checkpoint inhibitor is selected from an anti- PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD- L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding T08741ANM - 85 - fragment thereof; the cytokine is interleukin- 12 (IL-12); and the at least one tumorspecific neo-antigen is selected from the group consisint of Aatf, Cpnel, Dpagtl, Wbp7, Nlel, Irgq, Zbtb40, Cryl, pl5E, Gtf2i, Med 12, N4bp212 and Sydel, or a combination thereof. In one embodiment, the at least one tumor-specific neo-antigen is a patient-specific antigen. In a preferred embodiment, the patient is a human subject. In one embodiment, the tumor-specific neo-antigen comprises one or more epitopes from HPV E6ZE7, Merkel Cell polyoma LTA epitope, Tumor Associated Antigens (TAA) (preferably a patient-specific TAA), lineage-restricted Differentiation antigen (such as melanoma MART-1 or TRP2), NY-ESO-1, p53 or Ras.

[0338] In all embodiments listed above, the pharmaceutical composition of the invention optionally comprises a pharmaceutically acceptable carrier and / or diluent. Additionally, the pharmaceutical composition can comprise adjuvants and / or immuno-modulators to boost the activity of the pharmaceutical composition and the subject’s response. Such adjuvants and / or immuno-modulators are understood by those skilled in the art, and are readily described in available published literature.

[0339] In one embodiment, the pharmaceutical composition of the invention contains at least 1, at least 10, at least 100, at least 1000, at least 1 x 104, at least 1 x 105or at least 1 x 106activated dendritic cells. The number of activated dendritic cells can e.g. be estimated from the volume of the whole blood, which became apheresed and flow chamber (e.g. plate or bag or hybrid) passed or flow chamber (e.g. plate or bag or hybrid) passed directly. The at least one mRNA can be provided encapsulated in nanoparticles (e.g. LNPs), with the pharmaceutical composition containing at least about 0.1 pg, 0.5 pg, 5 pg, 10 pg, 100 pg, 500 pg or 1000 pg of encapsulated RNA, in particular mRNA.

[0340] In one embodiment, the pharmaceutical composition comprises at least one population T08741ANM - 86 - of LNPs, wherein each population of LNPs comprises at least one mRNA encoding at least one amino acid sequence. Hence, the pharmaceutical composition may comprise two or more populations of LNPs, wherein each population of LNP comprises one mRNA encoding one amino acid sequence comprising one protein. Alternatively or additionally, the pharmaceutical composition may comprise one population of LNPs, wherein the population of LNPs comprises two or more mRNAs each encoding one amino acid sequences each comprising one protein. Alternatively or additionally, the pharmaceutical composition may comprise one population of LNPs, wherein the population of LNPs comprises one mRNA encoding one amino acid sequence comprising two or more different proteins.

[0341] The pharmaceutical composition of the invention optionally comprises a pharmaceutically acceptable carrier and / or diluent. Additionally, the pharmaceutical composition can comprise adjuvants and / or immuno-modulators to boost the activity of the pharmaceutical composition or vaccine composition and the subject’s response. Such adjuvants and / or immuno-modulators are understood by those skilled in the art, and are readily described in available published literature. The pharmaceutical composition of the invention may contain one or more T cell activating agents.

[0342] In all embodiments of the invention, the pharmaceutical composition is suitable for the prevention of a disease, such as cancer. Hence, in one aspect of the invention, the pharmaceutical composition of the invention, as described in detail above, is for use in therapy. In one embodiment, the invention provides the pharmaceutical composition of the invention, as described in detail above, for use in a method of treating cancer. In another embodiment, the invention provides the pharmaceutical composition of the invention, as described in detail above, for use in a method of treating cancer, wherein the treatment does not induce autoimmunity. In one embodiment, the invention provides the pharmaceutical composition of the invention, as described in detail above, for use in a method of treating cancer, wherein the activation of autoimmune T cells is T08741ANM - 87 - reduced compared to treatment with a pharmaceutical composition comprising a soluble checkpoint inhibitor alone, preferably a soluble anti-PD-1 antibody. In one embodiment, the invention provides the pharmaceutical composition of the invention, as described in detail above, for use in a method of treating cancer, wherein the toxicity of the treatment is reduced compared to treatment with a pharmaceutical composition comprising a soluble checkpoint inhibitor alone, preferably a soluble anti-PD-1 antibody. In one embodiment, the invention provides the pharmaceutical composition of the invention, as described in detail above, for use in a method of treating cancer, wherein the side effects of the treatment is reduced compared to treatment with a pharmaceutical composition comprising a soluble checkpoint inhibitor alone, preferably a soluble anti-PD-1 antibody.

[0343] In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response includes induction or secretion of interferon-gamma (IFN-y) and / or tumor necrosis factor alpha (TNF-a). In a particular embodiment, the pharmaceutical composition acts by one or more of the following: (i) increasing cytotoxic T lymphocytes such as CD8+ (CTL) to attack and kill the disease causing particles which express the respective antigen; (ii) increasing T helper cell responses; (iii) increasing inflammatory responses via IFN-y and / or TNF-a; i(v) increasing natural killer cell responses; (v) increasing central memory T cell and stemlike T cell subsets. In one embodiment, the pharmaceutical composition acts by all of the aforementioned.

[0344] In one embodiment of the invention, the pharmaceutical composition of the invention for use in the treatment of disease does not induce diabetes. In one embodiment, the pharmaceutical composition of the invention for use in the treatment of disease does not induce type-1 diabetes. In one embodiment, the pharmaceutical composition of the invention for use in a method of treating cancer does not induce diabetes. In one T08741ANM - 88 - embodiment, the pharmaceutical composition of the invention for use in the treatment of disease does not activate autoimmune T cell, such as islet-reactive T cells.

[0345] Hence, in one embodiment, the invention relates to the pharmaceutical composition of the invention for use in preventing checkpoint inhibitor-induced type-1 diabetes.

[0346] The present invention provides an article of manufacture, such as a kit, that provides activated dendritic cells in one container and the at least one exogenous mRNA encoding a checkpoint inhibitor in another container. The container may be pre-treated glass or plastic vials or ampules. The article of manufacture may include instructions for use. The kit may also optionally comprise further containers comprising at least one exogenous mRNA encoding an immune modulator and / or at least one exogenous mRNA encoding at least one antigenic peptide.

[0347] III. Exemplary preparation of activated dendritic cells

[0348] In one example, activated dendritic cells can be prepared from monocytes (e.g. obtained from a donor). For instance, activated dendritic cells can be generated by subjecting monocytes (e.g. obtained from a donor) to a physical force. A physical force can e.g. be generated by passing the monocytes through a flow chamber. In one embodiment of the present invention, activated dendritic cell are obtained by platepassage of monocytes using known means, e.g. by use of shear stress in a a flow chamber or blood bag. The flow chamber can be a plate in one embodiment. A plate for preparing activated dendritic cells has been previously described in the literature, see e.g. Durazzo et al., 2014 (“Induction of Monocyte-to-Dendritic Cell Maturation by Extracorporeal Photochemotherapy: Initiation via Direct Platelet Signaling”) or Ventura et al., 2018 (“Extracorporeal Photochemotherapy Drives Monocyte-to- Dendritic Cell Maturation to Induce Anticancer Immunity”). Methods and devices for extracorporeal activation of monocytes and generation of dendritic cells therefrom are described in WO2014 / 106629 Al, W02014 / 106631 Al, W02016 / 001405 Al, and T08741ANM - 89 -

[0349] WO20 17 / 005700 Al, each of which is incorporated herein by reference in its entirety. Monocytes may be derived from a blood sample or a fraction thereof are exposed to mechanical stress (e.g., a physical force in the form of shear force) and plasma components (e.g., platelets) or derivatives or mimics thereof, thereby activating the monocytes to differentiate into healthy, physiologic dendritic cells which are also termed activated dendritic cell herein. However, providing monocytes and subjecting the monocytes to a physical force is sufficient for activation and differentiation of the monocytes into activated dendritic cells. Activation and differentiation of monocytes into activated dendritic cell, may be performed in a flow chamber (e.g., a THERAKOS® CELLEX® device); a Transimmunization plate (e.g. as described in W02017 / 005700 Al); or in a bag (e.g. flexible bag or plastic bag); or in a combination of any of the afore-mentioned.

[0350] The bag can be made of any material that does not leak liquids such as e.g. rubber, silicone or plastic. Preferably, the material is able to bend easily without breaking. Optionally, the bag or plate is made of a plastic material. Suitable plastic materials comprise polyolefin, polyethylene, fluoropolymer, polyvinyl chloride, ethylene-vinyl acetate-copolymer, ethylene vinyl alcohol, polyvinylidene fluoride, and / or other plastic comprising materials approved for medical use. The preferred plastic material is ethylen-vinyl acetate-copolymer. The bag or plate may be made of a material that provides a degree of transparency such that the sample or cell mixture can be irradiated with visible or UV light.

[0351] In one embodiment, the flow chamber can be hybrid flow chamber or a combination of a chamber and a bag, wherein each of the components are as described herein.

[0352] The inventors found that activated dendritic cell obtained by the method described above are advantageous as compared to DC obtained by other methods such as incubation of blood monocytes with cytokines or direct isolation from a donor, as activated dendritic cell are generated physiologically (without the need for chemicals such as cytokines and / or apoptotic agents) with greater reproducibility and controllability under precise in vitro laboratory conditions. Benefits of the above T08741ANM - 90 - described method for generating activated dendritic cells compared to other methods such as incubation with cytokines include one or more of higher yields, a faster process, better intracellular antigen processing and more effective priming of diseasespecific cytotoxic T cells. In one embodiment, the activated dendritic cells of the invention are not produced by incubation with cytokines.

[0353] Activated dendritic cells of a donor are obtained by subjecting monocytes contained in a blood sample (obtained from the donor) to a shear force by passing the blood sample or fraction thereof through a flow chamber. Preferably, platelets are present in the flow chamber which can be either derived from the donor' s blood sample or a fraction thereof or provided separately. Additionally or alternatively, plasma components can be present in the flow chamber which can be either derived from the donor' s blood sample or a fraction thereof or provided separately. After activated dendritic cells have been obtained they can be stored frozen until further use, e.g. combination with the at least one disease associated antigen or at least one mRNA encoding at least one antigenic protein.

[0354] A monocyte of a donor may be obtained by any suitable means, e.g., from a blood sample or a fraction thereof. The fraction of the blood sample may be, e.g., a buffy coat including white blood cells and platelets. Alternatively, the fraction of the blood sample may be an isolated peripheral blood mononuclear cell (PMBC). PMBCs may be isolated from a blood sample using, e.g., centrifugation over a Ficoll-Hypaque gradient (Isolymph, CTL Scientific). In another example, the fraction of the blood sample may be a purified or enriched monocyte preparation. Monocytes may be enriched from PBMCs using, e.g., one, two, or all three of plastic adherence; CD14 magnetic bead positive selection (e.g., from Miltenyi Biotec); and a Monocyte Isolation Kit II (Miltenyi Biotec).

[0355] Any suitable volume of blood can be used. The blood sample (e.g., the blood sample from which the fraction is derived) may be between about 1 pL and about 500 mL, e.g., between about 1 pL and about 10 mL, between about 1 pL and about 5 mL, between about 1 pL and about 1 mL, between about 1 pL and about 750 pL, between T08741ANM - 91 - about 1 pL and about 500 pL, between about 1 pL and about 250 pL, between about 10 mL and about 450 mL, about 20 mL and about 400 mL, about 30 mL and about 350 mL, about 40 mL and about 300 mL, about 50 mL and about 200 mL, or about 50 mL and about 100 mL. In some embodiments, the blood sample or the fraction thereof or the additional blood sample or the fraction thereof is less than or equal to about 100 mL (e.g., about 50 mL to about 100 mL). In one embodiment, the monocytes are derived from a blood sample obtained from the donor. However, monocytes can be derived from other sources in the donor that provide monocytes, e.g. bone marrow, spleen or other peripheral tissues. In one embodiment, monocytes are derived from peripheral blood mononuclear cells (PBMC) obtained from the donor.

[0356] The skilled person is well aware of methods to distinguish dendritic cells including activated dendritic cell from monocytes, such as e.g. by assessing gene expression. For example, CD80, CD83, and CD86 are markers expressed in higher levels by mature dendritic cells as compared to monocytes. In one embodiment, the donor is mammalian. Mammals include for example, but are not limited to, humans, nonhuman primates, pigs, dogs, cats and rodents. In a preferred embodiment, the donor is human.

[0357] IV. Definitions

[0358] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting” is considered to be a preferred embodiment of the term “comprising”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments.

[0359] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. activated dendritic cells are defined to be obtainable by a specific method, this is also to be understood to T08741ANM - 92 - disclose activated dendritic cells, which are obtained by this method.

[0360] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The term "or" means, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, and even more preferably ±5 %.

[0361] Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or “(i)”, “(ii)”, “(iii)”, “(iv)” etc.and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or “(i)”, “(ii)”, “(iii)”, “(iv)” etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps unless indicated otherwise, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.

[0362] The term "about" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30, 25, 20, 15, 30 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. The term "about" in the context of the present invention denotes an interval of accuracy that the person skilled T08741ANM - 93 - in the art will understand to still ensure the technical effect of the feature in question. In general, the term "about" is intended to modify a numerical value above and below the stated value by a variance of ± 10%.

[0363] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0364] Ex vivo activated dendritic cell

[0365] “Dendritic cells,” also referred to herein as “DCs,” are antigen-presenting immune cells that process antigenic material and present it to other cells of the immune system, most notably to T cells. DCs function to capture and process antigens. When DCs endocytose antigens, they process the antigens into smaller fragments, generally peptides, that are displayed on the DC surface, where they are presented to, for example, antigen-specific T cells through MHC molecules. After uptake of antigens, DCs migrate to the lymph nodes. During maturation, DCs can be prompted by various signals, including signaling through Toll-like receptors (TLR), to express costimulatory signals that induce cognate effector T cells (Teff) to become activated and to proliferate, thereby initiating a T-cell mediated immune response to the antigen. Alternatively, DCs can present an antigen to antigen-specific T cells without providing co-stimulatory signals (or while providing co-inhibitory signals), such that Teff are not properly activated. Such presentation can cause, for example, death or anergy of T cells recognizing the antigen, or can induce the generation and / or expansion of regulatory T cells (Treg). The term “dendritic cells” includes differentiated dendritic cells, immature, and mature dendritic cells. These cells can be characterized by expression of certain cell surface markers (e.g., CD11c, MHC class II, and at least low levels of CD80 and CD86), CDl lb, CD304 (BDCA4)). In some embodiments, DCs express CD8, CD103, CDld, etc. Other DCs can be identified by the absence of lineage markers such as CD3, CD14, CD19, CD56, etc. In addition, dendritic cells can T08741ANM - 94 - be characterized functionally by their capacity to stimulate alloresponses and mixed lymphocyte reactions (MLR).

