Therapeutic RNA

[0001] This application claims the benefit of priority to United States Provisional Application No.62/464,981, filed February 28, 2017; United States Provisional Application No.62/597,527, filed December 12, 2017; and European Patent Application No.17306089.8, filed August 23, 2017; all of which are incorporated by reference in their entirety.

[0002] This disclosure relates to the field of therapeutic RNA to treat solid tumors. The National Cancer Institute defines solid tumors as abnormal masses of tissue that do not normally contain cysts or liquid areas. Solid tumors include benign and malignant

(cancerous) sarcomas, carcinomas, and lymphomas, and can be physically located in any tissue or organ including the brain, ovary, breast, colon, and other tissues. Cancer is often divided into two main types: solid tumor cancer and hematological (blood) cancers. It is estimated that more than 1.5 million cases of cancer are diagnosed in the United States each year, and more than 500,000 people in the United States will die each year from cancer.

[0003] Solid tumor cancers are particularly difficult to treat. Current treatments include surgery, radiotherapy, immunotherapy and chemotherapy. Surgery alone may be an appropriate treatment for small localized tumors, but large invasive tumors and most metastatic malignancies are usually unresectable by surgery. Other common treatments such as radiotherapy and chemotherapy are associated with undesirable side effects and damage to healthy cells.

[0004] While surgery and current therapies sometimes are able to kill the bulk of the solid tumor, additional cells (including potentially cancer stem cells) may survive therapy. These cells, over time, can form a new tumor leading to cancer recurrence. In spite of multimodal conventional therapies, disease-free survival is less than 25% for many types of solid tumors. Solid tumors that are resistant to multi-modal therapy or that have recurred following therapy are even more difficult to treat, and long-term survival is less than 10%.

[00287] To evaluate the effect of the cytokine mRNA mixture on the development of immunological memory, a re-challenge experiment was performed using the CT26 tumor model. Therefore, CT26 tumor bearing mice were treated with a cytokine mRNA mixture of IL-15sushi, IL-12sc, GM-CSF, and IFNα (Mod B; SEQ ID NOs: 53, 41, 59, and 47). A portion of the cytokine mRNA treatment CT26 tumors completely regressed leading to tumor free animals. Three tumor free animals were then re-challenged with CT26 tumor cells and three tumor free animals were then re-challenged with CT26 tumor cells, in which the gp70 epitope (CT26- ^gp70) was knocked out.9 and 10, respectively, naïve mice were implanted with CT26 tumor cells and CT26- ^gp70 as a positive control for tumor engraftment. On day 21 after tumor inoculation 8 out of 9 naïve mice had engrafted with CT26 tumor cells developed tumors and all 10 naïve mice engrafted with CT26- ^gp70 tumor cells developed tumors whereas all three tumor-free mice in each group rejected the CT26 and CT26- ^gp70 cells and did not exhibit growth of CT26 tumors and CT26- ^gp70, respectively (FIG 12D). This experiment shows that immunological memory upon cytokine mRNA injection in the CT26 tumor model is not restricted to T-cells specific for the immunodominant epitope gp70. Example 5– Systemic Anti-tumor Activity of Cytokine mRNA

[00288] To evaluate the ability of local intratumoral cytokine mRNA to exert a systemic anti-tumor response, mice were engrafted with B16F10 tumor cells on both the left and right flanks (FIG 13A). Mice bearing bilateral B16F10 tumors received four intratumoral injections with control mRNA encoding luciferase or a cytokine mRNA mixture encoding IL-15 sushi, IL-12sc, GM-CSF and IFNα (ModB; SEQ ID NOs: 53, 41, 59, and 47). The right tumor was injected with mRNA at three different dose levels (80 μg, 8 μg, and 0.8 μg mRNA corresponding to 20 μg, 2 μg and 0.2 μg mRNA/target), while tumors on the left flank were untreated. Dose dependent anti-tumor activity was observed in both the injected (FIG 13B) and uninjected (FIG 13C) tumors with tumor growth inhibition ranging from 88% in the uninjected tumor to 96% in the injected tumor. Groups treated with cytokine mRNA treatment had increased median survival compared to groups treated with the Luciferase control mRNA (FIG 13D).