[0366] As used herein, the terms “ex vivo activated dendritic cell” or “activated dendritic cell (aDC)” are used interchangeably, and refer to dendritic cells which are produced by activating monocytes by shear stress, e.g. plate passing, a flow chamber, blood bag or extracorporeal photopheresis (ECP). Monocytes may be present in a blood sample, wherein the blood sample may comprise whole blood or a fraction thereof. The activated dendritic cells encompass dendritic cells of different stages of maturation or differentiation, including immature activated dendritic cells and mature activated dendritic cells. Immature activated dendritic cells have the capacity to further mature in vivo or in vitro to mature activated dendritic cells. The activated dendritic cells have also previously been referred to as “physiological dendritic cells”, “physiologic dendritic cells” or “phDC” in the literature, see e.g. Ventura et al., 2018 “Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity”, WO 2014 / 106629, WO 2016 / 001405 or Hanlon et al., 2020 “Rapid Production of Physiologic Dendritic Cells (phDCs) for Immunotherapy”. These terms refer to the same cells as the term “activated dendritic cell” used herein. The term “activated” in this context refers to the availability of the dendritic cell for highly effective and rapid loading or arming with nucleic acid sequences encoding proteins of interest, such as LNPs comprising mRNAs encoding checkpoint inhibitors described herein. The term “ex vivo” refers to outside-the-body production of the activated dendritic cells. Notably, activated dendritic cells are different from dendritic cells produced by other methods, in particular dendritic cells made by conventional methods rely on the use of cytokines to differentiate dendritic cells. In contrast, the activated dendritic cells of the invention are produced without the need for addition of a molecular cocktail comprising cytokines such as e.g. IL-4, GM-CSF, LPS, IFN-y, IL-ip and / or TNF-a. The activated dendritic cells more closely resemble the dendritic cells that are activated under physiological conditions. T08741ANM - 95 -

[0367] These activated dendritic cells may be loaded or comprise at least one nucleic acid sequence encoding a checkpoint inhibitor; these cells are referred to as “activated dendritic cell of the invention”. The loading of activated dendritic cells may comprise the incubation of the activated dendritic cells with the nucleic acid sequence encoding a checkpoint inhibitor, optionally wherein the nucleic acid (e.g. mRNA) is comprised in an LNP. The incubation may be performed for various time periods. The term activated dendritic cell or activated dendritic ell of the invention encompasses all incubation periods, including shorter incubation times (such as less than 16 h, such as 0.1 h-5 h, 4-6 h, 6-8 h, 8-10 h or 10-12 h), and longer incubation periods (such as 16 h or more).

[0368] Checkpoints and Checkpoint Inhibitors

[0369] Immune checkpoints are signaling pathways that regulate the immune system and can either be stimulatory or inhibitory. Inhibitory immune checkpoints are commonly upregulated on immune cells in the tumor microenvironment, thereby suppressing the immune system and helping tumors evade the immune response. Checkpoints contemplated herein are CTLA-4 (Cytotoxic T-Lymphocyte- Associated protein 4 and also called CD152),PD-1 (Programmed Death 1 (PD-1) receptor) and its ligand PD- Ll, ), and T-cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3) and T cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif (ITIM) domain (TIGIT).

[0370] The terms “Immune checkpoint inhibitors” or “checkpoint inhibitors” as used herein refer to molecules or drugs, such as small molecule compound, antibodies or antibody fragments, that inhibit and / or block inhibitory checkpoint molecules. The effect of a checkpoint inhibitor is that an inhibitory checkpoint, which may be upregulated in cancers, is blocked, and the immune system is in turn stimulated. Several Imune modulators exhibit antitumor properties in preclinical models, slowing tumor growth either by directly inhibiting proliferation and promoting T08741ANM - 96 - apoptosis, or indirectly by mobilizing an antitumor immune response. The activated dendritic cells of the invention, i.e. comprising and expressing a checkpoint inhibitor combines the anti-tumor effect of checkpoint inhibitors and the activated dendritic cells. Hence, the cytostatic, cytotoxic, and / or anti-angiogenic effects on tumors of checkpoint inhibitors is combined with the tumor targeting properties, enhanced tumor antigen presentation and activation of T cells, and boosting of natural killer (NK) cells, and reduction of regulatory T cells (Tregs). Without being bound by theory, this specific combination is believed to result in high therapeutic efficacy while minimizing adverse side effects that have been observed with a systemic administration of checkpoint inhibitors.

[0371] “T cells” as referred to herein, comprise CD8+ T cells, y5 T cells, natural killer T cells, CD4+ T cells, memory CD8+ T cells and memory CD4+ T cells. In a preferred embodiment T cells comprise CD4+ T cells, CD8+ T cells, memory CD4+ T cells and memory CD8+ T cells.

[0372] “CD4+ T cells” (also called T helper cells, Th cells) as referred to herein, are activated after interaction with antigen-MHC complex and differentiate into specific subtypes depending mainly on the cytokine milieu of the microenvironment. Besides the classical T-helper 1 and T-helper 2, other subsets have been identified, including T-helper 17, regulatory T cell, follicular helper T cell, and T-helper 9, each with a characteristic cytokine profile. Thl T cells, for example, mainly secrete IFNy, lymphotoxin a (Lfa), and IL2, while Th2 T cells mainly secrete IL4, IL5, IL9, IL13, IL10, IL25, and amphiregulin. Thl7 T cells mainly secrete IL17A, IL17F, IL21, and IL22. Main effector cytokines of Treg cells include IL10, TGF-P, and IL35.

[0373] Follicular helper T cells secrete IFNy, IL4 and IL10. The mentioned cytokines can be used to characterize the immune response according to the methods of the invention. “CD8+ (cytotoxic) T cells” (also referred to as CTLs), as referred to herein, express the T-cell receptor like CD4+ T cells. However, rather than the CD4 molecule, cytotoxic T cells express a dimeric co-receptor, CD8, usually composed of one CD8a and one CD8P chain. CD8+ T cells recognise peptides presented by MHC Class I T08741ANM - 97 - molecules, found on all nucleated cells. CD8+ T cells are very important for immune defence against intracellular pathogens, including viruses and bacteria, and for tumour surveillance. When a CD8+ T cell recognises its antigen and becomes activated, it will secrete primarily TNF-a and IFN-y, which have anti -turn our and anti-viral microbial effects.

[0374] CD8+ and CD4+ T cells can also be present in the form of memory T cells which maintain their antigen-specificity for the whole life-span of the host without further antigenic stimulation and can confer immunological protection to the subject against the antigen, memory T cells inter alia express the marker CD45RO, which may be used to characterize the immune response according to the methods of the invention. Memory T cells can also be further characterized using the markers CD44 and CD62L. CD44 is a cell surface glycoprotein involved in cell adhesion and migration. It mediates interactions with the extracellular matrix, particularly with hyaluronic acid. It is generally used to distinguish activated or memory T cells from naive T cells, which express low levels of CD44. CD44 expression levels can also be used to define subsets of memory cells. Herein, subsets of memory cells are indicated as memory 1 (higher expression of CD44) and memory 2 (lower expression of CD44, but still CD44 positive). High expression of CD44 in memory T cells after an immune response has concluded (e.g. once an infection is cleared) can be indicative of cells that have previously encountered antigen and are more likely to react faster upon subsequent exposures. CD62L, or L-selectin, is a molecule essential for the homing of T cells to secondary lymphoid organs (such as lymph nodes). It assists in the recirculation of T cells between the blood and lymphoid tissues.

[0375] A subset of T memory cells are T memory stem cells, a type of long-lived memory T cells with the ability to reconstitute diverse memory and effector T cell subpopluations as well as self-renewal. These cells are known to express high levels of CD62L, and IL-7Ra (Interleukin-7 receptor subunit alpha, also known as CD127), among others. T08741ANM - 98 -

[0376] “Effector cells” are generally CD62L low and CD44 high, and can be further categorized into effector 1 (higher expression of CD44) and effector 2 (lower expression of CD44.

[0377] “KLRG1” (Killer cell lectin-like receptor subfamily G member 1) is a co-inhibitory receptor expressed predominantly on late-differentiated effector and effector memory CD8+T and NK cells.

[0378] “Tim3” (T-cell immunoglobulin and mucin-domain containing-3) is a immune checkpoint receptor and mediates T-cell exhaustion together with other inhibitory receptors including PD-1.

[0379] “Natural Killer (NK) cells” as referred to herein, are lymphocytes in the same family as T cells, coming from a common progenitor. However, as cells of the innate immune system, NK cells are classified as group I innate lymphocytes (ILCs; ILCs comprise NK cells and ILCls, both capable of secreting IFNy) and respond quickly to a wide variety of pathological challenges. NK cells are best known for killing virally infected cells, and detecting and controlling early signs of cancer. Activated NK cells secrete a wide variety of cytokines such as IFN-y, TNF-a, GM-CSF, IL-10, IL-5, and IL-13 and chemokines such as MIP-la, MIP-ip, IL-8, and RANTES. The mentioned cytokines and chemokines can be used to characterize the immune response according to the methods of the invention.

[0380] “Gamma delta (y5) T cells” as referred to herein, are the prototype of ‘unconventional’ T cells and represent a relatively small subset of T cells in peripheral blood. They are defined by expression of heterodimeric T-cell receptors (TCRs) composed of y and 5 chains. This sets them apart from the classical CD4+ T cells and CD8+ T cells that express aP TCRs. y5 T cells mainly secrete IFN-y and TNF-a.

[0381] “Natural killer T (NKT) cells” as referred to herein, are a subset of CD Id-restricted T cells at the interface between the innate and adaptive immune system. NKT cells can be subdivided into functional subsets that respond rapidly to a wide variety of glycolipids and stress-related proteins using T- or natural killer (NK) cell-like T08741ANM - 99 - effector mechanisms. Because of their major modulating effects on immune responses via secretion of cytokines, NKT cells are also considered important players in tumor immunosurveillance. Upon activation, NKT cells produce IFN-y, IL-4, TNF-a, IL-5, IL-6, IL-10, IL-13 and TGF-p. Type I NKT cells synthesize growth factors such as IL-2 and GM-CSF and chemokines such as RANTES, MIP-la, and MIP-ip. The mentioned cytokines, growth factors and chemokines can be used to characterize the immune response according to the methods of the invention.

[0382] “IFNy producing killer dendritic cells” as referred to herein, are a subset of DCs appearing to be a chimera of NK cells and DCs, namely interferon-producing killer dendritic cells (IKDCs). IKDCs not only secrete type I and type II interferons to recognize and kill tumor cells effectively, but also express MHC-II molecules to present antigens. Thus, IKDCs are considered as important immunosurveilance cells for tumors, providing a link between innate and adaptive immunity.

[0383] Cells capable of secreting fFNy can be isolated from a subject (e.g. mammal, or human subject) using any suitable method known in the art. For example, cells capable of secreting IFNy (e.g. T cells) can be isolated from peripheral blood from a subject (e.g. mammal, or human subject) by using density centrifugation over a step gradient consisting of a mixture of the carbohydrate Ficoll™ and the dense iodine-containing compound metrizamide. This results in a population of mononuclear cells, called peripheral blood mononuclear cells (PBMCs), that have been depleted of red blood cells and most polymorphonuclear leukocytes or granulocytes, and consists mainly of lymphocytes and monocytes. T lymphocytes can be isolated from PBMCs by binding a sample to antibody-coated plastic surfaces, which is known in the art as “panning,” or by killing unwanted cells by treatment with a specific antibody and complement. Alternatively, PBMCs can be passed over columns of antibody- and nylon-coated steel wool, and different populations differentially eluted. Also, T lymphocytes can be isolated from PBMCs using flow cytometry or fluorescence-activated cell sorting (FACS). Methods for isolating lymphocytes from mammals, particularly humans, are further described in Janeway et al.. T08741ANM - 100 -

[0384] As used herein, the term "antibody" or "Ab" means an immunoglobulin molecule (or antigen-binding fragment thereof) that recognizes and specifically binds to a target (such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations thereof) through at least one antigen binding site or “epitope” within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact monoclonal antibodies, intact polyclonal antibodies, bi- or multispecific antibodies generated from at least two intact antibodies, human antibodies, humanized antibodies, chimeric antibodies, modified antibodies, single chain antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (dsFv), antibody fragments (such as Fab, F(ab'), F(ab')2, and Fv fragments, fragments produced by a Fab expression library, and fragments comprising either a VL or VH domain), anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to the TF antibodies disclosed herein), intracellularly-made antibodies (i.e., intrabodies), and antigenbinding antibody fragments.

[0385] The antibodies described herein can be of any of the five major classes of immunoglobulins (i.e., IgA, IgD, IgE, IgG, and IgM), or subclasses (isotypes) thereof (i.e., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), based on the identity of their heavychain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. In some embodiments, the immunoglobulin is an IgGl isotype. In some embodiments, the immunoglobulin is an IgG2 isotype. In some embodiments, the immunoglobulin is an IgG4 isotype. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. The antibodies described herein can be unmodified or conjugated to other molecules such as toxins, radioisotopes, etc.

[0386] The term “antibody fragment” or “antigen-binding fragment” is used herein in the broadest sense and comprises a portion of a full-length antibody, preferably comprising the antigen-binding or variable region thereof. An antibody fragment retains the original specificity of the parent immunoglobulin. Examples of antibody T08741ANM - 101 - fragments include, e.g., Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragment(s). Preferably, the antibody fragment is a Fab fragment. Antibody fragments may also be derived from multispecific antibodies and comprise, for example, two or more antigen-specific Fab fragments connected by a linker, or two or more antigen-specific single-chain antibody fragments connected by a linker.

[0387] As used herein, the term "monoclonal antibody" is intended to be used as in the art, and refers to an antibody obtained from a population of substantially homogenous antibodies that bind to the same antigenic determinants (epitope). "Substantially homogeneous" means that the individual antibodies are identical except for possibly naturally-occurring mutations that may be present in minor amounts. This is in contrast to polyclonal antibodies that typically include different antibodies directed against various, different antigenic determinants (epitopes). The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. "Monoclonal antibodies" and antigen-binding fragments thereof are made in any number of manners including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0388] Immune modulators

[0389] As used herein, the term “immune modulator” refers to a compound, composition, or substance that can regulate and / or adjust the response of the innate and / or adaptive immune system. Immunomodulation includes induction, amplification, attenuation or prevention of immune responses. The goal of immunomodulation is to treat or prevent disease. T08741ANM - 102 -

[0390] Immune modulators include cytokines, which are small secretory proteins, such as interferons, interleukines, chemokines, lymphokines, tumor necrosis factors, such as TNF-a, and hematopoietic growth factors such as erythropoietin, IL-11, GM-CSF and G-CSF; Bacillus Calmette-Guerin (BCG); and immunomodulatory drugs, also called biological response modifiers, such as thalidomide, lenalidomide, pomalidomide and imiquimod.

[0391] Immune modulators also include co-stimulatory molecules such ICOSL (inducible T- cell costimulatory (ICOS) ligand) ,CD70, CD30L, CD40, CD80, CD86, OX40L (Tumor necrosis factor receptor superfamily, member 4 (0X40) ligand), 4-1BBL (4- 1BB / CD137 ligand), GITRL (Glucocorticoid-induced tumor necrosis factor receptor- related protein (GITR) ligand), and TIM-4 (T-cell immunoglobulin and mucin domain containing 4, ligand for TIM-1).

[0392] Antigenic peptides

[0393] As used herein, the term “antigen” or “antigenic peptide” refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen or antigenic protein reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term antigen is used interchangeably with the term "immunogen”. The term "antigen" or “antigenic protein” include all related antigenic epitopes. The terms “antigen”, “antigenic molecule / protein” or “immunogen” include fragments thereof that are still capable of acting as an antigen. The term “antigenic protein” is used interchangeably with “immunogen” or “disease-associated antigen” throughout the present application. An “antigenic protein” comprises a number of amino acids ranging from small peptides to large proteins, e.g. 4 to 2000 amino acids or more, 4 to 1800 amino acids, 4 to 1600 amino acids or 4 to 1400 amino acids. In one embodiment, the antigenic protein, for which the RNA (e.g. mRNA) encodes, comprises at least 100, at least 200, T08741ANM - 103 - at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1200, at least 2000 or at least 3000 amino acids. In one embodiment, the antigenic protein, for which the RNA (e.g. mRNA) encodes, comprises at least 50 amino acids. In one embodiment, the antigenic protein, for which the RNA (e.g. mRNA) encodes, comprises at least 500 amino acids. In one embodiment, the antigenic protein, for which the RNA (e.g. mRNA) encodes, comprises at least 1000 amino acids. In one embodiment, the antigenic protein comprises 100 to 1500 amino acids. In one embodiment, the antigenic protein comprises 200 to 1300 amino acids. In one embodiment, the antigenic protein comprises 400 to 1300 amino acids.

[0394] "Epitope" refers to a site on an antigen to which B and / or T cells respond.

[0395] Nucleic acid sequence

[0396] The term “nucleic acid sequence” is used herein relates to RNA or DNA. In one embodiment, the exogenous nucleic acid sequence comprises or consists of RNA. In some embodiments, the RNA comprises mRNA. The DNA or RNA may comprise naturally occurring sequences, synthetic sequences or a combination thereof. RNA, such as mRNA, may comprise at least one chemical modification (i.e. the chemical modification from one RNA to another can be different). For the purposes of the present disclosure, chemical modification means that one of the four naturally- occuring standard nucleosides which occur in RNA (adenosine (A), guanosine (G), uridine (U), and cytidine (C)) are replaced by modified forms thereof wherein the modification affects the base moiety within the nucleoside. Modified nucleosides can be naturally-occurring or non-naturally-occurring modified nucleosides.

[0397] With respect to RNA (e.g. mRNA), the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein. In one embodiment, after combination of the RNA (e.g. mRNA) described herein, e.g., formulated as RNA (e.g. mRNA) lipid particles, with activated dendritic cells, at least a portion of the RNA T08741ANM - 104 -

[0398] (e.g. mRNA) is delivered to the activated dendritic cells. In one embodiment, the RNA (e.g. mRNA) is translated by the activated dendritic cells to produce the peptide or protein it encodes. RNA (e.g. mRNA) particles such as RNA (e.g. mRNA) lipid particles described herein may be used for delivering RNA to activated dendritic cells. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, RNA or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a RNA (e.g. mRNA) sequence may encode a protein (e.g. antigen) if translation of the RNA (e.g. mRNA) occurs in a cell. In some embodiments, the RNA (e.g. mRNA) comprises one coding sequence of a protein (e.g. an antigen). In some embodiments, the RNA (e.g. mRNA) comprises more than one coding sequence of a protein (e.g. an antigen), such as two coding sequences, three coding sequences, four coding sequences or more. The coding sequences for more than one protein (e.g. antigen), may be comprised on the same RNA (e.g. mRNA) molecule, or on multiple RNA molecules. As an example, the coding sequence for a checkpoint inhibitor and a coding sequence for an immune modulator may be present on the same RNA molecule or on at least two different RNA molecules.

[0399] Terms such as "reduce", “effectively reduce”, "decrease", "inhibit" or "impair" as used herein relate to an overall reduction or the ability to cause an overall reduction, preferably of at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or even more, in the level. These terms include a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. Terms such as "increase", "enhance" or "exceed" preferably relate to an increase or enhancement by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or even more. T08741ANM - 105 -

[0400] According to the disclosure, the term "peptide" comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "protein" or "polypeptide" refers to large peptides, in particular peptides having at least about 150 amino acids, but the terms "peptide", "protein" and "polypeptide" are used herein as synonyms if not mentioned otherwise.

[0401] Pharmaceutical composition

[0402] The term “composition” as used herein refers to a mixture comprising a therapeutically effective amount of the agent according to the present invention, i.e. a viral plasmid or recombinant virus of the invention, and one or more excipients. The term “excipient” as used herein may also be referred to as “pharmaceutically acceptable carrier”, or “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,”, or “pharmaceutically acceptable vehicle,” used interchangeably herein, refer to a nontoxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type.

[0403] A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier will not inhibit otherwise adversely affect the function of the agent according to the present invention. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, and any combination thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the formulation.

[0404] As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but T08741ANM - 106 - is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.).

[0405] The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subj ect that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease.

[0406] For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by 1 %-100% as compared to a control. In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (such as a genetic disease or disorder). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by 1 %-l 00% as compared to a control. In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is T08741ANM - 107 - understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder.

[0407] An "individual" or "subject" treated in accordance with this invention refers to vertebrates, particularly members of a mammalian species, and includes but is not limited to domestic animals, sports animals, and primates, including humans. In one embodiment, the subject treated in accordance with this invention is a mammal. In one embodiment, the subject treated in accordance with this invention is a human. In another embodiment, the subject treated in accordance with this invention is a nonhuman mammal.

[0408] “Wild-type”, “control” or “reference” gene expression, mRNA or protein levels are determined by a control sample, cell or organisms, or by averaging the expression levels from multiple control samples, cells or organisms. In the context of the present invention, the term “wild-type” or "control" refers to a cell or organism that is healthy or a sample from a subj ect that is healthy or to a cell or organism with a specific disease that is different from the disease to be treated.

[0409] Diseases

[0410] The terms "disease" or “disorder” refer to any disease or disorder which implicates an antigen or antigenic protein, e.g. a disease which is characterized by the presence of an antigen or antigenic protein. The disease can, e.g., be an infectious disease or a tumor disease (cancer). In one embodiment, a disease involving an antigen is a disease involving cells expressing an antigen, preferably on the cell surface.

[0411] The term “tumor” refers to a disease in which some of the body’s cells grow uncontrollably and spread to other parts of the body. Tumors can be cancerous or not cancerous (benign). In the context of the present invention, the terms tumor and cancer are used interchangeably. Cancerous tumors spread into, or invade, nearby tissues and can travel to distant places in the body to form new tumors (also called metastasis). T08741ANM - 108 -

[0412] Cancerous tumors may also be called malignant tumors. Many cancers form solid tumors, but cancers of the blood, such as leukemias, do not. Benign tumors do not spread into, or invade, nearby tissues. Blood cancer includes leukemia, lymphoma, myelodysplastic syndromes (MDS), myeloproliferative disorder (MPD), multiple myeloma and all subtypes thereof.

[0413] The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that can be afflicted with or is susceptible to a disease or disorder. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In some embodiments, the term "subject" includes humans of age of at least 50, at least 55, at least 60, at least 65, at least 70, or older. In some embodiments, the term "subject" includes humans of age of at least 65, such as 65 to 80, 65 to 75, or 65 to 70. In some embodiments of the present invention, the "individual" or "subject" is a "patient".

[0414] The term "patient" means an individual or subject for treatment or preventive treatment. The present invention relates to the preventive treatment, i.e. vaccination.

[0415] The invention is now described with respect to some specific examples which, however, are for illustrative purposes and not to be construed in a limiting manner.

[0416] Embodiments

[0417] The invention further relates to the following embodiments:

[0418] 1. A first ex vivo activated dendritic cell, wherein the activated dendritic cell comprises at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor. T08741ANM - 109 -

[0419] 2. The activated dendritic cell of embodiment 1, wherein the checkpoint inhibitor is capable of specifically binding to an immune checkpoint molecule, optionally wherein the immune checkpoint molecule is at least partially on a cell surface.

[0420] 3. The activated dendritic cell of any one of embodiments 1 or 2, wherein the checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA4 or a combination thereof.

[0421] 4. The activated dendritic cell of any one of the preceding embodiments, wherein the checkpoint inhibitor comprises an antibody or an antigen-binding fragment thereof.

[0422] 5. The activated dendritic cell of any one of the preceding embodiments, wherein the antibody or an antigen-binding fragment thereof is capable of binding to PD-1, PD-L1 or CTLA-4 or a combination thereof.

[0423] 6. The activated dendritic cell of any one of embodiments 4 or 5, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof.

[0424] 7. The activated dendritic cell of any one of the preceding embodiments, wherein the checkpoint inhibitor is selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof.

[0425] 7a. The activated dendritic cell of any one of the preceding embodiments, wherein the activated dendritic cell comprises exogenous nucleic acid sequences encoding two checkpoint inhibitors.

[0426] 7b. The activated dendritic cell of embodiment 7a, wherein the two checkpoint inhibitors comprise an anti-PD-1 antibody or a PD-1 binding fragment thereof, and an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof.

[0427] 7c. The activated dendritic cell of embodiment 7a or 7b, wherein the exogenous nucleic acid sequences encoding the two checkpoint inhibitors are comprised on the same or different nucleic acid molecules. T08741ANM - 110 -

[0428] 8. The activated dendritic cell of any one of the preceding embodiments, wherein the checkpoint inhibitor is secreted by the activated dendritic cell.

[0429] 9. The activated dendritic cell of any one of the preceding embodiments, wherein the checkpoint inhibitor is attached or anchored to the cell surface of the activated dendritic cell.

[0430] 10. The activated dendritic cell of any one of the preceding embodiments, wherein said at least one exogenous nucleic acid sequence comprises RNA or DNA.

[0431] 11. The activated dendritic cell of embodiment 10, wherein the RNA comprises mRNA.

[0432] 12. The activated dendritic cell of embodiment 11, wherein the mRNA encoding the checkpoint inhibitor comprises modified nucleotides .

[0433] 13. The activated dendritic cell of any one of the preceding embodiments, wherein said at least one exogenous nucleic acid sequence is comprised in at least one lipid nanoparticle (LNP).

[0434] 14. The activated dendritic cell of embodiment 13, wherein the at least one LNP comprises at least one lipid selected from the group consisting of acKK-E12 lipid, a SM-102 lipid, C12-(2-3-2), DMG-PEG-2K, cholesterol, DPPC, or a MC3 lipid.

[0435] 15. The activated dendritic cell of any one of the preceding embodiments, wherein the immune checkpoint molecule is PD-1, PD-L1 and / or CTLA-4.

[0436] 16. The activated dendritic cell of any one of the preceding embodiments, wherein the activated dendritic cell comprises at least one further exogenous nucleic acid sequence encoding an immune modulator.

[0437] 16a. The activated dendritic cell of embodiment 16, wherein the at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor and the at least one further exogenous nucleic acid sequence encoding an immune modulator are comprised on the same nucleic acid sequence or on separate nucleic acid sequences.

[0438] 17. The activated dendritic cell of embodiment 16, wherein the immune modulator is a cytokine. T08741ANM - I l l -

[0439] 18. The activated dendritic cell of any one of embodiments 16 or 17, wherein the immune modulator is at least one cytokine selected from the group consisting of interleukin- lb (IL- lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL- 4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin- 21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof.

[0440] 19. The activated dendritic cell of any one of embodiments 16 to 18, wherein the cytokine is interleukin -12 (IL-12).

[0441] 20. The activated dendritic cell of any one of the preceding embodiments, wherein the activated dendritic cell comprises at least one further exogenous nucleic acid sequence encoding an antigenic peptide or protein.

[0442] 20a. The activated dendritic cell of embodiment 20, wherein the at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor, the at least one further exogenous nucleic acid sequence encoding an immune modulator, and at least one further exogenous nucleic acid sequence encoding an antigenic peptide or protein are comprised on the same nucleic acid sequence, on separate nucleic acid sequences or a combination thereof.

[0443] 21. The activated dendritic cell of embodiment 20, wherein the at least one antigenic peptide or protein is a tumor-associated peptide or protein, a viral antigenic protein, a bacterial antigenic protein, or a fungal antigenic protein.

[0444] 22. The activated dendritic cell of any one of embodiments 20 or 21, wherein the at least one antigenic peptide or protein is a tumor-associated peptide or protein, and wherein the tumor-associated peptide or protein is a tumor-specific peptide or protein, preferably wherein the tumor-specific peptide or protein comprises at least one tumor-specific neo-antigen.

[0445] 23. The activated dendritic cell of any one of embodiments 20 to 22, wherein the at least one tumor-specific neo-antigen comprises HPV E6ZE7, Merkel Cell polyoma T08741ANM - 112 -

[0446] LTA epitope, a Tumor Associated Antigens (TAA), a lineage-restricted Differentiation antigen, melanoma MART- 1, TRP2), NY-ESO-1, p53 or Ras.

[0447] 24. A pharmaceutical composition comprising the activated dendritic cell of any one of embodiments 1 - 15 and a pharmaceutically acceptable carrier.

[0448] 25. The pharmaceutical composition of embodiment 24, further comprising an immune modulator.

[0449] 26. The pharmaceutical composition of any one of embodiments 24 or 25, wherein the immune modulator is a cytokine.

[0450] 27. The pharmaceutical composition of embodiment 26, wherein the cytokine is capable of inducing CD8+ T cells.

[0451] 28. The pharmaceutical composition of any one of embodiments 26 or 27, wherein the cytokine comprises at least one of interleukin-lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL-12), interleukin- 15 (IL-15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL- 23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof..

[0452] 29. The pharmaceutical composition of embodiment 28, wherein the cytokine is interleukin- 12 (IL- 12).

[0453] 30. The pharmaceutical composition of any one of embodiments 24 to 29, further comprising a second activated dendritic cell comprising an exogenous nucleic acid sequence encoding the immune modulator.

[0454] 31. The pharmaceutical composition of embodiment 30, wherein the second activated dendritic cell is capable of expressing and / or secreting the immune modulator.

[0455] 32. The pharmaceutical composition of any one of embodiments 24 to 31, further comprising a third activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide or protein. T08741ANM - 113 -

[0456] 33. The pharmaceutical composition of embodiment 32, wherein the exogenous nucleic acid sequence encoding the at least one antigenic peptide or protein is exogenous RNA, optionally mRNA.

[0457] 34. The pharmaceutical composition of any one of embodiments 32 or 33, wherein said at least one antigenic peptide or protein is a tumor-associated peptide or protein, a viral antigenic protein, a bacterial antigenic protein, or a fungal antigenic protein.

[0458] 35. The pharmaceutical composition of embodiment 34, wherein the tumor- associated peptide or protein is a tumor-specific peptide or protein.

[0459] 36. The pharmaceutical composition of embodiment 35, wherein the tumorspecific peptide or protein comprises at least one tumor-specific neo-antigen.

[0460] 36a. The pharmaceutical composition of embodiment 36, wherein the at least one tumor-specific neo-antigen comprises HPV E6ZE7, Merkel Cell polyoma LTA epitope, a Tumor Associated Antigens (TAA), a lineage-restricted Differentiation antigen, melanoma MART- 1, TRP2), NY-ESO-1, p53 or Ras.