[00289] To further evaluate the ability of local intratumoral cytokine mRNA to exert a systemic anti-tumor response, mice were engrafted with B16F10 tumor cells on the right flank and received an IV injection of Luciferase-expressing B16F10 cells for induction of tumors in the lung (FIG 19A). On day 11, 14 and 18 after SC tumor implantation mice bearing B16F10 tumors received in total three intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα (ModB; SEQ ID NOs: 53, 41, 59, and 47) into the flank tumor only, while tumors in the lung were untreated. The control group received intratumoral injection of equal amounts of control mRNA without any coding sequence. Mice were sacrificed for endpoint analysis on day 20, at which time lungs were taken out and weighed. Figure 19B shows exemplarily bioluminescence measurements of four animals and pictures of the according lungs taken out in order to visualize the dark tumor nodes. Tumor growth of SC tumors was strongly suppressed by injection of cytokine mRNA mixture, whereas tumors injected with control mRNA grew progressively as depicted in

Figure 19C showing mean tumor volume of 15 mice in each group. Lung tumor growth was suppressed in animals which received intratumoral cytokine mRNA injection in SC tumors when compared to animals treated with control mRNA; Figure 19D shows total flux analysis of bioluminescence measurements of all 15 animals per group on day 20, which is a correlate for tumor burden due to Luciferase-expressing tumor cells; line indicates median and asterisk indicates p < 0.05 analyzed by T-test. Additionally, lungs of animals treated with cytokine mRNA had significantly less weight (FIG 19E, line indicates median). Higher weight of lungs of animals treated with control RNA resulted from higher tumor burden.

Example 6– Human Cytokine mRNA

[00290] To evaluate in vitro expression of the human cytokine mRNA, an mRNA mixture encoding the human cytokines IL-15 sushi, IL-12sc, GM-CSF, and IFNα2b (SEQ ID Nos: 26, 18, 29, and 23) (ModB) were transfected into the HEK293 cell line along with four melanoma tumor cell lines (A375, A101D, A2058 and Hs294T) (FIG 14A). The cytokine mRNA mixture exhibited dose dependent expression and secretion across a panel of five human cell lines (FIG 14B-F).

[00291] The pharmacodynamic effects of the human mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα2b were evaluated in vitro with human peripheral blood mononuclear cells (PBMC). In short, human cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα2b (ModB) or the individual cytokine mRNAs encoding IL-12sc, IFNα2b, IL-15 sushi or GM-CSF (ModB) were transfected in HEK293 cells and the conditioned media was collected at 24 hrs, diluted and added to human PBMC (FIG 15A). The median IFNγ levels from 6 donors treated with the cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα2b was 5623 pg/mL, while treatment with the individual cytokine mRNA for IL-12sc, IFNα2b, IL-15 sushi or GM-CSF induced median IFNγ levels of 534, 67, 17, and 4 pg/mL, respectively (FIG 15B).

[00292] In vivo expression of the human cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα2b (ModB) and IL-2, IL-12sc, GM-CSF and IFNα2b (ModB) was monitored in the A375 human melanoma xenograft. Tumor bearing mice received a single injection of cytokine mRNA and tumor samples were collected at 2 hrs, 4 hrs, 8 hrs, 24 hrs, 48 hrs and 72 hrs after mRNA injection. Tumor lysates were prepared and expression of the individual human cytokines IFNα2b (FIG 16A), IL-2 (FIG 16B), IL-12sc (FIG 16C), IL-15 sushi (FIG 16D), GM-CSF (FIG 16E) were evaluated.

[00293] Time dependent expression was observed for each of the individual cytokines with the maximal concentration (Cmax) occurring between 2-8 hrs for the mixtures of IL-15 sushi, IL-12sc, GM-CSF and IFNα2b (Table 4) and IL-2, IL-12sc, GM-CSF and IFNα2b (Table 5).

Table 4: Pharmacokinetic results for the IL-15 sushi mixture

Table 5: Pharmacokinetic results for the IL-2 mixture

[00294] Induction of the human interferon alpha genes ISG15, ISG54 and MX1 were monitored in the A375 tumors as a pharmacodynamics marker at 2h, 4h, 8h, 24h, 48h and 72h following mRNA injection of the cytokine mRNA mixtures of IL-15 sushi, IL-12sc, GM-CSF and IFNα2b (ModB) and IL-2, IL-12sc, GM-CSF and IFNα2b (ModB). Compared to control treated tumors, A375 tumors treated with cytokine mRNA displayed greater than 100-fold induction of ISG15 (FIG 17A), ISG54 (FIG 17B) and MX1 (FIG 17C) with peak induction occurring by 8 hrs after intratumoral mRNA injection.

Example 7– Interferon effect

[00295] B16F10-tumor-bearing mice received intratumoral injections of ModA (“standard”) cytokine mRNA encoding IL-2, Flt3 ligand (FLT3L), 41BBL (also known as CD137L or tumor necrosis factor superfamily member 9), and CD27L-CD40L (this comprises a fusion protein of the soluble domain of CD27L also known as CD70, and CD40L; both the CD27L and the CD40L is comprised of three soluble domains of either CD27L or CD40L, all separated by GS-Linker sequences (FIG 18A, SEQ ID NOs: 32, 62, 68, and 74) or ModB (“modified”) cytokine mRNA encoding IL-2, FLT3L, 41BBL, and CD27L-CD40L (FIG 18B, SEQ ID NOs: 35, 65, 71, and 77). In addition, either ModA mRNA encoding IFNα (SEQ ID NO: 44) or ModB mRNA encoding IFNα (SEQ ID No: 47) was added to the ModA or ModB mRNA mixes, respectively (FIGS 18D and 18E). Anti-tumor activity was assessed.