[0461] 37. A pharmaceutical composition comprising the activated dendritic cell of any one of embodiments 16 to 23 and a pharmaceutically acceptable carrier.

[0462] 38. The ex vivo activated dendritic cell of any one of embodiments 1 to 21 or the pharmaceutical composition of any one of embodiments 22 to 37 for use in therapy.

[0463] 39. The ex vivo activated dendritic cell of any one of embodiments 1 to 21 or the pharmaceutical composition of any one of embodiments 22 to 37 for use in a method of treating cancer in a subject in need thereof.

[0464] 40. The activated dendritic cell or pharmaceutical composition for use of embodiment 39, wherein the method comprises administering a therapeutically effective amount of the activated dendritic cell or the pharmaceutical composition to the subject.

[0465] 41. The activated dendritic cell or pharmaceutical composition for use of embodiment 39 or 40, wherein the treatment prolongs the median survival of the T08741ANM - 114 - subject compared to a subject having cancer and being treated with an activated dendritic cell or a checkpoint inhibitor alone.

[0466] 42. The activated dendritic cell or pharmaceutical composition for use of any one of embodiments 39 to 41, wherein the cancer is a solid cancer or a hematopoietic cancer.

[0467] 43. The activated dendritic cell or pharmaceutical composition for use of any one of embodiments 39 to 42, wherein the cancer is a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or a germ cell tumor.

[0468] 44. The activated dendritic cell or pharmaceutical composition for use of any one of embodiments 39 to 43, wherein the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, and pancreatic cancer .

[0469] 45. The activated dendritic cell or pharmaceutical composition for use of any one of embodiments 39 to 44, wherein the method comprises administering to the subject a therapeutically effective amount of the activated dendritic cells.

[0470] 46. The activated dendritic cell or pharmaceutical composition for use of any one of embodiments 39 to 45, wherein the activated dendritic cell or the pharmaceutical composition is administered locally or systemically to the subject .

[0471] 47. The activated dendritic cell or pharmaceutical composition for use of any one of embodiments 39- 46, wherein the activated dendritic cell inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces T08741ANM - 115 - tumor growth rate, eradicates the tumor or tumor cells, and / or prolongs the median survival period relative to treatment with an activated dendritic cell or a checkpoint inhibitor alone .

[0472] 48. The activated dendritic cell or pharmaceutical composition for use of any one of embodiments 39 to 47, wherein the method further comprises administration of a further checkpoint inhibitor.

[0473] 49. The pharmaceutical composition of any one of embodiments 30 to 36 for use in a method of treating cancer in a subject in need thereof, wherein the first activated dendritic cell is administered separately, simultaneously or sequentially from the second or third activated dendritic cell.

[0474] 50. The pharmaceutical composition for use of embodiment 49, wherein the first activated dendritic cell, the second activated dendritic cell and the third activated dendritic cell are administered in a pre-defined order.

[0475] 51. The pharmaceutical composition for use of any one of embodiments 49 or 50, wherein the first activated dendritic cell, the second and / or the third activated dendritic cell are administered repeatedly to the subject.

[0476] 52. The pharmaceutical composition for use of embodiment 51, wherein the first activated dendritic cell, the second and / or the third activated dendritic cell are administered at least twice, 3-times, 4-times, 5-times, 6-times or more to the subject.

[0477] 53. The pharmaceutical composition for use of any one of embodiments 49 to 52, wherein the combined treatment with the first activated dendritic cell in conjunction with the second and / or the third activated dendritic cell prolongs the median survival of the treated subject compared to a subject being treated with any one of the first activated dendritic cell, the pharmaceutical composition, the second or the third activated dendritic cell alone.

[0478] 54. The pharmaceutical composition for use of any one of embodiments 49- 53, wherein the combined treatment with the first activated dendritic cell with the second and / or the third activated dendritic cell inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces tumor growth rate, eradicates T08741ANM - 116 - the tumor or tumor cells, and / or prolongs the median survival period relative to treatment with the activated dendritic cell, the pharmaceutical composition, the second and / or the third activated dendritic cell alone.

[0479] 55. The activated dendritic cell of any one of embodiments 16-23 for use in a method of treating cancer in a subject in need thereof.

[0480] 56. The activated dendritic cell for use of embodiment 55, wherein the treatment with the activated dendritic cell co-expressing and / or secreting a checkpoint inhibitor with one or both of an immune modulator and / or an antigenic peptide or protein prolongs the median survival of the treated subject compared to a subject being treated with any one of the first activated dendritic cell, the pharmaceutical composition, the second or the third activated dendritic cell alone.

[0481] 57. The activated dendritic cell for use of embodiment 55, wherein the treatment with the activated dendritic cell co-expressing and / or secreting a checkpoint inhibitor with one or both of an immune modulator and / or an antigenic peptide or protein inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces tumor growth rate, eradicates the tumor or tumor cells, and / or prolongs the median survival period relative to treatment with the activated dendritic cell, the pharmaceutical composition, the second and / or the third activated dendritic cell alone.

[0482] 58. A method for preparing the ex vivo activated dendritic cell of any one of embodiments 1-15, the method comprising the step of combining the activated dendritic cell with at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor .

[0483] 59. The method of embodiment 58, wherein the exogenous nucleic acid sequence is mRNA and comprised in a lipid nanoparticle (LNP).

[0484] 60. The method of embodiment 59, wherein the method comprises a further step of incubating said LNP comprising the at least one mRNA with said activated dendritic cells for a time period sufficient for the activated dendritic cells to take up the LNP. T08741ANM - 117 -

[0485] 61. The method of embodiment 60 wherein the time period is at least 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h or 24 h.

[0486] 62. An ex vivo activated dendritic cell obtainable by the method according to any one of embodiments 58-61.

[0487] 63. A kit-of-parts comprising a plurality of ex vivo activated dendritic cells and LNPs comprising at least one exogenous mRNA encoding at least one checkpoint inhibitor.

[0488] 64. The kit according to embodiment 63, wherein the kit further comprises at least one exogenous mRNA encoding at least one cytokine.

[0489] 65. The kit according to any one of embodiments 63 or 64, wherein the kit further comprises at least one exogenous mRNA encoding at least one antigenic peptide or protein.

[0490] 66. The kit according to embodiment 65, wherein the at least one exogenous mRNA encoding at least one cytokine and the at least one exogenous mRNA encoding at least one antigenic peptide or protein are either

[0491] (i) expressed by the same activated dendritic cell as the at least one exogenous mRNA encoding a checkpoint inhibitor, or

[0492] (ii) each expressed by a different activated dendritic cell.

[0493] EXAMPLES

[0494] Example 1. Preparation of aDC transduced with LNP particles

[0495] Isolation of murine peripheral blood mononuclear cells (PBMC)

[0496] Peripheral blood (100-200uL / mouse) is collected from experimental and control mice (as well as any additional bleeder mice if needed) as required for the experiment; eg, on day 1 and day 7 for preventive vaccination and booster studies, or twice a week for the duration of treatment for therapeutic tumor treatment studies. Whole blood is collected into 1 : 100 5,000 U / mL heparin (McKesson Packaging Services). Plateletcontaining peripheral blood mononuclear cells (PBMC) are isolated from peripheral T08741ANM - 118 - whole blood via Lympholyte M gradient separation (Cedarlane Labs). Autologous serum is collected from a separate cohort of syngeneic donor mice and reserved for subsequent steps (overnight culture).

[0497] Transimmunization chamber

[0498] The miniaturized ECP device suitable for work in animal models, called the Transimmunization (TI) chamber, was designed and created for Dr. Edelson's laboratory by Transimmune AG in collaboration with Fraunhofer Institute for Biomedical Engineering, Saarland, Germany. The sterile polystyrene TI chamber has the external dimensions of 25 * 75 mm, with the flow path of 18 * 66 mm, and the flow passage height of 290 + / - 15um.

[0499] PBMC TI treatment protocol

[0500] Isolated murine platelet-containing PBMC are resuspended in fetal bovine serum (FBS). The cells are then is incubated in the TI chamber for 1 hour at 37C. This step allows for platelet-activating plasma protein deposition in the chamber, and platelet adherence to the coated chamber surfaces, as confirmed by light microscopy. The cells are subsequently passed through the TI chamber using a syringe pump, at a rate of 0.09 mL / min. Following plate passage, cells are collected, and the TI chamber washed with 100% FBS at 0.49 mL / min while being physically perturbed by flicking or tapping the plate surface to help detach any adherent cells from the chamber. The collected cells are washed and cultured overnight at standard conditions in RPMI without phenol red (Gibco) supplemented with 15% autologous mouse serum and 1% penicillin / streptomycin / L-glutamine (Invitrogen). mRNA-containing LNP transfection of aDC

[0501] The desired amount of LNP (cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 35:46.5:2.5: 16 (Santangelo laboratory, Emory University) containing mRNA for the antigen of interest (such as SARS-CoV-2 Spike protein, or a sample tumor T08741ANM - 119 - antigen), is added directly to the overnight aDC culture, at the time of setting up the culture. In our experience, LNP amounts can vary from 20ug to lOng, depending on the LNP and antigen used. aDC re-introduction into experimental animals

[0502] The following day, LNP-transfected cells are harvested by scraping, washed, resuspended in sterile PBS (Gibco), and administered intravenously at lOOuL / animal via the retro-orbital plexus.

[0503] Example 2. aDC internalization of viral antigen-containing LNP

[0504] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Example 1. To the plate-passed PBMC in culture is added the desired amount of LNP containing mRNA for the viral antigen of interest (SARS-CoV-2 Spike protein mRNA), such as 20ug / mL of cKK-E12 based LNP containing SARS-CoV-2 Spike protein (LNP prepared and provided by the Santangelo laboratory, Emory University, mRNA sequence corresponds to SEQ ID NO: 33). Plate-passed PBMC are incubated with the LNP overnight, to allow for LNP uptake and antigen expression.

[0505] Antigen expression is monitored after overnight incubation by using an appropriate fluorescently labeled detection antibody (anti-human SARS-CoV-2 Spike protein antibody, kindly provided by Santangelo laboratory, Emory University), as well as any necessary antibodies to identify cells of interest (eg CDl lb+, Ly6G' for murine aDC within the plate-passed PBMC; CDl lb Biolegend clone MI / 70; Ly6G Biolegend clone 1A8).

[0506] Antibody binding to aDC can be detected by either flow cytometry (Cytoflex cytometer, analysis using FlowJo vlO software) or by confocal microscopy. T08741ANM - 120 -

[0507] Results

[0508] Murine aDC, among all other immune cell subsets contained in the PBMC, specifically internalize the cKK-E12 LNP, and efficiently express Spike protein encoded by mRNA contained in such LNP. FACS analysis shows Spike protein positivity specifically in CDl lb+aDCa, as compared to cultures without LNP transfection (Figure 1 A, B). This is confirmed by confocal microscopy analysis, showing Spike protein cell surface and cytoplasmic expression specifically in CDl lb+Ly6G' aDCs (projected Z-stack images, Figure 1 C, D; Z-plane slice image, Figure 1 E.

[0509] Example 3. Therapeutic aDC administration in the EG7-OVA lymphoma mouse model using tumor antigen mRNA-containing LNP (ovalbumin (OVA) antigen)

[0510] The experimental design is schematically described in Figure 2A.

[0511] For tumor induction, 3xl06EG7-OVA tumor cells are injected subcutaneously in 100 mL into the right flanks of recipient wild-type C57BL / 6J mice. Therapeutic treatment is initiated on day 2 following tumor implantation. For each treatment, aDC are prepared from the blood of respective experimental group (200uL blood collected per animal) by standard protocol described in Example 1. Blood from groups not treated with aDC is discarded.

[0512] To the plate-passed PBMC in culture is added lOng (0.5ug / kg of LNP, dose equivalent to COVID-19 Pfizer mRNA LNP vaccine in current human vaccine setting) of cKK- E12 based LNP containing mRNA for the tumor antigen of interest, here the model antigen ovalbumin (OVA) that has been introduced into the EL4 lymphoma cells to produce the EG7-OVA tumor model (SEQ ID NO: 34, also used for all following experiments relating to OVA mRNA containing LNPs). All mRNA and LNPs prepared and provided by the Santangelo laboratory, Emory University. T08741ANM - 121 -

[0513] After overnight incubation, cells co-cultured with LNP containing OVA mRNA are collected, and injected intravenously in a lOOuL volume of sterile PBS into the retro- orbital plexus of the “aDC”-treated experimental group animals. At the same time, an equivalent dose of LNP containing OVA mRNA in 10-50ul volume of sterile PBS is injected into the thigh of each animal in the intramuscularly (“IM”)-treated experimental group. The animals in the “Untreated” (“No Tx”) group do not receive any therapy.

[0514] Treatment is repeated twice a week until the end of the experiment, which is determined by the speed of largest tumor growth up to the maximal limit permitted by the animal care facility. Mice are usually bled for aDC therapeutic manufacture on Mondays and Thursdays, with aDC or i.m. treatments taking place on Tuesdays and Fridays. Typically, 5-6 bi-weekly therapeutic immunization treatments can be carried out.

[0515] Tumor volume is monitored via biweekly measurement of perpendicular tumor diameters and height using a caliper, and tumor volume calculated as (tumor length x width x height) / 2.

[0516] Splenocytes are harvested at the end of the experiment (Day 30 post tumor inoculation) from all treatment and control groups for further analysis, to characterize the resulting anti-tumor immune response. Elispot analysis: CD8+ splenic T cells from treated mice (negative selection using a Miltenyi CD8 T cell isolation kit) are immediately placed into an 18hr IFN-g Elispot assay at l*105cells per well in the presence or absence of lOug / mL SIINFEKL peptide. Antigen (SIINFEKL)-specific CD8 T cell evaluation: CD8 (BioRad clone KT1.5) and specific H2d-SIINFEKL dextramer staining (Immudex) followed by flow cytometry (Cytoflex). Tern (effector) / Tcm (central memory) type phenotype analysis: staining for CD44 (Biolegend clone IM7) and CD62L (Biolegend clone MEL-14) expression on antigen-specific T cells followed by flow cytometry (Cytoflex). Stem-like T cell analysis: staining for IL7Ra (Biolegend T08741ANM - 122 - clone A7R34) and SCA-1 (Biolegend clone E13-161.7) expression on antigen-specific T cells followed by flow cytometry (Cytoflex). T cell exhaustion marker evaluation: PD-1 (Biolegend clone 29F.1A12) expression on antigen-specific T cells followed by flow cytometry (Cytoflex).

[0517] Results

[0518] Compared to the control untreated (“No Tx”) group, therapeutic aDC treatment using the clinically relevant dose of lOng OVA mRNA-containing LNPs (0.5ug / kg of LNP, dose equivalent to COVID-19 Pfizer mRNA LNP vaccine in current human vaccine setting) successfully controlled the growth of EG7-0VA tumors, while direct intramuscular administration of the same amount of OVA mRNA-containing LNPs (“IM” group) did not significantly alter the kinetics of tumor growth (Figure 2B).

[0519] To better understand the therapeutic effect of the aDC vs the IM treatment methods, splenocytes were collected at the end of the experiment (Day 30 post tumor inoculation) and further analyzed (Figure 3A-C, experiment schematic). Overall reactivity against the OVA immunodominant peptide SIINFEKL, measured by IFNg Elispot analysis of splenocytes from the different groups, showed some response in the control untreated group as expected of an OVA-expressing tumor-bearing mouse; a stronger response in the aDC-treated group, and, intriguingly, the strongest response in the IM-treated group (Figure 3D-E), even though this response was clinically unproductive (Figure 2B).

[0520] It thus became apparent that it was not the overall quantity of the model tumor antigen OVA-specific T cell response that controls tumor growth. To account for the strong therapeutic effect of aDC treatment, it therefore had to be the quality of the T cell response generated. The quality of the T cell response at Day 30 in the splenocytes of tumor-bearing treated mice was further investigated. This analysis demonstrated that antigen-specific T cells in aDC-treated mice showed a phenotype characterized by the T08741ANM - 123 - presence of central memory T cell and stem-like T cell subsets (Figure 4A) that have been linked to productive anti-cancer immune responses in human and mouse studies. In contrast, the T cells from the IM-treated group showed a strong effector T celldominant phenotype (Figure 4A), which is consistent with the strong response seen in the IFNg Elispot assay (Figure 3D-E).

[0521] This effector-like response generated by IM treatment appears to be clinically unproductive in a tumor setting; perhaps due to the possibly exhausted state of such effector T cells, as demonstrated by their high levels of PD1 expression compared to antigen-specific T cells from aDC-treated animals (Figure 4B).

[0522] Example 4. Analysis of spontaneous immune response in prophylactic aDC vaccination with LNP containing ovalbumin (OVA) sample antigen mRNA

[0523] Methods

[0524] For each treatment, aDC are prepared from the blood of healthy C57BL / 6 mice from respective experimental group (200uL blood collected per animal) by standard protocol described in Example 1. Blood from groups not treated with aDC is discarded.

[0525] To the plate-passed PBMC in culture is added 6ug of cKK-E12 based LNP containing mRNA for the tumor antigen of interest, here the model antigen ovalbumin (OVA). All LNPs prepared and provided by the Santangelo laboratory, Emory University.

[0526] Plate-passed PBMC are incubated with the LNP overnight, to allow for LNP uptake and antigen expression. On the following day, the PBMC are collected and prepared as described for Example 1, and administered intravenously at lOOuL / animal via the retro-orbital plexus to the “aDC [ova]” -vaccinated experimental group. In parallel, an T08741ANM - 124 - equivalent dose of LNP containing OVA mRNA is injected in 10-50ul volume of sterile PBS into the thigh of each animal in the intramuscularly (“IM [ova]”)- vaccinated experimental group. The animals in the “Untreated” group do not receive any therapy.

[0527] Mice are bled on experiment Days -8 and -1, and prophylactically vaccinated with either aDC or with intramuscular LNP on Days -7 and 0. On Days 0, 5, and 13 post vaccination, whole undifferentiated splenocytes are collected for analysis of the spontaneous immune response by IFNg Elispot. Elispot analysis: whole undifferentiated splenocytes from all experimental groups are immediately placed into an 18hr IFN-g Elispot assay at l*105cells per well in the absence of any additional stimulation. To further identify cells responsible for IFNg production, some Day 13 samples were further fractionated into CD8 T cells (negative selection using a Miltenyi CD8 T cell isolation kit), or into NK cells (positive selection using Miltenyi NK1.1 cell isolation kit).

[0528] Results

[0529] Interestingly, animals prophylactically vaccinated and boosted with aDC, but not with intramuscular LNP injection, demonstrate spontaneous IFNg production in the absence of any additional stimulation (Figure 5A-C). This spontaneous response is detectable at least as long as 13 days post vaccination (Figure 5C).

[0530] Further dissection of the source of this spontaneous IFNg signal, by isolation of either CD8 T cells or NK cells, identified NK cells as the IFNg-secreting cell subset. This suggests that aDC vaccination not only initiates the antigen-specific T and B cell responses (Figures 4, 5), but also broadly engages the innate immune system, such as NK cells.

[0531] Example 5. Prophylactic aDC vaccination in the EG7-OVA lymphoma mouse T08741ANM - 125 - model using tumor antigen mRNA-containing LNP (ovalbumin (OVA) antigen)

[0532] The experimental design is schematically described in Figure 6A.

[0533] For each vaccination treatment, aDC are prepared from the blood of respective experimental group (200uL blood collected per animal) by standard protocol described in Example 1. Blood from groups not treated with aDC is discarded.

[0534] For the initial vaccination (Day -14), to the plate-passed PBMC in culture is added lug of cKK-E12 based LNP containing mRNA for the tumor antigen of interest, here the model antigen ovalbumin (OVA) that has been introduced into the EL4 lymphoma cells to produce the EG7-0VA tumor model. All LNPs prepared and provided by the Santangelo laboratory, Emory University. After overnight incubation, cells cocultured with LNP containing OVA mRNA are collected, and injected intravenously in a lOOuL volume of sterile PBS into the retro-orbital plexus of the “aDC”-treated experimental group animals. At the same time, 50ug of soluble OVA protein in lOOuL volume of sterile PBS is injected into the retro-orbital plexus of the “Soluble Ova”- treated experimental group animals.

[0535] The initial vaccination treatment is followed 1 week later (Day -7) by a booster vaccination, carried out in the identical manner as that described above.

[0536] For tumor challenge at Day 0, IxlO6EG7-OVA tumor cells are injected subcutaneously in 100 mL into the right flanks of mice from all treatment groups. Tumor volume is monitored via biweekly measurement of perpendicular tumor diameters and height using a caliper, and tumor volume calculated as (tumor length x width x height) / 2.

[0537] Results

[0538] Prophylactic aDC vaccination using OVA mRNA-containing LNPs successfully prevented the growth of EG7-OVA tumors, while vaccination with soluble OVA T08741ANM - 126 -

[0539] (“Soluble Ova” group) did not protect against EG7-0VA tumor challenge (Figure 6B)

[0540] Experiment 6. Detection of OVA protein in OVA mRNA LNP transduced aDC

[0541] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Experiment 1. To the plate-passed PBMC in culture is added the desired amount of LNP containing mRNA for antigen of interest, such as lug / mL of cKK-E12 based LNP containing OVA or SARS-CoV-2 Spike protein mRNA (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC are incubated with the LNP overnight, to allow for LNP uptake and antigen expression.

[0542] Antigen expression is monitored after overnight incubation by staining the cells intracellularly with fluorescently labeled anti-OVA antibody (Rockland Immunochemicals), as well as any necessary antibodies to identify cells of interest (eg CDl lb+ for murine aDC within the plate-passed PBMC; CDl lb Biolegend clone MI / 70).

[0543] Antibody binding to aDC can be detected by flow cytometry (Cytoflex cytometer, analysis using FlowJo vlO software).

[0544] Results

[0545] Murine aDC, among all other immune cell subsets contained in the PBMC, specifically express OVA protein encoded by mRNA contained in cKK-E12 LNP. FACS analysis shows OVA protein positivity specifically in CDl lb+ aDCs, as compared to cultures with mock (Spike protein) LNP transfection (Figure 7).

[0546] Experiment 7. Detection of SIINFEKL bound MHC I (H-2Kb) on mouse aDC T08741ANM - 127 - loaded with soluble OVA, OVA expressing tumor cells or OVA mRNA LNPs

[0547] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Experiment 1. To the plate-passed PBMC in culture is added the antigen source, such as soluble OVA protein (lOug / mL; 50ug / mL); EG7-0VA tumor cells treated with 8-MOP / UVA (200ng / mL 8-MOP, UVADEX, Therakos; 2 or 4 J / cm2 UVA); or LNP containing OVA protein mRNA (lug / mL or 5ug / mL of cKK-E12 based LNP) (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC are incubated with the respective antigen sources overnight, to allow for antigen uptake and / or expression, and processing.

[0548] Antigenic OVA peptide SIINFEKL presentation on aDC H-2Kb MHCI molecules is detected after overnight incubation by staining the cells with fluorescently labeled 25. DI TCR-like antibody (Biolegend, clone 25-D1.16), as well as any necessary antibodies to identify cells of interest (eg CD1 lb+, Ly6G- for murine aDC within the plate-passed PBMC; CD1 lb Biolegend clone MI / 70; Ly6G Biolegend clone 1 A8).

[0549] Antibody binding to aDC can be detected by flow cytometry (Cytoflex cytometer, analysis using FlowJo vlO software).

[0550] Results

[0551] Murine aDC, among all other immune cell subsets contained in the PBMC, specifically process and present antigenic OVA SIINFEKL peptide in their MHCI molecules. Antigenic peptide presentation is significantly higher when OVA protein is expressed via mRNA LNP, as opposed to delivery via soluble OVA protein, or dying tumor cells expressing OVA protein (Figure 8).

[0552] Experiment 8. Time kinetics of surface SIINFEKL-MHC I complex expression (via 25.D1 ab stain) in aDC transduced with OVA mRNA LNP T08741ANM - 128 -

[0553] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Experiment 1. To the plate-passed PBMC in culture is added the desired amount of LNP containing mRNA for antigen of interest, such as lug / mL of cKK-E12 based LNP containing OVA protein mRNA (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC are incubated with the LNP for 2, 4, 6, 8, 12, or 20hrs, then washed and fixed for staining.

[0554] Antigenic OVA peptide SIINFEKL expression on aDC H-2Kb MHCI molecules is detected after 2-20hr incubation by staining the cells with fluorescently labeled 25. DI TCR-like antibody (Biolegend, clone 25-D1.16), as well as any necessary antibodies to identify cells of interest (eg CDl lb+ for murine aDC within the plate-passed PBMC; CD1 lb Biolegend clone MI / 70).

[0555] Antibody binding to aDC can be detected by flow cytometry, and antibody-positive cell percentages / mean fluorescence intensity (MFI) of positive cells quantitated (Cytoflex cytometer, analysis using FlowJo vlO software).

[0556] Results

[0557] The percentage of aDC displaying antigenic OVA SIINFEKL peptide bound to MHCI molecules on their surface, as well as the amount of SIINFEKL / MHCI per cell, increases with aDC incubation time with OVA mRNA LNP (Figure 9). The 25. DI expression level is shown in CD1 lb+ cells at various time points (FACS plot, upper panel). Percentage of 25. DI positive cells and MFI level of 25. DI in CD1 lb+ subset is shown in the bar graph (lower panel).

[0558] Experiment 9. Detection of SIINFEKL bound MHC I (H-2Kb) on mouse aDC or BMDC transduced with OVA mRNA LNP (at reduced LNP concentration) T08741ANM - 129 -

[0559] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Experiment 1. BMDC were cultured from healthy C57BL / 6 donor mouse bone marrow according to standard protocol. Briefly, bone marrow cells were plated in cell culture media containing GM-CSF (20 ng / ml); after 5 days, nonadherent cells were washed and replated in fresh GM-CSF-supplemented media and incubated for 48 hours more; subsequently nonadherent BMDC cells were removed, washed, and used immediately for experiments.

[0560] To the plate-passed PBMC or BMDC in culture is added the desired amount of LNP containing mRNA for antigen of interest, such as O.lug / mL of cKK-E12 based LNP containing OVA protein mRNA (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC or cultured BMDC are incubated with the LNP overnight, to allow for LNP uptake and antigen expression.

[0561] Antigenic OVA peptide SIINFEKL expression on aDC H-2Kb MHCI molecules is detected after incubation by staining the cells with fluorescently labeled 25. DI TCR- like antibody (Biolegend, clone 25-D1.16), as well as any necessary antibodies to identify cells of interest (eg CD1 lb+ for murine aDC within the plate-passed PBMC; CD1 lb Biolegend clone MI / 70; CD11c for murine BMDC, Biolegend clone N418).

[0562] Antibody binding to aDC or BMDC can be detected by flow cytometry (Cytoflex cytometer, analysis using FlowJo vlO software).

[0563] Results

[0564] The percentage of aDC displaying antigenic OVA SIINFEKL peptide bound to MHCI molecules on their surface was significantly higher than the percentage of BMDC displaying the same, suggesting that aDC are superior at either transfection with OVA T08741ANM - 130 - mRNA LNP, or antigen expression and processing, or all of the above, from LNP mRNA antigen source (Figure 10).

[0565] Experiment 10. Surface 25.D1 stain in aDC transduced with SIINFEKL peptide mRNA vs OVA protein mRNA

[0566] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Experiment 1. To the plate-passed PBMC in culture is added the desired amount of LNP containing mRNA for antigen of interest, such as lug / mL of cKK-E12 based LNP containing OVA protein mRNA, or 0.05, O.lug, 0.5ug, lug, or 5ug of LNP containing immunogenic OVA SIINFEKL peptide mRNA (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC are incubated with the LNP overnight, to allow for LNP uptake and antigen expression.

[0567] Antigenic OVA peptide SIINFEKL expression on aDC H-2Kb MHCI molecules is detected after overnight incubation by staining the cells with fluorescently labeled 25. DI TCR-like antibody (Biolegend, clone 25-D1.16), as well as any necessary antibodies to identify cells of interest (eg CDl lb+ for murine aDC within the plate- passed PBMC; CD1 lb Biolegend clone MI / 70).

[0568] Antibody binding to aDC or BMDC can be detected by flow cytometry (Cytoflex cytometer, analysis using FlowJo vlO software).

[0569] Results

[0570] The percentage of aDC displaying antigenic OVA SIINFEKL peptide bound to MHCI molecules on their surface is higher at the same LNP concentration (lug / mL) for aDC transduced with SIINFEKL peptide mRNA LNP, than for aDC transduced with OVA protein mRNA LNP (Figure 11). aDC transduction with SIINFEKL peptide mRNA T08741ANM - 131 -

[0571] LNP is also efficient at much lower LNP concentrations, displaying surface antigen / MHCI complexes with as little as 0.05ug / mL LNP used. Together, these data show that loading of aDC with immunogenic peptides alone, rather than with whole proteins, as antigens, can be highly efficient.

[0572] Experiment 11. OT1 proliferation assay: aDC pulsed with titrating amounts of LNP or soluble OVA protein cultured with OT1 T cells

[0573] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Experiment 1. To the plate-passed PBMC in culture is added the antigen source, such as soluble OVA protein (50ug / mL; 200ug / mL); or LNP containing OVA protein mRNA (1, 0.1, 0.01, or O.OOlug / mL of cKK-E12 based LNP) (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC are incubated with the respective antigen sources overnight, to allow for antigen uptake and / or expression, and processing.

[0574] After overnight incubation, antigen-loaded cells are harvested and cocultured for 3 days at 2*105cells / mL (of note, aDC are not purified; the cell number is for total PBMC, of which 3-10% are CD11+ aDC; aDC cell numbers within PBMC are therefore at most ~2*104cells / mL) under standard conditions in 96-well plates with l*105cells / mL CFSE-labeled OVA-specific OT1 CD8 T cells (isolated from spleens of C57Bl / 6-Tg (TcraTcrb)1100Mjb / J mice that recognize OVA peptide residues 257 to 264 in the context of H-2Kb, Jackson Laboratory). At the end of coculture, cells are stained with anti-CD8 antibody to identify antigen-reactive T cells (Biolegend, clone 53-6.7).