[00296] Mice treated with this combination of ModA mRNA had 4/9 mice tumor-free without IFNα (FIG 18B) and 3/9 mice tumor-free with IFNα (FIG 18D). Therefore, treatment with IFNα mRNA did not appear to increase the response to the cytokines when mRNA was dosed in the ModA form.

[00297] In contrast, mice treated with the combination of ModB mRNA had 1/9 mice tumor-free without IFNα (FIG 18C) and 7/9 mice tumor-free with IFNα (FIG 18E). Thus, treatment with IFNα mRNA increased the response to the mixture of cytokines when mRNA was dosed in the ModB form.

Example 8– Cytokine mRNA in Combination with Antibodies

[00298] To evaluate the effect of intratumoral injection of cytokine mRNA in combination with systemic administration of antibodies, mice were engrafted with B16F10 or MC38 tumor cells on both the left and right flanks. Mice received four intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα (ModB; SEQ ID NOs: 53, 41, 59, and 47) into only one of the flank tumors on Days 11, 15, 19, 23, while the other flank tumor was left untreated. Mice also received intraperitoneal injection anti-PD1 antibody (Sanofi murinized version of rat IgG2a anti-mouse PD-1 clone RMP1-14 at 5mg/kg) on Days 10, 13, 16, 19, 22, 25. Groups were as follows: 1) control mRNA (80 μg total mRNA; 50 µL intratumoral injection at 1.6mg/mL plus control isotype antibody (clone MOPC-21 (BioLegend); 5mg/kg): 2) control mRNA plus anti-PD1 antibody; 3) cytokine mRNA plus control isotype antibody; and 4) cytokine mRNA plus anti-PD1. Overall survival was monitored in both the B16F10 (FIG 20A) and MC38 (FIG 20B) tumor models. In both tumor models the highest overall survival was observed with the combination of cytokine mRNA and anti-PD-1 treatment with 60% of mice bearing B16F10 and 80% of MC38 bearing mice tumor free at the end of the study. In the B16F10 tumor model 10% of mice treated with anti-PD-1 or cytokine mRNA alone were tumor free, while in the MC38 model 40% of mice treated with anti-PD-1 and 30% of mice treated with cytokine alone were tumor free. The results indicate strong antitumor activity associated with cytokine mRNA and PD-1 combination.

[00299] To further evaluate the ability of local intratumoral cytokine mRNA in combination with the PD-1 antibody to exert a systemic anti-tumor response, mice were engrafted with B16F10 tumor cells on the right flank and received one day later an IV injection of Luciferase-expressing B16F10 cells for induction of lung metastasis. On day 11, 14 and 17 after IV tumor implantation mice bearing B16F10 tumors received in total three intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα (ModB; SEQ ID NOs: 53, 41, 59, and 47) into the flank tumor only, while tumors in the lung were untreated. On the same day mice also received intraperitoneal (IP) injections of PD-1 antibody (Sanofi murinized version of rat IgG2a anti-mouse PD-1 clone RMP1-14 at 10 mg/kg). Groups were as follows: 1) control mRNA (40 µg total mRNA; 50 µL intratumoral injection of control isotype antibody (clone MOPC-21 (BioLegend); 10 mg/kg) (FIG 20C); 2) control mRNA plus anti-PD1 antibody (FIG 20D); 3) cytokine mRNA plus control isotype antibody (FIG 20E); and 4) cytokine mRNA plus anti-PD1 (FIG 20F). Tumor growth of SC tumors was monitored (FIGS 20C-F) as well as survival (FIG 20G). Overall survival in this model was determined by tumor burden due to SC tumors as well as lung pseudometastasis tumor (not shown in this Figure); in some mice the SC tumor was rejected, while lung metastasis grew progressively. The highest overall survival was observed with the combination of cytokine mRNA and anti-PD-1 treatment. 6-7 % of mice treated with cytokine mRNA alone were tumor free, while mice that had received anti-PD-1 alone or control mRNA + isotype antibody were all sacrificed at day 22 due to high tumor burden. The results indicate strong antitumor activity associated with cytokine mRNA and PD-1 combination in this B16F10 tumor model with lung pseudo-metastasis, while anti-PD-1 antibody alone did not show any anti-tumor activity.