[0575] Antigen-specific CD8 T cell proliferation, as indicated by CFSE dilution in OT1 CD8+ T cells, can be assessed by flow cytometry (Cytoflex cytometer, analysis using FlowJo T08741ANM - 132 - vlO software).

[0576] Results

[0577] The percentage of proliferated OVA-reactive OT1 CD8 T cells when cultured with OVA protein mRNA LNP -transduced aDC is very high, even at the lowest LNP concentration (O.OOlug / mL, Figure 12). This demonstrates that LNP -transduced aDC are extremely potent at stimulating antigen-specific CD8 T cell responses. Furthermore, aDC transduction with the lowest LNP concentration (O.OOlug / mL) was at least as potent at stimulating CD8 T cell response as aDC loading with the highest soluble OVA protein concentration (200ng / mL), suggesting that mRNA LNP are a significantly superior antigen source.

[0578] Experiment 12. OT1 proliferation assay: OT1 T cells cultured with titrating amount of aDC (at 1 ug / mL LNP)

[0579] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Experiment 1. To the plate-passed PBMC in culture is added the antigen source, such as LNP containing OVA protein mRNA (lug / mL of cKK-E12 based LNP) (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC are incubated with the respective antigen sources overnight, to allow for antigen uptake and / or expression, and processing.

[0580] After overnight incubation, cells are harvested and cocultured for 3 days at 2*105, l*105, 5*104, 2.5* 104, or 1.2*104cells / mL (of note, aDC are not purified; the cell number is for total PBMC, of which 3-10% are CD11+ aDC; aDC cell numbers within PBMC are therefore at most ~2* 104, l*104, 5*103, 2.5* 103, or 1.2*103cells / mL) under standard conditions in 96-well plates with 1 * 105cells / mL CFSE-labeled OVA-specific T08741ANM - 133 -

[0581] 0T1 CD8 T cells (isolated from spleens of C57Bl / 6-Tg (TcraTcrb)1100Mjb / J mice that recognize OVA peptide residues 257 to 264 in the context of H-2Kb, Jackson Laboratory). At the end of coculture, cells are stained with anti-CD8 antibody to identify antigen-reactive T cells (Biolegend, clone 53-6.7).

[0582] CD8 T cell proliferation as indicated by CFSE dilution in CD8+ T cells can be assessed by flow cytometry (Cytoflex cytometer, analysis using FlowJo vlO software).

[0583] Results

[0584] The percentage of proliferated OVA-reactive OT1 CD8 T cells when cultured with titrating amounts of OVA protein mRNA LNP -transduced aDC remains notably high, with as few as 1,200 aDC per mL (120 cells per well in a 96-well plate) retaining the ability to stimulate proliferation in up to 47% of OT1 CD8 T cells (Figure 13). This again indicates that mRNA LNP -transduced aDC are extremely potent and efficient stimulators of antigen-specific CD8 T cell response.

[0585] Experiment 13. Therapeutic aDC administration in the EG7-OVA lymphoma mouse model using tumor antigen mRNA-containing LNP (ovalbumin (OVA) antigen)

[0586] Methods

[0587] The experimental protocol is as described for Experiment 3, with the sole difference that control mice, instead of remaining untreated, received bi-weekly intramuscular injection of the same amount (lOng / mouse) of mock mRNA LNP (expressing SARS- CoV-2 Spike protein, irrelevant in the EG7-OVA tumor model setting).

[0588] The experimental groups therefore are:

[0589] Mock (SARS-CoV-2 Spike protein) mRNA LNP intramuscular vaccination OVA mRNA LNP intramuscular vaccination T08741ANM - 134 -

[0590] OVA mRNA LNP aDC retro-orbital vaccination

[0591] With treatments administered on days 4, 8, 11, 15, 18, and 25 post EG7-0VA tumor implantation (Figure 14 a).

[0592] Results

[0593] Compared to the control mock-treated (“Mock mRNA IM”) group, therapeutic aDC administration (“OVA mRNA aDC”) using the clinically relevant dose of lOng OVA mRNA-containing LNPs (0.5ug / kg of LNP, dose equivalent to COVID-19 Pfizer mRNA LNP vaccine in current human vaccine setting) successfully controlled the growth of EG7-0VA tumors, while direct intramuscular administration of the same amount of OVA mRNA-containing LNPs (“OVA mRNA IM (no aDC)” group) did not significantly alter the kinetics of tumor growth (Figure 14 b).

[0594] Experiment 14. SIINFEKL tetramer analysis and adoptive T cell transfer

[0595] Methods

[0596] Splenocytes are harvested at the end of Experiment 13 described above (were harvested at the end of the in vivo tumor monitoring period (Day 32 after tumor inoculation) from all treatment and control groups for further analysis, to characterize the resulting anti-tumor immune response.

[0597] Antigen (SIINFEKL)-specific CD8 T cell evaluation: CD8 (BioRad, clone KT1.5) and specific H2d-SIINFEKL dextramer (Immudex) staining of splenocytes isolated above, followed by flow cytometry and analysis (Cytoflex).

[0598] Adoptive T cell transfer: CD3+ T cells isolated from splenocytes of the three experimental groups of Experiment 13 (Miltenyi T cell isolation kit) are transferred intravenously at 1.5*107T cells / mouse into antigen-naiive C57BL / 6 mice, freshly T08741ANM - 135 - inoculated with EG7-0VA tumors (tumor inoculation as described for Experiment 3), and tumor development was monitored over the course of 19 days.

[0599] Results

[0600] Therapeutic OVA protein mRNA LNP -transduced aDC vaccination (“OVA aDC”) and intramuscular OVA protein mRNA LNP vaccination (“OVA IM”) both successfully induced OVA antigen-specific T cells, as measured by percentage of H- 2Kb SIINFEKL tetramer-positive CD8 T cells (Figure 15 a). Mock-treated animals (“Mock IM”) also showed some tetramer positivity, reflecting the presence of background, natural T immunity in EG7-0VA tumor bearing mice.

[0601] However, despite the presence of tumor-reactive T cells in all groups, only T cells isolated from the spleens of mice treated with OVA protein mRNA LNP -transduced aDC (“OVA aDC”) conferred protective immunity against EG7-0VA tumors in antigen-naiive, untreated mice (Figure 15 b). This indicates that only DC therapy provides true anti-tumor immunity.

[0602] Experiment 15: Spike protein ELISpot detection of SARS-CoV-2 reactive T cells

[0603] Spike protein mRNA-containing LNP transduction of human aDC

[0604] Plate-passed PBMC containing nascent aDC from healthy human donors, produced per methods described in Experiment 1, are plated at 5*105cells per well, in triplicate per experimental group, in human IFNy ELISpot plates. To each experimental well is added 62.5-250ng / well of cKK-E12 based LNP (cKK-E12, cholesterol, C14-PEG 2000-PE and DOPE at a ratio of 35:46.5:2.5: 16) containing Spike protein mRNA (LNPs prepared and provided by the Santangelo laboratory, Emory University, RNA sequence corresponds to SEQ ID NO: 33) in 200uL of culture medium consisting of RPMI without phenol red (Gibco) supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen). Positive control wells are treated with an overlapping pool of Spike Class I & II peptides (Miltenyi Biotec, T08741ANM - 136 -

[0605] PepTivatorSARS-CoV-2 Prot-S Complete). Negative control wells are treated with 62.5-250ng / well of cKK-E12 based LNP containing an irrelevant (eg Nanoluciferase) protein mRNA.

[0606] Elispot assay read-out

[0607] After overnight incubation, wells are washed and IFNy spots are detected with biotinylated anti-human IFNy mAb, streptavidin-ALP and BCIP / NBT-plus substrate per manufacturer’s protocol (MAbTech, 3420-2AST-2). Spot forming units (SFU) for each experimental condition are quantitated using an ELISpot plate reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). By convention, an ELISpot signal is deemed positive if it is above 50 SFU / million cells, and / or the experimental group signal is at least 2-fold higher than the control (mock) group signal.

[0608] CD4 and CD8 T cell depletions

[0609] To test the contribution of T cell subsets to cytokine production, CD4 or CD8 T cells may be selectively depleted from PBMC after plate passage, but prior to overnight incubation with LNPs in Elispot wells. T cells are depleted using standard depletion kits (Miltenyi; CellSep).

[0610] Results

[0611] Figure 16A: An example of a aDC ELISpot using PBMC isolated from a single human donor, 4 weeks post SARS-CoV2 infection. IFNy release was significantly elevated in the presence of aDC [Spike], when compared to negative controls. This demonstrates detection of human Spike specific T cells via mRNA transduced aDC in a Covid convalescent donor.

[0612] Figure 16B: An example of an aDC ELISpot dose response using PBMC from a single human donor, 4 weeks post SARS-CoV2 infection. IFNy release increased to levels T08741ANM - 137 - above 150 SFU / million when LNP [Spike] antigen was added at 62.5ng / well. T cell activation improved further at higher doses but showed evidence of a response plateau at 250ng / well. This demonstrates aDC LNP Spike dose response detectable at range as low as 62.5ng / well.

[0613] Figure 17 A: 18 previously vaccinated human donors were screened in aDC ELISpot against SARS-CoV-2 Spike antigen. Donors were separated into two cohorts based on whether they were previously infected with SARS-CoV-2 (black) or not (blue). Statistical analysis utilized an unpaired, two-tailed, Mann-Whitney U test. This demonstrates that human aDC transduced with LNP [Spike] can differentiate T cell responses associated with natural immunity from the response associated with vaccination alone.

[0614] Figure 17B: 11 previously vaccinated and convalescent human donors were screened in aDC ELISpot against SARS-CoV-2 Spike antigen. Dotted line represents the standard threshold cutoff for positive response (50 SFU / Million cells). This demonstrates that 1) the response strength is generally negatively correlated w / convalescent period; 2) the aDC induced IFNy response is durable and detectable out to 1 year post Covid infection.

[0615] Figure 17C: Vaccinated and convalescent donors’ plate passed PBMC were depleted of either CD8 or CD4 T cells prior to incorporation into the standard 18hr aDC [Spike] ELISpot IFNy assay. Depletion of CD8 T cells eliminates the majority of aDC [Spike] response, while CD4 depletion has minimal effect. This demonstrates that convalescent aDC [Spike] IFNy response is largely driven by CD8+ T cells.

[0616] Figure 17D: An example of an aDC ELISpot using PBMC isolated from a single human donor prior to, and six weeks post, SARS-CoV2 infection. IFNy release increased above the positive threshold of 50 SFU / Million cells following infection. T08741ANM - 138 -

[0617] This demonstrates the aDC induced IFNy T cell response increases following natural Covid infection, thus showing the feasibility and utility of longitudinal immune monitoring.

[0618] Example 16. OT1 proliferation assay: murine activated dendritic cells transduced with LNP containing anti-PDl antibody construct mRNA, cocultured with OT1 T cells

[0619] Methods

[0620] Activated dendritic cells are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described above. To the plate-passed PBMC in culture is added the antigen source, such as soluble OVA protein (150-200ug / mL) or [OVA] (lOng / mL of cKK-E12 based LNP); and LNP containing mRNA encoding aPDl surface- anchored or secreted antibody construct (lug / mL of cKK-E12 based LNP) or the same amount of irrelevant [mock] control (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC are incubated with the respective antigen sources overnight, to allow for antigen uptake and / or expression, and processing.

[0621] After overnight incubation, antigen-loaded cells are harvested and co-cultured for 3 days at 2*105cells / mL (of note, activated dendritic cell are not purified; the cell number is for total PBMC, of which 3-10% are CD11+ activated dendritic cells; activated dendritic cell numbers within PBMC are therefore at most ~2*104cells / mL) under standard conditions in 96-well plates with l*105cells / mL CFSE- labeled OVA-specific OT1 CD8 T cells (isolated from spleens of C57Bl / 6-Tg (TcraTcrb)l lOOMjb / J mice that recognize OVA peptide residues 257 to 264 in the context of H-2Kb; Jackson Laboratory). At the end of co-culture, cells are stained with anti-CD8 antibody to identify antigen-reactive T cells (Biolegend, clone 53- T08741ANM - 139 -

[0622] 6.7); as well as aCD44 (BioLegend, IM7), aCD62L (BioLegend, MEL-14), and aPDl (BioLegend, 29F.1A12) antibodies.

[0623] Antigen-specific CD8 T cell proliferation, as indicated by CFSE dilution in OT1 CD8+ T cells, can be assessed by flow cytometry (Cytoflex cytometer, analysis using FlowJo vlO software). T cell activation status can be further monitored by expression of CD44, CD62L, and PD1.

[0624] Results

[0625] Activated dendritic cells transduced with [anti-PDl] constructs significantly improve antigen-specific OT1 T cell proliferation and activation , validating the functionality of these antibody constructs in activated dendritic cells.

[0626] Example 17. Systemic activated dendritic cell vaccination in the MC38 colon carcinoma mouse model using anti-PDl antibody construct mRNA-containing LNP

[0627] The experimental design is schematically described in Figure 18A.

[0628] For tumor induction, IxlO5MC38 tumor cells are injected subcutaneously in lOOuL into the right flanks of recipient wild-type C57BL / 6J mice. Treatment is initiated on day 3 following tumor implantation.

[0629] For local treatment, activated dendritic cell are prepared from the blood of respective experimental group (200uL blood collected per animal) by standard protocol described in Example 1, with the adjustment that the activated dendritic cell culture, and co-culture with LNP, is reduced to 3 hours instead of overnight incubation, activated dendritic cell are transduced with surface-anchored or secreted [anti-PDl] T08741ANM - 140 - antibody construct (500ug / mouse of cKK-E12 based LNP), and injected into the flank intratumorally / peritumorally, at ~5xl05cells / mouse.

[0630] For systemic treatment, activated dendritic cell are prepared as for local treatment above, but administered by injection into the retro-orbital plexus.

[0631] All LNPs are prepared and provided by the Santangelo laboratory, Emory University.

[0632] Positive control animals receive intraperitoneal injections of commercially available anti-PDl antibody (200ug / mouse, BioXcell, RMP1-14) on the same schedule as local and systemic activated dendritic cell treatments.

[0633] Treatment is repeated twice a week until the end of the experiment, which is determined by the speed of largest tumor growth up to the maximal limit permitted by the animal care facility. Mice are usually bled for activated dendritic cell vaccine manufacture on Mondays and Thursdays, with activated dendritic cell vaccinations taking place on Tuesdays and Fridays. Typically, 5-6 bi-weekly therapeutic immunization treatments can be carried out.

[0634] Tumor volume is monitored via biweekly measurement of perpendicular tumor diameters and height using a caliper, and tumor volume calculated as (tumor length x width x height) / 2. At the end of the experiment, surviving animals do not receive any more treatments but continue to be monitored for tumor growth until / if they reach the maximal permitted tumor volume limit. This allows for survival curve creation for each treatment group.

[0635] To monitor for persistent immunity and immunological memory to tumor challenge, animals that fully reject the initial tumor and are tumor-free may be re-challenged with a similar additional inoculum of the same tumor cell line. Growth of any T08741ANM - 141 - resulting tumors is then monitored as above. Re-challenges typically take place at days 80 and / or 120 after the original tumor challenge.

[0636] Results

[0637] Therapeutic systemic activated dendritic cell vaccination using [anchored anti-PDl] significantly controlled the growth of MC38 tumors and improved animal survival. However, all treated animals still succumbed to the tumor (Figure 18A-D).

[0638] Example 18. Systemic activated dendritic cell treatment in the YUMMER1.7 mouse model using anti-PDl antibody construct mRNA-containing LNP

[0639] The experimental design is schematically described in Figure 19A. Tumor induction, treatment, monitoring and analysis was performed as described above (Experiment 17) with the difference that the dose of the activated DC was reduced to 200 ng / animal.

[0640] Results

[0641] Therapeutic systemic activated dendritic cell treatment using anchored as well as secreted anti-PDl significantly slowed down the growth of YUMMI1.7 tumors, improved animal survival and resulted in complete responses (CRs) in 3 / 10 animals. (Figure 19A-D).

[0642] Example 19: Systemic activated dendritic cell treatment using anti-PDl antibody construct mRNA and IL-12 mRNA-containing LNP

[0643] This experiment was performed to assess a multilateral tumor targeting approach via antigenic vaccination and immune-biologic augmentation using the activated T08741ANM - 142 - dendritic cells of the invention comprising a checkpoint inhibitor, a cytokine and / or tumor neo-antigens. .

[0644] The experimental design is schematically described in Figure 20A. Tumor induction, treatment, monitoring and analysis was performed as described above (Experiment 17) with the difference that the dose of the activated DC was reduced to 150 ng / animal.

[0645] For systemic injections, total dose limit was set at 200ng / mouse / dose.

[0646] Systemic injections (ie., Strings and anti-PD-1) began on Day+4, for a total of 6 treatments (see schematic of Fig. 20A). mIL-12 injection #1 given concurrently w / tumor inoculum on DayO, followed #2 injection given on Day+4 i.t.

[0647] In addition to loading the aDC with LNPs comprising mRNA encoding for the PD-1 checkpoint inhibitor, in some conditions, the activated dendritic cells were co-loaded with LNPs comprising mRNA encoding IL- 12 (lOOng / animal) was administered intra tumorally twice.

[0648] Further, the aDC was for the triple anti-tumor approach treated with aDCs loaded with mRNA encoding for tumor neo-antigens (“Neostrings”) the sequences of which are shown in Table 1. For the Neo-antigens, a 1 :1 mix of cKK-E12 LNP[8x+pl5] and LNP[4x] (25ng of each LNP) was used.

[0649] Results Treatment with NeoStrings alone in aDC conferred a small degree of antitumor activity. The co-treatment with anchored anti-PDl antibody in aDC (anc-

[0650] PD1) resulted in strong anti -turn or effects, including increasing probability of survival, reducing tumor volume and increasing the rate of complete responders. . Similar effects were also observed with the animals were treated with a combination of the anti-PDl antibody and IL-12 in aDCs. Moreover, the treatment using tumor antigens, anti-PDl antibody and IL- 12 resulted in a stronger anti -turn origenic effect (Figure 21). T08741ANM - 143 -

[0651] Example 20: Systemic activated dendritic cell treatment using anti-CTLA-4 antibody mRNA and anti-PDl antibody mRNA-containing LNP .

[0652] The experimental set-up was as described in Ex. 19 wherein the animals were treated with a combination of anti-PDl and anti-CTLA-4 antibody expressed in aDCs.

[0653] Treatment with aDCs loaded with anti-CTLA-4 encoding mRNA-LNPs in conjunction with tumor neo-antigens reduced tumor burden and increased survival and the rate of complete responders. Interestingly, the actiated DCs of the invention comprising tumor antigen and anti-CTLA-4 antibody demonstrated substantial synergy with standard soluble anti-PDl antibody (Figure 22).

[0654] Example 21: Type 1 Diabetes model using non-obese diabetic (NOD) mice.

[0655] It has been reported that cancer treatment using anti-PDl antibodies induced autoimmune diabetes mellitus. The progression of type 1 diabetes (T1D) was typically rapid. In addition, patients treated with PD-1 inhibitors combined with CTLA-4 inhibitors progressed to diabetes at an even earlier time (Lin et al., PD-1 inhibitor-associated type 1 diabetes: A case report and systematic review. Front Public Health. 2022 Aug 5;10:885001. doi: 10.3389 / fpubh.2022.885001).

[0656] NOD mice are an established animal model to investigate T1D, wherein male and female mice differ in the timing of diabetes onset (Figure 23A).

[0657] Similar to the T1D onset seen in checkpoint inhibitor treatment patients, the NOD mouse model shows an abrupt and rapid onset of T1D when treated with anti-PD-1 antibody (Figure 23), wherein no significant difference was present between male and female treated NOD mice. T08741ANM - 144 -

[0658] NOD mice (male, 7wk) were treated with standard anti-PD-1 antibody (lOmg / kg) or either an anti-PDl -antibody loaded activated dendritic cell (aDCfaPDl]) or an activated dendritic cell loaded with an anti-PD-1 antibody and anti-CTLA-4 antibody [aPDl] / CTLA4], anchored version, 150ng was administered per animal on day 0). Treatments were given twice / week for total of six treatments (same as the treatment protocol used in the cancer treatment model above).

[0659] Blood glucose level was monitored three times a week using ACCU-CHEK Guide glucometer via tail vein blood. Onset of T1D was determined via two consecutive readings of blood glucose level of >250 mg / dl. (n = 5 mice per group)

[0660] Results: Standard anti-PD-1 antibody treatment induced rapid and premature T1D onset in NOD mice. In contrast, no T1D induction was detected in mice treated with activated dendritic cells loaded with anti-PD-1 antibody. Even animals treated with activated dendritic cell loaded with an anti-PD-1 antibody and anti-CTLA-4 antibody had no early T1D induction, wherein in humans the dual treatment with anti-PD-1 antibody and anti-CTLA-4 antibody aggravates T1D onset compared to anti-PD-1 antibody treatment alone, resulting in T1D onset at an even earlier time point. Hence, the invention provides an effective immune checkpoint inhibitor treatment with reduced side effects, i.e. reduced toxicity (Figure 24).

[0661] Example 22. Systemic activated dendritic cell treatment using IL-12 mRNA- containing LNP.

[0662] An EliSpot assay was performed as described above.

[0663] IFNy release of mock transfected (aDC alone) or aDCs that were loaded with IL-12 mRNA LNPs was assessed, which serves as a readout of mounting a T cell response. The treatment with IL-12 loaded aDCs resulted in a strong IFNy T cell activation (Figure 25). T08741ANM - 145 -

[0664] Example 23. Systemic activated dendritic cell treatment using IL-12 mRNA- containing LNP.

[0665] This experiment was performed as described above in Ex. 19, with the difference that the aDCs were loaded only with IL-12 or left unloaded (mock). The animals received six consecutive treatments as shown in Figure 26A.

[0666] Treatment with IL- 12 loaded aDC resulted in a reduction of the tumor volume and extended the probability of survival (Figure 26).

[0667] Example 24. Systemic activated dendritic cell treatment using IL-12 mRNA- containing LNP and CpG co-stimulation.

[0668] This experiment was performed as described above in Ex. 23, wherein the dosage was reduced to two doses and CpG co-stimulation was used (Figure 27 A).

[0669] Repeated low dose of IL- 12 loaded aDC resulted in a significant reduction of the tumor volume and extension of the probability of survival, both of which were further enhanced with co-stimulation using CpG (Figure 27).

[0670] Example 25. Murine and human aDC expression of CCR7 aDC are prepared from the blood of healthy C57BL / 6J mice, or from healthy volunteer human donor blood, by standard protocols.

[0671] Plate-passed PBMC in culture are transduced with lOug-lOng of cKK-E12 (mouse) or SMI 02 (human) based LNP containing mRNA for CCR7 protein (denoted as [CCR7]) or irrelevant mRNA (denoted as [mock]). All LNPs prepared and provided by the Santangelo laboratory, Emory University. T08741ANM - 146 -

[0672] Cells are collected after incubation (time course of 4hrs up to 7 days), and analyzed by flow cytometry for aDC content and CCR7 expression (mouse Ly6G-, CD1 lb+, CCR7+ cells; human CD14+, CDl lb+, CDl lc+, CCR7+ cells).

[0673] Results

[0674] Murine and human aDC transduced with [CCR7], compared to [mock], are expected to show increased expression of CCR7 on their surface. The time course is expected to provide insight into CCR7 expression kinetics, and durability of expression.

[0675] Example 26. CCR7-transduced aDC migration to chemotactic cytokines aDC are prepared from the blood of healthy C57BL / 6J mice, or from healthy volunteer human donor blood, by standard protocols.

[0676] Plate-passed PBMC in culture are transduced with lOug-lOng of cKK-E12 (mouse) or SMI 02 (human) based LNP containing mRNA for CCR7 protein (denoted as [CCR7]) or irrelevant mRNA (denoted as [mock]). All LNPs prepared and provided by the Santangelo laboratory, Emory University.

[0677] Cells are placed for culture in the top well of a transwell system, containing in the bottom well culture medium alone, or culture medium supplemented with CCL19 or CCL2E After 1-3 days of culture, cells are collected from the bottom wells, counted, and analyzed by flow cytometry for aDC content and CCR7 expression (mouse Ly6G-, CDl lb+, CCR7+ cells; human CD14+, CDl lb+, CDl lc+, CCR7+ cells).

[0678] Results T08741ANM - 147 -

[0679] Murine and human aDC transduced with [CCR7], compared to [mock], are expected to show increased migration to CCL19 and CCL21, helping to establish that CCR7 expression in aDC is functional.

[0680] Example 27. Tracking transduced aDC accumulation in murine lymphoid tissues / tumor aDC are prepared from the blood of healthy or tumor-bearing mice (ex. day 7 postimplantation MC38 tumors, with IxlO5MC38 tumor cells injected subcutaneously in lOOuL into the right flanks of recipient wild-type C57BL / 6J mice) by standard protocol.

[0681] Plate-passed PBMC in culture are transduced with lOug-lOng of cKK-E12 based LNP containing mRNA for CCR7 protein and nanoluciferase (denoted as [CCR7- nanoLuc]) or irrelevant mRNA and nanoluciferase (denoted as [mock-nanoLuc]). All LNPs prepared and provided by the Santangelo laboratory, Emory University.

[0682] After overnight incubation, cells co-cultured with [CCR7-nanoLuc] or [mock- nanoLuc] are collected, and injected intravenously in a lOOuL volume of sterile PBS into the retro-orbital plexus of healthy or tumor-bearing mice.

[0683] Mice are subsequently sacrificed in a time course (immediately following aDC injection, to day 7-10 post injection), and luciferase signal is monitored by luminescence detection throughout the body (lung, liver, spleen, lymph nodes, tumor etc).

[0684] Results T08741ANM - 148 - aDC transduced with [CCR7-nanoLuc], compared to [mock-nanoLuc], are expected to show increased luciferase signal in lymphoid organs (spleen, lymph nodes) and possibly in tumor, indicating preferential homing to these organs conferred by functional expression of CCR7, a chemokine receptor known to be the primary mediator of dendritic cell homing to lymphoid tissues.

[0685] Example 28. Systemic aDC vaccination in the MC38 colon carcinoma mouse model using CCR7 mRNA-containing LNP

[0686] For tumor induction, IxlO5MC38 tumor cells are injected subcutaneously in lOOuL into the right flanks of recipient wild-type C57BL / 6J mice. Treatment is initiated on day 3 following tumor implantation. aDC are prepared from the blood of respective experimental group (200uL blood collected per animal) by standard protocol. aDC are transduced with [CCR7] or [mock] construct (lOug-lOng / mouse of cKK-E12 based LNP), in conjunction with relevant tumor antigens, and injected into the retro-orbital plexus. All LNPs are prepared and provided by the Santangelo laboratory, Emory University.

[0687] Treatment is repeated twice a week until the end of the experiment, which is determined by the speed of largest tumor growth up to the maximal limit permitted by the animal care facility. Mice are usually bled for aDC vaccine manufacture on Mondays and Thursdays, with aDC vaccinations taking place on Tuesdays and Fridays. Typically, 5-6 bi-weekly therapeutic immunization treatments can be carried out.

[0688] Tumor volume is monitored via biweekly measurement of perpendicular tumor diameters and height using a caliper, and tumor volume calculated as (tumor length x T08741ANM - 149 - width x height) / 2. At the end of the experiment, surviving animals do not receive any more treatments but continue to be monitored for tumor growth until / if they reach the maximal permitted tumor volume limit. This allows for survival curve creation for each treatment group.

[0689] To monitor for persistent immunity and immunological memory to tumor challenge, animals that fully reject the initial tumor and are tumor-free may be re-challenged with a similar additional inoculum of the same tumor cell line. Growth of any resulting tumors is then monitored as above. Re-challenges typically take place at days 80 and / or 120 after the original tumor challenge.

[0690] Results

[0691] Therapeutic systemic aDC vaccination using [CCR7], by augmenting aDC presence in the lymphoid tissues and possibly in the tumor itself, is expected to enhance aDC engagement with anti-tumor T cells, promote anti-tumor T cell responses, and thus limit tumor growth and enhance the survival of tumor-bearing animals. In the long term, CCR7 expression on aDC may enhance tumor-free survival and promote resistance to tumor re-challenge by generating long-term immunological memory.

[0692] Example 29: OT1 proliferation assay: aDC pulsed with titrating amounts of LNP[anti-PD-l ab], LNP[sec anti-PD-1 ab] or LNP[IL-12] and soluble OVA protein cultured with OT1 T cells

[0693] Methods aDC are prepared from healthy C57BL / 6 donor mouse blood by standard protocol described in Experiment 1. Plate-passed PBMC in culture are pulsed with soluble OVA protein (200ug / mL); and one of three LNPfmRNA]:

[0694] • LNP containing mRNA encoding anchored anti-PD-1 antibody (LNP[a-PD- i]); T08741ANM - 150 -

[0695] • LNP containing mRNA encoding secreted anti- PD-1 antibody (LNPfsec a- PD1]);

[0696] • or LNP containing mRNA encoding IL-12p70 (LNP[IL-12p70]).

[0697] LNP containing an mRNA encoding sOVA was used as a control (see Figure 28).

[0698] Each LNPfmRNA] is used at three different concentrations (0.1, 1 or 5 ug / mL of cKK-E12 based LNP) (LNP prepared and provided by the Santangelo laboratory, Emory University). Plate-passed PBMC are incubated with the respective antigen sources overnight, to allow for antigen uptake and / or expression, and processing.

[0699] After overnight incubation, antigen-loaded cells are harvested and cocultured for 3 days at 2*105 cells / mL under standard conditions in 96-well plates with 1*105 cells / mL CFSE-labeled OVA-specific OT1 CD8 T cells (isolated from spleens of C57Bl / 6-Tg (TcraTcrb)l lOOMjb / J mice that recognize OVA peptide residues 257 to 264 in the context of H-2Kb, Jackson Laboratory). At the end of coculture, cells are stained with anti-CD8 antibody to identify antigen-reactive T cells (Biolegend, clone 53-6.7).