[00300] To further evaluate the effect of intratumoral injection of cytokine mRNA in combination with systemic administration of antibodies, mice were engrafted with CT26 tumor cells on right flanks. Mice received four intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα (ModB; SEQ ID NOs: 53, 41, 59, and 47) on day 11, 14, 18 and 21 after tumor inoculation. On the same day mice also received intraperitoneal (IP) injections of an anti-CTLA-4 antibody (100 µg/200 µL per mouse; clone 9H10 from InVivoMAb) or the isotype control antibody (100 µg/200 µL per mouse;

Armenian hamster IgG from BioXCell). Groups were as follows: 1) cytokine mRNA plus anti-CTLA-4 antibody (FIG 21A); 2) cytokine mRNA plus isotype control antibody (FIG 21B); 3) control mRNA plus anti-CTLA-4 antibody (FIG 21C) and 4) control mRNA plus isotype control antibody (FIG 21D). Combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 12 tumor-free mice out of 16 mice on day 55 after tumor inoculation (FIG 21A). Treatment with either cytokine mRNA plus isotype control antibody (FIG 21B) or control mRNA plus anti-CTLA-4 antibody (FIG 21C) induced less anti-tumoral activity with 5 and 7 tumor-free mice at the end of the study, respectively. In comparison, in the group that received control mRNA plus isotype control antibody (FIG 21D), only one tumor-free mouse remained at the conclusion of the study.

[00301] To further evaluate the effect of intratumoral injection of cytokine mRNA in combination with an anti-CTLA-4 antibody, mice were engrafted with B16F10 tumor cells on right flanks. Mice received three intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα (ModB; SEQ ID NOs: 53, 41, 59, and 47) on day 13, 17 and 20 after tumor inoculation. On day 13, 17, 20 and 24 after tumor inoculation mice also received intraperitoneal (IP) injections of an anti-CTLA-4 antibody (100 µg/200 µL per mouse; clone 9H10 from InVivoMAb) or the isotype control antibody (100 µg/200 µL per mouse; Armenian hamster IgG from BioXCell). Tumor growth of SC tumors as well as survival was monitored. Groups were as follows: 1) cytokine mRNA plus anti-CTLA-4 antibody (FIG 21 E); 2) cytokine mRNA plus isotype control antibody (FIG 21F); 3) control mRNA plus anti-CTLA-4 antibody (FIG 21G) and 4) control mRNA plus isotype control antibody (FIG 21H). Combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 6 tumor-free mice out of 9 mice on day 60 after tumor inoculation (FIG 21E). Treatment with cytokine mRNA plus isotype control antibody (FIG 21F) induced less anti-tumoral activity with 2 tumor-free mice out of 9 mice. In comparison, in the two groups that either received control mRNA plus anti-CTLA-4 antibody (FIG 21G) or control mRNA plus isotype control antibody (FIG 21H), no tumor-free mouse remained at the conclusion of the study. Percent survival is depicted in FIG 21I, showing the highest overall survival in the combination of cytokine mRNA and anti-CTLA-4 treatment with 67% of mice bearing tumors at the end of the study (day 70), while 33% of mice treated with cytokine mRNA alone were tumor free. The results indicate strong antitumor activity associated with cytokine mRNA alone and cytokine mRNA and anti-CTLA-4 antibody in this B16F10 tumor model, in which anti-CTLA-4 antibody alone did not show any anti-tumor activity.

Example 9– mRNA cytokine injection in multiple cancer types

[00302] To evaluate the effect of intratumoral injection of cytokine mRNA in various types of cancer, five xenograft mouse models– KM12 (CRC), RPMI8226 (Myeloma), NCI-N87 (Gastric), A375 (Melanoma), and NCI-H1975 (NSCLC)– were tested as described in Example 1. Mice bearing KM12 (CRC), RPMI8226 (Myeloma), NCI-N87 (Gastric), A375 (Melanoma), and NCI-H1975 (NSCLC) tumors received a single intratumoral injection with 8 μg (2 μg/target) human cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and

IFNα (ModB; SEQ ID NOs: 26, 18, 29, and 23) and the encoded cytokines were assessed in the tumor at 24 hours. Expression of each of the 4 cytokines of IL-15 sushi (FIG 22D), IL-12sc (FIG 22A), GM-CSF (FIG 22C) and IFNα (FIG 22B) was detected in all five of the xenograft models with the highest cytokines levels observed in NCI-H1975, followed by A375, NCI-N87, RPMI8226, and KM12.

Example 10– Dose dependent serum expression of cytokines after intratumoral mRNA cytokine injection

[00303] The effect of different intratumoral mRNA doses on the expression of the encoded cytokines was examined in the serum of mice engrafted with a single A375 tumor on the right flank. Mice received a single intratumoral injection of a cytokine mRNA mixture of human IL-15 sushi, IL-12sc, GM-CSF and IFNα (ModB; SEQ ID NOs: 26, 18, 29, and 23). At 6 hours after intratumoral mRNA injection, serum was collected and cytokine expression was analyzed by Meso Scale Discovery assay. Dose dependent expression of each of the mRNA encoded cytokines was observed in the serum from the highest dose of 80 μg (20 μg) to the lowest dose of 0.0256 μg (0.0064 μg). Results are shown in Figures 23A-D.