[0700] Antigen-specific CD8 T cell proliferation, as indicated by CFSE dilution in OT1 CD8+ T cells, can be assessed by flow cytometry (Cytoflex cytometer, analysis using FlowJo vlO software).

[0701] Results

[0702] The percentage of proliferated OVA-reactive OT1 CD8 T cells when cultured with aDC(LNP[mRNAl-3])-transduced aDC were higher than the control (see Figure 29), aDC(LNP[sOVA]), in a dose-dependent matter. This demonstrates that aDCs loaded with LNPs comprising anti-PD-1 antibody and IL-12 stimulate antigen-specific CD8 T cell responses.

[0703] Example 30: Ex vivo aDC-TIL interaction assay T08741ANM - 151 -

[0704] KPN tumors were implanted sub-cutaneously into B6 mice. 18 days post implant, tumors were collected to harvest tumor cell / tumor infiltrating lymphocyte (TIL) cell suspension prep. In parallel, aDCs were generated from donor naive B6 mice according to the standard protocol of Example 1, which were then transfected with LNPfmRNA] according to the standard over-night transfection protocol. Post transfection, aDCs were added into the tumor / TIL mix, which were then analyzed via FACs (1-day post co- culture) for T cell characterization. The LNPs used for transfection were loaded with either an mRNA encoding Glycoprotein 33 (LNP[GP33]) or with an mRNA encoding anti-PDl antibody (LNPfaPDl]). A schematic overview of the experiment is shown in Figure 30.

[0705] Using FACS, GP33-specific T cells can be gated via tetramer positivity (Tet+), as shown in Figure 32. Note that the Tet+cell numbers in the aDC groups are lower than in the control, due to a larger number of CD8+cells present in the PBMC prep. The absolute number of Tet+cells between all groups was similar.

[0706] FACS was then used to isolate effector and memory T cell subsets using a gating strategy shown in Figure 31. effector and memory T cell subsets are identified based on their expression levels of the markers CD44 and CD62L.

[0707] The cells were then sub-categorized using CD44 / CD62L staining profile. The majority of cells were positive for CD44. Depending on CD62L status, cells were largely subdivided into effector or memory population. Both effector and memory populations were comprised of two sub-population, based on CD44 expression level (Effector 1 and memory 1 showing very high CD44 expression).

[0708] Results

[0709] Memory T-cell (CD62L+) subsets were increased in aDC(LNP[GP33]) group.

[0710] Additionally carrying LNPfaPDl] further increases the memory population, mainly T08741ANM - 152 - by boosting the memory 1 population (CD62L+, CD44 high). Addition of recombinant IL-2 may further increase this population. See Figure 32.

[0711] No difference in expression of selected markers was observed between the three aDC groups. In all groups, the memory 1 subset showed higher IL7R expression (one of the marker to identify stem-like T cells) compared to memory 2 subset. A...

Claims

1. T08741ANM - 153 -CLAIMS1. A first ex vivo activated dendritic cell, wherein the activated dendritic cell comprises at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor.

2. The activated dendritic cell of claim 1, wherein the checkpoint inhibitor is capable of specifically binding to an immune checkpoint molecule, optionally wherein the immune checkpoint molecule is at least partially on a cell surface.

3. The activated dendritic cell of any one of claims 1 or 2, wherein the checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof.

4. The activated dendritic cell of any one of the preceding claims, wherein the checkpoint inhibitor comprises an antibody or an antigen-binding fragment thereof.

5. The activated dendritic cell of any one of the preceding claims, wherein the antibody or an antigen-binding fragment thereof is capable of binding to PD-1, PD- L1 or CTLA-4 or a combination thereof.

6. The activated dendritic cell of any one of claims 4 or 5, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof.

7. The activated dendritic cell of any one of the preceding claims, wherein the checkpoint inhibitor is selected from an anti-PD-1 antibody or a PD-1 binding fragment thereof, an anti-PD-Ll antibody or a PD-L1 binding fragment thereof, or an anti-CTLA-4 antibody or a CTLA-4 binding fragment thereof.

8. The activated dendritic cell of any one of the preceding claims, wherein the checkpoint inhibitor is secreted by the activated dendritic cell.

9. The activated dendritic cell of any one of the preceding claims, wherein the checkpoint inhibitor is attached or anchored to the cell surface of the activated dendritic cell.

10. The activated dendritic cell of any one of the preceding claims, wherein said at least one exogenous nucleic acid sequence comprises RNA or DNA.

11. The activated dendritic cell of claim 10, wherein the RNA comprises mRNA.T08741ANM - 154 -12. The activated dendritic cell of claim 11, wherein the mRNA encoding the checkpoint inhibitor comprises modified nucleotides.

13. The activated dendritic cell of any one of the preceding claims, wherein said at least one exogenous nucleic acid sequence is comprised in at least one lipid nanoparticle (LNP).

14. The activated dendritic cell of claim 13, wherein the at least one LNP comprises at least one lipid selected from the group consisting of acKK-E12 lipid, a SM-102 lipid, C12-(2-3-2), DMG-PEG-2K, cholesterol, DPPC, or a MC3 lipid.

15. The activated dendritic cell of any one of the preceding claims, wherein the immune checkpoint molecule is PD-1, PD-L1 and / or CTLA-4.

16. The activated dendritic cell of any one of the preceding claims, wherein the activated dendritic cell comprises at least one further exogenous nucleic acid sequence encoding an immune modulator.

17. The activated dendritic cell of claim 16, wherein the immune modulator is a cytokine.

18. The activated dendritic cell of any one of claims 16 or 17, wherein the immune modulator is at least one cytokine selected from the group consisting ofinterleukin-lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL-23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof.

19. The activated dendritic cell of any one of claims 16 to 18, wherein the cytokine is interleukin -12 (IL-12).

20. The activated dendritic cell of any one of the preceding claims, wherein the activated dendritic cell comprises at least one further exogenous nucleic acid sequence encoding an antigenic peptide or protein.T08741ANM - 155 -21. The activated dendritic cell of claim 20, wherein the at least one antigenic peptide or protein is a tumor-associated peptide or protein, a viral antigenic protein, a bacterial antigenic protein, or a fungal antigenic protein.

22. The activated dendritic cell of any one of claims 20 or 21, wherein the at least one antigenic peptide or protein is a tumor-associated peptide or protein, and wherein the tumor-associated peptide or protein is a tumor-specific peptide or protein, preferably wherein the tumor-specific peptide or protein comprises at least one tumor-specific neo-antigen.

23. The activated dendritic cell of any one of claims 20 to 22, wherein the at least one tumor-specific neo-antigen comprises HPV E6ZE7, Merkel Cell polyoma LTA epitope, a Tumor Associated Antigens (TAA), a lineage-restricted Differentiation antigen, melanoma MART- 1, TRP2), NY-ESO-1, p53 or Ras.

24. A pharmaceutical composition comprising the activated dendritic cell of any one of claims 1 - 23 and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, further comprising an immune modulator.

26. The pharmaceutical composition of any one of claims 24 or 25, wherein the immune modulator is a cytokine.

27. The pharmaceutical composition of claim 26, wherein the cytokine is capable of inducing CD8+ T cells.

28. The pharmaceutical composition of any one of claims 26 or 27, wherein the cytokine comprises at least one of interleukin- lb (IL-lb), interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL-15), interleukin-21 (IL-21), interferons (IFNs) such as IFNy, interleukin- 18 (IL-18), interleukin-23 (IL-23), interleukin-36y (IL-36y), IL- 23 / IL36g, and tumor necrosis factor alpha (TNFa) or a combination thereof. .

29. The pharmaceutical composition of claim 28, wherein the cytokine is interleukin- 12 (IL- 12).T08741ANM - 156 -30. The pharmaceutical composition of any one of claims 24 to 29, further comprising a second activated dendritic cell comprising an exogenous nucleic acid sequence encoding the immune modulator.

31. The pharmaceutical composition of claim 30, wherein the second activated dendritic cell is capable of expressing and / or secreting the immune modulator.

32. The pharmaceutical composition of any one of claims 24 to 31, further comprising a third activated dendritic cell comprising an exogenous nucleic acid sequence encoding at least one antigenic peptide or protein.

33. The pharmaceutical composition of claim 32, wherein the exogenous nucleic acid sequence encoding the at least one antigenic peptide or protein is exogenous RNA, optionally mRNA.

34. The pharmaceutical composition of any one of claims 32 or 33, wherein said at least one antigenic peptide or protein is a tumor-associated peptide or protein, a viral antigenic protein, a bacterial antigenic protein, or a fungal antigenic protein.

35. The pharmaceutical composition of claim 34, wherein the tumor-associated peptide or protein is a tumor-specific peptide or protein.

36. The pharmaceutical composition of claim 35, wherein the tumor-specific peptide or protein comprises at least one tumor-specific neo-antigen, preferably wherein the at least one tumor-specific neo-antigen comprises HPV E6ZE7, Merkel Cell polyoma LTA epitope, a Tumor Associated Antigens (TAA), a lineage- restricted Differentiation antigen, melanoma MART- 1, TRP2, NY-ESO-1, p53 or Ras.

37. A pharmaceutical composition comprising the activated dendritic cell of any one of claims 16 to 23 and a pharmaceutically acceptable carrier.

38. The ex vivo activated dendritic cell of any one of claims 1 to 21 or the pharmaceutical composition of any one of claims 22 to 37 for use in therapy.

39. The ex vivo activated dendritic cell of any one of claims 1 to 21 or the pharmaceutical composition of any one of claims 22 to 37 for use in a method of treating cancer in a subject in need thereof.T08741ANM - 157 -40. The activated dendritic cell or pharmaceutical composition for use of claim 39, wherein the method comprises administering a therapeutically effective amount of the activated dendritic cell or the pharmaceutical composition to the subject.

41. The activated dendritic cell or pharmaceutical composition for use of claim 39 or 40, wherein the treatment prolongs the median survival of the subject compared to a subject having cancer and being treated with an activated dendritic cell or a checkpoint inhibitor alone.

42. The activated dendritic cell or pharmaceutical composition for use of any one of claims 39 to 41, wherein the cancer is a solid cancer or a hematopoietic cancer.

43. The activated dendritic cell or pharmaceutical composition for use of any one of claims 39 to 42, wherein the cancer is a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or a germ cell tumor.

44. The activated dendritic cell or pharmaceutical composition for use of any one of claims 39 to 43, wherein the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, and pancreatic cancer .

45. The activated dendritic cell or pharmaceutical composition for use of any one of claims 39 to 44, wherein the method comprises administering to the subject a therapeutically effective amount of the activated dendritic cells.T08741ANM - 158 -46. The activated dendritic cell or pharmaceutical composition for use of any one of claims 39 to 45, wherein the activated dendritic cell or the pharmaceutical composition is administered locally or systemically to the subject .

47. The activated dendritic cell or pharmaceutical composition for use of any one of claims 39- 46, wherein the activated dendritic cell inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces tumor growth rate, eradicates the tumor or tumor cells, and / or prolongs the median survival period relative to treatment with an activated dendritic cell or a checkpoint inhibitor alone .

48. The activated dendritic cell or pharmaceutical composition for use of any one of claims 39 to 47, wherein the method further comprises administration of a further checkpoint inhibitor.

49. The pharmaceutical composition of any one of claims 30 to 36 for use in a method of treating cancer in a subject in need thereof, wherein the first activated dendritic cell is administered separately, simultaneously or sequentially from the second or third activated dendritic cell.

50. The pharmaceutical composition for use of claim 49, wherein the first activated dendritic cell, the second activated dendritic cell and the third activated dendritic cell are administered in a pre-defined order.

51. The pharmaceutical composition for use of any one of claims 49 or 50, wherein the first activated dendritic cell, the second and / or the third activated dendritic cell are administered repeatedly to the subject.

52. The pharmaceutical composition for use of claim 51, wherein the first activated dendritic cell, the second and / or the third activated dendritic cell are administered at least twice, 3-times, 4-times, 5-times, 6-times or more to the subject.

53. The pharmaceutical composition for use of any one of claims 49 to 52, wherein the combined treatment with the first activated dendritic cell in conjunction with the second and / or the third activated dendritic cell prolongs the median survival of the treated subject compared to a subject being treated with any one of the firstT08741ANM - 159 - activated dendritic cell, the pharmaceutical composition, the second or the third activated dendritic cell alone.

54. The pharmaceutical composition for use of any one of claims 49- 53, wherein the combined treatment with the first activated dendritic cell with the second and / or the third activated dendritic cell inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces tumor growth rate, eradicates the tumor or tumor cells, and / or prolongs the median survival period relative to treatment with the activated dendritic cell, the pharmaceutical composition, the second and / or the third activated dendritic cell alone.

55. The activated dendritic cell of any one of claims 16-23 for use in a method of treating cancer in a subject in need thereof.

56. The activated dendritic cell for use of claim 55, wherein the treatment with the activated dendritic cell co-expressing and / or secreting a checkpoint inhibitor with one or both of an immune modulator and / or an antigenic peptide or protein prolongs the median survival of the treated subject compared to a subject being treated with any one of the first activated dendritic cell, the pharmaceutical composition, the second or the third activated dendritic cell alone.

57. The activated dendritic cell for use of claim 55, wherein the treatment with the activated dendritic cell co-expressing and / or secreting a checkpoint inhibitor with one or both of an immune modulator and / or an antigenic peptide or protein inhibits cancer progression, inhibits increase in the tumor volume, reduces tumor volume, reduces tumor growth rate, eradicates the tumor or tumor cells, and / or prolongs the median survival period relative to treatment with the activated dendritic cell, the pharmaceutical composition, the second and / or the third activated dendritic cell alone.

58. A method for preparing the ex vivo activated dendritic cell of any one of claims 1-15, the method comprising the step of combining the activated dendritic cell with at least one exogenous nucleic acid sequence encoding a checkpoint inhibitor.T08741ANM - 160 -59. The method of claim 58, wherein the exogenous nucleic acid sequence is mRNA and comprised in a lipid nanoparticle (LNP).

60. The method of claim 59, wherein the method comprises a further step of incubating said LNP comprising the at least one mRNA with said activated dendritic cells for a time period sufficient for the activated dendritic cells to take up the LNP.

61. The method of claim 60 wherein the time period is at least 0.1 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h or 24 h.

62. An ex vivo activated dendritic cell obtainable by the method according to any one of claims 58-61.

63. A kit-of-parts comprising a plurality of ex vivo activated dendritic cells and LNPs comprising at least one exogenous mRNA encoding at least one checkpoint inhibitor.

64. The kit according to claim 63, wherein the kit further comprises at least one exogenous mRNA encoding at least one cytokine.

65. The kit according to any one of claims 63 or 64, wherein the kit further comprises at least one exogenous mRNA encoding at least one antigenic peptide or protein.

66. The kit according to claim 65, wherein the at least one exogenous mRNA encoding at least one cytokine and the at least one exogenous mRNA encoding at least one antigenic peptide or protein are either(i) expressed by the same activated dendritic cell as the at least one exogenous mRNA encoding a checkpoint inhibitor, or(ii) each expressed by a different activated dendritic cell.

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