Example 11– Cytokine mRNA leads to expansion of gp70+ CD8 T cells

[00304] Mice bearing a single CT26 tumor on one flank received a four intratumoral injections of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFNα, and IL-12sc (ModB; SEQ ID NOs: 53, 41, 59, and 47). Blood was collected 13 days after first intratumoral mRNA administration and T cells specific for the gp70 tumor antigen were quantified by flow cytometry. Frequency of T cells specific for the gp70 tumor antigen in blood were strongly increased in mice upon intratumoral injection of mRNA cytokines compared to mice that had received control RNA.

Example 12– Cytokine mRNA induces multiple pro-inflammatory pathways and increases immune infiltrate in both treated and untreated tumors.

[00305] Mice bearing B16F10 tumors on the left and right flank received a single intratumoral injection of 80 μg of mRNA (20 μg/target) into right tumor which was initiated with 0.5 x 10^6 cells (treated), while the tumor initiated with 0.25 x 10^6 cells remained untreated. At seven days after intratumoral injection of mRNA, both tumors were collected, and RNA was isolated and subjected to RNA sequencing analysis. As shown in FIGS.27A-C, treatment with a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFNα, and IL-12sc (SEQ ID NOs: 59, 53, 41, and 47) upregulated multiple proinflammatory pathways including a range of IFNgamma response genes. The upregulation of proinflammatory/IFNgamma

related pathways occurred in both the treated and untreated tumors, supporting the notion that local intratumoral treatment has systemic immune modulatory effects.

[00306] Causal network analysis (part of Ingenuity pathway analysis tool) was performed on 3298 genes that were differentially expressed (1699 up-regulated and 1599 down-regulated) between cytokine mRNA treatment and control in injected flank and 4973 genes (2546 up-regulated and 2427 down-regulated) in un-injected flank to identify changes in signaling pathways that could explain the observed changes in gene expression. Z scores were calculated to indicate the changes in pathways, with signs of score indicating the direction of change (positive sign suggests the activation whereas negative sign suggests inhibition).

[00307] Hierarchical clustering on expression of 328 genes regulated by IFNG (295 up-regulation and 33 down-regulation) was performed in both control and cytokine mRNA treated samples in both injected and un-injected tumors. Expression of each gene across samples were z-score normalized. The similarity metric is based on Pearson’s correlation coefficient and complete-linkage is used to generate dendrogram. See, Table 6.

[00308] Relative abundance of infiltrated immune cells is determined by calculating the average expression of immune cell–type specific gene signatures.

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Example 13– Cytokine mRNA Increases CD4+ and CD8+ T Cells in Both Treated and Untreated Tumors

[00310] Mice bearing B16F10 tumors on the left and right flank received a single intratumoral injection of 80 μg of mRNA (20 μg/target) into right tumor which was initiated with 0.5 x 10^6 cells only one of the tumors (treated), while the other tumor initiated with 0.25 x 10^6 cells remained untreated. At seven days post intratumoral mRNA injection, both the tumors were collected and processed for IHC (Immunofluorescence microscopy) staining with antibodies for CD4+, CD8+, and FoxP3+ cells. Mice from tumors in Figures 28A and 28B were treated with cytokine mRNA, while mice from tumors in Figures 28C and D were treated with control mRNA. Panels A and C are from the tumors injected with mRNA, while panels B and D are from the corresponding contralateral tumor not injected. (FIGS 28A-D). For both the cytokine mRNA treated and control mRNA treated groups 5 tumors injected with RNA and the corresponding 5 contralateral tumors uninjected were subjected to immunofluorescent staining for CD4+, CD8+, FOXP3+ cells. The relative frequency and ratio of cells are plotted in Figures 28E, F, G. The results indicate that a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFNα, and IL-12sc (SEQ ID NOs: 59, 53, 41, and 47) increases CD8+ and CD4+ T cells infiltration leading to altering the CD8+/Treg ratio. An increase in immune infiltration occurred in both the treated and untreated tumors, supporting the notion that local intratumoral treatment has systemic immune modulatory effects.

Example 14– mRNA is expressed in both tumor and infiltrating immune cells

[00311] Mice bearing a single B16F10 tumor received a single intratumoral injection with mRNA encoding the Thy1.1 protein (FIG.29A–G). Approximately 18hrs after intratumoral injection the tumor was dissociated, stained with a panel of antibodies and flow cytometry was performed to define cells expressing Thy1.1 protein. The results indicate that both tumor and immune cells take up and express the mRNA.

Example 15– Dose dependent tumor expression and PD response following

intratumoral cytokine mRNA injection

[00312] Mice bearing B16F10 tumors received a single mRNA injection with 80, 8 or 0.8 µg of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFNα, and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47). Approximately 6 hrs after the intratumoral injection, the tumor was removed and lysed, and levels of IL-15 sushi, GM-CSF, IFNα, and IL-12sc, IFNgamma and IP-10 were quantified in the tumor lysate. Figures 30A-F show that the cytokine mRNA was expressed intratumorally in a dose-dependent manner.

[00313] In a separate experiment, mice bearing B16F10 tumors received a single mRNA injection with 80, 8 or 0.8ug of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFNα, and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47). At seven days following intratumoral cytokine mRNA injection, the tumors were dissociated, stained with a panel of antibodies, and analyzed by flow cytometry. The antibodies used were against murine: CD45, CD4, CD3, CD8, CD279, IFNgamma, TNFalpha, FOXP3, Granzyme B). The results indicate that treatment with the cytokine mRNA mixture altered the CD8+/Treg ratio (FIG 31A-B), led to increased frequency of polyfunctional CD8+ T cells in the tumor microenvironment (FIG 31C-D), increased PD-L1 on infiltrating myeloid cells (FIG 31E), and increased levels of PD-1 on infiltrating CD8+ T cells (FIG 31F).

[00314] In a further experiment, mice bearing B16F10 tumors on the left and right flank received a single intratumoral injection of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFNα, and IL-12sc (SEQ ID Nos: SEQ ID NOs: 59, 53, 41, and 47) or control mRNA into only one of the tumors (treated), while the other tumor remained untreated. At seven days post intratumoral mRNA injection, the injected tumor was collected and processed for flow cytometry staining with antibodies for CD45+, CD8+, CD3+, and Granzyme B. The results indicate that the cytokine mRNA mixture increased the frequency of intratumoral Granzyme B CD8+ T cells in the tumor (FIG 31G-H).

Example 16– Intratumorally injected mRNA is primarily expressed in the tumor

[00315] Mice bearing B16F10 tumors received a single intratumoral injection of 50 μg mRNA encoding firefly luciferase. At 6 and 24-hour time points, 3 mice were sacrificed and tumor, liver, spleen, tumor draining lymph node (TDLN) and non-tumor draining lymph node (NDLN) were analyzed ex vivo for luciferase expression. Figures 32A-B show that luciferase expression was highest in the tumor, in which expression was greater than 100-fold above any other tissue.

Example 17– CD4+, CD8+, and NK cells contribute to the anti-tumor activity of cytokine mRNA in B16F10 model

[00316] Groups of mice bearing B16F10 tumors were treated with 100 μg of depleting antibodies (anti-CD4, anti-CD8, anti-NK1.1) by intraperitoneal injection once a week for 4 weeks total. Antibody mediated cellular depletion was initiated one day prior to treatment with an 80 μg cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFNα, and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47). The effect of antibody depletion on overall survival was monitored. The results, shown in Figure 34, indicate that individual depletion CD8+, CD4+, or NK cells reduced, to varying degrees, the anti-tumor activity and overall survival of the cytokine mRNA.

Example 18– Antitumor activity of cytokine mRNAs is not observed in IFN-gamma-deficient mice

[00317] WT C57BL6J mice and C57BL6J mice deficient for the murine IFNγ (IFNγ KO) were implanted with B16F10 tumor cells as described in Example 1. Mice were treated by intratumoral injection with 80 μg (20 μg/target) cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFNα, and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47) or 80 μg control mRNA, and overall survival was monitored. The results, depicted in Figure 35, indicate that mice lacking IFNγ did not exhibit a detectable antitumor response when treated with the cytokine mRNA.

We claim:

1. A composition or medical preparation comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNα protein, and RNA encoding a GM-CSF protein.

2. The composition according to claim 1.

3. The medical preparation or composition of claim 1 or 2, wherein the IFNα protein is an IFNα2b protein.

4. The medical preparation or composition of any one of claims 1- 3, wherein (i) the RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18 and/or (ii) the IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.

5. The medical preparation or composition of any one of claims 1-4, wherein (i) the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26 and/or (ii) the IL- 15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24.

6. The medical preparation or composition of any one of claims 1-5, wherein (i) the RNA encoding an IFNα protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or (ii) the IFNα protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.

7. The medical preparation or composition of any one of claims 1-6, wherein (i) the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or (ii) the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid

sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.

8. The medical preparation or composition of any one of claims 1-7, wherein at least one RNA comprises a modified nucleoside in place of at least one uridine.

9. The medical preparation or composition of any one of claims 1-7, wherein at least one RNA comprises a modified nucleoside in place of each uridine.

10. The medical preparation or composition of any one of claims 1-7, wherein each RNA comprises a modified nucleoside in place of at least one uridine.

11. The medical preparation or composition of any one of claims 1-7, wherein each RNA comprises a modified nucleoside in place of each uridine.

12. The medical preparation or composition of any one of claims 8-11, wherein the

modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl- pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

13. The medical preparation or composition of any one of claims 7-11, wherein at least one RNA comprises more than one type of modified nucleoside, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl- pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

14. The medical preparation or composition of claim 12, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

15. The medical preparation or composition of any one of claims 1-14, wherein at least one RNA comprises the 5’ cap m27,3’-OGppp(m12’-O)ApG or 3´-O-Me- m7G(5')ppp(5')G.

16. The medical preparation or composition of any one of claims 1-15, wherein each RNA comprises the 5’ cap m27,3’-OGppp(m12’-O)ApG or 3´-O-Me-m7G(5')ppp(5')G. 17. The medical preparation or composition of any one of claims 1-16, wherein at least one RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.

18. The medical preparation or composition of any one of claims 1-17, wherein each RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.

19. The medical preparation or composition of any one of claims 1-18, wherein at least one RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.

20. The medical preparation or composition of any one of claims 1-19, wherein each RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.

21. The medical preparation or composition of any one of claims 1-20, wherein at least one RNA comprises a poly-A tail.

22. The medical preparation or composition of any one of claims 1-21, wherein each RNA comprises a poly-A tail.

23. The medical preparation or composition of claim 21 or 22, wherein the poly-A tail comprises at least 100 nucleotides.

24. The medical preparation or composition of claim 21 or 22, wherein the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 78.

25. The medical preparation or composition of any one of claims 1-24, wherein one or more RNA comprises:

a. a 5’ cap comprising m27,3’-OGppp(m12’-O)ApG or 3´-O-Me-m7G(5')ppp(5')G; b. a 5’ UTR comprising (i) a nucleotide sequence selected from the group

consisting of SEQ ID NOs: 2, 4, and 6, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6; c. a 3’ UTR comprising (i) the nucleotide sequence of SEQ ID NO: 8, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8; and

d. a poly-A tail comprising at least 100 nucleotides.

26. The medical preparation or composition of claim 25, wherein the poly-A tail

comprises SEQ ID NO: 78.

27. The medical preparation or composition of any one of claims 1 to 26, which is a

pharmaceutical composition comprising the RNAs.

28. The medical preparation or composition of claim 27, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

29. The medical preparation or composition of any one of claims 1 to 28, wherein the RNA is formulated as a liquid, formulated as a solid, or a combination thereof.

30. The medical preparation or composition of any one of claims 1 to 29 for

pharmaceutical use.

31. The medical preparation or composition of claim 30, wherein the pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder.

32. The medical preparation or composition of any one of claims 1 to 31 for use in a method for treating or preventing a solid tumor.

33. The medical preparation or composition of claim 32, wherein the solid tumor is a sarcoma, carcinoma, or lymphoma.

34. The medical preparation or composition of any one of claims 32 or 33, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.

35. The medical preparation or composition of any one of claims 32-34, wherein the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.

36. The medical preparation or composition of any one of claims 1-35, wherein the RNA is for intra-tumoral or peri-tumoral administration.

37. The medical preparation or composition of any one of claims 1-36, wherein the RNA is formulated for injection.

38. The medical preparation or composition of any one of claims 1-36, wherein the RNA is for use by injection.

39. The medical preparation or composition of any one of claims 1-38, which is for

administration to a human.

40. The medical preparation or composition of any one of claims 32-39, wherein treating or preventing the solid tumor comprises reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject. 41. A kit comprising the composition of any one of claims 1-29.

42. The medical preparation of any one of claims 1 or 3-29, wherein the medical preparation is a kit.

43. The medical preparation of claim 42, wherein the RNAs are in separate vials.

44. The kit of any one of claims 40-43, further comprising instructions for use of the composition for treating or preventing a solid tumor.

45. RNA for use in a method for treating or preventing a solid tumor in a subject, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNα protein, and RNA encoding a GM-CSF protein.

46. Use of RNA encoding an IL-12sc protein, an IL-15 sushi protein, an IFNα protein, and a GM-CSF protein for the treatment of solid tumor.

47. The RNA or use of any one of claims 45-46, wherein the IFNα protein is an IFNα2b protein.

48. The RNA or use of any one of claims 45-47, wherein (i) the RNA encoding an IL- 12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18 and/or (ii) the IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.

49. The RNA or use of any one of claims 45-48, wherein (i) the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26 and/or (ii) the IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24.

50. The RNA or use of any one of claims 45-49, wherein (i) the RNA encoding an IFNα protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or (ii) the IFNα protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.

51. The RNA or use of any one of claims 45-50, wherein (i) the RNA encoding a GM- CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or (ii) the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.

52. The RNA or use of any one of claims 45-51, wherein at least one RNA comprises a modified nucleoside in place of at least one uridine.

53. The RNA or use of any one of claims 45-52, wherein at least one RNA comprises a modified nucleoside in place of each uridine.

54. The RNA or use of any one of claims 45-53, wherein each RNA comprises a modified nucleoside in place of at least one uridine.

55. The RNA or use of any one of claims 45-54, wherein each RNA comprises a modified nucleoside in place of each uridine.

56. The RNA or use of any one of claims 52-55, wherein the modified nucleoside is

independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

57. The RNA of any one of claims 52-55, wherein at least one RNA comprises more than one type of modified nucleoside, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl- uridine (m5U).

58. The RNA or use of claim 56, wherein the modified nucleoside is N1-methyl- pseudouridine (m1ψ).

59. The RNA or use of any one of claims 45-58, wherein at least one RNA comprises the 5’ cap m27,3’-OGppp(m12’-O)ApG or 3´-O-Me-m7G(5')ppp(5')G.

60. The RNA or use of any one of claims 45-59, wherein at each RNA comprises the 5’ cap m27,3’-OGppp(m12’-O)ApG or 3´-O-Me-m7G(5')ppp(5')G.

61. The RNA or use of any one of claims 45-60, wherein at least one RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.

62. The RNA or use of any one of claims 45-61, wherein each RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.

63. The RNA or use of any one of claims 45-62, wherein at least one RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.

64. The RNA or use of any one of claims 45-63, wherein each RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.

65. The RNA or use of any one of claims 45-64, wherein at least one RNA comprises a poly-A tail.

66. The RNA or use of any one of claims 43-65, wherein each RNA comprises a poly-A tail.

67. The RNA or use of claim 65 or 66, wherein the poly-A tail comprises at least 100 nucleotides.

68. The RNA or use of claim 65 or 66, wherein the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 78.

69. The RNA or use of any one of claims 45-68, wherein one or more RNA comprises: a. a 5’ cap comprising m27,3’-OGppp(m12’-O)ApG or 3´-O-Me-m7G(5')ppp(5')G; b. a 5’ UTR comprising (i) a nucleotide sequence selected from the group

consisting of SEQ ID NOs: 2, 4, and 6, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6; c. a 3’ UTR comprising (i) the nucleotide sequence of SEQ ID NO: 8, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8; and

d. a poly-A tail comprising at least 100 nucleotides.

70. The RNA or use of claim 69, wherein the poly-A tail comprises SEQ ID NO: 78. 71. The RNA or use of any one of claims 45 to 70, wherein the method further comprises administering a further therapy.

72. The RNA or use of claim 71, wherein the further therapy comprises one or more selected from the group consisting of: (i) surgery to excise, resect, or debulk a tumor, (ii) immunotherapy, (iii) radiotherapy, and (iv) chemotherapy.

73. The RNA or use of claim 71 or 72, wherein the further therapy comprises

administering a further therapeutic agent.

74. The RNA or use of claim 73, wherein the further therapeutic agent is an anti-cancer therapeutic agent.

75. The RNA or use of claim 73 or 74, wherein the further therapeutic agent is a

checkpoint modulator.

76. The RNA or use of claim 75, wherein the checkpoint modulator is an anti-PD1

antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.

77. The RNA or use of any one of claims 45-76, wherein the solid tumor is a sarcoma, carcinoma, or lymphoma.

78. The RNA or use of any one of claims 45-77, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.

79. The RNA or use of any one of claims 45-78, wherein the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.

80. The RNA or use of any one of claims 45-79, wherein the RNA is administered intra- tumorally or peri-tumorally.

81. The RNA or use of any one of claims 45-80, wherein the RNA is formulated for injection.

82. The RNA or use of any one of claims 73-81, wherein the further therapeutic agent is administered systemically.

83. The RNA or use of any one of claims 45-82, wherein the subject is a human.

84. The RNA or use of any one of claims 45-83, wherein the RNAs are administered at the same time.

85. The RNA or use of any one of claims 45-84, wherein the RNAs are administered via injection, wherein the RNAs are mixed together in liquid solution prior to injection. 86. The RNA or use of any one of claims 45-85, wherein the RNAs are administered by administering a composition comprising a combination of the RNAs.

87. The RNA or use of any one of claims 45 to 86, wherein treating or preventing a solid tumor comprises reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject.

88. A method for treating or reducing the likelihood of a solid tumor comprising

administering to a subject in need thereof a first RNA, wherein the first RNA encodes an IL-12sc protein, an IL-15 sushi protein, an IFNα protein, or a GM-CSF protein and the subject is further treated with additional RNA, wherein:

a. if the first RNA encodes an IL-12sc protein, then the additional RNA encodes an IL-15 sushi protein, an IFNα protein, and a GM-CSF protein; b. if the first RNA encodes an IL-15 sushi protein, then the additional RNA encodes an IL-12sc protein, an IFNα protein, and a GM-CSF protein; c. if the first RNA encodes an IFNα protein, then the additional RNA encodes an IL-15 sushi protein, an IL-12sc protein, and a GM-CSF protein; and d. if the first RNA encodes a GM-CSF protein, then the additional RNA encodes an IL-15 sushi protein, an IFNα protein, and an IL-12sc protein.